Cyclic peptidomimetics, compositions containing them and their use in the treatment of diseases associated with angiogenesis

Abstract

The present invention relates to novel cyclic peptidomimetics, pharmaceutical compositions containing them and their use in the treatment of diseases associated with angiogenesis especially tumors and chronic inflammation in psoriasis, diabetes, degenerative diseases of the eye (ARMD), nephropathy and neuropathy.

Claims

1. A cyclic peptidomimetic of general formula I: ##STR00012## where m=from 0 to 4, n=from 0 to 4, i=3 or 4, and where A is selected from the group: CONH; NHCO; SS; HNCONH, CH.sub.2CH.sub.2; CH.sub.2NH; NHCH.sub.2; ##STR00013## B is selected from the group: (CH.sub.2).sub.d-NH.sub.2, where d=from 0 to 4; ##STR00014## where k=3 or 4, wherein, each chiral center may have L or D and/or R or S configuration, and pharmaceutically acceptable salts, hydrates or other pharmaceutically acceptable complexes thereof.

2. The cyclic peptidomimetic of claim 1, which inhibits VEGF 165 and NRP-1.

3. The cyclic peptidomimetic of claim 1, which exhibits antiangiogenic properties.

4. The cyclic peptidomimetic of claim 1, which is a monomer with a general formula: ##STR00015## where m=from 0 to 4, n=from 0 to 4, i=3 or 4, and where A is selected from the group: CONH; NHCO; SS; HNCONH, CH.sub.2CH.sub.2; CH.sub.2NH; NHCH.sub.2, B is selected from the group: (CH.sub.2)d-NH.sub.2, where d=from 0 to 4; ##STR00016## where k=3 or 4, wherein each chiral center may be L or D configuration or the R or S, and pharmaceutically acceptable salts, hydrates or other pharmaceutically acceptable complexes thereof.

5. The cyclic peptidomimetic of claim 1, which is a dimer of a general formula: ##STR00017## where m=from 0 to 4, n=from 0 to 4, i=3 or 4, and B is selected from the group: (CH.sub.2).sub.dNH.sub.2, where d=from 0 to 4; ##STR00018## where k=3 or 4, wherein each chiral center may be L or D configuration or the R or S, and pharmaceutically acceptable salts, hydrates or other pharmaceutically acceptable complexes thereof.

6. The cyclic peptidomimetic of claim 1, selected from the group consisting of the compounds of formulas:
(c[Lys-Pro-Glu]-Arg-OH).sub.2
(c[Dab-Pro-Glu]-Arg-OH).sub.2
(H-c[Dab-Pro-Glu]-Arg-OH).sub.2
(c[Arg-Pro-Glu]-Arg-OH).sub.2
H-c[Lys-Pro-Glu]-Arg-OH.

7. A pharmaceutical composition comprising, as an active ingredient, the cyclic peptidomimetic of claim 1 and a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable additive.

8. The composition of claim 7, which is for the treatment of tumors and/or chronic inflammation, psoriasis, diabetes, degenerative diseases of the eye, nephropathy and neuropathy.

9. A method for treating tumors or a disease associated with angiogenesis by administering a medicament wherein the cyclic peptidomimetic of claim 1 is used as an active ingredient in the medicament.

10. The method of claim 9, wherein the disease associated with angiogenesis is selected from, chronic inflammation, psoriasis, diabetes, degenerative eye diseases, nephropathy and neuropathy.

11. The method of claim 10, wherein the chronic inflammation is rheumatoid arthritis or inflammatory bowel disease.

12. The method of claim 9, wherein the medicament is in a dosage form adapted for infusion or intravenous injections or implants.

13. The method of claim 10, wherein the neurodegenerative diseases of the eye is age-related macular degeneration (ARMD).

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIG. 1 shows the general scheme of the synthesis of peptidomimetics of the invention.

(2) The Synthesis of the Compounds of the Invention

(3) The cyclic peptidomimetics of the invention may be obtained using well known peptide synthesis on the polymeric support (Solid Phase Peptide Synthesis, SPPS), where the building block's functional groups are protected by orthogonal protective groups cleavable in acidic or basic conditions.

(4) All used protecting groups should be stable during peptide bond or its isostere synthesis, while their removal should not lead to the destruction of the growing peptide chain or racemization of any chiral center.

(5) The preferred N--protecting groups are: 9-fluorenylmethyloxycarbonyl group (Fmoc) or tert-butyloxycarbonyl group (Boc). Other protecting groups proposed for the protection of chemical moieties located in the side chains of building blocks are: 2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl group (Pbf), 2,2,5,7,8-pentamethylchromane-6-sulfonyl group (Pmc), 4-methoxy-2,3,6-trimethylbenzylsulfonyl group (Mtr), p-toluenesulfonyl group (Tos), Boc, Fmoc, 4-methyltrityl group (Mtt), 4-methoxytrityl group (Mmt), benzyloxycarbonyl group (Cbz, Z), 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methyl-butyl group (ivDde), 2-chlorobenzyloxycarbonyl group (2-Cl-Z). For the synthesis of all cyclic peptidomimetics C-terminal amino acids are attached to a polymeric support, which is chemically inert and insoluble in the reaction media used. The preferred resin for the peptide synthesis in the Fmoc strategy is the 4-(hydroxymethyl)phenoxymethyl linker, which is directly attached to a polystyrene base matrix (Wang resin). The chloromethylpolystyrene with 1% of divinylbenzene (Merrifeild resin) and 4-Hydroxymethyl-phenylacetamidomethyl (PAM resin) are preferred resins for the peptide synthesis in the Boc strategy.

(6) Peptide bonds were obtained by using the following coupling reagents: N,N-Dicyclohexylcarbodiimide (DCC) with addition of 1-Hydroxybenzotriazole (HOBt), N,N-Diisopropylcarbodiimide (DIC) with addition of 1-Hydroxybenzotriazole (HOBt), N,N-dicyclohexylcarbodiimide with addition of hydroxybenzotriazole (HOBt), N,N-diisopropylcarbodiimide with addition of HOBt, N,N,N,N-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU), N,N,N,N-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), (1-cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethyl-amino-morpholino-carbenium hexafluorophosphate (COMU), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

(7) After deprotection of carboxylic group and amine group located in the side chains of amino acids in the position 2 and 4 cyclization on the resin was carried out using uronium salt (TBTU, HATU).

(8) The final step during the synthesis was peptide cleavage from the solid support. This process depends on the used strategy during peptide synthesis. The preferred is to use liquid hydrogen fluoride (HF) with the addition of anisole or a mixture of trifluoroacetic acid/water/triisopropylsilane (95:2.5:2.5; v/v/v). The crude products may be purified using high performance liquid chromatography on a reverse phase column packed with a C-12 or C-18 with the use of gradient method 0%-30% (B) over 30 minutes, where phase (A) is 0.05% TFA in H.sub.2O and the phase (B) is 0.05% TFA in ACN.

(9) The obtained products can be converted into a desired pharmaceutically acceptable salt by a conventional method.

EXAMPLE 1

(10) The Synthesis of the Compound of the Formula 1

(11) wherein A:

(12) ##STR00009##
B=(CH.sub.2).sub.4NH.sub.2, m=2, n=4; compound (c[Lys-Pro-Glu]-Arg-OH).sub.2

(13) ##STR00010##

(14) 333 mg (0.2 mmol/g) Boc-Arg-PAM resin with loading 0.37 mmol/g was stirred in methylene chloride (DCM). Boc protecting group was deprotected by 55% TFA in DCM (2, 5 i 25 min.). After that resin was washed by DCM (3), 5% DIPEA in DCM (2), DCM (3), IPA (3), DCM (3). The next step was a Kaiser test procedure. For this purpose to test tube were added dropwise equal volumes (a few drops) of three solutions [(A): 5 g of ninhidrine in 100 ml of ethanol; (B): 80 g of phenol in 20 ml of ethanol; (C): 2 ml 0.001M aqueous KCN in 98 ml of pyridine], then small amount of resin beads was added and after that sample was warmed to 100 C. and left for 5 minutes. When positive result was achieved (navy blue color) the next step of synthesis was carried out. Coupling of the next amino acid Fmoc-Glu(t-Bu)OH 249 mg (0.6 mmol) was carried out in DMF using 187 mg (0.6 mmol) TBTU and 271 l (1.2 mmol) DIPEA (2 h). After that time, the resin was washed with DMF (3) and the Kaiser test was performedit was negative. The Fmoc protecting group was removed by 20% piperidine in DMF (5 and 20 min). Next, the resin was washed with DMF (3), IPA (3), DMF (3). The Kaiser test was positive. Further, amino acid in a sequence Fmoc-Pro-OH 187 mg (0.6 mmol) was coupled in DMF (2 h) with TBTU and 271 l (1.2 mmol) DIPEA. The result of Kaiser test was negative. The Fmoc protecting group was removed by 20% piperidine in DMF (5 and 20 min). After that, the resin was washed with DMF (3), IPA (3), DMF (3). This step of the synthesis was followed by the chloranil test. For this purpose a few resin beads were placed in small test tube and few drops (equal volumes) of the two solutions [(A): 2% acetaldehyde in DMF; (B): 2% chloranil in DMF] were added. After short mixing the mixture was left at room temperature for 5 minutes. Subsequently, the dark green color of beads of resin indicated the completion of the Fmoc cleavage from Proline residue. Next, amino acid Boc-Lys(Fmoc)-OH, 274 mg (0.6 mmol) was coupled in DMF (2 h) with 187 mg (0.6 mmol) TBTU and 271 l (1.2 mmol) DIPEA. The result of the obtained chloranil test was negative. Next, the t-Bu protecting group from carboxylic group from glutamic acid and Boc protecting group from alfa-amine group from lysine was removed. For this purpose the peptide resin was washed with DCM (2) and 55% TFA in DCM (2) was added to the resin. Next, the resin was washed with DCM (3), 5% DIPEA w DCM (2), DCM (3), IPA (3), DCM (3). The Kaiser test was positive. After that, the amide bond was synthesized to obtain cyclic product. Here we use 187 mg TBTU (0.6 mmol), 102 mg 6-Cl-HOBt (0.6 mmol) i 271 l DIPEA (1.2 mmol). The reaction was performed in DMF for 4 h. After washing the resin with DMF (3) the Kaiser test was proceededit was positive. Another portion of the reagents were weighed and the reaction was continued for 12 h. After this time, the resin was washed with DMF (3). The Kaiser test was negative. Fmoc protecting group from the -amine group of lysine was removed by 20% piperidine in DMF (2). Next, the resin was washed with DMF (3), IPA (3), DMF (3). The Kaiser test was positive. In order to prepare the peptide resin to the final step of the synthesis it was washed with DCM (3) and placed in a vacuum desiccator for 24 h. The peptide was cleaved from the resin with anisol:HF (1:9, v/v) in use of the standard protocol. The reaction was carried out for 3 h. The peptide was purified by preparative high performance liquid chromatography (HPLC) using the C-12 reverse phase column. Elution was achieved by linear gradient 0%-30% (A) was 0.05% TFA in water and buffer (B) was 0.05% TFA in ACN. The fractions were analyzed using an analytical RP-HPLC on C-12 column.

(15) Rf=15, 97 (gradient 0-30% B in 30 minutes);

(16) MS[M+H]+ calc: 511.3, found. 511.3.

(17) [M+2H]2+ calc: 256.2, found. 256.2.

EXAMPLE 2

(18) Monomeric compound H-c[Lys-Pro-Glu]-Arg-OH was obtained according to the above scheme, but using Boc-Arg(Tos)-PAM resin in a loading 0.23 mmol/g.

(19) Analytical Data:

(20) Rf=15, 97, (gradient 0-30% B for 30 min)

(21) MS[M+H]+ calc: 511.3, found. 511.3.

(22) ##STR00011##

(23) Selected compounds were tested in vitro by measuring the inhibition binding of VEGF165 to NRP-1. This assay allows the determination of percentage inhibition of the tested compound at a predetermined concentration.

(24) Determination of the Inhibitory Activity of the Tested Compounds.

(25) The evaluation of the inhibitory effect of peptidomimetics was performed using an enzymatic method denoting the spectrophotometric displacement of the VEGF.sub.165 ligand from the specific receptor by the evaluated compound. The studies were performed using polystyrene 96-well plates (Maxisorb, Nunc.).

(26) The Procedure for Determining the Inhibition of VEGF165/NRP-1.

(27) To evaluate the biological inhibitory activity of selected molecules in each well of flat bottom polystyrene plate of 96-wells (Maxisorb, Nunc,) a 100 l of a solution containing 2 g/ml of anti-Fc IgG (Sigma-Aldrich) dissolved in a phosphate buffer PBS (PBS, Sigma) was placed. The plate was allowed to stand overnight at 4 C.

(28) The next day, wells were washed three times with 100 l PBS, and then saturated with 2% bovine serum albumin (BSA, Sigma) in PBS in order to eliminate non-specific interactions. After 2 hours of incubation at 37 C. the following solutions were added portionwise:

(29) 50 l of purified recombinant rat NRP1-Fc (R&D Systems, Abingdon, UK) in a solution of 20 ng/well protein dissolved in PBS-BSA 0.1% tween-80 0.005% (PBT),

(30) 50 l of compound solution in PBT in an appropriate concentration

(31) 50 l of biotinylated VEGF165 in a concentration 1 nM (R&D Systems) dissolved in PBT containing 2 g/ml heparin (Sigma). The total volume of added solution was 150 l.

(32) After an overnight incubation at 4 C., the wells were washed with PBT and treated with streptavidin-HRP polymer (horseradish peroxidase, Sigma). The plate was incubated for 1 hour at room temperature. Next, the wells were washed with 200 l PBT and substrate ABTS (2,2-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) (Sigma) was added.

(33) After 2 hours, the absorbance was measured at wavelength of 415 nm with respect to 470 nm.

(34) To determine IC50 for the best compounds measurements were made at concentrations from 0.03 to 10 M or 0.15 to 5 mM. In the control wells the tested compound was absent, but the respective concentration of DMSO was maintained. The heptapeptide A7R (ATWLPPR (SEQ ID NO:1)) was used as a positive control on every 96-well plate.

(35) TABLE-US-00001 TABLE 1 The results of inhibition studies of the heptapeptide A7R. Concentration Code 100 M 10 M 3 M 1 M 0.3 M A7R 87.8% 74.9% 55.1% 36.3% 14.6

(36) TABLE-US-00002 TABLE 2 Test results of inhibition of cyclic peptidomimetics of the invention. Exemplary cyclic peptidomimetics Concentration (dimers) 5 M 1.5 M 0.5 M 0.15 M (c[Lys-Pro-Glu]-Arg-OH).sub.2 81.2 72.6 58.4 43.9 (H-c[Dap-Pro-Glu]-Arg-OH).sub.2 64.0 43.4 39.9 21.3 c[Dap-Pro-Glu]-Arg-OH 63.5 34.6 30.7 22.2 (H-c[Lys-D-Pro-Glu]-Arg-OH).sub.2 69.8 51.8 48.5 29.9 (c[Dab-Pro-Glu]-Arg-OH).sub.2 74.8 63.5 54.8 40.6 (H-c[Dab-Pro-Glu]-Arg-OH).sub.2 72.1 64.3 46.8 40.7 (c[Arg-Pro-Glu]-Arg-OH).sub.2 80.4 72.6 66.1 53.3 (c[hArg-Pro-Glu]-Arg-OH).sub.2 80 63.7 40.7 31.7 Exemplary cyclic peptidomimetics Concentration (monomers) 10 M 3 M 1 M 0.3 M H-c[Lys-Pro-Glu]-Arg-OH 94.7 86.7 76.5 61.7 H-c[D-Lys-Pro-Glu]-Arg-OH 14.7 12.5 11.8 NS H-c[Lys-Pro-Asp]-Arg-OH 46.6 35.4 27.8 14.6 c[Orn-Pro-Glu]-Arg-OH 23.1 5.1 0.3 NS

(37) This test indicates potential antiangiogenic effects. The novel compounds of the invention are significantly better than standard peptide A7R, because they show inhibitory activity at nM concentrations.

(38) In comparison to the heptapeptide A7R, which exhibits rapid loss of activity with decreasing concentrations (reduction of concentrations of 10-0.3 M result in a 5-fold decrease in inhibition) in the case of the compounds of the invention at similar concentrations in the range 5-0.15 PM is only about 2-fold decrease in inhibitiononly half (inhibition decreases from 70-80% to 41-55%).

(39) For some compounds the IC.sub.50 was determined:

(40) (c[Lys-Pro-Glu]-Arg-OH).sub.2, IC.sub.50=0.46 M

(41) H-c[Lys-Pro-Glu]-Arg-OH, IC.sub.50=0.18 M