BORONATES AS ARGINASE INHIBITORS

专利摘要:
boronates as arginase inhibitors. compounds according to formula i are potent inhibitors of the activity of arginase i and ii: (i) wherein r1, r2, r3, r4, d, w, x, y and z are defined in the specification. the invention also provides pharmaceutical compositions of the compounds and methods of their use in treating or preventing a disease or condition associated with arginase activity.
公开号:BR112013010099B1
申请号:R112013010099-0
申请日:2011-10-19
公开日:2021-08-10
发明作者:Michael Van Zandt；Gunnar Erik Jagdmann Jr.
申请人:Mars, Incorporated；
IPC主号:

专利说明:

FUNDAMENTALS OF THE INVENTION
The present invention is generally related to arginase inhibitors and their use for the treatment of pathological conditions. Two arginase isoforms have been identified to date. Arginase I (ARG I), which is expressed in the cytosol, and Arginase II (ARG II), which is expressed in mitochondria. Arginase enzymes, together with NOS enzymes, play an important role in regulating free arginine levels in cells.
Arginases are implicated as participants in various disease states. These include, for example, erectile dysfunction, pulmonary hypertension, hypertension, atherosclerosis, kidney disease, asthma, T cell dysfunction, ischemia reperfusion injury, neurodegenerative diseases, wound healing and fibrotic diseases. However, the mechanism of action of arginase enzymes in these disease states is still the subject of ongoing research, and several studies imply that arginase enzymes are often up-regulated during disease states.
For example, it is postulated that upregulation of arginase activity results in reduced levels of arginine, which, in turn, reduces the level of NO, a physiologically important signaling molecule that is necessary for cell division, stimulation of increased blood flow and for the control of muscular and neurological signal transduction.
In addition to its role in regulating NO levels, arginase also affects the production of crucial polyamines such as putrescine, spermidine and spermine. As arginase consumes arginine it produces ornithine. Ornithine is subsequently converted to putrescine, spermidine and spermine via ornithine decarboxylase, spermidine synthase and spermine synthase, respectively. In this way, arginase enzymes control physiological signaling events by controlling intracellular levels of polyamine signal transducers. See Wang, J-Y; and Casero, Jr., R.A., Ed; Humana Press, Totowa, NJ, 2006.
These results therefore imply a role for therapeutic substances that are candidates for arginase inhibitors for the treatment of various disease states. The present invention provides compounds of Formula I as inhibitors of arginase activity, as well as methods for using the compounds of the invention in treatment. SUMMARY OF THE INVENTION
The present invention provides compounds that inhibit arginase activity and pharmaceutically acceptable formulations of these compounds, as therapeutic agents for the treatment of various disease states associated with an imbalance or dysfunction of the arginase enzymes. Specifically, the present invention provides compounds according to Formula I.
For compounds of Formula I, R1 is selected from the group consisting of -OH, ORa and NRbRc.
Substituent Ra is selected from the group consisting of hydrogen, (C1-C6) straight or branched chain alkyl, (C3-C8) cycloalkyl, (C3-C14) aryl, (C3-C14) heterocycloalkyl-(C1-C6) alkylene-, (C3-C14) heteroaryl-(Ci-Cε) alkylene- and (C3-C14) aryl(Ci-Cβ) alkylene-.
Each of Rb and Rc is independently selected from the group consisting of H, -OH, (Ci-Ce) linear or branched alkyl, -SO2-(Ci-Cg) alkyl, -SO2-(C3-C14) aryl, ( C3-C14) heterocycloalkyl-(Cx-C6)alkylene- and (C3-C14)heteroaryl-(C1-Cε)alkylene-.
The R2 substituent is selected from the group consisting of H, (CI-C6) straight or branched alkyl, and (C1-6 )alkyl-C(O)-. The variables W, X, Y and Z are each independently selected from the group consisting of a bond, -C (R''')2-, -CR'''-, -NR'''-, - N-, -O-, -C(O)- and -S-. Alternatively, the variables W, X, Y and Z are each independently -C(R')(R''')~. Furthermore, at most three of W, X, Y and Z can simultaneously represent a bond and none of two adjacent members of W, X, Y and Z are simultaneously -0-, -S-, -N- or -NR' ''-.
The subscripts 1, m, n, and p in Formula I are each independently integers between 0 and 2, inclusive, such that at least one of 1, m, n, or p is not 0.
Also, the dashed line
in Formula I indicates the option of having one or more double bonds. The R 3 and R 4 substituents in Formula I are each independently selected from hydrogen, (C 1 -C 6 ) linear or branched alkyl, and C(O)-R'. Alternatively, R3 and R4, together with the boron atom to which they are attached, form a 5- or 6-membered ring that is either fully saturated or partially saturated.
For compounds according to Formula I, D is selected from the group consisting of linear or branched (C3-C5)alkylene, (C2-Cg) linear or branched alkenylene, (C2-Cg) linear or branched alkynylene, (C3- C14) arylene and (C3-C14) cycloalkylene. In some embodiments, one or more -CH 2 -θm D groups are optionally and independently substituted with a portion selected from the group consisting of 0, NR', S, SO, SO 2 and CR'R. However, two adjacent -CH2- groups in D are not simultaneously 0, NR', S, SO or SO2.
For certain compounds of Formula I, D conforms to one of the formulas -L1-L2-CH2-CH2-, -CH2-L1-L2-CH2-, -CH2-CHs-L1-! -, -L1-CH2 -CH2-L2-, -L1-CH2-L2-CH2- OR -CH2-L1-CH2-L-. Variables L1 and L2 are independently selected from the group consisting of 0, NR', S, SO, SO2 and CR'R'', where R' and R'' are as defined below. In modalities when -L and -L are adjacent to each other, however, L and L are not simultaneously 0, NR' , S, S0 or an SO2 group. In another modality, L1 and L2 are not present simultaneously. According to that aspect of the invention, linker D is selected from the group consisting of -L1-CH2-CH2-, -CH2-L1-CH2-, -CH2-CH2-L1-, L2-CH2-CH2-, - CH2-L2-CH2-, -CH2-CH2-L2-.
In yet another embodiment, any two adjacent -CH2- groups of D optionally represent two members of a (C3-C14)-cycloalkylenyl group.
The R', R'' and R''' substituents are each independently selected from the group consisting of H, OH, S(O)Rd, S(0)2 Rd, (Ci-Cg)alkyl, ( C 3 -C 6 aryl, -NH 2 , - NH(C 1 -C 6 )alkyl, -N [(C 1 -C 6 )alkyl] 2, -C(O)NRdRe, -C(O)(C 1 -C 6 )alkyl, - C(0)(C3-C14) aryl, -C(0) 0 (C1 -C6) alkyl, -C(O)O(C3-C1Jaryl, (C3-C6) cycloalkyl, (C3-C14) heterocycloalkyl, C (0) (C3-C14) heterocycloalkyl, (C3-C14) heteroaryl, (C3-C14) aryl-(C1-Cg)alkylene-, -C(0)(C3-C14)aryl-(C1-C6)alkylene -, -C(O)(C3-C14) aryl, (C3-C6) cycloalkyl-(C1-6)alkylene-, (C3-C14)heteroaryl-(C1-4)alkylene-, (C3-C14)heterocycle -(Ci-Cβ)alkylene- Any alkyl, alkylene, aryl, heteroaryl, cycloalkyl or heterocycloalkyl is optionally substituted with one or more members selected from the group consisting of halogen, oxo, -COOH, -CN, -NO, -OH , -NRdRe, -NRgS(O)2Rh, (Cx-C6) alkoxy, (C3-C14) aryl, (C1 -C6 ) haloalkyl and (C3 -C14 ) aryloxy.
For compounds of Formula I, Rd, Re, Rg and Rh are each independently selected from the group consisting of -H, (Ci-Cg) linear or branched alkyl, (C3-C14) aryl (Ci-Cε) alkylene - optionally substituted, optionally substituted (C 3 -C 4 )aryl, (C 1 -C 6 )hydroxyalkyl, (C 1 -C 6 )aminoalkyl, H 2 N (C 1 -C 6 )alkylene-, optionally substituted (C 3 -C 6 )cycloalkyl, (C 3 -C 14 ) optionally substituted heterocycloalkyl, optionally substituted (C3-C14)heteroaryl, (C3-C14) aryl-(C1-C6) alkylene- optionally substituted, NR'R''C(0)- and (C3-C')aryl-(C3 -C14)-cycloalkylene-.
It should be understood that, despite the description of Formula I given herein, Formula I does not include 1-amino-2-(3-boronopropyl)cyclohexane carboxylic acid, more specifically (1S,2S)- or (1S,2R) acid carboxylic -1-amino-2-(3-boronopropyl)cyclohexane.
The present invention also provides pharmaceutically acceptable salts, stereoisomers, tautomers, and prodrug forms of compounds of Formula I.
Compounds and pharmaceutical formulations according to this invention are useful for the treatment of various diseases and conditions, including, without limitation, pulmonary hypertension, erectile dysfunction (ED), hypertension, atherosclerosis, kidney disease, asthma, T-cell dysfunction, injury. by reperfusion of ischemia, neurodegenerative diseases, wound healing and fibrotic diseases.
An embodiment of the present invention, therefore, is a pharmaceutical composition comprising a therapeutically effective amount of at least one of the compounds according to Formula I, its pharmaceutically acceptable salt, stereoisomer, tautomer or prodrug and a pharmaceutically acceptable carrier.
The invention also provides, in another embodiment, a method for inhibiting Arginase I, Arginase II, or a combination thereof, in a cell which comprises contacting the cell with at least one compound according to Formula 25 I.
According to another embodiment, the invention provides a method for treating or preventing a disease or condition associated with the expression or activity of Arginase I, Arginase II, or a combination thereof, in an individual, which comprises administering to the individual. of a therapeutically effective amount of at least one compound of Formula I.
According to another embodiment and as noted above, the invention provides a compound of Formula I for the treatment or prevention of a disease or condition associated with the expression or activity of Arginase I, Arginase II, or a combination thereof, in an individual. Also described is the use of a compound of Formula I for the same purpose, in addition to the use of compounds of Formula I in the manufacture of a medicament for the treatment or prevention of a disease or condition associated with the expression or activity of Arginase I, Arginase II, or a combination of both enzymes in cells. DETAILED DESCRIPTION
The compounds of the invention are arginase inhibitors.
Specifically, compounds of the invention according to Formula I are cyclic analogs of α-amino acids which contain at least one boron-containing group. The present invention also encompasses formulations of compounds of Formula I, in addition to formulations comprising prodrug forms of the compounds, for example, esters, amides and dioxaborolanes, which, after administration, are cleaved through metabolic processes to generate free compounds of Formula I.
The compounds of the invention and their pharmaceutical compositions are useful in treating or preventing diseases or conditions that are associated with arginase expression or activity. Examples of disease states for which compositions of the compounds of the invention have therapeutic applications include, without limitation, pulmonary hypertension, erectile dysfunction (ED), hypertension, atherosclerosis, kidney disease, asthma, T cell dysfunction, reperfusion injury. ischemia, neurodegenerative diseases, wound healing and fibrotic diseases. Definitions
"Alkyl" refers to straight, branched-chain, or cyclic hydrocarbyl groups that include from 1 to about 20 carbon atoms. For example, an alkyl can have 1 to 10 carbon atoms or 1 to 5 carbon atoms. Exemplary alkyl groups include straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like, and also include branched chain isomers of groups straight chain alkyl, for example, without limitation, -CH(CH3)2, -CH(CH3)(CH2CH3), -CH(CH2CH3)2, C(CH3)3, -C(CH2CH3)3, -CH2CH( CH3)2, -CH2CH(CH3)(CH2CH3), CH2CH(CH2CH3)2, -CH2C(CH3)3, -CH2C(CH2CH3)3, CH(CH3)CH(CH3)(CH2CH3), -CH2CH2CH(CH3) 2, CH2CH2CH(CH3)(CH2CH3), -CH2CH2CH(CH2CH3)2, -CH2CH2C(CH3)3, CH2CH2C(CH2CH3)3, -CH(CH3)CH2CH(CH3)2, -CH(CH3)CH(CH3) CH(CH3)2, and the like. Thus, alkyl groups include primary alkyl groups, secondary alkyl groups and tertiary alkyl groups.
The phrase "substituted alkyl" refers to alkyl substituted at one or more positions, for example 1, 2, 3, 4, 5 or even 6 positions, the substituents of which are attached to any suitable atom to produce a stable compound, with replacement as described here.
"Optionally substituted alkyl" refers to alkyl or substituted alkyl. The terms "halogen", "halide", and "halo" each refer to -F, -Cl, -Br or -I.
The terms "alkylene" and "substituted alkylene" refer to divalent alkyl and substituted divalent alkyl, respectively. Examples of alkylene include, without limitation, ethylene (-CH 2 -CH 2 -) • "Optionally substituted alkylene" refers to alkylene or substituted alkylene.
"Alkene" refers to straight, branched-chain, or cyclic hydrocarbyl groups that include from 2 to about 20 carbon atoms that have 1-3, 1-2 or at least one carbon to carbon double bond. "Substituted alkene" refers to alkene substituted at 1 or more, e.g., 1, 2, 3, 4, 5, or even 6 positions, whose substituents are attached at any suitable atom to produce a stable compound, with substitution as described here. "Optionally substituted alkene" refers to alkene or substituted alkene.
The term "alkenylene" refers to divalent alkene. Examples of alkenylene include, without limitation, ethenylene (-CH=CH-) and all stereoisomeric and conformational isomeric forms thereof. "Substituted alkenylene" refers to divalent substituted alkene. "Optionally substituted alkenylene" refers to alkenylene or substituted alkenylene.
"Alkyne or "alkynyl" refers to a straight or branched chain unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. Examples of a (C2-C8) alkynyl group include, without limitation, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octine, 2-octine, 3-octine and 4-octine An alkynyl group may be unsubstituted or optionally substituted with one or more substituents, as described herein below.
The term "alkynylene" refers to a divalent alkyne. Examples of alkynylene include, without limitation, ethynylene, propynylene. "Substituted alkynylene" refers to a divalent substituted alkyne.
The term "alkoxy" refers to an -O-alkyl group having the indicated number of carbon atoms. For example, a (Ci-Ce)alkoxy group includes -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-sec-butyl, -0-tert-butyl , -O-pentyl, -O-isopentyl, -O-neopentyl, -O-hexyl, -O-isohexyl, and -O-neohexyl.
The term "aryl", alone or in combination, refers to a monocyclic or bicyclic aromatic ring system such as, for example, phenyl or naphthyl. "Aryl" also includes aromatic ring systems that are optionally fused to a cycloalkyl ring, as defined herein.
A "substituted aryl" is an aryl that is independently substituted with one or more substituents attached at any suitable atom to produce a stable compound, wherein the substituents are as described herein. "Optionally substituted aryl" refers to a substituted aryl or aryl.
"Arylene" means divalent aryl, and "substituted arylene" refers to divalent substituted aryl. "Optionally substituted arylene" refers to a substituted arylene or arylene.
The term "heteroatom" refers to N, O and S. Compounds of the invention which contain N or S atoms can be optionally oxidized to the corresponding N-oxide, sulfoxide or sulfone compounds.
"Heteroaryl", alone or in combination with any other moiety described herein, refers to an aromatic monocyclic ring structure that contains 5 or 6 ring atoms, or a bicyclic aromatic group that has 8 to 10 atoms, which contains one or plus, for example, 1-4, 1-3 or 1-2, heteroatoms independently selected from the group consisting of 0, S and N. Heteroaryl is also intended to include oxidized S or N, for example, sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon or heteroatom is the point of attachment of the heteroaryl ring structure such that a stable compound is produced. Examples of heteroaryl groups include, without limitation, pyridinyl, pyridazinyl, pyrazinyl, quinoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, or isoxazolyl, , tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl and indolyl.
A "substituted heteroaryl" is a heteroaryl which is independently substituted, unless otherwise indicated, with one or more, for example, 1, 2, 3, 4 or 5, also 1, 2 or 3 substituents, also 1 substituent , attached at any suitable atom to produce a stable compound, wherein the substituents are as described herein. "Optionally substituted heteroaryl" refers to heteroaryl or substituted heteroaryl.
"Heteroarylene" refers to a divalent heteroaryl, and "substituted heteroarylene" refers to a divalent substituted heteroaryl. "Optionally substituted heteroarylene" refers to a substituted heteroarylene or heteroarylene.
"Heterocycloalkyl" means a monocyclic, bicyclic, tricyclic or polycyclic non-aromatic saturated or unsaturated ring system having from 5 to 14 atoms, wherein 1 to 3 ring carbon atoms are replaced by heteroatoms of 0, S or N. A heterocycloalkyl is optionally fused with 5-6 ring membered benzo or heteroaryl, and includes oxidized S or N, for example, sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. The point of attachment of the heterocycloalkyl ring is at a carbon or heteroatom such that a stable ring is retained. Examples of heterocycloalkyl groups include, without limitation, morpholino, tetrahydrofuranoyl, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydrobenzofuryl and dihydroindolyl.
"Optionally substituted heterocycloalkyl" means heterocycloalkyl which is substituted with 1 to 3 substituents, for example 1, 2 or 3 substituents, attached to any suitable atom to produce a stable compound, wherein the substituents are as described herein.
"Heteroalkyl" means a saturated alkyl group having 1 to about 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms, where 1 to 3 carbon atoms. carbon are substituted by heteroatoms of O, S or N. Heteroalkyl is also intended to include oxidized S or N, for example, sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. The point of attachment of the heteroalkyl substituent is at one atom such that a stable compound is formed. Examples of heteroalkyl groups include, without limitation, N-alkylaminoalkyl (for example, CH3NHCH2-), N,N-dialkylaminoalkyl (for example, (CH3)2NCH2-), and the like.
"Heteroalkylene" refers to a divalent heteroalkyl. The term "optionally substituted heteroalkylene" refers to a heteroalkylene which is substituted with 1 to 3 substituents, for example 1, 2 or 3 substituents, attached to any suitable atom to produce a stable compound, wherein the substituents are as described herein. .
"Heteroalkene" means an unsaturated alkyl group having 1 to about 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms, where 1 to 3 carbon atoms. carbon are substituted by heteroatoms of 0, S or N, and having 1-3, 1-2 or at least one carbon-to-carbon double bond or carbon-to-heteroatom double bond.
"Heteroalkenylene" refers to a divalent heteroalkene. The term "optionally substituted heteroalkenylene" refers to a heteroalkenylene that is substituted with 1 to 3 substituents, for example 1, 2 or 3 substituents, attached at any suitable atom to produce a stable compound, wherein the substituents are as described herein. .
The term "cycloalkyl" refers to monocyclic, bicyclic, tricyclic or polycyclic 3- to 14-membered ring systems, which are saturated, unsaturated or aromatic. The heterocycle can be attached via any atom. Cycloalkyl also contemplates fused rings in which the cycloalkyl is fused to an aryl or heteroaryl ring, as defined above. Representative examples of cycloalkyl include, without limitation, cyclopropyl, cycloisopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropene, cyclobutene, cyclopentene, cyclohexene, phenyl, naphthyl, anthracil, benzofuranyl, and benzothiophenyl. A cycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below.
The term "cycloalkylene" refers to a divalent cycloalkylene. The term "optionally substituted cycloalkylene" refers to cycloalkylene which is substituted with 1 to 3 substituents, for example 1, 2 or 3 substituents, attached at any suitable atom to produce a stable compound, wherein the substituents are as described herein.
The terms "nitrile" or "cyano" can be used interchangeably and refer to a -CN group that is attached to a carbon atom of a heteroaryl ring, aryl ring and a heterocycloalkyl ring.
The term "oxo" refers to an =0 atom attached to a saturated or unsaturated (Cs-C') cyclic moiety or to an acyclic (Ci-Cg) moiety. The =O atom can be attached to a carbon, sulfur and nitrogen atom that is part of the cyclic or acyclic portion.
The term "amine or amino" refers to a -NRdRe group where each Rd and Re refers independently to a hydrogen, (Ci-Ce)alkyl, aryl, heteroaryl, heterocycloalkyl, (Ci-C8)haloalkyl, and (Ci-Ci) group. -C6) hydroxyalkyl.
The term "amide" refers to a -NR'R''C(O)- group where each R' and R'' independently refers to a hydrogen, (C 1 -C 8 )alkyl, or (C 1 -C 8 )alkyl.
The term "carboxamido" refers to a group -C(O)NR'R'' where each R' and R'' independently refers to a hydrogen, (C 1 -C 8 )alkyl or (C 3 -C 6 )aryl.
The term "aryloxy" refers to an -O-aryl group having the indicated number of carbon atoms. Examples of aryloxy groups include, without limitation, phenoxy, naphthoxy and cyclopropenoxy.
The term "haloalkoxy" refers to an -O-(C1 -C8 )alkyl group in which one or more hydrogen atoms in the C1 -C8 alkyl group is replaced with a halogen atom, which may be the same or different. Examples of haloalkoxy groups include, without limitation, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 4-chlorobutoxy, 3-bromopropyloxy, pentachloroethoxy, and 1,1,1-trifluoro-2-bromo-2-chloroethoxy.
The term "hydroxyalkyl" refers to an alkyl group having the indicated number of carbon atoms in which one or more of the hydrogen atoms of the alkyl group is replaced with an -OH group. Examples of hydroxyalkyl groups include, without limitation, -CH2OH, -CH2CH2OH, CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2OH, CH2CH2CH2CH2CH2CH2OH, and branched versions thereof.
The term "alkylsulfonyl" refers to a (C1 -C6 )alkyl group in which one or more hydrogen atoms in the C1 -C6 alkyl group is replaced with an -S(O)a group. The subscript "a" can be 1 or 2, so as to generate an alkyl sulfoxide (sulfinyl group), or an alkyl sulfone, respectively. Examples of alkylsulfonyl groups include, without limitation, dimethylsulfoxide, ethylmethylsulfoxide and methyl vinylsulfoxide.
The term "haloalkyl" refers to a (C 1 -C 6 )alkyl group in which one or more hydrogen atoms in the C 1 -C 6 alkyl group is replaced with a halogen atom, which may be the same or different. Examples of haloalkyl groups include, without limitation, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropylyl, pentachloroethyl and 1,1,1-trifluoro-2-bromo-2-chloroethyl.
The term "aminoalkyl" refers to a (C 1 -C 6 )alkyl group in which one or more hydrogen atoms in the C 1 -C 6 alkyl group is replaced with an -NRdRe group, where Rd and Re may be the same or different, by For example, each Rd and Re refers independently to a hydrogen, (C1 -C6 )alkyl group, aryl, heteroaryl, heterocycloalkyl, (C1 -C6 ) haloalkyl, (C1 -C6 )cycloalkyl, and (C1 -C6 )hydroxyalkyl group. Examples of aminoalkyl groups include, without limitation, aminomethyl, aminoethyl, 4-aminobutyl and 3-aminobutylyl.
The term "thioalkyl" or "alkylthio" refers to a (C1 -C6 )alkyl group in which one or more hydrogen atoms in the C1 -C6 alkyl group is replaced with an -SRj group, where Rj is selected from the group that consists of hydrogen, (Ci-Cg) alkyl and (C3-C14) aryl.
"Amino(C1-6 )alkylene" refers to a divalent alkylene in which one or more hydrogen atoms in the C1-6 alkylene group is replaced with an -NRdRe group. Examples of amino(Ci-Cβ)alkylene include, without limitation, aminomethylene, aminoethylene, 4-aminobutylene and 3-aminobutylylene.
The term "sulfonamide" refers to a -NRgS(O)2Rh group where Rg and Rh each independently refer to a hydrogen, (Ci-Cg) alkyl, aryl, heteroaryl, heterocycloalkyl, (Ci-Cg) haloalkyl and (C 1 -C 6 ) hydroxyalkyl group.
The term "sulfoxide" refers to an -S(0)- group in which the sulfur atom is covalently attached to two carbon atoms.
The term "sulfone" refers to a chemical compound that contains a sulfonyl functional group (S(0)2) attached to two carbon atoms. The central hexavalent sulfur atom is double bonded to each of two oxygen atoms and is covalently attached via single bonds to each of two carbon atoms. A "hydroxyl" or "hydroxy" refers to an -OH group.
The term "(C 3 -C 14 )aryl-(C 1 -C 6 )alkylene" refers to a divalent alkylene in which one or more hydrogen atoms in the C 1 -C 6 alkylene group is replaced by a (C 3 -C 6 )alkylene group. (C3-C14) aryl-(C1 -C6 )alkylene include, without limitation, 1-phenylbutylene, phenyl-2-butylene, 1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene and naphthylethylene.
The term "(C 3 -C 14 )heteroaryl-(C 1 -C 6 )alkylene" refers to a divalent alkylene in which one or more hydrogen atoms in the C 1 -C 6 alkylene group is replaced with a (C 3 -C 14 )heteroaryl group. Examples of (C 3 -C 14 )heteroaryl-(C 1 -C 6 )alkylene groups include, without limitation, 1-pyridylbutylene, quinolinyl-2-butylene and 1-pyridyl-2-methylpropylene.
The term "(C 3 -C 14 )heterocycloalkyl-(C 1 -C 6 )alkylene" refers to a divalent alkylene in which one or more hydrogen atoms in the C 1 -C 6 alkylene group is replaced by a (C 3 -C 14 )heterocycloalkyl group. Examples of (C 3 -C 14 )heterocycloalkyl-(C 1 -C 6 )alkylene groups include, without limitation, 1-morpholinopropylene, azetidinyl-2-butylene and 1-tetrahydrofuranoyl-2-methylpropylene.
The term "(C3-C14)heteroaryl-(C1-C14)hetercycloalkylene" refers to a divalent heterocycloalkylene in which one or more hydrogen atoms in the C1 -C6 heterocycloalkylene group is replaced by a (C3-C14 )heteroaryl group. Examples of (C3-C14)heteroaryl-(Ci-C')heterocycloalkylene groups include, without limitation, pyridylazetidinylene and 4-quinoline-1-piperazinylene.
The term "(C3-C14)aryl-(C1-C14)heterocycloalkylene" refers to a divalent heterocycloalkylene in which one or more hydrogen atoms in the C1-C14 heterocycloalkylene group is replaced by a (C3-C14)aryl group. Examples of (C3-C14)aryl-(C1-C14)heterocycloalkylene groups include, without limitation, 1-naphthylpiperazinylene, phenylazetidinylene and phenylpiperidinylene.
The term "(C3-C14)aryl-(C1-Cg)alkyl-(C1-C14)heterocycloalkylene" refers to a divalent heterocycloalkylene in which one or more hydrogen atoms in the C1-C14 heterocycloalkylene group is replaced by a group ( C1 -C6 alkyl which is further substituted by replacing one or more hydrogen atoms of the (C1 -C6 )alkyl group with a (C3 -C14 )aryl group.
The term "(C3-C14)heteroaryl-(C1-C6)alkyl-(:ci-C14)heterocycloalkylene" refers to a divalent heterocycloalkylene in which one or more hydrogen atoms in the C1-C14 heterocycloalkylene group is replaced by a group (C 1 -C 6 )alkyl which is further substituted by replacing one or more hydrogen atoms of the (C 1 -C 6 )alkyl group with a (C 3 -C 4 ) heteroaryl group.
The term "(C3-C14)heterocycloalkyl-(C1-C6)alkyl-(C1-C14)heterocycloalkylene" refers to a divalent heterocycloalkylene in which one or more hydrogen atoms in the C1-C14 heterocycloalkylene group is replaced by a group ( C1 -C6 alkyl which is further substituted by replacing one or more hydrogen atoms of the (C1 -C6 )alkyl group with a (C3 -C14 ) heterocycloalkyl group.
The term "(C3-C14)aryl-(C1-C14)cycloalkylene" refers to a divalent cycloalkylene that is monocyclic, bicyclic or polycyclic and in which one or more hydrogen atoms in the (C1-C14)cycloalkylene group is replaced by a (C3-C14)aryl group. Examples of (C3 -C14 )aryl-(C1 -C14 )cycloalkylene groups include, without limitation, phenylcyclobutylene, phenyl-cyclopropylene and 3-phenyl-2-methylbutylene-1-one.
The -CO2H substituent can be replaced with bioisosteric substitutions such as:
and the like, wherein R has the same definition as defined herein. See, for example, "The Practice of Medicinal Chemistry"' (Academic Press: New York, 1996), on page 203.
The compound of the invention can exist in various isomeric forms, including configurational, geometric and conformational isomers, including, for example, cis or trans conformations. The compounds of the present invention may also exist in one or more tautomeric forms, including both simple tautomers and mixtures of tautomers. The term "isomer" is intended to encompass all isomeric forms of a compound of this invention, including tautomeric forms of the compound. The compounds of the present invention can also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms can result from water loss. The specific composition of the open-chain and cyclized forms can depend on how the compound is isolated, stored or administered. For example, the compound may exist primarily in an open-chain form under acidic conditions, but it cyclizes under neutral conditions. All forms are included in the invention.
Some compounds described herein may have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound of the invention can be in the form of an optical isomer or a diastereomer. Accordingly, the invention encompasses compounds of the invention and their uses, as described herein, in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the invention can be obtained by known techniques such as, for example, asymmetric synthesis, chiral chromatography, simulated moving bed technology or by means of chemical separation of stereoisomers by employing optically active resolving agents.
Unless otherwise noted, "stereoisomer" means a stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having a chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound that has two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises more than about 80% by weight of a stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, e.g. more than about 90% by weight of a stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, or more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure predominates. Additionally, if the stereochemistry of a structure or portion of a structure is not indicated, for example, in bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all of its stereoisomers. In some cases, however, when there is more than one chiral center, structures and names can be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the technique of organic synthesis will know whether compounds are prepared as single enantiomers from the methods used to prepare them.
In that description, a "pharmaceutically acceptable salt" is a pharmaceutically acceptable organic or inorganic acid or base salt of a compound of the invention. Representative pharmaceutically acceptable salts include, for example, alkali metal salts, alkaline earth metal salts, ammonium salts, water-soluble and water-insoluble salts such as, for example, the acetate, amsonate salts (4,4-diaminostilbene-2, 2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate , gluconate, glutamate, glycolylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylsulfate, naphthoate, methyl ammonium compound from N-methylglucamine, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate , polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, theoclate, tosylate, triethiodide and valerate. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In that case, the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.
The terms "treat", "treat" and "treatment" refer to the amelioration or eradication of a disease or symptoms associated with a disease. In certain embodiments, these terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic agents to a patient with that disease.
The terms "avoid", "prevent" and "prevention" refer to preventing the onset, recurrence or spread of the disease in a patient resulting from the administration of a prophylactic or therapeutic agent.
The term "effective amount" refers to an amount of a compound of the invention or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in treating or preventing a disease or to delay or minimize symptoms associated with a disease. Furthermore, a therapeutically effective amount with respect to a compound of the invention means that amount of therapeutic agent alone, or in combination with other therapies, which provides a therapeutic benefit in treating or preventing a disease. Used in connection with a compound of the invention, the term can encompass an amount that improves overall therapy, reduces or prevents symptoms or causes of disease, or enhances therapeutic effectiveness or synergies with another therapeutic agent.
The terms "modulate", "modulation" and the like refer to the ability of a compound to increase or decrease the function or activity of, for example, Arginase I or Arginase II. "Modulation", in its various forms, is intended to encompass inhibition, antagonism, partial antagonism, activation, agonism and/or partial agonism of the activity associated with arginase. Arginase inhibitors are compounds that, for example, bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize or down-regulate signal transduction. The ability of a compound to modulate arginase activity can be demonstrated in an enzyme assay or in a cell-based assay.
A "patient" or "individual" includes an animal, for example, a human, cow, horse, sheep, lamb, pig, chicken, turkey, coati, cat, dog, mouse, rat, rabbit or guinea pig. The animal can be a mammal such as a non-primate and a primate (eg ape and human). In one modality, the patient is a human, for example, a baby, a child, a teenager, or an adult.
The term "prodrug" refers to a precursor of a drug, which is a compound that, after administration to a patient, must undergo chemical conversion by metabolic processes before becoming an active pharmacological agent. Exemplary prodrugs of compounds according to Formula I are esters, dioxaborolanes and amides. COMPOUNDS
The present invention is directed to cyclic α-amino acid analogs. More particularly, the compounds of the invention contain at least one boron-containing group as shown in Formula I:

For compounds of Formula I, D is selected from the group consisting of linear or branched (C3-C5)alkylene, linear or branched (C2-Cs)alkenylene, linear or branched (C2-Cs)alkynylene, (C3-C14) arylene and (C3-C14)cycloalkylene. In one embodiment, one or more -CH2- groups in D are optionally and independently substituted with a portion selected from the group consisting of 0, NR', S, SO, SO2 and CR'R''. For compounds according to Formula I, however, two adjacent -CH 2 - groups in D cannot be simultaneously 0, NR', S, SO or SO 2 .
According to one embodiment, D has the formula -L1-L2-CH2-CH2-, -CH2-L1-L2-CH2-, -CH2-CH2-L1-L2-, -L1-CH2-CH2-L2-, -L1-CH2-L2-CH2- OR -CH2-L1-CH2-L2-. Variables L1 and L2 are independently selected from the group consisting of 0, NR', S, S0, S02 and CR'R'', where R' and R'' are as defined below. In embodiments where -L1 and -L2 are adjacent to each other, -L1 and -L2 are not simultaneously 0, NR', S, SO or an SO2 group.
In another modality, L1 and L2 are not present simultaneously. According to that aspect of the invention, linker D is selected from the group consisting of -L1-CH2-CH2-, -CH2-L1-CH2-, -CH2-CH2-L1-, L2-CH2-CH2-, - CH2-L2-CH2-, -CH2-CH2-L2-.
In another embodiment, D contains a (C3-C14)-cycloalkylenyl ring, where two ring members constitute two adjacent -CH2 groups in D, each having one hydrogen atom removed. Thus, for example, when D is propylene each of the atoms C2 and C3 omits a hydrogen atom so as to couple with a -CH2- group to form a cyclopropyl ring, as illustrated by the portion
Following.
For compounds of Formula I, R1 may be -OH, 0Ra or NRbRc, R2 is selected from the group consisting of H, (Ci-C6) linear or branched alkyl, and (Ci-Cg)alkyl-C(0)- and W , X, Y and Z are each independently selected from the group consisting of a bond, -C(R')(R'")-, -C(R'")2-, -CR'''- , -NR'"-, -N-, -0-, -C(0)- and -S-.
In one embodiment, the invention provides compounds of Formula I in which D is propylene, R1 is -OH, each of R2, R3 and R4 is hydrogen, each of W, Y, and Z is -CH2 and 1 + m + n + p = 3. Alternatively, any one of W, Y, or Z is -0-, -S- or -NH- and the remaining two groups are each independently -CH2. As prescribed, therefore, compounds according to Formula I include tetrahydrofuran, tetrahydrothiophene and pyrrolidine analogues, respectively.
In another embodiment, compounds of Formula I are provided in which D is propylene, R1 is -OH, each of R2, R3 and R4 is hydrogen, each of W, Y, and Z is a -CH2 group, X is -NH el+m+n+p=4. Alternatively, each of W, X, Y and Z is a -CH2 group to provide compounds of Formula I that are analogs of 1-aminocyclohexane carboxylic acid.
Also contemplated are compounds of Formula I in which R1 is -ORa or NRbRc, R2 is selected from the group consisting of a linear or branched (C3-C6) alkyl group, (C3-C6)cycloalkyl-C(0)-, ( C3-C6 cycloalkyl and (Ci-Cg)alkyl-C(O)-, and each of R3 and R4 is independently selected from (Ci-Cg) linear or branched alkyl or C(O)-R', or R3 and R4, together with the boron atom to which they are attached, form a 5- or 6-membered ring that is fully saturated, or partially saturated, wherein the Ra, Rb and Rc substituents are as defined.
Thus, for certain compounds of Formula I, the substituent Ra may be selected from the group consisting of hydrogen, (Ci-Cg) straight or branched chain alkyl, (C3-C8) cycloalkyl, (C3-C14) aryl, ( C3 -C14 ) heterocycloalkyl-(C1-6 )alkylene-, (C3 -C14 )heteroaryl-(C1-6 )alkylene- and (C3 -C14 ) aryl (C1-6 )alkylene- and each of Rb and Rc is independently selected from the group consisting of H, -OH, (C 1 -C 8 ) linear or branched alkyl, - SO 2 - (C 1 -C 6 ) alkyl, (C 3 -C 4 )aryl-SO 2 -, (C 3 -C 14 )heterocycloalkyl-( C 1 -C 6 alkylene- and (C 3 -C 14 ) heteroaryl-(C 1 -C 6 )alkylene-.
In one embodiment, W, X, Y, and Z, together with the carbon atoms to which they are attached, form a ring. The ring may contain one or more double bonds that are 11 kJ represented by a dashed line

The R', R'' and R''' substituents for compounds of Formula I are each independently selected from the group consisting of H, OH, -S(O)Rd, -S(0)2Rd, (Ci -C8) alkyl, (C3-C6)aryl, -NH2, -NH(Ci-C6) alkyl, -N[(CX-C6)alkyl]2, -C(0)(Ci-C6) alkyl, -C (0) (C3-C14) aryl, C (0) 0 (C1 -C6 )alkyl, -C (0) 0 (C3 -C14 ) aryl, (C3-Cg )cycloalkyl, (C3-C14 )heterocycloalkyl, ( C3-C14) heteroaryl, (C3-C14) aryl-(C1-C6) alkylene-, (C3-C6) cycloalkyl-(C1-C6) alkylene-, (C3-014) heteroaryl-(C1-C6) alkylene- and (C3-C14)heterocycle-(C1-C6)alkylene-.
Furthermore, for compounds according to Formula I, any alkyl, alkylene, aryl, heteroaryl, cycloalkyl or heterocycloalkyl is optionally substituted with one or more members selected from the group consisting of halogen, oxo, -COOH, -CN, -NO2 , -0H, -NRdRe, -NRgS(O)2Rh, (Ci-Cβ) alkoxy and (C3-C14) aryloxy, with each of Rd, Re, Rg and Rh being independently selected from the group consisting of -H, (Ci-Cg) linear or branched alkyl, (C3-C14) aryl (Ci-Cβ)alkylene- optionally substituted, (C3-C4 )aryl optionally substituted, (Ci-Cβ) hydroxyalkyl, (Ci-Cβ) aminoalkyl, H2N (C 1 -C 6 )alkylene-, (C 3 -C 6 )cycloalkyl optionally substituted, (C 3 -C 14 )heterocycloalkyl optionally substituted, (C 3 -C 14 )heteroaryl optionally substituted, (C 3 -C 14 ) aryl-(C 1 -C 6 )alkylene- optionally substituted, NR'R''C(O)- and (C3-C6)-aryl-(C3-C14)-cycloalkylene-.
It should be understood that, despite the definitions above, Formula I does not include 1-amino-2-(3-boronopropyl)cyclohexane carboxylic acid, for example (1S,2S)-1-amino-2-(3- boronopropyl)cyclohexane carboxylic acid and (1S, 2R)-1-amino-2-(3-boronopropyl.) cyclohexane carboxylic acid.


Exemplary Formula I compounds include, without limitation, those shown below in Table 1. Table 1







PHARMACEUTICAL COMPOSITIONS AND DOSAGES
The compounds of Formula I are administered to a patient or individual in need of treatment alone or in combination with other compounds that possess similar or different biological activities. For example, the compounds and compositions of the invention can be administered in a combination therapy, that is, simultaneously in single or separate dosage forms or in separate dosage forms within hours or days of each other. Examples of such combination therapies include the administration of the Formula I compositions and compounds with other agents used to treat erectile dysfunction, pulmonary hypertension, hypertension, asthma, inflammation, ischemia reperfusion, myocardial infarction, arteriosclerosis, immune response, psoriasis and scarring. wounds. Compounds suitable for use in combination therapy include, without limitation: Erectile dysfunction: sildenafil, vardenafil, tadalafil and alprostadil.
Pulmonary hypertension/Hypertension: epoprostenol, iloprost, bosentan, amlodipine, diltiazem, nifedipine, ambrisentan and warfarin.
Asthma: fluticasone, budesonide, mometasone, flunisolide, beclomethasone, montelukast, zafirlukast, zileuton, salmeterol, formoterol, theophylline, albuterol, levalbuterol, pirbuterol, ipratropium, prednisone, methylprednisolone, chromomatin, omatically.
Arteriosclerosis: atorvastatin, lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, gemfibrozil, fenofibrate, nicotinic acid, clopidogrel.
The invention also provides a pharmaceutical composition comprising one or more compounds according to Formula I or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer or prodrug, in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practice for manufacturing pharmaceutical compositions, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, dyes, buffers, flavoring agents.
In one embodiment, the pharmaceutical composition comprises a compound selected from those illustrated in Table 1 or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer or prodrug thereof, and a pharmaceutically acceptable carrier.
The compositions of the invention can be administered orally, topically, parenterally, by inhalation or spray, or rectally in unit dosage formulations. The term "parenteral", as used herein, includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
Suitable oral compositions according to the invention include, without limitation, tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs.
Encompassed within the scope of the invention are pharmaceutical compositions suitable for single unit dosages comprising a compound of the invention, its stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, hydrate or tautomer and a pharmaceutically acceptable carrier.
Compositions of the invention suitable for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. For example, liquid formulations of the compounds of the invention contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations of the arginase inhibitors.
For tablet compositions, the active ingredient mixed with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Exemplary of such excipients include, without limitation, inert diluents, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or can be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide a sustained therapeutic action over a desired period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin or as soft gelatin capsules in which the active ingredient it is mixed with water or an oily medium, for example peanut oil, liquid paraffin or olive oil.
For aqueous suspensions, the compound of the invention is mixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include, without limitation, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.
Oral suspensions may also contain dispersing or wetting agents, for example, naturally occurring phosphatide, for example, lecithin or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or ethylene oxide condensation products with long-chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as, for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters fatty acid derivatives and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as, for example, sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
Sweetening agents, such as those presented above, and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, eg sweetening, flavoring and coloring agents, may also be present.
Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents can be naturally occurring gums, for example, acacia gum or gum tragacanth, naturally occurring phosphatides, for example, soybean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. Emulsions can also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. These formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. Pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. Such a suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any mixture of fixed oils can be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injectables.
The compounds of general Formula I can also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. These materials are cocoa butter and polyethylene glycols.
Compositions for parenteral administration are administered in a sterile medium. Depending on the vehicle used and the concentration of drug in the formulation, the parenteral formulation can be a suspension or a solution containing dissolved drug. Adjuvants such as, for example, local anesthetics, preservatives and buffering agents can also be added to the parenteral compositions. COMPOUND SYNTHESIS
Compounds of the invention are prepared using general synthetic methods, as discussed in more detail below. The choice of an appropriate synthetic methodology is guided by the choice of the desired compound of Formula I and the nature of functional groups present in the intermediate and final product. Thus, selective protection/deprotection protocols may be necessary during synthesis, depending on the specific desired functional groups and protecting groups being used. A description of these protective groups and how to introduce and remove them is found in "Protective Groups In Organic Synthesis" Third Edition, T.W. Green and P.G.M. Wuts, John Wiley and Sons, New York 1999.
Exemplary general synthetic methodologies for producing compounds of Formula I are provided below. More specific syntheses of illustrative Formula I compounds are also provided.
Scheme A below illustrates a general method for the synthesis of the cyclic framework of many compounds according to Formula I. According to that method, α-allyl ketone A-3 is prepared from readily available starting materials using different methods. For example, A-3 is obtained through a sequence of reaction steps starting from the corresponding α-carboxylic acid ester Al. , A-3 is synthesized by transesterification of the ester to allyl ester A-2, which undergoes a decarboxylative rearrangement in the presence of a palladium catalyst to generate the target intermediate A-3.
Alternatively, if carboxylic acid is available it can be converted to a simple ester or an allyl ester directly using several known methods. One such method uses allyl bromide in acetone with aqueous potassium carbonate to directly synthesize allyl ester A-2.
According to an alternative approach, α-allyl ketone A-3 is prepared directly from cyclic ketone A-4 using allyl bromide and a base such as, for example, lithium diisopropylamide (LDA), or sodium hydride. In some cases, α-allyl ketone A-3 can be prepared from ketone A-4 via the corresponding allyl carbonate A-5. Thus, the reaction of cyclic ketone A-4 with allyl chloroformate in the presence of a base such as NaHMDS in a polar aprotic solvent such as THE at -78 °C results in allyl carbonate corresponding A-5, which is then converted to the N-allyl ketone A-3 in the presence of a metal reagent such as palladium acetate or tetrakis(triphenylphosphine)palladium.
Often additives such as tetramethylethylene diamine (TMEDA) are used to facilitate the formation of allyl carbonate. This synthetic methodology is versatile and suitable for the enantioselective preparation of ketone A-3 using some chiral ligands. A description of this approach can be found in Trost, B.M et al., J. Am. Chem. Soc. 2009, 131, 18,343-18,357.
In this way, α-allyl' ketone A-3 is easily converted to the protected cyclic amino acid A-6 using a Ugi reaction, by treating A-3 with tert-butylisocyanate (t-BuNC) and NH4OAc using trifluoroethanol as a solvent . For a review of Ugi's reaction, see; Doemling, A., Chem. Rev. 2006, 106, 17-89. Alternative solvents, as well as other isocyanate, amine sources and carboxylic acid sources, can be used. The choice of amine, isocyanate and carboxylic acid used further determines the nature of the protecting group present in the resulting amino acid. In some embodiments, enantiomeric selectivity is achieved by using an optically active amine or carboxylic acid.
As an alternative to the Ugi reaction, the α-substituted cyclic amino acid A-6 is synthesized from the corresponding ketone A-3 using the Strecker reaction. Many examples of the Strecker reaction and the modified Strecker reaction are available in the literature (Ma, D., Tian, H., Zou, G., J. Org. Chem. 1999, 64, 120-125; Murata, Y. , Statake, K., Synthetic Communications, 2008, 38, 1485-1,490). The final product containing a boronic acid group is obtained by reacting the protected amino acid A-6 with a borane source such as pinacol borane in the presence of iridium or rhodium as a catalyst. The resulting borane intermediate A-7 is hydrolyzed using aqueous acid to generate target compounds A-8.
Another convenient method for preparing the allyl ketone intermediate is based on the reaction of carboxylic acid B1 with oxalyl chloride and (N-isocyanimino)triphenylphosphorane (CNNPh3), followed by quenching the reaction mixture to generate the corresponding chlorohydrozone B-2, as illustrated in Scheme B.
Treatment of intermediate B-2 with ZnBr3 and a base (eg, diisopropylamine) results in the formation of a diazoketone B-3, which is subjected to cyclization using Cu(AcAc) to generate the desired allyl ketone intermediate B-4 .
Intermediate B-4 is then converted to the target compound using a Ugi reaction, followed by hydroboration and deprotection, as described in Scheme A. See Padwa, A.; Weingarten, D.Chem. Rev. 1996, 96, 223-269 for an overview of carbin-mediated cyclization reactions.
Alternatively, the allyl ketone intermediate is obtained using the Dieckmann condensation as shown in Scheme C below. According to this method, allyl diester Cl is cyclized with a base such as sodium hydride or LDA to form the desired allyl ketone C-2, which is converted to the target boronic acid compound using methodologies illustrated in the Scheme A. When the C1 starting diallyl ester is not commercially available, it is easily prepared from the corresponding diacid using known methods.
Scheme C
Conseqiientemente, a cetona D-l é condensada com o auxiliar de hidrazina na presença de ácido toluenossulfônico em tolueno para formar hidrazona D-2. Frequentemente a reação de condensação é realizada na presença de peneiras moleculares ou por utilização de um aparelho de Dean-Stark para remover água. Os substituintes do anel no carbono adjacente à hidrazona' D-2 são introduzidos de forma enantiosseletiva sob condições básicas. Por exemplo, quando o substituinte desejado, é um grupo -CH2-CH2-CH2-B (OH) 2, ele facilmente introduzido por reação da hidrazona com brometo de alila para gerar a hidrazona substituída D-3 que é hidroborada para gerar o composto de dioxaborolano D-4. Alternativamente, hidrazona D-2 pode ser diretamente alquilada usando 2-(4-bromobutil)- 4,4,5,5-tetrametil-l,3,2-dioxaborolano (não mostrado), para gerar D-4.The enantioselective synthesis of compounds according to Formula I is easily obtainable from several methods that prescribe the use of chiral auxiliaries. According to one such method for the synthesis of many compounds of Formula I, (R)-(+)-1-amino-2-(methoxy)pyrrolidine is used as the chiral auxiliary. See Scheme D and procedures disclosed by Enders, D. et al., Organic Synthesis 1987, 67 and Enders, D. et al., Synthesis 2005, 20, 3517-3530. Consequently, ketone D1 is condensed with the hydrazine auxiliary in the presence of toluenesulfonic acid in toluene to form hydrazone D-2. Often the condensation reaction is carried out in the presence of molecular sieves or by using a Dean-Stark apparatus to remove water. Ring substituents on the carbon adjacent to the D-2 hydrazone are enantioselectively introduced under basic conditions. For example, when the desired substituent is a -CH2-CH2-CH2-B(OH)2 group, it is easily introduced by reacting the hydrazone with allyl bromide to generate the D-3 substituted hydrazone which is hydroborated to generate the compound. of dioxaborolane D-4. Alternatively, hydrazone D-2 can be directly alkylated using 2-(4-bromobutyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (not shown) to generate D-4.
After enantioselective side chain incorporation, hydrazone D-4 is treated with ozone, followed by reduction of the ozonide with triphenylphosphine or dimethyl sulfide to generate optically active ketone D-5. Ketone D-5 is converted to the corresponding cyclic amino acid using the Ugi or Strecker reaction as described above. Deprotection of the boron-containing cyclic amino acid D-6 with an aqueous mineral acid provides a compound of Formula I D-7.
For some examples, hydrazone intermediates D-3 or D-4 are used to introduce a ring substituent, where Z is CRR' and R and/or R' are introduced via a second and possibly a third alkylation. The synthetic methodology illustrated in Scheme D also allows for the enantioselective introduction of substituent groups R and R' at the Z position.
In one embodiment, compounds of Formula I which have substituent groups on Z are obtained using the appropriately substituted ketone D-1. Based on the general method illustrated in Scheme D, it is evident to those skilled in the chemical arts that the illustrative synthetic methodology can be readily practiced by varying the starting materials and reaction conditions to arrive at the compounds encompassed by the present invention. In some cases, protection of certain reactive functionality may be needed to get some of the above transformations. In general, the need for any protecting groups, as well as the conditions necessary to attach and remove those groups, will be evident to those skilled in the technique of organic synthesis.
Another useful approach for preparing compounds of the invention uses Corey-Link amino acid synthesis. See Gorey et al., J. Am. Chem. Soc., 114, (1992), pages 1906-1908. In this approach, the amino acid is formed from a ketone using a carboxylic acid equivalent such as chloroform and an amine source such as sodium azide. When an optically pure amine source such as methyl benzylamine is used, diastereomers are formed in the reaction and end products as single enantiomers can often be obtained. As illustrated in Scheme E, the intermediate allyl ketone E1 is treated with the chloroform anion generated using a base such as LiHMDS in THE to form the corresponding carbinol E-2. Subsequent treatment with sodium hydroxide produces the dichloroepoxide, which is opened with an amine source such as sodium azide to generate the derivatized amino acid E-3. The carboxylic acid can be protected as an ester (E- 4) or amide prior to hydroboration of the double bond to generate intermediate E-5. Group reduction and hydrolysis generate the target compounds E-6. Depending on the functionality required, additional steps or alternative procedures may be required or preferred. Scheme E
The disclosures of all articles and references mentioned in that application, including patents, are hereby incorporated by reference. The preparation of compounds of the present invention is further illustrated by the following examples, which are not to be considered as limiting the invention in scope or spirit to the specific procedures and compounds described therein. EXAMPLES
In general, intermediates and target compounds that contain chiral centers are stereospecifically named. This designation is used primarily to distinguish relative stereochemistry and does not indicate optical purity. It will be obvious to those skilled in organic synthesis which compounds are optically pure from the methods used to prepare them. The compounds described in the methods and examples sections can also be isolated as hydrates or salts (eg, hydrochloric acid salts), but are not necessarily so designated. The compounds described in this invention are generally named using common names, IUPAC names or names generated using the naming algorithm in ChemDraw 10.0. Example 1. Preparation of (IS,2S)-1-amino-2-(3-boronopropyl)cyclopentanecarboxylic acid (antiisomer, racemic)
Example 1 describes the multi-step synthetic protocol used to produce an illustrative compound of Formula I. Step 1. Method A: Allyl 2-oxocyclopentanecarboxylate (transesterification)
A stirred solution of methyl 2-oxocyclopentanecarboxylate (4.26 g, 30 mmol) and allyl alcohol (10.2 ml, 150 mmol) in anhydrous toluene (25 ml) was treated with zinc powder (0.40 g, 6 mmol) ), refluxed for 48 h, and cooled to room temperature. The suspension was filtered, the filter cake rinsed with toluene, and the filtrate concentrated to give allyl 2-oxocyclopentanecarboxylate (5.01 g, 99%) as a colorless oil. 1H-RMN (CDC13, 300 MHz) δ 5.89 (ddt, J2 = 15.9 Hz, J2 = 10.5 Hz, J3 = 4.8 Hz, 1H), 5.33 (dtd, J2 = 15, 9 Hz, J2 = 2.7 Hz, J3 = 1.4 Hz, 1H), 5.23 (dtd, J2 = 10.5 Hz, J2 = 2.7 Hz, J3 = 1.4 Hz, 1H), 4.83-4.75 (m, 1H), 3.18 (t, J=9.0Hz, 1H), 2.41-2.23 (m, 4H), 2.22-2.07 (m, 1H), 1.94-1.80 (m, 1H); MS (+CI): m/z for C9H12O3: 168.1 expected; found 169.1 (M+H)+.
Step 1. Method B: Allyl 2-oxocyclopentanecarboxylate (Dieckman) A stirred solution of diallyl adipate (4.53 g, 20 mmol) in anhydrous tetrahydrofuran (100 ml) was cooled to 0°C and treated with bis(trimethylsilyl)amide from lithium (40 ml, 1.0 N in THF, 40 mmol). After the addition was complete, the solution was warmed to room temperature and stirred for 2 h. The solution was cooled back to 0°C and treated with acetic acid (2.53 ml, 44 mmol) dropwise. The cloudy mixture was warmed to room temperature and filtered. The filtrate was concentrated, dissolved in minimal dichloromethane and purified by flash column chromatography (silica gel, dichloromethane) to give allyl 2-oxocyclopentanecarboxylate (2.62 g, 78%) as a colorless oil.
The characterization data are the same as those observed by method A. Step 2: Synthesis of 2-allylcyclopentanone
A stirred solution of palladium(II) acetate (51 mg, 0.23 mmol) and triphenylphosphine (0.24 g, 0.9 mmol) in anhydrous THF (20 ml) was heated under a nitrogen atmosphere to 65° Ç. To the hot solution is added a solution of allyl 2-oxocyclopentanecarboxylate (2.52 g, 15 mmol) in anhydrous THF (rapid bubbling on addition). After 45 minutes at 65°C, the reaction mixture is cooled and concentrated. The resulting yellow residual oil was dissolved in minimal dichloromethane and purified by flash column chromatography (silica gel, dichloromethane) to give 2-allylcyclopentanone (1.32 g, 71%) as a colorless oil. 1H-NMR (CDC13, 300 MHz) δ 5.72 (ddt, J=17.1Hz, J2=10.2Hz, J3=7.2Hz, 1H), 5.09-4.98 (m, 2H), 2.55-2.46 (m, 1H), 2.35-2.22 (m, 1H), 2.22-1.91 (m, 5H), 1.87-1.70 ( m, 1H), 1.63-1.48 (m, 1H).
Uma mistura em agitação de 2-alilciclopentanona (0,993 g, 8 mmol) e acetato de amónio (1,54 g, 20 mmol) em 2,2,2- trifluoretanol (2,5 ml) foi tratada com t-butil isocianeto (1,81 ml, 16 mmol) . A mistura de reação foi agitada em temperatura ambiente por 4 dias, e depois a mistura foi dissolvida em uma quantidade minima de diclorometano e purificada por cromatografia instantânea em coluna (silica gel, acetato de etila 60% em heptano) para gerar ,o antiisômero de (IS,2S)-l-acetamido-2-alil-N-terc- butilciclopentanocarboxamida (0,771 g, 36%) como um sólido branco. O isômero syn que migra mais tarde é obtido como um sólido branco (0,851 g, 40%), por aumento da concentração do solvente polar, acetato de etila, até o 80%. Antiisômero: 1H-RNM (CDCI3, 300 MHz) δ 7,26 (br s, NH, 1H) , 6,82 (br s, NH, 1H), 5,88-5,72 (m, 1H) , 5,07 (br d, J = 17 Hz, 1H) , 5,00 (br d, J = 12 Hz, 1H) , 2,56-2,41 (m, 1H) , 2,36-2,17 (m, 3H) , 2,01 (s, 3H) , 1,97-1,81 (m, 2H) , 1,72- 1,60 (m, 2H), 1,32 (s, 9H) ; MS (+CI): m/z para C15H26N2O2: esperado 266,2; encontrado 266,2 (M+H)+.Step 3: (IS,2S)-1-acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide (antiisomer, racemic) A stirred mixture of 2-allylcyclopentanone (0.993 g, 8 mmol) and ammonium acetate (1.54 g, 20 mmol) in 2,2,2-trifluoroethanol (2.5 ml) was treated with t-butyl isocyanide ( 1.81 ml, 16 mmol). The reaction mixture was stirred at room temperature for 4 days, then the mixture was dissolved in a minimal amount of dichloromethane and purified by flash column chromatography (silica gel, 60% ethyl acetate in heptane) to generate the antiisomer of (1S,2S)-1-Acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide (0.771 g, 36%) as a white solid. The later migrating syn isomer is obtained as a white solid (0.851 g, 40%) by increasing the concentration of the polar solvent, ethyl acetate, to 80%. Antiisomer: 1H-NMR (CDCl3, 300 MHz) δ 7.26 (br s, NH, 1H), 6.82 (br s, NH, 1H), 5.88-5.72 (m, 1H), 5 .07 (br d, J = 17 Hz, 1H), 5.00 (br d, J = 12 Hz, 1H), 2.56-2.41 (m, 1H), 2.36-2.17 ( m, 3H), 2.01 (s, 3H), 1.97-1.81 (m, 2H), 1.72-1.60 (m, 2H), 1.32 (s, 9H); MS (+CI): m/z for C15H26N2O2: 266.2 expected; found 266.2 (M+H)+.
Uma solução agitada de (IS,2R)-l-acetamido-2-alil-N- terc-butilciclopentanocarboxamida (0,599 g, 2,25 mmol) em cloreto de metileno anidro (9 ml) sob nitrogênio foi tratada com Ir2C12(COD)2 (45 mg, 0,07 mmol) e DiPhos (54 mg, 0,136 mmol) e agitada em temperatura ambiente por 30 min. 4,4,5,5-Tetrametil-[1,3,2]dioxaborolano (0,65 ml, 4,48 mmol) foi adicionado gota a gota e a solução agitada em temperatura ambiente por 20 h. A mistura de reação foi derramada em água (20 ml) e extraida com acetato de etila (40 ml, depois 2 x 15 ml) , e a solução orgânica combinada foi lavada com cloreto de sódio aquoso saturado (30 ml), seca sobre MgSO4, e concentrada. 0 residuo foi dissolvido em diclorometano minimo e purificado por cromatografia instantânea em coluna (silica gel, acetato de etila 40-50% em heptano) para gerar (1S,2S)-l-acetamido-N-terc-butil-2- (3-(4,4,5,5-tetrametil-l,3,2-dioxaborolan-2-il)propil) ciclopentanocarboxamida (0,695 g, 78%) como um sólido branco. XH-RNM (CDC13, 300 MHz) δ 6,72 (br s, NH, 1H) , 5,68 (br s, NH, 1H), 2,46-2,29 (m, 2H), 2,10-2,19 (m, 1H), 2,02 (s, 3H) , 2,00-1,88 (m, 1H) , 1,75-1, 60 (m, 3H) , 1,52-1,38 (4H), 1,32 (s, 9H) , 1,31 (s, 12H) , 0,81-0,70 (m, 2H) ; MS (+CI) : m/z para C2IH39BN2O4: esperado 394,3; encontrado 395,2 (M+H)+. Etapa 5: Ácido (1S,2R) -l-amino-2- (3-boronopropil) ciclopentano-carboxílico (antiisômero, racêmico) Step 4: (1,2R)-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (antiisomer, racemic) A stirred solution of (IS,2R)-1-acetamido-2-allyl-N- tert -butylcyclopentanecarboxamide (0.599 g, 2.25 mmol) in anhydrous methylene chloride (9 ml) under nitrogen was treated with Ir 2 Cl 2 (COD) 2 (45mg, 0.07mmol) and DiPhos (54mg, 0.136mmol) and stirred at room temperature for 30 min. 4,4,5,5-Tetramethyl-[1,3,2]dioxaborolane (0.65 ml, 4.48 mmol) was added dropwise and the solution stirred at room temperature for 20 h. The reaction mixture was poured into water (20 ml) and extracted with ethyl acetate (40 ml, then 2 x 15 ml), and the combined organic solution was washed with saturated aqueous sodium chloride (30 ml), dried over MgSO4 , and concentrated. The residue was dissolved in minimal dichloromethane and purified by flash column chromatography (silica gel, 40-50% ethyl acetate in heptane) to give (1S,2S)-1-acetamido-N-tert-butyl-2-(3 -(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (0.695 g, 78%) as a white solid. XH-NMR (CDCl3 , 300 MHz) δ 6.72 (br s, NH, 1H), 5.68 (br s, NH, 1H), 2.46-2.29 (m, 2H), 2.10 -2.19 (m, 1H), 2.02 (s, 3H), 2.00-1.88 (m, 1H), 1.75-1.60 (m, 3H), 1.52-1 .38 (4H), 1.32 (s, 9H), 1.31 (s, 12H), 0.81-0.70 (m, 2H); MS (+CI): m/z for C2IH39BN2O4: expected 394.3; found 395.2 (M+H)+. Step 5: (1S,2R)-1-amino-2-(3-boronopropyl) cyclopentane carboxylic acid (antiisomer, racemic)
A stirred mixture of (1S,2R)-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) cyclopentanecarboxamide (0.627g, 1.59mmol) in 6N HCl (15ml) was heated to 90°C for 20h, cooled to room temperature and diluted with water (15ml). The mixture was extracted with dichloromethane (2 x 15 ml) and concentrated. Water (20 ml) was then added to the concentrated crude mixture and the aqueous solution is reconcentrated in vacuo (2x)' to remove excess HCl. The resulting residue was dissolved in methanol (5 ml) and diluted to a volume of 40 ml with ether and stirred for 1 hour to remove solid tert-butylamine hydrochloride by filtration. The resulting filtrate, after concentration, was treated with concentrated ammonium hydroxide solution (15 ml), followed by removal of excess ammonium hydroxide under vacuum (3x). The resulting white solid residue was triturated using acetonitrile and dried to give the target compound (1S,2R)-1-amino-2-(3-boronopropyl)cyclopentanecarboxylic acid (0.397 g, 93%) as a white powder which exists as a 1:1 mixture of cyclized and uncyclized forms. XH-NMR (DMSO, 300 MHz) δ 6.38 (br d, J = 11.4 Hz, NH, 0.5H), 5.97 (br d, J = 12 Hz, NH, 0.5H), 2.22-2.05 (m, 1H), 1.98-1.42 (m, 6H), 1.43-1.00 (m, 3H), 0.95-0.88 (m, 1H) ), 0.60-0.42 (m, 1H), 0.38-0.20 (m, 1H); MS (+CI): m/z for C9H18BNO4: 215.1 expected; found 215.3 (M+H) + . Example 2. Preparation of (IS,2S)-1-amino-2-(3-boronopropyl)cyclopentanecarboxylic acid (syn isomer, racemic)
Step 1: (IS, 2S)-1-Acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide The target intermediate, (IS, 2S)-1-acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide, was also characterized using 1H-NMR spectroscopy and represents the other isomer which is formed using stereoselective synthetic method described in step 3 of Example 1. 1H-NMR spectroscopy (CDCl3, 300 MHz) δ 6.44 (br s, NH, 1H), 6.06 (br s, NH, 1H), 5.80-5.65 (m, 1H), 5.05-4.96 (m, 2H), 2.54-2.56 (m, 2H) , 2.30-2.18 (m, 1H), 2.10-1.95 (m, 1H), 1.99 (s, 3H), 1.91-1.72 (m, 5H), 1 .58-1.43 (m, 1H), 1.32 (s, 9H); MS (+CI): m/z for C15H26N2O2: expected 266.2; found 267.2 (M+H)+.
Step 2: (1,2S)-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (syn isomer)
A stirred solution of syn-(IS,2S)-1-acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide (0.600 g, 2.25 mmol) in anhydrous methylene chloride (9 ml) under nitrogen was treated with Ir 2 Cl 2 ( COD) 2 (45 mg, 0.07 mmol) and DiPhos (54 mg, 0.136 mmol) and the resulting mixture was stirred at room temperature for 30 min. 4,4,5,5-Tetramethyl-[1,3,2]dioxaborolane (0.65 ml, 4.48 mmol) was then added dropwise and the solution stirred at room temperature for a further 20 h. The reaction mixture was poured into water (20 ml), extracted with ethyl acetate (40 ml, then 2 x 15 ml), and the combined organic layers were washed with saturated aqueous sodium chloride (30 ml), and dried using anhydrous MgSO4, prior to concentration. The residue obtained was dissolved in a minimal volume of dichloromethane and purified using flash column chromatography (silica gel, 40-80% ethyl acetate in heptane) to give syn-(IS,2S)-1-acetamido-N-tert- butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentarth carboxamide as a white solid (0.710 g, 80%). 1H-NMR (CDCl3, 300 MHz) δ 6.21 (br s, NH, 1H), 6.06 (br s, NH, 1H), 2.40-2.31 (m, 2H), 2.09 -1.99 (m, 1H), 1.98 (s, 3H), 1.98-1.41 (m, 1H), 1.86-1.73 (m, 2H), 1.58-1 , 48 (m, 3H), 1.32 (s, 9H), 1.35-1.16 (m, 2H), 1.23 (s, 12H), 0.80-0.71 (m, 2H) ) ; MS (+CI): m/z for C2IH39BN2O4: expected 394.4; found 395.2 (M+H)+.
Step 3: (1S,2S)-1-amino-2-(3-boronopropyl) cyclopentane carboxylic acid (syn isomer)
A stirred mixture of syn-(IS,2S)-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) propyl)cyclopentanecarboxamide (0.641 g, 1.625 mmol) in 6N HCl (15 ml) was heated to 90°C for 20 h, cooled to room temperature and diluted with water (15 ml). The mixture was extracted with methylene chloride (2 x 15 ml) and concentrated. Water (20 ml) was added twice, and removed twice in vacuo to remove excess HCl. The residue obtained after concentration was dissolved in methanol (5 ml) and diluted to a volume of 40 ml with ether, and stirred for 1 h to remove solid tert-butylamine hydrochloride which is filtered off. The resulting filtrate was concentrated and treated with concentrated ammonium hydroxide (15 ml, 3x). Removal of excess ammonium hydroxide in vacuo gave a white solid residue which was triturated using acetonitrile and dried to give (1,2S)-1-amino-2-(3-boronopropyl)cyclopentanecarboxylic acid as a white powder containing a 1:1 mixture of cyclized and uncyclized forms (0.276 g, 63%). 1H-NMR (DMSO, 300 MHz) δ 6.49-6.37 (m, NH, 2H), 2.0-1.85 (m, 2H), 1.83-1.58 (m, 6H) ), 1.53-1.25 (m, 3H), 1.00-0.80 (m, 1H), 0.49-0.40 (m, 1H), 0.40-0.31 (m , 1H); MS (+CI): m/z for C9H18BNO4: 215.1 expected; found 215.3 (M+H)+. Example 3: Preparation of (2R,3S)-3-amino-2-(3-boronopropyl)tetrahydrofuran-3-carboxylic acid
A uma solução de ácido 3-(propen-3-ilóxi)propiônico (0,976 g, 7,5 mmol) e N,N-dimetilformamida anidra (0,1 ml) em 1,2-dicloroetano anidro (40 ml) sob uma atmosfera de nitrogênio foi adicionado cloreto de oxalila (5,5 ml, 2,0 N em diclorometano, 11 mmol). A mistura de reação foi agitada por 1 h em temperatura ambiente, e depois concentrada a 4 0 °C sob vácuo para obter um volume final de 5 ml. Dicloroetano anidro (20 ml; 3x) foi adicionado e a mistura de reação foi concentrada após cada adição de 20 ml de diclorometano. A solução residual obtida após a reconcentração final foi diluida com dicloroetano anidro (20 ml) e adicionada gota a gota a uma solução agitada gelada a 4 °C de N-isocianoimino-trifenilfosfoforano (3,33 g, 11 mmol) em um dicloroetano anidro (20 ml). Após agitação a 4 °C por 1 h, a reação é aquecida até a temperatura ambiente e é deixada em agitação por mais 3 h. A reação é interrompida por extinção com água (10 ml) e a mistura de água-diclorometano é agitada por mais 16 h em temperatura ambiente.Step 1: 4-(Allyloxy)-2-oxobutanehydrazonoyl chloride To a solution of 3-(propen-3-yloxy)propionic acid (0.976 g, 7.5 mmol) and anhydrous N,N-dimethylformamide (0.1 ml) in anhydrous 1,2-dichloroethane (40 ml) under a nitrogen atmosphere was added oxalyl chloride (5.5 ml, 2.0 N in dichloromethane, 11 mmol). The reaction mixture was stirred for 1 h at room temperature, then concentrated at 40 °C under vacuum to obtain a final volume of 5 ml. Anhydrous dichloroethane (20 ml; 3x) was added and the reaction mixture was concentrated after each addition of 20 ml of dichloromethane. The residual solution obtained after the final reconcentration was diluted with anhydrous dichloroethane (20 ml) and added dropwise to a stirred ice cold 4°C solution of N-isocyanoimino-triphenylphosphophorane (3.33 g, 11 mmol) in an anhydrous dichloroethane (20 ml). After stirring at 4 °C for 1 h, the reaction is warmed to room temperature and allowed to stir for a further 3 h. The reaction is quenched with water (10 ml) and the water-dichloromethane mixture is stirred for a further 16 h at room temperature.
To the quenched reaction mixture, water (25 ml) is added, so as to separate the aqueous layer from the organic layer. The aqueous layer was extracted with methylene chloride (2 x 15 ml) and the combined organic layers were dried over anhydrous Na 2 SO 4 prior to concentration and purification by flash column chromatography (silica gel, 5% ethyl acetate in dichloromethane) to give 1-chloro-1,2-diketo-4-(propen-3-yloxy)butane-1-hydrazone as a pale yellow oil (1.05 g, 74%). 1H-NMR (CDCl3 , 300 MHz) δ 6.64 (br s, 2H), 5.87 (ddt J = 17 Hz, J2 = 10 Hz, J3 = 5.4 Hz, 1H), 5.30-5 .14 (m, 2H), 4.00-3.93 (m, 2H), 3.78 (t, J = 6.5 Hz, 2H, ), 3.12 (t, J = 6.5 Hz, 2H); MS (+CI): m/z for C7H11CIN2O2: 190.1 expected; found 191.2 (M+H)+.
Enquanto sob uma atmosfera de nitrogênio, a uma solução agitada de 1-cloro-l,2-diceto-4-(propen-3- ilóxi)butano-l-hidrazona (2,10 g, 11 mmol) em cloreto de metileno anidro (40 ml) foi adicionado brometo de zinco anidro (0,563 g, 2,5 mmol), seguido pela adição gota a gota de N,N-diisopropilamina anidra (2,1 ml, 15 mmol). A mistura de reação foi agitada por 1 h, e depois tratada com sal tetrassódico de ácido etilenodiaminatetraacético 1% aquoso (EDTA) (30 ml), e agitada por mais 15 min. As camadas aquosas e orgânicas formadas após a adição de EDTA aquoso foram separadas e a camada aquosa foi extraida com. cloreto de metileno (2 x 15 ml) . As camadas orgânicas combinadas foram secas sobre Na2SO4 anidro e concentradas antes da purificação usando cromatografia instantânea em coluna (silica gel, acetato de etila 10% em diclorometano), para gerar l-diazo-2-ceto-4-(propen-3-ilóxi)butano (1,42 g, 84%) como um óleo amarelo. 1H-RNM (CDCI3, 300 MHz) δ 5,95-5,80 (m, 1H) , 5,36 (s, 1H) , 5,28 (dq, J = 15,6 Hz, J2 = 1,6 Hz, 1H) , 5,19 (dq, J = 11,7 Hz, J2 = 1,6 Hz, 1H) , 5,36 (br s, 1H), 5,15-5,30 (m, 2H) , 3,98 (dt, = 5,5 Hz, J2 = 1,5 Hz, 2H), 3,71 (t, J = 6,5 Hz, 2H) , 2,57 (br s, 2H) ; MS (+CI) : m/z para C7H10N2O2: esperado 154,1; encontrado 155,1 (M+H)+.Step 2: 4-(Allyloxy)-1-diazobutan-2-one While under a nitrogen atmosphere, to a stirred solution of 1-chloro-1,2-diketo-4-(propen-3-yloxy)butane-1-hydrazone (2.10 g, 11 mmol) in anhydrous methylene chloride (40 ml) was added anhydrous zinc bromide (0.563 g, 2.5 mmol), followed by dropwise addition of anhydrous N,N-diisopropylamine (2.1 ml, 15 mmol). The reaction mixture was stirred for 1 h, then treated with 1% aqueous ethylenediaminetetraacetic acid tetrasodium salt (EDTA) (30 ml), and stirred for a further 15 min. The aqueous and organic layers formed after the addition of aqueous EDTA were separated and the aqueous layer was extracted with. methylene chloride (2 x 15 ml). The combined organic layers were dried over anhydrous Na 2 SO 4 and concentrated prior to purification using flash column chromatography (silica gel, 10% ethyl acetate in dichloromethane) to give 1-diazo-2-keto-4-(propen-3-yloxy )butane (1.42 g, 84%) as a yellow oil. 1H-NMR (CDCl3, 300 MHz) δ 5.95-5.80 (m, 1H), 5.36 (s, 1H), 5.28 (dq, J = 15.6 Hz, J2 = 1.6 Hz, 1H), 5.19 (dq, J = 11.7 Hz, J2 = 1.6 Hz, 1H), 5.36 (br s, 1H), 5.15-5.30 (m, 2H) , 3.98 (dt, =5.5Hz, J2 =1.5Hz, 2H), 3.71 (t, J =6.5Hz, 2H), 2.57 (br s, 2H); MS (+CI): m/z for C7H10N2O2: expected 154.1; found 155.1 (M+H)+.
Uma solução de 4-(aliloxi)-l-diazobutan-2-ona (1,505 g, 9,76 mmol) em cloreto de metileno anidro (150 ml) foi adicionada gota a gota a uma solução em refluxo de acetilacetonato de cobre (II) (0,13 g, 0,50 mmol) em cloreto de metileno anidro (150 ml). Após refluxo da mistura resultante por 2 horas, a solução foi concentrada e purificada usando cromatografia instantânea em coluna (silica gel, diclorometano) para gerar 2-alildiidrofuran- 3(2H)-ona (1,147 g, 93%) como um óleo incolor. 1H-RNM (CDCI3, 300 MHz) δ 5,90-5,75 (m, 1H) , 5,20-5,05 (m, 2H) , 4,33-4,29 (m, 1H) , 4,07 (q, J = 9 Hz, 1H) , 3,79 (dd, = 7,5 Hz, J2 = 4,5 Hz, 1H) , 2, 60-2,45 (m, 3H) , 2,34 (q, J = 7,5 Hz, 1H) ; MS (+CI): m/z para C7H10O2: esperado 126,1; encontrado 127,2 (M+H)+, 149,1 (M + Na).Step 3: 2-Allyldihydrofuran-3(2H)-one A solution of 4-(allyloxy)-1-diazobutan-2-one (1.505 g, 9.76 mmol) in anhydrous methylene chloride (150 ml) was added dropwise to a refluxing solution of copper(II) acetylacetonate. ) (0.13 g, 0.50 mmol) in anhydrous methylene chloride (150 ml). After refluxing the resulting mixture for 2 hours, the solution was concentrated and purified using flash column chromatography (silica gel, dichloromethane) to give 2-allyldihydrofuran-3(2H)-one (1.147 g, 93%) as a colorless oil. 1H-NMR (CDCl3, 300 MHz) δ 5.90-5.75 (m, 1H), 5.20-5.05 (m, 2H), 4.33-4.29 (m, 1H), 4 .07 (q, J = 9Hz, 1H), 3.79 (dd, =7.5Hz, J2 =4.5Hz, 1H), 2.60-2.45 (m, 3H), 2. 34 (q, J = 7.5 Hz, 1H); MS (+CI): m/z for C7H10O2: 126.1 expected; found 127.2 (M+H)+, 149.1 (M + Na).
Step 4: (2R,3S)-3-acetamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide (racemic)

A solution of 2-allyldihydrofuran-3(2H)-one (1.2 g, 9.5 mmol) and ammonium acetate (4.39 g, 57.0 mmol) in 2,2,2-trifluoroethanol (5 ml) ) was treated with tert-butyl isocyanide (2.37 g, 3.23 ml, 28.5 mmol). After stirring at room temperature for 5 days, the reaction mixture was diluted with water (50 ml) and extracted using ethyl acetate (2 x 50 ml). The combined organic layers were washed with saturated aqueous sodium chloride, dried over MgSO4, filtered and concentrated. Purification by flash column chromatography (silica gel, 50% ethyl acetate in methylene chloride) gave (2R,3S)-3-acetamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide as a colorless oil ( 930mg, 36%) and (2S,3S)-3-acetylamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide as a colorless oil (650mg, 26%). Analytical data for (27', 3S)-3-acetylamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide: XH-NMR (CDCl3 , 300 MHz) δ 6.65 (s, NH, 1H), 6 .06 (s, NH, 1H), 5.86 (m, 1H), 5.16 (m, 2H), 4.20 (dd, J = 9 Hz, J2 = 4 Hz, 1H), 3.82 (m, 2H), 2.54 (m, 2H), 2.20-2.40 (m, 2H), 2.03 (s, 3H), 1.32 (s, 9H); MS (+CI): m/z for C14H24N2O3: expected 268.2; found 269.2 (M+H) + .
Step 5: (2R,3S)-3-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)tetrahydrofuran -3-carboxamide
A solution of (2R,3S)-3-acetamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide (930 mg, 3.47 mmol) in dichloromethane (20 ml) was treated with chloro-1 dimer. ,5-cyclooctadiene-iridium (I) (70 mg, 3 mol%) and 1,2-bis(diphenylphosphino)-ethane (83 mg, 6 mol%). The solution was stirred at room temperature for 30 minutes, then 4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1.01 ml, 6.94 mmol) was added dropwise, and the reaction was stirred overnight at room temperature. The next day, the reaction mixture was poured into water and the aqueous solution was extracted using ethyl acetate (3x).
The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated. Purification by flash column chromatography (silica gel, 50-80% ethyl acetate in dichloromethane) gave (2R,3S)-3-acetamido-N-tert-butyl-2-(3-(4,4,5, 5-Tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)tetrahydrofuran-3-carboxamide as a colorless oil (864 mg, 63%). 1H-NMR (CDC13, 300 MHz) δ 7.01 (s, NH, 1H), 6.10 (s, NH, 1H), 4.38 (m, 1H), 4.12 (m, 1H), 4.00 (m, 1H), 2.96 (m, 1H), 2.02-2.18 (m, 2H), 1.99 (s, 3H), 1.42-1.62 (m, 3H), 1.36 (s, 9H), 1.22 (s, 12H), 0.76 (m, 2H); MS (+CI): m/z for C20H37BN205: expected 396.3; found 397.4 (M+H)+. Step 6: (2R, 3S)-3-amino-2-(3-boronopropyl) tetrahydrofuran-3-carboxylic acid
A solution of (2R,3S)-3-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)tetrahydrofuran -3-carboxamide (860 mg) in 6N HCl (15 ml) was stirred at 90°C for 1 day. After cooling to room temperature, the reaction mixture was transferred to a separatory funnel, diluted with deionized water (10 ml) and washed with dichloromethane (3x). The aqueous solution was concentrated. Purification by RP-HPLC (10-100% acetonitrile in water) gave (2R,3S)-3-amino-2-(3-boronopropyl)tetrahydrofuran-3-carboxylic acid as a white solid (269 mg). 1H-NMR (D2O, 300 MHz) δ 3.97 (m, 2H), 3.82 (m, 1H), 2.66 (ddd, J = 15 Hz, J2 = 9.5 Hz, J3 = 5. 5Hz, 1H), 2.14 (ddd, J = 15Hz, J2 = 9Hz, J3 = 6.5Hz, 1H), 1.22-1.49 (m, 4H), 0.67 (m , 2H); MS (+CI): m/z for CgHigBlNOs: 217.1 expected; found 435.3 (2M+H)+, 417.2 (2M+H-H2O)+, 217.7 (M+H)+, 200.0 (M+H-2H2O)+. Example 4: Preparation of (2S,3S)-3-amino-2-(3-boronopropyl)tetrahydrofuran-3-carboxylic acid
(2S,3S)-3-acetamido-2-alil-N-terc- butiltetrahidrofurano-3-carboxamida foi obtida em conjunto com seu isômero na etapa 4 do Exemplo 3. 1H-RNM (CDCI3, 300 MHz) δ 6,99 (s, NH, 1H) , 6,09 (s, NH, 1H) , 5,78 (m, 1H) , 5,08 (m, 2H) , 4,49 (dd, J = 9 Hz, J2 = 5 Hz, 1H) , 4,17 (q, J = 9 Hz, 1H) , 4,04 (td, = 9 Hz, J2 = 3,5 Hz, 1H), 2,97 (ddd, Ji = 12,5 Hz, J2 = 9 Hz, J 5 = 3,5 Hz, 1H) , 2,04-2,36 (m, 3H) , 1,99 (s, 3H) , 1,38 (s, 9H) ; MS (+CI): m/z para Ci4H24N2θ3: esperado 268,2; encontrado 269, 2 (M+H) + .Step 1: (2S,3S)-3-acetamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide (2S,3S)-3-acetamido-2-allyl-N-tert-butyltetrahydrofuran-3-carboxamide was obtained together with its isomer in step 4 of Example 3. 1H-NMR (CDCl3, 300 MHz) δ 6.99 (s, NH, 1H), 6.09 (s, NH, 1H), 5.78 (m, 1H), 5.08 (m, 2H), 4.49 (dd, J=9Hz, J2= 5Hz, 1H), 4.17 (q, J = 9Hz, 1H), 4.04 (td, =9Hz, J2 = 3.5Hz, 1H), 2.97 (ddd, Ji =12, 5Hz, J2 = 9Hz, J5 = 3.5Hz, 1H), 2.04-2.36 (m, 3H), 1.99 (s, 3H), 1.38 (s, 9H); MS (+CI): m/z for C14H24N2θ3: 268.2 expected; found 269.2 (M+H) + .
Step 2: (2S,3S)-3-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)tetrahydrofuran -3-carboxamide (2S,3S)-3-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) tetrahydrofuran-3-carboxamide was prepared using the method described in Step 5 of Example 3. The title compound was isolated as a colorless oil (496 mg), 1H-NMR (CDCl3, 300 MHz) δ 6.57 (s, NH , 1H), 5.83 (s, NH, 1H), 4.04 (m, 1H), 3.88 (m, 2H), 2.62 (m, 1H), 2.48 (m, 1H) , 2.05 (s, 3H), 1.40-1.58 (m, 4H), 1.33 (s, 9H), 1.24 (s, 12H), 0.83 (t, J=6 .5Hz, 2H); MS (+CI): m/z for C20H37BN2O5: expected 396.3; found 397.3 (M+H)+.
Step 3: (2S,3S)-3-Amino-2-(3-boronopropyl)tetrahydrofuran-3-carboxylic acid (2S,3S)-3-amino-2-(3-boronopropyl)tetrahydrofuran-3-carboxylic acid was prepared using the method described in Step 6 of Example 3. The title compound was isolated as a white solid (91 mg); 1H-NMR (D2O, 300 MHz) δ 4.00 (m, 1H), 3.80 (m, 1H), 3.73 (m, 1H), 2.58 (m, 1H), 2.04 ( ddd, J=13.5Hz, J2=8Hz, J3=4Hz, 1H), 1.34-1.49 (m, 3H), 1.28 (m, 1H), 0.66 (m, 2H); MS (+CI): m/z for CgHigB1NOg: 217.1 expected; found 417.1 (2M+H-H2O)+, 399.3 (2M+H-2H2O)+, 217.7 (M+H)+, 200.0 (M+H-2H2O)+.
Ácido 3-amino-2-(3-boronopropil)tetrahidrotiofeno-3- carboxilico foi preparado de forma análoga àquela apresentada no Exemplo 3, exceto que ácido 3- (aliltio)propanóico foi usado ao invés de ácido 3-(propen- 3-ilóxi)propiônico na Etapa 1 do Exemplo 3. O composto do titulo foi isolado como um sólido branco como uma inseparável (aproximadamente 1:1) de diastereoisômeros (51 mg); aH-RNM (D20, 300 MHz) δ 3,91 (m, 0,5H), 3,70 (m, 0,5H), 3,05 (m, 1H) , 2,80 (m, 1H) , 2,50 (m, 1H) , 2,38 (m, 1H) , 1,52 (m, 1H) , 1,16-1,42 (m, 3H) , 0,64 (m, 2H) ; MS (+CI) : m/z para C8HI6B1NO4 S: esperado 233,1; encontrado 234,0 (M+H)+, 216,1 (M+H-H2O) + .Example 5: Preparation of 3-amino-2-(3-boronopropyl)tetrahydrothiophene-3-carboxylic acid 3-Amino-2-(3-boronopropyl)tetrahydrothiophene-3-carboxylic acid was prepared analogously to that shown in Example 3, except that 3-(allylthio)propanoic acid was used in place of 3-(propen-3- yloxy)propionic in Step 1 of Example 3. The title compound was isolated as a white solid as an inseparable (approximately 1:1) of diastereoisomers (51 mg); aH-NMR (D20, 300 MHz) δ 3.91 (m, 0.5H), 3.70 (m, 0.5H), 3.05 (m, 1H), 2.80 (m, 1H), 2.50 (m, 1H), 2.38 (m, 1H), 1.52 (m, 1H), 1.16-1.42 (m, 3H), 0.64 (m, 2H); MS (+CI): m/z for C8HI6B1NO4 S: 233.1 expected; found 234.0 (M+H)+, 216.1 (M+H-H2O) + .
Ácido (3R, 4S)-4-amino-3-(3-boronopropil)piperidina-4- carboxilico foi preparado de forma análoga àquela apresentada no Exemplo 1, exceto que 1-terc-butil 3-metil 4-oxopiperidina-l,3-dicarboxilato foi usado ao invés de metil 2-oxociclopentanocarboxilato na Etapa 1 do Exemplo 1. A análise da mistura de reação indica a presença de três produtos isoméricos. 1H-RNM (DMSO, 300 MHz) δ 3,40-3,17 (m, 3H) , 3,04 (t, J = 12,9 Hz, 1H) , 2,22-2,18 (m, 1H) , 2,01- 1,68 (m, 2H), 1,45-0, 83 (m, 4H) , 0,75-0,40 (m, 2H) ; MS (+CI) : m/z para C9H19BN2O4: esperado 230,1; encontrado 231,0 (M+H)+.Example 6: Preparation of (3R,4S)-4-amino-3-(3-boronopropyl)piperidine-4-carboxylic acid (3R,4S)-4-Amino-3-(3-boronopropyl)piperidine-4-carboxylic acid was prepared analogously to that shown in Example 1, except that 1-tert-butyl 3-methyl 4-oxopiperidine-1, 3-dicarboxylate was used in place of methyl 2-oxocyclopentanecarboxylate in Step 1 of Example 1. Analysis of the reaction mixture indicates the presence of three isomeric products. 1H-NMR (DMSO, 300 MHz) δ 3.40-3.17 (m, 3H), 3.04 (t, J = 12.9 Hz, 1H), 2.22-2.18 (m, 1H) ), 2.01-1.68 (m, 2H), 1.45-0.83 (m, 4H), 0.75-0.40 (m, 2H); MS (+CI): m/z for C9H19BN2O4: expected 230.1; found 231.0 (M+H)+.
Ácido (3S,4S)-4-amino-3-(3-boronopropil)piperidina-4- carboxilico foi preparado de forma análoga àquela apresentada no Exemplo 1, exceto que 1-terc-butil 3-metil 4-oxopiperidina-l,3-dicarboxilato foi usado ao invés de metil 2-oxociclopentanocarboxilato na Etapa 1 do Exemplo 1. A análise da mistura de reação indica a presença de três produtos isoméricos. 1H-RNM (DMSO, 300 MHz) δ 3,40-3,20 (m, 2H) , 2,90-2,61 (m, 2H) , 2,28-2,19 (m, 2H) , 2,01-1,91 (m, 1H), 1,27-0,95 (m, 4H) , 0, 75-0, 40 (m, 2H) ; MS (+CI) : m/z para C9H19BN2O4: esperado 230,1; encontrado 231,0 (M+H) + .Example 7: Preparation of (3S,4S)-4-amino-3-(3-boronopropyl)piperidine-4-carboxylic acid (3S,4S)-4-Amino-3-(3-boronopropyl)piperidine-4-carboxylic acid was prepared analogously to that shown in Example 1, except that 1-tert-butyl 3-methyl 4-oxopiperidine-1, 3-dicarboxylate was used in place of methyl 2-oxocyclopentanecarboxylate in Step 1 of Example 1. Analysis of the reaction mixture indicates the presence of three isomeric products. 1H-NMR (DMSO, 300 MHz) δ 3.40-3.20 (m, 2H), 2.90-2.61 (m, 2H), 2.28-2.19 (m, 2H), 2 .01-1.91 (m, 1H), 1.27-0.95 (m, 4H), 0.75-0.40 (m, 2H); MS (+CI): m/z for C9H19BN2O4: expected 230.1; found 231.0 (M+H) + .
Etapa I, Método A: terc-butil 3-alil-4-oxopirrolidina- 1-carboxilato Uma solução agitada gelada (3°C) de N-boc-glicina, alil éster (6,46 g, 30 mmol) em tetrahidrofurano anidro (60 ml) sob nitrogênio foi tratada com 1 N de bis(trimetilsililamida) de litio/tetrahidrofurano (33 ml, 33 mmol) em uma taxa para manter a temperatura do vaso abaixo de 10°C, e depois agitada a 3°C por 15 min e resfriada (-40°C). Uma solução de alil acrilato (4,45 ml, 35 mmol) em tetrahidrofurano anidro (25 ml) foi adicionada gota a gota, e foi permitido que a mistura alcançasse temperatura ambiente, agitada 1 h, e ela foi refluida por 2 h. A mistura foi resfriada até a temperatura ambiente, extinta com ácido acético glacial (2,5 ml), e concentrada. O óleo residual foi dissolvido em cloreto de metileno (300 ml) e a solução lavada com água e bicarbonato de sódio saturado (150 ml cada), seco (NajSC^) , e concentrado. O residue foi dissolvido em um minimo de cloreto de metileno e carregado sobre uma coluna de gel de silica (volume de 350 ml) e eluido com 55:30:15 de heptano/cloreto de metileno/acetato de etila para gerar 3-alil 1-terc-butil 4- oxopirrolidina-1,3-dicarboxilato (5,07 g, 63%) como um óleo incolor. RNM (CDCI3) : δ 5,85-6, 00 (m, 1H) , 5,20-5,40 (m, 2H) , 4,60-4,75 (m, 2H) , 4,20 (m, 1H) , 3,95-4,10 (m, 1H) , 3,87 (d, J= 6,5 Hz, 1H) , 3,62 (t, J = 8 Hz, 1H) , 1,48 (s, 9H). MS (m + 1): 270,4; MS (m - bu + 1): 214,2. Esse composto (4,12 g, 15,3 mmol) foi dissolvido em tetrahidrofurano anidro (25 ml) e adicionado a uma solução agitada de Pd(PPhs)4 (0,36 g, 0,31 mmol) em tetrahidrofurano anidro (40 ml) sob nitrogênio, agitada por 4 h, e concentrada. O residuo foi dissolvido em um minimo de cloreto de metileno e carregado sobre uma coluna de gel de silica (volume de 350 ml) e eluido com 60:35:5 de heptano/cloreto de metileno/acetato de etila para gerar terc-butil 3-alil-4-oxopirrolidina-l-carboxilato (4,16 g, 60%) como um óleo amarelo muito pálido. RNM (CDCI3) : δ 5,65-5,80 (m, 1H) , 5,05-5,15 (m, 2H) , 4,02 (m, 1H) , 3,89 (br d, J = 20 Hz, 1H) , 3,67 (br d, J = 20 Hz, 1H) , 3,31 Mt (dd, J = 11 Hz, J2 = 8,5 Hz, 1H) , 2,60-2,70 (m, 1H), 2,50- V 2,60 (m, 1H), 2,10-2,30 (m, 1H) , 1,48 (s, 9H) . MS (m + 1): 226,1; MS (m- bu + 1): 170,1.Example 8: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid Step I, Method A: tert-butyl 3-allyl-4-oxopyrrolidine-1-carboxylate An ice-cold stirred solution (3°C) of N-boc-glycine, allyl ester (6.46 g, 30 mmol) in anhydrous tetrahydrofuran (60 ml) under nitrogen was treated with 1 N lithium bis(trimethylsilylamide)/tetrahydrofuran (33 ml, 33 mmol) at a rate to maintain the vessel temperature below 10°C, and then stirred at 3°C for 15 min and cooled (-40°C). A solution of allyl acrylate (4.45 ml, 35 mmol) in anhydrous tetrahydrofuran (25 ml) was added dropwise, and the mixture was allowed to reach room temperature, stirred 1 h, and refluxed for 2 h. The mixture was cooled to room temperature, quenched with glacial acetic acid (2.5 ml), and concentrated. The residual oil was dissolved in methylene chloride (300 ml) and the solution washed with water and saturated sodium bicarbonate (150 ml each), dried (NajSO^), and concentrated. The residue was dissolved in a minimum of methylene chloride and loaded onto a silica gel column (350 ml volume) and eluted with 55:30:15 heptane/methylene chloride/ethyl acetate to give 3-allyl 1 -tert-butyl 4-oxopyrrolidine-1,3-dicarboxylate (5.07 g, 63%) as a colorless oil. NMR (CDCl3 ): δ 5.85-6.00 (m, 1H), 5.20-5.40 (m, 2H), 4.60-4.75 (m, 2H), 4.20 (m) , 1H), 3.95-4.10 (m, 1H), 3.87 (d, J=6.5Hz, 1H), 3.62 (t, J=8Hz, 1H), 1.48 (s, 9H). MS (m+1): 270.4; MS (m - bu + 1): 214.2. This compound (4.12 g, 15.3 mmol) was dissolved in anhydrous tetrahydrofuran (25 ml) and added to a stirred solution of Pd(PPhs)4 (0.36 g, 0.31 mmol) in anhydrous tetrahydrofuran (40 ml) under nitrogen, stirred for 4 h, and concentrated. The residue was dissolved in a minimum of methylene chloride and loaded onto a silica gel column (350 ml volume) and eluted with 60:35:5 heptane/methylene chloride/ethyl acetate to give tert-butyl 3 -allyl-4-oxopyrrolidine-1-carboxylate (4.16 g, 60%) as a very pale yellow oil. NMR (CDCl3 ): δ 5.65-5.80 (m, 1H), 5.05-5.15 (m, 2H), 4.02 (m, 1H), 3.89 (br d, J = 20 Hz, 1H), 3.67 (br d, J = 20 Hz, 1H), 3.31 Mt (dd, J = 11 Hz, J2 = 8.5 Hz, 1H), 2.60-2.70 (m, 1H), 2.50-V 2.60 (m, 1H), 2.10-2.30 (m, 1H), 1.48 (s, 9H). MS (m+1): 226.1; MS (m-bu + 1): 170.1.
Step 1, Method B: Tert-butyl 3-allyl-4-oxopyrrolidine-5 1-carboxylate A solution of 1-tert-butyl 3-methyl 4-oxopyrrolidine-1,3-dicarboxylate (48.65 g, 0.20 mol), allyl alcohol (300 ml), and dibutyltin oxide (5.0 g, 20 mmol) in anhydrous toluene (800 ml) was refluxed for 20 h under a 10 Dean-Stark apparatus with portioned solvent removal ( 200 ml total) over the first 6 hours, followed by addition of more allyl alcohol (75 ml) at the end of the first 6 hours. The reaction mixture was concentrated, dissolved in a minimum of methylene chloride, and loaded onto a silica gel column 15 (700 ml volume) and eluted with methylene chloride, 10%, then 15%, then 20% acetate of ethyl/methylene chloride to give 3-allyl 1-tert-butyl 4-oxopyrrolidine-1,3-dicarboxylate (48.6 g, 90%) as a pale pink oil (NMR and MS as above). This compound (48.47 g, 0.18 mol) was dissolved in anhydrous tetrahydrofuran (200 ml) and added to a stirred solution of Pd(PPh3)4 (4.16 g, 3.6 mmol) in anhydrous tetrahydrofuran ( 400 ml) under nitrogen, stirred for 4 h, and concentrated. The residue was dissolved in heptane and loaded onto a silica gel column (1000 ml volume) and eluted with 60:35:5 heptane/methylene chloride/ethyl acetate to give tert-butyl 3-allyl-4 -oxopyrrolidine-1-carboxylate (27.93, 69%) as a pale yellow oil (NMR and MS as above).
Step 2: (3R, 4S)-tert-butyl 3-acetamido-4-allyl-3-(tert-butylcarbamoyl)pyrrolidine-1-carboxylate ■ A stirred mixture of tert-butyl 3-allyl-4-oxopyrrolidine-1- carboxylate (13.23 g, 58.7 mmol) and ammonium acetate (11.95 g, 155 mmol) in 2,2,2-trifluoroethanol (25 ml) under nitrogen was treated with t-butylisonitrile (12.25 ml). , 106 mmol), and then stirred at room temperature for 4 days and concentrated. The residue was partitioned between water (100 ml) and methylene chloride (200 ml), and the aqueous layer was extracted with methylene chloride (2 x 75 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (100 ml each), dried (Na 2 SO 4 ) and concentrated to an off-white solid. This was recrystallized twice from ethyl acetate (150 ml each) to give a portion of the title product (8.36 g) as a white solid. The combined mother liquors were concentrated, dissolved in a minimum of methylene chloride and loaded onto a silica gel column (650 ml volume). This was eluted with 60%, then 70%, then 90% ethyl acetate/heptane to generate additional product (3.84 g). The total yield of (3R,4S)-tert-butyl 3-acetamido-4-allyl-3-(tert-butylcarbamoyl)pyrrolidine-1-carboxylate was 12.22 g (57%) as a white solid. NMR (CDCl 3 ) δ 6.30-6.70 (m, 2H), 5.60-5.75 (m, 1H), 4.95-5.10 (m, 2H), 3.94 (d, J = 30 11.5 Hz, 1H), 3.75 (d, J = 11.5 Hz, 1H), 3.60 (m, 1H), 3.00-3.20 (m, 2H), 2 .20-2.30 (m, 1H), 2.00 (s, 3H), 1.80-1.90 (m, 1H), 1.44 (s, 9H), 1.33 (s, 9H) ) . MS (m+1): 368.3; MS (m - bu +1): 312.1; MS (m - boc + 1): 268.3.
Step 3: (3R,4S)-tert-butyl 3-acetamido-3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)propyl)pyrrolidine-1-carboxylate A stirred solution of (3R,4S)-tert-butyl 3-acetamido-4-allyl-3-(tert-butylcarbamoyl)pyrrolidine-1-carboxylate (5.51 g, 15 mmol ) in anhydrous methylene chloride (80 ml) under nitrogen' was treated with chloro-1,5-cyclooctadiene-iridium dimer (0.252 g, 0.375 mmol) and 1,2-bis(diphenylphosphino)ethane (0.299 g, 0. 75 mmol), stirred for 30 min and cooled (-20°C) . 4,4,5,5-Tetramethyl-1,3,2-dioxaborolane (3.30 ml, 22.5 mmol) was added dropwise, and the solution was placed in an ice bath and allowed to reach room temperature overnight (18 h) . The mixture was quenched with water (75 ml), stirred 15 min and extracted with ethyl acetate (400 ml, then 2 x 100 ml). The combined organic solution was washed with saturated aqueous sodium chloride (150 ml), dried (MgSO4 ) and concentrated. The solid was recrystallized (2 collections) from acetonitrile to give (3R,4S)-tert-butyl 3-acetamido-3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl-1). ,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1-carboxylate (6.13 g, 82%) as a white solid. NMR (CDCl 3 ) δ 6.40-6.60 (m, 1H), .6.23 (s, 1H), 3.95-4.05 (m, 1H), 3.65-3.75 (m) , 2H), 2.90-3.20 (m, 2H), 2.00 (s, 3H), 1.45 (s, 9H), 1.30-1.45 (m, 4H), 1. 33 (s, 9H), 1.22 (s, 12H), 0.70-0.80 (m, 2H). MS (m+1): 496.4.
Step 4:(3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine -3-carboxamide A 250 ml 3-neck round bottom flask was charged with (3R,4S)-tert-butyl 3-acetamido-3-(tert-butylcarbamoyl)—4—(3—(4,4,5) ,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1-carboxylate (4.95g, 10mmol) and 4N HCl/dioxane (50ml). After stirring for 3 h, the solution was diluted with ether (125 ml), stirred for a few minutes, filtered, and the solid rinsed with ether, collected and dried in vacuo to give an HCl salt of the title compound. This was dissolved in water (30 ml) and treated with sodium hydroxide (0.5 g, 12.5 mmol). The aqueous solution was treated with enough potassium carbonate to render the free base product insoluble. The mixture was extracted with methylene chloride (75 ml, then 3 x 50 ml) and the combined organic solution was washed with saturated aqueous sodium chloride (30 ml), dried (Na2SO4) and concentrated to give (3R,4S) -3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxamide (3.40 g, 86%) as a white solid. NMR (CDCl3 ) δ 8.26 (br s, 1H), 7.25 (s, 1H), 3.86 (d, J = 9.5 Hz, 1H), 3.53 (t, J = 9, 5Hz, 1H), 3.20 (d, J = 10Hz, 1H), 2.98 (m, 1H), 2.74 (dd, =10Hz, J2 = 7.5Hz, 1H), 2 1.02 (s, 3H), 1.50 (m, 1H), 1.33 (s, 9H), 1.20-1.40 (m, 3H), 1.22 (s, 12H), 0. 70-0.80 (m, 2H). MS (m+1): 396.0.
Step 5: (3R,4S)-3-Amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid A solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxamide (198 mg, 0.5 mmol) in 2:1:1 concentrated HCl: acid Glacial acetic:water (8 ml) in a pressure bottle was stirred for 2 h at 60°C, then capped and stirred for 18 h at 130°C, cooled to room temperature and uncapped. The solution was diluted with water (20 ml), then extracted with methylene chloride (20 ml) and concentrated. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in water (40 ml) and treated with DOMEX® 550A-OH resin (3 g) which had been rinsed with methanol. The mixture was stirred for 40 min and then filtered, and the resin washed successively with water, methanol, methylene chloride, water, methanol and methylene chloride. The resin was then shaken four times with 1N HCl (15 ml) and filtered, and the combined filtrates were concentrated. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in 1.5-2.0 ml of water and subjected to HPLC gradient purification as follows: 0-25% of B with A = 0.1% trifluoroacetic acid/water and B = 0.1% trifluoroacetic acid/acetonitrile. Appropriate fractions were concentrated, treated three times with 1 N HCl (10 ml) and concentrated, treated three times with water (10 ml) and concentrated, then dissolved in water (10 ml), frozen and lyophilized overnight. the other to give (3R,4S)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (114 mg, 79%) as a pale amber glass. NMR (D2O) δ 3.90 (d, J = 13 Hz, 1H), 3.71 (dd, = 11.5 Hz, J2 = 8.5 Hz, 1H), 3.46 (d, J = 13 Hz, 1H), 3.21 (t, J = 12 Hz, 1H), 2.50-2.65 (m, 1H), 1.5-1.65 (m, 1H), 1.10-1 0.40 (m, 3H), 0.60-0.75 (m, 2H). MS (m+1): 217.3; MS (m - H2O + 1): 199.1.
Ácido (3R,4R)-3-amino-4-(3-boronopropil)pirrolidina-3- carboxilico foi preparado de forma análoga àquela apresentada no Exemplo 8, exceto que (3R,4R)-terc-butil 3- acetamido-4-alil-3-(terc-butilcarbamoil)pirrolidina-l- carboxilato da Etapa 2 (isômero menor) foi usado. MS (+CI): m/z para CgHnBN^: esperado 216,1; encontrado 217,3 (M+H)+, 199,1 (M - H2O + H)+.Example 9: (3R,4R)-3-Amino-4-(3-boronopropyl)-pyrrolidine-3-carboxylic acid (3R,4R)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 8, except that (3R,4R)-tert-butyl 3-acetamido-4 -allyl-3-(tert-butylcarbamoyl)pyrrolidine-1-carboxylate from Step 2 (minor isomer) was used. MS (+CI): m/z for CgHnBN4: 216.1 expected; found 217.3 (M+H)+, 199.1 (M - H2O + H)+.
Example 10: (3R,34R)-3-Amino-4-(3-boronopropyl)-pyrrolidine-3-carboxylic acid
(3R,4R)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 8, except that tert-butyl 2-allyl-3-oxopyrrolidine-1-carboxylate was used in place of tert-butyl 3-allyl-4-oxopyrrolidine-1-carboxylate in Step 2. MS (+CI): m/z for C8H17BN2O4: expected 216.1; found 217.3 (M+H)+, 199.1 (M - H2O + H) + .
Example 11: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-isobutylpyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and isobutyraldehyde (0.060 ml, 0 .65 mmol), stirred for 2.5 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-isobutylpyrrolidine-3-carboxylic acid (44 mg , 26%) as a white semi-solid. NMR (D2O) δ 3.90 (m, 1H), 3.50 (m, 1H), 2.90-3.20 (m, 4H), 2.50 (m, 1H), 1.90 (m , 1H), 1.50 (m, 1H), 0.80-1.30 (m, 9H), 0.65 (m, 2H). MS (m+1): 273.2; MS (m - H2O + 1): 255.2; MS (m -2 H2O + 1): 237.2.
Uma solução agitada de (3R,4S)-3-acetamido-N-terc- butil-4-(3-(4,4,5,5-tetrametil-l,3,2-dioxaborolan-2- il)propil)pirrolidina-3-carboxamida (Exemplo 8, Etapa 4)(166 mg, 0,42 mmol) em 1,2-dicloroetano anidro (5 ml) foi tratada com sulfato de sódio anidro (2 g) e benzaldeido (69 mg, 0,65 mmol), agitada por 2,5 h, e depois tratada com triacetoxiborohidreto de sódio (212 mg, 1,0 mmol) e ácido acético glacial (2 gotas) e agitada por 18 h. Carbonato de sódio aquoso (10%, 5 ml) foi adicionado, e a mistura agitada por poucos minutos e extraida com acetato de etila (30 ml, depois 2 x 10 ml). A solução orgânica combinada foi lavada com água e cloreto de sódio aquoso saturado (20 ml cada), seca (MgSO4) , e concentrada. 0 material bruto foi desprotegido e isolado usando o método descrito para o Exemplo 8, Etapa 5, para gerar ácido (3R,4S)-3-amino-l- benzil-4-(3-boronopropil)pirrolidina-3-carboxilico (70 mg, 44%) como um semi-sólido branco. RNM (d6-DMSO) δ 7,62 (br s, 2H), 7,44 (br s, 3H), 4,30-4,60 (m, 2H) , 3,60-3,90 (m, 2H), 3,35-3,50 (m, 2H) , 2,85 (m, 1H), 2,50 (m, 1H), 1,20- 1,80 (m, 3H), 0,60-0,80 (m, 2H). MS (m + 1): 307,3; MS (m - H2O + 1) : 289,2.Example 12: Preparation of (3R,4S)-3-amino-1-benzyl-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (166 mg, 0.42 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (2 g) and benzaldehyde (69 mg, 0 .65 mmol), stirred for 2.5 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-1-benzyl-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (70 mg, 44%) as a white semi-solid. NMR (d6-DMSO) δ 7.62 (br s, 2H), 7.44 (br s, 3H), 4.30-4.60 (m, 2H), 3.60-3.90 (m, 2H), 3.35-3.50 (m, 2H), 2.85 (m, 1H), 2.50 (m, 1H), 1.20-1.80 (m, 3H), 0.60 -0.80 (m, 2H). MS (m+1): 307.3; MS (m - H2O + 1): 289.2.
Uma solução agitada de (3R,4S)-3-acetamido-N-terc- butil-4-(3-(4,4,5,5-tetrametil-l,3,2-dioxaborolan-2- il)propil)pirrolidina-3-carboxamida (Exemplo 8, Etapa 4) (175 mg, 0,443 mmol) em 1,2-dicloroetano anidro (5 ml) foi tratada com sulfato de sódio anidro (1 g) e piridina-3- carboxaldeido (75 mg, 0,70 mmol), agitada por 2 h, e depois tratada com triacetoxiborohidreto de sódio (212 mg, 1,0 mmol) e ácido acético glacial (2 gotas) e agitada por 20 h. Carbonato de sódio aquoso (10%, 5 ml) foi adicionado, e a mistura agitada por poucos minutos e extraida com acetato de etila (30 ml, depois 2 x 10 ml). A solução orgânica combinada foi lavada com água e cloreto de sódio aquoso saturado (20 ml cada), seca (MgSO4) , e concentrada. O material bruto foi desprotegido e isolado usando o método descrito para o Exemplo 8, Etapa 5, para gerar ácido (3R,4S)-3-amino-4-(3-boronopropil)-1-(piridin-3- ilmetil)pirrolidina-3-carboxilico (115 mg, 68%) como um semi-sólido branco. RNM (dβ-DMSO) δ 8,90 (m, 1H), 8,74 (m, 1H) , 8,00 (m, 1H) , 7,12 (m, 1H) , 4,55-4, 85 (m, 2H) , 3,10- 4,00 (m, 3H) , 2,70-3,00 (m, 1H) , 1,70 (m, 2H) , 0,90-1,50 (m, 3H) , 0,55-0,70 (m, 2H) . MS (m + 1): 308,4; MS (m - H2O + 1) : 290, 4.Example 13: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(pyridin-3-ylmethyl)pyrrolidine-3-carboxylic acid A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (175 mg, 0.443 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and pyridine-3-carboxaldehyde (75 mg). , 0.70 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 20 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(pyridin-3-ylmethyl)pyrrolidine acid -3-carboxylic (115 mg, 68%) as a white semi-solid. NMR (dβ-DMSO) δ 8.90 (m, 1H), 8.74 (m, 1H), 8.00 (m, 1H), 7.12 (m, 1H), 4.55-4.85 (m, 2H), 3.10-4.00 (m, 3H), 2.70-3.00 (m, 1H), 1.70 (m, 2H), 0.90-1.50 (m) , 3H), 0.55-0.70 (m, 2H). MS (m+1): 308.4; MS (m - H2O + 1): 290, 4.
Example 14: Preparation of (3R,4S)-3-amino-1-(2-aminocyclopentyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and 2-(N-BOC-amino)cyclopentan-1-one (0.199 g, 1.0 mmol) in 1,2- Anhydrous dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 1.5 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (276 mg, 1.3 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4), and concentrated in vacuo. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-1-(2-aminocyclopentyl)-4-(3-boronopropyl)pyrrolidine-3 acid -carboxylic (116 mg, 57%) as a white powder. NMR (D20) δ 3.85-4.05 (m, 4H), 3.77 (d, J = 12.5 Hz, 1H), 3.42 (dt, = 11.5 Hz, J2 = 4 Hz , 1H), 2.50-2.65 (m, 1H), 2.10-2.35 (m, 2H), 1.75-1.95 (m, 4H), 1.55-1.65 (m, 1H), 1.15-1.40 (m, 3H), 0.63-0.73 (m, 2H). MS (m+1): 300.0; MS (m - H2O + 1): 281.9.
Example 15: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-4-ylmethyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and N-boc-piperidine- 4-carboxaldehyde (149 mg, 0.7 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-4-ylmethyl)pyrrolidine acid -3-carboxylic acid (66 mg, 31%) as an amber granular solid. NMR (D2O) δ 3.70-4.00 (m, 2H), 3.30-3.45 (m, 4H), 3.25 (d, J=7Hz, 2H), 2.92 (br t, J = 13Hz, 2H), 2.50-2.65 (m, 1H), 2.00-2.15 (m, 1H), 1.85-2.00 (m, 2H), 1 .50-1.65 (m, 1H), 1.35-1.50 (m, 2H), 1.15-1.35 (m, 3H), 0.65-0.75 (m, 2H) . MS (m - H2O + 1): 296.3; MS (m-2 H2O+1): 278.1.
Example 16: Preparation of (3R, 4S)-3-amino-4-(3-boronopropyl)-1-(3-(4-carboxyphenyl)propyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and 3—(4—( trifluoromethyl)phenyl)propionaldehyde (142 mg, 0.7 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(3-(4-carboxyphenyl) acid propyl)pyrrolidine-3-carboxylic (12 mg, 5%) as a white bulky powder. NMR (D2O) δ 7.86 (d, J = 8 Hz, 2H), 7.29 (d, J = 8 Hz, 2H), 3.70-4.00 (m, 2H), 3.50 ( m, 1H), 3.10-3.30 (m, 3H), 2.70 (t, J = 7.5Hz, 2H), 2.40-2.60 (m, 1H), 2.00 (m, 2H), 1.55 (m, 1H), 1.10-1.35 (m, 3H), 0.65 (m, 2H). MS (m - H2O + 1): 361.0; MS (m = 2. H 2 O + 1) : 343, 0.
Example 17: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(3-(dimethylamino)-2,2-dimethylpropyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and 3-dimethylamino-2. 2-dimethylpropionaldehyde (91 mg, 0.7 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(3-(dimethylamino)- acid 2,2-dimethylpropyl)pyrrolidine-3-carboxylic acid (36 mg, 16%) as a white granular solid. NMR (D2O) δ 3.80-4.00 (m, 2H), 3.35-3.50 (m, 3H), 3.20 (s, 2H), 2.89 (s, 6H), 2 1.50-2.65 (m, 1H), 1.50-1.65 (m, 1H), 1.25-1.35 (m, 2H), 1.21 (s, 6H), 0.65 -0.75 (m, 2H). MS (m - H2O + 1): 312.0; MS (m - 2H20 + 1): 294.4.
Example 18: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-3-ylmethyl)pyrrolidine-3-α-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and N-boc-piperidine- 3-carboxaldehyde (149 mg, 0.7 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-3-ylmethyl)pyrrolidine acid -3-carboxylic acid (62 mg, 29%) as a pale amber granular solid. NMR (D2O) δ 3.70-4.00 (m, 2H), 3.20-3.45 (m, 5H), 2.65-2.90 (m, 2H), 2.50-2, 65 (m, 2H), 2.15-2.30 (m, 1H), 1.75-2.00 (m, 2H), 1.50-1.75 (m, 2H), 1.10- 1.40 (m, 4H), 0.65-0.75 (m, 2H). MS (m - H2O + 1): 296, 3; MS (m - 2 H2O + 1): 278.1.
Example 19: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(quinolin-4-ylmethyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and quinoline-4-carboxaldehyde ( 110 mg, 0.7 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate acid
(3R,4S)-3-amino-4-(3-boronopropyl)-1-(quinolin-4-ylmethyl)pyrrolidine-3-carboxylic acid (49 mg, 23%) as a pale amber solid. NMR (D2O) δ 9.12 (d, J = 5.5 Hz, 1H), 8.41 (d, J = 8.5 Hz, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.10-8.20 (m, 2H), 8.00 (t, J = 8.5 Hz, 1H), 5.30 (m, 2H), 4.06 (d, J = 12 .5Hz, 1H), 3.83 (m, 2H), 3.51 (t, J=11.5Hz, 1H), 2.50-2.70 (m, 1H), 1.60 (m , 1H), 1.15-1.30 (m, 3H), 0.60-0.70 (m, 2H). MS (m - H2O + 1): 340.3; MS (m - 2H2O + 1): 322.1.
Example 20: Preparation of (3R,4S)-1-((1H-imidazol-4-yl)methyl)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and imidazole-4-carboxaldehyde ( 110 mg, 0.7 mmol), stirred for 2 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 16 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate acid
(3R,4S)-1-((1H-imidazol-4-yl)methyl)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic (38 mg, 21%) as a pale yellow granular solid . NMR (D20) 8.74 (s, 1H), 7.71 (s, 1H), 4.60-4.80 (m, 2H), 3.96 (d, J= 12.5 Hz, 1H) , 3.80 (dd, J2 = 11 Hz, J2 = 8 Hz, 1H), 3.71 (d, J = 12.5 Hz, 1H), 3.40 (t, J = 11.5 Hz, 1H) ), 2.50-2.65 (m, 1H), 1.50-1.65 (m, 1H), 1.15-1.35 (m, 3H), 0.60-0.70 (m , 2H). MS (m - H2O + 1): 279.0; MS (m - 2 H2O + 1): 261.3.
Uma solução agitada de (3R,4S)-3-acetamido-N-terc- butil-4-(3- (4,4,5,5-tetrametil-l,3,2-dioxaborolan-2- il)propil)pirrolidina-3-carboxamida (Exemplo 8, Etapa 4) (198 mg, 0,5 mmol) em 1,2-dicloroetano anidro (5 ml) foi tratada com sulfato de sódio anidro (1 g) e cbz-piperidina- 2-carboxaldeido (173 mg, 0,7 mmol), agitada por 3 h, e depois tratada com triacetoxiborohidreto de sódio (212 mg, 1,0 mmol) e ácido acético glacial (2 gotas) e agitada por 18 h. Carbonato de sódio aquoso (10%, 5 ml) foi adicionado, e a mistura agitada por poucos minutos e extraida com acetato de etila (30 ml, depois 2 x 10 ml). A solução orgânica combinada foi lavada com água e cloreto de sódio aquoso saturado (20 ml cada), seca (MgSO4) , e concentrada. 0 material bruto foi desprotegido e isolado usando o método descrito para o Exemplo 8, Etapa 5, para gerar ácido (3R,4S)-3-amino-4-(3-boronopropil)-1-(piperidin-2- ilmetil)pirrolidina-3-carboxilico (156 mg, 74%) como um sólido âmbar pálido. RNM (D2O) 3,85-4,05 (m, 2H), 3,76 (d, J = 12,5 Hz, 1H) , 3, 50-3, 65 (m, 3H) , 3,39 (m, 2H) , 2,95 (dt, = 12,5 Hz, J2 = 3 Hz, 1H) , 2,55-2,70 (m, 1H), 2,00 (m, 1H) , 1,80 (m, 2H) , 1,45-1, 65 (m, 4H) , 1,15-1,35 (m, 3H) , 0, 65-0,72 (m, 2H) . MS (m + 1): 314,1; MS (m - H2O + 1): 296,2.Example 21: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-2-ylmethyl)pyrrolidine-3-carboxylic acid A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and cbz-piperidine-2- carboxaldehyde (173 mg, 0.7 mmol), stirred for 3 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-2-ylmethyl)pyrrolidine acid -3-carboxylic acid (156 mg, 74%) as a pale amber solid. NMR (D2O) 3.85-4.05 (m, 2H), 3.76 (d, J = 12.5 Hz, 1H), 3.50-3.65 (m, 3H), 3.39 ( m, 2H), 2.95 (dt, = 12.5Hz, J2 = 3Hz, 1H), 2.55-2.70 (m, 1H), 2.00 (m, 1H), 1.80 (m, 2H), 1.45-1.65 (m, 4H), 1.15-1.35 (m, 3H), 0.65-0.72 (m, 2H). MS (m+1): 314.1; MS (m - H2O + 1): 296.2.
Example 22: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(3-(4-chlorophenyl)propyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and 3-(4-chlorophenyl) ) propionaldehyde (118 mg, 0.7 mmol), stirred for 2.5 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R, 4S)-3-amino-4-(3-boronopropyl)-1-(3-(4-chlorophenyl) acid propyl)pyrrolidine-3-carboxylic (84.3 mg, 38%) as a white powder. NMR (D2O) 7.25 (d, <J = 8 Hz, 2H), 7.13 (d, J = 8 Hz, 2H), 3.75-4.10 (m, 2H), 3.40- 3.60 (m, 1H), 3.10-3.30 (m, 3H), 2.60 (t, J = 7Hz, 2H), 2.40-2.55 (m, 1H), 1 .85-2.00 (m, 2H), 1.50-1.65 (m, 1H), 1.10-1.40 (m, 3H), 0.60-0.70 (m, 2H) . MS (m+1): 369, 1; MS (m - H2O+1) : 351.2.
Example 23: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(7H-purin-6-yl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl )pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 2-propanol (5 ml) was treated with 6-chloropurine (92 mg, 0.6 mmol) and diisopropylethylamine (0.174 ml, 1.0 mmol), heated to 80°C for 18 h, and then diluted with methylene chloride (20 ml). The mixture was filtered through Celite® and the filtrate concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(7H-purin-6-yl acid) )pyrrolidine-3-carboxylic acid (41 mg, 20%) as a pale amber solid. NMR (D2O) δ 8.20-8.45 (m, 2H), 4.20-4.60 (m, 2H), 3.50-4.00 (m, 2H), 2.60-2, 80 (m, 2H), 1.65 (m, 1H), 1.20-1.55 (m, 3H), 0.65-0.75 (m, 2H). MS (m+1)': 335.2; MS (m - H20 + 1): 317.1.
Example 24: Preparation of (3R,4S)-3-amino-1-(2-aminoethyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and N-bominoacetaldehyde (111 mg) , 0.7 mmol), stirred for 3 h, then treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and glacial acetic acid (2 drops) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-1-(2-aminoethyl)-4-(3-boronopropyl)pyrrolidine-3 acid -carboxylic (64 mg, 35%) as a yellow solid. NMR (D2O) δ 3.60-4.00 (m, 4H), 3.15-3.50 (m, 4H), 2.60 (m, 1H), 1.40-1.65 (m, 1H), 1.10-1.35 (m, 3H), 0.63-0.73 (m, 2H). MS (m+1): 260, 2; MS (m - H2O + 1): 242.3.
Example 25: Preparation of 5-((3R,4S)-3-amino-4-(3-boronopropyl)-3-carboxypyrrolidin-1-yl)nicotinic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and ethyl 5-bromonicotinate (138 mg, 0.60 mmol) in anhydrous toluene (2.5 ml) was degassed under nitrogen by 15 min, and then treated with palladium diacetate (14 mg, 0.06 mmol), rac-binap (75 mg, 0.12 mmol) and cesium carbonate (0.65 g, 2 mmol). The mixture was heated to 80°C for 18 h, cooled to room temperature, diluted with methylene chloride (20 ml), filtered through Celite® and the filtrate concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate 5-((3R,4S)-3-amino-4-(3-boronopropyl)-3-carboxypyrrolidin-1-yl acid ) nicotinic (82 mg, 40%) as a yellow solid. NMR (D2O) δ 8.37 (s, 1H), 8.00 (s, 2H), 3.99 (d, J = 11 Hz, 1H), 3.88 (t, J = 10 Hz, 1H) , 3.68 (d, J = 11 Hz, 1H), 3.26 (t, J = 10 Hz, 1H), 2.60-2.75 (m, 1H), 1.60 (m, 1H) , 1.20-1.50 (m, 3H), 0.65-0.75 (m, 2H). MS (m+1): 338.2; MS (m - H2O + 1): 320.1.
Example 26: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-4-yl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-CBZ-piperidine-4-one (0.233 g, 1.0 mmol) in anhydrous 1,2-dichloroethane (5 ml) ) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 2.5 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (265 mg, 1.25 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-4-yl)pyrrolidine acid -3-carboxylic acid (146 mg, 71%) as a pale amber solid. NMR (D2O) õ 4.02 (d, J = 13Hz, 1H), 3.90 (m, 1H), 3.77 (m, 1H), 3.45-3.65 (m, 3H), 3.36 (m, 1H), 3.00 (m, 2H), 2.50-2, 60 (m, 1H), 2.30-2.40 (m, 2H), 1.75-1. 95 (m, 2H), 1.55-1.65 (m, 1H), 1.15-1.40 (m, 3H), 0.62-0.70 (m, 2H). MS (m+1): 300.0; MS (m - H2O + 1): 282.2.
Example 27: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1,3'-bipyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-BOC-pyrrolidin-3-one (0.185 g, 1.0 mmol) in anhydrous 1,2-dichloroethane (5 ml) ) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 2.5 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (276 mg, 1.3 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1,3'-bipyrrolidine-3-carboxylic acid (160 mg, 81%) as an amber solid. NMR (D20) δ 4.25-4.35 (m, 1H), 4.00 (dd, J = 12 Hz, J2 = 5 Hz, 1H), 3.70-3.95 (m, 3H), 3.25-3.60 (m, 4H), 2.45-2.65 (m, 2H), 2.15-2.25 (m, 1H), 1.50-1.65 (m, 1H) ), 1.15-1.35 (m, 3H), 0.60-0.70 (m, 2H). MS (m+1): 286.1; MS (m - H20 + 1): 268.3.
Example 28: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-3-yl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-BOC-piperidine-3-one (0.199 g, 1.0 mmol) in anhydrous 1,2-dichloroethane (5 ml) ) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 3 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (276 mg, 1.3 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(piperidin-3-yl)pyrrolidine acid -3-carboxylic (138 mg, 68%) as an amber solid. NMR (D2O) δ 3.80-4.10 (m, 2H), 3.60-3.80 (m, 2H), 3.30-3.50 (m, 2H), 3.00-3, 15 (m, 1H), 2.80-2.95 (m, 1H), 2.45-2.60 (m, 1H), 2.30 (m, 1H), 1.95-2.10 ( m, 1H), 1.50-1.85 (m, 4H), 1.10-1.40 (m, 3H), 0.60-0.70 (m, 2H). MS (m+1): 300.1; MS (m - H20 + 1): 282.1.
Example 29: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(pyridin-2-ylmethyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and pyridine-2-carboxaldehyde (0.199 g, 1.0 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 3 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (276 mg, 1, 3 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(pyridin-2-ylmethyl)pyrrolidine acid -3-carboxylic acid (141 mg, 74%) as a white solid. NMR (D2O) δ 8.63 (m, 1H), 8.20 (m, 1H), 7.65-7.85 (m, 2H), 4.50-4.65 (m, 2H), 3 .86 (d, J = 12.5 Hz, 1H), 3.70 (m, 2H), 3.22 (t, J = 11 Hz, 1H), 2.55 (m, 1H), 1.55 (m, 1H), 1.15-1.35 (m, 3H), 0.60-0.70 (m, 2H). MS (m+1): 308.0; MS (m - H2O + 1): 290.2.
Example 30: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-carboxycyclohexyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and 4-carbethoxycyclohexanone (0.170 g, 1.0 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with sulphate. anhydrous sodium (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 3 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (276 mg, 1.3 mmol) ) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4 ), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-carboxycyclohexyl)pyrrolidine-3 acid -carboxylic (135 mg, 65%) as a white solid. NMR (D2O) δ 3.97 (dd, Ji = 13Hz, J2 = 7Hz, 1H), 3.75-3.90 (m, 2H), 3.15-3.35 (m, 2H), 2.20-2.70 (m, 2H), 1.90-2.20 (m, 4H), 1.50-1.65 (m, 3H), 1.15-1.45 (m, 5H) ), 0.60-0.70 (m, 2H). MS (m+1): 343.1; MS (m - H2O+1) : 325.0.
Example 31: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-((1-methyl-1H-imidazol-2-yl)methyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and 1-methylimidazol-2-carboxaldehyde (83 mg, 0.75 mmol) in anhydrous 1,2-dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at room temperature for 3 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (212 mg, 1 .0 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSCh), and concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-((1-methyl-1H-) acid imidazol-2-yl)methyl)pyrrolidine-3-carboxylic acid (140 mg, 67%) as a pale yellow solid. NMR (D2O) δ 7.37 (s, 2H), 4.35-4.50 (m, 2H), 3.80 (s, 3H), 3.40-3.60 (m, 3H), 2 , 75-2.85 (m, 1H), 2.43 (m, 1H), 1.48 (s, 1H), 1.15-1.30 (3H), 0.60-0.70 (m) , 2H). MS (m+1): 311.0; MS (m - H2O + 1): 293.1.
Example 32: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-methylpyridin-3-yl)pyrrolidine-3-5-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and 3-bromo-4-methylpyridine (0.258 g, 1.5 15 mmol) in anhydrous toluene (5 ml) was degassed under nitrogen for 15 min, then treated with Pd 2 dba 3 (46 mg, 0.05 mmol), rac-binap (50 mg, 0.075 mmol), and sodium t-butoxide (0.18 g, 1.87 mmol) ) . The mixture was heated to 70°C for 18 h, cooled to room temperature, diluted with methylene chloride (20 ml), filtered through Celite® and the filtrate concentrated. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to give (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-methylpyridin-3-yl acid) )pyrrolidine-3-carboxylic acid (22 mg, 12%) as a pale amber solid. NMR (D20) δ 7.99 (d, J = 6 Hz, 1H), 7.95 (s, 1H), 7.57 (d, J = 6 Hz, 1H), 3.80 (d, J = 5 Hz, 2H), 3.72 (t, J = 9.5 Hz, 1H), 3.25 (t, J = 9.5 Hz, 1H), 2.49 (s, 3H), 2.40 -2.60 (m, 1H), 1.50-1.60 (m, 1H), 1.20-1.45 (m, 3H), 0.65-0.75 (m, 30 2H). MS (m - H20 + 1) : 290.0; MS (m - 2 H20 + 1) : 271, 9.
Example 33: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(piperidin-1-yl)ethyl)pyrrolidine-3-carboxylic acid
A cooled (0°C) solution of N-(2-hydroxyethyl)piperidine (90.5 mg, 0.70 mmol) and diisopropylethylamine (0.30 ml, 1.7 mmol) in anhydrous acetonitrile (12 ml) under nitrogen was treated with methanesulfonyl chloride (80.2 mg, 0.70 mmol), stirred 2 h, and treated with (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4 ,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and heated to 60°C for 15 H. The reaction mixture was concentrated, deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(piperidine) acid -1-yl)ethyl)pyrrolidine-3-carboxylic acid (140 mg, 64%) as a colorless glassy solid. NMR (D2O) δ 3.85-4.05 (m, 2H), 3.65-3.80 (m, 3H), 3.35-3.55 (m, 5H), 2.85-3, 00 (m, 2H), 2.60 (m, 1H), 1.80-1.95 (m, 2H), 1.55-1.75 (m, 4H), 1.10-1.50 ( m, 4H), 0.63-0.73 (m, 2H). MS (m+1): 328.3; MS (m - H2O+1) : 310.0.
Example 34: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(diethylamino)ethyl)pyrrolidine-3-carboxylic acid
A cooled (0°C) solution of N,N-diethylethanolamine (82 mg, 0.70 mmol) and diisopropylethylamine (0.30 ml, 1.7 mmol) in anhydrous acetonitrile (12 ml) under nitrogen was treated with hydrogen chloride. methanesulfonyl (80.2 mg, 0.70 mmol), stirred 2.5 h, and treated with (3R,4S)-3-acetamido-N- tert-butyl-4-(3-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and heated to 60 °C for 15 h. The reaction mixture was concentrated, deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(diethylamino) acid )ethyl)pyrrolidine-3-carboxylic acid (120 mg, 57%) as a white foam. NMR (D2O) δ 3.97 (d, J = 12.5 Hz, 1H), 3.87 (m, 1H), 3.65-3.80 (m, 3H), 3.35-3.55 (m, 3H), 3.19 (q, J=7.5Hz, 4H), 2.55-2.65 (m, 1H), 1.50-1.65 (m, 1H), 1. 20 (t, J=7.5Hz, 6H), 1.15-1.35 (m, 3H), 0.63-0.73 (m, 2H). MS (m - H2O + 1): 298.3; MS (m - 2 H20 + 1): 280.1.
Example 35: Preparation of (3R,4S)-4-(3-boronopropyl)-3-(methylamino)pyrrolidine-3-carboxylic acid
(3R,4S)-4-(3-boronopropyl)-3-(methylamino)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 8, except that methylamine and acetic acid were used in place of ammonium acetate in Step 2. LC-MS ESI” MS found for CgH19BN2O4 m/z 230, 1: (m + 1) : 231.1; MS (m - H20 + 1): 213.1.
Example 36: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-((1-methylpiperidin-2-yl)methyl)pyrrolidine-3-carboxylic acid
A cooled (0°C) solution of 1-methylpiperidine-2-methanol (97 mg, 0.75 mmol) and diisopropylethylamine (0.31 ml, 1.75 mmol) in anhydrous acetonitrile (12 ml) under nitrogen was treated with methanesulfonyl chloride (86 mg, 0.75 mmol), stirred 3 h, and treated with (3R,4S)-3-acetamido-N- tert-butyl-4-(3-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and heated to 60 °C for 15 h. The reaction mixture was concentrated, deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-((1-methylpiperidine) acid -2-yl)methyl)pyrrolidine-3-carboxylic acid (121 mg, 55%) as a white foam. NMR (D2O) δ 3.60-4.00 (m, 4H), 3.15-3.55 (m, 3H), 2.90 (m, 2H), 2.67 (s, 3H), 2 .50-2.80 (m, 2H), 2.00-2.30 (m, 1H), 1.45-1.95 (m, 5H), 1.10-1.40 (m, 3H) , 0.63-0.73 (m, 2H). MS (m+1): 328.1; MS (m - H20 + 1): 310.1.
Example 37: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(pyrrolidin-2-ylmethyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-BOC-piperidine-2-carboxaldehyde (0.15 g, 0.75 mmol) in anhydrous 1,2-dichloroethane ( 5 ml) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at room temperature for 3 h, then cooled to room temperature and treated with sodium triacetoxyborohydride ( 212 mg, 1.0 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4), and concentrated in vacuo. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(pyrrolidin-2-ylmethyl)pyrrolidine acid -3-carboxylic acid (144 mg, 70%) as a white foam. NMR (D2O) δ 3.85-4.05 (m, 3H), 3.60-3.80 (m, 3H), 3.40 (m, 1H), 3.30 (m, 2H), 2 1.55-2.70 (m, 1H), 2.20-2.35 (m, 1H), 1.85-2.10 (m, 2H), 1.75 (m, 1H), 1.60 (m, 1H), 1.15-1.40 (m, 3H), 0.63-0.73 (m, 2H). MS (m+1): 300.1; MS (m - H2O + 1): 282.1.
Example 38: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(pyrrolidin-1-yl)ethyl)pyrrolidine-3-carboxylic acid
A cooled (0°C) solution of N-(2-hydroxyethyl)pyrrolidine (92 mg, 0.80 mmol) and diisopropylethylamine (0.32 ml, 1.8 mmol) in anhydrous acetonitrile (12 ml) under nitrogen was treated with methanesulfonyl chloride (92 mg, 0.80 mmol), stirred 3 h, and treated with (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5 - tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and heated to 60°C for 15.h. The reaction mixture was concentrated, deprotected e. isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(pyrrolidin-1-yl)ethyl)pyrrolidine-acid 3-carboxylic (127 mg, 60%) as a white foam. NMR (D∑O) δ 3.80-4.00 (m, 2H), 3.50-3.80 (m, 4H), 3.40 (t,'J' = 11Hz, 1H), 3 2.05 (m, 2H), 2.55-2.75 (m, 4H), 2.05 (m, 2H), 1.90 (m, 2H), 1.60 (m, 1H), 1. 10-1.40 (m, 3H), 0.63-0.73 (m, 2H). MS (m+1): 314.0; MS (m - H20 + 1): 296.2.
Example 39: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl)pyrrolidine-acid 3-carboxylic
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-BOC-tetrahydroisoquinoline-3-carboxaldehyde (0.196 g, 0.75 mmol) in anhydrous 1,2-dichloroethane (5 ml) ) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at room temperature for 3 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (212 mg , 1.0 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO 4 ), and concentrated in vacuo. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(((S)-1, 2,3,4-tetrahydroisoquinolin-3-yl)methyl)pyrrolidine-3-carboxylic acid (146 mg, 62%) as a white solid. NMR (D2O) δ 7.10-7.30 (m, 4H), 4.39 (br s, 2H), 3.85-4.10 (m, 3H), 3.60-3.80 (m , 3H), 3.20-3.50 (m, 2H), 2.90-3.10 (m, 1H), 2.62 (m, 1H), 1.60 (m, 1H), 1. 15-1.30 (m, 3H), 0.63-0.73 (m, 2H). MS (m+1): 362.4; MS (m - H2O + 1): '344.0.
Example 40: Preparation of (3R,4S)-3-amino-1-(2-(benzylamino)ethyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-benzyl-N-BOC-glycinaldehyde (0.187 g, 0.75 mmol) in anhydrous 1,2-dichloroethane (5 ml) ) was stirred at room temperature for 30 min, then cooled in an ice bath and treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and stirred for 3 h at room temperature. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4 , and concentrated in vacuo.) The crude material was deprotected and isolated using the method described for Example 8, Step 5, to give acid (3R, 4S)-3-amino-1-(2-(benzylamino)ethyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (112 mg, 49%) as a white solid. NMR (D2O) δ 7.30 -7.45 (m, 5H), 4.21 (s, 2H), 3.94 (d, 1H, J=12Hz, 1H), 3.84 (t, J=9.5Hz, 1H ), 3.60-3.75 (m, 3H), 3.30-3.50 (m, 3H), 2.50-2.65 (m, 1H), 1.50-1.65 (m , 1H), 1.10-1.35 (m, 3H), 0.63-0.73 (m, 2H) MS (m+1): 350.4; MS (m - H20 + 1): 332.1.
Example 41: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(3,4-dichlorobenzylamino)ethyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and N-(3,4-dichlorobenzyl)-N-BOC-glycinaldehyde (0.240 g, 0.75 mmol) in 1.2 -anhydrous dichloroethane (5 ml) was stirred at room temperature for 30 min, then cooled in an ice bath and treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and stirred for 3 h at room temperature. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4), and concentrated in vacuo. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(3,4-) acid dichlorobenzylamino)ethyl)pyrrolidine-3-carboxylic acid (111 mg, 42%) as a white solid. NMR (D2O) δ 7.50-7.60 (m, 2H), 7.28 (dd, J = 8.5 Hz, J2 = 2 Hz, 1H), 4.20 (s, 2H), 3, 94 (d, J=12Hz, 1H), 3.84 (dd, J=11.5Hz, J2=8Hz, 1H), 3.60-3.75 (m, 3H), 3.30- 3.45 (m, 3H), 2.50-2.65 (m, 1H), 1.50-1.65 (m, 1H), 1.10-1.35 (m, 3H), 0. 62-0.72 (m, 2H). MS (m+1): 417.9; MS (m - H2O+1) : 400.3.
Example 42: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-chlorophenylcarbamoyl)pyrrolidine-3-carboxylic acid
(3R,4S)-3-Amino-4-(3-boronopropyl)-1-(4-chlorophenylcarbamoyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 46, except that 1-chloro-4- isocyanatobenzene was used as the acylating agent in Step 4 . LC-MS ESI" MS found for C15H21BCIN3O5 m/z 396, 2: (m+1): 397.1; MS (m - H2O+1): 379.0.
Example 43: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-3-carboxylic acid
(3R,4S)-3-Amino-4-(3-boronopropyl)-1-((S)-pyrrolidine-2-carbonyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 46, except that (S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid was used as the acylating agent in Step 4. LC-MS ESI" MS found for C13H24BN3O5 m/z 313.2: (m+1): 314.1; MS (m - H2O + 1): 296.0.
Example 44: Preparation of (3R,4S)-3-amino-1-(2-aminocyclohexyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.5 mmol) and 2-(N-BOC-amino)cyclohexane-1-one (0.213 g, 1.0 mmol) in 1,2- Anhydrous dichloroethane (5 ml) was treated with anhydrous sodium sulfate (1 g) and glacial acetic acid (30 mg, 0.5 mmol), stirred at 40°C for 1 h, then cooled to room temperature and treated with sodium triacetoxyborohydride (276 mg, 1.3 mmol) and stirred for 18 h. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4), and concentrated in vacuo. The crude material was deprotected and isolated using the method described for Example 8, Step 5, to generate (3R,4S)-3-amino-1-(2-aminocyclohexyl)-4-(3-boronopropyl)pyrrolidine-3 acid -carboxylic (156 mg, 74%) as a white powder. NMR (D2O) δ 3.65-4.05 (m, 3H), 3.05-3.60 (m, 3H), 2.90-3.10 (m, 1H), 1.95-2, 10 (m, 2H), 1.45-1.75 (m, 5H), 1.15-1.40 (m, 5H), 0.62-0.72 (m, 2H). MS (m+1): 314.1; MS (m - H2O + 1): 296.1.
Example 45: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(4-chlorophenyl)acetyl)pyrrolidine-3-carboxylic acid
(3R,4S)-3-Amino-4-(3-boronopropyl)-1-(2-(4-chlorophenyl)acetyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 46, except that acid 2-(4-chlorophenyl)acetic was used as the acylating agent in Step 4. LC-MS ESI" MS found for C16H22BCIN2O5 m/z 368.1: (m+1): 369.1; MS (m - H2O + 1): 352.1.
Example 46: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-fluorobenzoyl)pyrrolidine-3-carboxylic acid
Step 1: tert-Butyl 4-allyl-3-[(2-nitrophenyl)carbamoyl]-3-[(trifluoroacetyl)amino]-pyrrolidine-1-carboxylate While under nitrogen, a stirred mixture of tert-butyl 3-allyl- 4-oxopyrrolidine-1-carboxylate (600 mg, 2.66 mmol), ammonium trifluoroacetate (698 mg, 5.33 mmol) and 2-nitrophenyl isocyanide (690 mg, 4.6 mmol) in 2.2.2 - trifluorethanol (2.7 ml) was placed in an oil bath at 60°C and stirred overnight. After cooling to room temperature, the mixture was diluted with ethyl acetate (40 ml), washed with water (3 x 20 ml) and the combined aqueous phase was extracted again with ethyl acetate (20 ml). The combined organic phase was washed with saturated aqueous sodium chloride (20 ml), dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel chromatography (90 g column, 0-5% ethyl acetate in methylene chloride) gave tert-butyl 4-allyl-3-[(2-nitrophenyl)carbamoyl]-3-[(trifluoroacetyl) )amino]-pyrrolidine-1-carboxylate (665 mg, 51%, 3:2 mixture of diastereomers) as an amber gum. LC-MS ESI+ MS found for C21H25F3N4O6 m/z 5Q9rQ (M + Na). LC-MS ESI" MS found for C21H25F3N4O6 m/z 485.1 (M - H).
Step 2: tert-Butyl 4-allyl-3-(IH-benzotriazol-1-ylcarbonyl)-3-[(trifluoroacetyl)amino]-pyrrolidine-1-carboxylate (trans, racemic) A solution of tert-butyl 4-allyl -3-[(2-nitrophenyl)carbamoyl]-3-[(trifluoroacetyl)amino]pyrrolidine-1-carboxylate (0.816 g, 1.68 mmol) in methanol (30 ml) was treated with ammonium chloride (0.897 g, 16.8 mmol) and zinc (2.19 g, 33.5 mmol). After stirring at room temperature for 40 min, the mixture was diluted with ethyl acetate (30 ml) and filtered through a pad of Celite. The block was washed with ethyl acetate and the filtrate was concentrated under reduced pressure. The residue was further diluted with ethyl acetate (25 ml) and water (20 ml), and the layers were separated. The organic phase was washed with water (10 ml) and saturated aqueous sodium chloride (10 ml), dried over Na 2 SO 4 and concentrated and dried under high vacuum for approximately 2 h to give the intermediate tert-butyl 4-allyl-3-[ (2-aminophenyl)carbamoyl]-3-[(trifluoroacetyl)amino]pyrrolidine-1-carboxylate (760 mg, 3:2 mixture of diastereomers) as an off-white foam which was used without further purification. While under nitrogen, the crude intermediate was dissolved in chloroform (30 ml) and treated with isoamyl nitrite (0.78 ml, 5.6 mmol). The homogeneous mixture was stirred at room temperature for 3 h, concentrated under reduced pressure and purified by silica gel chromatography (90 g column, 2.5-7% ethyl acetate in methylene chloride) to give tert-butyl 4 -allyl-3-(1H-benzotriazol-1-ylcarbonyl)-3-[(trifluoroacetyl)amino]-pyrrolidine-1-carboxylate (347 mg, 44%, 95:5 trans/cis) as an off-white foam. For the intermediate mixture of anilines (mixture of diastereomers), LC-MS ESI” MS found for C21H27F3N4O4 m/z 455.2 (M - H) . For benzotriazole (isolated as the diastereomer shown), LC-MS ESI+ MS found for C21H24F3N5O4 m/z 490. 0 (M + Na). 1H-NMR (CDCl3, 400 MHz) δ 8.30 (m, 1H), 8.17 (m, 1H), 7.77 (m, 1H), 7.60 (m, 1H), 5.45 ( m, 1H), 4.90 (m, 2H), 4.60-4.15 (m, 2H), 3.95-3.60 (m, 2H), 3.19 (m, 1H), 2 .10 (m, 2H), 1.53 (s, 9H) (rotamers present).
Step 3: 1-tert-Butyl 3-methyl (3R, 4S)-4-allyl-3-[(trifluoroacetyl)amino]pyrrolidine-1,3-dicarboxylate (racemic) A solution of tert-butyl 4-allyl-3 -1H-benzotriazol-1-ylcarbonyl)-3-[(trifluoroacetyl)amino]pyrrolidine-1-carboxylate (340 mg, 0.727 mmol, 95:5 trans/cis) in methylene chloride (6 ml) and methanol ( 3 ml) was treated with Et3N (0.0203 ml, 0.145 mmol) and stirred at room temperature for 45 min. The mixture was concentrated under reduced pressure and purified by silica gel chromatography (40 g column, 2-5% ethyl acetate/methylene chloride) to give 1-tert-butyl 3-methyl (3R,4S)-4 -allyl-3-[(trifluoroacetyl)amino]pyrrolidine-1,3-dicarboxylate (racemic) (208 mg, 75%) as a partially crystalline film. LC-MS ESI' MS found for C16H23F3N2O5 m/z 379.1 (M - H). 1H-NMR (CDCl3, 400 MHz) δ 7.20 (m, 1H), 5.73 (m, 1H), 5.13 (m, 2H), 4.02 (d, J=11.7Hz, 1H), 3.88 (s, 3H), 3.80 (m, 2H), 3.25-2.85 (m, 2H), 2.18-1.93 (m, 2H), 1.49 (s, 9H) (rotamers present).
Step 4: Methyl (3R,4S)-4-allyl-1-(4-fluorobenzoyl)-3-[(trifluoroacetyl)amino]-pyrrolidine-3-carboxylate (racemic) ■ While under nitrogen, a 1-tert solution -butyl 3-methyl (3R, 4S)-4-allyl-3-[(trifluoroacetyl)amino] pyrrolidine-1,3-dicarboxylate racemic (Step 3, 0.207 g, 0.544 mmol) in methylene chloride (11 ml) was treated with trifluoroacetic acid (838 µl, 10.9 mmol). After stirring for 2 h, the mixture was concentrated and dried under high vacuum overnight to give methyl (3R, 4S)-4-allyl-3-[(trifluoroacetyl)amino]pyrrolidine-3-carboxylate trifluoroacetate as a light yellow foam which was used without further purification. A stirred solution of this intermediate in dry methylene chloride (11 ml) under nitrogen was treated with EtsN (0.341 ml, 2.45 mmol), followed by 4-fluorobenzoyl chloride (0.0979 ml, 0.816 mmol). After stirring at room temperature for 1.5. h, the solution was diluted with methylene chloride (15 ml), washed successively with water (10 ml), saturated aqueous NaHCOb (10 ml) and saturated aqueous sodium chloride (5 ml). The resulting organic phase was dried over Na 2 SO 4 and concentrated, and purification by radial chromatography (2000 micron silica gel rotor, 40-75% ethyl acetate in hexanes) gave methyl (3R,4S)-4-allyl- 1-(4-fluorobenzoyl)-3-[(trifluoroacetyl)amino]-pyrrolidine-3-carboxylate (racemic) (220 mg, 100%) as a light yellow film. Rf = 0.33 (50% ethyl acetate in hexanes). For intermediate pyrrolidine trifluoracetate, LC-MS ESI+ MS found for C11H15F3N2O3 m/z 281.1 (M + H) . For the amide product, LC-MS ESI' MS found for C18H18F4N2O4 m/z 401.1 (M - H).
Step 5: Methyl (3R,4S)-1-(4-fluorobenzoyl)-4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl] 3- [(trifluoroacetyl)amino]pyrrolidine-3-carboxylate (racemic) Methyl (3R,4S)-1-(4-fluorobenzoyl)-4-[3 - (4,4,5,5-tetramethyl-1,3,2 -dioxaborolan-2-yl)propyl]-3-[(trifluoroacetyl)amino]pyrrolidine-3-carboxylate (racemic) is prepared analogously to that given in Example 1, Step 3, except that methyl (3R,4S)-4 -allyl-1-(4-fluorobenzoyl)-3-[(trifluoroacetyl)amino]-pyrrolidine-3-carboxylate (racemic) is used as the substrate. ESI+ MS found for C24H31BF4N2O6 m/z 531.2 (M + H). Rf = 0.28 (50% ethyl acetate in hexanes).
Uma solução agitada de metil (3R,4S)-l-(4- fluorbenzoil)—4—[3—(4,4,5,5-tetrametil-l,3,2-dioxaborolan- 2-il)propil]-3-[(trifluoracetil)amino]pirrolidina-3- carboxilato racêmico (200 mg, 0,377 mmol) em THF (4,2 ml) e água (3,7 ml) foi borrifada com nitrogênio por 5 min. Monoidrato de hidróxido de litio (33,2 mg, 0,792 mmol) foi adicionado, e a mistura foi borrifada com nitrogênio por 10 min e agitada sob nitrogênio em temperatura ambiente por 4 dias, quando então monoidrato de hidróxido de litio 5 adicional (39,6 mg, 0,943 mmol) foi adicionado. A mistura foi diluida com água (5 ml) e acidificada até o pH < 1 com 3 M de HC1 aquoso. A solução resultante foi lavada com acetato de etila (2 x 20 ml) e cloreto de metileno (2 x 20 ml) . A fase aquosa foi concentrada sob pressão reduzida e 10 purificada por HPLC de fase reversa. Frações de produto foram reunidas em pool, concentradas, dissolvidas novamente em 1 M de HC1 aquoso, concentradas e liofilizadas para gerar cloridrato de ácido (3R,4S)-3-amino-4-[3- (diidroxiboril)propil]-1-(4-fluorbenzoil)pirrolidina-3- carboxilico racêmico (42 mg, 30%) como um sólido amorfo branco. MS (ESI + ) m/z 321 (M-H2O + H+) , 303 (M - 2H2O + H +) e MS (ESI”) m/z 319 (M - H2O - H+) .Step 6: (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-(4-fluorobenzoyl)pyrrolidine-3-carboxylic acid, racemic hydrochloride) A stirred solution of methyl (3R,4S)-1-(4-fluorobenzoyl)-4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]- Racemic 3-[(trifluoroacetyl)amino]pyrrolidine-3-carboxylate (200 mg, 0.377 mmol) in THF (4.2 ml) and water (3.7 ml) was sparged with nitrogen for 5 min. Lithium hydroxide monohydrate (33.2 mg, 0.792 mmol) was added, and the mixture was sparged with nitrogen for 10 min and stirred under nitrogen at room temperature for 4 days, then additional lithium hydroxide monohydrate (39, 6mg, 0.943mmol) was added. The mixture was diluted with water (5 ml) and acidified to pH < 1 with 3 M aqueous HCl. The resulting solution was washed with ethyl acetate (2 x 20 ml) and methylene chloride (2 x 20 ml). The aqueous phase was concentrated under reduced pressure and purified by reverse phase HPLC. Product fractions were pooled, concentrated, re-dissolved in 1 M aqueous HCl, concentrated and lyophilized to give (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-acid hydrochloride Racemic (4-fluorobenzoyl)pyrrolidine-3-carboxylic acid (42 mg, 30%) as a white amorphous solid. MS (ESI+) m/z 321 (M-H2O + H+), 303 (M - 2H2O + H+) and MS (ESI') m/z 319 (M - H2O - H+).
Ácido (3R,4S)-3-amino-4-(3-boronopropil)-1-(4- metoxibenzoil)pirrolidina-3-carboxilico foi preparado de forma análoga àquela apresentada no Exemplo 46, cloreto de metoxibenzoila foi usado como o agente de acilação na Etapa 4 . Exemplo 48: Preparação de ácido (3R,4S)-3-amino-4-(3- boronopropil)-1-(4-fluorfenilcarbamoil)pirrolidina-3- carboxilico Example 47: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-methoxybenzoyl)pyrrolidine-3-carboxylic acid (3R,4S)-3-Amino-4-(3-boronopropyl)-1-(4-methoxybenzoyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 46, methoxybenzoyl chloride was used as the agent of acylation in Step 4 . Example 48: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(4-fluorophenylcarbamoyl)pyrrolidine-3-carboxylic acid
Stage. 1: (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid dihydrochloride(racemate) tert-Butyl(3R,4S)-3-acetamido-3-(tert -butyl carbamoyl) —4—[3—(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-1-carboxylate (racemic) (1.60 g, 3 .23 mmol) was placed in an Ace pressure tube, carefully treated with concentrated HCl (22 ml) and warmed to -120°C. After 16 h the mixture was cooled to room temperature, added slowly to ice water (45 ml) and washed with methylene chloride (3 x 40 ml). The aqueous phase. was concentrated and redissolved in deionized water and lyophilized to give racemic (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid dihydrochloride as an off-white solid. The solid could be azeotroped by toluene several times to remove additional water. 1H- NMR (D20, 400 MHz) δ 3.87 (d J = 12.8 Hz, 1H), 3.72 (dd, J = 11.7, 8.5 Hz, 1H), 3.44 (d , J= 12.8 Hz, 1H), 3.23 (br t, J= 11.7 Hz, 1H), 2.53 (m, 1H), 1.60 (m, 1H), 1.31 ( m, 2H), 1.19 (m, 1H), 0.68 (m, 2H). ESI+ MS found for C8H17BN2O4 m/z 199.1 (M - 18 + H), 181.8 (M - 36 + H); ESI" MS found for C8H17BN2O4 m/z 197.3 (M - 18 - H).
Enquanto sob nitrogênio, uma mistura agitada de dicloridrato de ácido (3R,4S)-3-amino-4-[3- (diidroxiboril)propil]pirrolidina-3-carboxilico (0,225 g, 0,623 mmol) em DMF seca (9 ml) foi tratada com Et3N (0,521 ml, 3,74 mmol) e 4-fluorfenil isocianato (92,1 μl, 0,810 mmol) . Após agitação por 1,5 h, a mistura de reação foi diluida com água (5 ml) e acidificada até o pH de aproximadamente 1 com 3 M de HC1 aquoso (5 ml) . A fase aquosa foi lavada com cloreto de metileno (2 x 25 ml) e acetato de etila (25 ml), concentrada e purificada por HPLC de fase reversa. Frações de produto foram reunidas em pool, concentradas, dissolvidas novamente em 1 M de HC1 aquoso, concentradas e liofilizadas para gerar cloridrato de ácido (3R,4S)-3-amino-4-[3-(diidroxiboril)propil]-1-[ (4- fluorfenil)carbamoil]pirrolidina-3-carboxilico (racemato) (65 mg, 27%) como um sólido amorfo branco. MS (ÉSI + ) m/z 336 (M- H2O + H+) , 318 (M - 2H20 + H+) e MS (ESI-) m/z 334 (M - H20 + H+) .Step 2: (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-f(4-fluorophenyl)carbamoyl]pyrrolidine-3-carboxylic acid hydrochloride (racemate) While under nitrogen, a stirred mixture of (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid dihydrochloride (0.225 g, 0.623 mmol) in dry DMF (9 ml) was treated with Et3N (0.521 ml, 3.74 mmol) and 4-fluorophenyl isocyanate (92.1 µl, 0.810 mmol). After stirring for 1.5 h, the reaction mixture was diluted with water (5 ml) and acidified to pH approximately 1 with 3 M aqueous HCl (5 ml). The aqueous phase was washed with methylene chloride (2 x 25 ml) and ethyl acetate (25 ml), concentrated and purified by reverse phase HPLC. Product fractions were pooled, concentrated, re-dissolved in 1 M aqueous HCl, concentrated and lyophilized to give (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-acid hydrochloride [(4-fluorophenyl)carbamoyl]pyrrolidine-3-carboxylic acid (racemate) (65 mg, 27%) as a white amorphous solid. MS (ESI+) m/z 336 (M-H2O + H+), 318 (M - 2H20 + H+) and MS (ESI-) m/z 334 (M - H20 + H+).
Etapa 1: Metil 2-amino-3- (4-clorofenil)propanoato cloridrato Enquanto sob nitrogênio, uma suspensão agitada de 4- clorofenilalanina (2,50 g, 12,5 mmol) em metanol anidro (18 ml) foi resfriada em um banho de água gelada e cuidadosamente tratada com cloreto de tionila (1,00 ml, 13,8 mmol). Após agitação por 10 min, e o banho de resfriamento foi removido e foi permitido que a mistura se aquecesse até a temperatura ambiente. Um condensador de refluxo foi anexado, e a borra foi aquecida até 55° C. Após agitação de um dia para o outro, a mistura foi resfriada até a temperatura ambiente, concentrada e seca sob pressão reduzida para gerar metil 2-amino-3-(4- clorofenil)propanoato cloridrato (3,13 g, 99%) como um sólido branco. ESI+ MS encontrado para C10H12CINO2 m/z 214,0 (M + H) . Example 49: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-((7-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl)pyrrolidine-acid 3-carboxylic (mixture of two diastereomers; each racemate) Step 1: Methyl 2-amino-3-(4-chlorophenyl)propanoate hydrochloride While under nitrogen, a stirred suspension of 4-chlorophenylalanine (2.50 g, 12.5 mmol) in anhydrous methanol (18 ml) was cooled in a ice-water bath and carefully treated with thionyl chloride (1.00 ml, 13.8 mmol). After stirring for 10 min, the cooling bath was removed and the mixture was allowed to warm to room temperature. A reflux condenser was attached, and the sludge was heated to 55°C. After stirring overnight, the mixture was cooled to room temperature, concentrated and dried under reduced pressure to give methyl 2-amino-3- (4-chlorophenyl)propanoate hydrochloride (3.13 g, 99%) as a white solid. ESI+ MS found for C10H12CINO2 m/z 214.0 (M+H).
Step 2: Methyl 3-(4-chlorophenyl)-2-(ethoxycarbonylamino) propanoate While under nitrogen, a stirred mixture of methyl 2-amino-3-(4-chlorophenyl)propanoate hydrochloride (3.13 g, 12.5 mmol ) in methylene chloride (42 ml) was cooled in an ice-water bath and carefully treated with pyridine (2.23 ml, 27.5 mmol) and ethyl chloroformate (1.27 ml, 13.3 mmol). After stirring for 1 h, the solution was diluted with ethyl acetate (50 ml) and water (50 ml), and the layers were separated. The aqueous phase was extracted again with ethyl acetate (2 x 25 ml), and the combined organic phase was washed with saturated aqueous sodium chloride (25 ml), dried over Na 2 SO 4 and concentrated under reduced pressure to give methyl 3-(4- -chlorophenyl)-2-(ethoxycarbonylamino)propanoate (3.56 g, 99%) as a white solid. 1H-NMR (CDCl3, 400 MHz) δ 7.28 (m, 2H), 7.08 (m, 2H), 5.12 (m, 1H), 4.65 (m, 1H), 4.13 ( q, J = 8 Hz, 2H), 3.74 (s, 3H), 3.10 (qd, J = 16, 4.0 Hz, 2H), 1.25 (t, J = 8 Hz, 3H) . LC-MS generates ESI+ MS found for C13H16CINO4 m/z 308.0 (M + Na), 286.0 (M + H).
Step 3: 2-Ethyl 3-methyl 7-chloro-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate A mixture of methyl 3-(4-chlorophenyl)-2-(ethoxycarbonylamino)propanoate (3.55 g , 12.4 mmol) in acetic acid (12 ml) and sulfuric acid (4 ml) was treated with paraformaldehyde (0.392 g, 13.0 mmol). After stirring at room temperature overnight, the mixture was added to ice (50-60 g), diluted with water (30 ml) and extracted with ethyl acetate (3 x approximately 50 ml). The combined organic phase was washed with saturated aqueous sodium chloride (25 ml), dried over MgSO4 and concentrated. Purification by silica gel chromatography (90 g column, 25-50% ethyl acetate in hexanes) gave 2-ethyl 3-methyl 7-chloro-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate ( 2.19 g, 59%) as a clear viscous oil. 1H-NMR (CDCl3, 400 MHz) δ 7.12 (m, 3H), 5.20 (m, approx. 0.6H), 4.98 (m, approx. 0.4H), 4.76 (m, approx. 1H), 4.53 (m, 1H), 4.25 (m, 2H), 3.65 (s, 3H), 3.20 (m, 2H), 1.35 (t, J = 7 Hz, approximately 1.8H), 1.28 (t, J = 7 Hz, approximately 1.2H) (mixture of rotamers). ESI+ MS found for C14H16CINO4 m/z 2.98.0 (M+H, weak).
Step 4: 7-Chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride A mixture of 2-ethyl 3-methyl 7-chloro-3,4-dihydroisoquinoline-2,3(1H)-dicarboxylate (2.19 g, 7.36 mmol) in 6 M aqueous HCl (30 ml) was heated to 100-105°C for 2 days. The resulting mixture was concentrated under reduced pressure and triturated with toluene (2 x 100 ml). After removing the supernatant, the solid residue was dried under high vacuum to give 7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride (1.71 g, 94%) as a light yellow solid. which was used in the next step without further purification. 1H-NMR (DMSO-d 4 , 400 MHz) δ 14.2 (bs, approximately 1H), 10.0 (bs, approximately 2H), 7.41 (s, 1H), 7.32 (m, 2H) , 4.40 (m, 1H), 4.32 (m, 2H), 3.32 (dd, J=17, 4.9 Hz, 1H), 3.10 (dd, J=17, 11 Hz) . LC-MS generates ESI+ MS found for CIOHIOC1N02 m/z 212.1 (M + H).
Step 5: 2-(tert-Butoxycarbonyl)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid A solution of 7-chloro-1,2,3,4-tetrahydroisoquinoline-3 acid hydrochloride -carboxylic acid (1.70 g, 6.85 mmol) in 1,4-dioxane (11 ml) and 1 M aqueous NaOH (22.3 ml, 22.3 mmol) was cooled in an ice-water bath and treated with a second solution of di-tert-butyldicarbonate (1.87 g, 8.56 mmol) in 1,4-dioxane (11 ml). After 15 min the cooling bath was removed and stirring continued for 2 h. The resulting mixture was diluted with water (25 ml) and washed with ethyl acetate (50 ml). The aqueous phase was adjusted to pH approximately 3 with 1 M citric acid and extracted with ethyl acetate (2 x 75 ml). The combined organic phase was washed with saturated aqueous sodium chloride (50 ml), dried over MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography (90 g column, 25-75% ethyl acetate in hexanes) to give 2-(tert-butoxycarbonyl)-7-chloro-1,2,3,4-tetrahydroisoquinoline acid -3-carboxylic (1.61 g, 79%) as a white solid. 1H-NMR (DMSO-dg, 400 MHz) δ 12.8 (s, 1H), 7.34 (m, 1H), 7.24 (m, 2H), 4.88 (m, approximately 0.5H) , 4.67 (m, approximately 0.5H), 4.50 (m, 2H), 3.10 (m, 2H), 1.46 (s, approximately 4.5H), 1.40 (s, approximately 4.5H) (mixture of rotamers) . LC-MS generates ESI" MS found for CI5HI8ClNO4 m/z 310.1 (M - H).
Step 6: tert-Butyl 7-chloro-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate While under nitrogen, a flame-dried flask was charged with 2-(tert-butoxycarbonyl)- acid 7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (400 mg, 1.28 mmol) and anhydrous THE (2.5 ml), cooled in an ice bath of saturated aqueous sodium chloride and carefully treated with a solution of borane in THE (1M, 2.63 ml, 2.63 mmol). The resulting mixture was stirred at 0°C for approximately 1.5 h and then at room temperature overnight. The mixture was then cooled in an ice-water bath, slowly quenched by dropwise addition of water until most gas evolution had stopped, diluted with additional water (15 ml) and extracted with ethyl acetate (2 x 25 ml ). The combined organic phase was washed with saturated aqueous NaHCO3 (10 ml), water (10 ml) and saturated aqueous sodium chloride (10 ml), dried over Na2 SO4 and concentrated under reduced pressure. Purification by silica gel chromatography (40 g column, 10-20% ethyl acetate in methylene chloride) gave tert-butyl 7-chloro-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H) - carboxylate (292 mg, 76%) as a clear gum. 1H-NMR (DMSO-dg, 400 MHz) δ 7.29 (m, 1H), 7.20 (m, 2H), 4.81 (m, 1H), 4.64 (d, J = 17 Hz, 1H), 4.20 (bm, 2H), 3.30 (m, 1H), 3.12 (m, 1H), 2.84 (m, 2H), 1.43 (s, 9H).
Step 7: tert-Butyl 7-chloro-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate While under nitrogen, a stirred solution of tert-butyl 7-chloro-3-(hydroxymethyl)-3,4 -dihydroisoquinoline-2(1H)-carboxylate (Step 6, 270 mg, 0.907 mmol) in methylene chloride (11 ml) was cooled in an ice-water bath and treated dropwise with a Dess-Martin periodinane solution ( 461 mg, 1.09 mmol) in methylene chloride (3 ml) over several minutes. The cooling bath was removed, and the resulting clear mixture was stirred at room temperature for 1.5 h. Once complete, the mixture was cooled again in an ice-water bath, quenched in portions with a 1:1 mixture of saturated aqueous Na2S2O3 and saturated aqueous NaHCOs (20 mL total) and stirred for 10 min at room temperature. The layers were separated, the aqueous phase was extracted again with methylene chloride (20 ml) and the combined organic phase was washed with saturated aqueous sodium chloride (10 ml), dried over Na 2 SO 4 and concentrated under reduced pressure. Purification by silica gel chromatography (40 g column, 10-30% ethyl acetate in hexanes) gave tert-butyl 7-chloro-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (162 mg, 60%) as a clear gum. 1H-NMR (DMSO-de, 400 MHz) δ 9.45 (bs, 1H), 7.34 (m, 1H), 7.26 (m, 2H), 4.78 (m, approximately 0.5H) , 4.64 (m, approximately 0.5H), 4.50 (m, 2H), 3.21 (m, 1H), 3.05 (m, 1H), 1.46 (s, approximately 4.5H ), 1.38 (s, approximately 4.5H) (mixture of rotamers).
Step 8: (3R,4S)-3-Acetamido-N-tert-butyl-4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine -3-carboxamide hydrochloride (racemate) While under nitrogen, a stirred solution of tert-(3R,4S)-3-acetamido-3-(tert-butylcarbamoyl)—4 —[3 — (4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-1-carboxylate (400 mg, 0.807 mmol) in anhydrous THE (5 ml) was treated with 4 M HCl in 1,4-dioxane (3 .03 ml, 12.1 mmol). After 2.5 h the mixture was diluted with diethyl ether (15 ml) and filtered, washed with additional ether and concentrated to give (3R,4S)-3-acetamido-N- tert -butyl-4-[3-( 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-3-carboxamide hydrochloride (340 mg, 97%) as a white solid. XH-NMR (D2O, 400 MHz) δ 4.11 (d, J = 12 Hz, 1H), 3.58 (dd, J = 11.5, 7.7 Hz, 1H), 3.19 (d, J = 12 Hz, 1H), 3.01 (t, J = 11.5, 1H), 2.42 (m, 1H), 1.92 (s, 3H), 1.52 (m, 1H), 1.30 (m, 2H), 1.19 (s, 9H), 1.16 (s, 12H), 1.08 (m, 1H), 0.75 (m, 2H). LC-MS generates ESI+ MS found for C20H38BN3O4 m/z 396.1 (M+H).
Step 9: tert-Butyl 3-({(3R,4S)-3-acetamido-3-(tert-butylcarbamoyl)-4-[3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)propyl]pyrrolidin-1-yl}methyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate
A stirred mixture of (3R,4S)-3-acetamido-N-tert-butyl-4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl] pyrrolidine-3-carboxamide hydrochloride (226 mg, 0.524 mmol) and Et3N (0.110 ml, 0.786 mmol) in methylene chloride (2.5 ml) was treated with a solution of tert-butyl 7-chloro-3-formyl -3,4-dihydroisoquinoline-2(1H)-carboxylate (155 mg, 0.524 mmol) in methylene chloride (2.5 ml). After stirring for 20 min, sodium triacetoxyborohydride (233 mg, 1.10 mmol) was added and stirring continued for 1 h. The resulting mixture was carefully quenched with saturated aqueous NaHCO3 (5 ml), diluted with saturated aqueous sodium chloride (15 ml) and extracted with methylene chloride (4 x 15 ml). The combined organic phase was dried over Na2SO4 and concentrated. Purification by silica gel chromatography (40 g column, 2-4% MeOH in ethyl acetate) gave tert-butyl 3-({(3R,4S)-3-acetamido-3-(tert-butylcarbamoyl)- 4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidin-1-yl}methyl)-7-chloro-3,4-dihydroisoquinoline-2 (1H)-carboxylate (296 mg, 84%, mixture of diastereomers) as a clear gum. LC-MS generates ESI+ MS found for C35H56BC1N4O6 m/z 675.4 (M + H).
Step 10: (3R,4S)-3-Amino-1-[(7-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboryl)propyl acid trihydrochloride ]pyrrolidine-3-carboxylic acid (mixture of 2 diastereomers, each racemate)
In an Ace pressure tube, a solution of tert-butyl 3-({(3R,4S)-3-acetamido-3-(tert-butylcarbamoyl)-4-[3-(4,4,5,5-tetramethyl) -1,3,2-dioxaborolan-2-yl)propyl]pyrrolidin-1-yl}methyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate (280 mg, 0.415 mmol) was treated with Concentrated HCl (8 ml) and stirred at room temperature. After 10 min, the tube was sealed, and the mixture was heated to approximately 118 °C for 16 h. After cooling to room temperature, the solution was carefully diluted with water (20 ml) and washed with methylene chloride (2 x 15 ml). The aqueous phase was concentrated under reduced pressure, and the resulting residue purified by reverse phase HPLC. Product fractions (mixture of diastereomers) were pooled and concentrated. The residue was reconstituted in 1 M HCl and re-concentrated. The resulting residue was diluted with deionized water and lyophilized to give (3R,4S)-3-amino-1-[(7-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-acid trihydrochloride 4-[3-(dihydroxyboryl)propyl]-pyrrolidine-3-carboxylic acid (mixture of 2 diastereomers, each racemate) (102 mg, 49%) as a light yellow amorphous solid. 1H-NMR (0.1M DC1 in D2O, 400 MHz) δ 7.23 (m, 1H), 7.15 (m, 2H), 4.37 (s, 2H.), 4.00 (m) , 3H), 3.84 (m, 2H), 3.69 (m, 1H), 3.43 (m, 1H), 3.22 (m, 1H), 2.98 (m, 1H), 2 1.68 (m, 1H), 1.60 (m, 1H), 1.25 (m, 3H), 0.64 (m, 2H). ESI+ MS found for C18H27BCIN3O4 m/z 378.1 (M - 18 + H), 360.1 (M - 36 + H); ESI” MS m/z 376, 2 (M - 18 - H).
Example 50: Preparation of (3R,4S)-3-amino-1-(2-aminophenylsulfonyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
(3R,4S)-3-Amino-1-(2-aminophenylsulfonyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 46, except that 2-nitrobenzene-1- sulfonyl chloride was used as the acylating agent in Step 4. LC-MS ESI” MS found for CI4H20BN3O8 S m/z 400.1 (MH). '
Tricloridrato de ácido (3R,4S)-3-amino-4-(3- boronopropil)-1-((6-cloro-l,2, 3,4-tetrahidroisoquinolin-3- il)metil)pirrolidina-3-carboxilico (mistura de dois diastereômeros, cada um racemato) é preparado de forma análoga àquela apresentada no Exemplo 49, exceto que 3- clorofenilalanina é usada no lugar de 4-clorofenilalanina na Etapa 1. 1H-RNM (0,1 M de DC1 em D20, 400 MHz) δ 7,23 (m, 2H), 7,09 (m, 1H), 4,37 (s, 2H), 4,00 (m, 3H), 3,82 (m, 2H) , 3,69 (m, 1H) , 3,43 (m, 1H) , 3,22 (m, 1H) , 3,00 (m, 1H) , 2,68 (m, 1H) , 1,60 (m, 1H) , 1,26 (m, 3H) , 0,66 (m, 2H) . ESI+ MS encontrado para C18H27BCI3O4 m/z 378,1 (M - 18 + H) , 360,0 (M - 36 + H) ; ESI’ MS m/z 376, 1 (M - 18 - H) .Example 51: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-((6-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl)pyrrolidine-acid 3-carboxylic (3R,4S)-3-amino-4-(3-boronopropyl)-1-((6-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl)pyrrolidine-3-carboxylic acid trihydrochloride (mixture of two diastereomers, each racemate) is prepared analogously to that shown in Example 49, except that 3-chlorophenylalanine is used in place of 4-chlorophenylalanine in Step 1. 1H-NMR (0.1M DC1 in D20 , 400 MHz) δ 7.23 (m, 2H), 7.09 (m, 1H), 4.37 (s, 2H), 4.00 (m, 3H), 3.82 (m, 2H), 3.69 (m, 1H), 3.43 (m, 1H), 3.22 (m, 1H), 3.00 (m, 1H), 2.68 (m, 1H), 1.60 (m , 1H), 1.26 (m, 3H), 0.66 (m, 2H). ESI+ MS found for C18H27BCl3O4 m/z 378.1 (M - 18 + H), 360.0 (M - 36 + H); ESI' MS m/z 376.1 (M - 18 - H).
Uma solução agitada de (3R,4S)-3-acetamido-N-terc- butil-4-(3-(4,4,5,5-tetrametil-l,3,2-dioxaborolan-2- il)propil)pirrolidina-3-carboxamida (Exemplo 8, Etapa 4) (198 mg, 0,50 mmol) e N-(4-fenil)benzil-N-BOC-glicinaldeido (244 mg, 0,75 mmol) em 1,2-dicloroetano anidro (5 ml) foi agitada em temperatura ambiente por 1 h, e depois resfriada por banho de gelo e tratada com triacetoxiborohidreto de sódio (212 mg, 1,0 mmol) e agitada por 18 h em temperatura ambiente. Carbonato de sódio aquoso (10%, 5 ml) foi adicionado, e a mistura agitada por poucos minutos e extraída com acetato de etila (30 ml, depois 2 x 10 ml) .Example 52: Preparation of (3R,4S)-3-amino-1-(2-(biphenyl-4-ylamino)ethyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid A stirred solution of (3R,4S)-3-acetamido-N-tert-butyl-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl) pyrrolidine-3-carboxamide (Example 8, Step 4) (198 mg, 0.50 mmol) and N-(4-phenyl)benzyl-N-BOC-glycinaldehyde (244 mg, 0.75 mmol) in 1,2- Anhydrous dichloroethane (5 ml) was stirred at room temperature for 1 h, then cooled in an ice bath and treated with sodium triacetoxyborohydride (212 mg, 1.0 mmol) and stirred for 18 h at room temperature. Aqueous sodium carbonate (10%, 5 ml) was added, and the mixture stirred for a few minutes and extracted with ethyl acetate (30 ml, then 2 x 10 ml).
The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4), and concentrated in vacuo. The crude material was deprotected and isolated using method 5 described for Example 8, Step 5, to generate (3R,4S)-3-amino-1-(2-(biphenyl-4-ylamino)ethyl)-4-acid (3-boronopropyl)pyrrolidine-3-carboxylic acid (72 mg, 27%) as a white bulky solid. NMR (D2O) δ 7.60-7.75 (m, 4H), 7.30-7.55 (m, 5H), 4.27 (s, 2H), 3.94 (d, J=12, 5Hz, 1H), 10. 3.82 (dt, J2 =11Hz, J2 = 8Hz, 1H), 3.60-3.75 (m, 3H), 3.45 (m, 2H), 3. 37 (t, J = 11 Hz, 1H), 2.50-2.65 (m, 1H), 1.50-1.65 (m, 1H), 1.10-1.35 (m, 3H) , 0.60-0.70 (m, 2H). MS (m+1): 426.1; MS (m - H2O + 1): 408.1.
Ácido (3R,4S)-3-amino-4-(3-boronopropil)-1-(1,2,3,4- tetrahidroisoquinolina-3-carbonil)pirrolidina-3-carboxilico foi preparado de forma análoga àquela apresentada no 25 Exemplo 46, exceto que ácido 2-(terc-butoxicarbonil)- 1,2,3,4-tetrahidroisoquinolina-3-carboxilico foi usado como o agente de acilação na Etapa 4 (mistura de dois diastereômeros; cada um racemato) (43 mg, 12% global para 3 etapas) como um sólido amorfo amarelo pálido. MS (ESI+) m/z 30 375 (M - H2O + H+) , 392.Example 53: Preparation of (3R, 4S)-3-amino-4-(3-15 boronopropyl)-1-(1,2,3,4-tetrahydroisoquinoline-3-carbonyl)pyrrolidine-3-carboxylic acid (3R,4S)-3-Amino-4-(3-boronopropyl)-1-(1,2,3,4-tetrahydroisoquinoline-3-carbonyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in 25 Example 46, except that 2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was used as the acylating agent in Step 4 (mixture of two diastereomers; each racemate) (43 mg , 12% overall for 3 steps) as a pale yellow amorphous solid. MS (ESI+) m/z 30 375 (M - H2O + H+), 392.
Example 54: Preparation of (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-[4-(trifluoromethyl)phenylalanyl]pyrrolidine-3-carboxylic acid dihydrochloride (mixture of two diastereomers; each racemate)
Step 1: 2-Oxo-2-phenylethyl (3R,4S)-3-azido-1-[N-(tert-butoxycarbonyl)-4-(trifluoromethyl)phenylalanyl]-4-[3-(4,4,5- ,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-3-carboxylate A mixture of N-(tert-butoxycarbonyl)-4-(trifluoromethyl)phenylalanine (268 mg, 0.804 mmol), hydrate of 1-hydroxybenzotriazole (123 mg, 0.804 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (154 mg, 0.804 mmol) in dry methylene chloride (10 ml) and DMF (3 ml) was stirred at room temperature. After 1 h, a second solution of 2-oxo-2-phenylethyl hydrochloride (3R,4S)-3-azido-4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan) Racemic -2-yl)propyl]pyrrolidine-3-carboxylate (350 mg, 0.73 mmol) in methylene chloride (3.5 ml) was added, followed immediately by Et3N (204 µl, 1.46 mmol), and the resulting homogeneous mixture was stirred at room temperature for 3.5 h. Once complete, the mixture was diluted with dichloromethane (8ml), washed successively with water (2x20ml), saturated aqueous NaHCO3 (2x20ml) and saturated aqueous sodium chloride, dried over Na2SO4 and concentrated. Purification by silica gel chromatography (90 g column, 15-35% ethyl acetate in hexanes) gave 2-oxo-2-phenylethyl (3R,4S)-3-azido-1-[N-(tert- butoxycarbonyl)-4-(trifluoromethyl)phenylalanyl]—4—[3—(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-3-carboxylate (233 mg, approximately 60% purity) as a white foam which was used without further purification. LC-MS ESI+ MS found for C37H47BF3N5O8 m/z 758.3 (M + H), 780.3 (M + Na); ESI” MS m/z 756.5 (M - H).
Step 2: (3R,4S)-3-amino-1-[N-(tert-butoxycarbonyl)-4-(trifluoromethyl)phenylalanyl]-4-[3-(4,4,5,5-tetramethyl-1] acid ,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-3-carboxylic acid A stirred solution of 2-oxo-2-phenylethyl(3R,4S)-3-azido-1-[N-(tert-butoxycarbonyl)- 4-(trifluoromethyl)phenylalanyl]— 4 — [3—(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-3-carboxylate (Step 1) in acetic acid (9 ml) was treated with zinc (201 mg, 3.08 mmol, 10 equivalents), and the heterogeneous mixture was stirred at room temperature. After 1.25 h, the reaction was filtered, the filter pad was washed with acetic acid and ethyl acetate, and the filtrate was concentrated and dried under reduced pressure to give (3R,4S)-3-amino-1 acid. -[N-(tert-butoxycarbonyl)-4-(trifluoromethyl)phenylalanyl]-4-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine- crude 3-carboxylic acid (242 mg) as a clear film which was used without further purification. LC-MS ESI + MS found for C29H43BF3N3O7 m/z 614.3 (M + H); ESI" MS m/z 612.3 (M - H).
Step 3: (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-[4-(trifluoromethyl)phenylalanyl]pyrrolidine-3-carboxylic acid dihydrochloride (mixture of two diastereomers; each one racemate) A solution of (3R,4S)-3-amino-1-[N-(tert-butoxycarbonyl)-4-(trifluoromethyl)phenylalanyl]-4-[3-(4,4,5,5-tetramethyl) acid Crude -1,3,2-dioxaborolan-2-yl)propyl]pyrrolidine-3-carboxylic acid (Step 2, 242 mg) in anhydrous THF (6 ml) under nitrogen was treated with 4N HCl in 1,4-dioxane (6 ml). After stirring for 2 h at room temperature, 1 M aqueous HCl (6 ml) was added and stirring continued for a further 1.5 h. The reaction mixture was diluted with water (15 ml) and washed successively with ethyl acetate (25 ml) and methylene chloride (25 ml). The aqueous phase was concentrated and the residue was purified by reverse phase HPLC. Product fractions (mixture of diastereomers) were pooled and concentrated. The residue was reconstituted in 1 M HCl and reconcentrated. The resulting residue was diluted with deionized water and lyophilized to give (3R,4S)-3-amino-4-[3-(dihydroxyboryl)propyl]-1-[4-(trifluoromethyl)phenylalanyl]-pyrrolidine-3 acid dihydrochloride -carboxylic (mixture of two diastereomers, each racemate) (43 mg, 12% overall for 3 steps) as a pale yellow amorphous solid. MS (ESI) m/z 414 (M - H20 + H+), 396 (M - 2H2O + H+); MS (ESI') m/z 412 (M - H20 - H+).
Ácido (3R,4S)-3-amino-4-(3-boronopropil)-1-( (7- (trifluormetil)-1,2,3,4-tetrahídroisoquinolin-3-il)metil) pirrolidina-3-carboxílico é preparado de forma análoga àquela apresentada no Exemplo 51, exceto que 4- (trifluormetil)-fenilalanina é usada no lugar de 4- clorofènilalanina na Etapa 1. 1H-RNM (0,1 M de DC1 em D20, 400 MHz) δ’7,53 (d, J= 8,0 Hz, 1H), 7,49 (s, 1H), 7,34 (d, J = 8,3 Hz, 1H) , 4,46 (s, 2H) ,' 4,08 (m, 2H) , 3,97 (m, 1H) , 3,83 (m, 2H) , 3,71 (m, 1H) , 3,44 (m, 1H) , 3,34 (m, 1H) , 3,07 (m, 1H) , 2,69 (m, 1H) , 1,60 (m, . 1H) , 1,26 (m, 3H) , 0,66 (m, 2H) . ESI” MS encontrado para Ci9H27BF3N3O4 m/z 412,1 (M - 18 + H), 394,1 (M - 36 + H); ESI” MS m/z 410,2 (M - 18 - H) .Example 55: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-((7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl) acid pyrrolidine-3-carboxylic acid (mixture of two diastereomers; each racemate) (3R,4S)-3-Amino-4-(3-boronopropyl)-1-((7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl)pyrrolidine-3-carboxylic acid is prepared analogously to that shown in Example 51, except that 4-(trifluoromethyl)-phenylalanine is used in place of 4-chlorophenylalanine in Step 1. 1H-NMR (0.1 M DC1 in D20, 400 MHz) δ' 7.53 (d, J = 8.0 Hz, 1H), 7.49 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 4.46 (s, 2H), 4.08 (m, 2H), 3.97 (m, 1H), 3.83 (m, 2H), 3.71 (m, 1H), 3.44 (m, 1H), 3.34 ( m, 1H), 3.07 (m, 1H), 2.69 (m, 1H), 1.60 (m, 1H), 1.26 (m, 3H), 0.66 (m, 2H) . ESI” MS found for C19H27BF3N3O4 m/z 412.1 (M - 18 + H), 394.1 (M - 36 + H); ESI” MS m/z 410.2 (M - 18 - H).
Example 56: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)pyrrolidine-3-carboxylic acid
Step 1: tert-butyl 3-((3R,4S)-4-allyl-3-azido-3-((2-oxo-2-phenylethoxy)carbonyl)pyrrolidine-1-carbonyl)-7-chloro-3, 4-dihydroisoquinoline-2(1H)-carboxylate A stirred mixture of 2-oxo-2-phenylethyl(3R,4S)-4-allyl-3-azidopyrrolidine-3-carboxylate racemic hydrochloride (228 mg, 0.650 mmol) and acid 2 -(tert-butoxycarbonyl)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (258 mg, 0.826 mmol) in methylene chloride (6 ml) was treated with Et 3 N (0.272 ml, 1.95 mmol), followed by N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU, 321 mg, 0.845 mmol), and the resulting nearly homogeneous mixture was stirred in room temperature. After 1 h, the reaction was diluted with methylene chloride (20 ml), washed successively with water (20 ml), 1 N aqueous HCl (20 ml), saturated aqueous NaHCO3 (20 ml) and saturated aqueous sodium chloride (10 ml), dried over Na2SO4, and concentrated. Purification by silica gel chromatography (40 g column, 15-25% ethyl acetate in hexanes) gave tert-butyl 3-({(3R, 4S)-4-allyl-3-azido-3-'[ (2-oxo-2-phenylethoxy)carbonyl]pyrrolidin-1-yl}carbonyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate (214mg, 54%) as a pale amber film which was used without further purification. LC-MS ESI+ MS found for Csif^ClNsOg- m/z 608.2 (M + H), 630.3 (M + Na); ESI" MS m/z 606.3 (M - H) .
Etapa 3: dicloridrato de ácido (3R, 4S)-3-amino-l-[ (7- cloro-1,2,3, 4-tetrahidroisoquinolin-3-il)carbonil]-4-[3- (diidroxiboril)propil]pirrolidina-3-carboxílico (mistura de dois diastereômeros; cada um racemato) Dicloridrato de ácido (3R,4S)-3-amino-l-[(7-cloro- 1,2,3,4-tetrahidroisoquinolin-3-il)carbonil]-4-[3- (diidroxiboril)propil]pirrolidina-3-carboxilico (mistura de dois diastereômeros; cada um racemato) é preparado de forma análoga àquela apresentada no Exemplo 54, Etapas 2-3, exceto que terc-butil 3_({(3R,4S)-3-azido-3-[(2-oxo-2- feniletoxi)carbonil]—4—[3—(4,4,5,5-tetrametil-l,3,2- dioxaborolan-2-il)propil]pirrolidin-l-il}carbonil)-7-cloro- 3,4-diidroisoquinolina-2(1H)-carboxilato (Etapa 2) é . usado como o substrato.Step 2: tert-butyl 3-((3R,45)-3-azido-3-((2-oxo-2-phenylethoxy)carbonyl)-4-(3-(4,4,5,5-tetramethyl-) 1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine-1-carbonyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate tert-Butyl 3-((3R,4S)-3 -azido-3-((2-oxo-2-phenylethoxy)carbonyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)pyrrolidine- 1-carbonyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate is prepared analogously to that shown in Example 1, Step 3, except that tert-butyl 3-({(3R,4S)- 4-allyl-3-azido-3-[(2-oxo-2-phenylethoxy)carbonyl]pyrrolidin-1-yl}carbonyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate is used as the substrate. After chromatography, the slightly impure material was used without further purification. LC-MS ESI+ MS found for C37H47BCIN5O8 m/z 736.3 (M + H). Step 3: (3R,4S)-3-amino-1-[(7-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)carbonyl]-4-[3-(dihydroxyboryl)propyl acid dihydrochloride ]pyrrolidine-3-carboxylic acid (mixture of two diastereomers; each racemate) (3R,4S)-3-amino-1-[(7-chloro-1,2,3,4-tetrahydroisoquinolin-3-yl) acid dihydrochloride )carbonyl]-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic (mixture of two diastereomers; each racemate) is prepared analogously to that given in Example 54, Steps 2-3, except that tert-butyl 3_({(3R,4S)-3-azido-3-[(2-oxo-2-phenylethoxy)carbonyl]—4—[3—(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)propyl]pyrrolidin-1-yl}carbonyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate (Step 2) is . used as the substrate.
Example 57: Preparation of (3R,4S)-3-amino-1-(2-amino-3-phenylpropyl)-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid (mixture of two diastereomers; each racemate)
(3R,4S)-3-Amino-1-(2-amino-3-phenylpropyl)-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid trihydrochloride (mixture of two diastereomers; each racemate) is prepared analogously to that given in Example 54, Steps 1-3, except that commercial N-(tert-butoxycarbonylamino)-phenylalanine is used in place of tert-butyl 7-chloro-3-(hydroxymethyl)-3,4- dihydroisoquinoline-2(1H)-carboxylate. 1H-RNM (0.1 M DC1 in D20, 400 MHz) δ 7.30 (m, 5H), 4.41 (s, 2H), 4.00 (m, 3H), 3.85 (m, 2H), 3.75 (m, 1H), 3.41 (m, 2H), 2.86 (m, 1H), 2.69 (m, 1H), 1.60 (m, 1H), 1. 27 (m, 3H), 0.67 (m, 2H). ESI+ MS found for CI7H28BN3O4 m/z 332.2 (M - 18 + H),. 314.2 (M - 36 (2 H2O)+ H); ESI-MS m/z 330.2 (M - 18 (H2O) - H).
Example 58: Preparation of (3R,4S)-3-amino-4-(3-boronopropyl)-1-(2-(methylamino)-3-phenylpropanoyl)pyrrolidine-3-carboxylic acid
(3R,4S)-3-Amino-4-(3-boronopropyl)-1-(2-(methylamino)-3-phenylpropanoyl)pyrrolidine-3-carboxylic acid was prepared analogously to that shown in Example 46, except that 2-(tert-butoxycarbonyl(methyl)amino)-3-phenylpropanoic acid was used as the acylating agent in Step 4. MS (ESI+) m/z 378 (M - H2O + H+), 395.
Etapa 1: Metil 2, 4-dicloro-N-(etoxicarbonil) fenilalaninato Metil 2,4-dicloro-N-(etoxicarbonil)fenilalaninato é preparado de forma análoga àquela apresentada no Exemplo 51, Etapas 1-2, exceto que 2,4-diclorofenilalanina é usada no lugar de 4-clorofenilalanina na Etapa 1. XH-RNM (DMSO- d6, 400 MHz) δ 7,71 (d, . J = 8,4 Hz, 1H) , 7,61 (s, 1H) , 7,38 (m, 1H), 4,30 (m, 1H), 3,92 (m, 2H), 3,64 (s, 3H), 3,19 (m, 1H) , 2,93 (m, 1H), 1,10 (t, J = 7,1 Hz, 3H) . LC-MS gera ESI+ MS encontrado para C13H15CI2NO4 m/z 320, 0 (M + H) . Example 59: Preparation of (3R,4S)-3-amino-1-[(5,7-dichloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-acid trihydrochloride (dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid (mixture of two diastereomers; each racemate) Step 1: Methyl 2,4-dichloro-N-(ethoxycarbonyl)phenylalaninate Methyl 2,4-dichloro-N-(ethoxycarbonyl)phenylalaninate is prepared in a manner analogous to that shown in Example 51, Steps 1-2, except that 2.4 -dichlorophenylalanine is used in place of 4-chlorophenylalanine in Step 1. XH-NMR (DMSO-d6, 400 MHz) δ 7.71 (d, . J = 8.4 Hz, 1H), 7.61 (s, 1H) ), 7.38 (m, 1H), 4.30 (m, 1H), 3.92 (m, 2H), 3.64 (s, 3H), 3.19 (m, 1H), 2.93 (m, 1H), 1.10 (t, J = 7.1 Hz, 3H). LC-MS generates ESI+ MS found for C13H15Cl2NO4 m/z 320.0 (M+H).
Step 2: 2-(tert-Butoxycarbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid A mixture of methyl 2,4-dichloro-N-(ethoxycarbonyl)phenylalaninate (100mg) , 0.312 mmol) and paraformaldehyde (10.3 mg, 0.344 mmol) in acetic acid (0.90 ml) in a microwave tube was treated with concentrated sulfuric acid (0.30 ml) (slight exotherm with mixing). The mixture was heated at 80°C in the microwave for a total of 8 h, and then aliquots were checked by HPLC. The reaction mixture was then added to water (8 ml), and that aqueous phase was washed with methylene chloride (2 x 20 ml) and concentrated to give an approximately 4:1 mixture of the 5,7-dichloro-acid product. desired 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and 2,4-dichlorophenylalanine by-product in residual sulfuric acid.
A slurry of crude 5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in water (15 ml) was cooled in an ice bath, adjusted to pH approximately 8.5 with NaOH 50% aqueous, diluted with 1,4-dioxane (10 ml) and treated with a solution of di-tert-butyldicarbonate (530 mg, 2.43 mmol) in 1,4-dioxane (8 ml) quickly dropwise. The heterogeneous mixture was stirred at 0°C for 10 min and then at room temperature overnight, generating a thick white mixture. HPLC indicated starting material remaining and therefore the reaction was treated with 2N aqueous NaOH (0.48 ml, 0.96 mmol) and additional di-tert-butyldicarbonate (204 mg, 0.935 mmol). After stirring for 4 h, the mixture was diluted with water (25 ml) and washed with Et∑O (2 x 25 ml). The aqueous phase was adjusted to pH approximately 3 with 1 M aqueous citric acid and extracted with ethyl acetate (2 x 40 ml). The combined organic phase (Et20/ethyl acetate) was washed with saturated aqueous sodium chloride (10 ml), dried over MgSO4 and concentrated. Purification by silica gel chromatography (40 g column, 15-45% ethyl acetate in hexanes) gave 2-(tert-butoxycarbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline acid- 3-carboxylic (265 mg, 41%) as a partially crystalline white solid. 1H-NMR (DMSO-dg, 400 MHz) δ 12.9 (s, 1H), 7.51 (m, 1H), 7.41 (m, 1H), 4.98 (m, approximately 0.5H) , 4.80 (m, approximately 0.5H), 4.50 (m, 2H), 3.29 (m, 1H), 2.98 (m, 1H), 1.46 (s, approximately 4.5H ), 1.41 (s, approximately 4.5H) (mixture of rotamers). LC-MS generates ESI” MS found for C15H17CI2NO4 m/z 344.1 (M - H) .
Step 3: (3R,4S)-3-amino-1-[(5,7-dichloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboryl) acid trihydrochloride )propyl]pyrrolidine-3-carboxylic acid (mixture of two diastereomers; each racemate)
(3R,4S)-3-amino-1-[(5,7-dichloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-4-[3-(dihydroxyboryl)propyl] acid trihydrochloride pyrrolidine-3-carboxylic acid (mixture of two diastereomers, each racemate) is prepared analogously to that shown in Example 49, steps. 1H-RNM (0.1 M DC1 in D20, 400 MHz) δ 7.43 (m, 1H), 7.17 (m, 1H), 4.41 (s, 2H), 4.00 (m, 3H), 3.85 (m, 2H), 3.75 (m, 1H), 3.41 (m, 2H), 2.86 (m, 1H), 2.69 (m, 1H), 1, 60 (m, 1H), 1.27 (m, 3H), 0.67 (m, 2H). ESI + MS found for C18H26BC12N3O4 m/z 412.2 (M - 18 + H), 394.2 (M - 36 + H); ESI" MS m/z 410.3 (M - 18 - H).
Example 60: Preparation of (3aR,4S,5S,6aR)-5-amino-4-(3-boronopropyl)octahydrocyclopenta[c]pyrrole-5-carboxylic acid
Step 1: 2-Oxo-2-phenylethyl(3R,4S)-4-allyl-3-azidopyrrolidine-3-carboxylate trifluoroacetate (racemic) While under nitrogen, a solution of racemic 1-tert-butyl 3-(2-oxo) -2-phenylethyl) (3R, 4S)-4-allyl-3-azidopyrrolidine-1,3-dicarboxylate (0.425 g, 1.02 mmol) in methylene chloride (4 ml) was treated with trifluoroacetic acid (1.2 ml, 15 mmol) quickly dropwise, and the resulting homogeneous mixture was stirred at room temperature. After 40 min, the mixture was concentrated under reduced pressure and dried under high vacuum for several hours to give racemic crude 2-oxo-2-phenylethyl(3R,4S)-4-allyl-3-azidopyrrolidine-3-carboxylate trifluoroacetate as a dark amber viscous oil that was used immediately in the next step. LC-MS ESI+ MS found for C16H18N4O3 m/z 315.2 (M + H). XH-NMR (CDCl3 , 400 MHz) δ 7.92 (m, 2H), 7.69 (m, 1H), 7.55 (m, 2H), 5.75 (m, 1H), 5.62 ( m, 2H), 5.24 (d, J = 17 Hz, 1H), 5.20 (d, J = 10.2 Hz, 1H), 4.08 (m, 1H), 3.80 (m, 1H), 3.55 (m, 2H), 2.80 (m, 1H), 2.63 (m, 1H), 2.27 (m, 1H).
Step 2: 2-Oxo-2-phenylethyl(3R,4S)-4-allyl-3-azido-1-[N-benzyl-N-(tert-butoxycarbonyl)glycyl]pyrrolidine-3-carboxylate A stirred mixture of 2 -oxo-2-phenylethyl(3R,4S)-4-allyl-3-azidopyrrolidine-3-carboxylate crude racemic trifluoroacetate (Step 1, approximately 1 mmol) and N-benzyl-N-(tert-butoxycarbonyl)glycine (337 mg , 1.27 mmol) in methylene chloride (9 ml) was treated with EtsN (0.495 ml, 3.55 mmol), followed by N,N,N',N'-tetramethyl-O-(7-azabenzotriazole hexafluorophosphate) -1-yl)uronium (HATU, 502 mg, 1.32 mmol), and the resulting mixture was stirred at room temperature and monitored by HPLC. At 1 h, the reaction was diluted with methylene chloride (20 ml), washed with water (20 ml), 1 N aqueous HCl (10 ml), saturated aqueous NaHCO3 (20 ml) and saturated aqueous sodium chloride (10 ml), dried over NasSCL, and concentrated under reduced pressure. Purification by silica gel chromatography (40 g column, 15-30% ethyl acetate in hexanes) gave 2-oxo-2-phenylethyl (3R,4S)-4-allyl-3-azido-1-[N -benzyl-N-(tert-butoxycarbonyl)glycyl]pyrrolidine-3-carboxylate (365 mg, 64%) as an amber gum which was used as such in the next step. LC-MS ESI+ MS found for C30H35N5O6 m/z 562.3 (M + H), 584.3 (M + Na). 1H-NMR (CDCl3, 400 MHz) δ 7.92 (m, 2H), 7.66' (m, 1H), 7.53 (m, 2H), 7.30 (m, 5H), 5.75 (m, 1H), 5.52 (m, 2H), 5.12 (m, 2H), 4.58 (m, 2H), 4.22-3.42 (m, 6H), 2.60 ( m, 1H), 2.50 (m, 1H), 2.12 (m, 1H), 1.48 (s, 9H).
Step 3: (3R,4S)-3-Amino-1-(N-benzylglycyl)-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid dihydrochloride (mixture of two diastereomers; each racemate) Dihydrochloride of (3R,4S)-3-amino-1-(N-benzylglycyl)-4-[3-(dihydroxyboryl)propyl]pyrrolidine-3-carboxylic acid (mixture of two diastereomers; each racemate) is similarly prepared to that shown in Example 23, Steps 2-3, except that 2-oxo-2-phenylethyl (3R,4S)-4-allyl-3-azido-1-[N-benzyl-N-(tert-butoxycarbonyl)glycyl] pyrrolidine-3-carboxylate (Step 2) is used in place of tert-butyl 3-({(3R,4S)-4-allyl-3-azido-3-[(2-oxo-2-phenylethoxy)carbonyl]pyrrolidine -1-yl}carbonyl)-7-chloro-3,4-dihydroisoquinoline-2(1H)-carboxylate. MS (ESI+) m/z 346 (M - H2O + H+), 328 (M-2 H2O + H+); MS (ESI-) m/z 344 (M - H2O - H+).
Example 61: Preparation of (IS,2S,4S)-1,4-diamino-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic)
Step 1: 2-Allylcyclopentanone A stirred solution of methyl 2-carboxycyclopentanone (35.5 g, 250 mmol) in acetone (500 ml) was treated with anhydrous potassium carbonate (138 g, 1.0 mol) and allyl bromide ( 100 ml, 1.15 mol) and refluxed for 5 h. The mixture was cooled and filtered through Celite® (rinse the filter cake with acetone), and the filtrate was concentrated in vacuo. The residual oil was dissolved in methanol (450 ml), treated with 6N HCl (250 ml) and refluxed for 40 h. The solution was cooled to room temperature, concentrated in vacuo to remove most of the methanol and diluted with water (200 ml). The aqueous solution was extracted with dichloromethane (3 x 250 ml), and the combined organic solution was washed with water, saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride (200 ml each), dried (Na 2 SO 4 ) and gently (volatile product ) concentrated in vacuo to a volume of 75 ml. This solution was loaded onto a silica gel column (approximately 500 cm3) and eluted with dichloromethane to give (after mild concentration of fractions) 2-allylcyclopentanone (27.3 g, 88%) as a colorless oil. NMR (CDCl 3 ): δ 5.65-5.75 (m, 1H), 4.90-5.05 (m, 2H), 2.40-2.50 (m, 1H), 2.20-2 .30 (m, 1H), 1.85-2.15 (m, 5H), 1.65-1.75 (m, 1H), 1.45-1.55 (m, 1H).
Step 2: 2-A1H-5-(phenylselanyl)cyclopentanone While under a nitrogen atmosphere, a solution of 2-allylcyclopentanone (12.4 g, 100 mmol) in anhydrous tetrahydrofuran (100 ml) was cooled to -70°C and treated with 1 N lithium bis(tri-methylsilyl) amide/tetrahydrofuran (200 ml, 200 mmol) at a rate to maintain vessel temperature below -55°C. After the addition was complete, the mixture was stirred at -60 to -70°C for an additional hour. A second solution of phenylselenyl chloride (19.5 g, 102 mmol) in anhydrous tetrahydrofuran (50 ml) was added dropwise, and the mixture stirred at -60 to -70°C for 30 min, then allowed to stir. its heating to 0°C. The reaction was quenched by adding the solution to a rapidly stirred mixture of ethyl acetate (500 ml) and 5% aqueous citric acid (200 ml), and the organic layer was separated. The aqueous solution was extracted with ethyl acetate (2 x 100 ml) and the combined organic solution was washed with saturated aqueous sodium chloride (200 ml), dried (MgSO4) and concentrated in vacuo. The residue was dissolved in heptane and loaded onto a silica gel column (approximately 600 cm 3 ) and eluted with 2:1 heptane/dichloromethane, then 1:1 heptane/dichloromethane to give 2-allyl- 5-(phenylselanyl)cyclopentanone (19.7 g, 71%) as a pale yellow oil. NMR (CDCl3 ): δ 7.40-7.50 (m, 2H), 7.05-7.25 (m, 3H), 5.50-5.70 (m, 1H)', 4.80- 4.95 (m, 2H), 3.45-3.75 (m, 1H), 2.30-2.50 (m, 1H), 1.80-2.25 (m, 5H), 1. 50-1.75 15 (m, 1H). MS (M+1): 279.1/280.9 (for 2 major Se isotopes).
Step 3: 5-Allylcyclopent-2-enone A stirred ice-cold (3°C) solution of 2-allyl-5-(phenylselanyl)cyclopentanone, mixture of isomers (12.0 g, 43 mmol) in dichloromethane (200 ml), in a 1 liter round bottom flask equipped to contain excessive boiling was treated with saturated aqueous ammonium chloride (45 ml), then dropwise with 30% aqueous hydrogen peroxide (22 ml), and slowly carefully warmed. to room temperature. At approximately 20°C, a vigorous exotherm began, and the ice bath was reapplied. The mixture was cooled to room temperature and stirred for 1 h, then the solution was washed with water (100 ml), stirred with 10% aqueous sodium thiosulfate pentahydrate (75 ml) per . 10 min, and separated. The organic solution was washed with saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride (75 ml each), dried (Na 2 SO 4 ) and concentrated to a volume of 30 ml. The solution was added to a silica gel column (approximately 400 cm3) and eluted with dichloromethane to give (after very mild concentration of appropriate fractions) 5-10 allylcyclopent-2-enone (3.95 g, 75%) as a very pale yellow oil. NMR (CDCl3 ): δ 7.61 (m, 1H), 6.12 (m, 1H), 5.60-5.75 (m, 1H), 4.90-5.05 (m, 2H), 2.70-2.80 (m, 1H), 2.45-2.55 (m, 1H), 2.30-2.40 (m, 2H), 2.05-2.15 (m, 1H) ) .
Step 4: tert-butyl 3-allyl-4-oxocyclopentylcarbamate A stirred solution of 5-allylcyclopent-2-enone (2.20 g, 18 mmol), t-butyl carbamate (4.70 g, 40 mmol) and bromide of tetra-n-butylammonium (6.45 g, 20 mmol) in anhydrous dichloromethane (40 ml) under nitrogen was cooled in an ice bath (3°C) and treated dropwise with boron trifluoride etherate (2, 22 ml, 18 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 18 h. Saturated aqueous sodium bicarbonate (40 ml) was added and then the mixture was stirred for 15 min and separated. The aqueous solution was extracted with dichloromethane (2 x 20 ml) and the combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (Na 2 SO 4 ) and concentrated in vacuo. The residue was dissolved in a minimum of dichloromethane and added to a silica gel column (approximately 300 cm 3 ) and eluted first with 85:15 heptane/ethyl acetate to give recovered starting material (0.35 g), and then with 4:1 heptane/ethyl acetate to generate a mixture of t-butyl carbamate and subject compound. This mixture was heated in heptane and filtered to remove most of the t-butyl carbamate, and then the filtrate was added to a silica gel column (approximately 300 cm3) and eluted with 6:3:1 heptane/dichloromethane /ethyl acetate to give tert-butyl 3-allyl-4-oxocyclopentylcarbamate 1.17 g (27%) as a pale amber solid. NMR (CDCl 3 ): δ 5.60-5.75 (m, 1H), 4.90-5.05 (m, 2H), 4.50 (br s, 1H), 4.00-4.25 15 (m, 1H), 2.30-2.80 (m, 3H), 2.20-2.30 (m, 1H), 1.85-2.15 (m, 3H), 1.38 (s , 9H). MS (M+1): 240.1.
Step 5: tert-butyl (1S,3S,4S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentylcarbamate While under nitrogen, a mixture of tert-butyl 3-allyl-4-oxocyclopentylcarbamate, mixture of isomers (1.08 25 g, 4.5 mmol) and ammonium acetate (1.39 g, 18 mmol) in 2,2,2-trifluoroethanol (5 ml) was treated with t-butylisonitrile (1.53 ml, 13.5 mmol) and stirred at room temperature for 3 days. The reaction mixture was concentrated in vacuo and dissolved in dichloromethane (50ml). The solution was washed with water (25 ml), dried (Na 2 SO 4 ) and added to a silica gel column (approximately 250 cm 3 ). This was eluted successively with 50%, 65%, 70%, and 75% ethyl acetate' in heptane to give tert-butyl(IS,3S,4S)-3-acetamido-4-allyl-3-(tert- butylcarbamoyl)cyclopentylcarbamate (0.99 g, 58%) as a white solid. NMR (CDCl 3 ): δ 7.72 and 7.04 (br s, 1H combined), 7.27 and 6.35 (br s, 1H combined), 5.84 and 5.06 (br s, 1H combined), 5 .55-5.70 (m, 1H), 4.85-4.95 (m, 2H), 3.90-4.15 (m, 1H), 2.10-2.90 (m, 4H) , 1.70-2.00 (m, 6H), 1.20-1.40 (m, 18H). MS (M+1): 382.2.
Step 6: tert-butyl (1S,3S,4S)-3-acetamido-3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-) 2-yl)propyl)cyclopentylcarbamate A stirred solution of tert-butyl (1S,3S,4S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentylcarbamate, mixture of isomers (0.954 g, 2.50 mmol ) in anhydrous dichloromethane (25 ml) under nitrogen was treated with chloro-1,5-cyclooctadiene-iridium dimer (54 mg, 0.08 mmol) and Diphos® (64 mg, 0.16 mmol), and cooled (- 25°C). After stirring 30 min, pinacolborane (0.55 ml, 3.8 mmol) was added dropwise via syringe, and the vessel temperature was allowed to warm to ice bath temperature and gradually warmed to room temperature overnight (18 h) . Water (10 ml) was added, and the mixture was stirred 20 min, then extracted with ethyl acetate (100 ml). The organic solution was washed with chloride. saturated aqueous sodium (50 ml), dried (MgSO4) and concentrated in vacuo. The residual solid was recrystallized several times from acetonitrile to give tert-butyl (IS, 3S, 4S)-3-acetamido-5 3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl) -1,3,2-.dioxaborolan-2-yl)propyl)cyclopentylcarbamate, and the concentrated mother liquor was chromatographed on silica gel (eluted with 3:2, then 4:1 ethyl acetate/heptane) and recrystallized several times by acetonitrile to generate more of the compound in question. The total yield was 0.52 g (41%) as a white solid. NMR (CDCl 3 ): δ 7.26 (br s, 1H), 6.56 (br s, 1H), 5.48 (br s, 1H), 4.13 (m, 1H), 2.65-2 .80 (m, 1H), 2.40-2.60 (m, 1H), 2.15-2.25 (m, 1H), 1.90-2.05 (m, 1H), 1.93 (s, 3H), 1.40-1.55 (m, 1H), 1.00-1.40 (m, 4H), 1.38 (s, 9H), 1.28 (s, 9H) , 1.16 (s, 12H), 0.60-0.75 (m, 2H). MS (M+1): 510.6.
Step 7: (IS,25, 4S)-1,4-Diamino-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic) A solution of tert-butyl(IS, 3S, 4S)-3-acetamido-3-( tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentylcarbamate (0.204 g, 0.40 mmol) in 2:1: 1 of concentrated HCl:glacial acetic acid:water (8 ml) in a pressure bottle was stirred for 2 h at 60°C, then capped and stirred for 18 h at 130°C, cooled to room temperature and uncapped. The solution was diluted with water (20 ml), extracted with dichloromethane (20 ml) and concentrated in vacuo. The resulting residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in water (40 ml) and treated with DOWEX® 550A-OH resin (3 g) 5 which had been rinsed with methanol. The mixture was stirred for 40 min and then filtered, and the resin washed successively with water, methanol, dichloromethane, water, methanol and dichloromethane. The resin was shaken four times with 1N HCl (15 ml) and filtered, and the combined filtrates were concentrated in vacuo. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in 1.5-2.0 ml of water. After purification by HPLC, the appropriate fractions were concentrated in vacuo, treated three times with 1N 15 HCI (10 ml) and concentrated, treated three times with water (10 ml) and concentrated, then dissolved in water (10 ml) , frozen and lyophilized overnight to give the subject compound (98 mg, 81%) as a white foam. NMR (D20) δ 4.00-4.10 (m, 1H), 2.82 (dd, J=10.5 20 Hz, J2 = 6 Hz, 1H), 2.35-2.45 (m, 1H), 2.05-2.15 (m, 2H), 1.90-2.00 (m, 1H), 1.52 (m, 1H), 1.40 (m, 1H), 1.27 (m, 1H), 1.09 (m, 1H), 0.65-0.75 (m, 2H). MS (M+1): 230.9; MS (M - H2O + 1): 213.1.
Example 62: Preparation of (IS,2S,4S)-1-amino-4-25(benzylamino)-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic)
Step 1: Benzyl 3-allyl-4-oxocyclopentylcarbamate A solution of 5-(propen-3-yl)cyclopent-2-enone (4.28 g, 35 mmol) in dichloromethane (15 ml) was treated with benzyl 5-carbamate ( 10.6 g, 70 mmol) and bismuth trinitrate pentahydrate (2.2 g, 4.5 mmol), stirred rapidly for 18 h, then diluted with dichloromethane (50 ml). The mixture was filtered through Celite® (rinsing with dichloromethane) and the filtrate added directly to a silica gel column (approximately 550 cm3). Elution, first with 3:2 petroleum ether/dichloromethane, gave the recovered starting material (1.01 g), and then 4:1 heptane/ethyl acetate gave the compound in question (4.54 g, 47.5% uncorrected yield) as a pale yellow oil. NMR (CDCl3 ): δ 7.28 (br s, 5H), 5.55 -5.70 (m, 1H), 5.03 (br s, 2H), 4.90-5.10 (m, 2H) ), 4.75-4.90 (m, 1H), 4.10-4.30 (m, 1H), 2.40-2.80 (m, 2H), 2.15-2.40 (m , 2H), 1.85-2.15 (m, 3H). MS (M + Na): 296.0; MS (M - H2O + 1): 256.0 (no M + 1 visible).
Step 2: Benzyl (IS,3S,4S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentylcarbamate A stirred mixture of benzyl 3-allyl-4-oxocyclopentylcarbamate, mixture of isomers (5.19 g , 19 mmol) and ammonium acetate (5.86 g, 76 µmmol) in 2,2,2-trifluorethanol (20 ml) under nitrogen was treated with t-butylisonitrile (6.50 ml, 57 mmol) and stirred at room temperature for 3 days. The reaction mixture was concentrated in vacuo, dissolved in dichloromethane and added to a silica gel column (approximately 550 cm 3 ). Successive elution with 60%, 70% and 80% ethyl acetate/heptane generated the compound in question together with an undesired isomer (total of 3.48 g). Recrystallization (2 crops) with minimal acetonitrile/ether gave the subject compound (1.83 g, 23%) as a white solid. NMR (CDCl3 ): δ 7.38 (br s, 5H), 7.10 (br s, 1H), 5.65-5.80 (m, 10 1H), 5.67 (br s, 1H), 4.95-5.15 (m, 4H), 4.25-4.35 (m, 1H), 2.85-3.00 (m, 1H), 2.60-2.70 (m, 1H) ), 2.20-2.35 (m, 2H), 1.85-2.15 (m, 3H), 2.03 (s, 3H), 1.40 (s, 9H). MS (M+1): 416.1.
Step 3: Benzyl (IS,3S,4S)-3-acetamido-3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-20-dioxaborolan-2) -yl)propyl)cyclopentylcarbamate While under nitrogen, a stirred solution of benzyl (IS,3S,4S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentylcarbamate (1.25 g, 3.00 mmol) in anhydrous dichloromethane (30 ml) was treated with chloro-1,5-25 cyclooctadiene-iridium dimer (70.5 mg, 0.105 mmol) and Diphos® (83.7 mg, 0.21 mmol) and cooled (-25 °C). After stirring 30 min, pinacolborane (0.65 ml, 4.5 mmol) was added dropwise via . syringe, and the vessel temperature was allowed to warm to ice bath temperature and gradually warmed to room temperature overnight (18 h). Water (5 ml) was added, the mixture stirred 20 min and then extracted with ethyl acetate (2 x 30 ml). The organic solution was washed with water and saturated aqueous sodium chloride (25 ml each), dried (MgSO4) and concentrated in vacuo. The residue was dissolved in a minimum of dichloromethane and loaded onto a silica gel column (approximately 250 cm3) and eluted with 2:1 ethyl acetate/heptane to give the subject compound (0.75 g, 46%) as a white solid. NMR (CDCl3 ): δ 7.20-7.35 (m, 6H), 5.30 (br s, 1H), 4.95-5.10 (m, 2H), 4.20 (m, 1H) , 2.73 (m, 1H), 2.46 (m, 1H), 2.23 (d, J = 12 Hz, 1H), 2.05 (m, 1H), 1.93 (s, 3H) , 1.45 (m, 1H), 1.05-1.35 (m, 4H), 1.28 (s, 9H), 1.15 (s, 12H), 0.60-0.75 (m , 2H). MS (M+1): 544.0.
Step 4: (1-S,2S,4S)-1-acetamido-4-amino-N-tert-butyl-2-(4,5,5-tetramethyl-1,3,2-dioxaborolan-2 -yl)propyl)cyclopentanecarboxamide While under nitrogen, a stirred solution of benzyl (IS,3S,4S)-3-acetamido-3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl) -1,3,2-dioxaborolan-2-yl)propyl)cyclopentylcarbamate (0.544 g, 1.00 mmol) in 4:1 ethyl acetate/methanol (20 ml) was treated with Pd(OH) 2 /C 20 % (0.30 g), purged with hydrogen, and stirred under a balloon for 18 h. The mixture was purged with nitrogen and carefully filtered through a pad of Celite® (without allowing the filter pad to dry) and the filtrate concentrated in vacuo to give 0.409 g (100%) of the subject compound as a white solid. NMR (CDCl3 ): δ 6.72 (br s, 1H), 6.67 (br s, 1H), 3.63 (m, 1H), 2.80 (m, 1H), 2.57 (m, 1H), 1.75-2.00 (m, 4H), 1.93 (s, 3H), 1.00-1.50 (m, 3H), 1.25 (s, 9H), 1.17 (s, 12H), 0.60-0.75 (m, 2H). MS (M+1): 410.5.
Step 5: (1S,2S,4S)-1-Amino-4-(benzylamino)-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic) A stirred solution of benzaldehyde (32 mg, 0.30 mmol) in methanol ( 2.5 ml) was treated with (1S,2S,4S)-1-acetamido-4-amino-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (102.4 mg, 0.25 mmol) and stirred for 1 h at 50°C, then cooled in an ice bath. Sodium borohydride (12 mg, 0.32 mmol) was added, and the mixture was stirred for 1 h at 3 °C, warmed to room temperature, stirred for 30 min, and quenched with water (1 ml). The crude product, in a pressure bottle, was dissolved in 2:1:1 concentrated HCl: glacial acetic acid: water (8 ml) and stirred for 2 h at 60°C, then capped and stirred for 18 h at 130° C, cooled to room temperature and uncapped. The solution was diluted with water (20 ml), extracted with dichloromethane (20 ml) and concentrated in vacuo. The resulting residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in water (40 ml) and treated with DOWEX® 550A-OH resin (3 g) which had been rinsed with methanol. . The mixture was stirred for 40 min and then filtered, and the resin washed successively with water, methanol, dichloromethane, water, methanol and dichloromethane. The resin was shaken four times with 1N HCl (15 ml) and filtered, and the combined filtrates were concentrated in vacuo. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in 1.5-2.0 ml of water. After purification by HPLC, the appropriate fractions were concentrated in vacuo, treated three times with 1N HCl (10 ml) and concentrated, treated three times with water (10 ml) and concentrated, then dissolved in water (10 ml) , frozen and lyophilized overnight to give the subject compound (30.7 mg, 31%) as a white foam. NMR (D2O) δ 7.40 (br s, 15 5H), 4.17 (br s, 2H), 4.00-4.10 (m, 1H), 2.78 (m, 1H), 2. 34 (m, 1H), 2.20 (m, 1H), 1.95-2.15 (m, 2H), 1.50 (m, 1H), 1.39 (m, 1H), 1.25 (m, 1H), 1.06 (m, 1H), 0.60-0.75 (m, 2H). MS (M+1): 321.1; MS (M - H2O + 1): 303.3; MS (M - 2H2O + 1): 285.4.
Example 63: Preparation of (IS,2S,4S)-1-amino-2-(3-boronopropyl)-4-(dimethylamino)cyclopentanecarboxylic acid (racemic)
A stirred mixture of (1S,2S,4S)-1-acetamido-4-amino-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)propyl)cyclopentanecarboxamide (102.4 mg, 0.25 mmol) and 37% aqueous formaldehyde (0.07 ml, 0.94 mmol) in 1,2-dichloroethane (2 ml) was treated with triethylamine ( one drop), then with sodium triacetoxyborohydride (0.20 g, 0.94 mmol) and stirred at room temperature for 2 days, then quenched with saturated aqueous sodium bicarbonate (15 ml). The mixture was extracted with dichloromethane (2 x 10 ml) and the combined organic extracts were washed with water and saturated aqueous sodium chloride (5 ml each), dried (Na2SO4) and concentrated in vacuo. The crude product, in a pressure bottle, was dissolved in 2:1:1 concentrated HCl 10: glacial acetic acid: water (8 ml) and stirred for 2 h at 60°C, then capped and stirred for 18 h. °C, cooled to room temperature and uncapped. The solution was diluted with water (20 ml), extracted with dichloromethane (20 ml) and concentrated in vacuo. The resulting residue 15 was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in water (40 ml) and treated with DOWEX® 550A-OH resin (3 g) which had been rinsed with methanol. The mixture was stirred for 40 min and then filtered, and the resin washed successively with water, methanol, dichloromethane, water, methanol and dichloromethane. The resin was shaken four times with 1N HCl (15 ml) and filtered, and the combined filtrates were concentrated in vacuo. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in 1.5-2.0 ml of water. After purification by HPLC, the appropriate fractions were concentrated in vacuo, treated three times with 1N HCl (10 ml) and concentrated, treated three times with water (10 ml) and concentrated, then dissolved in water (10 30 ml) , frozen and lyophilized overnight to give the subject compound (24.5 mg, 30%) as a white foam. NMR (D2O) δ 4.00-4.10 (m, 1H), 2.81 (s, 3H), 2.79 (s, 3H), 2.70 (dd, J = 11 Hz, J2 = 7 Hz, 1H), 2.20-2.35 (m, 2H), 2.00-2.15 (m, 2H), 1.50 (m, 1H), 1.38 (m, 1H), 1 .25 (m, 1H), 1.06 (m, 1H), 0.60-0.75 (m, 2H). MS (M+1): 259, 3; MS (M - H2O + 1): 241.5; MS (M - 2H2O + 1): 223, 4.
Etapa 1: 2-A111-4-(nitrometil)ciclopentanona Uma solução agitada de 5-(propeno-3-il)ciclopent-2- enona (0,428, 3,5 mmol) em nitrometano (2 ml) sob nitrogênio foi tratada com resina 550A-OH de DOWEX® (0,80 g, enxaguada com metanol e parcialmente seca ao ar) , e aquecida até 60°C por 2 h. A mistura foi resfriada até a temperatura ambiente, diluída com diclorometano (20 ml) e filtrada. 0 filtrado foi concentrado in vacuo, redissolvido em um mínimo de diclorometano e adicionado a uma coluna de gel de sílica (aproximadamente 100 cm3) e eluído com diclorometano para gerar o composto em questão (0,368 g, 57%) como um óleo incolor. RNM (CDC13) : δ 5,65-5,80 (m, 1H) , 5,00-5,15 (m, 2H) , 4,40-4,50 (m, 2H) , 2,85-3,15 (m, 1H) , 2,30-2,70 (m, 4H) , 1, 90-2,20 (m, 3H) . MS (M + 1): 183,9.
Example 64: Preparation of (1S,2S,4R)-1-amino-4-(aminomethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid Step 1: 2-A111-4-(nitromethyl)cyclopentanone A stirred solution of 5-(propen-3-yl)cyclopent-2-enone (0.428, 3.5 mmol) in nitromethane (2 ml) under nitrogen was treated with DOWEX® 550A-OH resin (0.80 g, rinsed with methanol and partially air dried), and heated to 60°C for 2 h. The mixture was cooled to room temperature, diluted with dichloromethane (20 ml) and filtered. The filtrate was concentrated in vacuo, redissolved in a minimum of dichloromethane and added to a silica gel column (approximately 100 cm 3 ) and eluted with dichloromethane to give the subject compound (0.368 g, 57%) as a colorless oil. NMR (CDCl3 ): δ 5.65-5.80 (m, 1H), 5.00-5.15 (m, 2H), 4.40-4.50 (m, 2H), 2.85-3 .15 (m, 1H), 2.30-2.70 (m, 4H), 1.90-2.20 (m, 3H). MS (M+1): 183.9.

Step 2: (1-Acetamido-2-allyl-N-tert-butyl-4-(nitromethyl)cyclopentanecarboxamide, A and B isomers A stirred solution of 2-allyl-4-(nitromethyl)cyclopentanone, mixture of isomers (0.366 g, 2.0 mmol) in 2,2,2-trifluoroethanol (1.5 ml) under nitrogen was treated with ammonium acetate (0.617 g, 8 mmol) and t-butylisonitrile (0.68 ml, 6.0 mmol) and stirred at room temperature for 2 days. The mixture was diluted with dichloromethane (20 ml) and added directly to a silica gel column (approximately 250 cm3) and eluted with 7:3 dichloromethane/ethyl acetate to first generate the two isomers with acetamino and allyl substituents on syn relative geometry, then isomer A (122 mg, 19%) as a white solid, and then isomer B (195 mg, 30%) as a white solid. For Isomer A: NMR (CDCl3) : δ 6.12 (br s, 2H), 5.65-5.80 (m, 1H), 5.00-5.15 (m, 2H), 4.53 (d, J = 7Hz, 1H), 4.35-4.50 (m, 1H), 2.80-3.00 (m, 1H), 2.45-2.60 (m, 1H), 2.25-2.35 (m, 2H), 1.90-2.20 (m, 2H), 2.00 (s, 3H), 1.20-1.60 (m, 2H), 1. 34 (s, 9H). MS (M+1): 326.0. For Isomer B: NMR (CDC13): δ 6.05-6.15 (m, 2H), 5.65-5.80 (m, 1H), 5.00-5.15 (m, 2H), 4.43 (d, <7=6.5Hz, 2H), 2.90-3.10 (m, 2H), 2.40-2.50 (m, 1H), 2.20-2.30 (m, 1H), 2.00 (s, 3H), 1.70-2.00 (m, 4H), 1.35 (s, 9H). MS (M +
Step 3: (1-S,2S,4R)-1-acetamido-N-tert-butyl-4-(nitromethyl)-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan -2-yl)propyl)cyclopentanecarboxamide (racemic) While under nitrogen, a stirred solution of (1,2S)-1-acetamido-2-allyl-N-tert-butyl-4-(nitromethyl)cyclopentanecarboxamide, isomer A (0.180 g, 0.553 mmol) in anhydrous dichloromethane (5 ml) was treated with chloro-1,5-cyclooctadiene-iridium dimer (12 mg, 0.018 mmol) and Diphos® (14 mg, 0.035 mmol) and cooled (-25°C). ). After stirring for 30 min, pinacolborane (0.123 ml, 0.85 mmol) was added dropwise via syringe, and the vessel temperature was allowed to warm to ice bath temperature and gradually warmed to the temperature. overnight environment (18 h) . Water (3 ml) was added, the mixture stirred 20 min, then extracted with ethyl acetate (25 ml, then 10 ml). The combined organic solution was washed with water and saturated aqueous sodium chloride (20 ml each), dried (MgSO4) and concentrated in vacuo. Recrystallization from acetonitrile (2 crops) gave 0.170 g (68%) of the subject compound as a white solid. NMR (CDCl 3 ): δ 6.08 (br s, 1H), 5.92 (br s, 1H), 4.46 (d, J = 5 Hz, 2H), 2.75-2.90 (m, 1H), 2.49 (dd, J1 = 11Hz, J2 = 6Hz, 1H), 2.00-2.15 (m, 3H), 1.95 (s, 3H), 1.20-1. 50 (m, 5H), 1.25 (s, 9H), 1.17 (s, 12H), 0.65-0.85 (m, 2H). MS (M+1): 454.5.
Step 4: (1S,2S,4R)-1-Amino-4-(aminomethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic) A stirred solution of (1S,2S,4R)-1-acetamido -N-tert-butyl-4-(nitromethyl)-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (racemic), isomer A (0.167 g, 0.368 mmol) in ethanol (5 ml) and tetrahydrofuran (2 ml) under nitrogen was treated with Raney nickel (0.30 g), purged with hydrogen and stirred under a balloon for 20 h. The vial was purged with nitrogen and the mixture filtered through Celite® (carefully not allowing the filter cake to dry), and the filtrate concentrated in vacuo. The crude product, in a pressure bottle, was dissolved in 2:1:1 concentrated HCl:glacial acetic acid:water (8 ml) and stirred for 2 h at 60°C, then capped and stirred for 18 h at 130° C, cooled to room temperature and uncapped. The solution was diluted with water (20 ml), extracted with dichloromethane (20 ml) and concentrated in vacuo. The resulting residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in water (40 ml) and treated with DOWEX® 550A-OH resin (3 g) which had been rinsed with methanol. . The mixture was stirred for 40 min and then filtered, and the resin washed successively with water, methanol, dichloromethane, water, methanol and dichloromethane. The resin was shaken four times with 1N HCl (15 ml) and filtered, and the combined filtrates were concentrated in vacuo. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in 1.5-2.0 ml of water. After purification by HPLC, the appropriate fractions were concentrated in vacuo, treated three times with 1N HCl (10 ml) and concentrated, treated three times with water (10 ml) and concentrated, then dissolved in water (10 ml). frozen and lyophilized overnight to give the subject compound (62 mg, 53%) as a white foam. NMR (D20) δ 2.95-3.10 (m, 2H), 2.50 (m, 1H), 2.10-2.30 (m, 4H), 1.35-1.55 (m, 2H), 1.10-1.35 (m, 3H), 0.65-0.75 (m, 2H). MS (M+1): 245.2; MS (M -H20 + 1): 227.2.
Ácido (IS,2S,4S)-l-amino-4-(aminometil)-2-(3- boronopropil)ciclopentano-carboxilico (racêmico) foi preparado de forma análoga ao Exemplo 64, exceto que o isômero B foi usado na etapa de hidroboração. RNM (D2O) δ 2,85-3,15 ' (m, 2H) , 2,45-2,80 (m, 2H) , 2,15-2,35 (m, 1H) , 1,75-2,00 (m, 2H), 1,10-1,75 (m, 5H), 0,65-0,80 (m, 2H). MS (M+l) : 245,2; MS (M - H20 + 1): 227,1.'Example 65: Preparation of (IS,2S,4S)-1-amino-4-(aminomethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic) (IS,2S,4S)-1-Amino-4-(aminomethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic) was prepared analogously to Example 64, except that isomer B was used in step of hydroboration. NMR (D2O) δ 2.85-3.15' (m, 2H), 2.45-2.80 (m, 2H), 2.15-2.35 (m, 1H), 1.75-2 .00 (m, 2H), 1.10-1.75 (m, 5H), 0.65-0.80 (m, 2H). MS (M+1): 245.2; MS (M - H20 + 1): 227.1.'
Example 66: Preparation of (IS,2S,4R)-1-amino-4-(2-aminoethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid (racemic)
Step 1: 2-(3-Allyl-4-oxocyclopentyl)acetonitrile While under nitrogen, a cooled (-70°C) solution of trimethylsilylacetonitrile (1.83 g, 15 mmol) in anhydrous tetrahydrofuran (180 ml) was treated dropwise drop via syringe with 2.3 N n-butyllithium/hexane (7.4 ml, 17 mmol), stirred for 30 min, then treated with DMPU (9 ml). After a further 30 min at -70°C, a solution of 5-(propen-3-yl)cyclopent-2-enone (1.83 g, 15 mmol) in anhydrous tetrahydrofuran (20 ml) was added dropwise, and the temperature maintained at -70°C for 30 min, warmed slowly to -35°C, and the mixture quenched with 5% aqueous citric acid (90 ml). The reaction mixture was extracted with ethyl acetate (500 ml, then 2 x 75 ml), and the combined organic solution washed with water, saturated aqueous sodium bicarbonate and brine (150 ml each), dried (MgSO4 ) and concentrated in vacuum. The residue was dissolved in acetonitrile (162 ml) and treated with a solution of potassium fluoride (1.05 g, 18 mmol) in water (18 ml), and stirred for one hour. The solution was concentrated in vacuo to remove most of the acetonitrile, diluted with water (150 ml), and the aqueous mixture extracted with ether (150 ml, then 3 x 75 ml). The combined organic solution was washed with water, saturated aqueous sodium bicarbonate and brine (100 ml each), dried (MgSOJ and concentrated in vacuo.) The residual oil was dissolved in minimal methylene chloride, loaded onto a column of gel. silica (approximately 225 cm 3 ) and eluted with methylene chloride to give 1.64 g (67%) of 3-cyanomethyl-5-(propen-3-yl)cyclopentanone, mixture of isomers, as a pale yellow oil. CDCl 3 : δ 5.65-5.80 (m, 1H), 5.05-5.15 (m, 2H), 2.60-2.70 (m, 1H), 2.40-2.60 (m, 5H), 2.10-2.25 (m, 2H), 1.95-2.10 (m, 2H) MS (M+1): 164.3.
Step 2: 2-(9-allyl-1,4-dioxaspiro[4.4]nonan-7-yl)acetonitrile A stirred solution of 2-(3-allyl-4-oxocyclopentyl)acetonitrile, mixture of isomers (0.816 g, 5 mmol) in anhydrous toluene (40 ml) under nitrogen was treated with ethylene glycol (0.56 ml, 10 mmol) and toluenesulfonic acid hydrate (40 mg, 0.21 mmol), refluxed under a Dean-Stark apparatus for 8 h, concentrated in vacuo, and the residue dissolved in ether (100 ml). The organic solution was washed with water, bicarbonate of; saturated aqueous sodium and brine (50 ml each), dried (MgSO4) and concentrated in vacuo. The residual oil was dissolved in a minimum of methylene chloride, loaded onto a silica gel column (approximately 200 cm 3 ) and eluted with 30% ethyl acetate/heptane to give 0.875 g (84%) of 3-cyanomethyl-5 -(propen-3-yl)cyclopentanone, ketal with ethylene glycol, as a pale yellow oil. NMR (CDCl3 ): δ 5.70-5.85 (m, 1H), 4.95-5.10 (m, 2H), 3.85-4.00 (m, 4H), 2.40 (m) , 2H) 2.25-2.35 (m, 2H), 1.90-2.20 (m, 4H), 1.65-1.80 (m, 1H), 1.60 (m, 1H) . MS (M+1): 208.0.
Step 3: tert-butyl 2-(3-allyl-4-oxocyclopentyl)ethylcarbamate
An ice-cold stirred solution (3°C) of 2-(9-allyl-1,4-dioxaspiro[4.4]nonan-7-yl)acetonitrile with ethylene glycol (0.829 g, 4.0 mmol) in anhydrous tetrahydrofuran (30 ml) ) was treated via syringe with 2N lithium aluminum hydride/tetrahydrofuran (6 ml, 12 mmol) and refluxed for 4 h (precipitate formed). The mixture was cooled in an ice bath and successively treated carefully with water (0.5 ml), 15% aqueous sodium hydroxide (0.5 ml) and water (1.5 ml), filtered, and the filtrate concentrated in vacuo. The residual oil was dissolved in methylene chloride (20 ml), treated with di-t-butyldicarbonate (1.09 g, 5 mmol), stirred for 3 h and concentrated in vacuo. The residue was dissolved in 5:1 acetone/water (30 ml), treated with montmorillonite K-10 clay (5 g), refluxed for 6 h, cooled to room temperature and filtered through Celite®. The filtrate was concentrated in vacuo, the residue was diluted with water (20 ml), and the aqueous solution extracted with methylene chloride (2 x 30 ml). The combined organic solution was washed with water (25 ml), dried (Na2SO>4), and concentrated in vacuo. The residue was dissolved in a minimum of methylene chloride, added to a silica gel column (approximately 150 cm3) and eluted with 5% ethyl acetate/methylene chloride to give 0.51 g (48%) of the compound in matter as a colorless oil. NMR (CDCl3 ): δ 5.60-5.75 (m, 1H), 4.90-5.05 (m, 2H), 4.47 (m, 1H), 3.10 (m, 2H), 2.40-2.50 (m, 1H), 2.20-2.40 (m, 2H), 2.10-2.20 (m, 1H), 1.90-2.10 (m, 2H) ), 1.65-1.90 (m, 2H), 1.45-1.60 (m, 2H), 1.37 (s, 9H). MS (M+1): 268.2; MS (M + Na): 290.1.
Step 4: tert-butyl 2-((1R,3S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentyl)ethylcarbamate
A stirred solution of tert-butyl 2-(3-allyl-4-oxocyclopentyl)ethylcarbamate, mixture of isomers (0.50 g, 1.87 mmol) in 2,2,2-trifluoroethanol (2 ml) under nitrogen was treated with ammonium acetate (0.62 g, 8 mmol) and t-butylisonitrile (0.68 ml, 6.0 mmol) and stirred at room temperature for 3 days. The mixture was diluted with methylene chloride (20 ml) and added directly to a silica gel column (approximately 250 cm3) and eluted with 1:1 ethyl acetate/heptane, then 2:1 ethyl acetate/ heptane to give tert-butyl 2-((1R,3S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentyl)ethylcarbamate (260 mg, 34%) as a white solid. NMR (CDCl3 ): δ 6.25 (br s, 1H), 6.00 (br s, 1H), 5.55-5.70 (m, 1H), 4.90-5.00 (m, 2H) ), 4.48 (m, 1H), 3.03 (m, 2H), 2.35-2.80 (m, 1H), 2.27 (m, 1H), 2.00-2.20 ( m, 2H), 1.70-2.00 (m, 2H), 1.93 (s, 3H), 1.45-1.65 (m, 4H), 1.36 (s, 9H),
Step 5: tert-butyl 2-((1R,3S)-3-acetamido-3-(tert-butylcarbamoyl)-4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2) -yl)propyl)cyclopentyl)ethylcarbamate
While under nitrogen, a stirred solution of tert-butyl 2-((IR,3S)-3-acetamido-4-allyl-3-(tert-butylcarbamoyl)cyclopentyl)ethylcarbamate (0.25 g, 0.61 mmol) in anhydrous methylene chloride (6 ml) was treated with chloro-1,5-cyclooctadiene-iridium dimer (14.5 mg, 0.021 mmol) and Diphos® (17 mg, 0.042 mmol) and cooled (-25°C). After stirring for 30 min, pinacolborane (0.134 ml, 0.92 mmol) was added dropwise via syringe, and the vessel temperature was allowed to warm to ice bath temperature and gradually warmed to the temperature. overnight environment (18 h) . Water (4 ml) was added, the mixture stirred for 20 min, then extracted with ethyl acetate (30 ml, then 20 ml). The combined organic solution was washed with water and brine (20 ml each), dried (MgSO4 ) and concentrated in vacuo. Recrystallization from acetonitrile (2 crops) gave 0.136 g of the subject compound as a white solid. Silica gel chromatography (eluted with 70:30 ethyl acetate/heptane) of the concentrated mother liquor gave an additional 0.102 g of the subject compound. The total yield was 0.238 g (73%). NMR (CDCl3 ): δ 5.95-6.10 (m, 2H), 4.50 (m, 1H), 3.03 (m, 2H), 2.00-2.85 (m, 4H), 1.92 (s, 3H), 1.00-1.70 (m, 8H), 1.37 (s, 9H), 1.25 (s, 9H), 1.17 (s, 12H), 0 , 60-0.75 (m, 2H). MS (M+1): 538.1; MS (M+Na): 560.4.
Step 6: (1R,2S,4R)-1-Amino-4-(2-aminoethyl)-2-(3-boronopropyl)cyclopentanecarboxylic acid 2-((1R,3S)-3-acetamido-3-( tert-butylcarbamoyl) —4—(3—(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentyl)ethyl carbamate (0.226 g, 0.42 mmol), in a pressure bottle, was dissolved in 2:1:1 concentrated HCl:glacial acetic acid:water (8 ml) and stirred for 2 h at 60°C, then capped and stirred for 18 h at 130°C, cooled to room temperature and uncapped. The solution was diluted with water (20 ml), extracted with dichloromethane (20 ml) and concentrated in vacuo. The resulting residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in water (40 ml) and treated with DOWEX® 550A-OH resin (3 g) which had been rinsed with methanol. . The mixture was stirred for 40 min and then filtered, and the resin washed successively with water, methanol, dichloromethane, water, methanol and dichloromethane. The resin was shaken four times with 1N HCl (15 ml) and filtered, and the combined filtrates were concentrated in vacuo. The residue was treated with water (20 ml) and concentrated three times to remove excess HCl, then dissolved in 1.5-2.0 ml of water. After purification by HPLC, the appropriate fractions were concentrated in vacuo, treated three times with 1N HCl (10 ml) and concentrated, treated three times with water (10 ml) and concentrated, then dissolved in water (10 ml). frozen and lyophilized overnight to give the compound in question (85 mg,. 61% )■ ■ as a white foam. NMR (D2O) δ 2.85-3.00 (m, 2H), 2.64 (br s, 1H), 2.35-2.60 (m, 1H), 2.05-2.25 (m , 2H), 1.65-1.85 (m, 3H), 1.35-1.55 (m, 2H), 1.10-1.30 (m, 3H), 0.65-0.80 (m, 2H). MS (M+1): 259.0; MS (M - H20 + 1): 241.2. METHODS AND USES
The compounds of the invention are useful for inhibiting the expression or activity of arginase I, arginase II or a combination of these enzymes. Enzymes of the arginase family play an important role in regulating the physiological levels of L-arginine, a precursor to the nitric oxide (NO) signaling molecule, as well as in regulating the levels of certain polyamines that are important physiological signal transducers.
More specifically, the invention provides methods and uses for inhibiting arginase I, arginase II, or a combination thereof, in a cell, which comprises contacting the cell with at least one compound according to Formula I or Formula II, or composition thereof. , as described here. In some embodiments, the invention provides a method for treating or preventing a disease or condition associated with the expression or activity of arginase I, arginase II, or a combination thereof, in an individual.
For example, the disease or condition is selected from the group consisting of heart disease, hypertension, sexual disorders, gastric disorders, autoimmune disorders, parasitic infections, lung disorders, smooth muscle relaxation disorders and: hemolytic disorders.
More specifically, hypertension includes systemic hypertension, pulmonary arterial hypertension (PAH), and pulmonary arterial hypertension at high altitude.
Exemplary sexual disorders are diseases or conditions selected from the group consisting of Peyronie's disease and erectile dysfunction (ED).
In one embodiment, an arginase inhibitor according to the present invention is suitable for the treatment of a pulmonary disorder selected from the group consisting of chemically induced pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease (COPD).
The compounds according to the present invention are also useful for the treatment of gastrointestinal disorders, for example, diseases or conditions selected from the group consisting of gastrointestinal motility disorders, gastric cancers, reduced hepatic blood flow disorders, inflammatory bowel disease, Crohn's disease, ulcerative colitis and gastric ulcers.
Organ transport increases the risk of ischemia (IR) reperfusion injury, eg, liver IR, renal IR, and myocardial IR. Compounds of Formula I or Formula II according to the present invention are useful in organ protection during organ transport.
In another embodiment, arginase inhibitors according to the present invention are used to treat hemolytic disorders selected from the group consisting of paroxysmal nocturnal hemoglobinuria (BNH), sickle cell disease, thalassemias, hereditary spherocytosis and stomatocytosis, microangiopathic hemolytic anemias, pyruvate deficiency kinase, ABO transfusion incompatibility reaction, paroxysmal cold hemoglobinuria, severe idiopathic autoimmune hemolytic anemia, infection-induced anemia, cardiopulmonary bypass, mechanical heart valve-induced anemia, and chemical-induced anemia. Furthermore, the compounds described herein are useful in treating malaria.
The compounds of the invention are useful in the treatment of autoimmune diseases selected from the group consisting of encephalomyelitis, multiple sclerosis, antiphospholipid 1 syndrome, autoimmune hemolytic anemia, chronic inflatory demyelinating polyradiculoneuropathy, dermatitis herpetiformis, dermatomyositis, myasthenia gravis, pemphigus, rheumatoid arthritis rigid person, type 1 diabetes and ankylosing spondylitis. In another embodiment, compounds of Formulas I or II are useful for the treatment of immune disorders selected from the group consisting of immune responses, T cell dysfunction, e.g., myeloid derivation suppressor cell (MDSC) mediated T cell dysfunction , human immunodeficiency virus (HIV) and autoimmune encephalomyelitis.
Other exemplary disease conditions for which the compounds described herein are candidate therapeutic substances are African sleeping disease, Chagas disease, smooth muscle relaxation disorders, e.g. smooth muscle disorders selected from the group consisting of a muscle disorder. gastrointestinal smooth muscle, anal sphincter smooth muscle, esophageal sphincter muscle, corpus cavernosum, sphincter of Oddi, arterial smooth muscle, cardiac smooth muscle, pulmonary smooth muscle, renal smooth muscle, uterine smooth muscle, vaginal smooth muscle, cervical smooth muscle, placental smooth muscle, and ocular smooth muscle.
Increased arginase levels in certain cancer patients imply a therapeutic role for the arginase inhibitors of the invention in the treatment of certain cancers, eg renal cell carcinoma, prostate cancer, colorectal cancer, breast cancer, cancer skin cancer, lung cancer, ovarian cancer, gastric cancer.
Advantageously, the compounds of the invention are especially useful in the treatment of conditions or disorders selected from the group consisting of arthritis, myocardial infarction and atherosclerosis, kidney disease, asthma, inflammation, psoriasis, leishmaniasis, sickle cell disease (SCD), neurodegenerative diseases, scarring of wounds, for example, infected and uninfected wound healing, hepatitis B virus (HBV), H. pylori infections, fibrotic diseases such as, for example, cystic fibrosis, candidiasis, periodontal disease, keloid, adenotonsillar disease, cerebral vasospasm and Goodpasture's syndrome.
More specific descriptions of diseases and conditions will be presented below. erectile dysfunction
The observation that there are differences in arginase activity in the penis of young versus older mice led to the conclusion that arginase may play a role in erectile dysfunction (ED). In this context, Champion et al., Am. J. Physiol. Heart Circ. Physiol. 292: 340-351, (2006) and Biochem. and Biophys. "Research Communications", 283: 923-27, (2001) observed an increase in mRNA and arginase protein expression levels in aged mice along with a reduction in constitutively active NOS activity.
Nitric oxide is implicated in non-adrenergic, non-cholinergic neurotransmission that leads to smooth muscle relaxation in the corpus cavernosum, which allows penile erection (New England Journal of Medicine, 326, (1992)). Thus, erectile dysfunction can often be treated by raising nitric oxide (NO) levels in penile tissue. This increase in tissue levels of nitric oxide (NO) can be achieved by inhibiting the arginase activity in the penile tissue of elderly individuals. In other words, it has been postulated that arginase depletes the pool of free L-arginine available to NOS in cells, which results in lower levels of nitric oxide (NO) and erectile dysfunction. See Christianson et al., Acc. Chem. Res., 38: 191-201, (2005), and Nature Structural Biol., 6(11): 1,043-1047, (1999). Arginase inhibitors, therefore, may participate in the treatment of erectile dysfunction.
In addition to its role in male sexual function, Christianson et al. (Biochemistry, 42: 8.445-51, (2003)) have proposed a role for ARG II in female sexual arousal. The underlying mechanism by which ARG II inhibition promotes arousal, however, appears to be the same as that for promoting male arousal. That is, inhibition of ARG II increases the level of free L-arginine available as a substrate for NOS. This causes increased levels of NO in the corpus cavernosum of the clitoris and thus leads to increased sexual arousal.
Pulmonary hypertension It has been proposed that alterations in arginine metabolism are involved in the pathogenesis of pulmonary hypertension (Xu et al., FASEB J., 18: 1.746-48, 2004). The proposition is based, in part, on the finding that arginase II expression and arginase activity are significantly elevated in pulmonary artery endothelial cells derived from pulmonary explants from patients with class I pulmonary hypertension.
Additionally, secondary pulmonary hypertension emerges as one of the main causes of mortality and morbidity in patients suffering from hemolytic anemias, for example, thalassemia and sickle cell disease. The underlying cause of secondary pulmonary hypertension is a deficit in nitric oxide bioavailability as a consequence of arginase release after hemolysis, which decreases the pool of free arginine that is required for nitric oxide (NO) synthesis. Consequently, inhibition of arginase activity may provide a potential therapeutic avenue for the treatment of pulmonary hypertension. Hypertension
Xu et al. (FASEB 2004, 14, 1746-8) proposed a key role for arginase II in regulating blood pressure. In this context, high levels of vascular arginase are correlated with a concomitant reduction in vascular nitric oxide (NO) in hypertensive animals. For example, upregulation of arginase activity precedes elevation of blood pressure in rats that were genetically predisposed to hypertension (ie, spontaneously hypertensive rats), but administration of the antihypertensive agent hydralazine reduced blood pressure with a decrease in vascular arginase expression levels, thus indicating a strong correlation between arginase activity and blood pressure (Berthelot et al. Life Sciences, 80: 1.128-34, (2008)). Similar administration of the known arginase inhibitor N®-hydroxy-nor-L-arginine (nor-NOHA) reduced blood pressure and increased the vascular response of blood flow resistance vessels and pressure in spontaneously hypertensive animals, thus enhancing arginase inhibitors as candidate therapeutic substances for the treatment of hypertension (Demougeot et al., J. Hypertension, 26: 1110-18, (2008)).
Arginase also participates in reflex cutaneous hypertension by reducing cellular levels of nitric oxide (NO). Nitric oxide causes vasodilation and nitric oxide (NO) levels are normally raised or lowered to maintain blood pressure at physiologically acceptable levels. Kenny et al., (J. of Physiology 581 (2007) 863-872) formulated the hypothesis that reflex vasodilation in hypertensive individuals may attenuate arginase inhibition, thus suggesting a role for arginase inhibitors in treatment of hypertension. Asthma
Arginase activity is also associated with airway hyperreactivity in asthma. For example, arginase I is up-regulated in human asthmatics and in mice suffering from acute and chronic asthma, whereas levels of arginase II and NOS isoforms remain unchanged (Scott et al., (Am. J. Physiol. Lung Cell. Lung Cell). Mol. Physiol. 296: 911-920 (2009).) Furthermore, the methacholine-induced responsiveness of the central airways in the chronic murine model was attenuated after administration of the arginase inhibitor .S-(2-boronoethyl)-L- The similarity between the expression profiles of ARG I in humans and in mice that have chronic asthma indicates that compounds capable of inhibiting arginase activity are candidate therapeutic substances for the treatment of asthma.
Other lines of evidence reveal additional correlations between increased arginase activity in asthmatic lung tissue and disease progression, for example, an upregulation for genes related to cationic amino acid metabolism, including Arginase I and II, in mice with asthma (Rothenberg and cols., (J. Clin. Invest., Ill: 1.863-74 (2003), and Meurs et al., (Expert Opin. Investig Drugs, 14 (10: 1.221-1,231, (2005)).
Furthermore, all amino acid levels are lower in the plasma of asthmatics, but arginine levels are significantly lower in plasma compared to those of a normal individual (Morris et al., Am. J. Respir. Crit. Care Med. , 170: 148-154, (2004)). Thus, arginase activity is significantly increased in the plasma of an asthmatic, and these high levels of arginase activity may contribute to the lower bioavailability of plasma arginine, which creates a deficiency of nitric oxide (NO), which is responsible for promotion of hyperreactive airways in asthmatics. Inflammation
Arginase activity is also associated with autoimmune inflammation (Chen et al., Immunology, 110: 141-148, (2003)). The authors identified upregulation of ARG I gene expression levels in murine spinal cells from animals suffering from experimental autoimmune encephalomyelitis (EAE). Administration of the arginase inhibitor amino-6-boronohexanoic acid (ABH), however, causes animals to develop a much milder form of EAE than in control animals. These results suggest a therapeutic role for arginase inhibitors in the treatment of autoimmune encephalomyelitis.
Furthermore, Horowitz et al. (American J. Physiol. Gastrointestinal Liver Physiol., 292: Gl.323-36 (2007)) suggest a role for arginase enzymes in vascular pathophysiology. For example, these authors indicate a loss of nitric oxide (NO) production in chronically inflamed intestinal blood vessels in patients suffering from irritable bowel disease (IBD), Crohn's disease, and ulcerative colitis. The loss of nitric oxide (NO) production was correlated with an upregulation of arginase expression and activity that reduced arginine levels, preventing nitric oxide synthase (NOS) from synthesizing nitric oxide (NO). Arginase activity inhibitors, therefore, can be candidate therapeutic substances for the treatment of vascular pathophysiology. Ischemia reperfusion
It is also suggested that arginase inhibition plays a cardioprotective role during reperfusion of ischemia. More specifically, arginase inhibition protects against myocardial infarction by a mechanism that may be dependent on NOS activity and the consequent bioavailability of nitric oxide (NO) (Pernow et al., Cardiovascular Research, 85: 147-154 (2010) )).
Myocardial infarction and arteriosclerosis Arginase I polymorphism is associated with myocardial infarction, along with an increased risk for the development of carotid artery intima media thickness, which is considered to be a reliable indicator of atherosclerosis, as well as others coronary artery disease (Brousseau et al., J. Med Genetics, 44: 526-531, (2007)). The increased activity of arginase elevates the levels of ornithine, which is biochemically involved in promoting the formation of matrix and cellular components of the atherosclerotic plaque. Id. Thus, arginase inhibitors can serve as candidate therapeutic substances for the treatment of arteriosclerosis. Berkowitz et al. (Circulation Res. 102, (2008)) have suggested a role for ARG II in plaque formation and arteriosclerosis. The LDLP oxidation that accompanies plaque formation increases arginase activity and reduces nitric oxide (NO) levels in endothelial cells. In particular, ARG II levels are elevated in atherosclerotic mice, which indicates a role for arginase inhibitors as candidate therapeutic substances for the treatment of arteriosclerosis.
Additionally, studies by Ming et al. (Current Hypertension Reports., 54:54-59, (2006)), indicate that arginase upregulation, rather than endothelial nitric oxide (NO) dysfunction, plays an important role in cardiovascular disorders, including arteriosclerosis. The fact that arginase is involved in cardiovascular disease is further supported by the observation that ARG I and ARG II activity is up-regulated in cardiac myocytes which, in turn, negatively affect NOS activity and myocardial contractility (see , Margulies et al., Am. J. Physiol. Heart Ciro. Physiol., 290: 1756-62, (2006)).
Immune response The arginine/nitric oxide (NO) pathway may also participate in the immune response, for example, after organ transplants. For example, it has been proposed that reperfusion of a prototopic liver transplant graft would cause a significant increase in ornithine levels as a consequence of upregulation of arginase activity in the graft ( Tsikas et al., Nitric oxide, 20: 61 - 67, (2009)). Elevated levels of hydrolytic and proteolytic enzymes in the graft can result in a less favorable end result for the grafted organ. Thus, the inhibition of arginase enzymes may present an alternative therapeutic avenue to improve the final result of a transplant.
Psoriasis It has been suggested that arginase participates in the pathogenesis of psoriasis. For example, ARG I is highly expressed in hyperproliferative psoriasis and, in fact, is responsible for the 'downregulation of nitric oxide (NO), an inhibitor of cell proliferation, by competition for the common substrate L-arginine, as postulated by D Bruch-Gerharz et al., American Journal of Pathology 162(1) (2003) 203-211. A more recent work by Abeyakirthi et al. (British J. Dermatology, (2010)), and Berkowitz et al., (WO/2007/005620) support the findings of low levels of nitric oxide (NO) in psoriatic keratinocytes. Abeyakirthi et al. found that psoriatic keratinocytes were poorly differentiated and hyperproliferative. It has been suggested that low differentiation results from low levels of nitric oxide (NO), not because of low NOS expression, but rather because of overexpression of arginase that competes with NOS for the substrate L-arginine. Thus, arginase inhibition may provide therapeutic relief from psoriasis. wound healing
Under normal physiological conditions, nitric oxide (NO) plays an important role in promoting wound healing. For example, Hulst et al. (Nitric Oxide, 21: 175-183, (2009) ) , studied the role of ARG I and ARG II in wound healing. Immediately after injury, it is desirable to elevate tissue levels of nitric oxide (NO) in order to promote angiogenesis and cell proliferation that are important for healing. Arginase inhibitors may therefore find utility as therapeutic substances for the treatment of wounds, as these compounds would elevate tissue levels of nitric oxide (NO). Additional support for the use of arginase inhibitors as candidate therapeutic substances for the treatment of wounds was provided by South et al. (Experimental Dermatology, 29: 664-668 (2004)), who found a 5-fold increase in arginase I in chronic wounds such as erosions and skin blisters. Cystic fibrosis
Cystic fibrosis (CF) is a multisystem disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Common symptoms of CF are persistent lung infection, difficulty breathing, pancreatic insufficiency, and elevated sweat chloride levels. CF can be fatal if left untreated, with lung diseases resulting from mucus formation and decreased mucociliary clearance being the leading cause of morbidity and mortality.
Patients with cystic fibrosis (CF) have been found to have increased arginase activity in plasma and sputum, with an associated decrease in plasma L-arginine levels (H. Grasemann et al., Am. J. Respir. Crit. Care Med. 172(12) (2005) 1.523-1.528). The increased activity of arginase, however, results in lower physiological levels of nitric oxide (NO) which can cause airway obstruction and decreased lung function in patients suffering from cystic fibrosis (CF) .
Deficiency of electric field-induced stimulation of smooth muscle relaxation in the airways of a mouse model of CF and the administration of L-arginine and NO reversed this effect, as proposed by M. Mhanna et al. (Am. J. Respir. Cell Mol. Biol. 24(5) (200) 1 621-626). Graesmann et al found a positive correlation between lung function and exhaled NO and NO metabolite concentrations in the sputum of patients with CF (Grasemann et al 1997, 1998).
Taken together, these results indicate that increased arginase activity in FC contributes to NO deficiency and pulmonary obstruction in FC by limiting the availability of L-arginine to NOS. Thus, arginase activity inhibitors are candidate therapeutic substances for the treatment of cystic fibrosis (CF). Organ protection
Another therapeutic avenue for compounds according to the present invention is the protection of organs during transport from the donor to a site where they will be transplanted into a recipient. Ischemic reperfusion (IR) injury as a result of exposure of transplant organs to a period of warm ischemia (time from removal of the donor to being rinsed with preservatives) and cold ischemia (hypothermic preservation) is frequently observed in patients who undergo transplant surgery. Ischemic reperfusion (IR) injury and accompanying primary graft dysfunction and/or acute or chronic rejection result from altered cellular activity of the L-Arginine/NO pathway.
It has been proposed that arginase 1 and arginase 2 are released by endothelial cells and apoptotic kidney cells within the first 24 hours after removal of the organ from the body. To counteract the released arginase, L-Arginine is added to the preservation media. Results with canine kidney transplants indicate that the addition of L-arginine reduced the incidence and severity of ischemia, resulting in a post-transplant with lower MDA levels at 1 hour, and reduced BUN and serum creatinine levels during the first 72 hours. See Erkasap, 2000.
Similar results were observed for canine lung grafts over a 24-hour period when the lungs were preserved in the University of Wisconsin solution supplemented with L-Arginine. Yen et al. observed that the addition of L-arginine to the preservation medium increased pulmonary endothelial protection and reduced the incidence of ischemia, when compared to a control that is preserved in medium that does not contain L-arginine (Yen Chu, 2004).
Koch et al. stated that myocardial contractility and relaxation in rat cardiac muscle after transplantation increased when hearts were preserved in HTK solution with L-Arginine and N-alpha-acetyl-histidine (Koch, 2009).
The addition of an arginase inhibitor, therefore, may be a candidate therapeutic approach for preservation and/or reduction in the incidence and risk of ischemic reperfusion injury by a synergistic increase in the organ's protective effect of the preservation media. Considering the low number of available organs that are suitable for transplantation and the loss and damage of organs as a result of the onset of ischemia, arginase inhibitors according to the present invention can be useful as therapeutic substances for the preservation of organs, increasing the availability of organs by reducing the amount of ischemic reperfusion injury during organ transport. leishmaniasis
Leishmaniasis is caused by a protozoan and manifests as cutaneous leishmaniasis (ie, a skin infection causing hypopigmented nodules) and visceral leishmaniasis (more severe, affecting internal organs). It is proposed that arginase participates in disease progression, as the parasite depends on arginase for the synthesis of cellular polyamines that are essential for pathogenesis. Inhibition of arginase, therefore, could reduce the cellular parasitic load and promote increased levels of nitric oxide (NO), increasing parasite clearance. See Liew F.Y. et al. Eur. J. Immunol. 21 (1991) 2489, Iniesta V. et al. Parasite Immunol. 24 (2002) 113-118 and Kane M.M. et al. J. Immunol. 166 (2001) 1,141-1,147. The compounds according to Formula I or Formula II, therefore, can be used as therapeutic substances for the treatment of leishmaniasis.
Myeloid Derivation Suppressor Cells (MDSC) MDSCs are potent immune modulators that limit immune responses by several pathways, eg, L-arginine depletion through the release of arginase 1 into the microenvironment (Rodriguez 2009 Cancer Res.), restricted suppression of MHC (Nagaraj 2007 Nat. Med.), induction of regulatory T cells (Serafmi 2008 Cancer Res.) and IL-10 production (Rodrigues 2010 Neuro. Oncol.) (Sinha 2007 J. Immunol.), for example.
Tumor development is postulated to be accompanied by an increase in the number of MDSCs, both peripherally and infiltrating within tumors. See Almand 2001 J. Immunol. and Gabrilovich 2004 Nat. Rev. Immunol. Treating mice with tumors with established chemotherapeutics such as gemcitabine and 5-fluorouracil eliminates MDSC immunosuppression and results in tumor growth retardation. See Le 2009 Int. Immunopharmacol. And Vincent 2010 Cancer Res., respectively. Furthermore, inhibition of arginase I increased antitumor immunity by reducing MDSC function. Thus arginase inhibitors, e.g. compounds according to the present invention, reduce or delay tumor growth and can be used in combination with established anti-cancer agents in the treatment of cancer.
Helicobacter pylori (H. pylori) Helicobacter pylori (H. pylori') is a Gram-negative bacterium that colonizes the human gastric mucosa. Bacterial colonization can lead to acute or chronic gastritis and is highly associated with peptic ulcer disease and stomach cancer. The observation that the addition of L-arginine to the coculture of H. pylori and macrophages increased nitric oxide (NO)-mediated killing of H. pylori (Chaturvedi 2007) supports the hypothesis that bacterial arginase competes with arginase of macrophages by free arginine which is required for the synthesis of nitric oxide (NO). See Gobert A.P. 2001. L-arginine is required for T cell activation and for the rapid clearance of bacteria from infected cells. By depleting free L-arginine pools in vivo, H. pylori reduces arginine-induced CD3-zeta expression on T cells and prevents T cell activation and proliferation. See Zabaleta J. 2004.
Inhibition of bacterial arginase using the known inhibitor NON, however, re-established CD3 expression on T cells (Zabaleta J 2004) and increased CD3 production.
NO by macrophages thereby promoting macrophage-measured clearance of bacteria from infected cells. See Chaturvedi 2007.
Furthermore, Lewis et al. suggested a role for arginase II in H. pylori infection. For example, these authors indicate that primary argill-/- macrophages incubated with H. pylori extracts showed increased NO production and, correspondingly, increased NO-mediated killing (approximately 15%) of bacterial cells (Lewis ND 2010) . Arginase activity inhibitors, therefore, can be candidate therapeutic substances for the treatment of vascular pathophysiology. Arginase activity inhibitors, therefore, may be candidate therapeutic substances for the treatment of H. pylori infections and for the treatment of gastric ulcers, peptic ulcers, and cancer.
Sickle Cell Disease (SCD) Sickle cell disease (SCD), or sickle cell anemia, or sickle cell disease, is a genetic blood disorder characterized by red blood cells that take on an abnormal, rigid, sickle cell shape. The sickle shape decreases the cells' flexibility and increases the risk of complications. An increase in the concentration of circulating reactive oxygen species (ROS) causes blood cell adhesion and NO consumption that results in a deficiency in vasodilation or inability to vasodilate blood vessels. The inability to vasodilate, together with the increased adherence of blood cells in SCD, results in vessel occlusive crisis and pain.
Low levels of plasma L-arginine are commonly detected in patients with SCD (Morris 2005 JAMA). According to these authors, lysis of erythrocytes (RBCs) in patients suffering from SCD causes the release of arginase and a subsequent reduction in physiological levels of L-Arginine. This sequence of biological events reduces physiological concentrations of nitric oxide (NO), a signaling molecule that participates in vasodilation. Other biological events also limit NO bioavailability. These include, for example, the uncoupling of nitric oxide synthase (NOS) and the subsequent decrease in physiological levels of NO, as well as the reaction of reactive oxygen species of superoxide (O-2) with NO to sequester the latter as ONOO".
Based on these observations, arginase inhibitors, especially arginase I inhibitors, are being proposed by the present inventors as candidate therapeutic substances for patients with sickle cell disease. As stated above, SCD causes the uncoupling of eNOS due to low physiological levels of L-arginine. Inhibition of arginase present in the bloodstream, however, can solve this problem by increasing physiological levels of L-arginine, the substrate of endothelial nitric oxide synthase (eNOS). This sequence of events, notably, is proposed by the present inventors as responsible for the increase in endothelial function and relief of the vasoconstriction associated with SCD. Human immunodeficiency virus (HIV)
HIV disease is caused by a virus that infects CD4+ helper T cells and causes severe lymphopenia that predisposes infected individuals to opportunistic infection. Although antiretroviral therapy (ART) is used extensively to combat HIV infection, the widespread use of antiretroviral drugs has resulted in the generation of resistant HIV strains.
There is a correlation between arginase activity in patients suffering from HIV and the severity of the HIV disease. That is, increased arginase activity was correlated with increased viral titers in HIV patients. These patients also show decreased serum arginine levels as well as decreased CD4+/CD8+ cell levels.
Taken together, these observations suggest a role for arginase inhibitors, eg compounds according to Formula I or II, as candidate therapeutic substances in the treatment of HIV infection.
Chronic hepatitis B virus (HBV) Chronic hepatitis B infection is a viral disease that is transmitted by contact with infected body fluids. Chronic HBV infections are characterized by liver inflammation and jaundice and, if left untreated, can cause cirrhosis of the liver that can progress to form hepatocellular carcinomas. Currently used antiviral drugs, however, have low efficacy against chronic HBV infections. Serum and liver homogenates from patients with chronic HBV infections show reduced arginine levels and increased arginase activity. For infected patients, in addition, increased arginase activity is correlated with a deficient cytotoxic T lymphocyte (CTL) response, with reduced IL-2 production and CD3z expression.
The restoration of serum arginine to physiologically acceptable levels, however, reconstituted the expression of CD3z and IL-2, suggesting a role for arginase forinhibitors as potential therapeutic substances in the treatment of chronic HBV infections. Routes of administration and dosage regimen
Despite ample evidence linking arginase inhibition with therapies for various diseases and conditions, only a limited number of compounds are known that are capable of inhibiting arginase activity. The present invention, therefore, provides compounds and pharmaceutical compositions thereof that are useful in treating an individual suffering from such a disease or condition, as more generally set out above.
In one modality, the individual receiving treatment is a mammal. For example, the methods and uses described herein are suitable for human medical use. Alternatively, the methods and uses are also suitable in a veterinary context, where the individual includes, without limitation, a dog, cat, horse, cow, sheep and lamb.
The compound or composition of the invention can be formulated as described herein above and is suitable for administration in a therapeutically effective amount to the individual in various ways. The therapeutically effective amount of a compound of Formula I or Formula II may depend on the amounts and types of excipients used, the specific amounts and types of active ingredients in a dosage form, and the route by which the compound is to be administered to patients. However, typical dosage forms of the invention comprise a compound, or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.
Typical dosage levels for compounds of Formula I or Formula II generally range from about 0.001 to about 100 mg per kg of patient body weight per day, which can be administered in single or multiple doses. An exemplary dosage is about 0.01 to about 25 mg/kg per day or about 0.05 to about 10 mg/kg per day. In other embodiments, the dosage level is about 0.01 to about 25 mg/kg per day, about 0.05 to about 10 mg/kg per day, or about 0.1 to about 5 mg /kg per day.
A dose typically ranges from about 0.1 mg to about 2000 mg per day, given as a single dose once daily or, alternatively, as divided doses throughout the day, optionally taken with food. In one modality, the daily dose is administered twice a day in equally divided doses. A daily dose range can be from about 5 mg to about 500 mg per day, for example, from about 10 mg to about 300 mg per day. In patient treatment, therapy can be started at a lower dose, perhaps from about 1 mg to about 25 mg, and increased, if necessary, to from about 200 mg per day to about 2,000 mg per day, administered as a single dose or divided into multiple doses, depending on the patient's overall response.
Depending on the disease to be treated and the condition of the individual, the compounds according to Formula I or Formula II may be administered by the oral, parenteral route of administration (eg intramuscular, intraperitoneal, intravenous, ICV, intrasternal injection or infusion, injection or subcutaneous implant), by inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal, local) administration. The compounds can be formulated, alone or together, in suitable unit dosage formulations which contain conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles, as described above, which are suitable for the route of administration. The invention also contemplates the administration of the compounds of the invention in a depot formulation, in which the active ingredient is released over a defined period of time. INHIBITION OF ARGINASE
The compounds of the invention inhibit human arginase I (ARG I) and arginase II (ARG II), as evidenced by an ex vivo assay presented by a published protocol (Baggio et al. J. Pharmacol. Exp. Ther. 1999, 290, 1409 - 1,416). The assay established the concentration of inhibitors that is required to reduce arginase activity by 50% (IC50), as shown in Table 2 below. test protocol
Inhibition of arginase I (ARG I) and arginase II (ARG II) by compounds of Formula I or Formula II is monitored spectrophotometrically at 530 nm. The compound to be tested was dissolved in DMSO at an initial concentration 50 times greater than its final concentration in a cuvette. Ten μl of the stock solution was diluted in 90 μl of assay buffer comprising 0.1 M sodium phosphate buffer containing 130 mM NaCl, pH 7.4, to which ovalbumin (OVA) is added at a concentration of 1 mg/ml. Arginase I and II solutions were prepared in 100 mM sodium phosphate buffer, pH 7.4 containing 1 mg/ml OVA to generate an arginase stock solution at a final concentration of 100 ng/ml.
To each well of a 96-well microtiter plate, 40 µl of enzyme, 10 µl of a compound of the invention and 10 µl of enzyme substrate (L-arginine + manganese sulfate) were added. For wells that were used as positive controls, only the enzyme and its substrate were added, while wells used as negative controls contained only manganese sulfate.
After incubating the microtiter plate at 37 °C for 60 minutes, 150 μl of a urea reagent obtained by combining equal proportions (1:1) of reagents A and B are added to each well of the microtiter plate to stop the reaction . The urea reagent is made immediately before use by combining Reagent A (10 mM o-phthaldialdehyde and 0.4% polyoxyethylene lauryl ether (23) (w/v) in 1.8 M sulfuric acid) with Reagent B (1.3 mM primaquine diphosphate, 0.4% polyoxyethylene lauryl ether (23) (w/v), 130 mM boric acid in 3.6 mM sulfuric acid). After extinction of the reaction mixture, the microtiter plate is left for another 10 minutes at room temperature to "allow color development. Arginase inhibition was computed by measuring the optical density (OD) of the reaction mixture at 530 nm and normalizing the DO value to the percent inhibition observed in the control. The normalized DO is then used to generate a dose-response curve by tabulating the normalized DO values against log [concentration] and using regression analysis to compute the values of the IC50.
Table 2 below ranks the potency of compounds of Formula I 5 on a scale of 1 to 5, that is, the most potent compounds are designated as 1 and the least potent compounds being designated as 5. Thus, a potency value of 1 refers to compounds of the invention with IC50 values in the range of 0.1 nM to 250 nM; a value of 10 power of 2 refers to compounds of the invention with IC 50 values in the range of 251 nM to 1,000 nM; compounds that have a potency value of 3 exhibit IC50 values in the range of 1.001 nM to 2000 nM; compounds of the invention' with IC50 values in the range of 2,001 nM to 5,000 15 nM receive a potency value of 4, and compounds with IC50 values above 5001 nM receive a potency value of 5.

















Power order (from highest to lowest): 1 = 0.1 nM - 250 nM; 2 = 251 nM - 1000 nM; 3 = 1001 nM - 2000 nM; 4 = 2001 nM - 5,000 nM; 5 = 5001 nM or greater and n.d. = not determined. relaxation of the arterial ring
The purpose of this example is to demonstrate that the arginase inhibitor compounds according to the invention show efficacy in the treatment of pulmonary hypertension in an ex vivo model of the disease. Thus, arginase inhibitors according to the present invention are evaluated for their effectiveness in increasing acetylcholine-induced relaxation of pre-contracted arterial tissue obtained from mice.
In this study, mice are randomly divided into two groups. A first group of mice that serve as controls and a second group of mice that are injected with a solution of monocrotaline, an agent that is used experimentally to induce increased arterial pressure in the pulmonary artery and vein of the heart.
Both groups of mice are caged for 3-4 weeks to establish pulmonary hypertension in the monocrotaline-treated group. At the end of this period and before being sacrificed, the mice from the control and monocrotaline groups are divided into two subgroups. After euthanasia, the main pulmonary artery and its left and right branches are removed from each animal, cleaned and maintained in a physiologically acceptable solution before being used for use in the relaxation study.
The arterial tissue obtained first is sliced to obtain segments of the arterial ring approximately 1.5-2 mm in length. Ring segments from each individual animal are then mounted in independent chambers of a myograph (Danish MyoTechnology) using a 200 µm stainless steel wire. After bathing the arterial ring segments in Kreb's buffer, each ring segment is set to a predetermined optimal passive tension (ie length/tension ratio) and acclimatization is allowed for at least 1 hour before determining viability of tissue using KC1 (60 mM).
Arterial tissue from mice in one of two subgroups within the control and monocrotaline groups is then incubated with an appropriate arginase inhibitor for 30 minutes at a molar concentration of 100 µM before addition of phenylephrine (PE), an agent known to be causes muscle contraction.
Arterial tissue that belongs to the second subgroup within the control and monocrotaline groups, however, is induced to contract by direct exposure of the tissue to a 1 μM solution of phenylephrine. To calculate the effectiveness of the arginase inhibitors of the invention to increase acetylcholine-induced relaxation of pre-contracted arterial tissue, a myograph is used to measure the change in tension for each segment of tissue exposed or not exposed to a compound of the invention in the groups. control and monocrotaline.
For the compounds of the invention, the study of arterial ring relaxation indicates that, for mice belonging to the control group (that is, for mice that did not receive monocrotaline administration), there is no difference in the percentage increase in the relaxation of segments of pre-contracted arterial tissue exposed to an arginase inhibitor or exposed to ab vehicle (buffer), prior to addition of acetylcholine (AC) to induce relaxation.
In contrast, for mice belonging to the monocrotaline group, exposure of arterial tissue to an arginase inhibitor at a concentration of 100 µM prior to inducing contraction using phenylephrine increases tissue relaxation by about 75%. For tissue from mice treated with monocrotaline that was exposed to the vehicle (buffer) prior to contraction, the addition of acetylcholine causes a minor increase in relaxation of about 40% to 45%.
Without ascribing to any specific hypothesis, the inventors believe that the compounds of the invention inhibit arginase by causing an increase in the intracellular pool of arginine which is then available as a substrate for cellular nitric oxide synthases (NOS). NOS converts arginine to nitric oxide (NO), an important physiological signaling molecule that supposedly participates in muscle relaxation. Consequently, the arginase inhibitors of the present invention are suitable as therapeutic substances for the treatment of diseases such as, for example, hypertension and erectile dysfunction.

权利要求:
Claims (25)
[0001]
opcionalmente representa uma ou mais ligações duplas; R3 e R4 são, cada um independentemente, selecionados de hidrogênio, (C1-C6)alquil linear ou ramificado e C(O)-R’, ou R3 e R4, juntos com o átomo de boro ao qual estão anexados, formam um anel de 5 ou 6 membros que é totalmente saturado, ou parcialmente saturado; D é selecionado do grupo que consiste em (C3- C5)alquileno linear ou ramificado, (C2-C8)alquenileno linear ou ramificado, (C2-C8)alquinileno linear ou ramificado, (C3- C14)arileno e (C3-C14)cicloalquileno, em que um ou mais grupos -CH2- em D são opcional e independentemente substituídos com uma porção selecionada do grupo que consiste em O, NR’, S, SO, SO2 e CR’R’’; ou em que quaisquer dois grupos -CH2- adjacentes opcionalmente representam dois membros de um grupo (C3-C14)- cicloalquilenil; e em que nenhum de dois grupos -CH2- adjacentes simultaneamente representa O, NR’, S, SO ou SO2; R’, R’’ e R’’’ são, cada um independentemente, selecionados do grupo que consiste em H, OH, S(O)Rd, S(O)2Rd, (C1-C8)alquil, (C3-C6)aril, -NH2, -NH(C1-C6)alquil, -N[(C1- C6)alquil]2, -C(O)NRdRe, -C(O)(C1-C6)alquil, -C(O)(C3-C14)aril, -C(O)O(C1-C6)alquil, -C(O)O(C3-C14)aril, (C3-C6)cicloalquil, (C3-C14)heterocicloalquil, -C(O)(C3-C14)heterocicloalquil, (C3-C14)heteroaril, (C3-C14)aril-(C1-C6)alquileno-, -C(O)(C3- C14)aril-(C1-C6)alquileno-, -C(O)(C3-C14)aril, (C3- C6)cicloalquil-(C1-C6)alquileno-, (C3-C14)heteroaril-(C1- C6)alquileno-, (C3-C14)heterociclo-(C1-C6)alquileno-; e em que qualquer alquil, alquileno, aril, heteroaril, cicloalquil ou heterocicloalquil é opcionalmente substituído com um ou mais membros selecionados do grupo que consiste em halogênio, oxo, -COOH, -CN, -NO2, -OH, -NRdRe, -NRgS(O)2Rh, (C1-C6)alcóxi, (C3-C14)aril, (C1-C6)haloalquil e (C3- C14)ariloxi; em que Rd, Re, Rg e Rh são, cada um independentemente, selecionados do grupo que consiste em H, (C1-C6)alquil linear ou ramificado, (C3-C14)aril(C1-C6)alquileno- opcionalmente substituído, (C3-C14)aril opcionalmente substituído, (C1-C6)hidroxialquil, (C1-C6)aminoalquil, H2N(C1- C6)alquileno-, (C3-C6)cicloalquil opcionalmente substituído, (C3-C14)heterocicloalquil opcionalmente substituído, (C3- C14)heteroaril opcionalmente substituído, (C3-C14)aril-(C1- C6)alquileno- opcionalmente substituído, NR’R’’C(O)- e (C3- C6)aril-(C3-C14)-cicloalquileno-, ou um sal farmaceuticamente aceitável, estereoisômero, tautômero ou pró-fármaco destes; desde que o composto de acordo com Fórmula I não seja ácido 1-amino-2-(3-boronopropil)ciclohexano carboxílico.1. Compound characterized by having the Formula I,
[0002]
A compound according to claim 1, characterized in that D is linear or branched (C3-C5)alkylene.
[0003]
A compound according to claim 2, characterized in that D is propylene.
[0004]
A compound according to claim 3, characterized in that R 1 is -OH.
[0005]
A compound according to claim 4, characterized in that each of R2, R3 and R4 is hydrogen.
[0006]
A compound according to claim 5, characterized in that each of W, X, Y and Z is -C(R')2-.
[0007]
A compound according to claim 6, characterized in that R' is H.
[0008]
A compound according to claim 7, characterized in that l + m + n + p is = 3 or 4.
[0009]
A compound according to claim 5, characterized in that any one of W, X, Y and Z is -NH- and each occurrence of the remaining three is -C(R')2-.
[0010]
A compound according to claim 5, characterized in that any one of W, X, Y and Z is -N- and II each of the remaining three is -CR'''-, is present and represents one or more bonds doubles.
[0011]
11. Compound according to claim 1, characterized in that it is selected from the following table:
[0012]
12. Compound characterized by being selected from the following table:
[0013]
em que R'" é selecionado a partir de H, OH, -S(O)Rd, -S(O)2Rd, (C1-C8)alquil, (C3-C6)aril, -NH2, -NH(C1-C6)alquil, -N[(C1- 5 C6)alquil]2, -C(O)NRdRe, -C(O)(C1-C6)alquil, -C(O)(C3-C14)aril, -C(O)O(C1-C6)alquil, -C(O)O(C3-C14)aril, (C3-C6)cicloalquil, (C3-C14)heterocicloalquil, -C(O)(C3-C14)heterocicloalquil, (C3-C14)heteroaril, (C3-C14)aril-(C1-C6)alquileno-, -C(O)(C3- C14)aril-(C1-C6)alquileno-, (C3-C6)cicloalquil-(C1- C6)alquileno-, (C3-C14)heteroaril-(C1-C6)alquileno-, e (C3- C14)heterociclo-(C1-C6)alquileno-; em que qualquer alquil, alquileno, aril, heteroaril, cicloalquil, ou heterocicloalquil é opcionalmente substituído com um ou mais membros selecionados dentre halogênio, oxo, -COOH, -CN, -NO2, -OH, -NRdRe, -NRgS(O)2Rh, (C1-C6)alcóxi, (C3-C14)aril, (C1-C6)haloalquil e (C3- C14)ariloxi; e em que Rd, Re, Rg e Rh são cada um, independentemente, selecionados a partir de H, (C1-C6)alquil linear ou ramificado, (C3-C14)aril(C1-C6)alquileno-, (C3-C14)aril, (C1- C6)hidroxialquil, (C1-C6)aminoalquil, H2N(C1-C6)alquileno-, (C3-C6)cicloalquil, (C3-C14)heterocicloalquil, (C3- Ci4)heteroaril, (C3-Ci4)aril-(Ci-C6)alquileno-, NR‘R"C(O)- e (C3-C6)aril-(C3-C14)-cicloalquileno-; ou um sal farmaceuticamente aceitável deste.A compound according to claim 1 wherein R'" is selected from H, OH, -S(O)Rd, -S(O)2Rd, (C1-C8)alkyl, (C3-C6)aryl, -NH2, -NH(C1- C6)alkyl, -N[(C1-C6)alkyl]2, -C(O)NRdRe, -C(O)(C1-C6)alkyl, -C(O)(C3-C14)aryl, -C (O)O(C1-C6)alkyl, -C(O)O(C3-C14)aryl, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, -C(O)(C3-C14)heterocycloalkyl, (C3-C14)heteroaryl, (C3-C14)aryl-(C1-C6)alkylene-, -C(O)(C3-C14)aryl-(C1-C6)alkylene-, (C3-C6)cycloalkyl-( C1-C6)alkylene-, (C3-C14)heteroaryl-(C1-C6)alkylene-, and (C3-C14)heterocycle-(C1-C6)alkylene-; wherein any alkyl, alkylene, aryl, heteroaryl, cycloalkylene , or heterocycloalkyl is optionally substituted with one or more members selected from halogen, oxo, -COOH, -CN, -NO2, -OH, -NRdRe, -NRgS(O)2Rh, (C1-C6)alkoxy, (C3-C14 )aryl, (C1-C6)haloalkyl, and (C3-C14)aryloxy; and wherein Rd, Re, Rg and Rh are each independently selected from H, linear or branched (C1-C6)alkyl, ( C3-C14)aryl(C1-C6)alkylene-, (C3-C14)aryl, (C1-C6)hydroxyalkyl, (C1-C6)a minoalkyl, H2N(C1-C6)alkylene-, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, (C3-C14)heteroaryl, (C3-C14)aryl-(C1-C6)alkylene-, NR'R "C(O)- and (C3-C6)aryl-(C3-C14)-cycloalkylene-; or a pharmaceutically acceptable salt thereof.
[0014]
14. Compound according to claim i3, characterized in that R'" is selected from H, OH, -S(O)Rd, -S(O)2Rd, (Ci-C8)alkyl, (C3- C6)aryl, -NH2, -NH(Ci-C6)alkyl, -N[(Ci-C6)alkyl]2, -C(O)(Ci-C6)alkyl, -C(O)(C3-Ci4) aryl, -C(O)O(Ci-C6)alkyl, -C(O)O(C3-Ci4)aryl, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, -C(O)(C3- C14-heterocycloalkyl, (C3-C14)heteroaryl, (C3-C14)aryl-(C1-C6)alkylene-, (C3-C6)cycloalkyl-(C1-C6)alkylene-, (C3-C14)heteroaryl-(C1-4) - C6) alkylene- and (C3-C14)heterocycle-(C1-C6)alkylene-; wherein any alkyl, alkylene, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl is optionally substituted with one or more members selected from halogen, oxo, -COOH, -CN, -NO2, -OH, -NRdRe, -NRgS(O)2Rh, (C1-C6)alkoxy, (C3-C4)aryl, (C1-C6)haloalkyl, and (C3-C4)aryloxy; and wherein Rd, Re, Rg and Rh are each independently selected from H, (C1 -C6 ) linear or branched alkyl, (C3 -C14 )aryl(C1 -C6 )alkylene-, (C3 -C14 ) ) aryl, (Ci-C6)hydroxy ialkyl, (Ci-C6)aminoalkyl, H2N(Ci-C6)alkylene-, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, (C3-C4)heteroaryl, (C3-C14)aryl-(C1-Cs) )alkylene-, NR'R"C(O)- and (C3-C6)aryl-(C3-C14)-cycloalkylene-; or a pharmaceutically acceptable salt thereof.
[0015]
15. Compound according to claim i3, characterized in that: R'" is selected from -C(O) (C1-Cg)alkyl, -C(O) (C3-Cy4)aryl and -C( O)(C3-Ci4)heterocycloalkyl; wherein any alkyl, aryl or heterocycloalkyl is optionally substituted with one or more members selected from halogen, oxo, -COOH, -CN, -NO2, -OH, -NRdRe, - NRgS(O)2Rh, (Ci-C6)alkoxy, (C3-Ci4)aryl, (Ci-C6)haloalkyl, and (C3-Ci4)aryloxy; and wherein Rd, Re, Rg and Rh are each independently i0 selected from H, (C 1 -C 6 )alkyl linear or branched, (C 3 -C 4 )aryl(C 1 -C 6 )alkylene-, (C 3 -C 4 )aryl, (C 1 -C 6 )hydroxyalkyl, (C 1 -C 6 ) aminoalkyl, H2N(C1-C6)alkylene-, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, (C3-C14)heteroaryl, (C3-C14)aryl-(C1-Cs)alkylene-, NR'R "C(O)- and i5 (C3-C6)aryl-(C3-C14)-cycloalkylene-; or a pharmaceutically acceptable salt thereof.
[0016]
A compound according to claim 13, characterized in that it has the formula: or a pharmaceutically acceptable salt thereof.
[0017]
ou um sal farmaceuticamente aceitávelor deste.A compound according to claim 13, characterized in that it has the formula: or a pharmaceutically acceptable salt thereof.
[0018]
18. Compound according to claim 17, characterized in that: R'" is selected from H, OH, -S(O)Rd, -S(O)2Rd, (C1-C8)alkyl, (C3-C6)aryl, -NH2, -NH(C1-C6)alkyl, -N[(C1-C6)alkyl]2, -C(O)(C1-C6)alkyl, -C(O)(C3 -C14)aryl, -C(O)O(C1-C6)alkyl, -C(O)O(C3-C14)aryl, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, -C(O) (C3-C14)heterocycloalkyl, (C3-C14)heteroaryl, (C3-C14)aryl-(C1-C6)alkylene-, (C3-C6)cycloalkyl-(C1-C6)alkylene-, (C3-C14)heteroaryl -(C1-C6)alkylene- and (C3-C14)heterocycle-(C1-C6)alkylene-; wherein any alkyl, alkylene, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl is optionally substituted with one or more members selected from halogen , oxo, -COOH, -CN, -NO2, -OH, -NRdRe, -NRgS(O)2Rh, (C1-C6)alkoxy, (C3-C14)aryl, (C1-C6)haloalkyl and (C3-C14) )aryloxy; and wherein Rd, Re, Rg and Rh are each independently selected from H, (C1-C6) linear or branched alkyl, (C3-C14)aryl(C1-C6)alkylene-, ( C3-C14) aryl, (C1-C6) hydroxyalkyl, (C1-C6)aminoalkyl, H2N(C1-C6)alkylene-, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, (C3-C4)heteroaryl, (C3-C14)aryl-(C1-C6) )alkylene-, NR'R"C(O)- and (C3-C6)aryl-(C3-C14)-cycloalkylene-; or a pharmaceutically acceptable salt thereof.
[0019]
19. Compound according to claim 18, characterized in that: R'" is selected from -C(O)(C1-Cg)alkyl, -C(O)(C3-Ci4)aryl and - C(O)(C3-Ci4)heterocycloalkyl; wherein any alkyl, aryl or heterocycloalkyl is optionally substituted with one or more members selected from halogen, oxo, -COOH, -CN, -NO2, -OH, -NRdRe, -NRgS (O)2Rh, (Ci-C6)alkoxy, (C3-Ci4)aryl, (Ci-C6)haloalkyl, and (C3-Ci4)aryloxy; and wherein Rd, Re, Rg and Rh are each independently selected from H, linear or branched (C 1 -C 6 )alkyl, (C 3 -C 4 )aryl(C 1 -C 6 )alkylene-, (C 3 -C 4 )aryl, (C 1 -C 6 )hydroxyalkyl, (C 1 -C 6 )aminoalkyl, H2N(C1-C6)alkylene-, (C3-C6)cycloalkyl, (C3-C14)heterocycloalkyl, (C3-C14)heteroaryl, (C3-C14)aryl-(C1-C6)alkylene-, NR'R"C (O)- and (C3-C6)aryl-(C3-C14)-cycloalkylene-; or a pharmaceutically acceptable salt thereof.
[0020]
A pharmaceutical composition characterized in that it comprises a therapeutically effective amount of at least one compound as defined in any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
[0021]
21. Use of a compound as defined in any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, characterized in that it is in the preparation of a medicament for the inhibition of arginase I, arginase II, or a combination thereof, in a cell.
[0022]
22. Use of a compound as defined in any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, characterized in that it is in the preparation of a medicament for the treatment or prevention of a disease or condition associated with the expression or activity of arginase I, arginase II or a combination of these in an individual.
[0023]
23. Use according to claim 22, characterized in that the disease is cancer.
[0024]
24. Use according to claim 22, characterized in that the disease or condition is selected from the group consisting of pulmonary hypertension, erectile dysfunction, hypertension, atherosclerosis, kidney disease, asthma, T-cell dysfunction, ischemia reperfusion injury , neurodegenerative diseases, wound healing and fibrotic diseases.
[0025]
25. Use according to claim 24, characterized in that the disease or condition is pulmonary hypertension or erectile dysfunction.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-12-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-02| B07E| Notice 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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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-05-18| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-10| 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 19/10/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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