PEPTIDOMIMETIC DERIVATIVES SUICIDAL INHIBITORS OF THE HIV PROLIFERATION

Abstract: Dipeptides of formula (I) wherein X is oxygen or a group SO2 and R is hydrogen or a protecting group, are suicidal inhibitors of HIV.

Description: Peptidomimetic derivatives suicidal inhibitors of the HIV proliferation. The present invention refers to peptidomimetic derivatives and the therapeutical use thereof. The patent application EP-0 094 815 (in the name of SmithKline Beecham) describes ohgopeptides containing an α-substituted glycine residue, for example phenylthiogly- cine, useful as pro-drugs for antitumoral and antimicrobial agents in view of their ability of crossing the cell membrane and releasing the active principle therein following an enzymatic degradation. These ohgopeptides must not bear protecting groups on the terminal positions or such groups must be in vivo detachable, and the α-substituted glycine must be of the L-serie, otherwise the ohgopeptides cannot get into the cell. Among the different active principles describe therein, thiophenol is said to be particularly useful for studying the protease/peptidase activity in biological systems. The patent application WO 91/10679 (in the name of Warner-Lambert) teaches renin- inhibitor peptide derivatives having the -NH-CH(S-phenyl)-CO- group useful, inter alia, in the treatment of diseases caused by retrovirus such as HTLV-I, -II and -III. The HIV-aspartyl-protease is said to act on natural substrates made by at least 9 ami- noacids only. For example, Niddam V. et al., Bioorg. & Med. Che . Letters, 6, No.6, 609-14 (1996) show thiophenoxy-peptides inhibitors of the HIV replication derived from a substrate specific for the HIV proteases and having the -Leu-(S)-Phe- (peptide α-glycine substituted) structural unit. The presence of the sulphur atom seems to make the structure resistant against the proteases action. The antiviral activity data attest the need of free peptide terminal groups, as already suggested by the patent application EP-0 094 815 above, and moreover the need of a peptide of at least 9-10 aminoacids is stressed, as both the tested thiophenol dipeptides showed to be inactive. The necessity of a peptide having at least 9 aminoacids, optionally containing a thiophenol group, is underlined also by the patent application WO 97/01576 (in the name of Laboratoire Laphal) disclosing peptides inhibiting the HIV replication by means of the inhibition of an aspartyl-protease dimer . It has been now surprisingly found that peptidomimetic derivatives bearing a thiophenol moiety as side chain and made of only two aminoacid residues are suicidal inhibitors of the HIV proliferation by selectively releasing said thiophenol moiety inside the infected cell by means of the HIV-protease activity Adversely to what taught by the prior art, the compounds of the invention have a small dimension (two aminoacid residues versus the at least 9 showed by WO 97/01576 and Niddam V et al supra) and are substituted on the N- and C-terminal positions (contrary to what taught in EP-0 094 815 supra) Nevertheless they show specificity for the HIV-protease and hence for the cells infected by this virus They are deacylated by the above said enzyme in this site only, and become sufficiently unstable to release the cytocidal agent, I e thiophenol The notion of suicidal inhibitor is known in pharmacology since a long time Contrary to the classic inhibitors, these molecules interact with the target structure just to be destroyed and thus release a portion thereof which kills the host cell It is clear that such substances can show their activity only with a target well defined and peculiar for the pathology to combat As for our knowledge, until now no suicidal inhibitor for the HIV virus was found and this is an important feature of the present invention Therefore the present invention relates to dipeptides of formula (I) wherein X is an oxygen atom or a SO 2 group, and R is hydrogen or a protecting group of the hydroxy moiety The compounds of formula I have asymmetric centres, thus can be in form of stereoi- somers Object of the present invention are the compounds of formula I m form of both stereoisomeric mixtures and single stereoisomers. The compounds of formula I are suicidal inhibitors of the HIV proliferation. They are able to release thiophenol, the cytocidal agent, inside the infected cell after having been selectively deacylated by the HIV-aspartyl-protease, and this ensures an activity selective for the HIV infected cells and, consequently, a negligible toxicity. For protecting group of the hydroxy moiety it is intended, for example, a trimethylsil- yl, triethylsilyl or acetyl group, preferably a trimethylsilyl group. The preferred configuration of the compounds of formula I is 1 S,2R for the 2-hy- droxy-indan-1-yl residue and 3S for the tetrahydro-3-furanyl residue. As shown hereinafter, one of the isomer of the compounds of formula I has shown a higher affinity for the enzyme and a greater ability of releasing the thiophenol moiety, but this is not preferred with respect nor to the other isomer neither to the racemate. The compounds of formula I may be obtained starting from L-phenyl-alanine (T j ) which is reacted with a suitable acylating agent such as, for example, di-t-butyldicar- bonate, benzylchloroformate, t-butyldimethylsilyloxy-chloroformate, optionally in the presence of an organic base, to yield a compound of formula III wherein Pg is a protecting group of the amino moiety, which in turn is condensed with a compound of formula IV wherein Rj is a lower alkyl group or a -CH 2 CC1 3 group, in the presence of a condensing agent such as, for example, 1 -hydroxybenzotriazole, N-hydroxysuccinimide, and dicyclohexylcarbodiimide. The compound of formula IV is prepared according to methods known from the literature. The condensation of the compounds of formula III and IV yields the compound of formula V wherein Pg and Rj are as defined above, which is hydrolysed in the presence of a strong base such as sodium, potassium or tetrabutylamrnoniurn hydroxide, and gives a compound of formula VI wherein Pg is as defined above, which in turn is condensed with cis-l -amino-2-indan- ol, in the presence of 1 -hydroxybenzotriazole, dicyclohexylcarbodiimide, or N-(3-di- methylaminopropyl)-N-ethylcarbodiimide chloride/N-methylmorpholine, to give a compound of formula VII wherein Pg is as defined above, which is hydrolysed in the presence of a strong acid, for example hydrochloric, hydrobromic or trifluoroacetic acid, and gives a compound of formula VIII The compound of formula VIII condensed with a compound of formula IX wherein X is as defined in formula I, in the presence of bases, leads to a compound of formula 1 wherein R is H A compound of formula I wherein R is a protecting group of the hydroxy moiety may be obtained starting from a compound of formula I wherein R is H according to techniques well known to the skilled in the art (see, for example, T W Greene and P G M Wuts, Protective groups in organic synthesis, John Wiley & Sons, New York) The compounds of formula I in optically active form are obtained by optical separa- tion or through stereospecific or stereoselective synthesis The compounds of formula I are suicidal inhibitors of the HIV as shown by the HIV- aspartyl-protease enzyme affinity test and by the thiophenol release test (example 13 and 14) As reference compound a structurally analogous compound comprised by the general formula of the patent application EP-0 094 815 discussed above was em- ployed This has shown a good affinity for the enzyme, whereas did not provide the release of thiophenol thus demonstrating not to be endowed with the features of the suicidal inhibitors of formula I of the invention It is apparent that these features of enzymatic selectivity and specificity make the compounds of formula I particularly suitable for the treatment of the viral infections generated by the action of a retro virus such as HIV As a consequence for the practical therapeutical uses, the compounds of formula I may be administered both parenterally and orally The therapeutical doses are generally comprised between 1 and 40 mg a day via par- enteral route, and between 20 and 200 mg for single administration via oral route A further object of the present invention are the pharmaceutical compositions contain- ing a therapeutically effective amount of the compounds of formula I or of the pharmaceutically acceptable salts thereof in admixture with a suitable carrier The pharmaceutical compositions object of the invention may be liquid, suitable for the enteral or parenteral administration, and, preferably, solid, such as tablets, capsules, granulates, suitable for the oral administration The preparation of the pharmaceutical compositions object of the invention may be carried out according to conventional techniques For better illustrating the present invention the following examples are now provided The chromatographic purifications were effected on silica gel columns (230-400 mesh) When no otherwise specified, the mass spectra were carried out under the following conditions, chemical ionisation, isobutane, positive ions The assignment of the compound names in the examples was effected following the IUPAC nomenclature through the ACD/Name system (Advanced Chemistry Development Inc , Toronto, Canada) Example 1 Synthesis of 2-(2S)-t-butoxycarbonylamino-3-phenyl-propionic acid A solution of L-phenyl-glycine (33 04 g, 0.2 moles) in NaOH IN (200 ml, 0 2 moles) under stirring was added with 300 ml of dioxane, then the solution was cooled to 0- 5°C and dropwise added, during 1 hour, with a solution of di-t butyldicarbonate (48 02 g; 0.22 moles) in dioxane (50 ml) The reaction mixture was then brought to room temperature and left under stirring overnight The dioxane was eliminated under vacuum and the mixture was acidified with KHSO 4 (30 g, 0.22 moles) dissolved in 100 ml of water The formed oil was extracted with ethyl acetate three times The joined organic phases were washed with 20% sodium chloride until neutral pH, then the solution was anhydrified and concentrated to yield an oil (58 g) which was extracted with petrolatum and concentrated to dryness There were obtained 53 g of 2-(2S)-t-butoxycarbonylamino-3-phenyl-propionic acid which was used as such in the next step Example 2 Synthesis of 2.5-dioxo-pyrrolidin-l-yl and (3S)-tetrahvdrofuran-3-yl carbonate A solution of (S)(+)-3-hydroxytetrahydrofuran (1 g, 11.35 mmoles) in methylene chloride (10 ml) under stirring and nitrogen, was added with N.N'-disuccinimidylcar- bonate (3.2 g, 12 84 mmoles) to yield a suspension wherein, at 18°C, a solution of triethylamine (1 74 ml, 12 48 mmoles) in methylene chloride (10 ml) was dropped The reaction mixture was left under stirring at room temperature overnight under nitrogen, then diluted with methylene chloride and washed with a solution of sodium chloride 3 times The organic phase was anhydrified and concentrated to give an oil which salified by seeding, and was separated with petrolatum, filtered and dried There were obtained 2.3 g of 2,5-dioxo-pyrrolidin-l -yl and (3S)-tetrahydrofuran-3-yl carbonate which were used as such in the next step Example 3 Synthesis of 2.2.2-trichloroethyl (2S)-2-(t-butoxycarbonylamino-3-phenyl-propionyl- aminoVphenylsulfanyl-acetate A solution of 2-(2S)-t-butoxycarbonylamino-3-phenyl-propionic acid (6 98 g, 0 0263 moles), prepared as described in example 1 , in methylene chloride (70 ml) was added with a solution of 1 -hydroxybenzotriazole (2 96 g, 0 0219 moles) in dry tetrahydrofu- ran (90 ml) obtained by weak heating, and subsequently with a solution of 2,2,2- trichloroethyl aminophenylsulfanylacetate (7 7 g, 0 0219 moles), prepared according to what taught by J A C S , 1992, 35, pages 1032-1042, in methylene chloride (80 ml) The mixture was stirred at 0-5°C and dropwise added with a solution of triethylamine (3.05 ml, 0 0219 moles) in methylene chloride (30 ml) in 30 minutes, followed by a solution of dicyclohexylcarbodiimide (4 53 g, 0 0219 moles) in methylene chloride (30 ml) The resulting mixture was left under stirring for further 30 minutes, then brought to room temperature and stirred overnight After filtering off the insoluble residue the mixture was concentrated under vacuum to yield a residue which was taken up with ethyl acetate and 20% sodium chloride After filtration, the organic phase was separated and washed with 5% citric acid, 5% NaCl, 5% NaHCO-, and fi- nally with 20% sodium chloride until neutral pH The solution was anhydrified and concentrated to give a reddish oil (13 5 g) which was taken up in methylene chloride The formed precipitate was filtered off and the solution concentrated to dryness again to give a residue which, dissolved in methylene chloride, was purified by flash chro- matography (eluent ethyl acetate/petrolatum from 40 60 to 20 80) There were obtained 8 8 g of 2,2,2-trichloroethyl (2S)-2-(t-butoxycarbonylamιno-3- phenyl-propionilamino)-phenylsulfanyl-acetate (yield 71 5%) 1H-NMR (200 MHz, CDC1 3 ) δ (ppm) 7 44-7 14 (m, 10H, Ar), 6 85-6 73 (m, IH, S- CH-*NH), 5 80-5 74 (m, IH, CHS), 4 85-4 74 (m broad, IH, NH-COO), 4 76-4 61 (m, 2H, COOCH 2 ), 4 44-4 30 (m, IH, *CH-NH-COO), 3 17-2 92 (m. 2H, *CH 2 -Ar), 1 38 and 1 36 (2s, 9H, t-bu) Mass 563 [M+H] Example 4 Synthesis of [(2S -2-(t-butoxycarbonylamιno-3-phenyl-propionylamιno)]phenylsul- fanyl-acetic acid A solution of 2,2,2-trichloroethyl (2S)-2-(t-butoxycarbonylamino-3-phenyl-propιonyl- amιno)-phenylsulfanyl-acetate (8 8 g, 15 66 mmoles), prepared as described in example 3, in dioxane (300 ml) and water (30 ml), under stirring at 15-20°C, under nitro- gen, was dropwise added with 0 IN NaOH ( 164 ml), and the resulting mixture was left standing overnight The dioxane was evaporated at room temperature and the mixture was brought to pH=8 with a 5% aqueous solution of sodium bicarbonate, then extracted with ethyl ether The aqueous phase was filtered on celite, acidified with 5% KHSO 4 , extracted with ethyl acetate, and the organic phase was anhydrified and concentrated to give a residue which was used as such in the next phase There were obtained 6 46 g of [(2S)-2-(t-butoxycarbonylamino-3-phenyl-propιonyl- amino)]phenylsulfanyl-acetic acid (yield 95%) Example 5 Synthesis of t-butyl [(lS -(l-{ [(2-hydroxy-ιndan-l -ylcarbamoyl)phenylsulfanyl-meth- yll-carbamoyl}-2-phenylethyl)]carbamate A solution of (2S)-2-(t-butoxycarbonylamino-3-phenyl-propionylamino)phenylsulfan- yl-acetic acid (5 g, 1 1 6 mmoles), prepared as described in example 4, in methylene chloride (50 ml) was added with a solution of ( l S,2R)(-)-cis-l-amino-2-indanol (1,83 g, 11.6 mmoles) in methylene chloride (50 ml) The mixture was stirred at room temperature then added with a solution of 1 -hydroxy-benzotriazole (1 57 g, 1 1.6 mmoles) in dry THF (60 ml) and, sequentially, with N-methyl-morpholine (1 4 ml, 12 8 mmoles) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide chloride (2 45 g, 12 8 mmoles), then the mixture was left under stirring at room temperature for 2 days, then concentrated under vacuum and the residue taken up with ethyl acetate, washed with 5% citric acid, 5% NaCl, 5% NaHCO^ and finally with a saturated solution of NaCl The organic phase was anhydrified and concentrated and the residue triturated in ethyl ether and petrolatum, finally dried under vacuum at room temperature There were obtained 6.21 g of t-butyl [( l S)-( l -{ [(2-hydroxy-indan-l -ylcarbamo- yl)phenylsulfanyl-methyl]-carbamoyl }-2-phenylethyl)]carbamate (yield- 95%) 'H-NJVLR (200 MHz, CDCK) δ (ppm) 7 44-7 05 (m, 16H, Ar, *NH-CH-CH-OH and *NH-CH-S), 5 98-5 69 (broad, IH, CH-S), 5 25-5 19 (m, IH, NH*-CH-CH-OH), 4 91-1 12 (broad, IH, NH-COO), 4 45-4 27 ( , 2H, *CH-OH e t-bu-NH-*CH-CH 2 ), 3 12-2 63 (m, 4H, 2*CH 2 -Ar), 1 29 and 1 35 (2s. 9H, t-bu) Mass 562 [M+H] Example 6 Synthesis of (2S)-2-amino-N-[(2-hydroxy-indan- 1 -ylcarbamoyl)-phenylsulfanyl-meth- vπ-3-phenyl-propionamide A suspension of t-butyl [(l S)-(l-{ [(2-hydroxy-indan-l-ylcarbamoyl)phenylsulfanyl- methyl]-carbamoyl }-2-phenylethyl)]carbamate (6 68 g, 1 1 89 mmoles), prepared as described in example 5, in ethyl acetate (70 ml) was added with HC1 in ethyl acetate (30 ml) The mixture was added with methanol (5 ml), left under stirring for 3 hours at room temperature, concentrated under vacuum The resulting residue was taken up with water (200 ml) and brought to acid pH with some drops of 1 N HC1 The precipi- tated impurities were filtered off, then the solution was basified (ρH=7 5-8) with a 5% solution of NaHCO 3 The precipitate was extracted with methylene chloride and the organic phase was anhydrified and brought to dryness The residue was triturated with ethyl ether/petrolatum, filtered, washed with petrolatum and dried under vacuum at room temperature There were obtained 4 05 g of (2S)-2-amιno-N-[(2-hydroxy-ιndan-l-ylcarbamoyl)- phenylsulfanyl-methyl]-3-phenyl-propionamide (yield 85 6%) 'H-NMR (200 MHz , CDCK) δ (ppm) 8 45-8 20 (broad, IH, CO-NH), 7 50-7 08 (m, 15H, Ar and CO-NH), 6 10-5 82 (m, IH, CHS), 5 35-5 19 (m, IH, CO-NH-*CH- CH), 3 57-3 50 (m, IH, *CH-NH 2 ), 3 20-2 45 (m, 4H, 2*CH 2 -Ar) Mass (liquid flow injection) 576 (M + H) * Example 7 Synthesis of (3S)-tetrahydro-3-furanyl [ l -( l S)-{ |Y( l S.2R)-2-hydroxy-ιndan- l -ylcar- bamoyl)phenylsulfanyl-methyl]-carbamoyl }-2-phenylethyl carbamate (Compound 1 ) A suspension of (2S)-2-amino-N-[(2-hydroxy-ιndan-l -ylcarbamoyl)-phenylsulfanyl- methyl]-3-phenyl-propionamide (3 g, 6 5 mmoles), prepared as described in example 6, in methylene chloride (30 ml), under stirring, was sequentially added with dry tetra- hydrofuran (30 ml), a solution of tπethylamine (0 9 ml, 6 5 mmoles) in methylene chloride (5 ml) and a solution of 2,5-dioxo-pvrrolιdιn-l -yl and (3S)-tetrahydrofuran- 3-yl carbonate (1 9 g, 6 5 mmoles), prepared as described in example 2, in methylene chloride (15 ml) The mixture was stirred overnight, concentrated under vacuum, and the residue taken up in ethyl acetate and a 20% aqueous solution of sodium chloride The resulting solid was filtered and washed with ethyl acetate and water The organic phase was separated, washed 3 times with a 20% aqueous solution of sodium chlo- ride, alternate first by 5% citric acid and then 5% sodium bicarbonate Then anhydrifi- cation and concentration were effected to yield a residue which was triturated with a mixture of ethyl ether and petrolatum, then dried under vacuum at room temperature There were obtained 2 9 g of (3S)-tetrahydro-3-furanyl [l-(l S)-{ [((l S,2R)-2-hydro- xy-indan- 1 -ylcarbarnoyl)phenylsulfanyl-methyl]-carbamoyl } -2-phenylethyl]-carbamate 'H-NMR (200 MHz, DMSO-d 6 ) δ (ppm) 8 89-8 28 (m, 2H, CH-CO-*NH-CH-CO- *NH), 7 54-7 1 (m, 15H, Ar and *NH-CH-CH 2 -Ph), 6 20 and 6 01 (2d, IH, CHS), 5.22-4 84 (m, 3H, *CH-NH, OH and CH-O-CO), 4 45-4 23 (m, 2H, *CH-OH and *CH-CH 2 -Ph), 3 75-2 38 (m, 8H, 2*CH 2 -O, *CH 2 -Ph and *CH 2 -CH-OH), 2 14-1 68 (m, 2H, O-CH 2 -*CH 2 -CH-O-CO) Mass (liquid flow injection) 576 (M + H) + Example 8 Separation of the isomers of (3S)-tetrahvdro-3-furanyl ri-(lS)-{ |Y(lS.2R)-2-hydroxy- indan- 1 -ylcarbamovDphenylsulfanyl-methyll-carbamoyl }-2-phenylethyl]-carbamate 1 (3S)-tetrahydro-3-furanyl [l-(l S)-{ [(( l S,2R)-2-hydroxy-ιndan-l -ylcarbamoyl)- phenylsulfanyl-methyl]-carbamoyl}-2-phenylethyl]-carbamate (1 7 g) dissolved in a mixture of methylene chloride/methanol was charged on a silica gel column by eluting with methylene chloride/methanol 95 5 (flash chromatography) The eluates containing the first eluting isomer were dried and the formed residue was triturated with ethyl ether/petrolatum, filtered, washed with the same mixture and dried under vacuum There were obtained 0 53 g of (3S)-tetrahydro-3-furanyl [l-(l S)-{ [((lS,2R)-2-hy- droxy-indan-l -ylcarbamoyl)phenylsulfanyl-methyl]-carbamoyl }-2-phenylethyl]-car- bamate isomer A (Compound 1 A) Η-NMR (200 MHz, DMSO-d 6 ) δ (ppm) 8 66 (d, IH, JHH=8 6 Hz, *NH-CHS), 8 31 (d, IH, JHH=8 4 Hz, *NH-CH-CH), 7 55-7 05 (m, 15H, Ar and *NH-COO), 6 01 (d, IH, CHS), 5 21-4 83 (m, 3H, NH-*CH-CH-OH and CH 2 -*CH-CH 2 ), 4 46- 4 40 (m, IH, *CH-OH), 4 33-4 21 (m, IH, *CH-CH 2 -Ar), 3 78-2 62 (m, 8H, 2*CH 2 - Ar and *CH 2 -O-*CH 2 ), 2 15-1 74 (m. 2H, CH-*CH 2 -CH 2 -S) Mass (liquid flow injection) 576 (M + H) ^ HPLC 98 7% 2. Operating in a manner similar to point 1 and collecting the slower eluting isomer there were obtained 0 61 g of (3S)-tetrahydro-3-furanyl [l-(l S)-{ [((lS,2R)-2-hy- droxy-indan- 1 -ylcarbamoyl)phenylsulfanyl-methyl]-carbamoyl }-2-phenylethyl]-car- bamate isomer B (Compound IB) 'H-NMR (200 MHz , DMSO-d 6 ) δ (ppm) 8 87 (d, IH, JHH=9 2 Hz, *NH-CHS), 8 57 (d, IH, JHH=8 6 Hz, *NH-CH-CH), 7 54-7 08 (m, 15H, Ar and *NH-COO), 6 20 (d, IH, CHS), 5 21-5 14 (m, 2H, NH-*CH-CH-OH), 4 95-4 90 (m, 2H, CH 2 - *CH-CH 2 ), 4 46-4 40 (m, IH, *CH-OH), 4 36-4 25 (m, IH, *CH-CH 2 -Ar), 3 76-2 37 (m, 8H, 2*CH 2 -Ar and *CH 2 -O-*CH 2 ), 2 1 1-1 68 (m, 2H, CH-*CH 2 -CH 2 -S) Mass (liquid flow injection) 576 (M + H) ' HPLC 97 1% Example 9 Synthesis and separation of the isomers of 1.1 -dιoxo-tetrahydro-l λ 6 -thiophen-3-yl [ 1 - ( 1 S)-( [((1 S,2R)-2-hydroxy-indan-l -ylcarbamoyl)-phenylsulfanyl-methyl " |-carbamo- yl }-2-phenylethyllcarbamate (Compound 2) A suspension of (2S)-2-amino-N-[(2-hydroxy-ιndan-l-ylcarbamoyl)-phenyl-sulfanyl- methyl]-3-phenyl-propionamide (400 mg, 0 866 mmoles), prepared as described in example 6, in methylene chloride (4 ml) and dry tetrahydrofuran (1 5 ml) was added with pyridine (74 μl, 0 91 mmoles) in methylene chloride (1 ml) and then dropwise added with a solution of 2,5-dioxo-pyrrolidin-l-yl and (3S)-l, l-dioxo-tetrahydro-l λ 6 - thιophen-3-yl (0 252 g, 0 91 mmoles), prepared as described in Bioorg & Med Chem Letters, 1995, 5, 2891-2896, in methylene chloride (3 ml) The resulting solu- tion was left under stirring at room temperature for 2 days, then concentrated and the residue taken up in aqueous ethyl acetate The formed solid was filtered off and the organic phase was sequentially washed with 5% citric acid (twice), a 20% solution of sodium chloride (twice), 5% aqueous solution of sodium bicarbonate (once) and water (twice), then concentrated under vacuum and the residue joined to the previously filtered solid The purification and simultaneous separation of the isomers were effected by flash chromatography (eluent ethyl acetate/petrolatum 80 20) 1 The dried fractions provided a solid which was triturated in ethyl ether, the solid was filtered, washed with ethyl ether and dried to provide 200 mg of isomer A (Compound 2 A) (yield 37%) Η-NMR (200 MHz, DMSO-d 6 +DCl) δ (ppm) 8 74 (d,lH,JHH=8 8Hz, *NH-CH-S), 8 30(d, lH,JHH=8 6Hz, *NH-CH-CH), 7 68(d, lH,JHH=8 8Hz, O-CO-N), 7 55-7 05 (m, 14H,Ar), 6 02(d. lH,CHS), 5.23-5 09 (m, 2H. CH-*CH-N, CH-O-CO), 4 46-4 40 (m, IH, *CH-OH), 4 31 -4 24 (m, IH, *CH-CH 2 -Ph), 3 40-2 64 (m, 8H, 2*CH 2 -SO 2 , *CH-Ph and *CH 2 -CH-OH), 2 45-2 10 (m, 2H, S-CH 2 -*CH 2 -CH-O-CO) Mass (liquid flow injection) 624 (M + H)+ HPLC 96 2% TLC (ethyl acetate/petrolatum 80.20) Rf=0 44 TLC (methylene chloride/methanol 95 5) Rf=0 48 2 Operating in a manner similar to point 1 from the low fraction there were obtained 165 mg of isomer B (Compound 2B) (yield 30 5%) Η-NMR (200 MHz, DMSO-d 6 +DCl) δ (ppm) 8 94 (d, lH,JHH=9 1Hz, *NH-CH-S), 8 56(d, lH,JHH=8 6Hz, *NH-CH-CH), 7 60(d, lH,JHH=8 8Hz, O-CO-N), 7 54-7 08 (m, 14H. Ar), 6 21 (d, IH, CHS), 5.21-5 08 (m, 2H, CH-*CH-N, CH-O-CO), 4 45- 4 40 (m, IH, *CH-OH), 4 34-4.27 (m, IH, *CH-CH 2 -Ph), 3 36-2 67 (m, 8H, 2*CH 2 - SO 2 , *CH-Ph and *CH 2 -CH-OH), 2 42-2 03 (m, 2H, S-CH 2 -*CH 2 -CH-O-CO) Mass (liquid flow injection) 624 (M + H)+ HPLC 97 8% TLC (ethyl acetate/petrolatum 80 20) Rf=0 3 TLC (methylene chloride/methanol 95 5) Rf=0 37 Example 10 Synthesis of (3S)-tetrahvdrofuran-3-yl (2-phenyl-(l S)-l-( phenylsulfanyl-(( lS.2R)-2- trimethylsilanyloxy-indan-l-yl-carbamoyl)-methyl]-carbamoyl] ^ethyl)-carbamate (Compound 3) A suspension of (3S)-tetrahydro-3-furanyl [ l-(l S)-{ [((l S,2R)-2-hydroxy-indan-l-yl carbamoyl)phenylsulfanyl-methyl]-carbamoyl}-2-phenylethyl]carbamate (0 575 g, 1 mmole), prepared as described in example 7, in methylene chloride (10 ml), under stirring, was added with dry tetrahydrofuran (5 ml) and, under nitrogen, was dropwise added with a 98% solution of trimethyl-silyl-isocyanate (0 141 g, 1.2 mmole) in meth- ylene chloride (5 ml) After 1 night under stirring at room temperature, triethylamine (0 6 ml) and trimethyl-silyl-isocyanate (0 3 ml) were added The suspension was heated in a water bath and left under stirring at room temperature for 2 days, under nitrogen After dilution with methylene chloride, the organic phase was sequentially washed with a 20% solution of sodium chloride, 5% citric acid, a 20%, solution of sodium chloride, a 5% aqueous solution of sodium bicarbonate and a 20%) solution of sodium chloride until neutral pH, then was concentrated under vacuum and the residue was purified by flash chromatography (eluent methylene chloride/methanol 95 5) There were obtained 0 3 g of (3S)-tetrahydrofuran-3-yl (2-phenyl-(l S)-l-{ [phenyl- sulfanyl-((l S,2R)-2-trimethylsilanyloxy-indan- l -yl-carbamoyl)-methyl]-carbamoyl] }- ethyl)-carbamate (Compound 3) (yield 46 4%) 'H-NMR (200 MHz , DMSO-d 6 ) δ (ppm) 8 78-8 35 (m, 2H, CH-CO-*NH-CH-CO- *NH), 7 52-7 08 (m, 15H, Ar and *NH-CH-CH 2 -Ph), 6 13 and 6 04 (m, IH, CHS), 5.24-4 79 (m, 2H, *CH-NH and CH-O-CO), 4 62-4 20 (m, 2H, *CH-OSi and *CH- CH 2 -Ph), 3 78-2.35 (m, 8H, 2*CH 2 -O, *CH 2 -Ph and *CH 2 -CH-OSi), 2 14-1 67 (m, 2H, O-CH 2 -*CH 2 -CH-O-CO) Mass (liquid flow injection) 648 (M + H) + Example 1 1 Synthesis of 2,2.2-trichloroethyl [(2S)-2-amino-3-phenyl-propionylamino]-phenylsul- fanyl-acetate A solution of 2,2,2-trichloroethyl (2S)-2-(t -butoxycarbonylamino-3-phenyl-propion- ylamino)phenylsulfanyl-acetate (8 8 g, 15 66 mmoles), prepared as described in example 3, in dry ethyl ether (50 ml), under magnetic stirring, was added with ethyl ether (50 ml) containing HC1 and the mixture was left to stand for 2 days Then it is con- centrated to dryness, taken up in hexane, triturated and filtered washing with hexane There were so obtained 7 2 g of 2,2,2-trichloroethyl [(2S)-2-amino-3-phenyl-propion- ylaminoj-phenylsulfanyl-acetate which was used as such in the next step Example 12 Synthesis of [(2S)-2-amino-3-phenyl-propionylamino]-phenylsulfanyl-acetic acid (Reference compound) A solution of 2,2,2-trichloroethyl [(2S)-2-amino-3-phenyl-propionylamino]phenylsul- fa yl-acetate (0.5 g, 1 mmole), prepared as described in example 11, in ethanol (20 ml), was dropwise added with 0 IN NaOH (20 ml, 2 mmoles), while keeping the tem- perature between 0 and 5°C The mixture was left under stirring at 10°C overnight Water was added and the aqueous phase was acidified with HC1, then concentrated, and the residue was taken up twice with acetonitrile and concentrated under vacuum There were so obtained 0 43 g of [(2S)-2-amino-3-phenyl-propionylamino]-phenylsul- fanyl-acetic acid Η-NMR (200 MHz , IN DCl) δ (ppm) 7.67-7 34 (m, 10H, Ar), 5 95 and 5 73 (2s, IH, CHS), 4 48-4 41 (m, H, CH-CON), 3 31-3 05 (m, 2H, CH 2 ) Example 13 Test for the evaluation of the affinity for the HIV-1 aspartyl protease enzyme The incubation mixture was made of 0.5 μg/sample of HIV- 1 aspartyl protease (Ba- chem AG, Switzerland, in 0 1M sodium acetate buffer, pH 5 5 + 10% (v/v) glycerol + 5% (v/v) ethylenglycole), lOμg/sample of HIV protease III substrate (H-His-Lys-Ala- Arg-Val-Leu-p-nitro-Phe-Glu-Ala-Nle-Ser-NH2 by Bachem). the compound under test at different concentrations and a reaction buffer (50mM sodium acetate pH 4,9 + 200mM NaCl + 5mM dithiotreitol + glycerol 10% v/v) to a final volume of 125 μl The reaction was carried out for 20 minutes at 37°C and quenched by adding 10 μl of 10% trifluoroacetic acid The hydrolysis of the substrate was evaluated by reversed phase HPLC using a C 18 column for proteins and peptides (Vydac, cat 218TP54) The products were eluted with a linear gradient of 10-60%) of acetonitrile+0 1% of trifluoroacetic acid in water, at a flow of 1 ml/min for 15 minutes The wavelength used was 220 nm The hydrolysis of substrate III occurs between the Leu and p-nitro-Phe residues, yielding two peaks eluting before the substrate The affinity of the compounds of the invention is expressed as the concentration able to inhibit at 50% the hydrolysis of the substrate (IC 5 n) The results are set forth in the following table. Table Example 14 Test for the evaluation of the thiophenol release The reaction mixture was made of HIV-1 aspartyl protease (0.5 μg/sample), a compound of the invention (at the 50μM standard concentration) and a reaction buffer to a final volume of 125 μl. The reaction was effected in hermetically sealed tubes for 120 minutes at 37°C and quenched by adding 85 μl of acetonitrile. The thiophenol release was evaluated by HPLC using a Nucleosil C18 column (Macherey Nahel) and eluting with phosphoric acid 0.2% w/v acetonitrile 40:60 at a flow of 1.2 ml/min. The wavelength for the evaluation of thiophenol was 238 nm. The results are set forth in the following table 2. The experiments above show how the compounds of the invention are able to interact with HIV aspartyl-protease and release thiophenol as a result of this interaction Example 15 Test for the evalutation of the cytotoxicitv The CEM ATCC cells (lymphoblastoid line) infected by the HIV-1 BRU virus (CEM ATCC/BRU) are particular as they produce the virus while multiplying This system put into evidence the cytotoxicity due to the compound tested and not due to the virus Such a toxicity is more evident for the treated infected cells then for the untreated ones used as a control In the same way such a toxicity is weaker for the uninfected CEM ATCC cells The CEM ATCC and CEM ATCC/BRU cells were at a concentration of 5xl0 5 cells/ lOOμl They were treated for 1 hour with 100 μl of a solution containing the compound to be tested at various concentrations At the day 4 a count with Trypan blue was carried out and the numer of cells was adjusted to 5xl0 5 cells/1 OOμl, then moved to a 24-well plate in 1 ml of the same compound From day 5 to day 7 the cells were counted each day with Trypan blue for evaluating the ratio between infected and uninfected control The results are shown in Table 3 They show that the compounds of the invention are consistently more effective on the infected cells than on the uninfected ones This difference is due to the capacity of the compound of the invention of binding to the HIV aspartyl protease and, as a consequence, of releasing the toxic thiophenol, as already proved by the previous experiments Table

Claims: Claims 1. A compound of formula I wherein X is an oxygen atom or a SO 2 group; and Ri is hydrogen or a protecting group of the hydroxy moiety. 2. A compound according to claim 1 wherein Ri is hydrogen or a trimethylsilyl group. 3. A pharmaceutical composition containing a therapeutically effective amount of a compound according to claim 1 in admixture with a suitable carrier. 4. Process for preparing a compound according to claim 1 wherein L-phenyl-alanine is reacted with a suitable acylating agent, optionally in the presence of an organic base, to give a compound of formula III wherein Pg is a protecting group of the amino moiety, which in turn is condensed with a compound of formula IV wherein R] is a lower alkyl group or a -CH 2 CC1 3 group, in the presence of a condensing agent and dicyclohexylcarbodiimide to give a compound of formula V wherein Pg and R] are as defined above, which is hydrolysed in the presence of a strong base and provides a compound of formula VI wherein Pg is as defined above, which in turn is condensed with cis-l-amino-2-in- danol, in the presence of 1 -hydroxybenzotriazole, dicyclohexylcarbodiimide or N- (3-dimethylaminopropyl)-N-ethylcarbodiimide chloride/N-methylmorpholine, to give a compound of formula VII wherein Pg is as defined above, which by hydrolysis in the presence of a strong acid gives a compound of formula VIII which condensed with a compound of formula IX wherein X is as defined in formula I, in the presence of bases, yields a compound of formula I wherein R is H, and, in the case, is duly deprotected on the R substituent. 5. Suicidal inhibitor capable of releasing thiophenol as cytocidal agent useful in the treatment of infection caused by HIV.

Inventors: CHERMANN JEAN-CLAUDE, KRAUS JEAN-LOUIS, PELLACINI FRANCO, ROMAGNANO STEFANO, UNGHERI DOMENICO, CHERMANN, JEAN-CLAUDE, KRAUS, JEAN-LOUIS, PELLACINI, FRANCO, ROMAGNANO, STEFANO, UNGHERI, DOMENICO
Applicants: CHERMANN JEAN CLAUDE, INST NAT SANTE RECH MED, KRAUS JEAN LOUIS, PELLACINI FRANCO, ROMAGNANO STEFANO, UNGHERI DOMENICO, ZAMBON SPA, CHERMANN, JEAN-CLAUDE, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), KRAUS, JEAN-LOUIS, PELLACINI, FRANCO, ROMAGNANO, STEFANO, UNGHERI, DOMENICO, ZAMBON GROUP S.P.A.
Application year: 1998
Main classification: C07K5
IPC codes: A61K38, C07K5