PURINE DERIVATIVES HAVING PHARMACOLOGICAL ACTION

Abstract: Compounds of formula (I), wherein R1 is OH or NH2 and R2 is H or NH2, R is H or lower alkyl, A is an alkyl chain optionally interrupted by heteroatoms and B is an amino acid, a di- or tri-peptide, are useful in human therapy.

Description: PURINE DERIVATIVES HAVING PHARMACOLOGICAL ACTION The present, invention relates to purine derivatives having an immunomodulating, antiviral activity and or. Central Nervous System, to a process for the preparation thereof and to pharmaceutical compositions containing them. The derivatives according to the invention can be represented by the following tautomeric general formula I wherein R is OH or NH and R is hydrogen or NH_, wir.i the proviso that F. and cannot be both NH croups hydroαer. or C ^ -C r alkvi, A is a grouo of- formula II 1 6 " (CH.) - CH,) - 2 rr, or a αrouo of formula -(CH. ) -CH- III 2 P I wherein X is oxygen, sulphur or selenium m is an integer from 0 to 4 n is an integer from 2 to 4 p is an integer from 0 to S R_, is hvdroqen or C -C. alkvl; J " 1 v ' B is a residue of a natural a ir.c aci G or series or a di- or tri-peptide residue in which the arninc acids which compose it are selected from the group consis¬ ting of alanine, arginine, glycine, isoglutamine, leucine, lysine, methionine, ornithine, proline arrd serine, with the proviso that, when A is a residue of formula III, B is always a di- or tri-peptide residue as above defined. In case amino acid B or N-terminal amino acid of the di- or tri-peptide is a basic amino acid (arginine, ornithine, lysine), the carbammate bond can involve either the amino group which is at the ° -position to the carbo- xy group or the amino group at the U~> -position to the carboxy group. The present invention also relates to non toxic salts of compounds of formula I . European patent N. 0 077 660 discloses purine deri¬ vatives in which an amino acid is linked to the purine ring at the 9-position by means of carbonyloxvalkvl or carbonylethylcarbonyloxyalkyl bridges . The derivatives according to the present inver.tior. show, in comparison to the known compounds, a different and surprising pharmacological activity range, particular¬ ly immunostimulatmg activity, antiviral activity and activity on Central Nervous System, so that they are va¬ luable for use in human therapy. Preferred compounds according to the invention sre: (a) compounds in which R is hydrogen, A is a group cf formula II wherein m is 0 or 1 , X is oxygen, n is I and B is an amino acid residue selected fro the group consisting of arginine, methionine, leucine cr the residue of a di- or tri-Deotide selected frorr. the group consisting of leucyl -methionine, ornithyl-orni- thine, glycyl-leucyl-methionine , leucyl-seryl-argini- ne, lysyl-arginine, alanyl-isoglutamme , lysyl-lysine, histidyl-leucyl-methionine, propyl-arginine; (b) compounds in which R is hydrogen, A is a residue of formula III in which p is 2 or 3, R is hydrogen and B is a di- or tri-peptide selected from the above defin¬ ed group. Compounds I are prepared by reaction of a compound of formula IV: R wherein R , R , R and A are as above defined, with phosge¬ ne and subsequent, condensation with an amino acid or di- or tri-peptides of formula H NB according to per se known procedures . Compounds of formula IV in which R is OK, F is hydrogen or NH and A is a residue of formula III wherein R and R are hydrogen, are prepared according to the procedures described in EP-B-77460. Compounds IV wherein R is OH, R is hydrogen and A is a residue of formula III can be prepared according to the process described ir. Italian Pat. Application N. 19083 A/87. Compounds IV in which R, is OH and R„ is NH„ can be 1 ... __ obtained according to the following reaction scheme: Q-OH wherein Q is a residue of formula -CH-A ■ i R in which R and A are as above defined. Comtoounds IV in which P. 2 is hv-droσ-en and R^, is NH , on the contrary, are obtained from compounds of for¬ mul V wherein R and A are as above defined, bv reaction with liαuid Nf-ϊ at. hiqh temDerature 3 - 5 - For the preparation of compounds IV in which A is a residue of formula II, reaction schemes analogous to the ones reported above and the ones known for the preparation of purine systems can be conveniently used. Thus, fcr example, , 6-dichloro-5-amino-pyrimidine can be reacted with a compound for formula H N(CH ) -X-(CH ) OH wherein 2 2 m 2 n X, and n are as above defined, to obtain the interme ¬ diates of formula VI: (CH_) -X-(CH_) OH 2 m 2 n which can then be transformed into corresponding compounds I by means of an appropriate combination of known rea¬ ctions, analogous to the above reported ones and anyhow known to those skilled in the art. The following examples further illustrate the inve¬ ntion without, limiting its spirit, and scope. EXAMPLE 1 N-/N-/_5- (hypoxanthin-9-yl )pentyloxycarbony1V-L-leucyl./-L- methionine -<>-CO- Ii-CH-CH__-CH(α-!,)- 5 ( 2 3 2 CO-NH-CH-CH-,-CH.,-S-CH. COCH 0.850 g (0.0032 mole) of leucyl-methionine and 0.824 ~ (0.0096 mole) of sodium bicarbonate were diss-olveα in 40 ml of bidistilled water, in a 150 ml flask. To the result¬ ing aqueous solution, having pH = 8, a suspension of 1.05 g (0.0032 mole) of 9-/__T-chlorocarbonyloxy)pent.yiyhypoxan- thine hydrochloride in 40 ml of toluene was added in ice bath. The reaction mixture was lef to react at room temperature under magnetic stirring for 5 hours. After that the two phases were separated; the solvent was evaporated from the aqueous phase to obtain 1.8 g of crude compound which was chromatographed on a silica gel column using as the eluen first a 1:1 ethyl acetate/ethancl mixture, then methanol . 1.450 g of compound were thus obtained. Elemental analvsis for C H N_O S MK 510.492 22 34 6 6 theoric found C 51.75 51.82 H 6.71 6.70 N 16.45 16.38 EXAMPLE 2 " - < _N -/_5-(hypoxanthin-9-yl )pentyloxycarbonyl_/-L-ornit.hi /- L-ornithine and 5 — — 2 N -/5-(hypoxanthin-9-yl )pentyloxycarbony_l/-N -L-ornithil-L- ornithine -O-OO-NH-(CH 2 ) -CH-NH 2 (CH ) -O-∞- H-(CH,)_,-CH-C0OH 2 3 CO NH I NH CO I I CH-COOH CH-NH- (CH 2 ) 3 NH.. NH, To a suspension of 1,05 g 0,0032 mole 9- ( 5-chloro-carbonyl-oxy-pentyl )-hypoxanthine hydrochiori- de in 40 ml of toluene, a solution of 1.6δ g (0,0032 mole) di ornithil-ornithine monohydrochloride-trihydrobro- mide and 1,9 g (0,0224 mole) of sodium bicarbonate in bidistilled water at pH = 8 was added, under magnetic stirring in ice bath. When the addition was over the reaction mixture was left to stand for 20 hours at room temperature. Controls were carried out by TLC (Thin Layer Chromathography ) , using methanol as the eluent.. At the end of the rection the two phases were separated, the aqueous phase was evaporated under vacuum and the resulting crude compound was chromatographed on silica gel, using methanol as the eluent. The mixture of the two products was thereby obtained, which products were separated by means of preparative HPLC. E.A. for C H N O MK = 494,21 21 34 8 6 theoric found found C 51,03 51 , O 5G, 98 H 6,87 6,89 C , 81 22,66 22,65 22, 63 EXAMPLES 3-9 The compounds hereinbelow reported were obtained by means of an analogous process, using corresponding amounts of the appropriate peptide. EXAMPLE 3 N-/N-/N-/5-(hypoxanthin-9-yl JpentyloxycarbonyiL/glycyy-L- leucyl-methionine E.A. for C 24 H 37 N 7°7 S EXAMPLE 4 N -/N/N-/5-(hypoxanthin-9-yl ) pent yloxycarbony 1_/-L- leucy^ ' ■ L-seryl -arginine E.A. for C 26 H 42 N- 1() 0 8 MK = i Z 2 , 591 theoric found C 50,15 50,12 H 6, 79 6, 73 N 22,48 22,44 EXAMPLE 5 2 -6 - - N -/_N -/_5-(hypoxanthin-9-yl )pentyloxycarbony_l/-L-lysyl/- arginine and 2 -2 - - N -/N -/J5-hypoxanthin-9-yl )pentyloxycarbony y-L-lysyl_/-I arginine N -_l ~ -/_5- {hypoxanthin-9-yl )pentyloxycarbony_l/-L-alany1/- isoglutamine 2 -6 _ N ~/K -/5-(hypoxanthin-9-yl )pentyloxycarbony_l/-L-lysy] L /-L- Ivsine anc 6 - N -/_ - (hypoxanth: '1 )pentyloxycarbonyl/-N " -L-lysyl -L-ly (CH_ ) , -CH-CO-i-ϊ-CH- (S. CH ) -NH- E.A. for C 23 H 38 K 8 0 6 theoric N 21,4. EXAMPLE 8 - -2 — N-/_N-/N - _5- (hypoxanthin-9-yl )pentyloxycarbony] -L-histi • dyl./-L-leucyl-L-methionine /N-/ ( hypoxanthin-9-yl ) pentyloxycarbony^/pyrrolyl_/-arginιne E.A. for C H N O MW = 519,484 22 33 9 6 theoric found C 50,86 50,83 H 6,40 6, 1 N 24,25 24,29 EXAMPLE 10 2 - - Preparation of N -_/2-/2-(hypoxantτ_in-9-yi Jet oxy/- ethoxy-carbonyj./arginine a). Preparation of 4-chloro-5-amino-6-/_(2-( 2-hydroxyethoxy * - ethyl/amino.pyrimidine. 508 ml di n-pentanol , 25 g (0,152 mole) of 4,6-dι- chloro-5-aminopyrimidine, 36,25 ml (0,152 mole) of tribu- tylamine, 11 ml (0,152 mole) of 2-(2-aminoethoxyJethanci and KI in catalytic amounts were placed into a three neck flask provided with reflux condenser and mechanical stir- rer, for 24 hours. The progress of the reaction was che¬ cked by TLC, using ethyl acetate as the eluent. At the end of the reaction pentanol was evaporated under reduced pressure and the resulting crude residue was chrorcatogra- phed on silica gel using 9:1 chloromethane/ethanol as the eluent. The compound was crystallized from ethyl acetate. M.P. = 121°C. b) Preparation of 9-/2-(2-hydroxyethoxy)ethyl/ ypoxar.tr.i- ne. 10 g (0,043 mole) of compound a) in 150 ml cf formic acid were placed into a 250 ml flask. The reactior. was refluxed for 24 hours. The progress of the reaction. was controlled by TLC using 8:2 ethanol/triethylamine as the eluent. At the end of the reaction, formic acid was evaporated under reduced pressure and the residue was crystallized from ethanol. Reaction yield: 55%. M.P. > 200°C. c) Preparation of 9-/_2-/_2-chlorocarbonyloxy )ethox_y/ethy /- hypoxanthine hydrochloride . 3 g (0,0133 mole) of compound b) were reacted with 30 ml of 20% phosgene in toluene in 80 ml of anhydrous toluene, under magnetic stirring. It is advisable to drop the phosgene solution. The reaction was controlled by TLC using ethyl acetate/ethanol in 1:1 ratio as the eluent. In order to visualize the product in the plate, a reaction sample was treated with a tert-butylamine excess to obtain corresponding urethane. At the end of the reaction solvent was evaporated off under reduced pressure to obtain the crude product which was directly reacted for: 2 - — d) preparation of N - _2 -/_ 2 - (hypoxanthin-9-yl ) -ethoxy-etho- xy-carbonyl/arginine . 0,927 g (0,0053 mole) of L-arginine was dissolved in 10 ml of water, under magnetic stirring. 0,86 g (0,002 mole) of compound C, suspended in 10 ml of toluene, was addedd to the above solution. The reaction mixture was left under magnetic stirring for 5 hours. At the end of the reaction the two phases were separated and the aqueous phase was evaporated under redu¬ ced pressure. The resulting crude residue was purified or. a silica gel column, using methanol as the eluent. 0,5 g of compound was obtained. EXAMPLE 11 Using 2-( 2-aminoethyl ) thio )ethanol , by means of a process analogous to the above one, the following compound was prepared: N ~/2/_2- (hypoxanthin-9-yl ) -ethylthio/ethoxyearbonyl/-L-ar- ginme, according to the following reaction scheme: Cl i 2 "5" i 2 _G ' : 2 α ic: HCO L-arginire * ODOK Biological activity of the above exemplified cσrr.- pounds was studied, and the compounds according tc the invention proved to be active as immunomoduiatcrs, r.e rc- transmitters and antiviral agents. Particularly, the com¬ pounds of the invention turned out. to be active n the following tests, showing an interaction on immunosyste : a) in vitro generation of Natural Killer cells from bone marrow precursors,- b) in vivo boosting of Natural Killer cells activity; c) in vitro cytotoxicity against YAC-1 target, cells; d) transplant, of bone marrow in mice; e) macrophages activation. Some of the compounds of the invention (example 3 and analogous), showed neurokinin-like activity in several in vitro models. They possibly act on neurokinin receptors such as NK-P, NK-A and NK-B. Moreover, the compounds of the invention proved to be active in in vitro antiviral activity tests on Vescicu- la Stomatitis Virus (VSV) and on encephalomyocarditis in L929 cells. From what above reported, it is evident that the compounds of the invention can be advantageously used in human therapy of deseases having viral, tumoral and bacterial origin, of neurologic syndromes or different pathologies in which immunosyste is recognizedly involved. The present invention also relates to all the ind - striallv applicable aspects related to the use of com¬ pounds I as therapeutical agent. Thus, an essential object of the invention is provided by pharmaceutical composi¬ tions containing as the active ingredient compounds I alone or in admixture with a pharmaceutical carrier, in form of tablets, sugar-coated pills, capsules, powders, granules for reconstitution in oral solutions or suspen¬ sions, syrups, injection vials, etc. The active ingredients can be alone in capsules. Otherwise, they can be formulated using traditional phar- maceutical carriers, for example excipients such as lacto¬ se or talcum, granulation agents such as methylcellulose and/or surface active agents such as polyoxyethylene st.e- arate; preservatives such as ethyl p-hydroxybenzoate and possibly flavoring agents. The pharmaceutical compositions of the preεer.- invention can preferably be formulated in form of unitary dosage containing 1 to 1000 mg of a compound of formula I in admixture with a pharmaceutical carrier. Said unitary doses can be administered one to more times a day, depen- ding on the pathology and the conditions of the par-ient.

Claims: CLAIMS 1. Compounds of formula I CH - A 0 CONHB wherein is OH or NH and R is hydrogen or NH , with the proviso that R and R cannot be both NH groups, R is hydrogen or C,-C, alkyl, A is a group of formula II 1 6 -(CH ) -X-(CH_) - (ID 2 m 2 n or a group of formula -(CH ■ CH- (Ill) 2 P R. wherein X is oxygen, sulphur or selenium, * m is an integer from 0 to 4 n is an integer from 2 to 4 p is an integer from 0 to 8 R is hydrogen or C -C alkyl; 3 1 6 B is a residue of a natural ammo acid of the Λ , d or d-E, series or a di- or tri-peptide residue in which the amino acids which compose it are selected from the group consi¬ sting of alanme, arginine, glycine, isoglutamine , leuci¬ ne, lysine, methionine, ornithine, prolme and serine, with the proviso that, when A is a residue of formula III, B is always a di- or tri-peptide residue as aoove defined, and non-toxic salts thereof. 2. A compound according to claim 1, selected from the group consisting of: N-/N-/5-(hypoxanthin-9-yl )pentyloxycarbonyy-L-leucyl ~ /-L- methionine; 2 -5 — N -/_N -_5-(hypoxanthin-9-yl )pentyloxycarbony /-L-ornithy_l - L-ornithine; 5 — - 2 N -/_5-(hypoxanthin-9-yl)pentyloxycarbonyl./-N -L-ornithyl-L- ornithine; N-/_N-/_N-/_5-(hypoxanthin-9-yl Jpentyloxycarbonyl/glycy^/.-L- leucyl-methionine; N -/_N-/N-/5-(hypoxanthin-9-yl )pentyloxycarbonyl7-L-leucyi/- L-seryl-arginine; o _ _ N -/_N -/_5-(hyρoxanthin-9-yl )pentyloxycarbony_l/-L-lysyl/-L- arginine; 2 -2 — - N -/_N -/_5-(hyρoxanthin-9-yl )pentyloxycarbony_l/-L-lysyl./-L- arginine; N -/N-/5-(hypoxanthin-9-yl)pentyloxycarbonyl/-L-alany_l/-L- isoglutamine; --> __ N -/_N -/_5-(hypoxanthin-9-yl )pentyloxycarbony_l_/-L-lysy] L /-L- lysine; 6 — 2 N -/_5-(hypoxanthin-9-yl Jpentyloxycarbony^Z- -L-lysyl-L-Iy- sine; _ _2 - N-/_N-/_N - _5-(hypoxanthin-9-yl )pentyloxycarbonyl/-L-histi- dyjy-L-leucyl-L-methionine; 2 — — - N -/N-/5-(hypoxanthin-9-yl )pentyloxycarbonyl/-L-pyrrolyI/- L-arginine; 2 — — N -/2-/2-(hypoxanthin-9-yl )ethylthio/ethoxycarbonyl/-L-ar- ginine 3. A process for the preparation of compounds I, in which compounds of formula IV wherein R , R , R and A are as above defined, are reacted with phosgene and subsequenthly condensed with an amino acid or a di- or tri-peptide of formula H NB, according t.c per se known procedures. 4. Pharmaceutical compositions containing as the acτ.ive ingredient, a compound according to claims 1-2. 5. Use of a compound according to claims 1-2 for prepar¬ ing a medicament having anti-viral and immunomodulating activity.

Inventors: CORNAGLIA FERRARIS PAOLO, STRADI RICCARDO, CORNAGLIA FERRARIS, PAOLO, STRADI, RICCARDO
Applicants: CO PHARMA CORP SRL, CO PHARMA CORPORATION S.R.L.
Application year: 1988
Main classification: C07K5
IPC codes: A61K38, A61P31, A61P37, C07K1, C07K5