WO2004005328A2

WO2004005328A2 - Peptidomimetic compound useful as inhibitor of integrins

Info

Publication number: WO2004005328A2
Application number: PCT/IT2003/000422
Authority: WO
Inventors: Mauro Marzi; Domenico Alloatti; Giuseppe Giannini
Original assignee: Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.
Priority date: 2002-07-09
Filing date: 2003-07-03
Publication date: 2004-01-15
Also published as: ITRM20020370A0; AU2003281226A1; WO2004005328A3; ITRM20020370A1; AU2003281226A8

Abstract

The compound 3-carbonyl-[Arg-Gly-Asp]-1methyleneamine-tetrahydro-beta-carboline, a process for its preparation and its activity as an inhibitor of αvβ3 integrins are described. This compound is useful for the preparation of medicaments for the treatment of diseases due to abnormal angiogenesis.

Description

"Peptido-mimetic compound useful as inhibitor of integrins"

The invention described herein relates to the compound 3-carbonyl- [Arg-Gly-Asp]-l-methyleneamine-tetrahydro-beta-carboline, processes for its preparation, pharmaceutical compositions containing it, and its use as a medicament.

Background to the invention

The integrins are a class of receptors involved in the cell adhesion phenomenon. They are glycoproteins consisting of two sub units α and β, combined to give different families. For greater details, see Kessler, H. et al., Angew. Chem. Int. Ed. Engl, 1997, 36, 1374-89.

All the integrins have a "universal cell recognition site" capable of recognising the common peptide sequence Arg-Gly-Asp, also known as RGD, from the one-letter symbols that identify the three amino acids, though every integrin preferentially recognises a different conformation of this tripeptide (Kessler, H. et al., J. Am. Chem. Soc, 1996, 118, 7461-72).

The family of the βι_ integrins plays an important role in the organisation of the tissues and the β₂ integrins are important for the immune system, while the β₃ integrins regulate the coagulation process and angiogenesis.

One objective of pharmaceutical chemistry is to make available to the physician compounds capable of interacting with the integrin family, but selectively on the various subtypes, in view of the diversity of roles that each of them plays at the physiopathological level.

The invention described herein is aimed at the integrins involved in angiogenesis mechanisms. The action of different growth factors stimulates the expression of integrin α_vβ3 (vitronectin receptor) on endothelial cells. During the consequent migration of the endothelial cells in the direction of angiogenesis stimulation, the membrane with the ct_vβ3 integrin receptor binds the tripeptide sequence RGD present in the various forms on the extracellular matrix. This binding leads to an accumulation of proteins - of the thalin, paxilin, -actinin, tensin, and vinculin types - of the cytoskeleton. This favours the process of migration, acting as an endothelial cell survival signal, with the formation of new blood vessels. The administration of soluble RGD analogues impedes the build-up of proteins on the receptors and leads to programmed cell death (apoptosis), counteracting the migration of endothelial cells and preventing neovascularisation (Giannis, A. et al., Angew. Chem. Int. Ed. Engl, 1997, 36, 588-90).

Among the many molecules selectively involved in agiogenesis, the integrins constitute a promising target in cancer therapy and in all those diseases caused by uncontrolled neovascularisation.

An initial scientific study of the subject (Saiki, I. et al. Jpn. J. Cancer Res., 1990, 81:668-675) reports the action of peptides containing an RGD sequence recognising integrin, thus inhibiting angiogenesis in tumours.

The RGD tripeptide is present in natural ligands of these receptors, such as vitronectin, fibronectin and fibrinogen.

More recent studies have shown that type α_vβ3 and α_vβs integrins increase in the angiogenesis of endothelial cell tumours and that inhibition of α_v integrins by means of antibodies, RGD cyclic peptides and RGD peptidomimetic agents, can block neovascularisation (Arap, W. et al. Current Opinion in Oncology, 1998, 10:560-565). βi integrins (αiβi and ot2βι), too, may also play a role in angiogenesis, though their role has yet to be thoroughly studied. The systemic administration of an anti-α_vβ3 antibody, e.g. the LM609 antibody (Nitaxin), blocks tumour angiogenesis and reduces the growth and invasive properties of human carcinoma of the breast (Brooks, P.C. et al., J. Clin. Invest., 1995, 96:1815-22).

Many integrins can be inhibited by small peptides incorporating the RGD sequence. The incorporation of this sequence in penta- or hexape- ptide cycles containing D-amino acids usually leads to molecules that are potent, selective integrin inhibitors (Haubneυ, R. et al. J. Am. Chem. Soc, 1996, 118:7881-91).

Vitronectin is a protein of the vascular matrix and a selective antagonist of the α_vβ3 receptors, while fibrinogen, another protein, presents selective binding to the αιibβ3 receptors.

To date, the search for RGD analogues has been directed mainly towards antagonists of the αιibβ3 receptors that are potent and selective and can be administered orally. Some of these non-peptide RGD analogues, used as anticoagulants, are currently being investigated in clinical trials.

As antiangiogenic agents, on the other hand, what are needed are RGD analogues capable of selectively inhibiting the α_vβ3 and/or α_vβs receptors without affecting the αιibβ3 receptors.

As regards examples of compounds inhibiting the α_vβ3 receptors and their applications, see EP 1 077 218, filed in the name of the applicant, to which specific reference is made, also for further discussion of the state of the art. In this reference peptidomimetic compounds are disclosed containing the RGD sequence and having a non-peptide bicyclic lactam structure ("template" or "scaffold") completely different from that of the compound described in the present invention, which has a tricyclic non-lactam "template". Examples of cyclic peptide structures containing the Arg-Gly-Asp (RGD) sequence are described in EP 0 596 350, Merck Patent; Wermuth, J. e al, J. Am. Chem. Soc, 1997, 119(6), 1328-1335; US 5.705.481, Merck Patent; WO 99/58162, Du Pont Pharmaceuticals; Liu, S. et al, Bioconjugate Chemistry (2001), 12(4), 559-568; WO 01/097860.

One of the aims of the invention described herein is to provide selective agonists for the α_vβ3 receptors that can be administered orally, which is a useful characteristic for long-term therapies.

Summary of the invention

It has now been found that the compound 3-carbonyl-[Arg-Gly-Asp]-l- methyleneamine-tetrahydro-beta-carboline (whose IUPAC denomination corresponds to [(lS,5S,llS,14S)-ll-(3-{[amino(imino)methyl]a- mino}propyl)-4,7,10,13-tetraoxo-l,2,3,4,5,6,7,8,9,10,ll, 12,13,14,15,20- hexadecahydro-l,14-epimino[l,4,7,10]tetraazacycloheptadecino[13,14- b]indol-5-yl]), both in the form of a racemic mixture, and its individual enantiomers, and also in the form of a pharmaceutically acceptable salt is endowed with inte grin-inhibiting activity, and particularly against integrins belonging to the α_vβ3 class. Said compound is therefore useful as a medicament, particularly for the treatment of the diseases due to abnormal angiogenesis. Examples of such diseases are metastases, retinopathy, acute kidney failure and osteoporosis.

The objects of the present invention are the above-mentioned compound, processes for its preparation, pharmaceutical compositions containing it, and its use for the preparation of a medicament useful for the treatment of subjects affected by abnormal angiogenesis.

Detailed description of the invention

According to the present invention, pharmaceutically acceptable salts are all those salts that an expert in the field will be capable of pre- paring, without the acid or base used giving rise to unwanted adverse effects, when said salts are used as medicaments.

The compound according to the present invention can be prepared according to the scheme here below, which is presented as a retrosynthesis scheme, which provides the expert in the field with a complete description of the process.

c d

As regards the details of the reaction conditions the reader is referred to the example below.

The process comprises the cyclisation reaction of the compound indicated in the scheme above by the term "scaffold" with peptide c and peptide d. Peptide c and dipeptide d can be prepared according to the schemes here below.

For the preparation of intermediate product 3, the reaction conditions consist in the reaction of Fmoc (9-fluorenylmethoxycarbonyl) chloride with aminoacetaldehyde dimethylketal in dioxane/water, or similar solvent system, at a temperature ranging from approximately 0°C to room temperature, for a time interval ranging from 4 to 16 hours, preferably approximately 4 hours, to give intermediate product 2. The product obtained is then reacted with trifluoroacetic acid (TFA), acetone/water, or similar solvent system, at a temperature ranging from 0°C to 30°C, preferably approximately 0°C, for a time interval ranging from 4 to 12 hours, preferably approximately 6 hours, to give intermediate product 3.

For the synthesis of the dipeptide, the preparation of intermediate product 7 is illustrated.

5

n

For the preparation of intermediate product 7, the reaction conditions consist in the reaction of PyBOP (benzotriazol-1-yloxytripyrrollidino- phosphonium hexafluoro-phosphate), HOBT (N-hydroxybenzotriazole) and collidine in methylene chloride, or a similar solvent, at a temperature ranging from approximately 0°C to room temperature, for a time interval ranging from 6 to 16 hours, preferably approximately 8 hours, to give intermediate product 6. The product obtained is then reacted with piperidine, in methylene chloride, or a similar solvent, at a temperature of approximately 0°C, for a time interval ranging from 2 to 8 hours, to give intermediate product 7.

For the synthesis of the "scaffold" compound the synthesis scheme here below can be followed.

*(cis e trans)"

10a^fc 10 b*

12

20

11

21

IV

13 a° + 13 b^α The first step involves the reaction between compound 8 and intermediate product 3, described above, to give intermediate product 9. The reaction takes place in acetic acid/sodium acetate, at a temperature ranging from 0°C to 25°C, preferably at room temperature, according to the known Pictet-Spengler condensation conditions. Compound 10a is obtained, in a mixture with compound 10b, treating compound 9 with Cbz (benzylchloroformate), sodium bicarbonate in dioxane, or some other equivalent solvent, at a temperature ranging from 0°C to 25°C, preferably at room temperature, for a time interval ranging from 1 to 4 hours, preferably 1.5 hours. Once isolated, compound 10a is treated with piperidine, in methylene chloride, or some other equivalent solvent, at a temperature ranging from 0°C to 25°C, preferably 0°C, for a time interval ranging from 4 to 16 hours, preferably 8 hours, for the purposes of freeing the primary amine group, which is condensed with compound 12 to give 13a. Dicyclohexylcarbodiimide, hydroxybenzotriazole, or similar systems can be used as the condensing system, at a temperature ranging from 0°C to 25°C, preferably 0°C, for a time interval ranging from 2 to 10 hours, preferably 5 hours. Compound 13a is transformed into compound 14 by means of trifluoroacetic acid, in methylene chloride, or some other equivalent solvent, at a temperature ranging from 0°C to 25°C, preferably 0°C, for a time interval ranging from 5 to 16 hours, preferably 7 hours. Compound 14 is condensed with compound 7, described above, to give compound 15. HATU (O-(7- azabenzoxytriazol-l-yl)-l,l,3,3-tetramethyl-uronium-hexa-fluoride phosphate), or similar systems, in a suitable solvent, for example, dimethylformamide, can be used as the condensing system, at a temperature ranging from -10°C to 4°C, preferably -10°C, for a time interval ranging from 1 to 8 hours, preferably 1 hour. Compound 15 is treated with piperidine, in dimethylformamide, or some other equivalent solvent, at a temperature ranging from -10°C to 4°C, preferably -10°C, for a time period ranging from 4 to 12 hours, preferably 6 hours, for the purpose of freeing the primary amine group, which is salified, for example with 6N hydrochloric acid, in tetrahydrofuran, or some other equivalent solvent, at a temperature ranging from 0°C to 25°C, preferably 0°C, for a time interval ranging from 8 to 24 hours, preferably 20 hours. Lastly, compound 17, thus obtained, is cyclised according to the known Carpino procedure. HATU, HOBt, or similar systems, in a suitable solvent, e.g. dimethylformamide, can be used as the condensing system, at a temperature ranging from -10°C to 23°C, preferably 0°C, for a time period ranging from 8 to 72 hours, preferably 20 hours. The nitroimino group is reduced to imino and the benzyloxy and carbobenzoxy protecting groups are eliminated simultaneously, e.g. by catalytic hydrogenation, with a suitable catalyst, e.g. Pd C, in a suitable solvent, such as methanol/acetic acid, at a temperature ranging from 4°C to 25°C, preferably room temperature, for a time interval ranging from 8 to 24 hours, preferably 20 hours. The crude reaction product is purified with conventional techniques, e.g. with preparative HPLC in a gradient of methanol in water at 0.1% TFA.

The starting compounds 1, 4, 5 and 8 are commerically available and, in any event, can be prepared with techniques known to the expert in the field.

An example of preparation is provided here below. The various intermediates and reaction products are identified by numbers with reference to the synthesis schemes above.

The meanings of the abbreviations are as follows: DCM - dichloromethane; Fmoc - N-(9-fluorenylmethoxycarbonyl); TFA — tri- fluoroacetic acid; DMF - dimethylformamide; PyBOP - benzotriazol-1- yl-oxytripyrrolidinophosphonium hexafluoro-phosphate; HOBt - N- hydroxy-7-azabenzotriazole; AcOH — acetic acid; AcONa — sodium acetate; DCU - dicyclohexylurea; THF - tetrahydrofuran; H-L-Trp-OMe - L-tryptophan methyl ester.

EXAMPLE

Preparation of dipeptide 7 9H-fluoren-9-yl-methyl-2,2-dimethoxyethylcarbamate (2)

2.43 g (23 mmol) of compound 1 (aminoacetaldehyde-dimethylacetal) and 2.43 g (23 mmol) of Fmoc-Cl; AcOH were dissolved in a 90 ml mixture of dioxane/water 4:1 containing 1.95 g of NaHCO₃ (23 mmol).

The reaction was stirred at 0°C for 15 minutes and then held at room temperature, reaching completeness in the following 4 hours. The dioxane was dried in vacuo and the residual aqueous phase was extracted with 200 ml of DCM. The organic phase separated was dried on anhydrous sodium sulphate and concentrated in vacuo at 40°C. The crude product obtained was purified by means of chromatography on an SiO₂ column with ethyl acetate/n-hexane 6:4 to give 7.1 g (21 mmol; 90%) of the desired product with Rf = 0.51 (TLC in ethyl acetate/n- hexane 1:1).

Tdec = 148°C; m.p. = 97.4°C;

MS (IS): M+Na⁺ = 350; M+K⁺ = 366; 2M+Na⁺ = 677.

NMR (300 MHz, CDCl₃,δ): 3.32-3.34 (τ, 2H, CH₂), 3.36-3.40 (s, 6H, 2xOCH₃), 4.20-4.22 (t, 1H, CH), 4.37-4.42 (m, 3H, CH +CH₂), 4.97 (br.s., 1H, NH), 7.29-7.34 (dd, 2H, CH ar.), 7.38-7.42 (dd, 2H, CHar.), 7.58-7.60 (d, 2H, CHar.), 7.75-7.78 (d, 2H, CHar.);

"C NMR (75,4 MHz, CDC13, δ): 42.8; 47.5; 54.7; 67.0; 103.1; 120.2; 125.2; 127.3; 127.9; 141.6; 144.5; 156.7.

9H-fluoren-9-yl-methyl-2-oxyethylcarbamate (3)

1 g of intermediate product 2, i.e. N-Fmoc-aminoacetaldehyde dimethylacetal (3.1 mmol), was dissolved in a solution of 25 ml of acetone. The reaction was stirred at 0°C in an inert gas atmosphere and 32 ml of a solution of TFA/H2O 1:1 were added drop wise in 6 hours with the formation of a single product with Rf = 0.41 (TLC in ethyl acetate/n-hexane 1:1). The reaction was dried in vacuo at room temperature and the residual aqueous phase was extracted with 200 ml of DCM.

The organic phase obtained was shaken several times with water until the pH of the aqueous washing phases was neutral. The organic phase was then dried on anhydrous sodium sulphate and concentrated in vacuo at 20°C. The residual crude product was purified by chromatography on an S1O2 column with ethyl acetate/n-hexane 1:1 to give 687 mg (2.4 mmol; 80%) of intermediate product 3.

MS (IS): M+NH ⁺ = 299;

ⁱH NMR (200 MHz CDCI3, δ): 4.1-4.3 (m, 3H, CH+CH₂), 4.4-4.5 (d, 1H, CH), 5.5 (br.s., IH, NH), 7.2-7.5 (m, 4H, CHar.), 7.5-7.6 (d, 2H, CHar.), 7.7-7.8 (d, 2H, CHar.), 9.7 (s, IH, CHald.).

Tert-butyl N-5-r(Z)-amino(nitroimino)methvn-N-2-r(9H-fluoren-9-yl- methoxy)carbonyll -L-ornithylglycinate (6)

2 g (4.5 mmol) of compound 4 (Fmoc-Arg(NO2)-H), 0.684 g (5.9 mmol; 1.3 eq.) of intermediate product 5, i.e. (glycine-t-butyl ester hydro- chloride - commercial compound), 3.54 g (6.75 mmol; 1.5 eq.) of PyBOP, then 0.303 g (2.2 mmol, 0.5 eq.) of HOBt and, lastly, 0.89 ml of collidine (6.75 mmol, 1.5 eq.) were dissolved in a mixture of 72 ml of DCM/DMF 90:10 stirred at 0°C for the first hour and then brought to room temperature.

The reaction took 8 hours to complete. The organic phase was washed with 5 ml of H₂O, and then with HC1 IN and with a saturated sodium chloride solution, subsequently with NaOH IN, and lastly with a saturated solution of NaCl, and then dried on anhydrous sodium sulphate and concentrated in vacuo at 40°C. The crude reaction product was purified by chromatography on an SiO₂ column with ethyl acteate to give 2.8 g (5 mmol; 90%) of protected dipeptide with Rf = 0.41 (TLC in ethyl acetate/n-hexane 9:1).

m.p. = 70°C; αD = 8° (c = 0.15 in DCM);

MS (IS): M+H⁺ = 555; M+Na⁺ = 577.

Η NMR (300 MHz, CDCl₃,δ): 1.22-1.37 (s, 9H, OtBu), 1.50-1.70 (br.s., 2H, CH₂), 1.72-1.90 (br.s., 2H, CH₂), 3.10-3.30 (br.s., 2H, CH₂), 3.70- 3.95 (ddd, 2H, CH₂), 4.03-4.15 (dd, IH, CH), 4.20-4.40 (d, 3H, CH+CH₂), 5.58-5.08 (d, IH, NH), 7.18-7.28 (m, 8H, 4xCHar.+NH₂), 7.40-7.55 (d, 2H, 2xCHar.), 7.80-7.90 (d, 2H, 2xCHar.), 8.23-8.55 (IH, NH).

ⁱ³C NMR (75.4 MHz, CDC13, δ): 24,778, 28,217, 40,804, 42,253, 47,281, 53,918, 67,426, 82,737, 120,231, 125,239, 127,335, 127,990, 141,471, 141,498, 143,909, 156,936, 159,614, 169,276, 172,407.

Tert-butyl N-5-[(Z)amino(nitroimino)methyll-l-ornithyl-glvcinate (7)

To a solution of 17 ml of DCM, containing 1.7 g of intermediate product 6 (3 mmol) stirred at 0°C, were added 5.9 ml of piperidine (60 mmol; 20 eq.).

The reaction was completed in 2 hours and was diluted with fresh DCM to an overall volume of 200 ml, which was washed with 50 ml of NaOH IN and then with a saturated solution of NaCl.

The separated organic phase was dried on anhydrous sodium sulphate and concentrated in vacuo at 40°C. The crude product obtained was purified by chromatography on an SiO2 column with ethyl acetate /MeOH 7:3 to give 0.92 g (2.2 mmol; 92%) of deprotected product with R_f = 0.35 (TLC in ethyl acetate/MeOH 1:1). Tdec - 36°C; αD = +2.9° (c = 0,5 in MeOH);

MS (IS): M+H⁺ = 333; M+Na⁺ = 355; 2M+H⁺ = 665; 2M+Na⁺ = 687; M-l

= 331; M+Cl- = 367; M+CH3COO- = 390.

Η NMR (300 MHz, CDCI3, δ): 1.25-1.55 (s, 9H, OtBu), 1.50-1.70 (br.s., 2H, CH₂), 1.72-1.90 (br.s., 2H, CH₂), 3.10-3.30 (br.s., 2H, CH₂), 3.80- 4.00 (3H, CH2+CH), 7.18-7.28 (m, 8H, 4x CHar.+NH₂), 7.60-8.00 (d, 2H, NH₂), 8.00-8.20 (d, 2H, NH₂).

ⁱ³C NMR (75.4 MHz, CDC13, δ): 24,985, 28,090, 31,108, 39,595, 39,862, 40,149, 40,423, 41,758, 53,818, 81,836, 159,794, 168,895.

Synthesis of carboline 9 "scaffold"

Tert-butyl (1S.3S) and tert-butyl (lR,3S)-l-({r(9H-fluoren-9-ylmethoxy) carbonyl]amino}methyl)-2,3,4,9-tetrahvdro-lH-beta-carborine-3-carbo- xylate (9)

To a solution of 40 ml of concentrated AcOH containing 0.9 g (11 mmol; 1.5 eq.) of AcONa were added 2.1 g (7.34 mmol) of intermediate 3 and 2.4 g (8.1 mmol; 1.1 eq.) of intermediate product 8 (L-tryptophan-t-bu- tyl ester hydrochloride). The reaction was stirred for 8 hours at ambient temperature under nitrogen and concentrated in vacuo at 40°C.

The dry residue was dissolved in 150 ml of DCM, which was shaken with 50 ml di NaOH IN and then with a saturated solution of NaCl and then dried on anhydrous sodium sulphate and subsequently concentrated in vacuo at 40°C. The crude product was purified by chromatography on silica gel with ethyl acetate/n-hexane 6:4 to give 2.7 g (5.1 mmol; 70%) of compound with R_f = 0.33 (TLC in ethyl acetate/n- hexane 6:4), which proved to be a mixture of two diastereo-isomers at HPLC in a ratio of 79:21.

αD = -47.5° (c = 0.5 in DCM),

MS (IS): M+H⁺ = 524; M+Na⁺ = 546;

HPLC Symmetry-C18 (3.5 μm 75 mm x 4.6 m H2O/CH3CN 60:40, flow rate 1 ml/min) r.t.: 11.12 and 12.22 min. for the two diastereoisomers.

Construction of the RGD portion

2-Benzyl 3-tert-butyl (1S.3S) and 2-benzyl 3-tert-butyl (lR,3S)-l-(2- {r(9H-fluoren-9-ylmethoxy)carbonyllaminolethyl)-l,3,4,9-tetrahydro- 2H-beta-carboline-2,3-dicarboxylate (10a and 10b)

0.88 g of NaHCO₃ (10 mmol; 2 eq.), 2.7 g (5.2 mmol) of intermediate product 9, and lastly 1.8 g (10.5 mmol; 2 eq.) of benzylchloroformate were solubilised in 217 ml of a mixture of dioxane/H2θ 4:1 stirred at 0°C. After 1.5 hours the reaction was completed. The reaction mixture was dried in vacuo at 40°C and the residual aqueous phase was subjected to extraction with 200 ml of DCM.

The separated organic phase was shaken with a saturated solution of NaCl, dried on anhydrous sodium sulphate and concentrated in vacuo at 40°C. The crude product obtained was purified by chromatography on a silica gel column with ethyl acetate/n-hexane 3:7 to give 2.1 g (3.3 mmol; 61%) of the more abundant diastereoisomer called 10a, with Rf = 0.65 (TLC in ethyl acetate/n-hexane 4:6) and 0.49 g (0.74 mmol; 14%) of the other diastereoisomer called 10b with Rf = 0.73 (TLC in ethyl acetate/n-hexane 1:1).

Analysis of compound 10a:

Tde_c=156°C αD = +76.4° (c = 0.5 in DCM)

MS (IS): M+H⁺= 658; M+Na⁺ = 681; M+NH₄ ⁺ = 675.

ⁱH NMR (300 MHz, CDCl₃,δ): 1.29 (d, 9H, OtBu), 3.07-3.15 (m, IH, CH2), 3.41-3.59 (m, IH, CH₂), 3.80-3.98 (m, 2H, CH₂), 3.90-4.06 (m, 2H, CH₂), 4.20-4.35 (m, IH, CH), 4.40-4.50 (m, 2H, CH₂), 5.03-5.19 (m, IH, CH), 5.16-5.34 (m, 2H, CH₂), 5.24-5.46 (m, IH, CH), 6.05 (m, IH, NH), 7.02-7.82 (m, 9H, CHar.), 7.06-7.45 (m, 9H, CHar.), 9.37-9.60 (d, IH, NH).

HPLC Symmetry-C18 (3.5 μm 75 mm x 4.6 m) (H2O/CH3CN 60:40, flow- rate 1 ml/min) r.t.: 21.60 min.

Analysis of compound 10b:

αD = +19.6° (c = 0.5 in DCM);

MS (IS): M+H⁺ = 658; M+Na⁺ = 681; M+NH₄ ⁺ = 675.

NMR (300 MHz, CDCI3, δ) 1.12 (d, 9H, OtBu), 3.04-3.40 (m, 2H, CH₂), 3.65-3.75 (m, 2H, CH₂), 4.20-4.35 (m, IH, CH), 4.40-4.50 (m, 2H, CH₂), 4.99-5.19 (m, IH, CH), 5.16-5.34 (m, 2H, CH₂), 5.24-5.46 (m, IH, CH), 5.65 (br.s, IH, NH), 7.02-7.82 (m, 17H, CHar.), 8.9 (d, IH, NH).

HPLC Symmetry-C18 (3.5 μm 75 mm x 4.6 m) (H2O/CH3CN 60:40, flow rate 1 ml/min) r.t.: 18.95 min.

2-Benzyl 3-tert-butyl (1S.3S) l-(2-aminoethyl)-L3,4,9-tetrahydro-2H- beta-carboline-2,3-dicarboxyate (11a)

0.2 g (0.3 mmol) of intermediate product 10a were dissolved in 2 ml of anhydrous DCM. After suitably cooling the solution at 0°C, 0.35 ml (3.5 mmol; 12 eq.) of piperidine were added. The reaction took 8 hours to complete.

For the working up of the reaction the process described in the synthesis of compound 7 was adopted. The purification was done by chromatography on a silica gel column with ethyl acetate/MeOH 9:1 and 0.092 g (0.21 mmol; 70%) of deprotected product were obtained with Rf = 0.72 (TLC in ethyl acetate/MeOH/ammonia 9:1:0.2). MS (IS): M+H⁺ = 436.

ⁱH NMR (300 MHz, CDCl₃,δ): 1.22 (s, 9H, OtBu), 2.45-2.75 (s, 2H, NH₂) 2.99-3.15 (dd, IH, CH₂), 3.18-3.38 (dd, IH, CH₂), 3.40-3.75 (m, 2H, CH₂), 4.99-5.19 (d, IH, CH), 5.16-5.34 (m, 3H, CH₂+CH), 5.24-5.46 (d, IH, NH), 5.65 (br.s, IH, NH), 7.02-7.56 (m, 9H, CHar.), 9.61-10.01 (d, IH, NH).

2-Benzyl 3-tert-butyl (1R.3S) l-(2-aminoethvι)-1.3,4.9-tetrahvdro-2H- beta-carboline-2,3-dicarboxylate (lib)

See synthesis of diastereoisomer 11a. Compound lib has Rf = 0.44 in ethyl acetate/MeOH 9:1 and is not sufficiently stable for adequate NMR characterisation.

MS (IS): M+H⁺ = 436; αD = +19.3° (c = 0.4 in DCM).

2-Benzyl 3-tert-butyl (lS,3S)-l-{r((2R)-4-0)enzyloxy)-2-{r(9H-fluoren-9- ylmethoxy)carbonyllamino|-4-oxybutanoyl)aminolmethyl|-l,3,4,9-te- trahydro-2H-beta-carboline-2,3-dicarboxylate (13a)

0.8 g (1.8 mmol) of intermediate product 11a (or lib for the synthesis of 13b), then 1.2 g (2.8 mmol; 1.5 eq.) of Fmoc-Asp(Obzl)-OH and 0.12 g (0.9 mmol; 0.5 eq.) of HOBt, and lastly 1.1 g of DCC (5.5 mmol; 3 eq.) were dissolved in 25 ml of anhydrous DCM at 0°C. After 4 hours the reaction was brought to room temperature and in the next hour the reaction was completed.

The organic phase was washed with 5 ml of HCl IN, then with 5 ml of saturated solution of NaCl, then with 5 ml of NaOH IN and lastly with a saturated solution of NaCl, filtered on celite to remove the DCU and dried on anhydrous sodium sulphate. The crude product was purified by chromatography on an SiO₂ column with ethyl acetate/n-hexane 4:6 to give 1.1 g (1.3 mmol; 70%) of condensed compound with Rf = 0.61 (TLC in ethyl acetate/n-hexane 1:1).

T_dec= 169°C; α_D = +60,1° (c = 0,4 in DCM);

MS (IS): M+H⁺ = 863 M+Na⁺ = 885; 2M+H⁺ = 1725; 2M+Na⁺ = 1747.

NMR (300 MHz, CDC1₃, δ): 1.29 (d, 9H, OtBu), 2.67-2.81 (m, IH, CH₂), 2.90-3.10 (m, IH, CH₂), 3.068-3.147 (m, IH, CH₂), 3.410-3.59 (m, IH, CH₂), 3.80-3.98 (m, 2H, CH₂), 3.90-4.06 (m, 2H, CH₂), 4.20-4.35 (m, IH, CH), 4.38-4.40 (m, IH, CH), 4.40-4.50 (m, 2H, CH₂), 4.81-4.90 (m, 2H, CH₂), 5.03-5.19 (m, IH, CH), 5.16-5.34 (m, 2H, CH₂), 5.24-5.46 (m, IH, CH), 5.72-5.92 (m, IH, NH), 7.00-7.20 (m, IH, NH), 7.06-7.80 (m, 22H, CHar.), 9.37-9.60 (d, IH, NH).

"C NMR (75.4 MHz, CDCI3, δ): 23,563, 23,690, 25,112, 25,793, 28,083, 28,137, 34,033, 36,470, 45,124, 45,211, 47,381, 49,571, 51,714, 53,771, 54,165, 54,252, 67,012, 67,446, 67,573, 67,947, 68,434, 82,490, 105,654, 111,590, 111,677, 118,087, 119,496, 120,224, 122,107, 122,174, 125,319, 126,654, 127,309, 127,963, 128,377, 128,417, 128,544, 128,764, 129,038, 131,101, 125,635, 136,510, 136,811, 141,538, 143,882, 156,121, 171,479, 171,613, 171,873.

HPLC Symmetry-C18 (3.5 μm 75 mm x 4.6 m) (H₂O/CH₃CN 30:70, flow rate 0.2 ml/min) r.t.: 3.17 min.

2-Benzyl 3-tert-butyl (lR.3S)-l-(r((2R)-4-(benzyloxy)-2-{r(9H-fluoren-9- ylmethoxy)carbonyl1aminol-4-oxybutanoyl)amino1methyι)-l,3,4,9-te- trahydro-2H-beta-carboline-2,3-dicarboxylate (13b)

See synthesis of 13a.

Analysis of 13b. Rf= 0.3 (TLC in ethyl acetate/n-hexane 1:1).

Tdec = 202°C αD = +8° (c = 0.11 in DCM)

MS (IS): M+H⁺ = 863; M+NH₄ ⁺ = 880; 2M+NH₄ ⁺ = 1741; 2M+Na⁺ =

1747; 2M+K⁺ = 1763.

Η NMR (300 MHz, CDCl₃,δ): 1.12 (d, 9H, OtBu), 2.80-3.09 (m, 2H, CH₂), 3.04-3.40 (m, 2H, CH₂), 3.65-3.75 (m, 2H, CH₂), 4.20-4.35 (m, IH, CH), 4.40-4.50 (m, 2H, CH₂), 4.99-5.19 (m, IH, CH), 5.16-5.34 (m, 2H, CH₂), 5.24-5.46 (m, IH, CH), 5.65 (br.s, IH, NH), 7.02-7.82 (m, 22H, CHar.), 8.9 (d, IH, NH).

HPLC Symmetry-C18 (3.5 μm 75 mm x 4.6 m) (H₂O/CH₃CN 60:40, flow rate 1 ml/min) r.t.: 2.48 min.

(lS.3S)-2-r(Benzyloxy)carbonyll-l-{r((2R)-4-(benzyloxy)-2-{r(9H-fluoren- 9-ylmethoxy)carbonyllamino|-4-oxobutanoyl)-amino1methyl|-2.3.4,9- tetrahydro-lH-beta-carboline-3- carboxylic acid (14)

0.86 g (1.0 mmol) of intermediate product 13a were solubilised in 10 ml of a mixture of DCM/TFA 8:2, previously cooled at 0°C, and the mixture was then brought to room temperature.

The reaction, stirred under nitrogen, was completed in 7 hours and was repeatedly diluted with 5 ml of dioxane and dried in vacuo to remove the TFA. The residual solid was then purified by chromatography on an SiO₂ column with ethyl acetate/n-hexane 8:2 to give 0.72 mg (0.9 mmol; 90%) with Rf = 0.6 (TLC in ethyl acetate/n-hexane 9:1).

Tdec = 176°C; αD = +100° (c = 0.2 in DCM);

MS (IS): M+H⁺ = 809; M-l = 807. ⁱH NMR (300 MHz, CDC1₃, δ): 2.67-2.81 (m, IH, CH₂), 2.90-3.10 (m, IH, CH₂), 3.07-3.15 (m, IH, CH₂), 3.410-3.59 (m, IH, CH₂), 3.80-3.98 (m, 2H, CH₂), 3.90-4.06 (m, 2H, CH₂), 4.20-4.35 (m, IH, CH), 4.38-4.40 (m, IH, CH), 4.40-4.50 (m, 2H, CH₂), 4,81-4,90 (m, 2H, CH₂), 5,03-5,19 (m, IH, CH), 5.16-5.34 (m, 2H, CH₂), 5.24-5.46 (m, IH, CH), 5.72-5.92 (m, IH, NH), 7.00-7.20 (m, IH, NH), 7.06-7.80 (m, 22H, CHar.), 9.55- 9.75 (d, IH, NH).

Benzyl(lS,3SV3-{r('(lS)-4-{r(E)-amino(nitroimino)methvn amino)-l-{r(2- tert-butoxy-2-oxoethyl)aminolcarbonyllbutyl)aminolcarbonyl|-l-{r((2s)- 4-Q enzyloxy)-2-{r(9H-fluoren-9-yl-methoxy) carbonyllaminol-4-oxobu- tanoyl)aminolmethyl}-l,3,4,9-tetrahydro-2H-beta-carboline-2-carboxy- late (15)

To 5 ml of anhydrous DMF stirred at -10°C under nitrogen were added 0.36 g (0.45 mmol) of intermediate product 14 and 0.22 g (0.67 mmol; 1.5 eq.) of intermediate product 7, and lastly 0.22 g (0.58 mmol; 1.3 eq.) of HATU. The reaction was completed in 1 hour with formation of the desired product with Rf = 0.67 (TLC in ethyl acetate).

The organic phase was extracted with 100 ml of fresh DCM and processed as described above in the synthesis of compound 6. The crude solid was purified by chromatography on a column packed with silica gel in ethyl acetate/n-hexane 9:1 to give 0.35 g (0.32 mmol; 70%) of intermediate product 15.

Tdec = 173°C; αD = -23° (c = 0.25 in MeOH);

MS (IS): M+H⁺ = 1121; M+Na⁺ = 1143; M+K⁺ = 1159.

Benzyl (lS,3S)-l-({r(2S)-2-amino-4-(Denzyloxy)-4-oxobu-tanoyl1aminol methyl)-3-{r((ls)-4-{[(E)-amino(nitroimino)methyll-aminol-l-{[(2-tert- butoxy-2-oxoethyl)aminolcarbonyl)butyl)aminolcarbonyl|-l,3,4,9-tetra- hydro-2H-beta-carboline-2-carboxylate (16) To 1 ml of DMF stirred at -10°C were added 0.02 g (0.018 mmol) of intermediate 15 and 0.004 ml (0.036 mmol; 2 eq.) of piperidine. To speed up the reaction linetics and avoid side reactions, after 2 hours another 2 equivalents of piperidine were added and at the end of the 5th hour a further equivalent of piperidine was added.

After 6 hours the reaction was complete with formation of a single product with R_f = 0.6 (TLC in ethyl acetate/MeOH 8.5:1.5). The reaction was extracted with 50 ml of fresh DCM and shaken with 10 ml of H₂O; the separated organic phase was then dried on sodium sulphate and concentrated at 40°C. The crude reaction product was purified by chromatography on an SiO₂ column with ethyl acetate/MeOH 95:5 to give 0.014 g (0.016 mmol) of intermediate product 16.

Tdec = 137°C; αD = -7,2° (c = 0.5 in MeOH);

MS (IS): M+H⁺ = 899; M+Na⁺ = 921;

HPLC RP-18 LiChrosorb 25 cm x 4.6 mm (CH₃CN/H₂O 55:45, flow rate

1 ml/min) r.t.: 6 min.

(2S)-l-r({(lS,3S)-3-{r((lS)-4-{r(E)-Amino(nitroimino)methyllamino}-l- {r(carboxymethyl)aminolcarbonyl}butyl)amino1carbonyl)-2-rθ3enzyl- oxy)carbonyll-2,3,4,9-tetrahvdro-lH-beta-carbolin-l-yl|methyl)amino]- 4-(F>enzyloxy)-l,4-dioxobutan-2-ammoniun chloride (17)

To 4 ml of a mixture of HCl 6N/THF 1:1 held at 0°C and stirred under nitrogen were added 0.35 g (0.39 mmol) of intermediate product 16. After 1 hour the reaction was brought to room temperature and monitored both by TLC and HPLC, in order to verify disappearance of the starting reactant and formation of the desired product over a reaction period of 20 hours.

The reaction was dried in vacuo at 40°C. The solid residue was taken up several times with 5 ml of dioxane and brought back to dryness in vacuo to remove all excess HCl. The crude reaction product (a pale straw-yellow solid) was used as is in the next reaction without any further purification.

MS (IS): M+H⁺ = 843; M+Na⁺ = 865;

HPLC RP-18 LiChrosorb 25 cm x 4.6 mm (CH3CN/H2O 55:45, flow rate

1 ml min) r.t.: 2.4 min.

Benzyl (1S.5S.1 IS.14S)- 1 l-(3-{r(Z)-amino(nitroimino)me-thvnaminol propyl)-5-r2-(benzyloxy)-2-oxoethvn-4,7,10,13-tetraoxo-l,2,3.4.5.6.7.8.9. 10.11.12,13.14,15,20-hexadeca-hvdro-1.14-epimino-ri, 4,7,101tetraaza- cvcloheptadecino|T3, 14-b1indol-21-carboxylate (18)

0.11 g of crude reaction product containing intermediate product 17, 0.091 g (0.4 mmol) of HATU, 0.033 g (0.24 mmol) of HOAt and lastly 0.032 ml (0.024 mmol) of collidine were solubilised in 5 ml of anhydrous DMF stirred at -10°C under nitrogen. The reaction was brought to room temperature after 2 hours and was monitored both by TLC and HPLC and was completed within the space of 72 hours. The reaction mixture was extracted with 100 ml of fresh DCM and processed as indicated above in the description of the synthesis of 7. The crude product was purified by chromatography on an SiO₂ column with ethyl acetate/MeOH 95:5 to give 0.060 (0.073 mmol) of cyclised product with Rf = 0.55 (TLC in ethyl acetate/MeOH 9:1).

Tdec = 200°C; αD = +81° (c = 0.3 in MeOH/DCM 7:3);

MS (IS): M+H⁺ = 825; M+NH₄ ⁺ = 842; M+Na⁺ = 847; M-l = 823;

HPLC RP-18 LiChrosorb 25 cm x 4.6 mm (CH3CN/H2O 55:45, flow rate

1 ml/min) r.t.: 13.5 min.

r(lS,5S,llS,14S)-ll-(3-{ramino(imino)methyllamino|propyi)-4,7,10.13- tetraoxo-1, 2,3,4.5,6,7,8.9,10,11, 12.13.14.15.20-hexadecahvdro-l.14-epi- minori,4,7,10]tetraazacyclohepta-decinori3,14-blindol-5-yll trifluoroa- cetate (19: also referred to as ST2449) 0.025 g (0.030 mmol) of intermediate product 18 were dissolved in 1 ml of a mixture of MeOH/concentrated AcOH 7:3; the solution was added with 10% palladium carbon in a catalytic amount and subjected to hydrogenation at 60 psi. The reaction was monitored by both TLC and

HPLC and took 20 hours to complete. Subsequently the reaction mixture was brought to dryness in vacuo at 40°C and the crude product was submitted to LC-MS, using a C-18 chromatography column, with a gradient of MeOH in H₂O (0.1% TFA). The fraction with Rt = 10.37 minutes corresponds to the desired product. The purification method was transferred on preparative HPLC, using UN absorption at 270 nm as the detection parameter. The fraction with r.t.

= 15 min was concentrated in vacuo at 40°C, obtaining a transparent oil, which was lyophilized to give 0.014 g (0.025 mmol; 83%) of product

19 (ST2449). αD = -8.5° (c = 0.5 in MeOH);

MS (IS): M+H⁺ = 556; M+Νa⁺= 577; M-l = 554;

HPLC RP-18 Luna 5 μm 150 x 4.60 mm (MeOH gradient in H₂O 0.1%

TFA, Flow rate 1 ml/min) r.t.=10,37 min.

HPLC RP-18 Waters 300 x 3.3 (MeOH gradient in H₂O 0.1% TFA,

Flow rate 8 ml/min) r.t. =15.00 min.

Alternative synthesis of 13a and 13b

Benzyl N-l-(2,2-dimethoxyethyl)-N-2-f(9H-fluoren-9-ylmethoxy)carbo- nyl -L-asparaginate (20)

To 50 ml of a mixture of DCM/DMF 95:5 stirred at 0°C were added 5 g (11 mmol) of Fmoc-L-Asp(Obzl)-OH, followed by 1.3 g (12 mmol; 1.1 eq.) of aminoacetaldehydedimethylacetal, then 0.9 g (6.7 mmol; 0.6 eq.) of HOBt and lastly 7.0 g (13 mmol; 1.2 eq.) of PyBOP.

The reaction was brought to room temperature after 2 hours and was completed in 10 hours. The working up method has already been indicated in the description of the synthesis of intermediate 6. The crude reaction product was purified by flash chromatography with ethyl acetate/n-hexane 1:1 to give 5.3 g (9.9 mmol; 90%) of condensed product with Rf = 0.52 (TLC in ethyl acetate/n-hexane 6:4).

Tdec = 161°C;

Tf = 84.57°C;

MS (IS): M+NH₄ ⁺ = 550; M+Na⁺ = 555; M+K⁺ = 573;

Η NMR (300 MHz, CDC1_3> δ): 2.65-2.75 (dd, IH, CH), 3.0-3.1 (d, IH,

CH), 3.4 (t, 8H, 2xOCH₃+CH₂), 4.19-4.21 (t, IH, CH), 4.26-4.36 (t, IH,

CH), 4.4-4.5 (d, 2H, CH₂), 4.5-4.6 (d, 2H, CH₂), 5.25 (d, 2H, CH₂), 5.85-

5.95 (d, IH, NH), 6.56 (br.s., IH, NH), 7.21-7.41 (m, 9H, CH ar.), 7.52-

7.58 (d, 2H, CHar.), 7.65-7.77 (d, 2H, CH ar.).

¹³C NMR (75.4 MHz, CDC1₃, δ): 26,775, 35,789, 36,543, 41,425, 47,374, 51,314, 54,546, 54,639, 65,670, 67,146, 67,513, 83,338, 87,251, 102,582, 120,271, 125,192, 127,329, 128,023, 128,464, 128,671, 128,858, 129,505, 130,013, 133,091, 135,542, 141,565, 143,875, 170,491.

Benzyl N-2-[(9H-fluoren-9-ylmethoxy)carbonyI1-N-l-(2-oxoethyι)-l- asparaginate (21)

0.73 g (1.4 mmol) of intermediate product 20, 0.84 ml (7 mmol; 5 eq.) of etherated BF3 and 0.5 ml of water were solubilised in 7.5 ml of DCM stirred at 0°C in an inert gas atmosphere. After 2 hours another 5 equivalents of etherated BF3 and 0.5 ml of water were added to bring the reaction to completeness in 5 hours.

The solution was diluted with fresh DCM to a total volume of 50 ml and shaken with water until the pH of the aqueous phase was neutral. The separated organic phase was dried on sodium sulphate and concentrated in vacuo at 20°C. The crude product obtained was purified by chromatography on an SiO₂ column in ethyl acetate/n- hexane 2:8 to give 0.68 g (1.4 mmol; 60%) of product 21 with R_f = 0.3 (TLC in ethyl acetate/n-hexane 6:4). The crude product was used as is in the next reaction without any further purification: the isolated product proved rather unmanageable and so unstable that it could not be adequately characterised.

MS (IS): M+Na⁺ = 541 (hemiketalic form).

Tert-butyl (lS.3S -l-{r((2R)-4-(benzyloxy)-2-{r(9H-fluoren-9-yl-methyl- oxy)carbonyllamino|-4-oxobutanoyl)aminolmethyl)-2,3,4,9-tetrahydro- lH-beta-carboline-3-carboxylate (22)

0.06 g (0.11 mmol) of intermediate product 21, 0.04 g (0.18 mmol; 1.1 eq.) of L-tryptophan t-butyl ester hydrochloride, and lastly 0.015 g (0.18 mmol; 1.5 eq.) of AcONa were dissolved in 1.2 ml of concentrated AcOH. The reaction mixture was stirred under nitrogen for 16 hours and monitored by TLC to verify the disappearance of the aldehyde compound and formation of the product desired with Rf = 0.2 (TLC in ethyl acetate/n-hexane 1:1). The working up of the reaction has already been described in the synthesis of intermediate product 9. The crude product obtained was used as is in the next reaction without any further purification.

MS (IS): M+H⁺ = 730; M+Na⁺ = 752; M+Cϊ = 763; M+AcO- = 788.

Alternative synthesis of 2-benzyl 3-tert-butyl(lR.3S) and (1S.3S)-1- {r((2R)-4-(benzyloxy)-2-{r(9H-fluoren-9-ylmethoxy)carbonyllamino|-4- oxybutanoyl)aminolmethyl}-l,3,4,9-tetra-hvdro-2H-beta-carboline-2,3- dicarboxylate (13a e 13b)

0.5 g of crude reaction product obtained by the synthesis of intermediate product 22, 0.1 g (1.2 mmol) of NaHCOβ, and lastly 0.2 ml (1.4 mmol) of benzylchloroformate were dissolved in 35 ml of a mixture of dioxane/water 4:1 stirred at 0°C. The reaction was monitored by TLC to verify the disappearance of the starting amine and the formation of the two diastereoisomers. The working up of the reaction has already been described in the synthesis of intermediate product 10. The crude reaction product was purified by means of the method described in the previous synthesis of compound 13a. 0.36 g (0.42 mmol) of the diastereoisomer called 13a and 0.065 g (0.075 mmol) of the other diastereoisomer called 13b were obtained.

The compound according to the present invention is endowed with interesting physiological properties, which make it useful as a medicament. In particular, said compound is a selective antagonist of the α β3 integrins. This antagonist activity enables the compound according to the invention to be used for the preparation of medicaments useful for inhibiting the action of the α_vβ3 integrins; in particular, said medicaments are used in the treatment of subjects, preferably mammals, and particular human subjects, suffering from altered angiogenesis. In particular, said medicaments are useful for the treatment of tumour metastases, retinopathy, acute kidney failure, and osteoporosis.

The inhibitory activity of the α_vβ3 integrins can be determined according to the assays described in the abovementioned patent application EP 1 077 218.

In keeping with another object of the present invention, the pharmaceutical compositions contain at least one formula (I) compound as an active ingredient, in an amount such as to produce a significant therapeutic effect. The compositions covered by the present invention are entirely conventional and are obtained with methods which are common practice in the pharmaceutical industry, such as, for example, those illustrated in Remington's Pharmaceutical Science Handbook, Mack Pub. N. Y. - latest edition. According to the administration route chosen, the compositions will be in solid or liquid form, suitable for oral, parenteral or intravenous administration. The compositions according to the present invention contain, along with the active ingredient, at least one pharmaceutically acceptable vehicle or excipient. These may be particularly useful formulation coadjuvants, e.g. solubilising agents, dispersing agents, suspension agents, and emulsifying agents.

Claims

1. Compound which is 3-carbonyl-[Arg-Gly-Asp]-l-methyleneami- ne-tetrahydro-beta-carboline, its racemic mixture, the single cis and trans enantiomers, and its pharmaceutically acceptable salts.

2. Use of the compound according to claim 1 as a medicament.

3. Use of the compound according to claim 1 for the preparation of a medicament with a selective anti-integrin α_vβ3 antagonist action.

4. Use of the compound according to claim 1 for the preparation of a medicament useful for the treatment of abnormal angiogenesis.

5. Use of the compound according to claim 1 for the preparation of a medicament useful for the treatment of a disease selected from the group consisting of tumour metastases, retinopathy, acute kidney failure and osteoporosis.

6. Pharmaceutical compositions containing the compound according to claim 1 in a mixture with at least one pharmaceutically acceptable excipient and/or vehicle.