cytotoxic compound and its use, conjugate and its use and pharmaceutical composition

专利摘要:
BENZODIAZEPINE COMPOUND CYTOTOTIC AND CONJUGATE, USE OF THE SAME TO INHIBIT ABNORMAL CELL GROWTH OR TREAT PROLIFERATIVE, AUTOIMMUNE, BONUS DESTRUCTIVE, NEURODEGENERATIVE, INFECTIOUS, HEALTHY, INFECTIOUS, VEGETABLE DISEASE, VARIOUS, DISEASES. The invention relates to compounds derived from benzodiazepine with antiproliferative activity, as well as conjugates thereof linked to a cell binding agent. The invention further provides compositions and uses of the compounds and conjugates to inhibit abnormal cell growth or to treat a proliferative disorder in mammal.
公开号:BR112013019913B1
申请号:R112013019913-0
申请日:2012-02-15
公开日:2021-01-05
发明作者:Wei Li；Michael Miller；Nathan Fishkin；Ravi V.J. Chari
申请人:Immunogen, Inc.；
IPC主号:

专利说明:

REFERENCE TO RELATED REQUESTS
[001] This application claims the benefit of the filing date in accordance with 35 USC §119 (e), of provisional application US 61 / 443,062, filed on February 15, 2011, and provisional application US 61 / 483,499, filed on 6 of May 2011, and provisional application US 61 / 443,092, filed on February 15, 2011, the complete contents of which, including all drawings, formulas, reports and claims, are incorporated herein by reference. FIELD OF THE INVENTION
[002] The present invention relates to new cytotoxic compounds and cytotoxic conjugates comprising these cytotoxic compounds and cell-binding agent. More specifically, this invention relates to new compounds derived from benzodiazepine thereof, intermediates thereof, conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as antiproliferative agents. BACKGROUND OF THE INVENTION
[003] Benzodiazepine derivatives are compounds useful to treat various disorders, and include medications such as antiepileptics (imidazo [2,1-b] [1,3,5] benzothiadiazepines, US patent 4,444,688; US patent 4,062,852) , antibacterials (pyrimido [1,2- c] [1,3,5] benzothiadiazepines, GB 1476684), diuretics and hypotensive (pyrrole (1,2-b) [1,2,5] benzothiadiazepine 5,5 dioxide, patent US 3,506; 646), hypolipidemic (WO 03091232), antidepressants (US patent 3,453,266); osteoporosis (JP 2138272).
[004] Recently, it has been demonstrated in animal tumor models that benzodiazepine derivatives, such as pyrrolobenzodiazepines (PBDs), act as antitumor agents (N-2-imidazolyl substituted alkyl 1,2,5-benzothiadiazepine-1,1-dioxide, patent US 6,156,746), benzo-pyrido or dipyrido thiadiazepine (WO 2004/069843), pyrrole [1,2-b] [1,2,5] benzothiadiazepines and pyrrole [1,2-b] [1,2,5 ] benzodiazepine derivatives (WO2007 / 015280), tomamycin derivatives (for example, pyrrolo [1,4] benzodiazepines), such as those described in WO 00/12508, WO2005 / 085260, WO2007 / 085930, and EP 2019104. Benzodiazepines are still known for affecting cell growth and differentiation (Kamal A., et al., Bioorg Med Chem. 2008 Aug 15; 16 (16): 7804-10 (and references cited in this); Kumar R, Mini Rev Med Chem. 2003 Jun; 3 (4): 323-39 (and references cited in this); Bednarski JJ, et al., 2004; Sutter A. P, et al., 2002; Blatt NB, et al., 2002), Kamal A. et al ., Current Med. Chem., 2002; two; 215-254, Wang J. J., J. Med. Chem., 2206; 49: 1442-1449, Alley M.C. et al., Cancer Res. 2004; 64: 6700-6706, Pepper C. J., Cancer Res 2004; 74: 6750-6755, Thurston D.E. and Bose D.S., Chem Rev 1994; 94: 433-465; and Tozuka, Z., et al., Journal of Antibiotics, (1983) 36; 1699-1708. General structure of PBDs is described in US publication 20070072846. PBDs differ in the number, type and position of substituents, in both aromatic rings A and pyrrole rings C, and in the degree of saturation of ring C. Their capabilities to form an adduct in the minor groove and cross-linked DNA allows them to interfere with DNA processing, thus their potential for use as antiproliferative agents.
[005] The first pyrrolobenzodiazepine to enter the clinic, SJG-136 (NSC 694501) is a potent cytotoxic agent that causes DNA cross-strand crosslinking (SG Gregson et al., 2001, J. Med. Chem., 44: 737 - 748; MC Alley et al., 2004, Cancer Res., 64: 6700-6706; JA Hartley et al., 2004, Cancer Res., 64: 6693-6699; C. Martin et al., 2005, Biochemistry. , 44: 4135-4147; S. Arnould et al., 2006, Mol. Cancer Ther., 5: 1602-1509). The results of a Phase I clinical assessment of SJG-136 revealed that this drug was cytotoxic at extremely low doses (maximum tolerated dose of 45 μg / m2, and several adverse effects were observed, including vascular leak syndrome, peripheral edema, toxicity liver damage and fatigue DNA damage was noted at all doses in circulating lymphocytes (D. Hochhauser et al., 2009, Clin. Cancer Res., 15: 2140- 2147). Thus, there is a need for improved benzodiazepine derivatives than they are less toxic and still therapeutically active to treat a variety of proliferative disease states, such as cancer. SUMMARY OF THE INVENTION
[006] Cytotoxic benzodiazepine dimers disclosed in the art have two imine functionalities in their free forms or reversibly protected form, such as a hydrate, alkoxylate or sulfonate. The presence of these two imine functionalities results in DNA cross-linking (S.G. Gregson et al., 2001, J. Med. Chem., 44: 737- 748). The present invention is partly based on the unexpected finding that cell-binding agent conjugates of new cytotoxic benzodiazepine derivatives, such as indolinobenzodiazapene dimers that are devoid of two imine functionalities (for example, an imine functionality and an amine functionality), and thus unable to cross-link DNA, have a much higher therapeutic index (ratio of maximum tolerated dose to minimum effective dose) in vivo compared to benzodiazepine derivatives that can generate DNA cross-linking that are previously described in the art.
[007] Thus an object of the invention is to provide cytotoxic compound comprising a linking group with a reactive group attached to it capable of covalently attaching the cytotoxic compound to a cell binding agent (CBA, see below), wherein the cytotoxic compound is represented by any of the following formulas:

[008] or a pharmaceutically acceptable salt thereof, where:
[009] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, or a linear or branched alkyl containing 1 to 4 carbon atoms , and when this is a single bond, X is -H, the bonding group with the reactive group attached to it, or a protective amine moiety;
[0010] Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', -NR'COR '', -NR'NR 'R' ', an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (eg, piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by -NR' (C = NH) NR'R ' ', an amino acid, or a peptide represented by -NRCOP', where P 'is an amino acid or polypeptide containing between 2 to 20 amino acid units, -SR, - SOR', -SO2M, -SO3M, -OSO3M, halogen , cyan and an azide; or,
[0011] Y is a sulfite (HSO3, HSO2 or a salt of HSO3-, SO32- or HSO2- formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52- formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a PO3S3-, PO2S23-, POS33- or PS43- salt formed with a cation), thiophosphate ester (RiO) 2PS (ORi), RiS-, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a S2O32- salt formed with a cation), dithionite (HS2O4 or a S2O42- salt formed with a cation), phosphorodithioate (P (= S) (ORk ') (S) (OH) or a salt of the same formed with a cation), hydroxamic acid (Rk'C (= O) NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2- or a salt of HOCH2SO2- formed with a cation, such as HOCH2SO2- Na +) or a mixture thereof, where Ri is straight or branched alkyl containing 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N (Rj) 2, -CO2H, -SO3H, and -PO3H; R1 can be optionally further substituted with a substituent for an alkyl described herein; Rj is straight or branched alkyl containing 1 to 6 carbon atoms; Rk 'is a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; preferably, Y is a bisulfite, hydrosulfite, or metabisulfite adduct, or salts thereof (such as sodium salt);
[0012] M is -H or a cation;
[0013] R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted heterocyclic ring from 3 to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[0014] R 'and R' 'are each independently selected from - H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, a polyethylene glycol - (CH2CH2O) n-Rc unit, and an optionally substituted 3 to 18 membered heterocyclic ring containing 1 to 6 hetero atoms independently selected from O, S, N and P;
[0015] Rc is -H or a substituted or unsubstituted straight or branched alkyl containing 1 to 4 carbon atoms, or the linking group with the reactive group attached to it;
[0016] n is an integer from 1 to 24;
[0017] W is selected from C = O, C = S, CH2, BH, SO and SO2;
[0018] X 'is selected from -H, an amine protecting group, the linking group with the reactive group attached to it, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms , a polyethylene glycol - (CH2CH2O) n-Rc unit, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[0019] Y 'is selected from -H, an oxo group, the linking group with the reactive group attached to it, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, an optionally substituted 6 to 18 membered aryl, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, an optionally substituted 3 to 18 membered heterocyclic ring containing 1 to 6 heteroatoms;
[0020] R1, R2, R3, R4, R1 ', R2', R3 'and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-Rc, halogen, guanidinium [- NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NCO, - NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by - SO2R', a sulfonate -SO3-M +, a sulfate -OSO3-M +, a sulfonamide represented by -SO2NR'R '', cyano, an azido, -COR ', -OCOR', - OCONR'R '' and the linking group with the reactive group attached to it;
[0021] R6 is -H, -R, -OR, -SR, -NR'R '', -NO2, halogen or the linking group with the reactive group attached to it;
[0022] Z and Z 'are independently selected from - (CH2) n'-, - (CH2) n'-CR7R8- (CH2) na'-, - (CH2) n'-NR9- (CH2) na'- , - (CH2) n'-O- (CH2) na'- and - (CH2) nS- (CH2) na-
[0023] n 'and na' are the same or different, and are selected from 0, 1, 2 and 3;
[0024] R7 and R8 are the same or different, and are each independently selected from -H, -OH, -SH, -COOH, -NHR ', a polyethylene glycol unit - (OCH2CH2) n-, an amino acid, a peptide unit containing 2 to 6 amino acids, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[0025] R9 is independently selected from -H, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-;
[0026] A and A 'are the same or different, and are independently selected from -O-, oxo (-C (= O) -), -CRR'O-, - CRR'-, -S-, -CRR'S -, -NR5 and -CRR'N (R5) -;
[0027] R5 for each occurrence is independently -H or an optionally substituted linear or branched alkyl containing 1 to 10 carbon atoms;
[0028] D and D 'are the same or different, and are independently absent or selected from the group consisting of an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an amino acid, a peptide containing 2 to 6 amino acids, and a polyethylene glycol (-OCH2CH2) n- unit;
[0029] L is absent, the linking group with the reactive group attached to it, a polyethylene glycol (-OCH2CH2) n- unit, a linear, branched or cyclic alkyl or alkenyl containing 1 to 10 carbon atoms, a phenyl group , a 3- to 18-membered heterocyclic ring or a 5- to 18-membered heteroaryl ring containing 1 to 6 heteroatoms independently selected from O, S, N and P, where the alkyl or alkenyl is optionally substituted with the linker group with the reactive group linked to it; phenyl or heterocyclic or heteroaryl ring can be optionally substituted, wherein the substituent can be the linking group with the reactive group attached thereto.
[0030] In certain embodiments, X is not the bonding group with the reactive group attached to it. In certain embodiments, the double line - between N and C represents a single bond, Y is not -H.
[0031] In certain embodiments, the compound is not any of the following compounds:


[0032] In certain modalities, Y is an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', - NR'COR '', -NR ' NR'R '', an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (eg piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by - NR '(C = NH) NR'R '', an amino acid, or a peptide represented by - NRCOP ', where P' is an amino acid or polypeptide containing between 2 and 20 amino acid units, -SR, -SOR ', -SO2M, -SO3M, - OSO3M, halogen, cyan and an azide.
[0033] A second object of the invention is to provide cell-binding agent conjugates with the new benzodiazepine compounds or derivatives thereof of the present invention. These conjugates are useful as therapeutic agents, which are released specifically for target cells and are cytotoxic.
[0034] Specifically, a conjugate of the invention may comprise: a cytotoxic compound and a cell binding agent (CBA), wherein the cytotoxic compound comprises a linker group that covalently bonds the cytotoxic compound to the CBA, and in which the compound cytotoxic is represented by any of the following formulas:

[0035] or a pharmaceutically acceptable salt thereof, where:
[0036] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, or a linear or branched alkyl containing 1 to 4 carbon atoms , and when this is a single bond, X is -H, the linking group, or a protective amine moiety;
[0037] Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', -NR'COR '', -NR'NR 'R' ', an optionally substituted 5- or 6-membered heterocycle containing nitrogen (for example, piperidine, tetrahydropyrrole, pyrazole, morpholine), a guanidinium represented by -NR' (C = NH) NR'R '', a amino acid, or a peptide represented by -NRCOP ', where P' is an amino acid or polypeptide containing between 2 to 20 units of amino acids, -SR, - SOR ', -SO2M, -SO3M, -OSO3M, halogen, cyan and an azido; or
[0038] Y is a sulfite (HSO3, HSO2 or a salt of HSO3-, SO32- or HSO2- formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52- formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a PO3S3-, PO2S23-, POS33- or PS43- salt formed with a cation), thiophosphate ester (RiO) 2PS (ORi), RiS-, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a S2O32- salt formed with a cation), dithionite (HS2O4 or a S2O42- salt formed with a cation), phosphorodithioate (P (= S) (ORk ') (S) (OH) or a salt of the same formed with a cation), hydroxamic acid (Rk'C (= O) NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2- or a salt of HOCH2SO2- formed with a cation, such as HOCH2SO2- Na +) or a mixture thereof, where Ri is straight or branched alkyl containing 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N (Rj) 2, -CO2H, -SO3H, and -PO3H; R1 can be optionally further substituted with a substituent for an alkyl described herein; Rj is straight or branched alkyl containing 1 to 6 carbon atoms; Rk 'is a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; preferably, Y is a bisulfite, hydrosulfite, or metabisulfite adduct, or salts thereof (such as sodium salt);
[0039] M is -H or a cation;
[0040] R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted heterocyclic ring from 3 to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[0041] R 'and R' 'are each independently selected from - H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, a polyethylene glycol - (CH2CH2O) n-Rc unit, and an optionally substituted 3 to 18 membered heterocyclic ring containing 1 to 6 hetero atoms independently selected from O, S, N and P;
[0042] Rc is -H or a substituted or unsubstituted straight or branched alkyl containing 1 to 4 carbon atoms, or the linking group;
[0043] n is an integer from 1 to 24;
[0044] W is selected from C = O, C = S, CH2, BH, SO and SO2;
[0045] X 'is selected from -H, an amine protecting group, the linking group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - ( CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3-membered heterocyclic ring to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[0046] Y 'is selected from -H, an oxo group, the linking group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an optionally substituted 6 to 18 aryl members, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, an optionally substituted 3-18 membered heterocyclic ring containing 1 to 6 heteroatoms;
[0047] R1, R2, R3, R4, R1 ', R2', R3 'and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-Rc, halogen, guanidinium [- NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NCO, - NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by - SO2R', a sulfonate -SO3-M +, a sulfate -OSO3-M +, a sulfonamide represented by -SO2NR'R '', cyan, an azido, -COR ', -OCOR', - OCONR'R '' and the linking group;
[0048] R6 is -H, -R, -OR, -SR, -NR'R '', -NO2, halogen or the linking group;
[0049] Z and Z 'are independently selected from - (CH2) n'-, - (CH2) n'-CR7R8- (CH2) na'-, - (CH2) n'-NR9- (CH2) na'- , - (CH2) n'-O- (CH2) na'- and - (CH2) nS- (CH2) na-
[0050] n 'and na' are the same or different, and are selected from 0, 1, 2 and 3;
[0051] R7 and R8 are the same or different, and are each independently selected from -H, -OH, -SH, -COOH, -NHR ', a polyethylene glycol unit - (OCH2CH2) n-, an amino acid, a peptide unit containing 2 to 6 amino acids, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[0052] R9 is independently selected from -H, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-;
[0053] A and A 'are the same or different, and are independently selected from -O-, oxo (-C (= O) -), -CRR'O-, - CRR'-, -S-, -CRR'S -, -NR5 and -CRR'N (R5) -,
[0054] R5 for each occurrence is independently -H or an optionally substituted straight or branched alkyl containing 1 to 10 carbon atoms;
[0055] D and D 'are the same or different, and are independently absent or selected from the group consisting of an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an amino acid, a peptide containing 2 to 6 amino acids, and a polyethylene glycol (-OCH2CH2) n- unit;
[0056] L is absent, the linking group, a polyethylene glycol (-OCH2CH2) n- unit, a linear, branched or cyclic alkyl or alkenyl containing 1 to 10 carbon atoms, a phenyl group, a 3 to heterocyclic ring 18 members or a 5- to 18-membered heteroaryl ring containing 1 to 6 heteroatoms independently selected from O, S, N and P, where the alkyl or alkenyl is optionally substituted with the linking group; phenyl or heterocyclic or heteroaryl ring can be optionally substituted, where the substituent can be the linking group.
[0057] In certain embodiments, X is not the liaison group. In certain embodiments, the double line - between N and C represents a single bond, Y is not -H.
[0058] In certain modalities, Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', - NR'COR '', -NR'NR'R '', an optionally substituted 5- or 6-membered heterocycle containing nitrogen (eg, piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by - NR '(C = NH) NR'R '', an amino acid, or a peptide represented by - NRCOP ', where P' is an amino acid or polypeptide containing between 2 to 20 amino acid units, -SR, -SOR ', -SO2M, -SO3M, - OSO3M, halogen, cyan and an azide. In certain embodiments, Y is not -H.
[0059] In certain embodiments, the conjugate compound is not any of the following compounds (the wavy bond represents the bond by which the compound is attached to CBA):



The present invention further includes the composition (for example, a pharmaceutical composition) comprising new benzodiazepine compounds, derived therefrom, or conjugates thereof, (and / or solvates, hydrates and / or salts thereof) and a carrier ( a pharmaceutically acceptable carrier). The present invention furthermore includes the composition (for example, a pharmaceutical composition) comprising new benzodiazepine compounds, derived therefrom, or conjugates thereof (and / or solvates, hydrates and / or salts thereof), and a carrier (a carrier pharmaceutically acceptable), still comprising a second therapeutic agent. The present compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human). The present compositions are useful for treating conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft-host disease (GVHD), transplant rejection, lupus, myositis, infection, immune deficiency such as AIDS, and inflammatory diseases in a mammal (for example, human).
The present invention includes a method for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human) comprising administering to said mammal a therapeutically effective amount of new benzodiazepine compounds, derived therefrom, or conjugates of the (and / or solvates and salts thereof) or a composition thereof, alone or in combination with a second therapeutic agent. The present invention includes a method for synthesizing and using new benzodiazepine compounds, derived therefrom, and conjugates thereof for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, organisms, or associated pathological conditions.
[0062] The compounds of this invention, derived therefrom, or conjugated thereto, and compositions comprising them, are useful for treating or reducing the severity of disorders, such as, characterized by abnormal cell growth (e.g., cancer). Other applications for compounds and conjugates of this invention include, among others, treating conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft-host disease (GVHD), transplant rejection, lupus, myositis, infection, immune deficiency such as AIDS and inflammatory diseases in a mammal (for example, human).
[0063] As used here, when referring to a group (eg Rc, L, X 'etc.) "is / is" (or "is not") the bonding group or the bonding group with the group reactive attached thereto, the group is understood to "comprise" (or "does not understand") the linking group or the linking group with the reactive group attached thereto. BRIEF DESCRIPTION OF THE FIGURES
[0064] FIGS. 1-6 show the schemes for the synthesis of benzodiazepine compounds and the corresponding linkable compounds suitable for conjugation of the present invention.
[0065] FIG. 7 shows the scheme for the synthesis of representative compounds with modified PEG linkers of the present invention.
[0066] FIG. 8 shows the scheme for the synthesis of representative compounds with a methylthio linker of the present invention.
[0067] FIGS. 9-10 show the schemes for the synthesis of representative compounds containing a tertiary amine of the present invention.
[0068] FIG. 11 shows the scheme for the synthesis of representative compounds with a peptide linker of the present invention.
[0069] FIGS. 12-19 show the schemes for the synthesis of representative compounds suitable for one-step conjugation methods of the present invention.
[0070] FIG. 20 shows the scheme for a two-step monoimine dimer synthesis.
[0071] FIG. 21 shows the scheme for a two-step reduced dimer synthesis.
[0072] FIG. 22 shows the scheme for the one-step synthesis of the representative antibody-drug conjugates.
[0073] FIG. 23 shows the scheme for the two-step synthesis of the representative antibody-drug conjugates.
[0074] FIG. 24 shows the in vitro cytotoxicity of the 1d methyldithium dimer against Namalwa, KB and HL60 / QC cell lines.
[0075] FIG. 25 shows the in vitro cytotoxicity and specificity of huMy9-6-SPDB-1f conjugates against various cell lines. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0076] FIG. 26 shows the in vitro cytotoxicity and specificity of huFOLR1-SPDB-1f conjugates.
[0077] FIG. 27 shows dimer conjugation does not reduce antibody binding affinity. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0078] FIG. 28 shows in vivo antitumor activity of huMy9-6 conjugate. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0079] FIG. 29 shows in vitro cytotoxicity of huMy9- 6-SPDB-1f conjugate against antigen positive cells. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0080] FIG. 30 shows synthetic scheme for preparing disulfide binder containing thioether 27e-h.
[0081] FIG. 31 shows a synthetic scheme for preparing 28c-f dimers.
[0082] FIG. 32 shows a synthetic scheme for preparing dimers bound to phenyl 29b-c.
[0083] FIG. 33 shows the scheme for a synthesis of two alternative steps for monoimine dimers.
[0084] FIG. 34 shows in vitro cytotoxicity for huMy9-6-SPDB- 1f (A), huMy9-6-sulfoSPDB-1f (B) and huMy9-6-BMPS-1f (C) against HL60 / QC (Ag +) cells with and without blocking of antigen binding sites. Note that in all three experiments (34A, 34B, and 34C), sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0085] FIG. 35 shows in vitro cytotoxicity for chB38,1-SPDB-1f (A), and chB38,1-sulfoSPDB-1f (B) against COLO205 cells (Ag +). Note that in both experiments, sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0086] FIG. 36 shows in vivo efficacy of huMy9-6-SPDB-1f in mice containing HL60 / QC. Note that sodium bisulfite was added to the conjugation reaction.
[0087] FIG. 37 shows in vivo efficacy of huFOLR1-SPDB-1f in mice containing KB tumor.
[0088] FIG. 38 shows synthetic scheme of compound 1.
[0089] FIG. 39 shows a synthetic scheme of compound 1d with 5-ethyl-2-methylpyridine borane (PEMB).
[0090] FIG. 40 shows a synthetic scheme of compound 1d with sodium triacetoxyborohydride (STAB).
[0091] FIG. 41 shows a synthetic scheme of compound 31a-c.
[0092] FIG. 42 shows a synthetic scheme of compound 32c, d.
[0093] FIG. 43 shows a synthetic scheme of compounds 1i and 12a.
[0094] FIG. 44 shows antiproliferative activity comparing (A) huMy9-6-SPDB-1f, (B) huMy9-6-sulfoSPDB-1f, and (C) huMy9-6-BMPS- 1f, against OCI-AML3 (Ag +) cells with and without blocking antigen binding sites. Note that in all three experiments, sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[0095] FIG. 45 shows an alternative scheme for synthesizing 4- (benzyloxy) -5-methoxy-2-nitrobenzoic acid used in the preparation of IBD monomer.
[0096] FIG. 46 is an alternative synthesis scheme for (5 - ((2- (2- (2-methoxyethoxy) ethoxy) ethyl) (2-methyl-2- (methyldisulfanyl) propyl) amino) -1,3-phenylene) dimethanol ( 1b).
[0097] FIG. 47 is an alternative synthetic scheme for (5 - ((2- (2- (2-methoxyethoxy) ethoxy) ethyl) (2-methyl-2- (methyldisulfanyl) propyl) amino) -1,3-phenylene) dimethanol ( 1b).
[0098] FIG. 48 is an alternative synthesis scheme for a two-step monoimine dimer synthesis.
[0099] FIG. 49 shows the potency of several conjugates against several cell lines. The IC50 values listed in the table are listed in the nM unit.
[00100] FIG. 50 shows in vivo efficacy of huMy9-6-sulfo-SPDB-1f in mice containing MOLM-13 tumor.
[00101] FIG. 51 shows in vivo efficacy of huMy9-6-sulfo-SPDB-1f in mice containing NB4 tumor.
[00102] FIG. 52 shows in vivo efficacy of huMy9-6-BMPS-1f in mice containing HL60 / QC tumor.
[00103] FIG. 53 shows in vivo efficacy of huMy9-6-BMPS-1f in mice containing MOLM-13 tumor. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[00104] FIG. 54 shows a representative synthesis scheme for a sulfonated Folate / cytotoxic compound conjugate.
[00105] FIG. 55 shows several representative sulfonated drug-antibody conjugates with different ligands.
[00106] FIG. 56 shows in vivo efficacy of huMy9-6-Drug 2 in mice containing HL60 / QC tumor. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[00107] FIG. 57 shows in vivo efficacy of huMy9-6-Drug 2 in mice containing MOLM-13 tumor. Note that sodium bisulfite was added to the conjugation reaction to prepare the conjugate.
[00108] FIG. 58 shows similar in vitro cytotoxicity of HuMy9-6-Drug 2 (conjugates prepared without and with sodium bisulfite against HL60 cells that express CD33 antigen.
[00109] FIG. 59 shows similar cytotoxicity in vitro of anti-CD22 Ab-Drug 2 conjugates prepared without and with sodium bisulfite against CD22 expressing BJAB cells.
[00110] FIG. 60 shows the preparation of huMy9-6-sulfo-SPDB-1d using the highly reactive ligand 4-nitroPy-sulfo-SPDB. DETAILED DESCRIPTION OF THE INVENTION
[00111] The reference will now be prepared in detail to certain modalities of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the listed modalities, it will be understood that the invention is not intended to be limited to these modalities. Otherwise, the invention is intended to cover all alternatives, modifications, and equivalents that may be included in the scope of the present invention, as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.
[00112] It should be understood that any combination of the modalities described here, including those described in different aspects of the invention (for example, compounds, compound-ligand molecules, conjugates, compositions, methods of preparation and use) and different parts of the specification ( including modalities described in the Examples only) can be combined with one or more other modalities of the invention, unless explicitly denied or improper. Combination of modalities are not limited to the specified combinations claimed through the various dependent claims. DEFINITIONS
[00113] "Straight or branched alkyl" as used herein refers to a straight or branched chain saturated monovalent hydrocarbon radical of one to twenty carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH2CH (CH3) 2), 2-butyl, 2-methyl-2- propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Preferably, the alkyl has one to ten carbon atoms. More preferably, the alkyl has one to four carbon atoms.
[00114] "Straight or branched alkenyl" refers to a monovalent straight or branched chain hydrocarbon radical of two to twenty carbon atoms with at least one unsaturation site, that is, a carbon-carbon double bond, in which the alkenyl radical includes radicals containing "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. Examples include, but are not limited to, ethylene or vinyl (-CH = CH2), allyl (-CH2CH = CH2), and the like. Preferably, alkenyl has two to ten carbon atoms. More preferably, the alkyl has two to four carbon atoms.
[00115] "Linear or branched alkynyl" refers to a linear or branched monovalent hydrocarbon radical of two to twenty carbon atoms with at least one unsaturation site, that is, a carbon-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably, alkynyl has two to ten carbon atoms. Most preferably, alkynyl has two to four carbon atoms.
[00116] The term "carbocycle," "carbocyclyl" and "carbocyclic ring" refers to an unsaturated, saturated or partially unsaturated monovalent ring containing 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles containing 7 to 12 atoms can be arranged, for example, as a [4,5], [5,5], [5,6], or [6,6] system, and bicyclic carbocycles containing 9 or 10 ring atoms can be arranged as a bicycle [5,6] or [6,6] system, or as bridged systems like bicycle [2,2.1] heptane, bicycle [2,2,2] octane and bicycle [3, 2.2] nonane. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1 -enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.
[00117] The terms "cyclic alkyl" and "cycloalkyl" can be used interchangeably. These refer to a saturated monovalent carbocyclic ring radical. Preferably, cyclic alkyl is a 3- to 7-membered monocyclic ring radical. More preferably, the cyclic alkyl is cyclohexyl.
[00118] The term "cyclic alkenyl" refers to a carbocyclic radical containing at least one double bond in the ring structure.
[00119] The term "cyclic alkynyl" refers to a carbocyclic radical containing at least one triple bond in the ring structure.
[00120] "Aryl" means a monovalent aromatic hydrocarbon radical of 6-18 carbon atoms derived by the removal of a hydrogen atom from a single carbon atom from a relative aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar." Aryl includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated, or aromatic carbocyclic ring or heterocyclic ring. Typical aryl groups include, but are not limited to, radicals derived from benzene (phenyl), substituted benzenes, naphthalene, anthracene, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Preferably, aryl is a phenyl group.
[00121] The terms "heterocycle," "heterocyclyl," and "heterocyclic ring" are used interchangeably here and refer to a saturated or partially unsaturated carbocyclic radical (that is, containing one or more double and / or triple bonds) within the ring) from 3 to 18 ring atoms where at least one atom in the ring is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the other atoms in the ring being C, where one or more atoms in the ring is optionally independently replaced with one or more substituents described below. A heterocycle can be a monocycle containing 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 hetero atoms selected from N, O, P, and S) or a bicycle containing 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 hetero atoms selected from N, O, P, and S), for example: a bicycle system [4,5], [5,5], [5,6], or [6,6] . Heterocycles are described in Paquette, Leo A .; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to the present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82: 5566. "Heterocyclyl" further includes radicals where the heterocyclic radicals are fused with a saturated, partially unsaturated, or aromatic carbocyclic ring or heterocyclic ring. Examples of heterocyclic ring include, but are not limited to, pyrrolidinyl, tetrahydrofuranoyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidine, morpholino, thiomorpholine, thioxanyl, piperazinyl, piperazinyl, piperazinyl, homiperazine , tietanila, homopiperidinila, oxepanila, tiepanila, oxazepinila, diazepinila, tiazepinila, 2-pyrrolinila, 3-pyrrolinila, indolinila, 2H-piranila, 4H-pyranyl, dioxanila, 1,3-dioxolanila, pyazolinila, ditianila, diitanila, ditanila, ditanila, diitanila, ditanila, diitanila, ditanila, ditanila, ditanila, diitanila, ditanila, diitanila. , dihydrothienyl, dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicico [3,1,0] hexanyl, 3-azabicyclo [4,1,0] heptanil, and azabicyclo [2,2,2] hexanil. Spiro fractions are further included within the scope of this definition. Examples of a heterocyclic group in which ring atoms are replaced with oxo fractions (= O) are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.
[00122] The term "heteroaryl" refers to a 5- or 6-membered monovalent aromatic radical in the ring, and includes fused ring systems (at least one of which is aromatic) of 5-18 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienol, thylazol, isolaazole, isola , isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, benzyl, benziazolazole, triazolezol, triazine , quinoxalinyl, naphthyridinyl, and furopyridinyl.
[00123] The heterocycle or heteroaryl groups can be carbon (bonded to carbon) or nitrogen (bonded to nitrogen) bonded where possible. By way of example and without limitation, carbon-linked heterocycles or heteroaryl are linked in position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2 , 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2 , 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline.
[00124] By way of example and without limitation, heterocycles or heteroaryl linked to nitrogen are attached in position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3- imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of an isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or O -carboline.
[00125] The heteroatoms present in heteroaryl or heterocyclyl include oxidized forms such as NO, SO, and SO2.
[00126] The term "halo" or "halogen" refers to F, Cl, Br or I.
[00127] The alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl described above can optionally be replaced with one or more (for example, 2, 3, 4, 5, 6 or plus) substituents.
[00128] If a substituent is described as being "substituted," a non-hydrogen substituent is in place of a hydrogen substituent on a carbon, oxygen, sulfur or nitrogen of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen substituent is in place of a hydrogen substituent on the alkyl substituent. To illustrate, monofluoralkyl is alkyl substituted with one fluor substituent, and difluoralkyl is alkyl substituted with two fluor substituents. It must be recognized that if there is more than one substitution in a substituent, each non-hydrogen substituent can be identical or different (unless otherwise stated).
[00129] If a substituent is described as being "optionally substituted," the substituent can be (1) unsubstituted, or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the carbon (as far as there is one) can separately and / or together be replaced with an optional substituent independently selected. If a substituent's nitrogen is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogens in the nitrogen (as far as there is one) can each be replaced with an independently selected optional substituent. An exemplary substituent can be described as -NR'R '', where R 'and R' 'together with the nitrogen atom to which they are attached, can form a heterocyclic ring. The heterocyclic ring formed from R 'and R' 'together with the nitrogen atom to which they are attached can be partially or fully saturated. In one embodiment, the heterocyclic ring consists of 3 to 7 atoms. In another embodiment, the heterocyclic ring is selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl and thiazolyl.
[00130] This specification uses the terms "substituent," "radical," and "group" interchangeably.
[00131] If a group of substituents is collectively described as being optionally substituted by one or more of a list of substituents, the group may include: (1) non-substitutable substituents, (2) substitutable substituents that are not substituted by optional substituents, and / or (3) substitutable substituents that are replaced by one or more of the optional substituents.
[00132] If a substituent is described as being optionally substituted with up to a particular number of non-hydrogen substituents, that substituent may be (1) unsubstituted; or (2) replaced by up to that particular number of non-hydrogen substituents or by up to the maximum number of replaceable positions in the substituent, whichever is less. Thus, for example, if a substituent is described as a heteroaryl optionally substituted with up to 3 non-hydrogen substituents, then any heteroaryl with less than 3 substitutable positions could be optionally substituted by up to as many non-hydrogen substituents as the heteroaryl has substitutable positions. Said substituents, in non-limiting examples, can be selected from a linear, branched, cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [-NH (C = NH) NH2 ], - OR100, NR101R102, -NO2, -NR101COR102, -SR100, a sulfoxide represented by -SOR101, a sulfone represented by -SO2R101, a sulfonate -SO3M, a sulfate -OSO3M, a sulfonamide represented by -SO2NR101R102, cyano azido, -COR101, -OCOR101, - OCONR101R102 and a polyethylene glycol unit (-OCH2CH2) nR101 where M is H or a cation (such as Na + or K +); R101, R102 and R103 are each independently selected from linear, branched or cyclic H, alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol (-OCH2CH2) n-R104 unit, where n is an integer from 1 to 24, an aryl containing from 6 to 10 carbon atoms, a heterocyclic ring containing from 3 to 10 carbon atoms and a heteroaryl containing 5 to 10 carbon atoms; and R104 is H or a straight or branched alkyl containing 1 to 4 carbon atoms, where the alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl in the groups represented by R100, R101, R102, R103 and R104 are optionally substituted with a or more (for example, 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO2 and unsubstituted linear or branched alkyl containing 1 to 4 carbon atoms. Preferably, the substituents for alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, optionally substituted described above include halogen, -CN, -NR102R103, -CF3, -OR101, aryl, heteroaryl heterocyclyl, -SR101, -SOR101, -SO2R101 and -SO3M.
[00133] The term "compound" or "cytotoxic compound," "cytotoxic dimer" and "cytotoxic dimer compound" are used interchangeably. They are intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof which has been incorporated by reference. The term further includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs, and prodrug salts of a compound of all the formulas disclosed in the present invention. The term further includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific mention of "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salt" "prodrug," "prophylactic salt," "conjugates," "conjugated salts," "solvate," "hydrate," or "polymorph" in certain aspects of the invention described in this application is not to be interpreted as an intended omission of these forms in other aspects of the invention where the term "compound" is used without mention of these other forms.
[00134] The term "conjugate" as used herein refers to a compound described herein or a derivative thereof which is attached to a cell-binding agent.
[00135] The term "linkable to a cell binding agent" as used herein refers to the compounds described herein or derivatives thereof comprising at least one linker group or a precursor thereof suitable for linking these compounds or derivatives thereof to a cell-binding agent.
[00136] The term "precursor" of a particular group refers to any group that can lead to that group by any deprotection reaction, chemical modification or coupling.
[00137] The term "attached to a cell binding agent" refers to a conjugate molecule comprising at least one of the compounds described here (for example, compounds of formula (I) - (IV) and (VIII) - (XI) and drug-linker compounds described herein), or derived from it attached to a cell-binding agent via an appropriate linker group or a precursor thereof.
[00138] The term "chiral" refers to molecules that have the property of not overlapping the mirror image partner, while the term "achiral" refers to molecules that are superimposed on mirror images of partners.
[00139] The term "stereoisomer" refers to compounds that have identical chemical constitution and connectivity, but different orientations of their atoms in space that cannot be interconverted by rotation around simple bonds.
[00140] "Diastereoisomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of another. Diastereoisomers have different physical properties, for example, melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereoisomers can be separated in high resolution analytical procedures such as crystallization, electrophoresis and chromatography.
[00141] "Enantiomers" refer to two stereoisomers of a compound that are not mirror images superimposed on one another.
[00142] Stereochemical definitions and conventions used here generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the invention can contain chiral or asymmetric centers, and therefore exist in different forms stereoisomeric. It is intended that all stereoisomeric forms of the compounds of the invention, including, among others, diastereoisomers, enantiomers and atropisomers, as well as mixtures thereof as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of polarized light. When describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule around its chiral center. The prefixes d and l or (+) and (-) are used to designate the rotation signal of the plane of light polarized by the compound, with (-) or l meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of another. A specific stereoisomer can further be referred to as an enantiomer, and a mixture of said isomers is generally called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic or racemate mixture, which can occur where there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
[00143] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganizing some of the electrons in the bond.
[00144] The term "prodrug" as used in this application refers to a precursor or form derived from a compound of the invention that is capable of being enzymatically or hydrolytically activated or converted to the most active parent form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (Ed.), Pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, ester-containing prodrugs, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted drugs, phenoxyacidamide optionally substituted, phenoxyacid optionally substituted, phenoxyacid optionally substituted. prodrugs containing optionally substituted phenylacetamide, prodrugs containing 5-fluorocytosine and other 5-fluoruridine that can be converted into the most active cytotoxic free drug. Examples of cytotoxic drugs that can then be derivatized into a prodrug form for use in this invention include, among others, compounds of the invention and chemotherapeutic agents as described above.
[00145] The term "prodrug" is still intended to include a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs can only become active in said reaction under biological conditions, or they can have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of the compounds of any of the formulas disclosed herein that comprise biohydrolyzable fractions such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbates, biohydrolyzable carbonates, biohydrolyzable ureas and phosphorus analogues. Other examples of prodrugs include derivatives of compounds of any of the formulas disclosed herein which comprise -NO, -NO2, -ONO, or -ONO2 fractions. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs."
[00146] A preferred form of the prodrug of the invention includes compounds (with or without any linker group) and conjugates of the invention comprising an adduct formed between an imine bond of the compounds / conjugates and an reactive imine reagent. Another preferred form of prodrug of the invention includes compounds such as those of formula (I) - (IV), where when the double line - between N and C represents a single bond, X is H or an amine protecting group, and the compound becomes a prodrug. The prodrug of the invention may contain one or both of the forms of prodrugs described here (for example, containing an adduct formed between an imine bond of the compounds / conjugates and an imine reactive reagent, and / or containing an leaving group Y when X is - H).
[00147] The term "reactive imine reagent" refers to a reagent that is capable of reacting with an imine group. Examples of the reactive imine reagent include, but are not limited to, sulfites (H2SO3, H2SO2 or a salt of HSO3-, SO32- or HSO2- formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52- formed with a cation), mono, di, tri, and tetra-thiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3 or a PO3S3-, PO2S23-, POS33- or PS43- salt formed with a cation), thio phosphate esters i ii iii ((RO) 2PS ( OR), RSH, R SOH, R SO2H, R SO3H), various amines (hydroxyl amine (eg NH2OH), hydrazine (eg NH2NH2), NH2O-Ri, Ri'NH-Ri, NH2-Ri), NH2-CO-NH2, NH2-C (= S) -NH2, thiosulfate (H2S2O3 or a salt of S2O32- formed with a cation), dithionite (H2S2O4 or a salt of S2O42- formed with a cation), phosphorodithioate (P ( = S) (ORk) (SH) (OH) or a salt of the same formed with a cation), hydroxamic acid (RkC (= O) NHOH or a salt formed with a cation), hydrazide (RkCONHNH2), formaldehyde sulfoxylate (HOCH2SO2H or a HOCH2SO2- salt formed with a cation, such as HOCH2SO2-Na +), glycated nucleotide (such as GDP-mannose), fl udarabine or a mixture thereof, where Ri and Ri 'are each independently linear or branched alkyl containing 1 to 10 carbon atoms and are substituted with at least one substituent selected from -N (Rj) 2, - CO2H, - SO3H, and -PO3H; Ri and Ri 'can be optionally further substituted with a substituent for an alkyl described herein; Rj is straight or branched alkyl containing 1 to 6 carbon atoms; and Rk is a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl (preferably, Rk is a linear or branched alkyl containing 1 to 4 carbon atoms; more preferably, Rk is methyl, ethyl or propyl). Preferably, the cation is a monovalent cation, such as Na + or K +. Preferably, the imine reactive reagent is selected from sulfites, hydroxyl amine, urea and hydrazine. Most preferably, the imine reactive reagent is NaHSO3 or KHSO3.
[00148] As used herein unless otherwise indicated, the terms "biohydrolyzable amide," "biohydrolyzable ester," "biohydrolyzable carbamate," "biohydrolyzable carbonate," "biohydrolyzable ureide" and "biohydrolyzable phosphate analog" means an amide, ester, carbamate, carbonate, ureide, or phosphate analog, respectively, which: 1) does not destroy the biological activity of the compound and gives that compound advantageous properties in vivo, such as absorption, duration of action, or onset of action; or 2) is itself biologically inactive but is converted in vivo to a biologically active compound. Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, α-amino acid amides, alkoxyalkyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyalkoxy esters, alkyl acylamino esters, and choline esters. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylene diamines, amino acids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines. Particularly favored prodrugs and salts of drugs are those that increase the bioavailability of the compounds of this invention when said compounds are administered to a mammal.
[00149] The phrase "pharmaceutically acceptable salt" as used herein, refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate , ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sucrate, format, benzoate, glutamate, methanesulfonate "mesylate," ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (ie 1,1'-methylene-bis-bis (2-hydroxy-3-naphthoate)) salts, alkali metal salts eg sodium and potassium), alkaline earth metal salts (eg magnesium), and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or another counterion. The counterion can be an organic or inorganic portion that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have more than one atom charged in its structure. Cases where several charged atoms are part of the pharmaceutically acceptable salt can have several counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.
[00150] If the compound of the invention is a base, the desired pharmaceutically acceptable salt can be prepared by any method available appropriate in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, to acid pyranosidyl, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.
[00151] If the compound of the invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any appropriate method, for example, treatment of the free acid with an organic or inorganic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium , potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
[00152] As used herein, the term "solvate" means a compound that further includes a stoichiometric or non-stoichiometric amount of solvent such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and dichloromethane ethanolamine, 2-propanol, or the like, bound by non-covalent intermolecular forces. Solvates or hydrates of the compounds are readily prepared by adding at least one molar equivalent of a hydroxyl solvent such as methanol, ethanol, 1-propanol, 2-propanol or water to the compound to result in solvation or hydration of the imine moiety.
[00153] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. "Abnormal cell growth," as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (for example, loss of contact inhibition). This includes, for example, the abnormal growth of: (1) tumor cells (tumors) that proliferate by expression of a mutated tyrosine kinase or overexpression of a tyrosine kinase receptor; (2) benign and malignant cells from other proliferative disorders in which abnormal tyrosine kinase activation occurs; (3) any tumor that proliferates by abnormal activation of tyrosine receptor kinases; (4) any tumor that proliferates by abnormal activation of serine / threonine kinase; and (5) benign and malignant cells from other proliferative diseases in which abnormal activation of serine / threonine kinase occurs.
[00154] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells, and / or benign or precancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and lymphoid leukemia or malignancies. More particular examples of such cancers include squamous cell cancer (eg, epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and squamous lung carcinoma , peritoneum cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer , endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penis carcinoma, acute leukemia, head / brain and neck cancer, cancers of lymphatic organs and haematological malignancy including leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), lymphocytic leukemia chronic disease (CLL), chronic myeloid leukemia (CML), acute monocytic leukemia (AMOL), hair cell leukemia (HCL), prolymphocytic T cell leukemia (T-PLL), large granular lymphocytic leukemia, adult T cell leukemia , Lymphoma (small lymphocytic lymphoma (SLL), Hodgkin's lymphoma (Nodular sclerosis, Mixed cellularity, Lymphocyte-rich, Depleted or non-depleted lymphocyte, and Hodgkin's lymphoma predominant in nodular lymphocyte), Non-Hodgkin's lymphoma (all subtypes), Leukemia chronic lymphocytic / small lymphocytic lymphoma, B-cell lymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms (Plasma cell myeloma, Plasmacytoma, immunoglobulin deposition diseases, immunoglobulin chain diseases extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle, diffuse large B-cell lymphoma, large mediastinal (thymic) B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, T-cell prolymphocytic leukemia, large granular T-cell lymphocytic leukemia , Aggressive NK cell leukemia, Adult T cell leukemia / lymphoma, Extranodal NK / T cell lymphoma (nasal type), Enteropathy type T cell lymphoma, Hepatosplenic T cell lymphoma, Blast NK cell lymphoma, Fungi mycosis / Syndrome de Sezary, Primary cutaneous CD30 positive T cell lymphoproliferative disorders, Primary cutaneous anaplastic large cell lymphoma, Lymphoid papulosis, Angioimmunoblastic T cell lymphoma, Peripheral T cell lymphoma (unspecified), Anaplastic large cell lymphoma), Multiple myeloma (plasma cell myeloma or Kahler's disease).
[00155] A "therapeutic agent" includes both a biological agent such as an antibody, a peptide, a protein, an enzyme or a chemotherapeutic agent.
[00156] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Novartis, Imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamicin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TY , GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs), and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as tiotepa® and CYTOANAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenomelamine, triethylenophosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including the synthetic topotecan analogue); briostatin; calistatin; CC-1065 (including their synthetic analogues adozelesin, carzelesin and bizelesin); cryptoficina (particularly cryptoficina 1 and criptoficina 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleuterobin; pancratistatin; a sarcodictiina; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mecloretamine, mecloretamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediin antibiotics (eg calicheamicin, especially calicheamicin gammall and calicheamicin omegall (Angew Chem. Intl. Ed. Engl. (1994) 33: 183-186); dinemicin, including dinemicin A; bisphosphonates, such as clodronate; a speramycin ; as well as chromophore neocarzinostatin and enedin related chromoprotein antibiotic), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, Caminomycin, carzinophylline, chromomycin, dactinomycin, daunorinicine-5-nororubin-5-nororubin, 5-diororin-oxine, 6 ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin as well, mycomycin, purine, mycomycin, in mycomycin, purine , rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; erogenous like calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutetimide, mitotane, trilostane; replenishing folic acid as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucila; bisanthrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; eliptinium acetate; an epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2.2 ', 2' '- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesina; dacarbazine; Mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinosideo ("Ara-C"); cyclophosphamide; thiotepa; taxoids, for example, TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle paclitaxel formulations (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE ® (doxetaxel; Rhona-Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR® (gemcitabine); 6- thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); new chair; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase RFS 2000 inhibitors; difluormethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
[00157] Also included in the definition of "chemotherapeutic agent" are: (i) anti-hormonal agents act to regulate or inhibit hormonal action in tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulate the production of estrogen in the adrenal glands, such as, for example, 4 (5) -imidazois, aminoglutetimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer) , formestania, fadrozol, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a nucleoside cytosine analog of 1,3-dioxolane); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in the signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes as inhibitors of VEGF expression (for example, ANGIOZYME®) and inhibitors of HER2 expression; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; a topoisomerase 1 inhibitor like LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and (x) pharmaceutically acceptable salts, acids and derivatives of any of the above. Other anti-angiogenic agents include MMP-2 inhibitors (matrix metalloproteinase 2), MMP-9 inhibitors (matrix metalloproteinase 9), COX-II inhibitors (cyclooxygenase II), and VEGF tyrosine kinase receptor inhibitors. Examples of said useful matrix metalloproteinase inhibitors that can be used in combination with the present compounds / compositions are described in WO 96/33172, WO 96/27583, EP 818442, EP 1004578, WO 98/07697, WO 98/03516 , WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, EP 606,046, EP 931,788, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO 99 / 07675, EP 945864, US patent 5,863,949, US patent 5,861,510, and EP 780,386, all of which are incorporated herein in their entirety by reference. Examples of VEGF tyrosine kinase receptor inhibitors include 4- (4-bromo-2-fluoranilino) -6-methoxy-7- (1-methylpiperidin-4-ylmethoxy) quinazoline (ZD6474; Example 2 in WO 01/32651), 4 - (4-fluor-2-methylindol-5-yloxy) - 6-methoxy-7- (3-pyrrolidin-1-ylpropoxy) -quinazoline (AZD2171; Example 240 in WO 00/47212), vatalanib (PTK787; WO 98 / 35985) and SU11248 (sunitinib; WO 01/60814), and compounds such as those disclosed in PCT publications WO 97/22596, WO 97/30035, WO 97/32856, and WO 98/13354).
[00158] Other examples of chemotherapeutic agents that can be used in combination with the present compounds include PI3K (phosphoinositide-3 kinase) inhibitors, such as those reported in Yaguchi et al (2006) Jour. of the Nat. Cancer Inst. 98 (8): 545-556; US patent 7,173,029; US patent 7,037,915; US patent 6,608,056; US patent 6,608,053; US patent 6,838,457; US patent 6,770,641; US patent 6,653,320; US patent 6,403,588; WO 2006/046031; WO 2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO 2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO 2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US 2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP 08176070; US patent 6,703,414; and WO 97/15658, all of which are incorporated herein in their entirety by reference. Specific examples of said PI3K inhibitors include SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis, Inc.).
[00159] Chemotherapeutic agents may also include any of the generic or biosimilar drugs of the branded drugs referenced here, or improvements thereof, including improved formulations, prodrugs, release means (sustained release, bioadhesive coating, targeted release, etc.), and dosage forms.
[00160] A "metabolite" is a product produced by the metabolism in the body of a specific compound, a derivative thereof, or a conjugate thereof, or salt thereof. Metabolites of a compound, a derivative thereof, or a conjugate thereof, can be identified using routine techniques known in the art and their activities determined using tests such as those described here. Said products can result, for example, from the oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound. Thus, the invention includes metabolites of compounds, a derivative thereof, or a conjugate thereof, of the invention, including compounds, a derivative thereof, or a conjugate thereof, produced by a process comprising contacting a compound, a derivative thereof , or a conjugate thereof, of this invention with a mammal for a period of time sufficient to generate a metabolic product thereof.
[00161] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated with it.
[00162] The term "protecting group" or "protecting moiety" refers to a substituent that is commonly used to block or protect a particular functionality while reacting with other functional groups in the compound, a derivative thereof, or a conjugate thereof . For example, an "amine protecting group" or an "amino protecting moiety" is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Such groups are well known in the art (see, for example, P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, chapter 7, J. Wiley & Sons, NJ) and exemplified by carbamates such as methyl and ethyl carbamate, FMOC, substituted ethyl carbamates, 1,6-e-eliminated carbamates (also called "auto immolative"), ureas, amides, peptides, alkyl and aryl derivatives. Suitable amino protecting groups include acetyl, trifluoracetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their uses, see P. G.M. Wuts & T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2007.
[00163] The term "exit group" refers to a group of loaded or unloaded portion that departs during a replacement or displacement. Said leaving groups are well known in the art and include, among others, halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates, nitriles, azide, carbamate, disulfides, thioesters, thioethers and diazonium compounds.
[00164] The term "bifunctional cross-linking agent," "bifunctional linker" or "cross-linking agents" refers to modifying agents that have two reactive groups; one of which is capable of reacting with a cell-binding agent while the other is reacting with the cytotoxic compound to link the two fractions. Such bifunctional crosslinkers are well known in the art (see, for example, Isalm and Dent in Bioconjugation chapter 5, p218-363, Groves Dictionaries Inc. New York, 1999). For example, bifunctional cross-linking agents that allow binding through a thioether bond include N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC) to introduce maleimido groups, or with N-succinimidyl-4 - (iodoacetyl) - aminobenzoate (SIAB) to introduce iodoacetyl groups. Other bifunctional cross-linking agents that introduce maleimido groups or haloacetyl groups into a cell binding agent are well known in the art (see US patent application 2008/0050310, 20050169933, available from Pierce Biotechnology Inc., PO Box 117, Rockland, IL 61105, USA) and include, but are not limited to, bis-maleimidopolyethylene glycol (BMPEO), BM (PEO) 2, BM (PEO) 3, N- (β-maleimidopropyloxy) succinimide ester (BMPS), Y- acid N-succinimidyl ester maleimidobutyric acid (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), NHS 5-maleimidovaleric acid, HBVS, N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy (6-amidocaproate) ), which is a "long chain" analog of SMCC (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4- (4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH) , N-succinimidyl 3- (bromoacetamido) propionate (SBAP), N-succinimidyl iodoacetate (SIA), K-maleimidoundecanoic acid N-succinimidyl ester (KMUA), Ns uccinimidyl 4- (p-maleimidophenyl) - butyrate (SMPB), succinimidyl-6- (β-maleimidopropionamido) hexanoate (SMPH), succinimidyl- (4-vinylsulfonyl) benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4 -bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4- (N-maleimido-methyl) cyclo- hexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl) aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N- (Y-maleimidobutyriloxide) sulfosucide sulfo-GMBS), N- (ε-maleimidocaproyloxy) sulfosuccimido ester (sulfo-EMCS), N- (k- maleimidoundecanoyloxy ester) sulfosuccinimide (sulfo-KMUS), and sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate (sulfo-SM) .
[00165] Heterobifunctional crosslinking agents are bifunctional crosslinking agents containing two different reactive groups. Heterobifunctional crosslinking agents containing both an N-hydroxysuccinimide amine-reactive group (NHS group) and a hydrazine carbonyl-reactive group can further be used to bind the cytotoxic compounds described herein with a cell binding agent (for example, antibody). Examples of said commercially available heterobifunctional crosslinking agents include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazidotherephthalate hydrochloride (SHTH) and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates containing a labile acid bond can further be prepared using a hydrazine-containing benzodiazepine derivative of the present invention. Examples of bifunctional cross-linking agents that can be used include succinimidyl-p-formyl benzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).
[00166] Bifunctional cross-linking agents that allow the binding of cell binding agent with cytotoxic compounds through disulfide bonds are known in the art and include N-succinimidyl- 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl- 4- (2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4- (2-pyridyldithio) butanoate (SPDB), N-succinimidyl-4- (2-pyridyldithio) 2-sulfobutanoate (sulfo-SPDB) to introduce groups dithiopyridyl. Other bifunctional cross-linking agents that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. patents 6,913,748, 6,716,821 and US Patent publications 20090274713 and 20100129314, all of which are incorporated herein by reference. Alternatively, crosslinking agents such as 2-iminothiolane, homocysteine thiolactone or S-acetylsuccinic anhydride that introduce thiol groups can still be used.
[00167] A "linker," "linker moiety," or "linker group" as defined herein refers to a moiety that connects two groups, such as a cell-binding agent and a cytotoxic compound, together. Typically, the linker is substantially inert under conditions to which the two groups it is connecting are linked. A bifunctional cross-linking agent can comprise two reactive groups, one at each end of a linker portion, so that a reactive group can be first reacted with the cytotoxic compound to provide a compound containing the linker portion and a second reactive group, which it can then react with a cell-binding agent. Alternatively, one end of the bifunctional cross-linking agent can first be reacted with the cell-binding agent to provide a cell-binding agent containing a binding moiety and a second reactive group, which can then be reacted with a cytotoxic compound. The binding portion may contain a chemical bond that allows the release of the cytotoxic portion at a particular location. Such chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds (see, for example, US Patents 5,208,020; 5,475,092; 6,441 .163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). Disulfide bonds, labile bonds to thioether and peptidase are preferred. Other binders that can be used in the present invention include non-cleavable binders, such as those described in detail in publication number US 20050169933, or charged binders or hydrophilic binders are described in US 2009/0274713, US 2010/01293140 and WO 2009/134976, each one of which is expressly incorporated herein by reference, each of which is expressly incorporated herein by reference.
[00168] In one embodiment, the bonding group with a reactive group attached at one end, such as a reactive ester, is selected from one of the following: - O (CR20R21) m (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -O (CR20R21) m (CR26 = CR27) m '(CR22R23) n (OCH2CH2) p (CR40R41) p" Y' '(CR24R25) q (CO ) tX '', -O (CR20R21) m (alkynyl) n '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y' '(CR24R25) q (CO) tX' ', -O (CR20R21) m ( piperazino) t '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y' '(CR24R25) q (CO) tX' ', -O (CR20R21) m (pyrrole) t' (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -O (CR20R21) mA" m "(CR22R23) n (OCH2CH2) p (CR40R41) p" Y '' (CR24R25) q (CO ) tX '', -S (CR20R21) m (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -S (CR20R21) m (CR26 = CR27) m '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y' '(CR24R25) q (CO) tX' ', -S (CR20R21) m (alkynyl) n' (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -S (CR20R21) m (piperazine) t '(CR22R23) n (OCH2CH2) p (CR40R41) p" Y' '(CR24R25) q (CO) tX '', -S (CR20R21) m (pyrrole) t '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y' '(CR24R25) q (CO) tX' ', -S (CR20R 21) mA "m" (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -NR33 (C = O) p" (CR20R21) m (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -NR33 (C = O) p" (CR20R21) m (CR26 = CR27) m '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', -NR33 (C = O) p" (CR20R21) m (alkynyl) n '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q- (CO) tX '', -NR33 (C = O) p" (CR20R21) m (piperazine) t '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y' ' (CR24R25) q (CO) tX '', -NR33 (C = O) p "(CR20R21) m (pyrrole) t '(CR22R23) n (OCH2CH2) p (CR40R41) p" Y' '(CR24R25) q- (CO) tX '', -NR33 (C = O) p "(CR20R21) mA" m "(CR22R23) n (OCH2CH2) p (CR40R41) p" Y '' (CR24R25) q (CO) tX '', - (CR20R21) m (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', - (CR20R21) m (CR26 = CR27) m '(CR22R23) n (OCH2CH2) ) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', - (CR20R21) m (alkynyl) n '(CR22R23) n (OCH2CH2) p (CR40R41) p" Y' '(CR24R25) q (CO) tX '', - (CR20R21) m (piperazine) t '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y' '(CR24R25) q (CO) tX' ', - (CR20R21) mA "m" (CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', - (CR20R21) m (CR29 = N-NR30) n" (CR22R23) n (OCH2CH 2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', - (CR20R21) m (CR29 = N-NR30) n" (CR26 = CR27) m '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q (CO) tX '', - (CR20R21) m (CR29 = N-NR30) n" (alkynyl) n '(CR22R23) n (OCH2CH2) p (CR40R41) p "Y '' (CR24R25) q- (CO) tX '', - (CR20R21) m (CR29 = N-NR30) n" A "m" (CR22R23) n (OCH2CH2) p (CR40R41) p "Y ' '(CR24R25) q (CO) tX' ',
[00169] where:
[00170] m, n, p, q, m ', n', t 'are integers from 1 to 10, or are optionally 0;
[00171] t, m ", n", and p "are 0 or 1;
[00172] X "is selected from OR36, SR37, NR38R39, where R36, R37, R38, R39 are H, or linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 20 carbon atoms and, or, one polyethylene glycol unit - (OCH2CH2) n, R37, optionally, is a thiol protecting group when t = 1, COX '' forms a reactive ester selected from N-hydroxysuccinimide esters, N-hydroxyphalimide esters, N-hydroxy sulfo-succinimized esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorfenyl esters and their derivatives, wherein said derivatives facilitate amide bond formation;
[00173] Y '' is absent or is selected from O, S, S-S or NR32, where R32 has the same definition given above for R; or
[00174] when Y "is not S-S and t = 0, X" is selected from a maleimido group, a haloacetyl group or SR37, where R37 has the same definition as above;
[00175] A "is an amino acid selected from glycine, alanine, leucine, valine, lysine, citrulline and glutamate or a polypeptide containing between 2 to 20 units of amino acids;
[00176] R20, R21, R22, R23, R24, R25, R26, and R27 are the same or different, and are -H or a linear or branched alkyl containing 1 to 5 carbon atoms;
[00177] R29 and R30 are the same or different, and are -H or alkyl of 1 to 5 carbon atoms;
[00178] R33 is -H or linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 12 carbon atoms, a polyethylene glycol unit R- (OCH2CH2) n-, or R33 is -COR34, -CSR34, -SOR34 , or - SO2R34, where R34 is H or linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 20 carbon atoms or, a polyethylene glycol unit - (OCH2CH2) n; and
[00179] one of R40 and R41 is optionally a negatively or positively charged functional group and the other is H or alkyl, alkenyl, alkynyl containing 1 to 4 carbon atoms.
[00180] Any of the above linker groups can be present in any of the compounds, drug-linker compounds, or conjugates of the invention, including substituting the linkage group of any of the formulas described herein.
[00181] The term "amino acid" refers to naturally occurring amino acids or non-naturally occurring amino acids represented by NH2- C (Raa'Raa) -C (= O) OH, where Raa and Raa 'are each independently H , an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl. The term "amino acid" further refers to the corresponding residue when a hydrogen atom is removed from the amine and / or carboxy end of the amino acid, such as -NH-C (Raa'Raa) -C (= O) O-.
[00182] The term "cation" refers to an ion with a positive charge. The cation can be monovalent (for example, Na +, K +, etc.), bi-valent (for example, Ca2 +, Mg2 +, etc.) or multi-valent (for example, Al3 + etc.). Preferably, the cation is monovalent.
[00183] The term "therapeutically effective amount" means the amount of active or conjugated compound that induces the biological response in a subject. Said response includes relief of symptoms of the disease or disorder being treated, prevention, inhibition or a delay in the recurrence of symptoms of the disease or the disease itself, an increase in the subject's longevity compared to the absence of treatment, or prevention, inhibition or delay in progression of the symptom of the disease or the disease itself. Determination of the amount effected is within the capacity of those skilled in the art, especially in light of the detailed disclosure provided here. The toxicity and therapeutic efficacy of compound I can be determined by standard pharmaceutical procedures in cell cultures and experimental animals. The effective amount of the compound or conjugate of the present invention or other therapeutic agent to be administered to a subject will depend on the stage, category and state of multiple myeloma and characteristics of the subject, such as general health, age, sex, body weight and tolerance to the drug. The effective amount of the compound of the present invention or other therapeutic agent to be administered will further depend on the route of administration and dosage form. The dosage amount and interval can be individually adjusted to provide plasma levels of the active compound that are sufficient to maintain the desired therapeutic effects. Cytotoxic Compounds
[00184] The present invention is directed to cytotoxic compounds described here (for example, compounds of formulas (I), (II), (III), and (IV)). In one embodiment, the cytotoxic compounds of the present invention do not include any compounds described in US 2010/0203007 (the full teaching of which is incorporated herein by reference), such as those specifically disclosed in the provision below.
[00185] In a first specific embodiment, the invention provides a cytotoxic compound comprising a linking group with a reactive group attached to it capable of covalently attaching the cytotoxic compound to a cell binding agent (CBA), wherein said cytotoxic compound is represented by any of the following formulas (I), (II), (III) or (IV):

[00186] or a pharmaceutically acceptable salt thereof, where:
[00187] the double line - between N and C represents a single bond or a double bond, since when this is a double bond, X is absent and Y is -H, or a linear or branched alkyl containing 1 to 4 atoms of carbon, and when this is a single bond, X is -H, the bonding group to the reactive group attached to it, or a protective amine moiety; preferably, the double line - between N and C represents a double bond;
[00188] Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', -NR'COR '', -NR'NR 'R' ', an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (eg, piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by -NR' (C = NH) NR'R ' ', an amino acid, or a peptide represented by -NRCOP', where P 'is an amino acid or polypeptide containing between 2 to 20 amino acid units, -SR, - SOR', -SO2M, -SO3M, -OSO3M, halogen , cyan and an azido, where M is -H or a cation; like Na + or K +. Preferably, M is -H or Na +. Preferably, Y is selected from -SO3M, -OH, -OMe, -OEt or - NHOH. More preferably, Y is -SO3M or -OH; or,
[00189] Y is a sulfite (HSO3, HSO2 or a salt of HSO3-, SO32- or HSO2- formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52- formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a PO3S3-, PO2S23-, POS33- or PS43- salt formed with a cation), thio phosphate ester (RiO) 2PS (ORi), RiS-, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a S2O32- salt formed with a cation), dithionite (HS2O4 or a S2O42- salt formed with a cation), phosphorodithioate (P (= S) (ORk ') (S) (OH) or a salt of the same formed with a cation), hydroxamic acid (Rk'C (= O) NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2- or a salt of HOCH2SO2- formed with a cation, such as HOCH2SO2- Na +) or a mixture thereof, where Ri is straight or branched alkyl containing 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N (Rj) 2, -CO2H, -SO3H, and -PO3H; R1 can be optionally further substituted with a substituent for an alkyl described herein; Rj is straight or branched alkyl containing 1 to 6 carbon atoms; Rk 'is a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; preferably, Y is a bisulfite, hydrosulfite, or metabisulfite adduct, or salts thereof (such as sodium salt);
[00190] R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted heterocyclic ring from 3 to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[00191] R 'and R "are the same or different, and are independently selected from -H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, a polyethylene glycol - (CH2CH2O) n-Rc unit, and an optionally substituted 3 to 18 membered heterocyclic ring containing 1 to 6 hetero atoms independently selected from O, S, N and P;
[00192] Rc is -H or a substituted or unsubstituted straight or branched alkyl containing 1 to 4 carbon atoms, or the linking group with the reactive group attached thereto;
[00193] n is an integer from 1 to 24;
[00194] W is selected from C = O, C = S, CH2, BH, SO, and SO2;
[00195] X 'is selected from the group consisting of -H, -OH, an amine protecting group, the linking group with the reactive group attached to it, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol - (CH2CH2O) n-Rc unit, an optionally substituted aryl containing 6 to 18 carbon atoms (eg phenyl), an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P. Preferably, X 'is -H, -OH, Me or the liaison group with the reactive group attached to it. More preferably, X 'is -H;
[00196] Y 'is selected from the group consisting of -H, an oxo group, the linking group with the reactive group attached to it, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 atoms carbon, an optionally substituted 6-18 membered aryl, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, an optionally substituted 3-18 membered heterocyclic ring containing 1 to 6 heteroatoms. Preferably, Y 'is selected from -H or oxo. More preferably, Y 'is -H;
[00197] R1, R2, R3, R4, R1 '. R2 '. R3 'and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n- Rc, halogen, guanidinium [- NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NCO, -NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by - SO2R ', a sulfonate -SO3-M +, a sulfate -OSO3-M +, a sulfonamide represented by -SO2NR'R' ', cyano, an azide, -COR', -OCOR ', - OCONR'R '' and the bonding group with the reactive group attached to it. Preferably, one of R2, R3, R2 'and R3' is the linking group with the reactive group attached to it and the others are -H;
[00198] R6 is -H, -R, -OR, -SR, -NR'R '', -NO2, halogen, the linking group with the reactive group attached to it, -ORc or -SRc, where Rc is - H, a straight or branched alkyl containing 1 to 4 carbon atoms. Preferably, R6 is -OMe or -SMe. Even more preferably, R6 is -OMe;
[00199] Z and Z 'are independently selected from - (CH2) n'-, - (CH2) n'-CR7R8- (CH2) na'-, - (CH2) n'-NR9- (CH2) na'- , - (CH2) n'-O- (CH2) na'- and - (CH2) nS- (CH2) na-
[00200] n 'and na' are the same or different, and are selected from 0, 1, 2 and 3;
[00201] R7 and R8 are the same or different, and are each independently selected from -H, -OH, -SH, -COOH, -NHR ', a polyethylene glycol unit - (OCH2CH2) n-, an amino acid, a peptide unit containing 2 to 6 amino acids, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00202] R9 is independently selected from -H, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-;
[00203] A and A 'are the same or different, and are independently selected from -O-, oxo (-C (= O) -), -CRR'O-, - CRR'-, -S-, -CRR'S -, -N (R5) - and -CRR'N (R5) -. Preferably, A and A 'are the same or different, and are selected from -O- and -S-. Most preferably, A and A 'are -O-;
[00204] R5 for each occurrence is independently -H or an optionally substituted linear or branched alkyl containing 1 to 10 carbon atoms;
[00205] D and D 'are the same or different, and are independently absent or selected from the group consisting of an amino acid, a peptide containing 2 to 6 amino acids, and a polyethylene glycol (-OCH2CH2) n- unit, an alkyl, optionally substituted linear, branched or cyclic alkenyl or alkynyl containing 1 to 10 carbon atoms, where the alkyl, alkenyl and alkynyl are optionally substituted with one or more (for example, 2, 3, 4, 5, 6 or more) substituents independently selected from the group consisting of halogen, -OR, -NR'COR '', -SR and -COR ';
[00206] Preferably, D and D 'are the same or different, and are independently selected from linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms. More preferably, D and D 'are straight or branched alkyl containing 1 to 4 carbon atoms. Even more preferably, D and D 'are the same or different, and are selected from linear alkyl containing 1 to 4 carbon atoms;
[00207] L is absent, the linking group with the reactive group attached to it, a polyethylene glycol (-OCH2CH2) n- unit, a linear, branched or cyclic alkyl or alkenyl containing 1 to 10 carbon atoms (for example, 1-6 carbon atoms), a phenyl group, a 3- to 18-membered heterocyclic ring or a 5- to 18-membered heteroaryl ring containing 1 to 6 heteroatoms independently selected from O, S, N and P, where the alkyl or alkenyl is optionally substituted with the linking group with the reactive group attached to it; phenyl or heterocyclic or heteroaryl ring can be optionally substituted, wherein the substituent can be the linking group with the reactive group attached thereto.
[00208] In certain embodiments, X is not the bonding group with the reactive group attached to it. In certain embodiments, the double line - between N and C represents a single bond, Y is not -H.
[00209] In certain embodiments, the cytotoxic compounds of the present invention are not any of the following compounds:


[00210] In certain modalities, Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', - NR'COR '', -NR'NR'R '', an optionally substituted 5- or 6-membered heterocycle containing nitrogen (eg, piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by - NR '(C = NH) NR'R '', an amino acid, or a peptide represented by - NRCOP ', where P' is an amino acid or polypeptide containing between 2 to 20 amino acid units, -SR, -SOR ', -SO2M, -SO3M, - OSO3M, halogen, cyan and an azide. Preferably, Y is sodium bisulfite adduct, Sodium Hydrosulfite adduct, or Sodium Metabisulfite adduct. In certain embodiments, Y is not -H.
[00211] In certain embodiments, L is absent, or is selected from an optionally substituted phenyl group and an optionally substituted pyridyl group, wherein the phenyl and pyridyl group contain the linking group with the reactive group attached to it, or L is an amine group containing the linking group with the reactive group attached to it (i.e., -N (linking group) -), or L is a linear, branched or cyclic alkyl or alkenyl containing from 1 to 6 carbon atoms and containing the linking group with the reactive group attached to it.
[00212] In a second specific modality, for cytotoxic dimers (I), (II), (III) and (IV), the variables are as described below:
[00213] the double line - between N and C represents a double bond;
[00214] Y is -H;
[00215] W is C = O;
[00216] R1, R2, R1 ', R2', R4 and R4 'are -H;
[00217] one of R3, or R3 'is optionally a linking group and the other is -H;
[00218] R6 is -OMe;
[00219] Z and Z 'are -CH2-;
[00220] X 'is -H;
[00221] Y 'is -H;
[00222] A and A 'are -O-; and the rest of the variables are as described in the first specific modality.
[00223] In a third specific modality, the cytotoxic dimers of formula (I), (II), (III) and (IV) are represented by the following formulas:

[00224] where:
[00225] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is selected from - H, the linking group with the reactive group attached to it, or an amine protecting group (preferably X is -H);
[00226] Y is selected from -H, -OR, -OCOR ', -SR, -NR'R ", -SO3M, - SO2M, or -OSO3M, where M is -H or a cation like Na +. K +. Preferably, Y is selected from -OH, -OMe, -OEt, -NHOH or -SO3M Even more preferably, Y is -OH or -SO3M, preferably M is -H or Na +;
[00227] R is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or a PEG - (CH2CH2O) n-Rc group, where n is an integer from 1 to 24 and Rc is straight or branched alkyl containing 1 to 4 carbon atoms;
[00228] R 'and R "are the same or different, and are selected from -H, -OH, -OR, -NRRg', -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, an optionally substituted aryl containing from 6 to 18 carbon atoms, an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms selected from O, S, N and P, a PEG group (CH2CH2O ) n-Rc, where n is an integer from 1 to 24, preferably n is 2, 4 or 8; and Rg 'is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms or a PEG - (CH2CH2O) n-Rc group;
[00229] X 'is selected from the group consisting of -H, -OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, phenyl, and an amine protecting group . Preferably, X 'is -H, -OH or -Me. More preferably, X 'is -H;
[00230] Y 'is selected from the group consisting of -H, an oxo group, an alkyl, alkenyl or linear, branched or cyclic substituted or unsubstituted alkyl containing 1 to 10 carbon atoms. Preferably, Y 'is selected from -H or -Me. Most preferably Y 'is -H;
[00231] R6 is -ORc or -SRc, where Rc is a straight or branched alkyl containing 1 to 4 carbon atoms. Preferably, R6 is -OMe or -SMe. Even more preferably, R6 is -OMe;
[00232] A and A 'are selected from -O- and -S-. Preferably, A and A 'are -O-;
[00233] L ', L' ', and L' '' are the same or different, and are independently selected from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms , a polyethylene glycol unit - (OCH2CH2) n-Rc, halogen, guanidinium [-NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by -SO2R', a sulfonate -SO3-M +, a sulfate - OSO3 + M-, a sulfonamide represented by SO2NR'R '', cyano, an azide, -COR ', -OCOR ', -OCONR'R' 'and the bonding group with the reactive group attached to it, provided that only one of L', L '', and L '' 'is the bonding group with the bonded reactive group to this. Preferably, L 'is the linking group with the reactive group attached to it. Alternatively, one of L ', L' 'or L "' is the linking group with the reactive group attached to it, while the others are -H. Most preferably, L 'is the linking group with the reactive group attached to this, and L '' and L '' 'are -H;
[00234] G is selected from -CH- or -N-; and the rest of the variables are as described in the first specific modality.
[00235] In certain embodiments, X is not the bonding group with the reactive group attached to it. In certain embodiments, the double line - between N and C represents a single bond, Y is not -H.
[00236] In certain embodiments, A and A 'are both -O-, R6 is -OMe, and G is -CH-.
[00237] In a fourth specific modality, for cytotoxic dimers of formula (IA), (IIA), (IIIA) or (IVA), L 'is represented for the formula: -W'-Rx-V-Ry-J ,
[00238] where:
[00239] W 'and V are the same or different, and are each independently absent or selected from -CReRe'-, -O-, -O- C (= O) -, -C (= O) -O- , -S-, -SO-, -SO2-, -CH2-S-, -CH2O-, -CH2NRe-, -O- (C = O) O-, -O- (C = O) N (Re) -, -N (Re) -, -N (Re) -C (= O) -, -C (= O) -N (Re) -, - N (Re) -C (= O) O-, - N (C (= O) Re) C (= O) -, -N (C (= O) Re) -, - (O-CH2-CH2) n-, - SS-, or -C (= O) -, or an amino acid, or a peptide containing 2 to 8 amino acids;
[00240] Rx and Ry are the same or different, and are each independently absent or an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an aryl containing 6 to 10 carbon atoms or a 3- to 8-membered heterocyclic ring containing 1 to 3 heteroatoms selected from O, N or S;
[00241] Re and Re 'are the same or different, and are selected from -H, a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk , where Rk is a -H, a linear, cyclic branched alkyl containing 1 to 6 carbon atoms, optionally containing a secondary amino group (for example, -NHR101) or tertiary amino group (-NR101R102) or a 5-containing nitrogen heterocycle or 6 members, such as piperidine or morpholine, where R101 and R102 are each independently a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms. Preferably, R101 and R102 are each independently a straight or branched alkyl containing 1 to 6 carbon atoms;
[00242] n is an integer from 1 to 24; and
[00243] J comprises the reactive group attached to it, and is selected from a maleimido, a haloacetamido, -SH, -SSRd, -CH2SH, -CH (Me) SH, -C (Me) 2SH, -NHRc1, -CH2NHRc1 , -NRc1NH2, -COOH, and -COE, where - COE represents a reactive ester selected from, among others, N-hydroxysuccinimide ester, N-hydroxy sulfosuccinimide ester, nitrophenyl (for example, 2 or 4-nitrophenyl) ester, dinitrophenyl (e.g. 2,4-dinitrophenyl) ester, sulfo-tetraflurophenyl (e.g. 4-sulfo-2,3,5,6-tetrafluorphenyl) ester, and pentafluorphenyl ester, and where Rc1 is -H or a linear alkyl or substituted or unsubstituted branched containing 1 to 4 carbon atoms, and
[00244] Rd is selected from phenyl, nitrophenyl (eg 2 or 4-nitrophenyl), dinitrophenyl (eg 2 or 4-nitrophenyl), carboxynitrophenyl (eg 3-carboxy-4-nitrophenyl), pyridyl or nitropyridyl (for example, 4-nitropyridyl).
[00245] In certain embodiments, J is -SH, -SSRd, a maleimide, or an N-hydroxysuccinimide ester.
[00246] In certain modalities, Re 'is -H or -Me; Re is straight or branched alkyl containing 1 to 6 carbon atoms or - (CH2-CH2-O) n-Rk; n is an integer from 2 to 8; preferably Rk is -H, -Me or -CH2CH2-NMe2, and the rest of the variables are as described above in the fourth specific modality.
[00247] In certain embodiments, V is an amino acid or a peptide containing 2 to 8 amino acids. In certain modalities, V is valine-citrulline, gly-gly-gly, or ala-leu-ala-leu.
[00248] In certain modalities,
[00249] W 'is -O-, -N (Re) - or -N (Re) -C (= O) -;
[00250] Re is H, a straight or branched alkyl containing 1 to 4 carbon atoms, or - (CH2-CH2-O) n-Rk;
[00251] Rx is a straight or branched alkyl containing 1 to 6 carbon atoms;
[00252] V is absent, - (O-CH2-CH2) n-, -C (= O) -NH-, -S-, -NH-C (= O) -;
[00253] Ry is absent or a straight or branched alkyl containing 1 to 4 carbon atoms; and
[00254] J is -SH, -SSRd or -COE (preferably, N-hydroxysuccinimide ester). The rest of the variables are as described in the fourth specific modality.
[00255] In certain modalities,
[00256] W 'is -O-, -N (Re) - or -N (Re) -C (= O) -;
[00257] Re is H, Me, or - (CH2-CH2-O) n-Me;
[00258] n is an integer from 2 to 6;
[00259] Rx is straight or branched alkyl containing 1 to 6 carbon atoms;
[00260] V and Ry are absent; and
[00261] J is -COE, preferably N-hydroxysuccinimide ester. The rest of the variables are as described in the fourth specific modality.
[00262] In a fifth specific modality, L 'is represented by the following formula: -W' - [CR1''R2 ''] a-V- [Cy] 0-1- [CR3''R4 ''] b-COE,
[00263] where:
[00264] R1 '', R2 '', and R3 '' are each independently -H or a straight or branched alkyl containing 1 to 4 carbon atoms, preferably -Me;
[00265] R4 '' is -H, a straight or branched alkyl containing 1 to 4 carbon atoms (preferably -Me), -SO3H, or -SO3-M +, where M + is a pharmaceutically acceptable cation;
[00266] a is an integer from 0-5 (for example, 0 to 2, 3, 4, or 5), and b is an integer from 0-6 (for example, 0 to 3, 4, 5, or 6); and,
[00267] Cy is an optionally substituted 5-membered heterocyclic ring containing a hetero N, preferably Cy is

[00268] In certain modalities, W 'is -N (Re) -.
[00269] In certain embodiments, such as in the fourth and / or fifth specific modality, Re is - (CH2-CH2-O) 2-6- Rk, where Rk is a -H, a linear, branched cyclic alkyl containing 1 to 6 carbon atoms.
[00270] In certain modalities, as in the fourth and / or fifth specific modality, V is -S- or -SS-.
[00271] In the sixth specific modality, as in the fourth and / or fifth specific modality, L 'is represented by the following formula: -NRe- [CR1''R2' '] aS- [CR3''R4' '] b-COE .
[00272] In certain embodiments, the compound is any of the following:

[00273] where Y is -H or -SO3M, and M is -H or a pharmaceutically acceptable cation. In certain modalities, Y is - SO3M.
[00274] In a specific seventh modality, as in the fourth and / or fifth specific modality, L 'is represented by the following formula: -NRe- [CR1''R2' '] aS-Cy- [CR3''R4' '] b-COE.
[00275] In certain embodiments, the compound is any of the following:

[00276] where Y is -H or -SO3M, and M is -H or a pharmaceutically acceptable cation. In certain modalities, Y is - SO3M.
[00277] In an eighth specific modality, the cytotoxic dimers of formula (I), (II), (III) and (IV) are represented by the following formulas:

[00278] where:
[00279] W 'is absent or selected from -O-, -N (Re) -, -N (Re) -C (= O) -, -N (C (= O) Re) -, -S- or -CH2-S-, -CH2NRe-;
[00280] Rx is absent or selected from a linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00281] Re is -H, a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear alkyl , cyclic branched containing 1 to 6 carbon atoms, optionally containing a secondary amino group (for example, -NHR101) or tertiary amino group (-NR101R102) or a 5- or 6-membered heterocycle containing nitrogen, such as piperidine or morpholine, where R101 and R102 are each independently a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms. Preferably, R101 and R102 are each independently a straight or branched alkyl containing 1 to 6 carbon atoms;
[00282] Zs is -H, -SRm;
[00283] Rm is Rd or a substituted or unsubstituted straight or branched alkyl containing 1 to 4 carbon atoms containing a reactive ester, selected from N-hydroxysuccinimide esters, N-hydroxyphalimide esters, N-hydroxy sulfo-succinimide esters, esters for - nitrophenyl, dinitrophenyl esters, pentafluorfenyl esters;
[00284] Rd is selected from phenyl, nitrophenyl, dinitrophenyl, carboxinitrophenyl, pyridyl or nitropyridyl; and,
[00285] n is an integer from 1 to 24; and the rest of the variables are as described above in the third specific modality.
[00286] Preferably, Rk is -H or -Me, and n is an integer from 2 to 8. Preferably, Rx is a straight or branched alkyl containing 1 to 6 carbon atoms; and the rest of the variables are as described above in the third, fourth, and / or fifth specific modality.
[00287] In a ninth specific modality, the cytotoxic dimers of formula (I), (II), (III) and (IV) are represented by the following formulas:


[00288] where:
[00289] the double line - between N and C represents a single bond or a double bond, since when this is a double bond, X is absent and Y is -H, and when this is a single bond, X is selected from -H, the linking group with the reactive group attached to it, or an amine protecting group (preferably X is -H or an amine protecting group; more preferably, X is -H);
[00290] Y is selected from -H, -OR, -OCOR ', -SR, -NR'R, "-SO3M, - SO2M or -OSO3M (for example, Y is -OR, -OCOR', -SR, -NR'R, "-SO3M, -SO2M or -OSO3M), where M is -H or a cation like Na + or K +;
[00291] R is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or a PEG - (CH2CH2O) n-Rc group, where n is an integer from 1 to 24, and Rc is straight or branched alkyl containing 1 to 4 carbon atoms;
[00292] R 'and R "are the same or different, and are selected from -H, -OH, -OR, -NRRg', -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, an optionally substituted aryl containing from 6 to 18 carbon atoms, an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms selected from O, S, N and P, a PEG group (CH2CH2O ) n-Rc, where n is an integer from 1 to 24, preferably n is 2, 4 or 8; and Rg 'is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms or a PEG - (CH2CH2O) n-Rc group;
[00293] X 'is selected from the group consisting of -H, -OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, phenyl, and an amine protecting group ;
[00294] Y 'is selected from the group consisting of -H, an oxo group, an alkyl, alkenyl or linear, branched or cyclic substituted or unsubstituted alkyl containing 1 to 10 carbon atoms;
[00295] A and A 'are selected from -O- and -S-;
[00296] W 'is absent or selected from -O-, -N (Re) -, -N (Re) -C (= O) -, -N (C (= O) Re) -, -S- or -CH2-S-, -CH2NRe-;
[00297] Rx is absent or selected from a linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00298] Re is -H, a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear alkyl , cyclic branched containing 1 to 6 carbon atoms, optionally containing a secondary amino group (for example, -NHR101) or tertiary amino (-NR101R102) or a 5- or 6-membered nitrogen-containing heterocycle, such as piperidine or morpholine, where R101 and R102 are each independently a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms;
[00299] G is selected from -CH- or -N-;
[00300] Zs is -H, or is selected from any of the following formulas:


[00301] where:
[00302] q is an integer from 1 to 5;
[00303] n is an integer from 2 to 6;
[00304] D is -H or -SO3M;
[00305] M is -H or a cation, like Na + or K +.
[00306] In certain modalities, Zs is represented by any of the following formulas:

[00307] In certain modalities, W 'is -N (Re) -.
[00308] In certain embodiments, Re is - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear, branched cyclic alkyl containing 1 to 6 carbon atoms.
[00309] In certain modalities, Rk is -H or -Me, n is 4, and q is 2.
[00310] In certain embodiments, Rx is a straight or branched alkyl containing 1 to 6 carbon atoms.
[00311] In certain embodiments, Rx may be - (CH2) p- (CRfRg) -, in which Rf and Rg are each independently selected from H or a linear or branched alkyl containing 1 to 4 carbon atoms; and p is 0, 1, 2 or 3.
[00312] In certain modalities, Rf and Rg are the same or different, and are selected from -H and -Me; and p is 1.
[00313] In a tenth specific modality, the compounds of formula (VIII), (IX), (X) and (XI) described in the ninth specific modality, the variables are as described below:
[00314] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is -H; Y is -H, -OH or -SO3M;
[00315] M is -H or a pharmaceutically acceptable cation (for example, Na +);
[00316] X 'and Y' are both -H;
[00317] A and A 'are both -O-;
[00318] R6 is -OMe; and
[00319] Rx is a straight or branched alkyl containing 1 to 6 carbon atoms.
[00320] In a related modality, Y is -OH or -SO3M.
[00321] In another embodiment, the compounds of formula (VIII), (IX), (X) and (XI) described in the ninth specific modality, the variables are as described below:
[00322] W 'is -O-, -N (Re) -, -N (Re) -C (= O) -, -N (CORe) -, -S- or -CH2-S-;
[00323] Rx is absent or selected from a linear, branched or cyclic alkyl containing 1 to 6 carbon atoms;
[00324] Re is -H, a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear alkyl , cyclic branched containing 1 to 6 carbon atoms, optionally containing a primary, secondary or tertiary amine group or a 5- or 6-membered heterocycle containing nitrogen, such as piperidine or morpholine;
[00325] n is an integer from 1 to 24; and the rest of the variables are as described above in the ninth specific modality.
[00326] Preferably, Rk is -H or -Me, and n is an integer from 2 to 8. Preferably, Rx is a straight or branched alkyl containing 1 to 6 carbon atoms.
[00327] Preferably, Rx is - (CH2) p- (CRfRg) -, wherein Rf and Rg are each independently selected from H or a linear or branched alkyl containing 1 to 4 carbon atoms; p is 0, 1, 2 or 3. More preferably, Rf and Rg are the same or different, and are selected from -H and -Me; and p is 1.
[00328] In another preferred embodiment, the linker is represented by any of the selected formulas of formulas (a1), (a4), (a5), (a10) and (a11) shown above; and the rest of the variables are as described above in the tenth specific modality.
[00329] In an eleventh specific modality, for the compounds of formula (IB), (IIB), (IIIB) and (IVB) described in the eighth specific modality, the variables are as described below:
[00330] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is -H; Y is -H, -OH or -SO3M (for example, Y is -OH or -SO3M);
[00331] M is -H or Na +;
[00332] X 'and Y' are both -H;
[00333] A and A 'are both -O-;
[00334] R6 is -OMe;
[00335] Rx is a straight or branched alkyl containing 1 to 6 carbon atoms; and the rest of the variables are as described above in the third, fourth, or fifth specific modality.
[00336] Preferably, Rx is - (CH2) p- (CRfRg) -, where Rf and Rg are each independently selected from H or a linear or branched alkyl containing 1 to 4 carbon atoms; p is 0, 1, 2 or 3. More preferably, Rf and Rg are the same or different, and are selected from -H and -Me; and p is 1.
[00337] In any of the specific modalities above (for example, the first to the 11th specific modalities), the double line - between N and C can represent a double bond.
[00338] In any of the specific modalities above (for example, the first to the 11th specific modalities), the double line - between N and C can represent a single bond, X is -H, the bonding group with the reactive group attached to it, or an amine protecting group (for example, X is -H or an amine protecting group); and Y is selected from -H, -OR, -OCOR ', -SR, -NR'R, "an optionally substituted 5- or 6-membered heterocycle containing nitrogen, - SO3M, -SO2M and a -OSO3M sulfate (for example, Y is -OR, -OCOR ', - SR, -NR'R, "an optionally substituted 5- or 6-membered heterocycle containing nitrogen, -SO3M, -SO2M and a -OSO3M sulfate).
[00339] In certain modalities, Y is selected from -H, - SO3M, -OH, -OMe, -OEt or -NHOH (for example, Y is -SO3M, -OH, - OMe, -OEt or -NHOH).
[00340] In certain embodiments, Y is -H, -SO3M or -OH (for example, Y is -SO3M or -OH).
[00341] In certain modalities, M is -H, Na + or K +.
[00342] In any of the above specific modalities (for example, the first to the 11th specific modalities), W, when present, is C = O.
[00343] In any of the specific modalities above (for example, the first to the 11th specific modalities), Z and Z ', when present, are -CH2.
[00344] In any of the above specific modalities (for example, the first through the 11th specific modalities), X 'is selected from the group consisting of optionally linear, branched or cyclic alkyl, alkenyl or alkynyl substituted containing 1 to 10 carbon atoms, phenyl, the linking group with the reactive group attached to it, and an amine protecting group.
[00345] In certain embodiments, X 'is -H, -OH, -Me or the linking group with the reactive group attached to it.
[00346] In certain embodiments, X 'is -H.
[00347] In any of the above specific modalities (for example, the first through the 11th specific modalities), Y 'is selected from the group consisting of -H, an oxo group, an alkyl, alkenyl or linear, branched or cyclic alkynyl substituted or unsubstituted containing 1 to 10 carbon atoms.
[00348] In certain embodiments, Y 'is -H or oxo.
[00349] In certain embodiments, Y 'is -H.
[00350] In any of the specific modalities above (for example, the first to the 11th specific modalities), A and A 'are the same or different, and are selected from O, S, NR5 and oxo (C = O). A and A 'can be the same or different and selected from -O- and -S-. Preferably, both A and A 'are -O-.
[00351] In any of the specific modalities above (for example, the first to the 11th specific modalities), D and D ', when present, are the same or different, and are independently selected from a polyethylene glycol unit (-OCH2CH2) n, where n is an integer from 1 to 24, an amino acid, a peptide containing 2 to 6 amino acids, or a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, where the alkyl, alkenyl and alkynyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, -OR, -NR'COR '', -SR and -COR '. Preferably, D and D 'are straight or branched alkyl containing 1 to 4 carbon atoms.
[00352] In a twelfth modality, the cytotoxic compound of the present invention as described in the first, third, and ninth modality is represented by the following:
[00353] the double line - between N and C represents a double bond;
[00354] Y is -H;
[00355] W is C = O;
[00356] R1, R2, R1 ', R2', R4 and R4 'are -H;
[00357] one of R3, or R3 'is optionally the linking group with the reactive group attached to it and the other is -H;
[00358] R6 is -OMe;
[00359] Z and Z 'are -CH2;
[00360] X 'is -H;
[00361] Y 'is -H; and
[00362] A and A 'are -O-.
[00363] In a thirteenth embodiment, the cytotoxic compound of the present invention is:

[00364] or a pharmaceutically acceptable salt thereof.
[00365] In one embodiment, compound 29b can be used in the methods of the present invention described here. In a preferred embodiment, compound 29b can be used to treat a proliferative disorder, such as cancer.
[00366] In another embodiment, compound 29b can be used to screen cell lines that are sensitive to benzodiazepine compounds, such as benzodiazepine derivatives described here. DRUG COMPOUNDS & DRUG COMPOUNDS- BINDING
[00367] The cytotoxic compounds described above comprise a linker group with a reactive group attached to it, whose compounds can result from the reaction of a bifunctional cross-linking reagent with "less linker" compounds to form so-called drug-linker compounds. Alternatively, drug compounds that are otherwise identical to drug-binding compounds, but without the binding portion are still included by the present invention.
[00368] Thus in certain embodiments, the invention provides a cytotoxic compound without a binding group, but may be able to react with a bifunctional cross-linking agent to form a compound of the invention, such as any of the 1st through 12th specific modalities described above ; or to form a cell-binding agent conjugate of the invention (such as those described below). An exemplary binder-free cytotoxic compound includes compound 29b of the 13th specific embodiment above. The binder-free cytotoxic compounds of the invention are represented by any of the following formulas (I '), (II'), (III ') or (IV'):


[00369] or a pharmaceutically acceptable salt thereof, where:
[00370] the double line - between N and C represents a single bond or a double bond, since when this is a double bond, X is absent and Y is -H, or a linear or branched alkyl containing 1 to 4 atoms of carbon, and when this is a single bond, X is -H, or a protective amine moiety; preferably, the double line - between N and C represents a double bond;
[00371] Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', -NR'COR '', -NR'NR 'R' ', an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (eg, piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by -NR' (C = NH) NR'R ' ', an amino acid, or a peptide represented by -NRCOP', where P 'is an amino acid or polypeptide containing between 2 to 20 amino acid units, -SR, - SOR', -SO2M, -SO3M, -OSO3M, halogen , cyan and an azido, where M is -H or a cation; like Na + or K +. Preferably, M is -H or Na +. Preferably, Y is selected from -SO3M, -OH, -OMe, -OEt or - NHOH. More preferably, Y is -SO3M or -OH; or
[00372] Y is a sulfite (HSO3, HSO2 or a salt of HSO3-, SO32- or HSO2- formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52- formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a PO3S3-, PO2S23-, POS33- or PS43- salt formed with a cation), thio phosphate ester (RiO) 2PS (ORi), RiS-, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a S2O32- salt formed with a cation), dithionite (HS2O4 or a S2O42- salt formed with a cation), phosphorodithioate (P (= S) (ORk ') (S) (OH) or a salt of the same formed with a cation), hydroxamic acid (Rk'C (= O) NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2- or a salt of HOCH2SO2- formed with a cation, such as HOCH2SO2- Na +) or a mixture thereof, where Ri is straight or branched alkyl containing 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N (Rj) 2, -CO2H, -SO3H, and -PO3H; R1 can be optionally further substituted with a substituent for an alkyl described herein; Rj is straight or branched alkyl containing 1 to 6 carbon atoms; Rk 'is a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; preferably, Y is a bisulfite, hydrosulfite, or metabisulfite adduct, or salts thereof (such as sodium salt);
[00373] R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted heterocyclic ring from 3 to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[00374] R 'and R "are the same or different, and are independently selected from -H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, a polyethylene glycol - (CH2CH2O) n-Rc unit, and an optionally substituted 3 to 18 membered heterocyclic ring containing 1 to 6 hetero atoms independently selected from O, S, N and P;
[00375] Rc is -H or a substituted or unsubstituted straight or branched alkyl containing 1 to 4 carbon atoms;
[00376] n is an integer from 1 to 24;
[00377] W is selected from C = O, C = S, CH2, BH, SO, and SO2;
[00378] X 'is selected from the group consisting of -H, -OH, an amine protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms (eg phenyl), an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P. Preferably, X 'is -H, -OH, or -Me. More preferably, X 'is -H;
[00379] Y 'is selected from the group consisting of -H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an optionally substituted 6 to 18 membered aryl , an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, an optionally substituted 3-18 membered heterocyclic ring containing 1 to 6 heteroatoms. Preferably, Y 'is selected from -H or oxo. More preferably, Y 'is -H;
[00380] R1, R2, R3, R4, R1 ', R2', R3 'and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-Rc, halogen, guanidinium [- NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NCO, - NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by - SO2R', a sulfonate -SO3-M +, a sulfate -OSO3-M +, a sulfonamide represented by -SO2NR'R '', cyan, an azido, -COR ', -OCOR', and - OCONR'R ''. Preferably, 1, 2, 3, or all of R2, R3, R2 'and R3' is - H;
[00381] R6 is -H, -R, -OR, -SR, -NR'R '', -NO2, halogen, -ORc or - SRc, where Rc is -H, a linear or branched alkyl containing 1 to 4 carbon atoms. Preferably, R6 is -OMe or -SMe. Even more preferably, R6 is -OMe;
[00382] Z and Z 'are independently selected from - (CH2) n'-, - (CH2) n'-CR7R8- (CH2) na'-, - (CH2) n'-NR9- (CH2) na'- , - (CH2) n'-O- (CH2) na'- and - (CH2) n'-S- (CH2) na'-;
[00383] n 'and na' are the same or different, and are selected from 0, 1, 2 and 3;
[00384] R7 and R8 are the same or different, and are each independently selected from -H, -OH, -SH, -COOH, -NHR ', a polyethylene glycol unit - (OCH2CH2) n-, an amino acid, a peptide unit containing 2 to 6 amino acids, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00385] R9 is independently selected from -H, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-;
[00386] A and A 'are the same or different, and are independently selected from -O-, oxo (-C (= O) -), -CRR'O-, - CRR'-, -S-, -CRR'S -, -N (R5) - and -CRR'N (R5) -. Preferably, A and A 'are the same or different, and are selected from -O- and -S-. Most preferably, A and A 'are -O-;
[00387] R5 for each occurrence is independently -H or an optionally substituted linear or branched alkyl containing 1 to 10 carbon atoms;
[00388] L ', L' ', and L' '' are the same or different, and are independently selected from -H, halogen, an optionally substituted linear, branched or cyclic alkyl, haloalkyl, alkoxy, haloalkoxy, -NO2, or -CN;
[00389] G is selected from -CH- or -N-.
[00390] In certain modalities, the double line - between N and C represents a single bond, Y is not -H.
[00391] In certain modalities, the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is selected from -H, or an amine protecting group (preferably X is -H); W is C = O; R1, R2, R3, R4, R1 ', R2', R3 ', and R4' are -H; Z and Z 'are -CH2-; A and A 'are both -O-; W is - (C = O) -; G is -CH-; R6 is -H, or optionally substituted linear C1-C10, branched C1-C10, or C3-C7 cyclic alkyl, -O-alkyl, or -O-halo-alkyl, such as -OMe; X 'is selected from the group consisting of -H, -OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, phenyl, and an amine protecting group; and Y 'is selected from the group consisting of -H, an oxo group, an alkyl, alkenyl or linear, branched or cyclic substituted or unsubstituted alkyl containing 1 to 10 carbon atoms.
[00392] Preferably, when Y is not -H, Y is selected from - OR, -OCOR ', -SR, -NR'R ", -SO3M, -SO2M, or -OSO3M, where M is -H or a cation as Na +. K +. Preferably, Y is selected from -H, -OH, -OMe, -OEt, -NHOH or -SO3M (for example, Y is -OH, -OMe, -OEt, -NHOH or -SO3M) Even more preferably, Y is -H, -OH or -SO3M (for example, Y is -OH or -SO3M), preferably M is -H or Na +.
[00393] In certain modalities, the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is selected from -H, or an amine protecting group (preferably X is -H); W is C = O; R1, R2, R3, R4, R1 ', R2', R3 ', R4', X 'and Y' are -H; Z and Z 'are -CH2-; A and A 'are both -O-; W is - (C = O) -; G is -CH-; R6 is -H, or optionally substituted linear C1-C10, branched C1- C10, or C3-C7 cyclic alkyl, -O-alkyl, or -O-halo-alkyl, as -OMe.
[00394] Bifunctional cross-linking agents can be any bifunctional binder known in the art. For example, bifunctional ligands can be used to prepare the drug-ligand compounds are those that form disulfide bonds, thioether bonds, labile acid bonds, photolabile bonds, labile peptidase bonds and labile esterase bonds with cytotoxic compounds (see for example, US Patent 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073, all of which are incorporated herein by reference). Preferably, the bifunctional cross-linking agents are those that form disulfide bonds, labile bonds to thioether and peptidase with cytotoxic compounds. Other bifunctional cross-linking agents that can be used in the present invention include non-cleavable binders, such as those described in US publication number 2005/0169933, or charged binders or hydrophilic binders and are described in US 2009/0274713, US 2010/01293140 and WO 2009/134976, each of which is expressly incorporated herein by reference. The bifunctional cross-linking agents that can be used to prepare the compounds (drug-linker) of the present invention further include those described in Thermo Scientific Pierce Crosslinking Technical Handbook, the complete teaching of which is incorporated herein by reference. SYNTHESIS OF CYTOToxic COMPOUNDS
[00395] Representative processes for preparing the cytotoxic dimer compounds of the present invention are shown in FIGS. 1- 11. The dimers were prepared by reacting a monomer with linker compounds that have two leaving groups such as halogen, triflate, mesylate, or tosylate such as those described for the synthesis of 1c in FIG. 1. Synthesis of representative dimers that contain a thiol or disulfide moiety allowing binding to the cell binding agent through reducible or non-reducible bonds are shown in FIGS. 1-5, 7, 8, and 10. In FIG. 1 a binder containing a small portion of polyethylene glycol and an alkyl disulfide was prepared by the reducing amination of 1a. The conversion of 1b to its corresponding mesylate and coupling with the IBD monomer unit (indolinobenzodiazepine) generated dimer 1c which was reduced to monoimine, converted to free thiol, and coupled with 2 to generate compound 1g of the present invention. In FIG. 3, a modified form of IBD monomer was prepared and coupled to generate a dimer of the present invention in which the reduced imine was converted to a linker. FIG. 4 describes a dimer processing a small portion of polyethylene glycol and a disulfide amide which has been reduced to the thiol 4c and converted to a reactive ester. FIG. 5 describes the synthesis of pyridyl disulfide containing linker 5e which was converted to the monoimine thiol 5i of the present invention before being converted to a reactive ester. The synthesis of representative dimers that have ligands that can react with cell-binding agent are prepared by converting methyl esters to the corresponding reactive esters of a leaving group such as, among others, N-hydroxysuccinimide esters, N-hydroxiftalimide esters, esters N-hydroxy sulfo-succinimide, para-nitrophenyl esters, pentafluorfenyl esters are shown in FIGS. 6, 9, and 11.
Representative processes for preparing the cytotoxic dimer compounds of the present invention suitable for one-step conjugation with a cell binding agent are shown in FIGS. 1 and 12-19. In all these examples, a dimer containing a thiol moiety is reacted with a bifunctional cross-linking reagent that has a reactive group such as, among others, a thiopyridyl, a maleimide, iodide, bromide, or tosylate on one side and an appropriate reactive substituent for reaction with a cell-binding agent such as, among others, N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters, paranitrophenyl esters, pentafluorfenyl esters.
[00397] Alternative synthetic processes for preparing representative cytotoxic dimer compounds of the present invention are shown in FIGS. 20-21. In FIG. 20, the synthesis of the reduced mono dimer (that is, containing an imine group) is obtained by a two-step coupling method, in which a reduced form of monomer is initially coupled to the linker followed by coupling with the IBD monomer or the dimer it is prepared using a mixture of both reduced monomer and IBD monomer in coupling with the reactive binder. While the reduced dimer is potentially a by-product of the second synthetic pathway described above, a more direct pathway is shown in FIG. 21 in which the reduced monomer is coupled to both with the binder directly. CELL BINDING AGENTS
[00398] The effectiveness of the conjugates of the invention as therapeutic agents depends on the careful selection of an appropriate cell-binding agent. Cell-binding agents can be of any type currently known, or become known and include peptides and non-peptides. These can usually be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins (such as folate, etc., which can bind to a cell surface receptor on the same, for example, a folate receptor), molecules nutrient carriers (such as transferrin), or any other cell-binding molecule or substance.
[00399] In certain embodiments, cell-binding agents are proteins or polypeptides, or compounds comprising proteins or polypeptides. Preferably, the proteins or polypeptides comprise one or more Lys residues with side chain groups -NH2. Alternatively or in addition, the proteins or polypeptides comprise one or more Cys residues. The -SH side chain groups of the Cys residues can be intact, or they can be in a disulfide bond that can be reduced. Preferably, reduction of disulfide bonds does not significantly negatively impact the cell-binding function of proteins or polypeptides (for example, in the case of antibody or antigen-binding portion thereof, reduction of disulfide bonds does not substantially increase the dissociation of light chains / heavy chains).
The side chain Lys groups -NH2 and / or the groups of Cys side chain -SH can be covalently linked to the linkers, which are, in turn, linked to the compound dimers of the invention, thus conjugation of binding agents to the cell to the compound dimers of the invention. Each cell-based protein-based agent can contain several Lys side chain -NH2 groups and / or the Cys side chain -SH groups available for binding the compounds of the invention through bifunctional crosslinkers.
[00401] Examples of more specific cell-binding agents that can be used include:
[00402] polyclonal antibodies;
[00403] monoclonal antibodies;
[00404] antibody fragments such as Fab, Fab ', and F (ab') 2, Fv, minibodies, diabodies, tribodies, tetrabodies (Parham, J. Immunol. 131: 2895-2902 (1983); Spring et al. J Immunol. 113: 470-478 (1974); Nisonoff et al. Arch. Biochem. Biophys. 89: 230-244 (1960), Kim et al., Mol, Cancer Ther., 7: 2486-2497 (2008) , Carter, Nature Revs., 6: 343-357 (2006));
[00405] interferons (for example, α, β, Y);
[00406] lymphokines such as IL-2, IL-3, IL-4, IL-6;
[00407] hormones like insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones, such as androgens and estrogens;
[00408] growth factors and colony stimulating factors such as EGF, TGF-alpha, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984));
[00409] transferrin (O'Keefe et al. J. Biol. Chem. 260: 932-937 (1985));
[00410] vitamins, such as folate;
[00411] Protein structures based on a consensus sequence of fibronectin type III (FN3) repeats (also known as Centyrins; see U.S. patent publication Patent publication 2010/0255056, incorporated herein by reference);
[00412] Designer Ankyrin Repeat Proteins (DARPins; US patent publications 20040132028; 20090082274; 20110118146; 20110224100, incorporated herein by reference), C. Zahnd et al. 2010, Cancer Res., 70; 1595-1605, incorporated herein by reference); and,
[00413] Fibronectin domain structure proteins (Adnectins: US Patent Publications 20070082365; 20080139791, incorporated herein by reference).
[00414] Monoclonal antibody techniques allow the production of extremely specific cell-binding agents in the form of specific monoclonal antibodies. Particularly well known in the art are techniques for creating monoclonal antibodies produced by mice, rats, hamsters or any other mammal immunizing with the antigen of interest such as the intact target cell, antigens isolated from the target cell, whose viruses, attenuated whole viruses , and viral proteins like viral cover proteins. Sensitized human cells can still be used. Any method for creating monoclonal antibodies is the use of phage libraries from scFv (single chain variable region), specifically human scFv (see for example, Griffiths et al., US Patents 5,885,793 and 5,969,108; McCafferty et al. , WO 92/01047; Liming et al., WO 99/06587). In addition, regeneration antibodies disclosed in US patent 5,639,641 can still be used, as can chimeric antibodies and humanized antibodies. The selection of the appropriate cell-binding agent is a matter of choice that depends on the particular cell population to be targeted, but in general human monoclonal antibodies are preferred as an appropriate one is available.
[00415] For example, the MY9 monoclonal antibody is a murine IgG1 antibody that specifically binds to the CD33 antigen {J.D. Griffin et al 8 Leukemia Res., 521 (1984)} and can be used if the target cells express CD33 as in acute myeloid leukemia (AML) disease. The cell-binding agent can be any compound that binds to a cell, in a specific or non-specific way. These can generally be antibodies (especially monoclonal antibodies and antibody fragments), interferons, lymphokines, hormones, growth factors, vitamins, nutrient-carrying molecules (such as transferrin), or any other cell-binding molecule or substance.
[00416] Where the cell-binding agent is an antibody, it binds to an antigen that is a polypeptide and can be a transmembrane molecule (eg, receptor) or a ligand as a growth factor. Exemplary antigens include molecules like renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin chain A; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as vmc factor, factor IX, tissue factor (TF), and von Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; a plasminogen activator, such as urokinase or human urine or tissue type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor alpha and beta; encephalinase; RANTES (T cell regulated activation normally expressed and secreted); inflammatory human macrophage protein (MIP-1-alpha); a serum albumin, such as human serum albumin; substance that inhibits Muellerian; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-associated peptide; a proteinamicrobial, such as beta-lactamase; DNase; IgE; a cytotoxic T lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin -3, -4, -5, or-6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor like aFGF and bFGF; fibroblast growth factor 2 receptor (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-Dβ5; insulin growth factor type I and II (IGF-I and IGF-II); des (1-3) -IGF- I (cerebral IGF-I), insulin-like growth factor-binding proteins, melanotransferrin, EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, receptors EphA, EphB receptors, folate receptor, FOLR1, mesothelin, crypto, alfavbeta6, integrins, VEGF, VEGFR, EGFR, tarnsferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55 , CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152 or an antibody that binds to one or more tumor-associated antigens or cell surface receptors disclosed in US publication 20080171040 or publication US 20080305044 and are incorporated in their entirety by reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon, such as alpha, beta, and gamma interferons; colony stimulating factors (CSFs), for example, M-CSF, GM-CSF, and G-CSF; interleukins (ILs), for example, IL-1 to IL-10; oxide dismutase; T cell receptors; surface membrane proteins; decay acceleration factor; viral antigen, such as a portion of the HIV envelope; transport proteins; homing receivers; adressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor-associated antigen such as the HER2, HER3 or HER4 receptor; endoglina, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, LGR5, B7H4, and fragments of any of the polypeptides listed above.
[00417] In addition, GM-CSF, which binds to myeloid cells can be used as a cell-binding agent for cells sick with acute myeloid leukemia. IL-2 that binds to activated T cells can be used for the prevention of transplant graft rejection, for therapy and prevention of graft-host disease, and for the treatment of acute T-cell leukemia. MSH, which binds to melanocytes, can be used to treat melanoma, as the antibodies can be directed towards melanomas. Folic acid can be used to target the folate receptor expressed in ovarian and other tumors. Epidermal growth factor can be used to target scaly cancers like lung and head and neck. Somatostatin can be used to target neuroblastomas and other types of tumor.
[00418] Breast and testicular cancers can be successfully targeted with estrogen (or estrogen analogs) or androgen (or androgen analogs) respectively as a cell-binding agent.
[00419] In one embodiment, the cell-binding agent is humanized monoclonal antibodies. In another embodiment, the cell-binding agent is huMy9-6, or other related antibodies, which are described in US patents 7,342,110 and 7,557,189 (incorporated herein by reference). In another embodiment, the cell binding agent is an anti-folate receptor antibody described in provisional application US 61 / 307,797, 61 / 346,595, 61 / 413,172 and application US 13 / 033,723 (published as US 2012-0009181 A1). The teachings of all these requests are hereby incorporated by reference in their entirety.
[00420] In certain embodiments, the cell-binding agent may be a monoclonal antibody or antigen-binding portions thereof sharing critical sequences for binding the antigen with an antibody disclosed here, such as huMy9-6 or its related antibodies described in the patents US 7,342,110 and 7,557,189 (incorporated herein by reference). These derived antibodies can have substantially the same or identical (1) light chain and / or heavy chain regions CDR3; (2) light chain and / or heavy chain regions CDR1, CDR2, and CDR3; or (3) light chain and / or heavy chain regions, compared to an antibody described herein. Sequences within these regions can contain conserved amino acid substitutions, including substituents within the CDR regions. Preferably, there are no more than 1, 2, 3, 4, or 5 conserved substitutions. In certain embodiments, the derived antibodies have a light chain region and / or a heavy chain region that is at least about 90%, 95%, 99% or 100% identical to an antibody described herein. These derived antibodies can have substantially the same binding specificity and / or affinity for the target antigen compared to an antibody described herein. Preferably, the Kd and / or koff values of the derived antibodies are within 10 times (greater or lesser), 5 times (greater or lesser), 3 times (greater or lesser), or 2 times (greater or lesser) than one antibody described here. These derived antibodies can be fully human antibodies, or humanized antibodies, or chimeric antibodies. Derived antibodies can be produced according to methods recognized in the art.
[00421] In one embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof that specifically binds to a human folate receptor 1, wherein the antibody comprises: (a) the heavy chain CDR1 comprising GYFMN (SEQ ID NO: 1); the CDR2 heavy chain comprising RIHPYDGDTFYNQXaa1FXaa2Xaa3 (SEQ ID NO: 2); and the heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO: 3); and (b) the CDR1 light chain comprising KASQSVSFAGTSLMH (SEQ ID NO: 4); the CDR2 light chain comprising RASNLEA (SEQ ID NO: 5); and the CDR3 light chain comprising QQSREYPYT (SEQ ID NO: 6); where Xaa1 is selected from K, Q, H, and R; Xaa2 is selected from Q, H, N, and R; and Xaa3 is selected from G, E, T, S, A, and V. Preferably, the heavy chain sequence CDR2 comprises RIHPYDGDTFYNQKFQG (SEQ ID NO: 7).
[00422] In another embodiment, the anti-folate receptor antibody is a humanized antibody or binding fragment antigen thereof that specifically binds to the receptor 1 for human folate comprising a heavy chain containing the QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHP amino acid sequence YDGDTFYNQKFQGKATETVDKSSNTAIIMEEESETSEDFAVYYCTRYDGSRAM DYWGQGlTVTVSSASrKGPSVFPLAPSSKSTSGGTAALGCXVKDYFPEPVrVSW NSGALTSGVI1TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY1CNVNHKPSNTKVDK KVEPKSCDKTI 1TCPPCPAPELLGGPSVFI .FPPKPKDTLMISRTPEVTC V VVD VSII EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVI TVIJIQDWI .NGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP VEWESNGQPENNYKTTPPVI SDIA, DSDGSFFI .YSKI .TVDKSR WQQ <iNVFSGSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8).
[00423] In another embodiment, the anti-folate antibody is a humanized antibody or antigen-binding fragment encoded by the DNA plasmid deposited with the ATCC on April 7, 2010 and containing ATCC deposit number PTA- 10772 and PTA-10773 or 10774.
[00424] In another embodiment, the anti-folate receptor antibody is a humanized antibody or fragment antigen same binding specifically binds to the human folate receptor 1 comprising the light chain containing the DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRA amino acid sequence SNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPYTFGGGTKLEI KRTVAAPSVFIFPPSDEQI KSGTASVVCI LNNFYPREAKVQWKVDNAI .QSGNSQ ESVTEQDSKDSTYSI SSTLTLSKADYEKHKVYACEVTIIQGLSSPVTKSFNRGEC-M "V. I>: or DIVLTQSPLSLAVSLCJQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLIYRA SNLEAG VPDRFSGSGSK TD1TL TISPVEAED AA TY YCQQSREYPY TFGGG TKLE1 KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSI -SSTI .ti SK ADYI IKI IK ACIiVTlIQ VY (JLSSPVTKSFNRGEC (SEQ ID NO: 10).
[00425] In another embodiment, anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 comprising the heavy chain containing the amino acid sequence of SEQ ID NO: 8, and the light chain containing the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. Preferably, the antibody comprises the heavy chain containing the amino acid sequence of SEQ ID NO: 8 and the light chain containing the amino acid sequence of SEQ ID NO: 10 (hu FOLR1).
[00426] In another embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment encoded by the DNA plasmid deposited with the ATCC on April 7, 2010 and containing deposit numbers ATCC PTA -10772 and PTA-10773 or 10774.
[00427] In another embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable domain at least about 90%, 95%, 99% or 100% identical to
[00428] QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVK QSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLS LTSEDFAVYYCTRYDGSRAMDYWGQRTVD:
[00429] and a light chain variable domain at least about 90%, 95%, 99% or 100% identical to
[00430] DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYH QKPGQQPRLLIYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAA TYYCQQSREYPYTFGGGTKLEIKR (SE) NO: 12; orDIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQ QPRLLIYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQ SREYPYTFGGGTKLEIKR (SEQ ID NO: 13) PHARMACEUTICAL CELL BINDING AGENT CONJUGATES
[00431] The present invention further provides cell-binding agent-drug conjugates comprising a cell-binding agent attached to one or more cytotoxic compounds of the present invention through a variety of linkers, including, but not limited to, disulfide ligands, ligands thioether, amide binding ligands, peptidase labile ligands, acid labile ligands, esterase labile ligands.
[00432] Representative conjugates of the invention are antibody / cytotoxic compound, antibody fragment / cytotoxic compound, epidermal growth factor (EGF) / cytotoxic compound, melanocyte stimulating hormone (MSH) / cytotoxic compound, thyroid stimulating hormone (TSH) / cytotoxic compound, somatostatin / cytotoxic compound, folate / cytotoxic compound, estrogen / cytotoxic compound, estrogen analog / cytotoxic compound, androgen / cytotoxic compound, and androgen analog / cytotoxic compound. A representative folate / cytotoxic compound conjugate is shown below, with the optional adduct -SO3-Na + at the imine bond of one of the drug monomers. A representative synthesis scheme for this conjugate is shown in Figure 54.

[00433] In a preferred embodiment, the present invention provides conjugates comprising an indolinobenzodiazepine dimer compound (e.g., compounds of formulas (I) - (IV), (IA) - (IVA) and (IB) - (IVB)) and the cell-binding agent linked by a covalent bond. The ligand can be cleaved at the site of the tumor / unwanted proliferating cells to release the cytotoxic agent to its target in a number of ways. The linker can be cleaved, for example, by low pH (hydrazone), reducing environment (disulfide), proteolysis (amide / peptide bond), or by an enzymatic reaction (esterase / glycosidase).
[00434] In a preferred aspect, representative cytotoxic conjugates of the invention are antibody / indolinobenzodiazepine dimer compound, antibody fragment / indolinobenzodiazepine dimer compound, epidermal growth factor (EGF) / indolinobenzodiazepine dimer compound, melanocyte stimulating hormone compound (MSH) / indolinobenzodiazepine, thyroid-stimulating hormone (TSH) dimer compound / indolinobenzodiazepine, somatostatin / indolinobenzodiazepine dimer compound, folate / indolinobenzodiazepine dimer compound, estrogen / indolinobenzodiazepine dimer compound, estrogen-specific analogue inhibitor compound and indolinobenzzepine indephazepine prostate (PSMA) / indolinobenzodiazepine, compound dimer matriptase inhibitor / indolinobenzodiazepine, compound ancherin repeat proteins drawn (DARPins) / indolinobenzodiazepine, compound androgen / indolinobenzodiazepine, and analogue compound d and androgen / indolinobenzodiazepine.
[00435] Thus in the fourteenth specific embodiment, the invention provides a conjugate comprising: a cytotoxic compound and a cell binding agent (CBA), wherein the cytotoxic compound comprises a linking group that covalently bonds the cytotoxic compound to the CBA, and wherein the cytotoxic compound is represented by any of the following formulas:

[00436] or a pharmaceutically acceptable salt thereof, where:
[00437] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, or a linear or branched alkyl containing 1 to 4 carbon atoms , and when this is a single bond, X is -H, the linking group, or a protective amine moiety;
[00438] Y is -H or an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', -NR'COR '', -NR'NR 'R' ', an optionally substituted 5- or 6-membered heterocycle containing nitrogen (for example, piperidine, tetrahydropyrrole, pyrazole, morpholine), a guanidinium represented by -NR' (C = NH) NR'R '', a amino acid, or a peptide represented by -NRCOP ', where P' is an amino acid or polypeptide containing between 2 to 20 units of amino acids, -SR, - SOR ', -SO2M, -SO3M, -OSO3M, halogen, cyan and an azido; or,
[00439] Y is a sulfite (HSO3, HSO2 or a salt of HSO3-, SO32- or HSO2- formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52- formed with a cation), mono-, di-, tri-, and tetra-thiophosphate (PO3SH3, PO2S2H2, POS3H2, PS4H2 or a PO3S3-, PO2S23-, POS33- or PS43- salt formed with a cation), thio phosphate ester (RiO) 2PS (ORi), RiS-, RiSO, RiSO2, RiSO3, thiosulfate (HS2O3 or a S2O32- salt formed with a cation), dithionite (HS2O4 or a S2O42- salt formed with a cation), phosphorodithioate (P (= S) (ORk ') (S) (OH) or a salt of the same formed with a cation), hydroxamic acid (Rk'C (= O) NOH or a salt formed with a cation), formaldehyde sulfoxylate (HOCH2SO2- or a salt of HOCH2SO2- formed with a cation, such as HOCH2SO2- Na +) or a mixture thereof, where Ri is straight or branched alkyl containing 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N (Rj) 2, -CO2H, -SO3H, and -PO3H; R1 can be optionally further substituted with a substituent for an alkyl described herein; Rj is straight or branched alkyl containing 1 to 6 carbon atoms; Rk 'is a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl; preferably, Y is a bisulfite, hydrosulfite, or metabisulfite adduct, or salts thereof (such as sodium salt);
[00440] M is -H or a cation;
[00441] R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5 to 18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted heterocyclic ring from 3 to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[00442] R 'and R' 'are each independently selected from - H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, a polyethylene glycol - (CH2CH2O) n-Rc unit, and an optionally substituted 3 to 18 membered heterocyclic ring containing 1 to 6 hetero atoms independently selected from O, S, N and P;
[00443] Rc is -H or a substituted or unsubstituted straight or branched alkyl containing 1 to 4 carbon atoms, or the linking group;
[00444] n is an integer from 1 to 24;
[00445] W is selected from C = O, C = S, CH2, BH, SO and SO2;
[00446] X 'is selected from -H, an amine protecting group, the linking group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing from 1 to 10 carbon atoms, a polyethylene glycol unit - ( CH2CH2O) n-Rc, an optionally substituted aryl containing 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3-membered heterocyclic ring to 18 members containing 1 to 6 heteroatoms independently selected from O, S, N and P;
[00447] Y 'is selected from -H, an oxo group, the linking group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an optionally substituted 6 to 18 aryl members, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, an optionally substituted 3-18 membered heterocyclic ring containing 1 to 6 heteroatoms;
[00448] R1, R2, R3, R4, R1 ', R2', R3 'and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, a polyethylene glycol unit - (OCH2CH2) n-Rc, halogen, guanidinium [- NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NCO, - NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by - SO2R', a sulfonate -SO3-M +, a sulfate -OSO3-M +, a sulfonamide represented by -SO2NR'R '', cyan, an azido, -COR ', -OCOR', - OCONR'R '' and the linking group;
[00449] R6 is -H, -R, -OR, -SR, -NR'R '', -NO2, halogen or the linking group;
[00450] Z and Z 'are independently selected from - (CH2) n'-, - (CH2) n'-CR7R8- (CH2) na'-, - (CH2) n'-NR9- (CH2) na'- , - (CH2) n'-O- (CH2) na'- and - (CH2) nS- (CH2) na-
[00451] n 'and na' are the same or different, and are selected from 0, 1, 2 and 3;
[00452] R7 and R8 are the same or different, and are each independently selected from -H, -OH, -SH, -COOH, -NHR ', a polyethylene glycol unit (OCH2CH2) n-, an amino acid, a peptide unit containing 2 to 6 amino acids, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00453] R9 is independently selected from -H, an optionally substituted linear, branched or cyclic alkyl containing 1 to 10 carbon atoms, a polyethylene glycol - (OCH2CH2) n- unit;
[00454] A and A 'are the same or different, and are independently selected from -O-, oxo (-C (= O) -), -CRR'O-, - CRR'-, -S-, -CRR'S -, -NR5 and -CRR'N (R5) -;
[00455] R5 for each occurrence is independently -H or an optionally substituted linear or branched alkyl containing 1 to 10 carbon atoms;
[00456] D and D 'are the same or different, and are independently absent or selected from the group consisting of an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an amino acid, a peptide containing 2 to 6 amino acids, and a polyethylene glycol (-OCH2CH2) n- unit;
[00457] L is absent, the linking group, a polyethylene glycol (-OCH2CH2) n- unit, a linear, branched or cyclic alkyl or alkenyl containing 1 to 10 carbon atoms, a phenyl group, a 3 to a heterocyclic ring 18 members or a 5- to 18-membered heteroaryl ring containing 1 to 6 heteroatoms independently selected from O, S, N and P, where the alkyl or alkenyl is optionally substituted with the linking group; phenyl or heterocyclic or heteroaryl ring can be optionally substituted, where the substituent can be the linking group.
[00458] In certain embodiments, X is not the liaison group. In certain embodiments, the double line - between N and C represents a single bond, Y is not -H.
[00459] In certain modalities, Y is an output group selected from -OR, -OCOR ', -OCOOR', -OCONR'R '', -NR'R '', - NR'COR '', -NR ' NR'R '', an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (eg piperidine, tetrahydropyrrole, pyrazole, morpholine, etc.), a guanidinium represented by - NR '(C = NH) NR'R '', an amino acid, or a peptide represented by - NRCOP ', where P' is an amino acid or polypeptide containing between 2 and 20 amino acid units, -SR, -SOR ', -SO2M, -SO3M, - OSO3M, halogen, cyan and an azide.
[00460] In certain embodiments, the compound is not any of the following compounds: J


[00461] In certain embodiments, the conjugates of the invention include the following:




[00462] where:
[00463] CBA is the cell binding agent, r is an integer from 1 to 10, Y is -H, a bisulfite adduct, a hydrosulfite, or a metabisulfite, or salts thereof, or -SO3M, and M is -H or a pharmaceutically acceptable cation.
[00464] In certain embodiments, L is absent, or is selected from an optionally substituted phenyl group and an optionally substituted pyridyl group, wherein the phenyl and pyridyl group contain the linking group, or L is an amine group containing the group bond (i.e. -N (linker group) -), or L is a linear, branched or cyclic alkyl or alkenyl containing 1 to 6 carbon atoms and containing the linking group.
[00465] In the fifteenth specific modality, the compound is represented by any of the following formulas:

[00466] where:
[00467] L ', L' ', and L' '' are the same or different, and are independently selected from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms , a polyethylene glycol unit - (OCH2CH2) n-Rc, halogen, guanidinium [-NH (C = NH) NH2], -OR, -NR'R '', -NO2, -NR'COR '', -SR, a sulfoxide represented by -SOR ', a sulfone represented by -SO2R', a sulfonate -SO3M, a sulfate - OSO3M, a sulfonamide represented by -SO2NR'R '', cyano, an azide, -COR ', -OCOR', -OCONR'R '' and the bonding group, provided that only one of L ', L' ', and L' '' is the bonding group; and
[00468] G is selected from -CH- or -N-. The other groups are as described in the fourteenth specific modality above.
[00469] In certain embodiments, one of L ', L' ', or L "' is the bonding group, while the others are -H. Preferably, L 'is the bonding group, and L' 'and L' '' are -H.
[00470] In certain embodiments, A and A 'are both -O-, R6 is -OMe, and G is -CH-.
[00471] In the sixteenth specific modality, L 'is represented by the following formula: -W'-Rx-V-Ry-J,
[00472] where:
[00473] W 'and V are the same or different, and are each independently absent or selected from -CReRe'-, -O-, -O- C (= O) -, -C (= O) -O- , -S-, -SO-, -SO2-, -CH2-S-, -CH2O-, -CH2NRe-, -O- (C = O) O-, -O- (C = O) N (Re) -, -N (Re) -, -N (Re) -C (= O) -, -C (= O) -N (Re) -, - N (Re) -C (= O) O-, - N (C (= O) Re) C (= O) -, -N (C (= O) Re) -, - (O-CH2-CH2) n-, - SS-, or -C (= O) -, or an amino acid, or a peptide containing 2 to 8 amino acids;
[00474] Rx and Ry are the same or different, and are each independently absent or an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, an aryl containing 6 to 10 carbon atoms or a 3- to 8-membered heterocyclic ring containing 1 to 3 heteroatoms selected from O, N or S;
[00475] Re and Re 'are the same or different, and are selected from -H, a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk , where Rk is a -H, a linear, cyclic branched alkyl containing 1 to 6 carbon atoms, optionally containing a secondary amino group (for example, -NHR101) or tertiary amino group (-NR101R102) or a 5 or 6 heterocycle nitrogen-containing members, such as piperidine or morpholine, where R101 and R102 are each independently a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms; preferably, R101 and R102 are each independently a straight or branched alkyl containing 1 to 6 carbon atoms;
[00476] n is an integer from 1 to 24; and
[00477] J is covalently linked to CBA, and is selected from a succinimide, an acetamido, -S-, -SS-, -CH2S-, -CH (Me) S-, -C (Me) 2S-, - NRc1 -, -CH2NRc1-, -NRc1N-, and -C (= O) -, where Rc1 is -H or a substituted or unsubstituted linear or branched alkyl containing 1 to 4 carbon atoms.
[00478] In certain embodiments, J is -S-, -SS-, a succinimide, or -C (= O) -.
[00479] In certain modalities, Re 'is -H or -Me; Re is straight or branched alkyl containing 1 to 6 carbon atoms or - (CH2-CH2-O) n-Rk; n is an integer from 2 to 8; and Rk is -H, -Me or -CH2CH2-NMe2, and the rest of the variables are as described above in the fifteenth specific modality.
[00480] In certain embodiments, V is an amino acid or a peptide containing 2 to 8 amino acids.
[00481] In certain modalities, V is valine-citrulline, gly-gly-gly, or ala-leu-ala-leu.
[00482] In certain modalities,
[00483] W 'is -O-, -N (Re) - or -N (Re) -C (= O) -;
[00484] Re is H, a straight or branched alkyl containing 1 to 4 carbon atoms, or - (CH2-CH2-O) n-Rk;
[00485] Rx is a straight or branched alkyl containing 1 to 6 carbon atoms;
[00486] V is absent, - (O-CH2-CH2) n-, -C (= O) -NH-, -S-, -NH-C (= O) -;
[00487] Ry is absent or a straight or branched alkyl containing 1 to 4 carbon atoms; and
[00488] J is -S-, -SS-, or -C (= O) -, and the other groups are as defined in the sixteenth specific modality.
[00489] In certain modalities,
[00490] W 'is -O-, -N (Re) - or -N (Re) -C (= O) -;
[00491] Re is -H, -Me, or - (CH2-CH2-O) n-Me;
[00492] n is an integer from 2 to 6;
[00493] Rx is straight or branched alkyl containing 1 to 6 carbon atoms;
[00494] V and Ry are absent; and
[00495] J is -C (= O) -. The other groups are as defined in the sixteenth specific modality.
[00496] In the seventeenth specific modality, L 'in the sixteenth specific modality is represented by the following formula: -W' - [CR1''R2 ''] aV- [Cy] 0-1- [CR3''R4 '' ] bC (= O) -,
[00497] where:
[00498] R1 '', R2 '', and R3 '' are each independently -H or a straight or branched alkyl containing 1 to 4 carbon atoms, preferably -Me;
[00499] R4 '' is -H, a straight or branched alkyl containing 1 to 4 carbon atoms (preferably -Me), -SO3H, or -SO3-M +, where M + is a pharmaceutically acceptable cation;
[00500] a is an integer from 0-5 (for example, from 0 to 2, 3, 4, or 5), and b is an integer from 0-6 (for example, from 0 to 3, 4, 5, or 6); and,
[00501] Cy is an optionally substituted 5-membered heterocyclic ring containing a hetero N, preferably Cy is

[00502] In certain modalities, as in the sixteenth or the seventeenth specific modality, W 'is -N (Re) -.
[00503] In certain embodiments, as in the sixteenth or seventeenth specific modality, Re is - (CH2-CH2-O) 2-6-Rk, where Rk is a linear or branched alkyl containing 1 to 6 carbon atoms .
[00504] In certain modalities, as in the sixteenth or the seventeenth specific modality, V is -S- or -SS-.
[00505] In the eighteenth specific modality, L 'in the sixteenth or seventeenth specific modality is represented by the following formula: NRe- [CR1''R2' '] aS- [CR3''R4' '] bC (= O ) -.
[00506] In certain modalities, as in the sixteenth to the eighteenth specific modalities, the conjugate is:


[00507] where r is an integer from 1 to 10, Y is -H or -SO3M (for example, Y is -SO3M), and M is -H or a pharmaceutically acceptable cation.
[00508] In certain modalities, such as in the sixteenth to the eighteenth specific modalities, the antibody is huMy9-6.
[00509] In the nineteenth specific modality, L 'in the sixteenth or seventeenth specific modality is represented by the following formula: NRe- [CR1''R2' '] aS-Cy- [CR3''R4' '] bC ( = O) -.
[00510] In certain modalities, as in the sixteenth, seventeenth, and the nineteenth specific modalities, the conjugate is:


[00511] where r is an integer from 1 to 10, Y is -H or -SO3M (for example, Y is -SO3M), and M is -H or a pharmaceutically acceptable cation.
[00512] In certain modalities, as in the sixteenth, seventeenth, and the nineteenth specific modalities, the antibody is huMy9-6.
[00513] In the twentieth specific modality, the compound is represented by the following formula:


[00514] where:
[00515] W 'is absent or selected from -O-, -N (Re) -, -N (Re) -C (= O) -, -N (C (= O) Re) -, -S-, -CH2-S-, or -CH2NRe-;
[00516] Rx is absent or selected from a linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00517] Re is -H, a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear alkyl , cyclic branched containing 1 to 6 carbon atoms, optionally containing a secondary amino group (for example, -NHR101) or tertiary amino (-NR101R102) or a 5- or 6-membered nitrogen-containing heterocycle, such as piperidine or morpholine, where R101 and R102 are each independently a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms;
[00518] Zs is linked to CBA, and is either a link, or -SRm-;
[00519] Rm is Rd or a straight or branched substituted alkyl containing 1 to 4 carbon atoms containing reactive ester, selected from N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters dinitrophenyl, and pentafluorfenyl esters;
[00520] Rd is selected from phenyl, nitrophenyl, dinitrophenyl, carboxinitrophenyl, pyridyl or nitropyridyl; and
[00521] n is an integer from 1 to 24; and the rest of the variables are as described above in the eighth or fifteenth specific modality.
[00522] In a twenty-first specific modality, the compound is represented by the following formula:

[00523] where:
[00524] W 'is absent or selected from -O-, -N (Re) -, -N (Re) -C (= O) -, -N (C (= O) Re) -, -S-, -CH2-S-, or -CH2NRe-;
[00525] Rx is absent or selected from a linear, branched or cyclic alkyl containing 1 to 10 carbon atoms;
[00526] Re is -H, a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms or - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear alkyl , cyclic branched containing 1 to 6 carbon atoms, optionally containing a secondary amino group (for example, -NHR101) or tertiary amino (-NR101R102) or a 5- or 6-membered nitrogen-containing heterocycle, such as piperidine or morpholine, where R101 and R102 are each independently a linear, branched or cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms;
[00527] n is an integer from 2 to 6;
[00528] Zs is connected to the CBA, and is selected from:
[00529] a connection


[00530] where:
[00531] q is an integer from 1 to 5; and,
[00532] M is -H or a cation, like Na + or K +.
[00533] In certain modalities, Zs is represented by any of the following formulas:

[00534] In certain modalities, W 'is -N (Re) -.
[00535] In certain embodiments, Re is - (CH2-CH2-O) n-Rk, where Rk is a -H, a linear, branched cyclic alkyl containing 1 to 6 carbon atoms.
[00536] In certain modalities, Rk is -H or -Me, n is 4, and q is 2.
[00537] In certain embodiments, Rx is a straight or branched alkyl containing 1 to 6 carbon atoms.
[00538] In certain embodiments, Rx is - (CH2) p- (CRfRg) -, where Rf and Rg are each independently selected from H or a linear or branched alkyl containing 1 to 4 carbon atoms; and p is 0, 1, 2 or 3.
[00539] In certain modalities, Rf and Rg are the same or different, and are selected from -H and -Me; and p is 1.
[00540] In a twenty-second specific modality, the conjugate of formula (VIII), (IX), (X) and (XI) described in the twenty-first specific modality, the variables are as described below:
[00541] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is -H; Y is -H, -OH or -SO3M (for example, Y is -OH or -SO3M);
[00542] M is -H or a pharmaceutically acceptable cation (for example, Na +);
[00543] X 'and Y' are both -H;
[00544] A and A 'are both -O-;
[00545] R6 is -OMe; and
[00546] Rx is a straight or branched alkyl containing 1 to 6 carbon atoms.
[00547] In the twenty-third specific modality, for the compounds of formula (IB), (IIB), (IIIB) and (IVB) described in the twenty-specific modality, the variables are as described below:
[00548] the double line - between N and C represents a single bond or a double bond, since when this is a double bond X is absent and Y is -H, and when this is a single bond, X is -H; Y is -H, -OH or -SO3M (for example, Y is -OH or -SO3M);
[00549] M is -H or Na +;
[00550] X 'and Y' are both -H;
[00551] A and A 'are both -O-;
[00552] R6 is -OMe;
[00553] Rx is a straight or branched alkyl containing 1 to 6 carbon atoms.
[00554] Preferably, Rx is - (CH2) p- (CRfRg) -, where Rf and Rg are each independently selected from -H or a linear or branched alkyl containing 1 to 4 carbon atoms; p is 0, 1, 2 or 3. More preferably, Rf and Rg are the same or different, and are selected from -H and -Me; and p is 1.
[00555] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, the double line - between N and C can represent a double bond.
[00556] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, the double line - between N and C can represent a single bond, X is -H, the bonding group , or an amine protecting group (for example, X is -H); and Y is -H or selected from -OR, -OCOR ', -SR, -NR'R ", an optionally substituted 5- or 6-membered heterocycle containing nitrogen, -SO3M, -SO2M and a sulfate - OSO3M. In certain embodiments , Y is not -H.
[00557] In certain modalities, Y is selected from -H, - SO3M, -OH, -OMe, -OEt or -NHOH (for example, Y is -SO3M, -OH, - OMe, -OEt or -NHOH).
[00558] In certain embodiments, Y is -H, -SO3M or -OH (for example, Y is -SO3M or -OH).
[00559] In certain modalities, M is -H, Na + or K +.
[00560] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, W, when present, is C = O.
[00561] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, Z and Z ', when present, are -CH2-.
[00562] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, X 'is selected from the group consisting of -H, -OH, an alkyl, alkenyl or linear alkynyl, optionally substituted branched or cyclic containing 1 to 10 carbon atoms, phenyl, the linking group, and an amine protecting group.
[00563] In certain embodiments, X 'is -H, -OH, -Me or the linking group.
[00564] In certain embodiments, X 'is -H.
[00565] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, Y 'is selected from the group consisting of -H, an oxo group, an alkyl, alkenyl or linear alkynyl , branched or cyclic substituted or unsubstituted containing 1 to 10 carbon atoms.
[00566] In certain embodiments, Y 'is -H or oxo.
[00567] In certain embodiments, Y 'is -H.
[00568] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, A and A 'are the same or different, and are selected from -O-, -S-, - N (R5) -, and oxo (C = O).
[00569] In certain modalities, A and A 'are the same or different, and are selected from -O- and -S-.
[00570] In certain modalities, A and A 'are -O-.
[00571] In any of the specific modalities for the conjugate of the above invention, such as the fourteenth to the twenty-third specific modalities, D and D ', when present, are the same or different, and are independently selected from a polyethylene glycol unit (-OCH2CH2) n, where n is an integer from 1 to 24, an amino acid, a peptide containing 2 to 6 amino acids, or a linear, branched, cyclic alkyl, alkenyl or alkynyl containing 1 to 10 carbon atoms, where alkyl, alkenyl and alkynyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, -OR, -NR'COR '', -SR and -COR '.
[00572] In certain embodiments, D and D 'are straight or branched alkyl containing 1 to 4 carbon atoms.
[00573] In the twenty-fourth specific modality, the conjugate of the present invention as described in the fourteenth, fifteenth, or twenty-first specific modality is represented by the following:
[00574] the double line - between N and C represents a double bond;
[00575] Y is -H;
[00576] W is C = O;
[00577] R1, R2, R1 ', R2', R4 and R4 'are -H;
[00578] one of R3, or R3 'is optionally the linking group and the other is -H;
[00579] R6 is -OMe;
[00580] Z and Z 'are -CH2;
[00581] X 'is -H;
[00582] Y 'is -H; and
[00583] A and A 'are -O-.
[00584] In certain modalities, the conjugate of any of the described modalities, such as the fourteenth to the twenty-fourth specific modalities, can comprise 1-10 cytotoxic compounds, 2-9 cytotoxic compounds, 3-8 cytotoxic compounds, 4-7 cytotoxic compounds, or 5-6 cytotoxic compounds, each cytotoxic compound comprising the linking group linking the cytotoxic compound to the CBA, and each cytotoxic compound in the conjugate is the same.
[00585] In any of the conjugate modalities, such as the fourteenth to the twenty-fourth specific modalities, the cell-binding agent can bind to the selected target cells of tumor cells, virus-infected cells, micro-infected cells organisms, parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; cells expressing the CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD44, CD56, EpCAM, CanAg, CALLA, or Her-2 antigens; Her-3 antigens; or cells that express insulin growth factor receptor, epidermal growth factor receptor, and folate receptor.
[00586] In any of the conjugate modalities, such as the fourteenth to the twenty-fourth specific modalities, the cell binding agent can be an antibody, a single chain antibody, an antibody fragment that specifically binds to the cell- target, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to the target cell, a domain of antibody, a fragment of antibody domain that specifically binds to the target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule.
[00587] The antibody can be a regenerating antibody, a regenerating single chain antibody, or a regenerating antibody fragment.
[00588] The antibody can be a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment thereof.
[00589] The antibody can be a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment.
[00590] The invention further provides a pharmaceutical composition comprising any of the conjugates described herein, and a pharmaceutically acceptable carrier.
[00591] The invention further provides a drug-linker compound comprising any of the object compounds covalently linked to a bifunctional linker.
[00592] The invention provides further conjugate comprising any of the object compounds, or the drug-binding object compounds, attached to a cell-binding agent.
[00593] The invention further provides a method for inhibiting abnormal cell growth or treating a proliferative disorder, an autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis or kidney disease in a mammal comprising administering to the mammal a therapeutically effective amount of any of the compounds (with or without any linker group) or conjugates of the invention, and, optionally, a second chemotherapeutic agent.
[00594] In certain embodiments, the compound or conjugate is:





[00595] where r is an integer from 1 to 10, Y is -H or -SO3M (for example, Y is -SO3M), and M is -H or a pharmaceutically acceptable cation.
[00596] In certain embodiments, the second chemotherapeutic agent is administered to the mammal sequentially or consecutively.
[00597] In certain modalities, the method is to treat a selected condition from cancer, rheumatoid arthritis, multiple sclerosis, graft-host disease (GVHD), transplant rejection, lupus, myositis, infection, and immune deficiency.
[00598] In certain modalities, the method or conjugate is to treat cancer.
[00599] In certain modalities, cancer is selected from breast cancer, colon cancer, brain cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, colorectal cancer, gastric cancer, squamous cancer, small cell lung cancer, non-small cell lung cancer, testicular cancer, Merkel cell carcinoma, glioblastoma, neuroblastoma, lymphatic organ cancers and hematological malignancy including leukemia (Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML ), Chronic lymphocytic leukemia (CLL), Chronic myeloid leukemia (CML), Acute monocytic leukemia (AMOL), Hair cell leukemia (HCL), Prolymphocytic T cell leukemia (T-PLL), Large granular lymphocytic leukemia, Large cell leukemia Adult T), Lymphoma (small lymphocytic lymphoma (SLL), Hodgkin's lymphoma (Nodular sclerosis, Mixed cellularity, Lymphocyte rich, Depleted or non-depleted lymphocyte, and Hodgki's lymphoma n predominant in nodular lymphocyte), Non-Hodgkin's lymphoma (all subtypes), Chronic lymphocytic leukemia / Small lymphocytic lymphoma, B-cell lymphocytic leukemia, Lymphoplasmacytic lymphoma (such as Waldenstrom macroglobulinemia), Splenic marginal zone lymphoma, Plasma cells Plasma cell myeloma, Plasmacytoma, Monoclonal immunoglobulin deposition diseases, Heavy chain diseases), Extranodal marginal zone B cell lingoma (MALT lymphoma), Nodal marginal zone B cell lymphoma (NMZL), Follicular lymphoma, Lymphoma mantle cell, diffuse large B-cell lymphoma, large mediastinal (thymic) B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell lymphocytic leukemia granular, aggressive NK cell leukemia, adult T cell leukemia / lymphoma, extranodal NK / T cell lymphoma (nasal type), T cell lymphoma type enteropathy, Hepatosplenic T cell lymphoma, Blast NK cell lymphoma, Mycosis fungoides / Sezary syndrome, T cell lymphoproliferative disorders for primary cutaneous CD30, Primary cutaneous anaplastic large cell lymphoma, Lymphomatoid papulosis, Angioimmunoblastic T cell lymphoma, Peripheral T cell lymphoma (unspecified), anaplastic large cell lymphoma), multiple myeloma (plasma cell myeloma or Kahler's disease). PRODUCTION OF PHARMACEUTICAL CELL BINDING AGENTS
[00600] To bind the cytotoxic compounds or derivatives thereof of the present invention to the cell binding agent, the cytotoxic compound may comprise a linking moiety with a reactive group attached thereto. In one embodiment, the bifunctional cross-linking reagent can first be reacted with the cytotoxic compound to provide the compound containing a linker portion with a reactive group attached to it (i.e., drug-linker compound), which can then be reacted with a binding agent. connection to the cell. Alternatively, one end of the bifunctional cross-linking reagent can first react with the cell binding agent to provide the cell binding agent containing a binding portion with a reactive group attached thereto, which can then react with a cytotoxic compound. The binding portion may contain a chemical bond that allows the release of the cytotoxic portion at a particular location. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds (see for example, US Patents 5,208,020; 5,475,092; 6,441. 163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). Preferred bonds are disulfide, labile bonds to thioether and peptidase. Other binders that can be used in the present invention include non-cleavable binders, such as those described in detail in publication number US 2005/0169933, or charged binders or hydrophilic binders and are described in US 2009/0274713, US 2010/01293140 and WO 2009 / 134976, each of which is expressly incorporated herein by reference, each of which is expressly incorporated herein by reference.
[00601] The compounds of formula (I) - (IV), (IA) - (IVA), and (IB) - (IVB) can be linked by R1, R2, R3, R4, R1 ', R2', R3 ', R4', L ', L' ', L' '', or X (when present). Of these, preferred bonding groups are R2 ', R3', R4 ', L', L '', L '' 'and most preferred bonding groups are R2', R3 ', and L'. Examples of linker groups for compounds of formula (I) - (IV), (IA) - (IVA), and (IB) - (IVB) are described above.
[00602] In one embodiment, a solution of an antibody in aqueous buffer can be incubated with a molar excess of an antibody modifying agent such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) or with N-succinimidil -4- (2-pyridyldithio) butanoate (SPDB) to introduce dithiopyridyl groups. The modified antibody is then reacted with the thiol-containing cytotoxic compound, as compound 2a, to produce a disulfide-indolinobenzodiazepine-bound antibody dimer conjugate. The cell-drug binding agent conjugate can then be purified using any purification method known in the art, such as that described in US Patent 7,811,572 and US publication 2006/0182750, both of which are incorporated herein by reference. For example, the cell-drug binding agent conjugate can be purified using tangential flow filtration, adsorptive chromatography, adsorptive filtration, selective precipitation, non-absorptive filtration or combination thereof. Preferably, tangential flow filtration (TFF, also known as cross flow filtration, ultrafiltration and diafiltration) and / or adsorptive chromatography resins are used for the purification of the conjugates.
[00603] Alternatively, the antibody can be incubated with a molar excess of an antibody modifying agent such as 2-iminothiolane, L-homocysteine thiolactone (or derivatives), or N-succinimidyl-S-acetylthioacetate (SATA) to introduce sulfhydryl groups . The modified antibody is then reacted with the appropriate disulfide-containing cytotoxic agent to produce an antibody bound to the disulfide-cytotoxic agent conjugate. The antibody-cytotoxic agent conjugate can then be purified by the methods described above. Cell binding can also be engineered to introduce thiol fractions, such as cysteine engineered antibodies disclosed in Patents 7,772,485 and 7,855,275.
[00604] In another embodiment, a solution of an antibody in aqueous buffer can be incubated with a molar excess of an antibody-modifying agent such as N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate maleimido groups, or with N-succinimidyl-4- (iodoacetyl) -aminobenzoate (SIAB) to introduce iodoacetyl groups. The modified antibody is then reacted with the thiol-containing cytotoxic agent to produce a thioether-bound antibody-cytotoxic conjugate. The antibody-cytotoxic conjugate can then be purified by the methods described above.
[00605] The number of cytotoxic molecules bound per antibody molecule can be determined spectrophotometrically by measuring the absorbance ratio at 280 nm and 330 nm. An average of 1-10 cytotoxic compound / antibody molecules can be linked by the methods described here. The preferred average number of cytotoxic compounds bound per antibody molecule is 2-5, and the most preferred is 2.5-4.0.
[00606] Cytotoxic agents containing ligands ending in an N-hydroxy succinimidyl ester (NHS), such as compounds 1g and 10, can react with the antibody to produce amide-linked conjugates such as huMy9-6-SPDB-1f or huMy9-6-BMPS -1f. The antibody-cytotoxic agent conjugate can then be purified by gel filtration by any method described above.
Representative processes for preparing the cell-drug binding agent conjugates of the present invention are shown in FIGS. 22 and 23. The cytotoxic dimer compound of the present invention can be conjugated to a cell-binding agent by a one-step or two-step conjugation method. In FIGs. 22a and 22b, representative examples are described, in which a dimer compound having a linker such as an N-hydroxysuccinimide ester is reacted directly with a cell binding agent, such as an antibody, generating the desired conjugate. In FIG. 22c linkable dimer 1g was first treated with sodium bisulfate to provide a modified dimer compound 26 before adding antibody to form the huMy9-6-SBDP-1f conjugate of the present invention.
[00608] A representative example of a two-step conjugation method is described in FIG. 23, in which an antibody is first modified with a bifunctional cross-linking agent resulting in an antibody that has a desired number of appropriate ligands for reaction with a dimer compound containing a portion of free thiol. In this example the huMy9-6 antibody was first modified with SPDB to generate an antibody with ligands containing the dithiopyridyl moiety. The modified antibody was then exposed to a free thiol, such as 2a, generating the desired huMy9-6-SPDB-2a conjugate.
[00609] Processes for synthesizing the drug-ligand and conjugate compounds of the invention are further described in provisional patent application US 61 / 443,092, filed on February 15, 2011, and a US utility application claiming the benefit of the filing date of the same and deposited on the same day of the present application, entitled "METHODS OF PREPARATION OF CONJUGATES" the complete contents of whose applications, including all drawings, formulas, synthesis schemes, specifications, and claims, are incorporated herein by reference.
[00610] The structures of the representative and conjugated compounds of the present invention are shown in tables 1-8. These compounds and conjugates can be prepared according to the methods described here. Table 1. Structures of representative compounds in the present invention.
Table 2. Structures of representative compounds in the present

Table 3. Structures of representative compounds in the present invention (Continued).

Table 4. Structures of representative compounds in the present invention (Continued).
Table 5. Structures of representative compounds in the present

Table 6. Structures of representative compounds in the present invention (Continued).

Table 7. Structures of representative compounds in the present invention (Continued).
Table 8. Representative conjugate structures of the present invention.
IN VITRO CITOTOXICITY OF COMPOUNDS AND CONJUGATES
[00611] The cytotoxic compounds and cell-binding agent conjugates of the invention can be evaluated for their ability to suppress proliferation of various cancer cell lines in vitro. For example, cell lines such as the COLO 205 human colon carcinoma line, the rhabdomyosarcoma cell line RH-30, and the multiple myeloma cell line MOLP-8 can be used for the cytotoxicity assessment of these compounds and conjugates. . The cells to be evaluated can be exposed to the compounds or conjugates for 1-5 days and the survival fractions of the cells measured in straight assays by known methods. IC50 values can then be calculated from the test results. Alternatively or in addition, an in vitro cell line sensitivity assay, such as that described by the US National Cancer Institute (see Voskoglou-Nomikos et al., 2003, Clinical Cancer Res. 9: 42227-4239, incorporated herein by reference) it can be used as one of the guides to determine the types of cancers that may be sensitive to treatment with the compounds or conjugates of the invention.
[00612] Examples of in vitro potency and target specificity of antibody-cytotoxic agent conjugates of the present invention are shown in FIGS. 25-26. All conjugates are extremely cytotoxic in cancer cells positive for antigens with an IC50 in the lower picomolar range. Antigen-negative cell lines remained viable when exposed to the same conjugates. The indolinobenzodiazepine dimers showed target specific potency being 160 times less potent when blocked with huMy9-6 unconjugated antibody (anti-CD33) and 40 less potent when blocked with unconjugated FOLR1 antibody (anti-folate receptor antibody). For example, the huMy9-6-SPDB-1f conjugate maintained antigen-positive HL60 / QC cells with an IC50 value of 10.5 pM, while the addition of an excess of unconjugated huMy9-6 antibody reduced this cytotoxic effect (IC50 = 1.69 nM), demonstrating antigen specificity (FIG. 25A). In addition, the huMy9- 6-SPDB-1f conjugate is still highly potent in the direction of both HL60 / ATCC cell line with an IC50 value of 21 pM and the NB-4 cell line with an IC50 value of 190 pM (FIGS 25B and 25C).
[00613] Similarly, the huFOLR1-SPDB-1f conjugate was highly potent, with an IC50 value of 55 pM for antigen positive KB cells (FIG. 26). The addition of an excess of unconjugated huFOLR1 antibody reduced this cytotoxic effect> 40 times, demonstrating antigen specificity.
[00614] The effect of conjugation on binding to the antibody was measured by comparing the binding of both huMy9- 6 unconjugated antibody and the huMy9-6-SPDB-1f conjugate towards the HL60 / QC cell line (FIG. 27). FACS analysis revealed that there is no change in the binding capacity of the conjugate to the naked antibody indicating that there is no compromise in binding due to the conjugation of the cytotoxic agent to the antibody.
[00615] In one example, in vivo efficacy of a cell-binding agent / cytotoxic agent conjugate was measured. Naked mice containing human HL60 / QC tumors were treated with huMy9-6-SPDB-1f conjugate and significant tumor regression was observed at various doses while in untreated mice the tumors grew rapidly (FIG. 28). Activity was observed at doses as low as 20 μg / kg, which is at least 35 times less than the maximum tolerated dose.
[00616] The imine saturation effect directs tolerability is shown in table 9. Diimine huFOLR1-drug1 has been tested in various doses all of which have been shown to be highly toxic leaving only survivors in the lowest tested group at 50 μg / kg. In contrast to the partially reduced monoimine huFOLR1-Drug 2 and huFOLR1-SPDB-IGN (huFOLR1-SPDB-1f) conjugates have been shown to have significantly improved tolerability with the huFOLR1-SPDB-IGN (huFOLR1-SPDB-1f) conjugate showing 100% of animal survival at the highest tested doses of 560 μg / kg. COMPOSITIONS AND METHODS OF USE
[00617] The present invention includes the composition (for example, a pharmaceutical composition) comprising new benzodiazepine compounds described herein (for example, indolinobenzodiazepine or oxazolidinobenzodiazepine), derivatives thereof, or conjugates thereof, (and / or solvates, hydrates and / or salts thereof) and a carrier (a pharmaceutically acceptable carrier). The present invention further includes the composition (for example, a pharmaceutical composition) comprising new benzodiazepine compounds described herein, derived therefrom, or conjugates thereof, (and / or solvates, hydrates and / or salts thereof) and a carrier (a pharmaceutically acceptable carrier), further comprising a second therapeutic agent. The present compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human). The present compositions are further useful for treating depression, anxiety, stress, phobias, panic, dysphoria, psychiatric disorders, pain and inflammatory diseases in a mammal (e.g., human).
[00618] The present invention includes a method for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (for example, human) comprising administering to said mammal a therapeutically effective amount of new benzodiazepine compounds described herein (for example, indolinobenzodiazepine or oxazolidinobenzodiazepine), derivatives thereof, or conjugates thereof, (and / or solvates and salts thereof) or a composition thereof, alone or in combination with a second therapeutic agent.
[00619] The present invention further provides treatment methods comprising administering to a subject in need of treatment an effective amount of any of the conjugates described above.
Similarly, the present invention provides a method for inducing cell death in selected cell populations comprising contacting target cells or tissue containing target cells with an effective amount of a cytotoxic agent comprising any of the cytotoxic compound-agents cell binding (e.g., indolinobenzodiazepine dimer or oxazolidinobenzodiazepine attached to a cell binding agent) of the present invention, a salt or solvate thereof. Target cells are cells to which the cell-binding agent can bind.
[00621] If desired, other active agents, such as other anti-tumor agents, can be administered together with the conjugate.
[00622] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those skilled in the art as the clinical situation warrants.
[00623] Examples of suitable carriers, diluents and / or excipients include: (1) Dulbecco phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg / mL to 25 mg / mL human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose; and it can also contain an antioxidant like tryptamine and a stabilizing agent like Tween 20.
[00624] The method for inducing cell death in selected cell populations can be practiced in vitro, in vivo, or ex vivo.
[00625] Examples of in vitro uses include autologous bone marrow treatments before their transplants in the same patient to kill sick or malignant cells: bone marrow treatments before their transplants to kill competent T cells and prevent host graft disease (GVHD); cell culture treatments to kill all cells except the desired variants that do not express the target antigen; or kill variants that express unwanted antigen.
[00626] Conditions for non-clinical use in vitro are readily determined by a person skilled in the art.
[00627] Examples of ex vivo clinical use are to remove tumor cells or lymphoid cells from bone marrow before autologous transplantation in the treatment of cancer or treatment of autoimmune disease, or to remove T cells and other lymphoid cells from autologous bone marrow or or tissue before transplantation to prevent GVHD. Treatment can be performed as follows. The bone marrow is collected from the patient or another individual and then incubated in a medium containing serum in which the cytotoxic agent of the invention is added, concentration ranges from about 10 μM to 1 pM, for about 30 minutes to about 48 hours in about 37 ° C. The exact conditions of concentration and incubation time, that is, the dose, are readily determined by a person skilled in the art. After incubation, bone marrow cells are washed with serum-containing medium and returned to the patient intravenously according to known methods. In circumstances where the patient receives another treatment with a course of ablative chemotherapy or full body irradiation between the time of marrow collection and reinfusion of treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard medical equipment.
[00628] For in vivo clinical use, the cytotoxic agent of the invention will be supplied as a solution or a lyophilized powder that are tested for sterility levels and for endotoxin. Examples of appropriate conjugate administration protocols are as follows. Conjugates are administered weekly for 4 weeks as an intravenous bolus each week. Bolus doses are administered in 50 to 1000 ml of normal saline to which 5 to 10 ml of human serum albumin can be added. Dosages will be 10 μg to 2000 mg per administration, intravenously (range of 100 ng to 20 mg / kg per day). After four weeks of treatment, the patient can continue to receive treatment on a weekly basis. Specific clinical protocols regarding the route of administration, excipients, diluents, dosages, times, etc., can be determined by a person skilled in the art as the clinical situation justifies.
[00629] Examples of medical conditions that can be treated according to the in vivo or ex vivo methods of inducing cell death in selected cell populations include malignancy of any type including, for example, lung cancer (small cell and non-small cell ), breast, colon, brain, prostate, kidney, pancreas, ovary, head and neck, skin (melanoma), Merkel cell carcinoma, glioblastoma, neuroblastoma, and lymphatic organ cancers; autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft rejections, such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; graft-host disease; viral infections, such as CMV infection, HIV infection, AIDS, etc .; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by a person skilled in the art.
[00630] Cancer therapies and their dosages, routes of administration and recommended use are known in the art and have been described in said literature as the Physician's Desk Reference (PDR). The PDR reveals the dosages of the agents that have been used to treat various cancers. The dosage and dosage regimen of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician. The contents of the PDR are expressly incorporated here in their entirety by reference. A person skilled in the art can review the PDR, using one or more of the following parameters, to determine the dosage regimen and doses of chemotherapeutic agents and conjugates that can be used in accordance with the teachings of this invention. These parameters include: Comprehensive index By manufacturer Products (by company drug name or trademark) Category index Generic / chemical index (common non-commercial drug names) Color images of drugs Product information, consistent with FDA labeling Chemical information Function / action Indications and contraindications Clinical research, side effects, alerts. ANALOGS AND DERIVATIVES
[00631] One skilled in the art of cytotoxic agents will readily understand that each of the cytotoxic agents described herein can be modified in such a way that the resulting compound still retains the specificity and / or activity of the starting compound. The person skilled in the art will understand that many of these compounds can be used in place of the cytotoxic agents described herein. Thus, the cytotoxic agents of the present invention include analogs and derivatives of the compounds described herein.
[00632] All references cited here and in the examples that follow are expressly incorporated by reference in their entirety.
EXAMPLES
[00633] The invention will now be illustrated with reference to the non-limiting examples. Unless stated otherwise, all percentages, reasons, parts, etc. are by weight. All reagents were purchased from Aldrich Chemical Co., New Jersey, or other commercial sources. Magnetic resonance spectra (1H NMR) were acquired on a Bruker 400 MHz instrument and mass spectra were acquired on a Bruker Daltonics Esquire 3000 instrument using electrospray ionization.

[00634] To a stirred solution of aniline 1a (1.55 g, 5.18 mmol) and 2- (methyldithio) -isobutyraldehyde (0.7 mL, 5.18 mmol) in anhydrous 1,2-dichloromethane (20 ml) sodium triacetoxy borohydride (1.1 g, 5.18 mmol) and zinc chloride powder (353 mg, 2.59 mmol) were added followed by the addition of anhydrous magnesium sulfate (800 mg). The mixture was stirred at room temperature (rt) for 6 hours then a second part of 2- (methyldithio) -isobutyraldehyde (0.7 mL, 5.18 mmol) and sodium triacetoxycoborohydride (1.1 g, 5.18 mmol ) have been added. Stirring continued at rt overnight. The reaction mixture was filtered through celite and washed with dichloromethane. The filtrate was concentrated and the rest was purified by silica gel chromatography (Column Combiflash, 40 g, dichloromethane / MeOH) to generate compound 1b (487 mg y = 22%) as a colorless oil. Aniline 1a unreacted starting material (1.02 g) was further recovered in 65% yield. 1H NMR (400 Hz, CDCl3): δ 6.76 (s, 2H), 6.63 (s, 1H), 4.55 (s, 4H), 3.65-3.51 (m, 14H), 3.35 (s, 3H), 2.44 (s, 3H), 1.33 (s, 6H); 13C NMR (400 Hz, CDCl3): δ 149.0, 142.35, 114.0, 111.1, 71.98, 70.7, 70.6, 70.5, 67.6, 65.5, 59.75, 59.1, 53.9, 51.9, 26.6, 25.7, 20.75; MS (m / z): found 456.2 (M + Na) +. See FIG. 1.
Compound 1c:
[00635] To a stirred solution of 1b (243 mg, 0.56 mmol) in anhydrous dichloromethane (3.5 mL) was added triethylamine (234 μl, 1.68 mmol). The mixture was cooled to -10 C and methanesuanesulfonyl chloride (113 μl, 1.46 mmol) was added slowly over 15 minutes with a syringe. The solution continued to be stirred for 60 minutes at -10 ~ -7 ° C and quenched by the addition of ice / water. It was diluted with ethyl acetate and washed with cold water. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated and subjected to high vacuum to generate the mesylates as a light yellow oil (340 mg). The mesylates were transferred to a 10 mL round bottom flask with ethyl acetate / dichloromethane, concentrated and subjected to high vacuum. IBD monomer (412 mg, 1.4 mmol) was added followed by the addition of anhydrous dimethylformamide (3 mL) and anhydrous potassium carbonate (232 mg, 1.68 mmol). The yellowish mixture obtained was stirred at room temperature overnight. It was diluted with dichloromethane and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was dissolved in dichloromethane and loaded onto a silica gel column and eluted with dichloromethane / methanol (15: 1 then 10: 1). The fractions containing compound 1c were combined and concentrated to generate 705 mg of crude product which was further purified by preparative reverse phase HPLC (column C18, eluted with acetonitrile / water) to generate compound 1c as a yellowish fuzzy solid (181 mg, y = 33%). 1H NMR (400 Hz, CDCl3): δ 8.28 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 3.6 Hz, 2H), 7.59 (s, 2H) , 7.31-7.26 (m, 4H), 7.12 (t, J = 7.6 Hz, 2H), 6.87-6.80 (m, 5H), 5.18 (dd, J1 = 20.8 Hz, J2 = 12.4 Hz, 4H), 4.50-4.47 (m, 2H), 3.99 (s, 6H), 3.75-3.48 (m, 18H) , 3.37 (s, 3H), 2.44 (s, 3H), 1.32 (s, 6H); MS (m / z): found 1025.9 (M + H2O + Na) +, 1043.9 (M + 2H2O + Na) +, 983.8 (M - H) -, 1055.8 (M + 4H2O - H)-. See FIG. 1.
Compound 1d:
[00636] To a stirring solution of compound 1c (112 mg, 0.114 mmol) in anhydrous dichloromethane (0.3 mL) and absolute ethanol (0.6 mL) was added sodium borohydride (0.9 mg, 0.023 mmol) at 0 ° C. The ice bath was removed after 5 minutes and the mixture was stirred at room temperature for 3 hours and then cooled to 0 ° C and quenched with saturated ammonium chloride, diluted with dichloromethane, separated and the organic layer was washed with brine, dried anhydrous Na2SO4 and filtered through celite and concentrated. The residue was purified by reverse phase HPLC (column C18, acetonitrile / water). The corresponding fractions were extracted with dichloromethane and concentrated to obtain products 1d, 1e and unreacted starting material 1c. Compound 1d: 37.1 mg (y = 33%), MS (m / z): found 1010.4 (M + Na) +, 1028.4 (M + H2O + Na) +, 1040.3 (M + 3H2O - H) -; compound 1e: 6.4 mg (y = 5.7%), MS (m / z): found 1012.4 (M + Na) +; compound 1c: 44.1 mg (y = 39%). See FIG. 1.
Compound 1g:
[00637] To a stirred solution of 1d (23.6 mg, 0.024 mmol) in acetonitrile (3 ml) and methanol (3 ml) was added freshly prepared TCEP solution (17 mg TCEP HCl salt was neutralized with sodium bicarbonate) saturated sodium at pH 6 ~ 6.5 then diluted with 0.5 mL of pH 6.5 phosphate buffer) at room temperature. The mixture was stirred at room temperature for 3 hours and then diluted with dichloromethane and deionized water, separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and subjected to high vacuum to generate 22 mg of 1f as a light yellow foam. Another 18 mg of 1f were prepared from 19 mg of 1d following the same procedure. The combined 40 mg (0.042 mmol) of 1f was dissolved in anhydrous dichloromethane (0.5 ml) and stirred. To this stirring solution was added SPDB NHS 2 ester (34.6 mg, 80% purity, 0.085 mmol) and diisopropylethylamine (15 μl, 0.085 mmol). It continued to be stirred at room temperature overnight, quenched with saturated ammonium chloride and diluted with dichloromethane, separated and washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by preparative reverse phase HPLC (column C18, acetonitrile / water). The product-containing fractions were combined, extracted with dichloromethane and concentrated to generate compound 1g as a white solid (29.7 mg, y = 60%). 1H NMR (400 Hz, CD3CN): δ 8.28-8.25 (m, 1H), 8.20-8.17 (m, 1H), 7.87-7.84 (m, 1H), 7 , 49 (d, J = 4.4 Hz, 1H), 7.39 (d, J = 4.4 Hz, 1H), 7.31-7.19 (m, 4H), 7.13-7, 01 (m, 2H), 6.92-6.87 (m, 3H), 6.77 (bs, 1H), 6.31-6.29 (m, 1H), 5.16-5.09 ( m, 2H), 5.00 (d, J = 4.4 Hz, 2H), 4.94 (bs, -NH), 4.48-4.43 (m, 1H), 4.40-4, 34 (m, 1H), 3.90 (d, J = 4.4 Hz, 3H), 3.77 (d, J = 4.4 Hz, 3H), 3.64-3.39 (m, 18H ), 3.26 (d, J = 4.4 Hz, 3H), 2.82-2.70 (m, 8H), 2.17 (d, J = 4.4 Hz, 1H), 2.08 -2.01 (m, 3H), 1.30 (d, J = 4.4 Hz, 6H); MS (m / z): found 1025.9 (M + H2O + Na) +, 1043.9 (M + 2H2O + Na) +, 983.8 (M - H) -, 1055.8 (M + 4H2O - H)-; MS (m / z), found 1179.5 (M + Na) +. See FIG. 1. Example 2
Compound 1e:
[00638] To a stirred solution of 1c (8 mg, 0.0081 mmol) in anhydrous 1,2-dichloromethane (0.2 mL) was added sodium triacetoxycoborohydride (3.8 mg, 0.018 mmol). The mixture was stirred at room temperature for 1.5 hours, then the mixture was diluted with dichloromethane and quenched with saturated sodium bicarbonate, separated and the organic layer was washed with brine, dried over sodium sulfate, and filtered. The filtrate was concentrated and the remainder was purified by reverse phase HPLC (column C18, acetonitrile / water) to generate compound 1e as a white solid (4.7 mg, y = 58%). MS (m / z), found 1012.4 (M +
Compound 2a:
[00639] To a stirred solution of compound 1e (12 mg, 0.012 mmol) in acetonitrile (1 mL) and methanol (3 mL) was added freshly prepared TCEP solution (11 mg TCEP HCl salt was neutralized with sodium bicarbonate saturated to pH ~ 6.5 then diluted with 0.4 mL of pH 6.5 phosphate buffer) at room temperature. The mixture was stirred at room temperature for 3.5 hours and then diluted with dichloromethane and deionized water, separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the remainder was purified by reverse phase HPLC (column C18, acetonitrile / water) to generate compound 2a as a white solid (4.9 mg, y = 43%). MS (m / z), found 966.4 (M + Na) +, 978.2 (M + 2H2O - H) -. See FIG. 2. Example 3
Compound 3b:
[00640] To a solution of compound 3a (830 mg, 1.9 mmol) in methanol (15 mL) was added Pd / C (10%, 204 mg, 0.19 mmol). The air in the flask was removed by vacuum and then replaced with hydrogen in a flask. The mixture was stirred at room temperature overnight. The mixture was filtered through celite and the celite / Pd / C washed with dichloromethane and methanol. The filtrate was concentrated and the residue diluted with dichloromethane and evaporated for a few cycles and then purified by silica gel chromatography (dichloromethane / methanol) to generate compound 3b as a light yellowish solid (558 mg, y = 98%). 1H NMR (400 Hz, CDCl3): δ 8.34 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.22 (dd, J1 = 8.0 Hz, J2 = 7.6 Hz, 1H), 7.17 (d, J = 7.2 Hz, 1H), 7.02 (dd, J1 = 7.2 Hz, J2 = 7.6 Hz, 1H), 6.16 (s, 1H), 4.37 (tt, J1 = 10.4 Hz, J2 = 7.2 Hz, 1H), 3.76 (s, 3H), 3.49-3.36 (m, 3H) , 2.73 (dd, J1 = 16.8 Hz, J2 = 3.6 Hz, 1H); 13C NMR (400 Hz, CDCl3): δ 167.0, 150.4, 142.6, 141.2, 140.8, 129.9, 127.7, 124.8, 123.96, 117.4, 113.7, 112.5, 104.7, 57.3, 56.3, 54.7, 33.0; MS (m / z), found 295.1 (M - H) -. See FIG. 3.
Compound 3c:
[00641] To a solution of 2- (methyldithio) -isobutyraldehyde (113 mg, 0.75 mmol) and compound 3b (148 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (2 mL) was added triacetoxycoborohydride sodium (212 mg, 1.0 mmol). The mixture was stirred at room temperature for 2 days. During the time, two other portions (0.05 mL, 0.5 mmol / portion) of 2- (methyldithio) -isobutyraldehyde along with a portion of sodium triacetoxyborohydride (106 mg, 0.5 mmol) were added. The reaction was quenched with saturated sodium bicarbonate, diluted with dichloromethane and water. The organic layer was washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography (Combiflash column, 24 g, hexanes / ethyl acetate) to generate compound 3c as a fuzzy white solid (92.5 mg, y = 43% ). Unreacted starting material 3b was still recovered (49.3 mg, y = 33%). 1H NMR (400 Hz, CDCl3): δ 8.30 (d, J = 8.0 Hz, 1H), 7.28 (dd, J1 = 6.8 Hz, J2 = 7.6 Hz, 1H), 7 , 25-7.20 (m, 2H), 7.07 (t, J = 7.6 Hz, 1H), 6.80 (s, 1H), 6.17 (s, 1H), 4.36- 4.28 (m, 1H), 3.89 (s, 3H), 3.78 (d, J = 14.4 Hz, 1H), 3.46-3.34 (m, 3H), 2.90 (d, J = 14.4 Hz, 1H), 2.73 (dd, J1 = 16.4 Hz, J2 = 2.8 Hz, 1H), 2.34 (s, 3H), 1.17 (s , 3H), 1.05 (s, 3H); 13C NMR (400 Hz, CDCl3): δ 167.2, 149.0, 142.5, 142.2, 141.9, 129.9, 128.0, 125.3, 124.5, 124.1, 117.1, 112.0, 108.5, 64.8, 61.4, 58.1, 56.3, 53.4, 32.0, 26.3, 25.7, 25.4; MS (m / z), found 453.3 (M + Na) +, 429.2 (M - H) -. See FIG. 3.
3d compound:
[00642] To a stirred solution of IBD monomer (125 mg, 0.425 mmol) and 1,5-diiodopentane (0.63 mL, 4.25 mmol) in anhydrous dimethylformamide (3 mL) was added potassium carbonate (59 mg, 0.425 mmol) and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane, washed with brine and dried over anhydrous sodium sulfate. This was filtered and concentrated. The residue was purified by silica gel chromatography (hexanes / ethyl acetate) to generate compound 3d as yellowish foam (94 mg, y = 45%). 1H NMR (400 Hz, CDCl3): δ 8.27 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 4.8 Hz, 1H), 7.56 (s, 1H) , 7.27 (dd, J1 = 8.4 Hz, J2 = 7.6 Hz, 2H), 7.10 (dd, J1 = 7.6 Hz, J2 = 7.2 Hz, 1H), 6.82 (s, 1H), 4.48 (dt, J1 = 10.8 Hz, J2 = 4.4 Hz, 1H), 4.15-4.07 (m, 2H), 3.96 (s, 3H) , 3.70 (dd, J1 = 16.8 Hz, J2 = 10.8 Hz, 1H), 3.49 (dd, J1 = 16.8 Hz, J2 = 4.0 Hz, 1H), 3.22 (t, J = 7.2 Hz, 2H), 1.96-1.87 (m, 4H), 1.64-1.57 (m, 2H); 13C NMR (400 Hz, CDCl3): δ 164.0, 163.2, 151.4, 148.3, 142.2, 140.3, 129.6, 128.3, 124.9, 120.5, 117.0, 112.0, 110.6, 68.8, 56.4, 55.1, 33.3, 32.7, 28.0, 27.2, 6.6; MS (m / z), found 513.3 (M + Na) +, 543.2 (M + 3H2O - H) -. See FIG. 3.
Compound 3e:
[00643] To a stirred solution of the starting materials 3c (91 mg, 0.21 mmol) and 3d (94 mg, 0.19 mmol) in anhydrous dimethylformamide (1 mL) was added potassium carbonate (29 mg, 0, 21 mmol) and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane, washed with brine and dried over anhydrous sodium sulfate. This was filtered, concentrated and the residue was purified by silica gel chromatography (hexanes / ethyl acetate) to generate compound 3e as a yellowish foam (89.1 mg, y = 58%). 1H NMR (400 Hz, CDCl3): δ 8.32-8.28 (m, 2H), 7.91 (bs, 1H), 7.57 (s, 1H), 7.36- 7.21 (m , 5H), 7.15-7.05 (m, 2H), 6.85 (s, 1H), 6.74 (s, 1H), 4.53-4.48 (m, 1H), 4, 37-4.31 (m, 1H), 4.21 - 4.03 (m, 4H), 3.98 (s, 3H), 3.88 (s, 3H), 3.86-3.70 ( m, 2H), 3.55-3.35 (m, 4H), 2.93 (d, J = 4.0 Hz, 1H), 2.73 (dd, J1 = 16.4 Hz, J2 = 2 , 4 Hz, 1H), 2.36 (s, 3H), 2.03-1.96 (m, 3H), 1.77-1.67 (m, 3H), 1.21 (s, 3H) , 1.06 (s, 3H); MS (m / z), found 815.3 (M + Na) +. See FIG. 3.

[00644] To a stirred solution of compound 3e (33.1 mg, 0.042 mmol) in acetonitrile (2 ml) and methanol (4 ml) was added freshly prepared solution of TCEP (36 mg of TCEP salt HCl was neutralized with bicarbonate of saturated sodium to pH ~ 6.5 then diluted with 0.4 mL of pH 6.5 phosphate buffer) at room temperature. The mixture was stirred at room temperature for 3 hours and then diluted with dichloromethane and deionized water, separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and subjected to high vacuum to generate 31 mg of compound 3f as a yellowish solid. This was dissolved in anhydrous dichloromethane (0.5 ml). SPDB NHS 2 ester (26 mg, 80% purity, 0.063 mmol) and diisopropylethylamine (11 μl, 0.063 mmol) were added subsequently. The mixture continued to be stirred at room temperature overnight, quenched with saturated ammonium chloride and diluted with dichloromethane, separated and washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by preparative reverse phase HPLC (column C18, acetonitrile / water). The product-containing fractions were combined, extracted with dichloromethane and concentrated to generate compound 3g as a yellowish solid (15.2 mg, y = 38%). MS (m / z), found 984.3 (M + Na) +, 1014.2 (M + 3H2O - H) -. See FIG. 3. Example 4
Compound 4b:
[00645] A stirred solution of compound 4a (111 mg, 0.108 mmol) in absolute ethanol (720 μL) and anhydrous dichloromethane (360 μL) was cooled to 0 ° C in an ice bath. Sodium borohydride (0.817 mg, 0.022 mmol) in 50 µl of absolute ethanol was added at 0 ° C. The reaction was stirred at room temperature for two hours. The mixture was cooled to 0 ° C in an ice bath, quenched with saturated ammonium chloride and extracted with dichloromethane. The organic extracts were washed with brine, dried over anhydrous sodium sulfate and filtered through Celite. The filtrate was concentrated under reduced pressure and the crude material was purified by RP-HPLC (C18 DI water / acetonitrile) to generate compound 4b (43mg, 38%). 1H NMR (400 Hz, CDCl3): δ8.26 (d, 1H, J = 8.0Hz), 8.18 (d, 1H, J = 8.0Hz), 7.77 (d, 1H, J = 4 , 4 Hz), 7.51 (s, 1H), 7.41 (s, 2H), 7.17 (m, 6H), 7.03 (t, 1H, J = 7.2Hz), 6.96 (t, 1H, J = 7.2Hz), 6.76 (s, 1H), 6.04 (s, 1H), 5.13 (m, 4H), 4.38 (m, 2H), 3, 90 (s, 3H), 3.81 (s, 3H), 3.79 (m, 2H), 3.63 (m, 1H), 3.51 (m, 8H), 3.43 (m, 6H ), 3.25 (s, 3H), 2.73 (dd, 1H, J = 3.6, 16.4Hz), 2.22 (s, 3H), 2.04 (m, 2H), 1, 81 (m, 2H), 1.18 (s, 6H); MS (m / z) found, 1051.9 (M + Na), 1069.9 (M + Na + H2O). See FIG. 4.
Compound 4c:
[00646] To a stirred solution of compound 4b (40 mg, 0.039 mmol) in methanol (4.45 mL) and acetonitrile (2.225 mL) was added TCEP.HCl (39.0 mg, 0.136 mmol) in sodium phosphate buffer (0.89mL, pH 6.5). The mixture is stirred at room temperature for 18 hours. The mixture was diluted with dichloromethane and washed with water and brine. The organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated. Purification by RP-hPLC (C18, DI water / acetonitrile) and extraction with dichloromethane generated compound 4c (26.5mg, 64%); MS (m / z) found, 1006.0 (M + Na). See FIG. 4.
Compound 4d:
[00647] To a stirred solution of compound 4c (24 mg, 0.024 mmol) in anhydrous dichloromethane (800 μL) was added PBA (11.18 mg, 0.049 mmol) and diisopropylethylamine (20.18 μL, 0.116 mmol). After stirring for 18 hours at room temperature, the reaction was diluted with dichloromethane and quenched with saturated ammonium chloride. The layers were separated and the organic was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. PTLC purification (5% methanol / dichloromethane) generated compound 4d (17mg, 63%); MS (m / z) found, 1123.9 (M + Na) 1139.9 (M + K); 1099.8 (MH) 117.9 (M-H + H2O). See FIG. 4.
Compound 4e:
[00648] To a mixture of compound 4d (15 mg, 0.014 mmol) and N-hydroxy succinimide (4.70 mg, 0.041 mmol) in anhydrous dichloromethane (1.0 mL) was added EDC.HCl (7.83 mg, 0.041 mmol). After stirring for 18 hours at room temperature, the reaction was diluted with dichloromethane and quenched with saturated ammonium chloride. The mixture was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by RP-HPLC (C18, DI water / acetonitrile). The product-containing fractions were pooled and extracted with dichloromethane, dried over anhydrous magnesium sulfate, filtered and concentrated to generate compound 4e (13 mg, 80%); MS (m / z) found, 1220.8 (M + Na) 1238.8 (M + Na + H2O), 1254.8 (M + K + H2O). See FIG. 4. Example 5
Compound 5a:
[00649] A mixture of hydrated chelidamic acid (3.0 g, 15.56 mmol) and sulfuric acid (0.6 mL, 11.26 mmol) in absolute ethanol (40 mL) was refluxed for 20 hours. The reaction was cooled to room temperature, neutralized with aqueous sodium carbonate, and then acidified with concentrated HCl. Water was added and the mixture was extracted with dichloromethane. The extracts were dried with anhydrous magnesium sulfate, filtered and concentrated. The crude material was purified by silica gel chromatography (5% methanol / dichloromethane) to generate diethyl 4-hydroxypyridine-2,6-dicarboxylate (5a) (2.5 g, 68%) as a white solid. See FIG. 5.
Compound 5c:
[00650] A solution of 4-methyl-4- (methyldisulfanyl) pentan-1-ol (5b) (2.0 g, 11.09 mmol) in anhydrous dichloromethane (55.5 mL) was cooled to 0 ° C in an ice bath. Triethylamine (5.41 ml, 38.8 mmol) and sulfonyl toluene chloride (3.17 g, 16.64 mmol) were added at 0 ° C. The reaction stirred for three hours at room temperature. The mixture was extracted with ethyl acetate and washed with brine. The organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated. Purification by silica gel chromatography (5% ethyl acetate / hexanes) resulted in 4-methylbenzenesuenesulfonate of 4-methyl-4- (methyldisulfanyl) pentyl (5c) (1.5g, 40%). 5b: 1H NMR (400 Hz, CDCl3): δ3.42 (m, 2H), 2.19 (s, 3H), 1.77 (bs, 1H), 1.43 (m, 4H), 1.09 (s, 6H). 5c: 1H NMR (400 Hz, CDCl3): δ7.66 (d, 2H, J = 7.6Hz), 7.22 (d, 2H, J = 8.0Hz), 3.90 (t, 2H, J = 6.4Hz), 2.32 (s, 3H), 2.23 (s, 3H), 1.60 (m, 2H), 1.44 (m, 2H), 1.11 (s, 6H) . See FIG. 5.
Compound 5d:
[00651] To a stirred solution of 4-methyl-4-methylbenzenesuenesulfonate of 4-methyl-4- (methyldisulfanyl) pentyl (5c) (0.48 g, 1.435 mmol) and diethyl 4-hydroxypyridine-2,6-dicarboxylate (5a) (0.343 g, 1.435 mmol) in anhydrous dimethylformamide (6.5 ml) Potassium carbonate (0.297 g, 2.152 mmol) was added. The reaction was stirred at 90 ° C for 18 hours. Then, allowed to cool to room temperature and quenched with saturated ammonium chloride. The mixture was extracted three times with ethyl acetate. The extracts were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (30% hexanes / ethyl acetate) gave 4- (4-methyl-4- (methylldisulfanyl) pentyloxy) pyridine-2,6-dicarboxylate (5d) (300 mg, 52%) ; 1H NMR (400 Hz, CDCl3): δ 7.70 (s, 2H), 4.40 (q, 4H, J = 7.2, 14.4 Hz), 4.07 (t, 2H, J = 6. Hz), 2.35 (s, 3H), 1.86 (m, 2H), 1.70 (m, 2H), 1.38 (t, 6H, J = 7.2Hz), 1.27 (s , 6H); MS (m / z), found 424.1 (M + Na), 440.1 (M + K). See FIG. 5.
Compound 5e:
[00652] To a stirred solution of diethyl 4- (4-methyl-4- (methyldisulfanyl) pentyloxy) pyridine-2,6-dicarboxylate (5d) (270 mg, 0.672 mmol) in absolute ethanol (7.0 mL) calcium chloride (224 mg, 2.017 mmol) and sodium borohydride (76 mg, 2.017 mmol) were added. The reaction was allowed to stir at room temperature for 90 minutes after which it was quenched with water and concentrated in vacuo to remove ethanol. The mixture was then extracted twice with dichloromethane. The organic extracts were combined, washed with water, dried with anhydrous magnesium sulfate and filtered through celite. The filtrate was concentrated under reduced pressure and the crude material was purified by silica gel chromatography eluting with 10% methanol / dichloromethane to generate (4- (4-methyl-4- (methyldisulfanyl) pentyloxy) pyridine-2,6-diyl ) dimethanol (5e) (75 mg, 35%); 1H NMR (400 Hz, CDCl3): δ 6.63 (s, 2H), 4.60 (s, 4H), 3.95 (t, 2H, J = 6.2Hz), 3.54 (bs, 2H ), 2.35 (s, 3H), 1.82 (m, 2H), 1.66 (m, 2H), 1.26 (s, 6H); MS (m / z), found 340.1 (M + Na). See FIG. 5.
Compound 5f:
[00653] A stirred solution of (4- (4-methyl-4- (methyldisulfanyl) pentyloxy) pyridine-2,6-diyl) dimethanol (5e) (51 mg, 0.161 mmol) in anhydrous dichloromethane (1.6 mL) it was cooled to -5 ° C in an acetone / ice bath. Triethylamine (0.112 ml, 0.803 mmol) and methanesuanesulfonyl chloride (0.031 ml, 0.402 mmol) were added. The mixture was stirred for 60 minutes at -5 ° C. The reaction was quenched with ice water and extracted with cold ethyl acetate. The organic extracts were washed with ice water, dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure to generate the dimesylate. To a stirring mixture of dimesylate intermediate (179 mg, 0.378 mmol) and IBD monomer (256 mg, 0.869 mmol) in anhydrous dimethylformamide (3.8mL) was added potassium carbonate (261 mg, 1.890 mmol) and potassium iodide ( 31.4 mg, 0.189 mmol). The reaction was allowed to stir at room temperature for 18 hours. The mixture was quenched with water and extracted three times with dichloromethane. The organic extracts were dried over sodium sulfate, filtered and concentrated. The crude material was redissolved in acetonitrile and purified on RP-HPLC (C18, deionized water / acetonitrile). The product-containing fractions were combined and extracted with dichloromethane, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to generate compound 5f (65 mg, 20%); 1H NMR (400 Hz, CDCl3): δ 8.20 (d, 2H, J = 8.0 Hz), 7.78 (m, 2H), 7.53 (s, 2H), 7.20 (m, 4H ), 7.04 (t, 2H, J = 7.4 Hz), 6.91 (m, 2H), 6.80 (s, 2H), 5.22 (s, 4H), 4.40 (m, 2H), 3.94 (s, 6H), 3.93 (m, 2H), 3.63 (m, 2H), 3.42 (dd, 2H, J = Hz), 2.32 (s, 3H ), 1.80 (m, 2H), 1.64 (m, 2H), 1.24 (s, 6H); MS (m / z), found 892.3 (M + Na) 910.3 (M + Na + H2O) 928.3 (M + Na + 2H2O). See FIG. 5.
Compound 5g and 5h:
[00654] A solution of compound 5f (74 mg, 0.085 mmol) in absolute ethanol (600 μL) and anhydrous dichloromethane (300 μL) was cooled to 0 ° C in an ice bath. Sodium borohydride (0.644 mg, 0.017 mmol) in 50 μL of absolute ethanol was added at 0 ° C. The mixture was allowed to stir at room temperature for two hours and was then cooled to 0 ° C. The reaction was quenched with saturated ammonium chloride and extracted with dichloromethane. The organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered through Celite and concentrated under reduced pressure. The crude material was redissolved in dimethylformamide and purified by RP-HPLC (C18 deionized water / acetonitrile). The fractions containing compounds 5g and 5h were combined separately and extracted with dichloromethane, dried with anhydrous magnesium sulfate, filtered and concentrated to generate compound 5g (20 mg, 27%) and compound 5h. 5g: 1H NMR (400 Hz, CDCl3): δ 8.25 (m, 1H), 8.18 (m, 1H), 7.77 (m, 1H), 7.51 (ss, 1H), 7, 40 (ss, 1H), 7.18 (m, 4H), 7.08 (m, 1H), 7.03 (m, 1H), 6.92 (m, 2H), 6.86 (ss, 1H ) 5.98 / 6.06 (ss, 1H), 5.24 (m, 4H), 4.40 (m, 1H), 4.30 (m, 1H), 3.94 (s, 3H), 3.92 (m, 2H), 3.84 (s, 3H), 3.62 (m, 1H), 3.37 (m, 4H), 2.65 (m or dd, 1H), 2.32 (ss, 3H), 1.77 (m, 2H), 1.64 (m, 2H), 1.24 (s, 6H). 5h: 1H NMR (400 Hz, CDCl3): δ 8.24 (d, 2H, J = 8.0Hz), 7.39 (s, 2H), 7.14 (m, 4H), 6.97 (m , 2H), 6.93 (m, 2H), 6.15 (ss, 2H), 5.25 (s, 4H), 4.37 (m or t, 2H, J = 9.8Hz), 4, 2 (bs, 2H), 3.94 (m, 2H), 3.83 (s, 6H), 3.40 (m, 6H), 2.72 (dd, 2H, J = Hz), 2.32 (s, 3H), 1.79 (m, 2H), 1.64 (m, 2H), 1.24 (s, 6H). See FIG. 5.
Compound 5i:
[00655] To a stirred solution of compound 5g (20 mg, 0.023 mmol) in methanol (5.25 mL) and acetonitrile (1.750 mL) was added TCEP.HCl (19.72 mg, 0.069 mmol) in phosphate buffer sodium (0.7mL, pH 6.5). The mixture was stirred for 3 hours at room temperature and then diluted with dichloromethane and water. The layers were separated and the organic was washed with brine. The crude product was purified by RP-HPLC (C18, deionized water / acetonitrile). The product-containing fractions were combined, extracted with dichloromethane and evaporated to generate compound 5i (7 mg, 37%). MS (m / z), found 848.3 (M + Na) 866.3 (M + Na + H2O) 880.3 (M + Na + MeOH). See FIG. 5.
Compound 5j:
[00656] To a stirred solution of compound 5i (7 mg, 8.47 μmol) and 2,5-dioxopyrrolidin-1-yl 4- (pyridin-2-ildisulfanyl) butanoate (8.64 mg, 0.021 mmol) in dichloromethane anhydrous (113 μL) diisopropylethylamine (3.69 μL, 0.021 mmol) was added. After stirring for 18 hours at room temperature, the reaction was quenched with saturated ammonium chloride solution and extracted with dichloromethane. The organic extracts were washed with brine, dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by preparative RP-HPLC (C18, deionized water / acetonitrile). Fractions containing the product were extracted with dichloromethane, filtered and evaporated to generate compound 5j (3 mg, 34%). MS (m / z), found 1063.3 (M + Na) 1081.3 (M + Na + H2O). See FIG. 5. Example 6 Preparation of antibody-SPDB-drug conjugate:
[00657] Compound 1g was pretreated with 3 molar equivalents of sodium bisulfite (using a freshly prepared solution of NaHSO3 in water) in 96-98% DMA in water for 4-5 hours at 25 ° C. For conjugation, the humanized antibody at 2 mg / mL was reacted with 5-7 molar equivalents of compound 1g (pretreated with NaHSO3) for 6 h at 25 ° C in 85-90% PBS, pH 7.4, aqueous buffer, or 50 mM HEPES, pH 8.5, aqueous buffer, containing 10-15% N, N-dimethylacetamide (DMA) and then purified by a G25 column of gel filtration in PBS, pH 7.4, to remove non-drug compound hydrolyzed reaction. The humanized antibody-SPDB-drug conjugates were dialyzed in 10 mM Histidine, 250 mM Glycine, 1% sucrose, pH 6.5 buffer. The Drug Antibody (DAR) ratio of the conjugates was measured to be 2.2-2.9 by UV absorbance measurements at 280 and 320 nm and using the drug and antibody extinction coefficients at 280 nm (215,000 M-1cm-1 ) and 320 nm (9137 M-1cm-1). The percentage of monomer in the conjugates was determined to be> 90% by SEC (size exclusion chromatography) using TSK-Gel G300SWXL column (7.8 mm x 300 mm, 5 μm particle size). Based on the UV absorbance of the monomer peak in SEC, it was further demonstrated that the conjugated monomer peaks had drug molecules attached. For the free drug (unconjugated) assay, the conjugate was extracted with acetone to remove protein, dried, and reconstituted in the mobile phase and injected into a VYDAC 208TP C8 HPLC reverse phase column (4.6 x 250 mm, 7 μm particle size) and compared to standards. The percentage of free drug compound in the conjugate was determined to be <0.5% of the conjugated drug compound. See FIG. 22. Preparation of humanized Ab-SPDB-2a conjugate:
[00658] Humanized Ab at 8 mg / mL was derivatized with 4-6 molar equivalents of bifunctional heterologous SPDB for 1.5 h at 25 ° C in 95% PBS, PH 7.4, containing 5% DMA (v / v), and then purified by a G25 desalting column in citrate buffer (35 mM citrate buffer, pH 5.5, containing 2 mM EDTA, 150 mM NaCl) to remove unreacted binder. LAR (Antibody Binding Ratio) was measured using UV absorbance at 280 and 343 nm without and with 50 mM addition of dithiothreitol (to measure total antibody and SPy released by dithiothreitol) and was determined to be 2.7-4.1 LAR. The modified SPDB-antibody at 2 mg / mL was reacted with 2 molar equivalents of compound 2a (HCl salt) by SPDB bound for 20 h at room temperature in 85% citrate buffer, 15% DMA (v / v) and then purified by a G25 desalting column in PBS, pH 7.4 to remove unconjugated drug compound. The DAR of the final humanized Ab-SPDB-2a conjugate was measured by UV spectrophotometry at 280 and 350 nm and calculated to be ~ 1.7-2.1 DAR. The percentage of monomer and drug compound bound to the monomer in the conjugate was determined by HPLC using SEC column (size exclusion chromatography). See FIG. 23. Example 7 In vitro potency of free and conjugated drugs:
[00659] General procedure used: Samples of unconjugated drug-free compounds were added to 96-well flat-bottom tissue culture plates and titrated using serial dilutions to cover the desired molar range. Antigen positive (Ag +) or antigen negative (Ag-) cells were added to the wells at specific cell densities so that they were triplicate samples for each drug concentration for each corresponding cell line. The plates were then incubated at 37 oC in an atmosphere of 5% CO2 for 4-5 days depending on the cell line. COLO 205 (1,000 cells / well), Namalwa (3,000 cells / well), HEL 92,1,7 (3,000 cells / well) - 4 days; RH30 (1,000 cells / well), HL60 / QC (5,000 cells / well), Ramos (10,000 cells / well), KB (2,000 cells / well), BJAB (2,000 cells / well), NB4 (3,000 cells / well) - 5 days, RPMI 8226 (8,000 cells / well) - 6 days.
[00660] At the end of the incubation period cytotoxic potencies were then assessed using a cell viability assay based on WST-8 and surviving cells were measured developing with WST-8 (2-7 hours). The absorbance in each well was measured and the portion of surviving cells at each concentration was plotted to reveal the cytotoxicity and / or antigen specificity (of the conjugates).
[00661] Using the general procedure described above, the cytotoxicity of the unconjugated free compounds of the drug was measured against seven cell lines: KB, a HeLa cell contaminant, HL60 / QC, an acute myeloid leukemia cell line, Namalwa, a Burkitt's lymphoma cell line, NB4, an acute promyelocytic cell line, HEL92.1.7, an erythroleukemia cell line, RPMI8226, a multiple myeloma cell line and BJAB, a B-cell leukemia cell line. results, shown in FIG. 24 and Table 10 demonstrate the high potency of these compounds across a wide range of cell types. The potency and specificity of the antibody-drug conjugates were measured against cells that express antigens, with and without the addition of an excess amount of unconjugated blocking antibody to show specificity of the death effect. The MY9-6-drug conjugate was extremely potent for three different cells that express the antigen: HL60 / ATCC, HL60 / QC and NB-4, despite the very low expression of antigen in NB4 cells. The specific potency could be blocked by adding excess unconjugated antibody, demonstrating that the cell's death effect is antigen specific. Similarly, the huFOLR1- drug conjugate was effective in killing KB cells that express antigen in a specific way. The results are illustrated in Figures 25 and 26. Table 10. Potency of free drugs against various cell lines. The IC50 values listed in the table are listed in the nM unit.

Similar results were also obtained using different cell lines and different conjugates of the invention, including: huMY9-6-SPDB-1f against HL60 / QC cells (Ag +), HL60 / ATCC cells (Ag +), and NB-4 cells ( Ag +) (FIG. 25); huFOLR1-SPDB-1f against KB (Ag +) cells (FIG. 26); huMY9-6-SPDB-1f against antigen positive HL60 / QC cells, HL60 / ATCC cells, NB-4 cells, and HEL 92.1.7 cells (FIG. 29); huMy9-6-SPDB-1f, huMy9-6-sulfoSPDB-1f, and huMy9-6-BMPS-1f against HL60 / QC (Ag +) cells (FIG. 34); chB38,1-SPDB-1f and chB38,1-sulfoSPDB-1f against COLO205 (Ag +) cells (FIG. 35); huMy9-6-SPDB-1f, huMy9-6-sulfoSPDB-1f, and huMy9- 6-BMPS-1f against OCI-AML3 (Ag +) cells (FIG. 44). See also FIG. 49 for the potency of the various conjugates against various cell lines, expressed as IC50 values (nM). Note that in FIGs. 25, 29, 34, 35, and 44, conjugates were prepared in the presence of sodium bisulfite.
[00663] To compare in vitro potency measurements for object conjugates prepared with and without reactive imine reagent, such as sodium bisulfite, huMy9-6-BMPS-1f, huMy9-6-sulfo-SPDB-1f, and huMy9-6- drug 2 were prepared with and without sodium bisulfite using the in situ sulfonation method (in which the respective compounds of the invention were first mixed with sodium bisulfite and a bifunctional cross-linker containing a reactive group, then the reaction mixture, without another purification, was reacted with the monoclonal antibody huMy9-6 as the cell binding agent). IC50s for the conjugates in HL60-QC cells are shown below. The data indicate that the inclusion of an imine reactive group (such as sodium bisulfite) in the conjugate preparation step does not negatively impact the in vitro potency of the object conjugates.

[00664] It is apparent that the pretreatment of drug compounds with sodium bisulfite (5 molar equivalents, 22 h, 4 ° C, 90:10 DMA pH 5.5 water) before conjugation with huMy9-6 had no effect significant in antigen-dependent or antigen-independent potency (blocking antigen with 1 μM of unconjugated huMy9-6) in vitro of the conjugates. Example 8
[00665] Antibody-Drug Conjugate Binding is similar to unmodified antibody:
[00666] The huMY9-6-drug conjugate binding was compared with that of unmodified huMy9-6 antibody against HL60 / QC cells expressing the antigen using flow cytometry. Briefly, antigen positive cells were incubated with conjugates or unmodified antibodies at 4 ° C, then with a secondary antibody-FITC conjugate at 4 ° C, fixed with formaldehyde (1% in PBS) and analyzed by flow cytometry. No significant difference was observed between the binding of the conjugate versus that of the unmodified antibody. An example is shown in FIG. 27, where a huMY9-6-drug conjugate bound to antigen-positive cells with a high affinity similar to that of unmodified antibody. Example 9 In vivo efficacy of huMy9-6-SPDB-1f conjugate in nude mice containing HL60 / QC tumor:
[00667] In this study, the anti-tumor activity of huMY9-6-SPDB-1f was investigated in nude female mice containing HL60 / QC tumors, a model of acute human myeloid leukemia. HL60 / QC tumor cells, 2 x 106 cells / mouse were subcutaneously inoculated in a volume of 0.1 ml / mouse in the area over the right shoulder of nude athymic female mice, 5 weeks old. Eight days after inoculation of mice with tumor cells were randomized into groups (n = 6 per group) by tumor volume. Treatment was started on the day of randomization, and the groups included a control group dosed with PBS (200 μL / injection), or a single treatment in several doses (5 to 100 μg / kg) of huMY9-6-SPDB-1f (50 μg / kg 1f dose corresponded to 2.5 mg / kg antibody dose). All treatments were well tolerated with average body weights comparable to the loss observed in control mice with PBS. Average tumor volume vs time is shown (FIGS. 28 and 36) with the data demonstrating a dose-dependent anti-tumor activity of huMY9-6-SPDB-1f conjugate. The minimum effective dose was estimated to be 20 μg / kg, which is about 35 times less than the maximum tolerated dose. Example 10
[00668] The tolerability of huFOLR-1 conjugates was investigated in female CD-1 mice. The animals were observed for seven days before the start of the study and demonstrated to be free of disease or illness. The mice were administered with a single iv injection of the conjugate and the animals were monitored daily for body weight loss, morbidity or mortality. Table 9 shows that for the huFOLR1-drug1 conjugate it was tolerated in only the smallest of those tested at 50 μg / kg. In contrast, both the huFOLR1-Drug2 and huFOLR1-SPDB-1f monoinimine conjugates have been shown to be better tolerated with a maximum tolerated dose of <198 μg / kg and> 560 μg / kg respectively. Table 9. Tolerability comparison data for (A) huFOLRI-drug conjugates, (B) huFOLR1-drug2, and (C) huFOLR1-SPDB-1f. THE)



Compound 10:
[00669] To a stirred solution of 1f (18 mg, 0.019 mmol) and N- (β-maleimidopropyloxy) succinimide (BMPS) ester (9.2 mg, 0.034 mmol) in anhydrous dichloromethane (0.3 mL) -anhydrous isopropylethylamine (5 μL, 0.029 mmol). The mixture was stirred at room temperature for 27 hours, quenched with saturated ammonium chloride and diluted with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC (column C18, CH3CN / H2O). The product-containing fractions were combined, extracted with dichloromethane and evaporated to generate compound 10 as a white solid (7.6 mg, y = 33%). MS (m / z): found 1208.3 (M + H) +. See FIG. 13. Example 12
Compound 12:
[00670] To a stirred solution of 1f (16.5 mg, 0.018 mmol) and sulfo-SPDB (14.2 mg, 0.036 mmol) in anhydrous dichloromethane (0.3 mL) was added anhydrous diisopropylethylamine (9 μL, 0.054 mmol). The mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC (column C18, CH3CN / H2O). The product-containing fractions were combined, extracted with dichloromethane and evaporated to generate 6.6 mg of compound 12 as yellowish foam. The aqueous layer was lyophilized to generate an additional 0.5 mg of compound 12 as a white solid. MS (m / z): found 1235.0 (M-H) -. See FIG. 15. Example 13 Preparation of humanized antibody-sulfo conjugate SPDB-1f
[00671] A reaction containing 2.5 mg / mL huMy9-6 antibody and 10 molar equivalents of 10 (pretreated with 5 times excess sodium bisulfite in 90:10 DMA: water) in 50 mM HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic) pH 8.5 buffer and 15% v / v DMA (N, N-dimethylacetamide) cosolvent was left to conjugate for 6 hours at 25 ° C. After the reaction, the conjugate was purified and buffer exchanged in 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween, 50 μM sodium bisulfite buffer formulation, using NAP desalting columns (Illustra Sephadex G-25 DNA Grade , GE Healthcare). Dialysis was performed in the same buffer for 4 hours at room temperature using Slide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO). The purified conjugate was found to have a DAR of 2.4 (by UV-Vis using molar extinction coefficients ε330 nm = 15.484 cm-1M-1 and ε280 nm = 30, 115 cm-1M-1 by 1f, and ε280 nm = 146,000 cm-1M-1 for My9-6 antibody), 96.7% monomer (by size exclusion chromatography), <1% unconjugated free drug compound (by acetone extraction / reverse phase HPLC) and a concentration final protein of 1.4 mg / mL.
[00672] The in vitro potency of antibody-sulfoSPDB-1f conjugates was measured according to the general procedure described in Example 7 and the data are shown in Figures 34 and 35. The antibody-sulfoSPDB-1f conjugates have comparable or greater potency than antibody-SPDB-1f conjugates.
[00673] Use of covalent imine reactants, such as sodium bisulfite, improves the specifications of the Ab-compound conjugate (for example,% monomer and drug load). In one experiment, adduct formation was performed with 5 molar equivalents of reactive imine on NHS-BMPS-lf in 90% DMSO / 10% PBS pH 7.4 for 4 h at 25 ° C. The reaction mixture was then added to the huMy9-6 antibody (4 molar equivalents IGN, 2 mg / ml, 10% v / v DMSO, 50 mM HEPES buffer, pH 8.5, 5 h, 25 ° C). Conjugates prepared using sodium hydrosulfite, sodium bisulfite, or sodium metabisulfite had similar ratios of IGN / Ab and% monomer, while conjugates prepared without additive treatment led to very low drug incorporation. See table below.
Example 14 Preparation of humanized antibody-BMPS-1f conjugate
[00674] A reaction containing 2.0 mg / mL huMy9-6 antibody and 5 molar equivalents of 12 (pretreated with 5 times excess sodium bisulfite in 90:10 DMA: water) in 50 mM HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic) pH 8.5 buffer and 15% v / v DMA (N, N-dimethylacetamide) cosolvent was allowed to react for 6 hours at 25 ° C. After the reaction, the conjugate was purified and buffer exchanged in 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween, 50μM μM sodium bisulfite buffer formulation, using NAP desalting columns (Illustra Sephadex G-25 DNA Grade , GE Healthcare). Dialysis was performed in the same buffer for 4 hours at room temperature using Slide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO). The purified conjugate was shown to have a DAR of 2.8 (by UV-Vis using molar extinction coefficients ε330 nm = 15.484 cm-1M-1 and ε280 nm = 30, 115 cm-1M-1 by 1f, and ε280 nm = 146,000 cm-1M-1 for My9-6 antibody), 91.7% monomer (by size exclusion chromatography), <1% unconjugated free drug compound (by acetone extraction / reverse phase HPLC) and a final concentration of 1.2 mg / mL protein.
[00675] The in vitro potency of antibody-BMPS-1f conjugates was measured according to the general procedure described in Example 7 and the data are shown in FIGS. 34 and 35. Antibody-BMPS-1f conjugates have potency comparable to antibody-SPDB-1f conjugates. Example 15 In vivo efficacy of hu conjugate FOLR1-SPDB-1f in nude mice containing KB tumor:
[00676] In this study, the antitumor activity of hu FOLR1-SPDB-1f was investigated in nude female mice containing KB tumors, a model of human cervical carcinoma. KB, 1 x 107 cells / mouse were inoculated subcutaneously in a volume of 0.1 ml / mouse in the area over the right shoulder of nude female athymic mice, 6 weeks old. Six days after tumor cell inoculation, mice were randomized into groups (n = 6 per group) by tumor volume. Treatment was started the day after randomization, and the groups included a control group dosed with PBS (200 μL / injection), or a single treatment in multiple doses (20 to 200 μg / kg) of hu FOLR1-SPDB-1f (50 μg / kg of bound drug dose corresponding to 2.8 mg / kg antibody dose). All treatments were well tolerated with no weight loss seen in any of the test groups. Average tumor volume vs time is shown (FIG. 37) with the data demonstrating a dose-dependent hu FOLR1-SPDB-1f antitumor activity. The minimum effective dose was estimated to be <50 μg / kg, which is about 14 times less than the maximum tolerated dose.
[00677] Similar in vivo results have been obtained using other conjugates of the invention against several other cancer models, including huMy9-6-sulfo-SPDB-1f in mice containing MOLM-13 tumor (FIG. 50); huMy9-6-sulfo-SPDB-1f in mice containing NB4 tumor (FIG. 51); huMy9-6-BMPS-1f in mice containing HL60 / QC tumor (FIG. 52); huMy9-6-BMPS-1f in mice containing MOLM-13 tumor (FIG. 53); huMy9-6-drug 2 in mice containing HL60 / QC tumor (FIG. 56); and huMy9-6- drug 2 in mice containing MOLM-13 tumor (FIG. 57). Note that in FIGs. 53, 54, 56, and 57, conjugates were prepared in the presence of sodium bisulfite.
[00678] To compare in vivo efficacy of object conjugates prepared with or without a reactive imine group, huMy9-6-drug 2 were formulated with or without 50 μM sodium bisulfite, and the conjugates were used to treat mice containing HL60- xenograft tumors QC. The data below show that the conjugate formulated with or without 50 μM sodium bisulfite showed T / C% comparable to ~ 20 μg / kg dose of the drug, indicating that the inclusion of sodium bisulfite in the conjugate preparation does not negatively impact the potency in live from the object conjugate.
Compound 27b:
[00679] (5 - ((2-mercapto-2-methylpropylthio) methyl) -1,3-phenylenophenylene) dimethanol: (5- (mercaptomethyl) -1,3-phenylene) dimethanol (0.163 g, 0.885 mmol) was dissolved in methanol (3 mL) in a small bottle and a stir bar was added. To this solution was added triethylamine (0.016 ml, 0.118 mmol) followed by 2,2-dimethylthiirane (0.058 ml, 0.590 mmol) and the resulting mixture was closed and stirred overnight (16 hrs) at room temperature. The reaction was then concentrated, redissolved in dichloromethane, loaded onto a ptlc silica plate (1000 micron) and the plate was developed using 10% methanol in dichloromethane. The band corresponding to the product was scraped, filtered with liquid ethyl acetate, and concentrated to generate (5 - ((2-mercapto-2-methylpropylthio) methyl) -1,3-phenylene) dimethanol (0.095 g, 0.349 mmol, 59 , 1% yield). 1H NMR (400 Hz, CDCl3): δ 7.26 (s, 3H), 4.69 (s, 4H), 3.82 (s, 2H), 2.74 (s, 2H), 2.17 ( s, 1H), 2.12 (br s, 2H), 1.43 (s, 6H); 13C NMR (400 Hz, CDCl3): δ 141.6, 138.9, 126.7, 124.3, 65.0, 49.0, 45.4, 38.4, 31.5; MS (m / z), expected: 272.4, found 295.0 (M + Na). See FIG. 30.
Compound 27c:
[00680] (5 - ((2-methyl-2- (methylldisulfanyl) propylthio) methyl) -1,3-phenylene) dimethanol: (5 - ((2-mercapto-2-methylpropylthio) methyl) -1,3- phenylene) dimethanol (0.120 g, 0.440 mmol) was dissolved in ethanol (5 mL) and 1.0 M potassium phosphate buffer (pH 7) (5.00 mL) and cooled in an ice bath (a ppt was formed, but was ignored). S-methyl methanesulfonothioate (0.083 mL, 0.881 mmol) was added and the mixture stirred overnight with gradual heating (for 30 minutes) at room temperature. The reaction was diluted with dichloromethane and the organic layer was removed, washed with water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was dissolved in dichloromethane and loaded onto a 500 micron ptlc plate and developed with 66% ethyl acetate in hexane. The band corresponding to the product was scraped, filtered using ethyl acetate, and concentrated to generate (5- ((2-methyl-2- (methylldisulfanyl) propylthio) methyl) -1,3-phenylene) dimethanol (0.091 g, 0.286 mmol , 64.9% yield). 1H NMR (400 Hz, CDCl3): δ 7.27 (s, 3H), 4.71 (s, 4H), 3.78 (s, 2H), 2.77 (s, 2H), 2.41 ( s, 3H), 1.94 (br s, 2H), 1.38 (s, 6H); 13C NMR (400 Hz, CDCl3): δ 141.6, 139.0, 126.7, 124.2, 65.0, 51.8, 44.0, 38.2, 26.7, 25.3; MS (m / z), expected: 341.5, found 341.1 (M + Na). See FIG. 30.
Compound 27d:
[00681] (5 - (((2-methyl-2- (methyldisulfanyl) propylthio) methyl) -1,3-phenylene) dimethanol (80 mg, 0.251 mmol) in anhydrous dichloromethane (1.75 mL) was cooled to -5 ° C ° C in a brine / ice bath. Triethylamine (105 μL, 0.753 mmol) was added followed by the addition of methanesuanesulfonyl chloride (50.7 μL, 0.653 mmol) at -5 ° C. The reaction was stirred at -5 ° C for one hour after which it was diluted with cold ethyl acetate and ice was added. The mixture was transferred to a separatory funnel and extracted with cold ethyl acetate. The organic extracts were washed with ice water and then dried with anhydrous magnesium and sodium sulfate, filtered and concentrated under reduced pressure. The resulting (5 - ((2-methyl-2- (methylldisulfanyl) propylthio) methyl) -1,3-phenylene) bis (methylene) dimethanesulfonate was used without further purification.
[00682] IBD monomer (177 mg, 0.602 mmol) in anhydrous N, N-dimethylformamide (1.75 mL) was added to (5 - ((2-methyl-2- (methyldisulfanyl) propylthio) methyl) -1,3 -phenylene) bis (methylene) dimethanesulfonate (119 mg, 0.251 mmol) at room temperature. Potassium carbonate (173 mg, 1.253 mmol) was added and the reaction was allowed to stir at room temperature for 20 hours. The reaction mixture was quenched with water and extracted with dichloromethane. The extracts were washed with brine and then dried with anhydrous sodium sulfate, filtered and concentrated in a high vacuum. The crude product was purified by flash chromatography on silica gel (neat DCM ^ 2% MeOH / DCM). The product-containing fractions were combined, concentrated and purified by RP-HPLC semi-prep (C18, A = DI water B = ACN, 20mL / min). The fractions containing the desired product were combined, extracted with dichloromethane, dried with anhydrous magnesium sulfate, filtered and concentrated to generate the desired product (46mg, 21%). 1H NMR (400 Hz, CDCl3): δ 8.19 (d, J = 8.0 Hz, 2H), 7.77 (m, d, J = 4.4 Hz, 2H), 7.50 (s, 2H), 7.34 (s, 1H), 7.31 (s, 2H), 7.19 (m, 4H), 7.03 (t, J = 7.2, 7.6 Hz, 2H), 6.77 (s, 2H), 5.14 (m, 4H), 4.40 (m, 2H), 3.91 (s, 6H), 3.70 (m, 2H), 3.63 (m , 2H), 3.41 (m, 2H), 2.65 (s, 2H), 2.29 (s, 3H), 1.26 (s, 6H). MS (m / z), calculated. 893.2 (M + Na) +; found 893.2 (M + Na) +, 911.2 (M + H2O + Na) +, 929.2 (M + 2H2O + Na) +, 945.1 (M + 2H2O + K) +. See FIG. 30.
Compound 27e and 27f:
[00683] To a cooled solution (0o C) of 27d (50 mg, 0.057 mmol) in anhydrous dichloromethane (225 μL) and ethanol (450 μL) was added sodium borohydride (0.651 mg, 0.017 mmol). The reaction was stirred for five minutes at 0 oC and then at room temperature for 2.5 hrs. The reaction mixture was cooled to 0 oC, quenched with saturated ammonium chloride, and extracted with dichloromethane. The organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered through Celite and concentrated. The crude material was purified by RP-HPLC semi-prep (C18, A = DI water B = ACN, 20mL / min). Fractions containing the desired product were combined, extracted with dichloromethane, dried over anhydrous magnesium sulfate, filtered and concentrated to generate the reduced mono amine 27e (11mg, 22%) MS (m / z), Calculated. 895.3 (M + Na) + found 895.2 (M + Na) +, 913.2 (M + H2O + Na) +, 929.2 (M + H2O + K) + and the reduced amine 27f (5mg, 10%) MS (m / z), Calculated. 897.3 (M + Na) +, found 897.3 (M + Na) +. See FIG. 30.
27g Compound:
[00684] To a stirred solution of 27e (10 mg, 0.011 mmol) in methanol (733 μL) and acetonitrile (880 μL) was added tris (2-Carboxyethyl) phosphine hydrochloride (9.85 mg, 0.034 mmol) in buffer 6 , 5 (147 μL). The mixture is stirred for 3 hours at room temperature. The reaction was diluted with dichloromethane. Water was added and the layers were separated. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to generate compound 27g (9mg, 95%). MS (m / z), Calculated. 849.3 (M + Na) +; found 849.2 (M + Na) +, 867.2 (M + K) +. See FIG. 30.
Compound 27h:
[00685] To a stirred solution of 27g (9 mg, 10.88 μmol) and 2,5-dioxopyrrolidin-1-yl 4- (pyridin-2-ildisulfanyl) butanoate (9.3mg, 0.023 mmol) in anhydrous dichloromethane ( 0.4 mL) anhydrous diisopropylethylamine (9 μl, 0.054 mmol) was added and the reaction was stirred at room temperature overnight. The mixture was quenched with saturated ammonium chloride solution and extracted with dichloromethane. The extracts were washed with brine, dried with anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC (column C18, CH3CN / H2O). The product containing fractions were combined, extracted with dichloromethane and evaporated to generate compound 27h (5mg, 44%). MS (m / z), Calculated. 1064.3 (M + Na) +; found 1064.1 (M + Na) +, 1082.1 (M + H2O + Na) +, 1098.1 (M + H2O + K) +. See FIG. 30. Example 17
Compound 28b:
[00686] (5- (methyl (2-methyl-2- (methylldisulfanyl) propyl) amino) -1,3-phenylene) dimethanol (52 mg, 0.172 mmol) was dissolved in anhydrous dichloromethane (1.7 mL) and cooled at -5 in an acetone / ice bath. First, triethylamine (0.120 ml, 0.862 mmol) was added followed by methanesulfonyl chloride (0.040 ml, 0.517 mmol). The mixture was stirred in the bath for 1 hour. The reaction was then diluted with cold ethyl acetate and washed with cold water three times and then dried over anhydrous magnesium sulfate. The dimesylate was filtered, concentrated in vacuo, and placed in high vacuum until completely dry. The product was used as such directly in the next step.
[00687] IBD monomer (115 mg, 0.39 mmol) in anhydrous N, N-dimethylformamide (1.5 mL) was added to (5- (methyl (2-methyl-2 (methyl-sulfanyl) propyl) amino dimethanesulfonate ) -1,3-phenylene) bis (methylene) (72 mg, 0.156 mmol) at room temperature. Potassium carbonate (108 mg, 0.780 mmol) was added and the reaction was allowed to stir at room temperature for 20 hours. Water (10mL) was added directly to a mixture with resulting stirring to form a white precipitate. The mixture was filtered and the solids were washed with more portions of water. The solid was then dissolved in dichloromethane, extracted with water, the organic layer was then dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to generate compound 28b (104 mg, 78%) which was used in the next step without further treatment . MS (m / z), found 912.1 (M + 2H2O + Na). See FIG. 31.
Compound 28c and 28d:
[00688] Compound 28b (55 mg, 0.064 mmol) was dissolved in an anhydrous mixture of dichloromethane (0.4mL) and ethanol (0.8 ml) and cooled to 0 ° C in an ice bath. A solution of sodium borohydride (0.731 mg, 0.019 mmol) dissolved in ethanol (100 μl) was then added and the mixture was stirred for 5 minutes and the ice bath was removed. The reaction was allowed to stir for 2 hours, quenched at low temperature by adding saturated ammonium chloride and dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by RP-HPLC semi-prep (C18, A = DI water B = ACN, 20mL / min). Fractions containing the desired products were extracted with dichloromethane and concentrated to generate monoimine 28c (19 mg, 32%) MS (m / z), expected: 855.1, found: 896.2 (M + H2O + Na ) and the reduced amine 28d (22 mg, 38%) MS (m / z), expected: 857.1, found: 880.2 (M + Na) +. See FIG. 31.
Compound 29b and 29c:
[00689] Compound 29a (60 mg, 0.043 mmol) was dissolved in an anhydrous mixture of dichloromethane (0.25mL) and ethanol (0.5 ml) and cooled to 0 ° C in an ice bath. A solution of sodium borohydride (0.493 mg, 0.013 mmol) dissolved in ethanol (50 μL) was then added and the mixture was stirred for 5 minutes and the ice bath was removed. The reaction was allowed to stir for 3 hours, quenched at low temperature by adding saturated ammonium chloride and dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by RP-HPLC semi-prep (C18, A = DI water B = ACN, 20mL / min). Fractions containing the desired products were extracted with dichloromethane and concentrated to generate monoimine 29b (20 mg, 33%) MS (m / z), expected: 715.7, found: 715.2 (M + Na) + , 733.2 (M + H2O + Na) +, 749.2 (M + H2O + K) + and the reduced di-amine 29c (12 mg, 20%) MS (m / z), expected: 694.7 , found: 717.2 (M + Na) +. See FIG. 32. Example 19
Compound 30a:
[00690] (5 - ((2- (2- (2-methoxyethoxy) ethoxy) ethyl) (2-methyl-2- (methyldisulfanyl) propyl) amino) -1,3-phenylene) bis (methylene) dimethanesulfonate (0.566 g, 0.960 mmol) was dissolved in acetone (30 ml) and a solution of sodium iodide (0.544 g, 3.63 mmol) dissolved in acetone (2 ml) was added with vigorous stirring. The reaction was monitored by tlc (50% ethyl acetate in hexane) and after 2 hours the reaction was filtered, concentrated in vacuo and dichloromethane was added to the residue. The solid salt left behind was filtered, the filtrate was concentrated and the resulting residue was purified on silica gel using a 3: 5: 2 mixture of ethyl acetate: hexane: dichloromethane to generate 3,5-bis (iodomethyl) - N- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) -N- (2-methyl-2- (methylldisulfanyl) propyl) aniline (0.550 g, 0.773 mmol, 74.5% yield) as a yellow oil . 1H NMR (400 Hz, CDCl3): δ 6.75 (s, 2H), 6.73 (s, 1H), 4.38 (s, 4H), 3.63 (m, 14H), 3.40 ( s, 3H), 2.50 (s, 3H), 1.38 (s, 6H); 13C NMR (400 Hz, CDCl3): δ 148.7, 140.3, 117.3, 113.4, 71.9, 70.7, 70.6, 67.2, 59.8, 59.1, 53.5, 53.4, 51.8, 26.5, 25.6, 6.11; MS (m / z), Calculated. 676.0 (M + Na) +; found 675.8 (M + Na) +. See FIG. 33.
Compound 30b:
[00691] IBD monomer (0.060 g, 0.204 mmol) was dissolved in acetone (4 ml) in a small flask, a stir bar was added, followed by 3,5-bis (iodomethyl) -N- (2- (2 - (2-methoxyethoxy) ethoxy) ethyl) -N- (2-methyl-2- (methyldisulfanyl) propyl) aniline (0.167 g, 0.255 mmol) and potassium carbonate (0.070 g, 0.510 mmol). The flask was sealed and stirred at room temperature overnight. The solids were filtered and the filtrate was concentrated. The residue was dissolved in dichloromethane, extracted with water, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to generate 108 mg of crude material. The crude material was purified on silica gel using 30% ethyl acetate to remove the starting material diiodine followed by 10% methanol in dichloromethane to generate the desired product 30b (21 mg, 0.026 mmol, 13%). MS (m / z), expected: 819.1, found: 858.0 (M + K) +, 890.0 (M + CH3OH + K) +. See FIG. 33. Compound 1d:
[00692] The reduced monomer 3b (4.16 mg, 0.014 mmol) was dissolved in acetone (2 ml) in a small flask, a stir bar was added, followed by 30b (10 mg, 0.012 mmol) and potassium carbonate (4.21 mg, 0.030 mmol). The flask was sealed and stirred at room temperature overnight. The reaction was concentrated to remove the acetone and then redissolved in dichloromethane, extracted with water, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by reverse phase C18 HPLC to generate 1d (2.1 mg, 2.125 μmol, 17.42% yield). MS (m / z): found 1010.4 (M + Na) +, 1028.4 (M + H2O + Na) +. See FIG. 33. Example 20: Synthesis of compound 1
Compound 1:
[00693] To a stirred suspension of 1f (226 mg, 0.24 mmol) in IPA (20 mL) and deionized water (10 mL) was added sodium bisulfite (50 mg, 0.48 mmol). The mixture was stirred vigorously at rt for 2 hours. It was frozen with dry ice / acetone and lyophilized. The white shaggy solid obtained was dissolved in CH3CN / H2O and purified by reverse phase HPLC (column C18, CH3CN / H2O). The fractions containing the desired product were combined and frozen with dry ice / acetone and lyophilized to generate the desired compound 1 as a fuzzy white solid (179.6 mg, 5 = 71.6%). MS (m / z): found 1022.0 (M - H) -. See FIG. 38.
Compound 9c:
[00694] To a stirred solution of 1c (60 mg, 0.061 mmol) in CH3CN (3 mL) was added freshly prepared TCEP solution (49 mg, 0.17 mmol of TCEP HCl salt was neutralized with saturated sodium bicarbonate until pH ~ 6.5 then diluted with 0.5 mL of pH 6.5 phosphate buffer) at room temperature. MeOH (2.5 mL) was added and the mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with dichloromethane and deionized water, separated and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered. The filtrate was peeled and subjected to high vacuum to generate 60 mg of 1h as a light yellow foam. MS (m / z): found 940.1 (M + H) +. This was dissolved in methanol (1.0 ml) and CH3CN (1.4 ml) followed by the addition of iodoacetic acid (24 mg, 0.13 mmol), deionized water (0.1 ml) and potassium carbonate (27 mg , 0.19 mmol). The mixture was stirred at rt overnight (monitored by LCMS). This was quenched with saturated ammonium chloride to prepare the acid solution then diluted with dichloromethane, separated and washed with brine, dried over anhydrous Na2SO4, filtered and separated to generate compound 9c (57.8 mg, y = 91%) which was directly used for the next step without purification. MS (m / z): found 998.1 (M + H) +. See FIG. 12A.
Compound 9a:
[00695] To a stirred solution of compound 9c (57.8 mg, 0.058 mmol) in anhydrous dichloromethane (0.2 mL) and absolute ethanol (0.6 mL) was added NaBH4 (2.5 mg, 0.066 mmol) at 0 ° C. The ice bath was removed and the mixture was stirred at room temperature for 3 hours and then quenched with saturated ammonium chloride, diluted with dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered through celite and separated . The residue was purified by reverse phase HPLC (column C18, CH3CN / H2O). The product fractions were extracted with dichloromethane and separated to generate compound 9a (13.0 mg, y = 22%). MS (m / z): found 1000.0 (M + H) +, 1015.9 (M + H2O - H) -. See FIG. 12A.
Compound 9a:
[00696] To a solution of 1f free thiol (45 mg, 0.048 mmol) and iodoacetic acid (18 mg, 0.096 mmol) in methanol (1.0 mL) and CH3CN (1.4 mL) was added deionized water (0, 1 mL) and potassium carbonate (20 mg, 0.14 mmol). The mixture was stirred at rt overnight (monitored by LCMS). This was quenched with saturated ammonium chloride to prepare the acid solution then diluted with dichloromethane, separated and washed with brine, dried over anhydrous Na2SO4, filtered and separated. The residue was purified by preparative HPLC reverse phase (C18, CH3CN / H2O). The pure product fractions (based on MS) were extracted with dichloromethane, separated to generate the desired acid 9a (18 mg, y = 38%). MS (m / z): found 1000.1 (M + H) +. See FIG. 12B. Example 22 Synthesis of compound 1d
Compound 1d:
[00697] To a stirred solution of compound 1c (178 mg, 0.18 mmol) in anhydrous dichloromethane (1.2 mL) and absolute ethanol or anhydrous methanol (0.1 mL) was added 5-ethyl-2-methylpyridine borane (PEMB, 0.017 mL, 0.11 mmol) under drip. The mixture was stirred at rt for 1 hour and quenched with 88% formic acid. This was basified with saturated NaHCO3 and diluted with dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered through celite and separated. The residue was dissolved in CH3CN / H2O / 88% HCOOH (5: 1: 0.05) and purified by reverse phase HPLC (C18, CH3CN / H2O). The fractions containing pure product were extracted with dichloromethane and separated to generate compound 1d (56 mg, y = 31%). MS (m / z): found 988.1 (M + H) +. See FIG. 39.
Compound 1d:
[00698] To a stirred solution of compound 1c (71 mg, 0.072 mmol) in anhydrous 1,2-dichloroethane (0.8 mL) was added sodium triacetoxycoborohydride (14 mg, 0.65 mmol). The mixture was stirred at rt for 2 hours and quenched with saturated NaHCO3 and diluted with dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered through celite and separated. The residue was dissolved in CH3CN / H2O / 88% HCOOH (5: 1: 0.05) and purified by reverse phase HPLC (C18, CH3CN / H2O). The fractions containing the pure product were extracted with dichloromethane and separated to generate compound 1d (17 mg, y = 24%). MS (m / z): found 988.1 (M + H) +. Unreacted starting material 1c was further recovered (24 mg, y = 34%). See FIG. 40. Example 23 Synthesis of compound 31c
Compound 31a:
[00699] To a solution of compound 1f (57.8 mg, 0.061 mmol) and methyl 4-b'omobutylate (22 mg, 0.12 mmol) in methanol (1.0 mL) and CH3CN (1.0 ml), deionized water (0.1 ml) and potassium carbonate (17 mg, 0.12 mmol) were added. The mixture was stirred at night, then quenched with saturated ammonium chloride and diluted with dichloromethane, separated and washed with brine, dried over Na2SO4, filtered. ada and sepa'ada. The 'waste was pu'ified by' HPLC p'epa'active phase 'eve'sa (C18, CH3CN / H2O) for ge'a' the desired product 31a (14 mg, y = 22%) as yellow foam. MS (m / z): found 1042.1 (M + H) +. See 'FIG. 41.
Compound 31b:
[00700] To a solution of methyl 31a (14 mg, 0.013 mmol) in anhydrous 1,2-dichloroethane (1.5 mL) was added t-methyl hydroxide (36 mg, 0.2 mmol). The mixture was stirred overnight in an 80 ° C oil bath until the starting material was completely consumed. This was' ground to room temperature, diluted with dichloromethane, washed with brine / drops 5% HCl and brine, dried and filtered. The filtrate was separated and purified with silica gel chromatography (dichloromethane / MeOH) to generate acid 31b as a yellowish solid (10.2 mg, y = 74%). MS (m / z): found 1028.2 (M + H) +, 1044.1 (M + H2O -H) -. See FIG. 41.
Compound 31c:
[00701] To a solution of 31b acid (10.2 mg, 0.0099 mmol) in anhydrous dichloromethane (0.5 ml) was added N-hydroxysuccinimide (3.4 mg, 0.03 mmol) and PL-DCC ( 26 mg, 0.04 mmol, 1.55 mmol / g). The mixture was stirred at room temperature overnight and filtered to remove the resin. The resin was washed with dichloromethane then ethyl acetate. The filtrate was separated and the residue was purified by reverse phase HPLC (C18, CH3CN / H2O). The product containing fractions were combined and lyophilized to generate NHS 31c ester as a white solid (3.6 mg, y = 32%). MS (m / z): found 1125.1 (M + H) +. See FIG. 41. Example 24 Synthesis of compound 32c
Compound 32a:
[00702] To a stirred solution of aniline 1a (339 mg, 1.1 mmol) in anhydrous tetrahydrofuran (4.0 mL) was added Boc anhydride (272 mg, 1.2 mmol). The mixture continued to be stirred at room temperature for three days. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (CH2Cl2 / MeOH) to generate compound 32a (405 mg, y = 90%) as a colorless oil. 1H NMR (400 Hz, CDCl3): δ 7.00 (s, 2H), 6.97 (s, 1H), 4.38 (s, 4H), 4.12 (s, 2h), 3.64 ( t, J = 5.6 Hz, 2H), 3.48-3.44 (m, 8H), 3.40-3.38 (m, 2H), 3.21 (s, 3H), 1.31 (s, 9H); 13C NMR (400 Hz, CDCl3): δ 154.65, 142.3, 142.1, 124.1, 122.7, 80.2, 71.6, 70.3, 70.1, 69.9, 68.5, 63.9, 58.65, 49.4, 28.1. See FIG. 42.
Compound 32b:
[00703] To a stirred solution of compound 32a (51 mg, 0.128 mmol) in anhydrous dichloromethane was added triethylamine (0.053 mL, 0.383 mmol) at -5 ~ -10 ° C. Methanesulfonyl chloride (0.026 mL, 0.332 mmol) was then added slowly over 15 minutes with a syringe. The mixture was stirred at -5-10 ° C for 1 hour (TLC, DCM / MeOH 10: 1). The reaction was quenched with ice / water, diluted with cold AcOEt, separated and the organic layer was washed with cold water, dried over anhydrous Na2SO4 / MgSO4, filtered and separated. The residue was transferred in a small reaction flask with dichloromethane, separated and subjected to high vacuum. It was dissolved in anhydrous DMF (0.8 ml) followed by the addition of IBD monomer (90 mg, 0.31 mmol) and potassium (53 mg, 0.38 mmol). The mixture was stirred at rt overnight. This was diluted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate, filtered and separated. The residue was purified by reverse phase HPLC (C18, CH3CN / H2O) to generate compound 32b (56 mg, 46%) as a yellowish solid. %). 1H NMR (400 Hz, CDCl3): δ 8.29 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 4.8 Hz, 2H), 7.60 (s, 2H) , 7.38-7.36 (m, 3H), 7.33-7.27 (m, 4H), 7.13 (t, J = 7.6 Hz, 2H), 6.88 (s, 2H ), 5.21 (dd, J1 = 20.0 Hz, J2 = 12.4 Hz, 4H), 4.49 (dt, J1 = 11.2 Hz, J2 = 4.0 Hz, 2H), 3, 99 (s, 6H), 3.83 (t, J = 6.0 Hz, 2H), 3.76-3.48 (m, 14H), 3.35 (s, 3H), 1.43 (s , 9H); MS (m / z): found 992.2 (M + H2O + Na) +, 1010.2 (M + 2H2O + Na) +. See FIG. 42.
Compound 32c:
[00704] To a stirred solution of compound 32b (56 mg, 0.059 mmol) in anhydrous dichloromethane (0.3 ml) and absolute ethanol (0.9 ml) was added NaBH4 (2.7 mg, 0.07 mmol) to 0 ° C. The ice bath was removed and the mixture was stirred at room temperature for 3 hours and then quenched with saturated ammonium chloride, diluted with dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous Na2SO4 and filtered through celite and separated . The residue was purified by reverse phase HPLC (column C18, CH3CN / H2O). Recovered starting material 32b weighed 12 mg which was again subjected to the reduction conditions and purified by reverse phase HPLC. All fractions containing the pure product were extracted with dichloromethane and separated to generate compound 32c (20.7 mg, y = 37%) as a light yellowish solid. MS (m / z): found 954.2 (M + H) +. See FIG. 42. Example 25
[00705] The tolerability of huMy9-6 conjugates was investigated in female CD-1 mice. The animals were observed for seven days before the start of the study and demonstrated to be free of disease or illness. The mice were administered with a single iv injection of the conjugate and the animals were monitored daily for body weight loss, morbidity or mortality. Table 10 shows that for the huMy9-6-SPDB-1c diimine disulfide conjugate containing conjugate it was tolerated at a dose of less than 300 μg / kg. In contrast, the huMy9-6-SPDB-1f and huMy9-6-sulfo-SPDB-1f monoimine disulfide conjugates have been shown to be better tolerated with a maximum tolerated dose of> 729 μg / kg and <750 μg / kg respectively. Table 10. Tolerability comparison data for conjugates (A) huMy9-6-SPDB-1c, (B) huMy9-6-SPDB-1f, (C) huMy9-6-sulfo-SPDB-1f, and (D) huMy9 -6-BMPS-1f.
Example 26 Compound 33b:
[00706] Compound 33a (20g, 77 mmol) was added as a slurry in anhydrous dichloromethane (100 ml) and was cooled to 0 ° C. Acetic acid (191mL) was added, resulting in a clear solution that was stirred at 0 oC until cool. Nitric acid (26mL, 581 mmol) was added slowly under drip through an addition funnel. The ice bath was removed and the solution continued to stir at room temperature. After 3 hours, the reaction was diluted with deionized water and extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and the filtrate concentrated in vacuo. The crude residue was recrystallized using ethyl acetate and hexanes. The solid was filtered and washed with hexanes to generate compound 33b as a shaggy yellow solid (13.8g, y = 59%). 1H NMR (400 Hz, CDCl3): δ 7.48-7.43 (m, 6H), 7.25 (s, 1H), 5.25 (s, 2H), 4.02 (s, 3H), MS (m / z): 326.1 (M + Na) +. See Figure 45.
3,5-bis ((((tert-butyldimethylsilyl) oxy) methyl) -N- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) aniline:
[00707] A mixture of 2- (2- (2-methoxyethoxy) ethoxy) ethyl (5-amino-1,3-phenylene) dimethanol (11.78 g, 77 mmol), ethyl (15.3 g , 48.1 mmol), and potassium carbonate (13.28 g, 96 mmol) in DMF (96 ml) was refluxed for 20 hours. The reaction was cooled to room temperature and diluted with dichloromethane. The mixture was filtered through celite and concentrated in vacuo. The resulting orange oil was dissolved in dichloromethane (240 ml) and t-butyldimethylsilyl chloride (18.09 g, 120 mmol) and imidazole (9.80 g, 144 mmol) were added. The reaction was stirred at room temperature for 20 hours when it was diluted with dichloromethane and filtered through celite. Purification by silica gel chromatography (EtOAc / Hex) generated 3,5-bis ((((tert-butyldimethylsilyl) oxy) methyl) -N- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) aniline (13g , 52%). 1H NMR (400 Hz, CDCl3): δ6.52 (s, 1H), 6.40 (s, 2H), 4.56 (s, 4H), 3.60 (t, 2H, J = 5.2 Hz ), 3.56 (m, 6H), 3.46 (m, 2H), 3.29 (s, 3H), 3.20 (t, 2H, J = 5.2 Hz), 0.84 (s , 18H), 0.00 (s, 12H). MS (m / z): found 550.1 (M + Na) +. See FIG. 46.
3.5- bis ((((tert-butyldimethylsilyl) oxy) methyl) -N- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) - N- (2-methyl-2- (methylsulfanyl) propyl) aniline:
[00708] To a solution of 3,5-bis ((((tert-butyldimethylsilyl) oxy) methyl) -N- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) aniline (6.7 g, 12, 69 mmol) in anhydrous 1,2-dichloroethane (50 ml) was added 2- (methyldithio) isobutyraldehyde (2.74 ml, 19.04 mmol), sodium triacetoxycoborohydride (2.8 g, 1eq), zinc chloride (II ) (0.865 g, 6.35 mmol) and magnesium sulfate (2.292 g, 19.04 mmol). The mixture was stirred for five hours at room temperature. Sodium triacetoxycoborohydride (2.8g, 1eq) was added. The reaction continued to stir at room temperature for 20 hours. The mixture was filtered through celite rinsing with dichloromethane and concentrated under reduced pressure then extracted with ethyl acetate and water. The organic extracts were washed with brine, dried over magnesium sulfate, filtered, concentrated and purified by combiflash (EtOAc / Hex) to generate 3,5-bis ((((tert-butyldimethylsilyl) oxy) methyl) -N- (2- ( 2- (2-methoxyethoxy) ethoxy) ethyl) -N- (2-methyl-2- (methyldisulfanyl) propyl) aniline (3.5g, 40%). 1H NMR (400 Hz, CDCl3): δ 6.73 (s, 2H), 6.59 (s, 1H), 4.56 (s, 4H), 3.65-3.51 (m, 14H), 3.30 (s, 3H), 2.38 (s, 3H), 1.28 (s, 6H), 0.84 (s, 18H), 0.00 (s, 12H). MS (m / z): found 684.2 (M + Na) +. See FIG. 46.
(5 - ((2- (2- (2-methoxyethoxy) ethoxy) ethyl) (2-methyl-2- (methyldisulfanyl) propyl) amino) -1,3-phenylene) dimethanol (1b):
[00709] Tetrabutylammonium fluoride (1M in THF) (10.57 ml, 10.57 mmol) was added under a drip to the stirring solution of 3,5-bis ((((tert-butyldimethylsilyl) oxy) methyl) -N- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) -N- (2-methyl-2- (methylldisulfanyl) propyl) aniline (3.5 g, 5.29 mmol) in anhydrous THF (65 ml) a 0 oC in an ice bath. After addition, the mixture was stirred at room temperature for two hours. The mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate. The extracts were washed with water and brine, dried with magnesium sulfate, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (MeOH / DCM) generated (5 - ((2- (2- (2-methoxyethoxy) ethoxy) ethyl) (2-methyl-2- (methyldisulfanyl) propyl) amino) -1,3 - phenylene) dimethanol (2g, 87%). 1H NMR (400 Hz, CDCl3): δ 6.76 (s, 2H), 6.63 (s, 1H), 4.55 (s, 4H), 3.65-3.51 (m, 14H), 3.35 (s, 3H), 2.44 (s, 3H), 1.33 (s, 6H); 13C NMR (400 Hz, CDCl3): δ 149.0, 142.35, 114.0, 111.1, 71.98, 70.7, 70.6, 70.5, 67.6, 65.5, 59.75, 59.1, 53.9, 51.9, 26.6, 25.7, 20.75; MS (m / z): found 456.2 (M + Na) +. See FIG. 46. Example 28
(5- (2-methyl-2- (methyldisulfanyl) propylamino) - 1,3-phenylene) dimethanol:
[00710] (5-amino-1,3-phenylene) dimethanol (2.5 g, 16.32 mmol) and 2- (methyldithio) isobutyraldehyde (2.347 ml, 16.32 mmol) were stirred at room temperature in absolute ethanol (82 ml) until completely dissolved (3 hours). The mixture was cooled to 0 ° C in an ice bath and sodium borohydride (0.741 g, 19.59 mmol) was added. The reaction was stirred for 1 hour at 0 oC, and was then slowly quenched with a cold 5% HCl solution. The mixture was diluted with dichloromethane and the pH was adjusted to pH = 8 with saturated sodium bicarbonate solution then extracted with dichloromethane and then washed with brine. The organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel chromatography (MeOH / DCM) generated (5- (2-methyl-2- (methylldisulfanyl) propylamino) - 1,3-phenylene) dimethanol (3g, 65%) as a white solid.
[00711] 1H NMR (400 Hz, CDCl3): δ6.62 (s, 1H), 6.54 (s, 2H), 4.53 (s, 4H), 3.13 (s, 2H), 2, 30 (s, 3H), 1.32 (s, 6H). See FIG. 47. Example 29
9-hydroxy-8-methoxy-6-oxo-12a, 13-dihydro-6H-benzo [5.6] [1.4] diazepine [1,2-a] indole-11 (12H) -carboxylate tert-butyl:
[00712] To a solution of 9-hydroxy-8-methoxy-11,12,12a, 13-tetrahydro-6H-benzo [5.6] [1.4] diazepine [1,2-a] indole- 6-one 3b (0.3 g, 1.012 mmol) in methanol (5.06 ml) were added di-tert-butyl dicarbonate (0.265 g, 1.215 mmol), triethylamine (0.212 ml, 1.519 mmol) and DMAP (6 , 18 mg, 0.051 mmol). After 5 hours of stirring at room temperature the reaction mixture was concentrated in vacuo. The residue was redissolved in dichloromethane and filtered through celite. Purification by silica gel chromatography (20% EtOAc / DCM) generated 9-hydroxy-8-methoxy-6-oxo-12a, 13-dihydro-6H-benzo [5.6] [1.4] diazepine [ 1,2-a] tert-butyl indole-11 (12H) -carboxylate (0.21g, 52%) as a white solid. 1H NMR (400 Hz, CDCl3): δ 8.25 (d, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.18 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.2 Hz, 1H), 6.98 (t, J = 7.2 Hz, 1H), 6.39 (s, 1H), 4.37 (m, 1H), 3 , 75 (s, 3H), 3.42 (m, 3H), 2.74 (dd, J = 3.6, 16.4 Hz, 1H), 1.47 (s, 9H). See FIG. 48. Example 30 Preparation and testing of huMy9-6-31c
Antibody conjugate
[00713] A reaction containing 2.0 mg / mL huMy9-6 antibody and 5 molar equivalents of compound 31c (pretreated with 5 times excess sodium bisulfite in 90:10 DMA: water) in 50 mM HEPES (acid 4 - Ethanesulfonic (2-hydroxyethyl) -1-piperazine) pH 8.5 buffer and 10% v / v DMA (N, N-Dimethylacetamide) cosolvent was left to conjugate for 6 hours at 25 ° C. After the reaction, the conjugate was purified and buffer exchanged in 250 mM Glycine, 10 mM Histidine, 1% sucrose, 0.01% Tween-20, 50μM sodium bisulfite formulation buffer, pH 6.2, using NAP desalting columns (Illustra Sephadex G-25 DNA Grade, GE Healthcare). Dialysis was performed in the same buffer for 4 hours at room temperature using Slide-a-Lyzer dialysis cassettes (ThermoScientific 20,000 MWCO).
[00714] The purified conjugate was shown to have an average of 3.1 IGN molecules bound by antibody (by UV-Vis using molar extinction coefficients ε330 nm = 15.484 cm-1M-1 and ε280 nm = 30, 115 cm-1M-1 per 1, and ε280 nm = 207,000 cm-1M-1 for My9-6 antibody), 98% monomer (by size exclusion chromatography), <0.2% unconjugated drug (by column reverse phase HPLC analysis double) and a final protein concentration of 0.4 mg / ml.
[00715] In vitro potency measurements for huMy9-6 conjugates with 31c in two different drug loads were shown below. Both conjugates were highly potent for antigen-positive HL60-QC cells, with IC 50 values between 1.3-1.8 pM. Blocking antigen with 1 μM of unconjugated huMy9-6 significantly decreased potency, demonstrating the antigen specificity of the cytotoxic effect.
Example 31 In vivo efficacy of various conjugates in nude mice containing tumor
[00716] In this study, the antitumor activity of several conjugates of the invention is investigated in immune compromised mice (nu or SCID), preferably female nude mice, containing several tumors. In some cases, in addition to or as an alternative, nude mice can be employed. The conjugates to be tested include any one or more of the conjugates described here. The various tumor cell lines that can be used to inoculate nude mice included HL60 / QC, MOLM-13, NB4, HEL92.1.7, OCI-AML3, KB, and / or any other cancer cell line recognized in the art as an appropriate model for a disease indication (eg cancer). Some criteria that can be applied for the selection of appropriate tumor lines for in vivo evaluation include: a) expression of the target antigen in the tumor cell, and, b) sensitivity of tumor cells to unconjugated drug in vitro. For example, an in vitro cell line sensitivity screening, such as cell line 60 screening described by the US National Cancer Institute (see Voskoglou-Nomikos et al., 2003, Clinical Cancer Res. 9; 42227-4239, incorporated herein by reference) can be used as one of the guidelines for determining the types of cancers that may be appropriate for treatment with the compounds of the invention. The potency of the various conjugates against the various tumor cell lines, as expressed by IC 50 values (nM), is therefore measured.
[00717] The various tumor cell lines are inoculated to the nude mice or SCID using substantially the same protocol as outlined in Example 15. For example, about 1 x 106 - 5 x 107 tumor cells (typically 1 x 107) cells / mice are inoculated subcutaneously in a volume of approximately 0.1-0.2 mL / mouse, in the area over the right shoulder of nude female athymic mice, 6 weeks old. When the tumor has reached an average size of ~ 100 mm3 (typically 6 to 8 days after tumor cell inoculation), mice are randomized into groups (for example, n = 5 - 8 per group) by tumor volume. Treatment starts the day after randomization, and the groups included a control group with the appropriate vehicle (200 μL / injection), or a single treatment in multiple doses (5 to 700 μg / kg) of the drug conjugates referenced above (50 μg / kg of bound drug dose corresponding to about 2 mg / kg of antibody dose). Calendars of various dosages (for example, treatment on day 1, 3, 5, or day 1, 4, 7) can still be used.
[00718] Median and mean tumor volume vs time is measured, with the data demonstrating a dose-dependent antitumor activity of the object conjugates. The minimum effective dose is then calculated and compared to the maximum tolerated dose. Example 32 Preparation of huMy9-6-sulfo-SPDB-1d using the 4-nitroPy-sulfo-SPDB linker
[00719] A reaction containing 6 mg / mL huMy9-6 antibody and 5 molar equivalents of the highly reactive N-succinimidyl-4- (4-nitropyridyl-2-dithio) butanoate (20 mM ethanol stock) was incubated for 3 ha 25 ° C in 50 mM EPPS buffer at pH 8. Unreacted binder was removed using a NAP desalting column (Illustra Sephadex G-25 DNA Grade, GE Healthcare). The ratio of ligand to antibody (LAR) was determined to be about 2.3 based on antibody concentration and concentration of nitropyridine-2-thione released by DTT by UV-Vis (ε394 nm = 14205 cm-1M-1 by 2 -thio-4-nitropyridone).
[00720] Modified ligand huMy9-6 was diluted to 2 mg / mL in 50 mM HEPES buffer at pH 8.5, 10% v / v DMA, and reacted with 2 molar equivalents of compound 1d per ligand (5 mM DMA stock 4.6 antibody equivalents) for 30 min at 25 ° C. The completion of the disulfide exchange reaction was determined by monitoring an increase in absorbance at 394 nm by UV.
[00721] After the reaction, the conjugate was purified and buffer exchanged in 250 mM glycine, 10 mM histidine, 1% sucrose, 0.01% Tween-20, 50 μM sodium bisulfite at pH 6.2 using the desalting column ( G-25 Sephadex, fine grade, GE Healthcare).
[00722] The purified conjugate was shown to have an average of 2.1 molecules of 1d bound by antibody (by UV-Vis using molar extinction coefficients ε330 nm = 15.484 cm-1M-1 and ε280 nm = 30, 115 cm-1M- 1 by 1d, and ε280 nm = 207,000 cm-1M-1 for huMy9-6), 98% monomer (by size exclusion chromatography), <1% unconjugated (by acetone extraction / reverse phase HPLC), at 70% protein yield, and an overall yield of 32% 1d. See FIG. 60.

权利要求:
Claims (24)
[0001]
1. Cytotoxic compound, characterized by the fact that it is represented by the following formula:
[0002]
2. Compound according to claim 1, characterized by the fact that Zs is -H.
[0003]
3. A compound according to claim 1 or 2, characterized by the fact that Rk is -H or -Me.
[0004]
A compound according to any one of claims 1 to 3, characterized by the fact that Rx is - (CH2) p- (CRfRg) -, in which Rf and Rg are each independently selected from -H or a straight or branched alkyl with 1 to 4 carbon atoms; and p is 0, 1, 2 or 3.
[0005]
5. Compound, according to claim 4, characterized by the fact that Rf and Rg are the same or different and are selected from -H and -Me; and p is 1.
[0006]
6. Compound according to claim 1, characterized by the fact that the compound is:
[0007]
7. Compound according to claim 6, characterized by the fact that the compound is represented by the following formula:
[0008]
A compound according to any one of claims 1 to 7, characterized in that Y is -SO3M.
[0009]
Compound according to any one of claims 1 to 8, characterized in that M is -H, Na + or K +.
[0010]
10. Conjugate, characterized by the fact that it comprises a cytotoxic compound and a cell binding agent (CBA), in which the cytotoxic compound comprises a linking group that covalently binds the cytotoxic compound to CBA, and in which said cytotoxic compound is represented by the following formula:
[0011]
11. Conjugate according to claim 10, characterized by the fact that Rk is -H or -Me.
[0012]
12. Conjugate according to claim 10 or 11, characterized by the fact that Rx is - (CH2) p- (CRfRg) -, in which Rf and Rg are each independently selected from H or a linear alkyl or branched with 1 to 4 carbon atoms; and p is 0, 1, 2 or 3.
[0013]
13. Conjugate, according to claim 12, characterized by the fact that Rf and Rg are the same or different and are selected from H and Me; and p is 1.
[0014]
14. Conjugate according to claim 10, characterized by the fact that the conjugate is:
[0015]
15. Conjugate according to claim 14, characterized by the fact that the conjugate is represented by the following formula:
[0016]
16. Conjugate according to any one of claims 10 to 15, characterized in that the antibody is huMy9-6.
[0017]
17. Conjugate according to any one of claims 10 to 16, characterized in that Y is -SO3M.
[0018]
18. Conjugate according to any one of claims 10 to 17, characterized by the fact that M is -H, Na + or K +.
[0019]
19. Conjugate according to any of claims 10 to 18, characterized in that the cell-binding agent is an antibody, a single chain antibody, an antibody fragment that specifically binds to the target cell, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to the target cell, a domain antibody, a domain antibody fragment that specifically binds to the target cell, a recapture antibody, a recapture single chain antibody, or a recapture antibody fragment, a monoclonal antibody, a single chain monoclonal antibody, or a monoclonal antibody fragment of the even, a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment, a lymphokine, a hormone, a vitamin, a cre factor a colony stimulating factor, or a nutrient transport molecule.
[0020]
20. Conjugate according to any one of claims 10 to 19, characterized in that the conjugate comprises 1 to 10 cytotoxic compounds, each cytotoxic compound comprising the linking group that binds the cytotoxic compound to CBA and each cytotoxic compound in the conjugate it's the same.
[0021]
21. Pharmaceutical composition, characterized by the fact that it comprises the compound, as defined in claim 1, or the conjugate, as defined in claim 10, or a pharmaceutically acceptable carrier.
[0022]
22. Use of a compound, as defined in any of claims 1 to 9, or a conjugate, as defined in any of claims 10 to 20, or a pharmaceutically acceptable cation thereof, characterized in that it is in the preparation of a medicine for the treatment of cancer.
[0023]
23. Use according to claim 22, characterized by the fact that the cancer is selected from breast cancer, colon cancer, brain cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, head and neck cancer , melanoma, colorectal cancer, gastric cancer, squamous cancer, small cell lung cancer, non-small cell lung cancer, testicular cancer, Merkel cell carcinoma, glioblastoma, neuroblastoma, lymphatic organ cancers and haematological malignancy including Leukemia (Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronic myeloid leukemia (CML), Acute monocytic leukemia (AMOL), Hair cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL) , Large granular lymphocytic leukemia, Adult T cell leukemia, Lymphoma (small lymphocytic lymphoma (SLL), Hodgkin's lymphomas (nodular sclerosis, Mixed cellularity, Lymphocyte rich, Exhausted or not exhausted lymphocyte and Hodgkin's lymphoma predominant in nodular lymphocytes), Non-Hodgkin's lymphomas (all subtypes), Chronic lymphocytic leukemia / Small lymphocytic lymphoma, L-lymphocytic leukemia, Lymphoplasmacytic lymphoma (such as Waldenstrom zone macroglobulinemia) splenic, Plasma cell neoplasms (Plasma cell myeloma, Plasmacytoma, Monoclonal immunoglobulin deposition diseases, Heavy chain diseases), Extranodal marginal zone B cell lymphoma (MALT lymphoma), Nodal marginal zone B cell lymphoma (NMZL ), Follicular lymphoma, Mantle cell lymphoma, Large diffuse B-cell lymphoma, Large mediastinal (thymic) B-cell lymphoma, Large intravascular B-cell lymphoma, Primary effusion lymphoma, Burkitt's lymphoma / leukemia, Prolymphocytic cell leukemia T, Large granular T cell lymphocytic leukemia, Aggressive NK cell leukemia, Adult T cell leukemia / lymphoma, NK / T cell lymphoma extranodal (nasal type), Enteropathy-type T-cell lymphoma, Hepatosplenic T-cell lymphoma, Blast NK cell lymphoma, Mycosis fungoides / Sezary syndrome, Primary cutaneous CD30 positive T-cell lymphoproliferative disorders, Primary cutaneous anaplastic large cell lymphoma , Lymphoid papulosis, Angioimmunoblastic T-cell lymphoma, Peripheral T-cell lymphoma (unspecified), Anaplastic large cell lymphoma, multiple myeloma (plasma cell myeloma or Kahler's disease).
[0024]
24. Use according to claim 23, characterized by the fact that the drug is for the treatment of acute myeloid leukemia (AML).

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 47/00 (2006.01), C07D 487/04 (2006.01), C07D |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-11-05| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-05| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-09-29| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-05| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/02/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161443092P| true| 2011-02-15|2011-02-15|

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US201161443062P| true| 2011-02-15|2011-02-15|

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US61/443,092|2011-02-15|

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US61/443,062|2011-02-15|

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US201161483499P| true| 2011-05-06|2011-05-06|

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US61/483,499|2011-05-06|

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PCT/US2012/025252|WO2012128868A1|2011-02-15|2012-02-15|Cytotoxic benzodiazepine derivatives|

---