WO2008139009A1 - Pyridothieneotriazines as antiangiogenic compounds - Google Patents

Abstract

Pyridothienotriazines as antiangiogenic compounds. The present invention concerns derivatives of pyridothienotriazines with antiangiogenic activity and use thereof in the preparation of pharmaceutical compositions or medicines for the treatment of diseases involving the angiogenesis process, such as cancer, obesity, diabetic retinopathias, haemangomas, arthritis, psoriasis and atherosclerosis.

Description

 Title

Pyridothienotriazines as antiangiogenic compounds

Technical sector

The present invention relates to the use of some derivatives of pyridothienotriazines as antiangiogenic compounds and their use in the manufacture of medicaments for the treatment of cancer, obesity, diabetic retinopathies, macular degeneration, hemangioma, arthritis, psoriasis and atherosclerosis.

Prior art

Angiogenesis is the generation of new capillaries from preexisting vessels. Under physiological conditions, angiogenesis is under very strict control and only takes place during embryonic development and in processes related to the female reproductive cycle, fracture repair and wound healing. However, in many pathological processes (for example, tumor growth and its spread in metastases, ophthalmic diseases such as diabetic retinopathy and macular degeneration, hemangiomas, arthritis, psoriasis, atherosclerosis ...), the disease has been linked to an deregulated angiogenesis and continuously active (J. Nat. Med. 1995, 1, 27-31).

Although all the mechanisms that lead to pathological angiogenesis are unknown, experimental evidence indicates that the final process is the result of a balance between pro-angiogenic factors and angiogenesis inhibitors. From J. Folkman's pioneering hypothesis that tumors are dependent on angiogenesis (J. New Eng. J. Med. 1971, 285, 1182-1186), it has been clinically proven that inhibition or prevention of angiogenesis is an attractive therapeutic alternative for the treatment of cancer and other angiogenesis-dependent diseases (Cancer Res. 1998, 152, 341-352; Angiogenesis 1998, 2, 295-307). The development of new vessels is a complex process that consists of several stages, including activation of endothelial cells by angiogenic stimulation, synthesis and release of proteases that will allow endothelial cells to migrate, proliferate and finally differentiate to generate New capillary tubes. Any of these steps can be a target for pharmacological intervention of angiogenesis. Although angiogenesis is a highly regulated process under physiological conditions, a lack of control in this regulation, leading to a permanent activation of angiogenesis is characteristic of a series of pathologies (which we could call "angiogenesis dependent diseases"), as we have commented previously. Thus, deregulation of angiogenesis can be the direct cause or exacerbate a certain pathological condition. For example, the growth of metastasis of a solid tumor is dependent on angiogenesis. Based on these findings, there is a continuing need for compounds that show an antiangiogenic activity, due to their potential use in the treatment of various pathologies, and among others cancer.

Pyridothienothriazine derivatives of general formula (Ia) or (Ib)

(Ia)

(Ib)

where X is a halogen atom, R is selected from a group consisting of hydrogen, and a substituted or unsubstituted phenyl group, and R ² is selected from a group consisting of NH ₂ , NH-lower alkyl, N- (lower alkyl ) ₂ , NH-benzyl, and piperidine-benzyl, are known compounds (Eur. J. Med. Chem. 1998, 33, 887-897, J. Med. Chem. 1999, 42, 4720-4724, and Eur. J Med. Chem. 2003, 38, 265-275) which have been described as inhibitors of nitric oxide and prostaglandin E ₂ generation by murine macrophages, as inhibitors of and the production of histamine by mast cells, and as antiprotozoa against Philasterides dicentrarchi, but the antiangiogenic activity of the pyridothienotriazine derivatives of the general formula (I) has not been previously described.

Disclosure of the invention

The present invention is directed to the use for the preparation of medicaments for the treatment of angiogenesis-dependent diseases (in particular cancer, ophthalmic diseases such as diabetic retinopathy and macular degeneration, hemangiomas, arthritis, psoriasis, atherosclerosis ...), of compounds of formula general (Ia) or (Ib), of a pharmaceutically acceptable salt, a derivative or prodrug.

Pyridothienotriazine derivatives of general formula (Ia) or (Ib)

(Ia)

(Ib)

where X is a halogen atom, R ¹ is selected from a group consisting of hydrogen, and a substituted or unsubstituted phenyl group, and R ² is selected from a group consisting of NH ₂ , NH-lower alkyl, N- (alkyl lower ^, NH-benzyl, and piperidine-benzyl. In the above definition of compounds of formula (Ia) or (Ib), the following terms have the meaning indicated below:

"Lower alkyl" refers to a straight or branched chain alkyl group of 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl . "Halogen" includes F, Cl, Br and I.

References to a substituted phenyl group in the compounds of the present invention refer to the phenyl group which can be substituted in one or more positions available by one or more suitable groups, such as F, Cl, Br and L; cyano; hydroxyl; nitro; azido; alkanoyl as a C 1-6 alkanoyl group as acyl and the like; carboxamide; alkyl groups that include those having 1 to 12 carbon atoms, or 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms; alkenyl and alkynyl groups, including those groups having one or more unsaturated bonds and 2 to 12 carbon atoms, or 2 to 6 carbon atoms; alkoxy groups having one or more oxygen and 1 to about 12 carbon atoms, or 1 to about 6 carbon atoms; aryloxy groups such as phenoxy; alkylthio groups, including those having one or more thioether bonds and from 1 to about 12 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfinyl groups including those having one or more sulfinyl bonds and from 1 to about 12 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfonyl groups including those having one or more sulfonyl bonds and from 1 to about 12 carbon atoms, or from 1 to about 6 carbon atoms; aminoalkyl groups such as groups containing one or more atoms of N and 1 to about 12 carbon atoms, or 1 to about 6 carbon atoms; aryl groups of 6 or more carbons, in particular phenyl or naphthyl and aralkyl groups such as benzyl. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and each substitution is independent of the other.

The term "a pharmaceutically acceptable salt, a derivative or a prodrug" refers to any pharmaceutically acceptable salt, ester, solvate, hydrate or any other compound that, upon administration to the recipient, is capable of producing (directly or indirectly) a compound as described here. The preparation of salts, prodrugs and derivatives may be carried out by known methods.

For example, pharmaceutically acceptable salts of the compounds of formula (Ia) or (Ib) are synthesized by conventional chemical methods from compounds that They contain an acidic or basic group. Generally these salts are synthesized, for example, by reacting the free acid or base forms of said compounds with appropriate stoichiometric amounts of the base or acid in water, or in an organic solvent, or in a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferable. Examples of acid addition salts are the addition salts of mineral acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate and the addition salts of organic acids such as, for example, acetate, maleate, fumarate, citrate, oxalate , succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of alkali addition salts are inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N ₅ N-dialkylene ethanolamine, triethanolamine and de basic amino acids

The term "prodrug" is used in the broadest sense and refers to those derivatives that are converted in vivo into the compounds of formula (Ia) or (Ib). Such derivatives include, for example, compounds in which a free hydroxyl group is converted into an esterified derivative.

The compounds of formula (Ia) or (Ib) may include enantiomers depending on their asymmetry or diastereoisomers. The use of isolated isomers or mixture of the isomers falls within the scope of the present invention. The pharmaceutical compositions comprise a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt, derivative, prodrug or stereoisomer together with a pharmaceutically acceptable carrier, adjuvant or carrier, for administration to a patient.

Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) for oral, topical or parenteral administration.

In a preferred application the pharmaceutical compositions will be in oral, solid or liquid form. Doses suitable for administration may be supplied in the form of tablets, capsules, syrups or solutions and may contain conventional excipients such as binding agents, for example syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; lubricants such as magnesium stearate; disintegrants such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate. Solid oral compositions can be prepared by conventional methods of mixing, filling or compressing. Remixing can be used to distribute the active agent in those compositions that use large amounts of filler. Such operations are conventional. The tablets may, for example, be prepared by dry or wet granulation and optionally covered by methods well known in pharmaceutical practice, in particular with an enteric coating.

The pharmaceutical compositions can also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the form suitable for the dose unit. Suitable excipients, such as fillers, buffering agents or surfactants, may be used.

The aforementioned formulations will be prepared using standard methods such as those described or referred to in Spanish or US pharmacopoeias and similar reference texts.

The administration of compounds of formula (Ia), (Ib) or their compositions may be by any suitable method, such as intravenous infusion, oral preparations, and intraperitoneal and intravenous administration. Oral administration is preferred due to the convenience for the patient and the chronic nature of the diseases to be treated.

Generally the effective amount administered of a compound of formula (Ia) or

(Ib) will depend on the relative efficacy of the chosen compound, the severity of the disease to be treated and the patient's weight. The compounds will typically be administered one or more times a day for example 1, 2, 3 or 4 times a day, with total daily doses in the range of 0.1 to 1000 mg / kg / day.

The compounds of formula (Ia), (Ib), and their corresponding combinations can be used with other drugs in a combination therapy. The other drugs may be part of the same composition, or be supplied as an independent composition for simultaneous administration or at a different time.

Particularly preferred compounds of those comprised in general formulas (Ia) or (Ib) are compounds I ₅ II, III, and IV:

Compound I Compound II

Compound III Compound IV

Compound (I) is preferably obtained as described in (Eur. J. Med. Chem. 1998, 33, 887-897), compounds II, III, and IV are preferably obtained as described in (J. Med. Chem. 1999, 42, 4720-4724). Similarly, any compound of formulas

(Ia) or (Ib) can be obtained through the standard procedures of organic synthesis and following the guidance of the aforementioned documents.

Ways of carrying out the invention

CELL CULTURES

Bovine aorta (BAE) cells were maintained in DMEM medium with glucose (lg / L), glutamine (2mM), penicillin (50 IU / mL), streptomycin (50 µg / mL), and amphotericin

(1.25 μg / mL) supplemented with 10% fetal bovine serum (FBS) (Br. J. Cancer 1995, 71, 770-

775). Human umbilical cord endothelial cells (HUVE) were isolated from umbilical cords by collagenase digestion (J. CeIl. Biol. 1988, 107, 1589-1598) and maintained in Medium 199 containing HEPES (10 mM), L -glutamine (2 mM), heparin (10 mg / mL), penicillin (50 IU / mL), streptomycin (50 µg / mL), and amphotericin (1.25 µg / mL), supplemented with 3 mg / L of endothelial cell growth supplement (ECGS, Sigma) and 20% FBS in 5% CO ₂ and 37 ⁰ C.

ACTIVITIES OF HV COMPOUNDS

Endothelial cell differentiation test: Formation of tubular structures on Matrigel

The wells of a 96 well plate with 50 uL of Matrigel (10.5 mg / mL) at 4 0C were covered and were allowed to polymerize at 37 ⁰ C for at least 30 min (FASEB J. 2002, 16, 261-263). 5x10 ⁴ BAE cells in 200 μL of DMEM were added to each well. For HUVE cells, 2.5x10 ⁴ cells in 200 μL Medium 199 supplemented with 5% FBS were added. Finally, different concentrations of each compound I-IV, incubating at 37 ⁰ C in a humidified chamber with 5% _CO2 were added. After 7 hours of incubation, the wells were observed and photographed with the help of an inverted NIKON DIAPHOT-TMD microscope (NIKON Corp., Tokyo, Japan).

Table I shows the results of the minimum concentration of compounds I-IV (MIC) that gave a complete inhibition of endothelial morphogenesis on Matrigel of BAE and HUVE cells.

Table I

Cell growth assay

The 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO) bromide assay was used in 96-well plates, as required. described in (Anticancer Res. 2001, 21, 3457-3460). 3x10 ³ BAE, 4x10 ³ HUVE were incubated in a total volume of 100 μL of their respective growth media with serial dilutions of compounds I-IV. After 3 days of incubation (37 ⁰ C, 5% CO ₂ in humid) atmosphere 10 .mu.l of MTT (5 mg / ml in PBS) was added to each well and the plate was incubated 4 hours (37 ⁰ C). The resulting formazan was dissolved in 150 μL of 0.04 N HC1-2 propanol and its absorbance was read at 550 nm with the aid of a plate reader. The IC ₅₀ value was calculated as the concentration of compound that gave 50% of the cell survival of the untreated control.

IC ₅₀ values for compounds I-IV against BAE and HUVE cells are shown in Table II:

Table II

Chorioallantoid Membrane Assay (CAM)

Table

Dose

Compound I Compound Il Compound III Compound IV (μg / CAM)

0 0 (0/20) 0 (0/20) 0 (0/20) 0 (0/20)

0.02 20 (1/5) 0 (0/3)

0.05 75 (3/4) 0 (0/4) 40 (2/5) -

0.1 100 (4/4) 80 (4/5) 40 (2/5) -

0.2 100 (3/3) 33 (2/6) 25 (1/4) -

0.5 100 (3/3) 60 (3/5) 20 (1/5) 0 (0/3)

1 100 (4/4) 100 (4/4) 67 (2/3) 33 (1/3)

2 100 (4/4) 100 (3/3) 100 (3/3) 100 (4/4)

5 100 (3/3) - - 25 (1/4)

The CAM test was carried out as described in (FASEB J. 2002, 16, 261-263).

Fertilized eggs from hen horizontally incubated at 38 ⁰ C in a humidified incubator, they were opened a window on day 3 of incubation and were processed at day 8. Test compounds were added to a solution of 0.7% methylcellulose in water . In a laminar flow chamber and on a Teflon surface, drops of 10 μL of dried this solution, which was then implanted on the CAM. After 48 hours of re-incubation, two people independently observed the CAM using a stereomicroscope. The trial was considered positive when both people reported a significant decrease in vascularization in the treated area.

Table III summarizes the evaluation of angiogenesis inhibition in vivo by the CAM assay for compounds I-IV. Data appears as percent inhibition. In brackets, the number of eggs with inhibited angiogenesis in their CAMs is indicated by the total number of eggs treated.

Claims

Claims 1.- Use of a compound of formula (Ia) or (Ib)

(Ia)

(Ib) where X is a halogen atom, R ¹ is selected from a group consisting of hydrogen, and a substituted or unsubstituted phenyl group, and R is selected from a group consisting of NH ₂ , NH-lower alkyl, N- (lower alkyl ) ₂ , NH-benzyl, and piperidine-benzyl, or a pharmaceutically acceptable salt, prodrug or solvate thereof; in the preparation of a pharmaceutical composition or medicament for the treatment of angiogenesis-dependent diseases. 2. The use according to claim 1 wherein the angiogenesis-dependent disease is cancer. 3. The use according to claim 1 wherein the angiogenesis-dependent disease is diabetic retinopathy. 4. The use according to claim 1 wherein the angiogenesis-dependent disease is macular degeneration. 5. The use according to claim 1 wherein the angiogenesis-dependent disease is hemangiomas. 6. The use according to claim 1 wherein the angiogenesis-dependent disease is arthritis. 7. The use according to claim 1 wherein the angiogenesis-dependent disease is psoriasis. 8. The use according to claim 1 wherein the angiogenesis-dependent disease is atherosclerosis. 9. The use according to claims 1-8 wherein the compound of formula (Ib) is

10. The use according to claims 1-8 wherein the compound of formula (Ia) is

1 L- The use according to claims 1-8 wherein the compound of formula (Ia) is

12. The use according to claims 1-8 wherein the compound of formula (Ia) is

13. Use of the pharmaceutical composition or medicament for the treatment of the diseases defined in claims 1-8, whose composition comprises the compound of formula (Ia) or (Ib) defined in claims 1, 9, 10, 11 and 12, or a pharmaceutically acceptable salt, prodrug or solvate thereof, characterized in that it involves the administration of a therapeutically effective amount of said compound or of the pharmaceutically acceptable salt, prodrug or solvate thereof.