西班牙专利ES2648538A1 Benzo-heterociclos of six members with atoms of oxygen and nitrogen with antitumoral activity (Machi

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专利摘要:
Benzo-six-member heterocycles with oxygen and nitrogen atoms with antitumor activity. The present invention relates to a new family of six-membered benzo-heterocycles with oxygen and nitrogen atoms, linked to purines, halogens and substituted triazoles which are useful in therapy against cancer, in particular in therapies whose therapeutic target is the cancer stem cells. The invention also describes the method of synthesis of said compounds. (Machine-translation by Google Translate, not legally binding)
公开号:ES2648538A1
申请号:ES201630714
申请日:2016-05-31
公开日:2018-01-03
发明作者:Joaquín María CAMPOS ROSA；María Eugenia GARCÍA RUBIÑO；Juan Antonio Marchal Corrales；Cynthia MORATA TARIFA；Alberto RAMÍREZ RIVERA；Elier Paz Rojas
申请人:CANVAX BIOTECH S L；Universidad de Granada；CANVAX BIOTECH SL；Universidad de Jaen；
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DESCRIPTION

BENZO-HETEROCICLOS OF SIX MEMBERS WITH OXYGEN AND NITROGEN ATOMS WITH ANTITUMORAL ACTIVITY
5 STATE OF THE TECHNIQUE
Cancer remains one of the leading causes of death in the civilized world. Despite the great progress that has been made in terms of understanding the molecular basis of cancer, detection and treatment, its mortality remains high and there is no definitive cure despite the improvements that have been made. Experienced the various therapies. Current regimes show limited survival rates when applied to most cancers in advanced stages. With the rise of bio-informatics and molecular biology, new techniques have been developed to analyze the global modifications of the genome and proteome of tumor cells. The detection by means of these techniques of the gene and protein modifications induced by new antitumor drugs, allow us to characterize the potential targets of these compounds and the most accurate knowledge of their mechanism of action. The deepening of the mechanisms for regulating these events supports the development of new therapeutic strategies based on the rational design of new selective antitumor agents against their molecular targets.

Purine use in cancer treatment

Among the various active principles for the treatment of cancer known in the state of the art are purines. In the state of the art closest to the present invention is patent ES200601538, which describes racemic mixtures and their uses in the treatment of cancer of structures 1 and 2.

These families of compounds 1 and 2 have antitumor activity against various cancers and low toxicity against a healthy cell line (normal breast epithelial line MCF-10A).
In particular, ES200601538 discloses compounds in which in the heptagonal ring the heteroatoms present are O and X, where X is selected from O, S and SO2, and their 5 inhibitory concentrations 50 (IC50) on the human cell line derived from cancer of breast MCF-7.
The antitumor activities of these compounds are remarkable and comparable to the active agents conventionally used in therapy such as the cytotoxic agent 5-fluorouracil (Díaz-Gavilán, M .; Gómez-Vidal, JA; Rodríguez-Serrano, F .; Marchal, 10 JA ; Caba, O .; Aránega, A .; Gallo, MA; Espinosa, A .; Campos, JMBioorg. Med. Chem. Lett. 2008, 18, 1457-1460).
In their effort to provide improved anti-proliferative agents, the inventors have subsequently disclosed new compounds, O, N-acetals and their synthesis in which X in the heptagonal ring is an N atom substituted with a nitrobenzenesulfonyl group (Diaz-Gavilan, M .; Choquesillo-Lazarte, D .; González-Pérez, JM; Gallo, MA; Espinosa, A .; Campos, JM Tetrahedron 2007, 63, 5274-5286).
The results on the anti-proliferative activities of various compounds O, N-acetals N-9 and N-7purinic or N-1 and N-3 pyrimidines in vitro against the human breast cancer cell line MCF-7 have been published ( Díaz-Gavilán, M .; Gómez-Vidal, 20 JA; Rodríguez-Serrano, F .; Marchal, JA; Caba, O .; Aránega, A .; Gallo, MA; Espinosa, A .; Campos, JM Bioorg. Med Chem. Lett. 2008, 18, 1457-1460). These results have shown, among other aspects, that the most potent antitumor compounds are purine derivatives. Compounds that have a 4,1-benzoxazepine ring compared to 25 compounds that have a 4,1-benzoxatiepine or 4,1-benzodioxepine ring also seem more potent in general.

On the other hand, ES200802431 describes (RS) -9- [1- (p-nitrobenzenesulfonyl) -1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl] -2.6 -dichlor-9H-purine. In a recent publication we have demonstrated the inhibitory effect on kinases involved in carcinogenesis, proliferation and angiogenesis (Ramírez A .; Boulaiz H .; Morata-Tarifa C .; Perán M .; Jiménez G .; Picón-Ruiz M .; Agil A , Cruz-López O .; Conejo-García A .; Campos JM .; Sánchez A .; García MA .; Marchal JA. Oncotarget2014, 5, 3590-3606). Said compound inhibited the signaling pathway of HER-2 and the kinases JNK and ERK1 / 2. It also had an inhibitory effect of AKT and VEGF, along with anti-migratory and anti-angiogenic activities. On the other hand, said compound
was able to inhibit the formation of mammo and colonospheres, and eliminated the sub-populations of positive CSCsaldehyde dehydrogenase (ALDH1 +) at low micromolar range. This action was due to the down-regulation of c-MYC, β-CATENINE and SOX2, and the excess regulation of the Hedgehog signaling pathway repressor, GLI-3. Finally, it also demonstrated anti-tumor and anti-metastatic efficacy in vivo in 5 induced tumors (xenotransplants) without presenting acute or sub-acute toxicity (Ramírez A .; Boulaiz H .; Morata-Tarifa C.; Perán M .; Jiménez G. ; Picón-Ruiz M.; Agil A, Cruz-López O .; Conejo-García A .; Campos JM .; Sánchez A .; García MA .; Marchal JA. Oncotarget 2014, 5, 3590-3606).
10
On the other hand, in ES201030415 the two enantiomers of (RS) -9- [1- (p-nitrobenzenesulfonyl) -1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl] - are presented 2,6-dichloro-9H-purine, with inhibition of EGFR, HER-2 and VEGF, with production of a high level of apoptosis in tumor cells and also, without manifestation of acute or chronic toxicity. fifteen

Finally, ES201430048 describes the preparation of secondary sulfonamides 3 (derived from secondary amines), and their use as medicaments for the treatment and prevention of cancer, in particular for the treatment of breast, lung, colorectal, pancreas, myelo-proliferative neoplasms and syndromes. twenty

Type 3 structures are related to bozepinib and derivatives, since they have purine units in their structure, however, as a differentiating aspect, they are acyclic structures opposite to the structure of the bozepinib that has a heterocyclic seven-membered ring. Another differentiating aspect is that in the family 3 there is no stereocenter than if it is present in the bozepinib, which simplifies the synthetic procedure.

The main difficulties presented by the compounds described in these patents are scalability and their enantiomeric synthesis. 30

Cancer Stem Cells

Currently, one of the most interesting concepts that is being explored in cancer research is the theory of cancer stem cells (hereinafter "CSCs") or tumor-initiating cells. CSCs can be defined as the sub-populations of cells within tumors that have the ability to self-renew and differentiate into the different cancer cell lines that make up the tumor. This type of cells is proposed as the promoter of resistance against antitumor therapies, being able to maintain benign and malignant tumors, as well as cause recurrences (Reya, T .; Morrison, SJ; 10 Clarke, MF; Weissman, IL Nature 2001, 414, 105-111). Compared to normal stem cells, CSCs are believed to have no control over their proliferation. These CSCs, present in tumors in small quantities, are characterized by their ability to remain quiescent for long periods of time, self-renewal capacity, growth maintenance and tumor heterogeneity, affinity for hypoxic environments, resistance to chemotherapy and development. of metastasis (Tirino, V .; Desiderio, V .; Paino, F .; De Rosa, A .; Papaccio, F .; La Noce, M .; Laino, L .; De Francesco, F .; Papaccio, G. FASEB J. 2013, 27, 13-24).
CSCs are characterized by their ability to form spherical colonies by being grown in suspension (Dontu, G .; Abdallah, WM; Foley, JM; Jackson, KW; Clarke, MF; Kawamura, MJ; Wicha, MS Genes Dev, 2003, 17, 1253-1270). Al-Hajjet al. Managed to demonstrate that the injection of 200 tumor cells expressing characteristic markers of CSCs was more effective in generating tumors in immuno-depressed mice than the injection of 50,000 tumor cells with 25 markers of differentiated cells thereof. histological line (Al-Hajj, M .; Wicha, MS; Benito-Hernández, A .; Morrison, SJ; Clarke. MF ProcNatlAcadSci USA, 2003, 100: 3983-3988). It has been found that multiple molecules related to the characteristic properties of stem cells such as self-renewal and pluripotency, and certain enzymatic activities are largely expressed in 30 CSCs, including c-MYC, β-CATENINE (Moumen, M .; Chiche, A .; Decraene, C.; Petit, V .; Gandarillas, A.; Deugnier, MA; Glukhova, MA; Faraldo, MM Mol Cancer, 2013,12,132-140), SOX-2 (Leis, O .; Eguiara, A .; López-Arribillaga, E .; Alberdi, MJ; Hernández-García, S .; Elorriaga, K .; Pandiella, A .; Rezola, R .; Martín, AG Oncogene 2012, 31, 1354-1365) , and aldehyde dehydrogenase (ALDH1) activity (Deng, S .; 35 Yang, X .; Lassus, H .; Liang, S .; Kaur, S .; Ye, Q .; Li C .; Wang, LP; Roby , KF; Orsulic, S .; Connolly, DC; Zhang, Y .; Montone, K .; Bützow, R .; Coukos, G .; Zhang, L.
PLoSOne 2010, 5, e10277, among others). Acting selectively on the over-regulated pathways or molecules in differentiated cancer cells and / or in CSC populations, but not in normal cells, they can provide new strategies for selective and effective cancer therapy.
5
Targeted therapies against Cancer Stem Cells

Currently, cancer treatment is directed to its proliferation potential and, therefore, most treatments are aimed at rapidly dividing cells. The presence of CSCs may explain the failure of treatments 10 to eradicate the disease or recurrence of cancer (Reya, T .; Morrison, S.J .; Clarke, M.F .; Weissman, I.L. Nature 2001, 414, 105-111). The property that CSCs have to maintain a quiescence state, a state in which the cell does not divide, remaining in the G0 phase of the cell cycle, (Arai, F .; Hirao, A .; Ohmura, M .; Sato , H .; Matsuoka, S .; Takubo, K .; Ito, K .; Koh, GY; Suda. T. Cell 2004, 118, 149-15 161) allows them to survive the majority of anti-cancer treatments. This characteristic makes relapse or recurrence possible, even decades after initial treatment, such as colon or breast cancer (Reya, T .; Morrison, SJ; Clarke, MF; Weissman, IL Nature 2001, 414, 105- 111; Dick JE Blood 2008, 112, 4793-4807). Although current treatments can reduce the size of the tumor, these effects are transient and generally do not improve patient survival outcomes. For tumors in which CSCs play a role, there are three possibilities. First, the mutation of normal stem cells or progenitor cells of CSCs can lead to the development of the primary tumor. Second, during chemotherapy, most of the 25 primary tumor cells can be destroyed, but if CSCs are not eradicated, they become refractory CSCs and can lead to tumor recurrence (Stockler, M .; Wilcken, N .; Ghersi, D .; Simes, RJCancerTreatRev 2000, 26, 151-168). Third, CSCs can migrate to distant sites of the primary tumor and cause metastasis (Jordan, CT; Guzman, MLOncogene 2004, 23.7178-7187; Jordan, CT; Guzman, 30 ML; Noble, M.New Eng. J Med 2006, 355, 1253-1261; Liu, HG; Chen, C .; Yang, H .; Pan, YF; Zhang, XH Transl Med 2011, 9, 50-58). Theoretically, the identification of the CSCs may allow the development of treatment modalities that are directed against said cells instead of towards rapidly dividing cells (Jayesh, S .; Chaib, B .; Sales, M .; Seifalian, A. Cancer Cell Int 2009, 7, 9-14). 35

One of the therapeutic approaches currently being studied to combat CSCs is related to the signaling pathways involved in the processes of their self-renewal, proliferation and differentiation. This is due to the fact that the loss of the regulation of pathways such as Hedgehog (Hh), Notch and Wnt / β-catenin, gives rise to the key processes involved in the characteristics of CSCs. Currently, 5 targeted therapy against these routes represents one of the most promising mechanisms of action against these tumor-initiating cells (Maugeri-Sacca M .; Zeuner A .; De Maria R. Front Oncol. 2011, 1, 10; Takebe N .; Harris PJ; Warren RQ; Ivy SP NatRevClinOncol2011, 8, 97-106).
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BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a new family of six-membered benzo-heterocycles with oxygen and nitrogen atoms, attached to purines and halogens, which are useful in cancer therapy, in particular in therapies whose therapeutic target 15 are cancer stem cells, and more particularly against melanoma, preferably metastatic melanoma, and breast, lung, pancreas and colorectal cancer. The enantiomers, isomers, salts, or solvates of said compounds will also be considered subject of the invention. The pro-drugs of the compounds of the invention are also subject of the invention. twenty

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Reaction scheme of the synthesis process of the compounds of the invention. 25

Figure 2. Isolation and characterization of CSCs sub-populations from MDA-MB 468 cells by flow cytometry separation according to the aldehyde dehydrogenase activity; A: use of the inhibitor, B: sub-population ALDH (+), C: sub-population ALDH (-). 30

Figure 3. Images obtained by optical microscope belonging to cultured mammals after the isolation of the CSCsALDH + sub-populations of the MDA-MB 468 cell line.
35
Figure 4. Western blot of the MDA-MB 468 cell line using 13 at a concentration of 5 µM for 2h, 4h, 8h, 12h, 16h and 24h. A. Effect on EGFR and ERK1 / 2 phosphorylated. B. Densitometric analysis normalized with the β-actin signal

Figure 5. Western blot of Sox2 (SOX-2) of the sub-populations of CSD ALDH + 5 isolated from the MDA-MB 468 cell line and treated with 13 at a concentration of 1.43 µM (IC50) and 2.86 µM (2 × IC50 ) for 24 hours.

Figure 6. Representation of the in vivo antitumor activity of 13 on the metastatic line of human melanoma A375. Mice used as a control were injected only with the vehicle () and those treated with 13 were divided into two groups, one treated with 50 mg / kg () and another with 100 mg / kg ().

Figure 7. Kaplan – Meier survival curve of mice treated with the vehicle (controls, ) and treated with 13 (: 50 mg / kg, : 100 mg / kg). fifteen

DETAILED DESCRIPTION OF THE INVENTION

Definitions
 Here the following terms have the meanings indicated: "Alkyl" refers to a linear or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, which does not contain unsaturations, having 1-12, preferably of one to eight, more preferably one to six carbon atoms, and which is attached to the rest of the molecule by a single bond, e.g. eg, methyl, ethyl, n-propyl, i-propyl, n-25 butyl, t-butyl, n-pentyl, etc.
 "Alkenyl" refers to a linear or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, which contains at least one unsaturation, having 2-12, preferably two to eight, more preferably two to six atoms of carbon, and that is attached to the rest of the molecule by a single bond.
• "Alkynyl" refers to a linear or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, which contains at least one carbon-carbon triple bond, conjugated or not, having two to twelve, preferably two to eight, preferably two to six carbon atoms, and which is attached to the rest of the molecule by a single bond, such as -CCH, -CH2CCH, -CCCH3, -CH2CCCH3.
 "Aryl" or "Ar" refers to an aromatic hydrocarbon radical such as phenyl, naphthyl or anthracil.
 "Arylalkyl" refers to an aryl group attached to the rest of the molecule through an alkyl group, such as benzyl and phenethyl.
 "Cycloalkyl" refers to a cyclic hydrocarbon chain radical consisting of carbon atoms and hydrogen, which does not contain unsaturation, having 3 to 8 carbon atoms, attached to the rest of the molecule by a single bond. " Alkoxy "refers to a radical of the formula -Oalkyl, e.g. ex. Methoxy, ethoxy, propoxy, etc. "Aryloxy" refers to a radical of the formula -Oaryl.
 "Heterocycloalkyl" refers to a stable ring of 3 to 15 members consisting of carbon atoms and one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, preferably a ring of 4 to 8 members with one to more heteroatoms, more preferably a 5 or 6-membered ring with one or more heteroatoms attached to the aniline-like nitrogen atom by an alkyl group, such as methyl, ethyl, propyl attached to any carbon atom of the heterocycle system. For the purpose of this invention, the heterocycle may be a monocyclic, bicyclic or tricyclic ring system, which may include condensed ring systems; and the nitrogen, carbon or sulfur atoms in the heterocycle may optionally be oxidized; the nitrogen atom may be optionally quaternized; and the heterocycle may be partially or fully saturated or aromatic. Examples of such heterocycles include, but are not limited to azepines, benzimidazole, benzothiazole, furan, isothiazole, imidazole, indole, piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran.
 "Acyl" refers to a group -C (= O) R4. 25
 "Alkoxyacyl" refers to a group -C (= O) OR4tals such as methyl carbamate, ethyl carbamate, benzyl carbamate, p-nitrobenzyl carbamate, tert-butyl carbamate, allyl carbamate, 2.2 carbamate , 2-trichlorethyl, 2- (trimethylsilyl) ethyl carbamate, and allyl carbamate.
 "Alkoxysulfonyl" refers to a group -SO2OR4 such as methyl sulfate, ethyl sulfate, benzyl sulfate, p-nitrobenzyl sulfate, tert-butyl sulfate, allyl sulfate, 2,2,2- trichlorethyl, 2- (trimethylsilyl) ethyl sulfate, and allyl sulfate.

In the above formulas R4 represents a group selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted aryl-alkyl, and heterocycle.

Organic functions that are bound to an oxygen atom are well known in the art and are representative of the following:
- Silyl ethers of the formula -Si (R4) 3, such as trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, tri-isopropylsilyl ether, diethylisopropylsilyl ether, texyl dimethyl ether, texyl dimethyl ether of triphenylsilyl, di-tert-butylmethylsilyl ether; 10
- Alkyl and arylalkyl ethers such as methyl ether, tert-butyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trityl ether; allyl ether;
- Alkoxymethyl and aryloxy ethers of the formula -CH2OR4, such as methoxymethyl ether, 2-methoxyethoxymethyl ether, benzyloxymethyl ether, p-15-methoxybenzyloxymethyl ether, 2- (trimethylsilyl) ethoxymethyl ether; tetrahydropyranyl and related ethers; methylthiomethyl ether;
- Esters of formula -C (= O) R4, such as acetate ester, benzoate ester, pivalate ester (dimethylacetate), methoxyacetate ester, chloroacetate ester, levulinate (4-oxopentanoate) ester; twenty
- Carbonates of the formula -C (= O) OR4, such as benzyl carbonate, p-nitrobenzyl carbonate, tert-butyl carbonate, 2,2,2-trichlorethyl carbonate, and 2- (trimethylsilyl) ethyl carbonate, allyl carbonate.
- Sulphonates of the formula -SO2R4, such as methyl sulphonates, tosylate, brosylate, mesylate. 25

In all of the above formulas R4 represents a group selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl and substituted or unsubstituted arylalkyl. 30

The term "treatment" or "treat" in the context of this document refers to the administration of a compound or a composition according to the invention to prevent, ameliorate or eliminate the disease, pathological condition or one or more symptoms associated with said disease or condition in a mammal, preferably in a human being. “Treatment” also covers the prevention, improvement or elimination of
physiological sequelae of the disease. Specifically, the concept "treat" can be interpreted as:
(i) Prevent the disease or pathological condition from occurring in a mammal, in particular, when said mammal has a pre-disposition for the pathological condition, but has not yet been diagnosed as having it. In particular, the method of the invention would allow, after the detection of CSCs, to begin a treatment that could prevent the development of the tumor in advance;
(ii) Inhibit the disease or pathological condition, that is, stop its development;
(iii) Relieve the disease or the pathological condition, that is, causes the regression of the disease or the pathological condition;
(iv) Stabilize the disease or pathological condition.

Throughout the description and claims the term "comprises", which may also be construed as "consists of", and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. Compounds of the invention 20
In a first aspect of the invention, the present invention relates to new compounds ("compounds of the invention"), of general formula IV

25

where
 R can be an alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heterocycloalkyl, -C (= O) R '', -C (= O) OR '', or the rest of benzenesulfonyl indicated below:

 R 'can be H, OH, 3,5-diOH, 2-NO2, 3-NO2, 4-NO2, 2-NH2, 3-NH2, 4-NH2, 4-Me, 4-OMe (Ts), 3,4-diOMe, 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 3,5-diCl, 3, 5-diBr, 3,5-diI or 3,5-diMe in a benzenesulfonyl moiety; 5
 R ’’ is selected from the group consisting of:

 X and W each independently represent each other, an electronegative atom 10 selected from the group consisting of -Cl, -Br, and -I;
 Z is a group of atoms that is integrated into various organic functions such as silyl ethers of the formula -Si (R4) 3, alkyl and arylalkyl ethers, alkoxymethyl and aryloxy ethers of the formula -CH2OR4; esters of formula -C (= O) R4; carbonates of formula -C (= O) OR4 and 15 sulphonates of formula -SO2R4.
 R '' '' may be H, acyl groups of the formula -C (= O) R4, such that the group -OC (= O) R4 may be an acetate ester, benzoate ester, pivalate ester (dimethylacetate), methoxyacetate ester, chloroacetate ester, levulinate ester (4-oxopentanoate), 20
 R4 represents a group selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and heterocycle.

In a particular embodiment, the compounds of the invention are presented in crystalline form as free compounds or solvates (for example hydrates) or as enantiomers, isomers, or salts of said compounds, all of these forms being within the scope of protection of the present invention . Solvation methods are generally known in the state of the art.
30
Also included within the scope of the present invention are pro-drugs of the compounds of the invention.
A particular embodiment of the first aspect of the invention relates to compounds of general formula IVa.

Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVb. 5

Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVc.
10 ONNNO2SXWNNR '
IVc

Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVc ’.
15 ONNNO2SXNNR'W
IVc ’
Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVd.

Another particular embodiment of the first aspect of the invention relates to compounds 5 of general formula IVe.

Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVf. 10

Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVg.
fifteen

Another particular embodiment of the first aspect of the invention relates to compounds of general formula IVh.

ONNXNNWN
IVh

Yet another particular embodiment of the first aspect of the invention relates to compounds of general formula IVh ’. 5

ONNWNNXN
IVh ’

Another particular embodiment of the first aspect of the invention relates to compounds 10 selected from the group consisting of the compounds detailed in Table 1:

Group Comp. R R ’’ W X
IVa 4 -Ts -CO2Et - -
IVb 5 -Ts -CH2OH - -
IVd 6 -Ts -CH2Cl - -
IVe 7 -Ts -CH2Ts - -
IVh ’ 8 -CH3 NNNNWX9 Cl H
9 -CH3 Br H
10 -CH3 Cl Cl
IVc 11 -Ts Cl H
12 -Ts Br H
13 -Ts Cl Cl
IVf 14 -Ts CH2N3 - -
IVg 15 -Ts - -

Table 1. Preferred embodiments of 3,4-dihydro-2H-1,4-benzoxazine.

Yet another particular embodiment of the first aspect of the invention relates to ONO2SR'R5
Where R5 is selected from the list consisting of-CO2Et, -CH2OH, -CH2Cl and -CH2Ts. 5

Yet another particular embodiment of the first aspect of the invention relates to compounds with the following general formula V.
ONR6R '' '
V 10
Where R ’’ is selected from the group consisting of:

, CH2N3,;

and where R6 is selected from the group consisting of a methyl group or the following group:
.

Pharmaceutical formulations
twenty
In a second aspect, the invention provides pharmaceutical formulations comprising as active ingredient at least one compound of the invention, in
in particular a compound of those detailed in the preferred embodiments of the first aspect of the invention. Said pharmaceutical formulations may contain one or more excipients and / or transport substances. Furthermore, said formulations may contain any other active ingredient in the treatment of cancer.
5
The excipients, transport substances and auxiliary substances must be pharmaceutically and pharmacologically tolerable, so that they can be combined with other components of the formulation or preparation and do not exert adverse effects on the treated organism. Pharmaceutical compositions or formulations include those that are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous), although the best route of administration depends on the patient's condition. The formulations can be in the form of single doses. The formulations are prepared according to methods known in the field of pharmacology. The amounts of active substances to be administered may vary depending on the particularities of the therapy. fifteen

Optionally the pharmaceutical composition may comprise another active ingredient. In addition to the requirement of therapeutic efficacy, which may require the use of therapeutic agents, in addition to the compounds of the invention, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of a compound of the invention and another therapeutic agent, such as in the treatment of diseases or conditions that directly or indirectly modulate the function of the substance.

The "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art and commonly used in the elaboration of therapeutic compositions.

Pharmaceutical formulations may contain one or more excipients and / or transport substances. In addition, said formulations may contain another 30 active ingredients with anti-cancer properties. The excipients, transport substances and auxiliary substances have to be pharmaceutically and pharmacologically tolerable, so that they can be combined with other components of the formulation or preparation and do not exert adverse effects on the treated organism. Pharmaceutical compositions or formulations include those that are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular or intravenous), although the best route of
Administration depends on the patient's condition. The formulations can be in the form of single doses. The formulations are prepared according to methods known in the field of pharmacology. The amounts of active substances to be administered may vary depending on the particularities of the therapy.
5
The compositions of the invention (see compositions of the first or second aspect of the invention) are prepared using usual methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.
10
Medical uses of the compounds of the invention

In a third aspect, the invention relates to the use of the compounds or compositions of the invention either in therapy or as a medicament. Specifically, the compounds or compositions of the invention for use in therapy or as a medicament are subject of the invention. In a particular embodiment, the invention relates to the use of the compounds or compositions of the invention for the treatment of cancer, preferably breast, pancreas, lung and colorectal cancers, as well as melanomas such as metastatic melanoma. In a preferred embodiment, its use is directed to the inhibition of CSCs. twenty

On the other hand, the use of one of the compounds or compositions of the invention in the preparation of a medicament is considered an object of the invention. In a particular embodiment, the compounds or compositions of the invention are employed in the preparation of a medicament for cancer, preferably of breast, pancreas, lung and colorectal cancers, as well as melanomas such as metastatic melanoma. In a preferred embodiment, its use is directed to the preparation of medicaments for the inhibition of CSCs.

Another aspect of the present invention relates to the use of the compounds or compositions of the invention for use as a medicament for the treatment of pathologies or diseases caused by neoplastic processes. More specifically, the invention relates to compounds for use as medicaments for the treatment of cancer. More preferably breast, pancreas, lung and colorectal cancer, as well as melanomas such as metastatic melanoma.

In another aspect, the present invention relates to a method for the treatment of patients affected by cancer through the use of the compounds or compositions of the invention. The anticancer effects of a treatment method of the present invention include, but are not limited to, the antitumor effects, the speed of response, the time of disease progression and the survival rate. The antitumor effects of a method of treatment of the present invention include, but are not limited to tumor growth inhibition, tumor growth retardation, tumor regression, tumor contraction, increased time for tumor growth again. at the end of treatment, and slowing the progression of the disease. It is expected that when a method 10 of treatment of the present invention is administered to an animal, preferably mammalian, and more preferably human, in need of anticancer treatment, the method of treatment will produce a measured effect, for example by one or more of the following characteristics: the extent of the antitumor effect, the speed of response, the time of disease progression and the survival rate. Anticancer effects include prophylactic treatment.

This treatment consists in the administration to the individuals affected by these diseases of therapeutically effective amounts of a compound of the invention, or a pharmaceutical composition that includes it. twenty

In the sense used in this description, the term "therapeutically effective amount" refers to that amount of a compound of the invention that when administered to a mammal, preferably a human, is sufficient to produce the treatment, as defined. below, of a disease or pathological condition of interest in the mammal, preferably a human. The amount of a compound of the invention that constitutes a therapeutically effective amount will vary, for example, depending on the activity of the specific compound employed; the metabolic stability and duration of action of the compound; the age, body weight, general state of health, sex and diet of the patient; the mode and time of administration; the rate of excretion, the combination of drugs; the severity of the particular disorder or pathological condition; and the subject who undergoes therapy, but can be determined by a specialist in the art according to their own knowledge and that description.
35
The administration of the compounds of the invention, in pure form or in an appropriate pharmaceutical composition, can be carried out by means of the administration modes of agents accepted to serve similar utilities.

5
Invention Procedure

In another aspect the invention relates to a process (Figure 1) for the preparation of the compounds of the present invention. The compounds of the invention, of general structure IV, comprising the formation of an ester from an intermediate of general formula I (Intermediates I).

In particular, the process for obtaining compounds of general formula IVa consists in the formation of an ester from an intermediate of general formula I.
fifteen
In another particular embodiment, the compounds of general formula IVb are obtained by a process consisting of the formation of an ester from an intermediate of general formula I and a subsequent reduction.

In an even more particular embodiment, this reduction is achieved by the action 20 of lithium borohydride in THF.

In another particular embodiment, the compounds of general formula IVc or IVc 'are obtained by a process that consists in carrying out a reaction of Mitsunobu on a compound of general formula IVb. 25

In another particular embodiment, the compounds of general formula IVd are obtained by a process that consists in carrying out a halogenation on a compound of general formula IVb.
30
In another particular embodiment, the compounds of general formula IVg are obtained by a process consisting of forming a substituted triazole on a compound of general formula IVb.

In another particular embodiment, the compounds of general formula IVe are obtained by a process consisting of treating a compound of general formula
IVb with an equivalent (the same amount of moles) of TsCl heating under reflux, preferably for 24 hours.

In another particular embodiment, the compounds of general formula IVf are obtained by treating a compound of general formula IVe with sodium azide. 5

In a preferred embodiment, the process for obtaining the different groups of compounds of general formula IV, comprises the following steps:

1. React o-aminophenol with benzenesulfonyl chloride with different substituents on the aromatic ring (donor electron groups or attractants) to produce intermediates I

2. React intermediates I with racemic ethyl 2,3-dibromopropionate to produce IVa 15

3. Reduce IVa to obtain intermediate IVb

The different variations to pass from IVb to IVc, IVc ’, IVd and Vg are described below:

d1) Mitsunobu reaction (IVc and IVc ’)

React the intermediates IVb and a mono- or di-substituted purine in the presence of dialkyl azodicarboxylate and triphenylphosphine in an organic solvent to obtain IVc (N-9) and / or IVc ’(N-7).

5 ONNNO2SXNNR'W
IVc

IVc ’

10

d2) halogenation (IVd);
Treat IVb in a particularization with thionyl chloride to replace alcohol with chlorine. In general, halogenation is carried out.
fifteen

d3) formation of substituted triazoles

d3a) Treat IVb with an equivalent (the same amount of moles) of TsCl by refluxing for 24 hours, to obtain IVe.

5
d3b) Treat IVe with sodium azide to obtain IVf.

d3c) Perform the chemistry click to IVf, to obtain the compounds IVg. 10

Next, the steps of the process of the invention are described in more detail:
fifteen
Stage a
The formation of intermediate compounds I is carried out by reaction between o-aminophenol (Mulzer, JJ Org. Chem., 2000, 65, 6540-6546) and sulfonyl chlorides according to previously described procedures (Díaz-Gavilán, M .; Rodríguez-Serrano, F .; Gómez-Vidal, JA; Marchal, JA; Aránega, A .; Gallo, MA; Espinosa, A .; 20 Campos, JMTetrahedron, 2004, 60, 11547-11557).

Intermediates I are substituted phenylsulfonamides in which R '= H, 4-OH, 3,5-diOH, 4-Me, 4-OMe, 3,4-diOMe, 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 3,5-diCl, 3,5-diBr, 3,5-diI, 3,5-diMe.

Stage b 5
Compounds IVa are synthesized from the corresponding intermediates by double exchange reaction of the bromine atoms of racemic ethyl 2,3-dibromopropionate, using potassium carbonate base in acetone as a base and heating at reflux. Compounds IVa are substituted phenylsulfonamides (R '= H, 4-OH, 3,5-diOH, 4-Me, 4-OMe, 3,4-diOMe, 2-Cl, 3-Cl, 4-Cl, 2- Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 3,5-diCl, 3,5-10 diBr, 3,5-diI, 3,5-diMe), in the that the nitrogen atom is part of an ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate residue.

Stage c
Compounds IVa are reduced, preferably with lithium borohydride, in THF at room temperature to give compound intermediates IVb. Compounds IVb are substituted phenylsulfonamides (R '= H, 4-OH, 3,4-diOH, 4-Me, 4-OMe, 3,4-diOMe, 2-Cl, 3-Cl, 4-Cl, 2- Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 3,5-diCl, 3,5-diBr, 3,5-diI, 3,5-diMe). in which the nitrogen atom is part of a 3,4-dihydro-2H-1,4-benzoxazine-2-hydroxymethyl moiety. twenty

Stage d1 (Mitsunobu)
Compounds IVb and a mono- or di-substituted halogen purine at positions 2 and / or 6 each representing, independently from each other, an atom selected from the group consisting of Cl, -Br, and -I, are reacted in presence of dialkyl azodicarboxylate and triphenylphosphine in an organic solvent to obtain compounds of families IVc or IVc '.

Stage d2 (halogenation)
Compounds IVb are reacted with thionyl chloride (to obtain the derivative with chlorine 6) or with carbon tetrabromide (to obtain the derivative with bromine) and from the chlorine derivative, compound 6, by the Finkelstein reaction, it is replaced chlorine for iodine to obtain the family of compounds IVd.

Stage d3 (formation of substituted triazoles) 35
d3a) Protection of compounds IVb with leaving group (IVe), d3b) formation of azide (IVf) and d3c) chemical click to form the substituted triazole ring.Finally we obtain the family of compounds IVg.

MODES OF EMBODIMENT 5

The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention:

Materials and Methods 10
Abbreviations and Acronyms (obtained from the Guide for Authors; Abbreviations and Acronyms J. Org. Chem. 2008, 78, 10A):
Ac: acetyl
Bu: butyl
cat .: catalytic 15
13CRMN carbon nuclear magnetic resonance
d: double
dd: double double
ddd: double double double
DMSO: dimethylsulfoxide 20
DIAD: diisopropylazodicarboxylate
dq: double quartet
dt: triplet doublet
Et: ethyl
g: gram 25
Hz: hertz
J: coupling constant measured in Hz
HRMS: high resolution mass spectroscopy
Ts: p-toluenesulfonyl (Tosyl)
mM: 30 millimolar
M +: molecular peak
Me: methyl
mg: milligram
mL: milliliter
mmol: millimol 35
m / z: mass / load ratio
Pf: melting point
ppm: parts per million
s: singlet
t: triplet
t.a .: room temperature
THF: tetrahydrofuran 5

The reagents and solvents used come from the Aldrich, Acros, SDS or Scharlau commercial houses, and were purified by usual procedures (see Armarego, W. L .; Perrin, D. D. Purification of Laboratory Chemicals; Butterworth-Heinemann: Oxford, 1996.) when necessary. 10

The purification of the reaction products was carried out by column chromatography under pressure (flash chromatography), using silica gel as a stationary phase (with a particle size 230-400 mesh) and as the mobile phase the solvents to be indicated in each case . The eluent used is indicated in each case and the 15 proportions of the solvent mixture used are always volume / volume.

The reactions were monitored by thin plate chromatography (TLC), using 60 F254 silica gel chromatopholios marketed by Merck. The plates were developed using a UV light viewer ( = 254 nm). twenty

The 1H and 13C nuclear magnetic resonance spectra (completely decoupled) were performed at room temperature in the solvent indicated in each case (CDCl3, and DMSO-d6) using the following devices: VarianINOVA-TM (300, 400, 500, 600 MHz) . The values of chemical shifts are expressed in 25 parts per million (δ, ppm), using the residual solvent signal as internal reference: CDCl3, 7.26 ppm (1HRMN) and 77.0 ppm (13CRMN); DMSO-d6, 2.50 ppm (1HRMN) and 41.1 ppm (13CRMN).

The 1 H-NMR spectra are described indicating the number of protons and the apparent multiplicity of each signal. The coupling constants (J) are the apparent ones and are expressed in Hz. Melting points (Pf) were measured in open capillaries in an Electrothermal brand apparatus. The data obtained from the mass spectra are expressed in units of mass (m / z). The molecular peak is specified as M +. Microwave-assisted small-scale synthesis has been carried out in 35 Initiator 2.0 instrument in a simple way, producing controlled irradiation at 2,450 GHz (Biotage AB, Uppsala). The reaction time refers to when
it reaches the expected temperature, and does not refer to the entire irradiation time. The temperature was measured by means of an IR sensor placed outside the reaction medium. The anhydrous conditions were carried out under an inert atmosphere of argon.

Examples of the synthesis procedure 5

I) Known starting reagents

2- (Ethoxycarbonyl) -3,4-dihydro-2H-1,4-benzoxazine
Obtained according to a known procedure (Augstein, J .; Monro, A.M .; 10 Hessey, G. W. G.B. Patent 1,057,568, 1967; Chem. Abstr. 1967, 66, 95058z).

2- (Ethoxycarbonyl) -4-methyl-3,4-dihydro-2H-1,4-benzoxazine
Obtained according to a known procedure (Augstein, J .; Monro, A.M .; 15 Hessey, G. W. G.B. Patent 1,057,568, 1967; Chem. Abstr. 1967, 66, 95058z).

2- (Hydroxymethyl) -4-methyl-3,4-dihydro-2H-1,4-benzoxazine
Obtained according to a known procedure (Zhou, D .; Harrison, BL; Shah, 20 U .; Andree, TH; Hornby, GA; Scerni, R .; Schechter, LE; Smith, DL; Sullivan, KM; Mewshaw, RE Bioorg. & Med. Chem. Lett. 2006,16, 1338-1341).

II) Preparation of starting substances 25

N- [2- (Hydroxyphenyl) -4-methylbenzenesulfonamide (Intermediate I)
To a solution of 2-aminophenol (8 g, 73.3 mmol) in anhydrous CH2Cl2 (250 mL), pyridine (7.09 mL, 88 mmol) and tosyl chloride (15.4 g, 80.6 mmol) are added. The reaction mixture is heated at reflux for 24 hours. After cooling the mixture of 30
reaction is washed with H2O (250mL). After drying the organic phase (anhydrous Na2SO4) and filtration, it is concentrated on a rotary evaporator and intermediate I is precipitated with hexane. White solid (19.1 g, 99%). Mp = 120-121 ° C; 1HRMN (400 MHz, DMSO-d6) δ 9.46 (m, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.31 ( dd, J = 7.9, 1.4 Hz, 1H), 7.09 (t, J = 8.67, 7.98 Hz, 1H), 6.88 (m, 2H), 3.52 (s, 1H) .13C NMR (100 MHz, DMSO-d6) δ 5 150.06, 142.72, 137.74, 129.27, 126.73, 126.01, 124.27, 124.19, 118.90, 115.51, 20.92. HRMS TOF MSES + (m / z): Calculated for C13H13NNaO3S (M + Na) +: 286.0514, found: 286.0515. Elemental Analysis: Theoretical C13H13NO3S (%): C 59.30; H 4.98, N 5.32; S 12.18; Obtained (%): C 59.40, H 4.88, N 5.00; S 12.32.
10
Synthesis Examples of Compounds of the Invention
The following examples are presented by way of illustration of the present invention:

Ethyl 4-Tosyl-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate (compound 4)
To a mixture of intermediate I with tosyl chloride (6 g, 22.79 mmol) and K2CO3 (7.66 g, 15 55.47 mmol), acetone (70 mL) is added first and then ethyl 2,3-dibromopropionate (5.92 g , 22.79 mmol). The reaction mixture is refluxed for 8 hours. After cooling the mixture is filtered, it is concentrated to rotary evaporation by the solvent leaving a crude which is suspended in H2O, filtered and the compound 4 is obtained as a white solid (8.2 g, 99%). Mp = 147-148 ° C; 1H 20 NMR (600 MHz, CDCl3) δ 7.83–7.78 (m, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 7.12–7.06 (m, 1H ), 6.99-6.93 (m, 2H), 4.51-4.46 (m, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.95 (dd, J = 9.5, 2.8 Hz, 1H), 3.53-3.46 ( m, 1H), 2.39 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 167.50, 145.88, 144.73, 135.45, 130.18, 127.38, 126.57, 124.45, 123.50, 121.69, 117.87, 70.37, 62.16, 45.82, 21.70, 14.25.HRMS TOF MSES + (m / z): Calculated 25 for C18H19NNaO5S (M + Na) +: 384.0882, found: 384.0883. Elemental Analysis: Theoretical C18H19NO5S (%): C 59.82; H 5.30, N 3.88; S 8.87; Obtained (%): C 59.85, H 5.28, N 3.96; S 8.68.

2-Hydroxymethyl-2-tosyl-3,4-dihydro-2H-1,4-benzoxazine (compound 5) 30
To a solution of compound 4 (8.23 g, 22.77mmol) in THF (90 mL), LiBH4 (0.60 g, 27.33mmol) is added at room temperature and dropwise. The reaction is instantaneous. The solvent is removed by rotary evaporation, H2O is added to obtain compound 5 as a white precipitate that is filtered and dried. Mp .: 133-134 ° C. 1H NMR (500 MHz, CDCl3) δ 7.84-7.79 (m, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.5 35 Hz, 2H), 7.07 (dd, J = 11.3 , 4.1 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H), 4.27 (dd, J = 14.4, 2.3 Hz, 1H), 3.76 (dd, J = 12.0, 4.1 Hz, 1H), 3.66 (dd, J = 12.0,
4.6 Hz, 1H), 3.53 (m, 1H), 3.38 (dd, J = 14.3, 10.0 Hz, 1H), 2.39 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 146.87, 144.47, 135.84, 130.11, 127.33, 126.38, 124.59, 123.76, 121.26, 117.53, 72.11, 62.80, 45.54, 29.86, 21.74, 1.17. HRMS (m / z): Calculated for C16H17NNaO4S TOF MSES + (M + Na) +: 342.0776, found: 342.0773. Elemental Analysis: Theoretical C16H17NO4S (%): C 60.17; H 5.37, N 4.39; S 10.04; Obtained (%): C 5 59.99, H 5.29, N 4.46; S 9.91.

2- (Chloromethyl) -4-tosyl-3,4-dihydro-2H-1,4-benzoxazine (compound 6)
To a solution of 2- (hydroxymethyl) -4-methyl-3,4-dihydro-2H-1,4-benzoxazine, compound 5 (0.26 g, 0.081mmol) in anhydrous CH2Cl2 (2.4 mL), is added 10 thionyl (0.071 mL, 0.9769mmol). Stir half an hour at room temperature. Subsequently, anhydrous pyridine (0.072 mL, 0.9769mmol) is added. The reaction mixture is irradiated in microwave at 100 ° C for 90min. The solvent is evaporated in vacuo and the reaction crude is purified by flash chromatography using only dichloromethane as eluent. White solid (0.27 g, 99%); mp = 120-121 ° C. 1H NMR 15 (500 MHz, CDCl3) δ 7.85 (dd, J = 8.3, 1.5 Hz, 1H), 7.55–7.51 (m, 2H), 7.25 (d, J = 8.0 Hz, 2H ), 7.08 (ddd, J = 8.3, 7.4, 1.6 Hz, 1H), 6.96 (ddd, J = 8.6, 7.4, 1.5 Hz, 1H), 6.83 (dd, J = 8.2, 1.5 Hz, 1H), 4.45 ( dd, J = 14.4, 2.4 Hz, 1H), 3.60 (dd, J = 11.2, 4.1 Hz, 1H), 3.57–3.50 (m, 1H), 3.46 (dd, J = 11.2, 6.7 Hz, 1H), 3.28 (dd, J = 14.4, 9.6 Hz, 1H), 2.40 (s, 3H) .13C NMR (125 MHz, CDCl3) δ 146.56, 144.61, 135.47, 130.16, 127.42, 20 126.51, 124.74, 123.61, 121.47, 117.51, 70.91, 46.47, 42.97, 29.85, 21.75. HRMS (m / z): Calculated for C16H16ClNNaO3STOF MSES + (M + Na) +: 360.0437, found: 360.0438. Elemental Analysis: Theoretical C16H16ClNO3S (%): C 56.89; H 4.77, N 4.15; S 9.49; Obtained (%): C 56.77, H 4.53, N 4.21; S 9.54.
25
4-Tosyl-3,4-dihydro-2H-1,4-benzoxazin-2-yl) methyl 4-methylbenzenesulfonate (compound 7)
To a solution of compound 5 (2 g. 6.26 mmol) in anhydrous CH2Cl2 (20 mL) is added successively anhydrous pyridine (0.6 g, 7.51mmol) and tosyl chloride (1.2 g, 6.26 mmol). After heating the solution at reflux for 24 hours, the solvent is removed at 30 rotary evaporation after cooling, the crude is suspended in H2O, filtered and purified by gradient flash chromatography (EtOAc / hexane: 2/8, 3/7, 4 / 6 and 1/1). After removal of the solvents, we obtain compound 7 (2.71 g, 56.8%), as a white solid; mp: 84-850C. 1H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 8.2 Hz, 3H), 7.35 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.90 (dt, J = 8.3, 35 4.6 Hz, 1H), 6.78 (t, J = 7.2 Hz, 1H), 6.56 (dd, J = 8.1, 0.9 Hz, 1H), 4.14 ( dd, J = 14.4, 2.3 Hz, 1H), 3.94 (dq, J = 11.0, 4.5 Hz, 2H), 3.43 (m, 1H), 3.07 (dd, J = 14.4, 10.1 Hz,
1H), 2.32 (s, 3H), 2.23 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 146.05, 145.25, 144.47, 135.10, 132.16, 129.97, 129.91, 127.85, 127.04, 126.22, 124.34, 123.15, 121.13, 117.32, 68.92, 68.25, 44.98, 30.74.HRMS (m / z ): Calculated for C23H24NO6S2 TOF MS ES + (M + H) +: 474.1040, found: 474.1040 Elemental Analysis: C23H23NO6S2 Theoretical (%): C 58.33; H 4.90, N 2.96; S 13.54; Obtained (%): C 58.19, H 4.80, N 3.08; 5 S 13.87.

2- (Azidomethyl) -4-tosyl-3,4-dihydro-2H-1,4-benzoxazin-2-yl) methyl (compound 14)
To a solution of compound 7 (3 g. 6.3mmol) in DMF (29 mL) is added sodium azide (0.6 g, 9.5mmol). After heating the reaction at reflux at 150 ° C for 5 hours, it is allowed to cool and the solvent is removed by rotary evaporation. The crude precipitates with methanol, is filtered with bushner and kitasate and compound 14 (1.57 g, 72%) is obtained, as a yellowish solid; mp: 136-140 ° C. 1H NMR (400 MHz, DMSO-d6) 1H NMR (400 MHz, dmso) δ 7.57 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.10 (m, 2H), 6.97 (m, 1H), 6.84 (dd, J = 8.2, 1.4 Hz, 1H), 4.33 (dd, J = 14.4, 2.5 Hz, 1H), 3.63 (dd, J = 12.7, 3.3 15 Hz, 1H), 3.55 (ddd, J = 9.2, 5.7, 2.7 Hz, 1H), 3.49 (dd, J = 12.7, 5.6 Hz, 1H), 3.33 (dd, J = 12.7, 5.6 Hz, 1H), 2.36 (s, 3H) . 13C NMR (100 MHz, DMSO-d6) δ 146.03, 144.57, 137.63, 134.69, 130.17, 127.02, 125.49, 123.68, 121.00, 117.28, 70.87, 50.88, 45.10, 20.99.HRMS (m / z): Calculated for C16H16N4O3NaSTOF MS ES + (M + Na) +: 367.0876, found: 367.0841. Elemental Analysis: C16H16N4O3 Theoretical (%): C 55.80, H 4.68, 20 N 16.27, S 9.31. Obtained (%): C 55.90, H 4.71, N 15.99, S 9.27.

3- (1 - ((4-Tosyl-3,4-dihydro-2H-1,4-benzoxazin-2-yl) methyl) -1H-1,2,3-triazol-4-yl) phenol (compound 15 )
To a solution of compound 14 (0.2 g. 0.58mmol) in DMF (5 mL) is added 3-4-25 95% hydroxyphenylacetylene (0.1 g, 0.75mmol), copper bromide (0.13 g, 0.58mmol) and propylamine (0.03 mL, 0.35mmol). The reaction is irradiated at 140 ° C for 30 minutes. The DMF is removed and the reaction crude is purified by flash chromatography using as eluent a gradient solvent mixture (EtOAc / hexane: 2/8, 3/7, 4/6 and 1/1). Compound 15 (1.57 g, 72%) is obtained, as a yellowish solid; mp: 153-30 160 ° C. 1H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.45 (s, 1H), 7.68 (dd, J = 8.3, 1.5 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H) , 7.29 (m, 5H), 7.09 (m, 1H), 6.97 (m, 1H), 6.87 (dd, J = 8.2, 1.4 Hz, 1H), 6.75 (dt, J = 6.4, 2.5 Hz, 1H), 4.78 (dd, J = 14.4, 4.4 Hz, 1H), 4.65 (dd, J = 14.5, 6.2 Hz, 1H), 4.46 (dd, J = 14.5, 2.4 Hz, 1H), 3.74 (dd, J = 6.3, 3.4 Hz, 1H), 3.32 (dd, J = 8.3, 1.5 Hz, 4H), 2.33 (s, 3H) .13C NMR (100 MHz, DMSO-d6) δ 35 157.77, 146.50, 145.77, 144.60, 134.45, 131.69 , 130.17, 129.97, 126.96, 126.21, 123.58, 123.18, 122.31, 121.17, 117.46, 116.07, 115.01, 111.89, 70.42, 50.41, 45.35,
20.98. HRMS (m / z): Calculated for C24H23N4O4STOF MS ES + (M + H) +: 463.1440, found: 463.1453. Elemental Analysis: C24H22N4O4S Theoretical (%): C 62.32, H 4.79, N 12.11, S 6.93. Obtained (%): C 62.38, H 4.70, N 12.30, S 6.78.

5
General methodology of synthesis of compounds 8-13
Under argon, a mixture of PPh3 (2.2 equiv.), Compound 5 (1 equiv.) And the corresponding purine (1 equiv.) In anhydrous THF (5 mL) is prepared and cooled to -20 ° C in a bath. of acetone and CO2 before adding DIAD (2.2 equiv.) drop by drop. The temperature of the reaction mixture is raised slowly to 5 ° C by adding acetone to the bath and irradiated in microwave at 140 ° C for 10min. The solvent is evaporated in vacuo and the reaction crude is purified by flash chromatography using as eluent a mixture of EtOAc: hexane solvents (1: 1) (Table 1).
fifteen
2 - [(6-Chloro-9H-purin-9-yl) methyl] -4-methyl-3,4-dihydro-2H-1,4-benzoxazine (compound 8)
Yellowish solid (60%). Mp: 143-144 ° C. 1H NMR (600 MHz, CDCl3) δ 6.89 (td, J = 7.7, 1.5 Hz, 1H), 6.79 (m, 1H), 6.71 (dd, J = 12.0, 4.6 Hz, 2H), 4.62 (dd, J = 14.6, 3.6 Hz, 1H), 4.53 (dd, J = 14.6, 7.6 Hz, 1H), 3.37 (dd, J = 11.7, 2.7 Hz, 1H), 3.01 (dd, J = 20 11.6, 6.0 Hz, 1H) . 13C NMR (150 MHz, CDCl3) 151.96, 151.18, 146.15, 142.29, 135.50, 131.46, 122.22, 119.03, 116.28, 112.73, 77.19, 76.98, 76.77, 71.19, 50.40, 45.75, 38.62. HRMS (m / z): Calculated for C15H15ClN5O TOF MS ES + (M + H) +: 316.0965, found: 316.0963. Elemental Analysis: Theoretical C15H14ClN5O (%): C 57.06, H 4.47, N 22.18. Obtained (%): C 57.23, H 4.71, N 22.00. 25

2 - [(6-Bromo-9H-purin-9-yl) methyl] -4-methyl-3,4-dihydro-2H-1,4-benzoxazine (compound 9)
Yellowish solid (70%). Mp: 137-138 ° C. 1 H NMR (600 MHz, CDCl 3) δ 8.73 (s, 1H), 8.23 (s, 1H), 6.92 (td, J = 7.9, 1.5 Hz, 1H), 6.81 (dd, J = 8.4, 1.4 Hz, 1H), 6.74 (dd, J = 30 11.6, 4.5 Hz, 2H), 4.70 (ddd, J = 9.7, 6.2, 3.2 Hz, 1H), 4.63 (dd, J = 14.6, 3.6 Hz , 1H), 4.54 (dd, J = 14.6, 7.6 Hz, 1H), 3.40 (dd, J = 11.7, 2.7 Hz, 1H), 3.03 (dd, J = 11.7, 6.0 Hz, 1H), 2.90 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 151.93, 150.72, 146.04, 143.28, 142.32, 134.50, 134.09, 122.25, 119.11, 116.32, 112.80, 71.17, 50.42, 45.77, 38.68. HRMS (m / z): Calculated for C15H15BrN5O TOF MS ES + (M + H) +: 360.0454, 35 found: 360.0456. Elemental Analysis: C15H14BrN5O Theoretical (%): C 50.02, H 3.92, N 19.44. Obtained (%): C 49.89, H 3.80, N 19.29.

2 - [(2,6-Dichloro-9H-purin-9-yl) methyl] -4-methyl-3,4-dihydro-2H-1,4-benzoxazine (compound 10)
Yellowish solid (85%). Mp: 123-124 ° C. 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 6.83 (m, 1H), 6.71 (dd, J = 8.2, 1.6 Hz, 1H), 6.64 (m, 2H ), 4.61 (ddd, J = 8.6, 5.9, 2.9 5 Hz, 1H), 4.55 (dd, J = 14.5, 3.3 Hz, 1H), 4.43 (dd, J = 14.5, 8.0 Hz, 1H), 3.35 (dd , J = 11.7, 2.8 Hz, 1H), 3.00 (dd, J = 11.7, 5.8 Hz, 1H), 2.84 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 153.26, 152.83, 151.63, 146.95, 142.05, 135.45, 130.54, 122.22, 118.91, 116.17, 112.69, 71.01, 50.23, 45.88, 38.57. HRMS (m / z): Calculated for C15H14Cl2N5O TOF MS ES + (M + H) +: 350.0570, found: 350.0573. Elemental Analysis: 10 C15H13Cl2N5O Theoretical (%): C 51.44, H 3.74, N 20.00. Obtained (%): C 51.58, H 3.81, N 20.30.

2 - [(6-Chloro-9H-purin-9-yl) methyl] -4-tosyl-3,4-dihydro-2H-1,4-benzoxazine (compound 11) 15
White solid (45%). Mp: 189-190 ° C. 1 NMR (400 MHz, CDCl 3) δ 8.77 (s, 1H), 8.14 (s, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 7.07 (t, J = 7.7 Hz, 1H), 6.95 (t, J = 7.7 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 4.52 (dd, J = 14.7, 3.6 Hz, 1H), 4.37 (m, 2H), 3.84 (m, 1H), 3.15 (dd, J = 14.3, 9.6 Hz, 1H), 2.34 (s, 3H ). 13C NMR (100 MHz, CDCl3) δ 152.28, 151.97, 151.53, 145.88, 145.72, 144.74, 135.51, 20 131.56, 130.23, 127.28, 126.65, 124.49, 123.54, 121.97, 117.56, 69.77, 45.96, 45.52, 21.71.HRMS ( m / z): Calculated for C21H19ClN5O3S TOF MS ES + (M + H) +: 456.0892, found: 456.0890. Elemental Analysis: C21H18ClN5O3S Theoretical (%): C 55.32, H 3.98, N 15.36, S 7.03. Obtained (%): C 55.49, H 4.08, N 15.51, S 6.89.
25
2 - [(6-Bromo-9H-purin-9-yl) methyl] -4-tosyl-3,4-dihydro-2H-1,4-benzoxazine (compound 12)
White solid (39%). Mp: 168-169 ° C. 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 8.15 (s, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 7.07 (t, J = 7.0 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 4.51 (dd, J = 14.7, 3.5 30 Hz, 1H), 4.36 (m, 2H), 3.83 (m, 1H), 3.15 (dd, J = 14.4, 9.6 Hz, 1H), 2.34 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 152.20, 150.72, 145.75, 145.71, 144.74, 143.60, 135.50, 134.16, 130.23, 127.27, 126.65, 124.49, 123.53, 121.97, 117.55, 69.75, 45.95, 45.53, 21.71. HRMS (m / z): Calculated for C15H15BrN5O TOF MS ES + (M + H) +: 500.0386, found: 500.0387. Elemental Analysis: C21H18BrN5O3S Theoretical (%): C 50.41, H 35 3.63, N 14.00, S 6.41. Obtained (%): C 50.40, H 3.44, N 13.89, S 6.29.

2 - [(2,6-Dichloro-9H-purin-9-yl) methyl] -4-tosyl-3,4-dihydro-2H-1,4-benzoxazine (13)
White solid (62.8%). Mp: 196-197 ° C. 1H NMR (500 MHz, CDCl3) δ 8.12 (s, 1H), 7.77 (dd, J = 8.3, 1.5 Hz, 1H), 7.51 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.2 Hz , 2H), 7.09 - 7.04 (m, 1H), 6.99-6.93 (m, 1H), 6.78 (dd, J = 8.2, 1.4 Hz, 1H), 4.51 (dd, J = 14.7, 3.3 Hz, 1H), 4.36 (dd, J = 14.4, 2.5 Hz, 1H), 4.29 (dd, J = 14.7, 7.4 Hz, 1H), 3.78 (ddt, J = 9.9, 5 7.3, 2.9 Hz, 1H), 3.20 (dd, J = 14.4, 9.5 Hz, 1H), 2.34 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 153.31, 153.18, 152.22, 146.60, 145.49, 144.87, 135.29, 130.70, 130.29, 127.23, 126.69, 124.51, 123.46, 122.04, 117.52, 69.41, 45.83, 45.70, 21.70.HRMS (m / z): Calculated for C21H18Cl2N5O3S TOF MS ES + (M + H) +: 490.0502, found: 490.0500. Elemental Analysis: C21H17Cl2N5O3S Theoretical (%): C 51.44, H 3.49, N 14.28, 10 S 6.54. Obtained (%): C 51.56, H 3.49, N 14.65, S 6.20.

Preservation of the drugs The compounds are dissolved in DMSO and stored at –20 ° C. The stock solution is diluted in the corresponding medium to obtain the required concentrations in each experiment, the final concentration always being less than 0.1% v / v of DMSO. As controls parallel cultures of the cell lines MCF-7, MDA-MB 468, SKBR-3, HCT-116, HT-29, RKO, A-375, NCI-H460, A-549 and BxPC-3 have been used in the middle. twenty Examples of tests with the compounds of the inventionCell lines used Cultures of the following cell lines MCF-7, MDA-MB 468, SKBR-3, HCT-116, HT-29, RKO, A-375, NCI-H460, A-549 and BxPC-3 have been used. 25

Cytotoxicity tests
The cells are seeded in 24-well plates (1,000 cells / well) in their respective medium. After allowing cell adhesion overnight, they are treated for 3 or 6 days with increasing concentrations of compounds 4-15. The medium 30 is changed at 3 days, in the case of experiments at 6 days, adding the respective concentrations of the compounds. Parallel cultures of cell lines without drug addiction and with DMSO are used as controls. After 3 or 6 days of treatment, staining with sulforrodamine B (SRB) is carried out to determine the survival rate. First, the cells are fixed with 10% acid-trichloroacetic acid for 20 min at 4 ° C, and after washing the plates 3 times with double-distilled water, they are allowed to dry at room temperature. Next is added
0.4% SRB in 1% acetic acid and after incubating 20 minutes at room temperature, the SRB is removed and washed three times with 1% acetic acid. Finally, the SRB is solubilized with a 10 mM Tris-base solution (pH 10.5) by gentle stirring for ten minutes. Subsequently, 100 μL is transferred to 96-well plates in triplicate and analyzed on a Multiscan Titertek colorimeter at 492 nm. 5
In this way, the data that were used to make the Optical Density (D.O.) graphs (y-axis of the graph) and concentration (x-axis) of the drug were obtained. The equation of the curve was calculated by means of a minimum square regression and hence the concentration of the drug corresponding to a D.O. (y axis) = 50.
Treatment of cell lines with drugs 10
Table 2 summarizes the results obtained in the tests performed for experiments with 6 days of incubation with each compound.

Comp. A-375 HCT-116 MCF-7
4 10.9 ± 0.012 13.6 ± 0.008> 20
5 1.34 ± 0.012 7.65 ± 0.005> 20
6 0.83 ± 0.003 0.47 ± 0.006 1.32 ± 0.018
7 4.04 ± 0.003 6.88 ± 0.004> 20
8 4.97 ± 0.004 6.88 ± 0.004> 20
9 2.96 ± 0.003 4.18 ± 0.004> 20
10 0.95 ± 0.010 1.05 ± 0.008 3.68 ± 0.016
eleven 0.63 ± 0.013 1.21 ± 0.024 4.78 ± 0.013
12 0.73 ± 0.037 1.30 ± 0.040 2.61 ± 0.024
13 0.41 ± 0.022 0.37 ± 0.006 1.15 ± 0.017

Table 2. Fifty inhibitory concentrations (IC50 M) of the substituted 3,4-dihydro-2H-1,4-15 benzoxazines in position 2 (4-13) against cell lines A-375, HCT-116 and MCF -7 (numbering of the compounds according to what is stated in table 1).

Table 3 summarizes the results obtained in the tests performed for experiments with 3 days of incubation with each compound:

Comp. HT-29 HCT-116 RKO NCI-H460 BxPC-3
6 2.54 +/- 0.6 3.70 +/- 0.9 3.62 +/- 1.2 2.96 +/- 1.4 1.79 ± 0.89
eleven 6.13 +/- 1.3 6.78 +/- 1.8 6.67 +/- 2.6 7.21 +/- 1.5 2.15 ± 0.95
12 5.38 +/- 1.1 5.8 +/- 1.5 43.18 +/- 8.3 8.53 +/- 1.6 2.92 ± 1.07
13 5.52 +/- 0.9 4.01 +/- 3 4.91 +/- 1.2 5.95 +/- 1.7 8.88 ± 1.64
fifteen 5.52 +/- 1.2 9.64 +/- 1.3 3.6 +/- 1.1 6.33 +/- 2.6 30.86 ± 5.33

Table 3. Fifty inhibitory concentrations (IC50 M) of the substituted 3,4-dihydro-2H-1,4-benzoxazines in position 2 (6, 11-13 and 15) against the NCI-H460, RKO, HCT- cell lines 116, HT-29 and BxPC-3, treated for 3 days (numbering of the compounds according to the table 1). 5
The fifty inhibitory concentrations, (IC50 µM) shown in Table 3, at 3 days were greater than at 6 days as appropriate but equivalent to those of drugs currently used in the clinic. In addition, the action of 13 has been studied in the SKBR-3 and MDA-MB 10 468 cell lines, which overexpress the HER-2 and EGFR surface receptors respectively, obtaining IC50 values of 1.17 +/- 0.005 µM and 1.43 +/- 0.006 µM.

Kinase Inhibition Assay 15
The inhibitory capacity of 13 on kinases involved in cancer-related signaling pathways was demonstrated in vitro by the western blot technique. For this, the MDA-MB 468 breast cancer cell line, which over-expresses the EGFR surface receptor, was used. Overexpression of this receptor in breast cancer is related to an increase in tumor size, a greater 20 expression of stem cell characteristics (stem) and a poor prognosis (Viale G, Rotmensz N, Maisonneuve P, et al. , ClinCancer Res 13: 4429-4434, 2007). In addition, 13 is able to inhibit signaling pathways involved in cell proliferation, which are altered in tumor cells (Yu S .; Cai X .; Wu C .; Wu L .; Wang Y .; Liu Y .; Yu Z .; Qin S .; Ma F .; Thiery JP .; Chen L. Oncotarget. 2014. Such is the case 25 of the ERK1 / 2 kinase, which plays a fundamental role in the invasion, migration and angiogenesis of different types of tumor cells, both in vivo and in vitro (Wang, XG; Meng, Q .; Qi, FM and Yang, QF European Review for Medical and Pharmacological Sciences. 2014, 18: 3844-3853.
Currently, kinase inhibitors are being studied to be used as a therapeutic option in the treatment of patients presenting with tumors with
mutations for BRAF, KRAS, or NRAS, including patients who had relapses and who were using inhibitory therapies against BRAF or MEK (Morris, EJ. et al., Cancer Discov. 2013, 3: 742-750.)
To carry out our study, overexpression of pEGFR was induced by adding to the EGF culture medium for two hours. After that time, 13 5 (5 µM) was added and its ability to inhibit pEGFR and pERK1 / 2 kinases was determined at different times. Protein extraction was performed from the culture of adherent cells and CSD ALDH + in 6-well plates (normal and low adhesion), after treatments with the compound. Subsequently, the culture medium was removed and the cells were washed with PBS. Next, 100 µL of the protein extraction solution was added and the cells were peeled off the surface of the plate by using a cell brush ("scraper"). In the case of CSCs, the medium was centrifuged and after being washed with PBS, the lysis solution was added, which was collected and stored in a 1.5 mL Eppendorf type tube, to be subsequently boiled at 100 ° C for 10 min in a thermoblockThermoStat 15 plus (Eppendorf Corp., Hamburg, Germany). The samples were finally stored at -20 ° C. After this, the proteins were separated based on their load and size by acrylamide gels (10%). The next step was the transfer of electrophoretically separated proteins to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a Trans-20 Blot® Turbo ™ Transfer System (Bio-Rad Laboratories, Inc. , Hercules, CA, USA), Once the nitrocellulose membrane was transferred, blockade was carried out to avoid nonspecific binding with the proteins and their subsequent labeling with the primary antibodies (p-EGFR and p-ERK; CellSignaling). After an incubation period, the proteins were labeled with the corresponding secondary antibody (Sigma Aldrich). 25 Finally, the membrane was developed using the ECL kit (Bonus, Amersham, Little Chalfont, UK), in a LAS-4000 image system, using the Image Reader LAS-4000 software.

Figure 4 shows that 13 significantly inhibited both kinases from 30 to 12 hours of treatment.

Similarly, a study was conducted on the ability of 13 to modulate signaling pathways related to stem cell properties (stem) of CSCs. The results indicate that 13 was able to inhibit the formation of spheres both in number and size thereof in the CSD ALDH +, as well as inhibit the expression of Sox-2 at very low concentrations (1.43 and 2.86 µM) after only 24 hours
of treatment. Sox-2 has been associated with the maintenance of pluripotency and is considered as a stem cell marker (stemcells) ( Lü, X .; Deng, Q .; Li H .; Suo Z. ExpTherMed. 2011 Sep; 2 (5): 821-826). In fact it has been shown that it plays a key role in the development of tamoxifen resistance in estrogen receptor (ER) -positive breast cancer cells. Overexpression 5 of Sox2 increases the proportion of stem cells by activating the Wnt signaling pathway, thereby making the cells resistant to chemotherapy. These findings, together with the observation that Sox2 levels are elevated in the primary tumors of patients who do not respond to endocrine therapy, suggest that Wnt signaling may be an attractive therapeutic target in these patients. The implication of these findings is that small Wnt signaling inhibitory molecules could be a new treatment to prevent recurrence in defined groups of breast cancer patients (Piva M .; Domenici G .; Iriondo O .; Radish M .; Simões BM .; Comaills V .; Barredo I .; López-Ruiz JA .; Zabalza I .; Kypta R .; Vivanco Md. EMBO Mol Med. 2014, 6, 66-79. In addition, it has been described that inhibition of Sox2 increases the levels of apoptosis of CSCs. In fact, it has recently been shown that Sox2 contributes to the proliferation and stem characteristics of pancreatic cancer through the regulation of genes that control transition from phase G1 to S of the cell cycle and phenotype of the mesenchymal epithelium transition (EMT) ( Herreros-Villanueva M .; Zhang JS .; Koenig A .; Abel EV .; twenty Smyrk TC .; Bamlet WR .; of Narvajas AA .; Gomez TS .; Simeone DM .; Bujanda L .; Billadeau DD. Oncogenesis 2013, 2; e61). All this indicates that the selective inhibition of Sox2 in CSCs by 13 is a promising therapeutic strategy.

In vivo toxicity tests 25

Acute toxicity studies in vivo were carried out with 13. The toxicity of the compound was evaluated using 6-week-old female BALBc mice (n = 40) and weighing about 20 mg to those administered orally. using a bucoesophageal probe a daily dose of up to 250 mg / kg / day (50/75/100/150/250) of the 30 compounds for 1 week followed by 1 week of rest. The compound was dissolved in 1% methylcellulose. Mice were inoculated with the same volume of 1% methylcellulose (control group) and DMSO only (DMSO group). The mice were kept under standard conditions and weighed. Systemic toxicity was evaluated: apathy, excitement, weight loss; and local: alopecia, skin reactions and motility of one leg every 24 hours. During the study period, no behavioral changes, clinical signs of toxicity, or deaths were observed in 100% of patients.
treated animals The macroscopic analysis of the organs studied did not show any data of interest, being its normal appearance.

Tests of antitumor activity in xenografts
To assess the effect of 13 on tumor growth, 5 heterotopic tumor xenografts were established using the A375 metastatic melanoma cell line. For this, tumor formation was induced by subcutaneous injection of 5 × 105 cells / mouse in female NOD scid gamma mice (NOD, Cg-Prkdcscid Il2rgtm1Wjl / SzJ, NSG) aged eight weeks. They were housed and maintained at 20 ° C to 24 ° C, 50% humidity, a light-dark cycle from 14 to 10 h with food and water 10 ad libitum. The tumors were allowed to grow to an average volume of 100 mm3, after which they were randomly assigned as control groups (1% methylcellulose) and treatment with 13 (50 mg / kg, dissolved in 1% methylcellulose) or 13 (100 mg / kg, dissolved in 1% methylcellulose) injected ip three times a week for 40 days. The tumor weight was calculated according to the formula: TW (mg) = tumor volume 15 (mm3) = d2 × D / 2, where d and D are the shortest and longest diameters, respectively. Treatment with 13 significantly inhibited the growth of melanoma compared to the control group from day 20 for both concentrations, and these significant differences were maintained for the concentration of 100 mg / kg until day 40 (Figure 6). These results indicate the potent antitumor activity of 13 even in highly tumorigenic and invasive tumor lines with high levels of CSCs and in tumors with few therapeutic alternatives.

Survival Rate 25
To carry out survival studies, the mice were closely monitored, at least three times per week, throughout the experimental period. As shown in Figure 7, treatment with 13 at both concentrations (50mg / kg and 100mg / kg) showed better survival in relation to the control group. This survival was better in the group of animals treated with 30 100mg / kg.

权利要求:
Claims (24)
[1]
1. A compound of general formula IV:

as well as racemic compounds, stereoisomers, salts or solvates thereof; 5
where
 R is an alkyl of 1 to 12 carbon atoms, an alkenyl of 2 to 12 carbon atoms, an alkynyl of 1 to 12 carbon atoms, an arylalkyl where the aryl group is selected from the list consisting of benzyl and phenethyl and the alkyl group has 1 to 12 carbon atoms, a 3 to 15 membered heterocycloalkyl consisting of carbon atoms and one to five heteroatoms selected from the group consisting of N, O2 and S, -C (= O) R '', -C (= O) OR '', or the rest of benzenesulfonyl indicated below:
fifteen
or where R 'is H, OH, 3,5-diOH, 2-NO2, 3-NO2, 4-NO2, 2-NH2, 3-NH2, 4-NH2, 4-Me, 4-OMe (Ts), 3,4-diOMe, 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 3,5-diCl, 3, 5-diBr, 3,5-diI or 3,5-diMe in a benzenesulfonyl moiety; twenty
 R ’’ ’is selected from the group consisting of:

or X and W each independently represent each other, an electronegative atom selected from the group consisting of -Cl, -Br, and -I;
or Z is a group of atoms that is integrated into various organic functions such as silyl ethers of the formula -Si (R4) 3, alkyl and arylalkyl ethers, alkoxymethyl ethers and
aryloxy of formula -CH2OR4; esters of formula -C (= O) R4; carbonates of the formula -C (= O) OR4 and sulphonates of the formula -SO2R4.
or R '' '' can be H, acyl groups of the formula -C (= O) R4, such that the group -OC (= O) R4 can be an acetate ester, benzoate ester, pivalate ester (dimethylacetate), Methoxyacetate ester, 5-chloroacetate ester, levulinate (4-oxopentanoate) ester,
or R4 represents a group selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and heterocycle. 10

[2]
2. The compound according to claim 1, wherein said compound comprises structural formula V:
fifteen
V
Where R ’’ is selected from the group consisting of:
, CH2N3,; twenty
and where R6 is selected from the group consisting of a methyl group or the following group:
.
25
Where R 'is H, OH, 3,5-diOH, 2-NO2, 3-NO2, 4-NO2, 2-NH2, 3-NH2, 4-NH2, 4-Me, 4-OMe (Ts), 3 , 4-diOMe, 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-I, 3-I, 4-I, 3,5-diCl, 3,5 -diBr, 3,5-diI or 3,5-diMe in a benzenesulfonyl moiety.

[3]
3. The compound according to claim 1, wherein said compound comprises the formula:
where R5 is selected from the list consisting of-CO2Et, -CH2OH, -CH2Cl and -5 CH2Ts.

[4]
4. The compound according to claim 3, wherein said compound comprises the following structural formula:
10

[5]
5. Compound according to previous claim characterized in that R 'is 4-methyl.

[6]
6. The compound according to claim 3, wherein said compound comprises the following structural formula:

[7]
7. Compound according to previous claim characterized in that R 'is 4-methyl.
[8]
8. The compound according to claim 2, wherein said compound 20 comprises the following structural formula:

[9]
9. Compound according to the preceding claim characterized in that R 'is 4 methyl, W is Cl or Br and X is H or Cl.

[10]
10. The compound according to claim 2, wherein said compound 5 comprises the following structural formula:
ONNNO2SWNNR'X

[11]
11. The compound according to claim 3, wherein said compound comprises the following structural formula:
ONO2SR'CH2Cl
[12]
12. Compound according to previous claim characterized in that R 'is 4-methyl.

[13]
13. The compound according to claim 3, wherein said compound comprises the following structural formula:
ONO2SR'CH2Ts

[14]
14. Compound according to previous claim characterized in that R 'is 4-methyl.

[15]
15. The compound according to claim 2, wherein said compound 5 comprises the following structural formula:
ONO2SR'CH2N3
[16]
16. Compound according to previous claim characterized in that R 'is 4-methyl.

[17]
17. The compound according to claim 2, wherein said compound 10 comprises the following structural formula:

[18]
18. Compound according to previous claim characterized in that R 'is 4-methyl and 15 OR "" is 3-OH.

[19]
19. The compound according to claim 7, wherein W is Cl, X is Cl and R 'is 4-methyl.

[20]
20. A pharmaceutical composition comprising at least one compound according to any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

[21]
21. Use of a compound according to any of claims 1 to 18, for the preparation of a medicament for the treatment of cancer.

[22]
22. Use according to claim 20, wherein the cancer is selected from the list consisting of breast, colon, colorectal, melanoma and lung cancer.

[23]
23. The pharmaceutical composition of claim 19, wherein said compound is the compound defined in claim 18.
10
[24]
24. The use according to claim 21 wherein said compound is the compound defined in claim 18 and the cancer is selected from the list consisting of breast cancer or melanoma, preferably metastatic melanoma.
fifteen

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ES201630714A|ES2648538B1|2016-05-31|2016-05-31|BENZO-HETEROCICLOS OF SIX MEMBERS WITH OXYGEN AND NITROGEN ATOMS WITH ANTITUMORAL ACTIVITY|ES201630714A| ES2648538B1|2016-05-31|2016-05-31|BENZO-HETEROCICLOS OF SIX MEMBERS WITH OXYGEN AND NITROGEN ATOMS WITH ANTITUMORAL ACTIVITY|

PCT/ES2016/070730| WO2017064350A1|2015-10-15|2016-10-17|Six-membered benzo-heterocycles having oxygen and nitrogen atoms with anti-tumour activity|

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