Antiherpes compositions

Abstract: Disclosed herein is a combination of an antiviral nucleoside analog and a ribonucleotide

Description: Field of the Invention This invention relates to an antiviral pharmaceutical composition comprising a combination of a nucleoside analog and a peptide derivative, useful for treating herpes infections in a mammal by administering the combination to the mammal. Background of the Invention Herpes viruses inflict a wide range of diseases against humans and animals. For instance; herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), are responsible for cold sores and genital lesions, respectively; varicella zoster virus (VZV) causes chicken pox and shingles; and the Epstein-Barr virus (EBV) causes infections mononucleosis. Over the past two decades, a class of compounds known as the purine and pyrimidine nucleoside analogs has been the subject of much attention by investigators in the search for new therapeutic agents for the treatment of herpes virus infections. As a result, several nucleoside analogs have been developed as antiviral agents. The most successful to date is acyclovir which is the agent of choice for treating genital herpes simplex infections. Other nucleoside analogs which are used therapeutically for the treatment of herpes infections include vidarabrine, idoxuridine, trifluridine and ganciclovir. The mode of action by which the nucleoside analogs exert their antiviral effect is thought to involve the inhibition of viral nucleic acid replication. In the case of herpes viruses, the production of new viral deoxyribonucleic acid (DNA), an essential stage of viral replication, depends on the interaction of the virally encoded enzyme, DNA polymerase, with cellular deoxynucleotides. The nucleoside analog, when converted enzymatically in vivo to its triphosphate derivative, acts as an alternate substrate (i.e. a "fraudulent substrate) for the viral DNA polymerase, and becomes incorporated into the growing viral DNA chain. Since the nucleoside analog either lacks and essential group, e.g. the 3'-hydroxyl, or has the wrong stereochemistry, it also acts as a "chain terminator" of the growing viral DNA chain. The net effect is that the nucleoside analog acts in vivo as an inhibitor of the viral DNA polymerase. Although the therapeutically useful nucleoside analogs have proven to be a valuable agents for combatting or controlling herpes infections, the agents are not without side effects. For example, skin rashes and renal impairment have been reported as side effects for acyclovir (see Physicians' Desk Reference, 44th ed., Medical Economics Inc., Oradell, N.J., USA, 1990, pp 819-821). For a recent review of the available antiviral drugs and their side effects, see M.C. Nahata, "Antiviral Drugs: Pharmacokinetics, Adverse Effects, and Therapeutic Use", J. Pharm. Technol., 3 , 100 (1987). Hence, safety as well as cost advantages would be realized if these agents were formulated in a manner which enhanced their therapeutic activity. We now have found that the antiviral activity of the nucleoside analogs can be enhanced synergistically, without concomitant enhancement of toxic effects, by combining the same with certain peptide derivatives having selective herpes ribonucleotide reductase inhibiting properties. Ribonucleotide reductase (RR) is the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. The role of RR in DNA biosynthesis has been reviewed recently by J. Stubbe, J. Biol. Chem. 265 , 5329 (1990). In 1985, T. Spector et al., Proc. Natl. Acad. Sci. USA, 82 , 4254 (1985) reported that a combination of acyclovir and a semicarbazone RR inhibitor, 2-acetylpyridine thiosemicarbazone, produced a synergistic antiherpes effect. However, the combination of acyclovir with the RR inhibitor hydroxyurea was toxic to the host cell and acyclovir combined with some related semicarbazone derivatives did not always potentiate the antiherpes activity of acyclovir. A three-way combination of acyclovir, bacitracin and an RR inhibiting nonapeptide has been reported by E.A. Cohen et al., US patent 4,795,740, January 3, 1989. Curiously, the antiherpes activity of the latter combination was indicated as being equal or less than acyclovir alone. Still other synergistic combinations containing a nucleoside analog as a component have been reported; for example: T.P. Zimmerman and G. Wolberg, European patent application 235931, published September 9, 1987 (nucleoside analogs plus nucleoside transport inhibitors); K.O. Smith, Canadian patent 1,239,093, issued July 12, 1988 (nucleoside analog plus an interferon); T. Spector et al., Proc. Natl. Acad. Sci. USA, 86 , 1051 (1989), (nucleoside analog plus RR inhibitor); T. Spector et al., US patent 4,758,572, issued July 19, 1988 (nucleoside analogs plus RR inhibitors); and T.A. Blumenkopf et al, European patent application 349,243, published January 3, 1990 (nucleoside analogs plus RR inhibitors). The combination of the present invention can be distinguished from the preceding combinations by its different composition and/or its relative lack of toxicity. Summary of the Invention Provided herein is a pharmaceutical composition for treating herpes infections in a mammal comprising a pharmaceutically or veterinarily acceptable carrier, and an effective amount of the combination of an antiviral nucleoside analog, being selected from acyclovir, vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, brovavir, fiacitabine, penciclovir, famciclovir and rociclovir, or a therapeutically acceptable salt thereof, and a ribonucleotide reductase inhibiting peptide derivative being selected from: PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-{(S)-NHCH[CH 2 C(CH 3 ) 3 ]CH 2 OH}, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH, [4-(Boc-NH)-Phe]CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, (4-NH 2 -Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-NH 2 , (4-NH 2 -Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(N-Me-piperazino)-Asp(cyPn)-Leu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyBu)-Leu-OH, and PhCH 2 CH 2 CO-N-Me-Val-Tbg-NHCH(2-cyclopentyl-2-oxoethyl)-CO-Asp(cyPn)-Leu-OH; or a therapeutically acceptable salt of any of these. The antiviral nucleoside analog employed in the combination is one which is enzymatically convertible (in vivo) to a viral DNA polymerase inhibitor of, and/or an alternative substrate for, a herpes DNA polymerase. Included within the scope of the invention is a cosmetic composition comprising a herpes viral prophylactic amount of the combination of an antiviral nucleoside analog as defined herein above, or a therapeutically acceptable salt thereof; a ribonucleotide reductase inhibiting peptide derivative as defined herein or a therapeutically acceptable salt thereof; and a physiologically acceptable carrier. According to a further aspect, the present invention provides a composition comprising a nucleoside analog, or a therapeutically acceptable salt thereof, and a ribonucleotide reductase inhibiting peptide derivative as hereinbefore defined, or a therapeutically acceptable salt thereof, in a form suitable for simultaneous or sequential administration in the treatment of herpes viral infections in a mammal. For the treatment or prophylaxis of herpes viral infections in a mammal an effective amount of a combination of an antiviral nucleosiede analog and the ribonucleotide reductase inhibiting peptide derivative or a therapeutically acceptable salt thereof is administered thereto. Brief Description of the Drawing Figure 1 is an isobologram showing the synergistic inhibition of HSV-2 replication by the peptide of formula 1, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-{(S)-NH-CH[CH 2 C(CH 3 ) 2 ]CH 2 OH} and acyclovir. The concentration of the peptide was varied and the inhibition of virus replication was assessed. The FIC60 (acyclovir) is the ratio of the concentration of acyclovir required to inhibit virus replication by 60% in the presence of a given concentration of the peptide. The x axis is the ratio of a given concentration of the peptide to the concentration of the peptide producing 60% inhibition of virus replication in the absence of acyclovir. Details of the Invention The antiviral nucleoside analogs, and their therapeutically acceptable salts, for use according to the present invention are a well known class of compounds. As noted above, the members of this class are characterized by the manner in which they mediate an antiviral effect against herpes viruses, i.e. by in vivo inhibition of viral DNA polymerase. Important members of this class are acyclovir and its analogs which are described by H.J. Schaeffer in US patent 4,199,574, issued April 22, 1980; see also H.J. Schaeffer et al., Nature (London), 272 , 583 (1978) and T.A. Krenitsk et al., Proc. Natl. Acad. Sci. USA, 81 , 3209 (1984). Other antiviral nucleosides contemplated for use according to the present invention include vidarabine (9-β-D-arabinofuranosyladenine monohydrate), see R.J. Whitley et al., N. Engl. J. Med., 307 , 971 (1982); idoxudine (2'-deoxy-5-iodouridine), see W.H. Prusoff, Biochim. Biophys. Acta, 32 , 295 (1959); trifluridine [2'-deoxy-5-(trifluoro-methyl)-uridine], see C. Heidelberger, US patent 3,201,387, issued August 17, 1965; ganciclovir 9- [(1,3-dihydroxy-2-propoxy)methyl]guanine, see J.P. Verheyden and J.C. Martin, US patent 4,355,032, issued October 19, 1982; edoxudine (5-ethyl-2'-deoxyuridine), see K.K. Gauri, US patent 3,553,192, issued January 5, 1971; brovavir [(E)-5-(2-bromovinyl)-2'-deoxyuridine], see Y. Benoít et al., Eur. J. Pediatrics, 143 , 198 (1985); fiacitabine (2'-fluoro-deoxy-5-iodouridine), see B. Leyland-Jones et al., J. Infect. Dis., 154 , 430 (1986), penciclovir (9-[4-hydroxy-3-(hydroxymethyl)butyl]guanine, see S.E. Fowler et al., Br. J. Clin. Pharmacol., 28 , 236P (1989); famciclovir (9-[4-acetoxy-3-(acetoxy-methyl)butyl]adenine, see R.A.V. Hodge et al., Antimicrob. Agents Chemotherap., 33 , 1765 (1989); and rociclovir (9-[(1,3-diisopropoxy-2-propoxy)methyl]adenine, see E. Winklemann et al., Arzneim.-Forsch., 38 , 1545 (1988). For convenience, the RR inhibiting peptide derivatives of this invention are sometimes designated hereinafter as the peptides. With reference to the peptides the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature, see European Journal of Biochemistry, 138 , 9 (1984). For instance, Gly, Val, Thr, Ala, Ile, Asp, Ser and Leu, represent the residues of glycine, L-valine, L-threonine, L-alanine, L-isoleucine, L-aspartic acid, L-serine and L-leucine, respectively. The asymmetric carbon atoms residing in the principal linear axis (i.e. the backbone) of the peptides of formula 1, exclusive of the terminal groups, have an S configuration. Asymmetric carbon atoms residing in the side chain of an amino acid or derived amino acid residue, including those in terminal groups, may also have the R configuration. Furthermore, with respect to disubstituted benzoyl and disubstituted phenyl(1-10C)alkanoyl as defined for X of peptides of formula 1, the substituents are selected on the basis that they do not interfere with each others presence. The term "residue" with reference to an amino acid or amino acid derivative means a radical derived from the corresponding α-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the α-amino group. Additional abbreviations or symbols used hereafter are: Boc 1,1-dimethylethoxycarbonyl or tertiary-butyloxycarbonyl Ph phenyl PhCH 2 CH 2 CO 1-oxo-3-phenylpropyl N-Me-Val N-methylvalyl residue Tbg 2(S)-amino-3,3-dimethylbutanoic acid residue N-Me-Tbg 2(S)-methylamino-3,3-dimethylbutanoic acid residue Asp(cyBu) (S)-α-amino-1-carboxycyclobutaneacetic acid residue Asp(cyPn) (S)-α-amino-1-carboxycyclopentaneacetic acid residue Asp(diMe) 2(S)-amino-3,3-dimethylbutanedioic acid residue γMeLeu 2(S)-amino-4,4-dimethylpentanoic acid residue The ribonucleotide reductase (RR) inhibiting peptides and their therapeutically acceptable salts, are prepared by processes described by J. Adams et al., in Canadian patent application SN 605,091, filed July 7, 1989, by P.L. Beaulieu et al., in Canadian patent application SN 605,062, filed July 7, 1989, and by R. Déziel and N. Moss in Canadian patent application SN 2,019,005, filed June 14, 1990. More specifically, the peptides of formula 1 can be prepared by processes which incorporate therein methods commonly used in peptide synthesis such as classical solution coupling of amino acid residues and/or peptide fragments, and if desired solid phase techniques. Such methods are described, for example, by E. Schröder and K. Lübke, "The Peptides", Vol. 1, Academic Press, New York, N.Y., 1965, pp 2-128, in the textbook series, "The Peptides: Analysis, Synthesis, Biology", E. Gross et al., Eds., Academic press, New York, N.Y., 1979-1987, Volumes 1 to 8, and by J.M. Stewart and J.D. Young in "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chem. Co., Rockford, IL, USA, 1984. A common feature of the aforementioned processes for the peptides is the protection of the reactive side chain groups of the various amino acid residues or derived amino acid residues with suitable protective groups which will prevent a chemical reaction from occurring at that site until the protective group is ultimately removed. Usually also common is the protection of an α-amino group on an amino acid or a fragment while that entity reacts at the carboxy group, followed by the selective removal of the α-amino protective group to allow subsequent reaction to take place at that location. Usually another common feature is the initial protection of the C-terminal carboxyl of the amino acid residue or peptide fragment, if present, which is to become the C-terminal function of the peptide, with a suitable protective group which will prevent a chemical reaction from occurring at that site until the protective group is removed after the desired sequence of the peptide has been assembled. In general, therefore, the peptides can be prepared by the stepwise coupling in the order of the sequence of the peptide of the amino acid or derived amino acid residues, or fragments of the peptide, which if required are suitably protected, and eliminating all protecting groups, if present, at the completion of the stepwise coupling to obtain the peptide . The term "pharmaceutically acceptable carrier" as used herein means a non-toxic, generally inert vehicle for the active ingredients, which does not adversely affect the active ingredients. The term "physiologically acceptable carrier" as used herein means an acceptable cosmetic vehicle of one or more non-toxic excipients which do not react with or reduce the effectiveness of the active ingredients contained therein. The term "veterinarily acceptable carrier" as used herein means a physiologically acceptable vehicle for administering drug substances to domestic animals comprising one or more non-toxic pharmaceutically acceptable excipients which do not react with the drug substance or reduce its effectiveness. The term "effective amount" means a predetermined antiviral amount of the antiviral agent, i.e. an amount of the agent sufficient to be effective against the viral organisms in vivo. The term "synergistic effect" when used in relation to the antiviral or antiherpes activity of the above defined combination of the nucleoside analog and peptide means an antiviral or antiherpes effect which is greater than the predictive additive effect of the two individual components of the combination. The antiviral activity of the combination of this invention can be demonstrated by biochemical, microbiological and biological procedures showing the inhibitory effect of the combination on the replication of HSV-1 and HSV-2, and other herpes viruses, for example, varicella zoster virus (VZV), Epstein-Barr virus (EBV), equine herpes virus (EHV) and pseudorabies virus (PRV). For example, a method for demonstrating the inhibitory effect of the combination on viral replication is the cell culture technique; see, for example, T. Spector et al., Proc. Natl. Acad. Sci. USA, 82 , 4254 (1985). This method in a modified form is exemplified hereinafter. A method for demonstrating the therapeutic effect of the combination is the guinea pig model for cutaneous herpes simplex viral infections; see, for example, S. Alenius and B. Oberg, Archives of Virology, 58 , 277 (1978). When utilizing the combination of this invention for treating viral infections, the combination is administered to warm blooded animals, e.g. humans, pigs or horses, in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the nucleoside analog and the peptide, chosen route of administration, standard biological practice, and by the relative amounts of the two active ingredients to provide a synergistic antiviral effect. Preferably, the combination is administered topically. For example, the two active agents (i.e. the antiviral nucleoside analog and the peptide or their therapeutically acceptable salts) can be formulated in the form of solutions, emulsions, creams, or lotions in pharmaceutically acceptable vehicles. Such formulation can contain 0.01-1.0%, preferably 0.05-0.5%, by weight of the nucleoside analog, or a therapeutically acceptable salt thereof, and about 0.5-20%, preferably 1-10%, by weight of the peptide or a therapeutically acceptable salt thereof. In any event, the two active agents are present in the pharmaceutical composition in amounts to provide a synergistic antiherpes effect. One preferred embodiment of this invention involves an antiviral pharmaceutical composition for treating herpes viral infections of the skin or part of the oral or genital cavity. This composition comprises a combination of 0.05-1.0% by weight of the nucleoside analog acyclovir, 1-10 % by weight of a ribonucleotide reductase inhibiting peptide derivative as defined herein together with a pharmaceutically acceptable carrier. Preferred carriers in this instance are water soluble ointment bases or water-oil type emulsions. Examples of suitable excipients or carriers for the above mentioned formulations are found in standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical Sciences", 18th ed, Mack Publishing Company, Easton, Penn., 1990. The dosage of the combination of this invention will vary with the form of administration and the particular active agents chosen for the combination. Furthermore, it will vary with the particular host under treatment. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the combination. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the combination is most desirably administered at a concentration level that will generally afford antiviral effective results against herpes virus without causing any harmful or deleterious side effects. The combinations is administered topically to the infected area of the body, eg. the skin or part of the oral or genital cavity, in an amount sufficient to cover the infected area. The treatment should be repeated, for example, every four to six hours until lesions heal. Although the method of treating herpes viral infections can be most advantageously practised by administering the combination of the nucleoside analog and the peptide simultaneously in a formulation, the separate or sequential administration on a daily basis of the two active agents is also encompassed within the scope of this invention. Another embodiment of this invention comprises a cosmetic composition comprising a herpes viral prophylactic amount of the combination of this invention, together with a physiologically acceptable cosmetic carrier. Additional components, for example, skin softeners, may be included in the formulations. The cosmetic formulation of this invention is used prophylactically to prevent the outbreak of herpetic lesions. They can be applied nightly and generally contain less of the two active agents of the combination than pharmaceutical preparations. A preferred range for the amount of each of the agents in the cosmetic composition is 0.01-0.1% by weight of the nucleoside analog and 0.1 to 1% by weight of the peptide. The following examples illustrate further this invention. Solution percentages or ratios express volume to volume relationship, unless stated otherwise. Abbreviations used in the examples include; Boc: t-butyloxy-carbonyl; BOP: (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; Bzl: benzyl; CH 2 Cl 2 : methylenedichloride; DIPEA: diisopropylethylamine; DCC: N,N-dicyclohexylcarbodiimide; DMF: dimethyl formamide; Et 2 O: diethyl ether; EtOAc: ethyl acetate; EtOH: ethanol; HOBt: 1-hydroxybenzotriazole; HPLC: high performance liquid chromatography; MeOH: methanol; NMM: N-methylmorpholine; TFA: trifluoroacetic acid; THF: tetrahydrofuran. Temperatures are given in degrees centrigrade. Example 1 a) Preparation of the Intermediate Boc-Asp(NEt 2 )-OH BOP (2.20 g, 5.0 mmol) was added under N 2 to a cooled (0 °) solution of Boc-Asp-OBzl (1.90 g, 4.6 mmol) in CH 2 Cl 2 (50 mL). After 3 min NHEt 2 .HCl (0.55 g, 5.0 mmol) and DIPEA (2.4 mL, 13.8 mmol) were added. The resultant solution was stirred at 20-22 ° for 18 h. The solution was washed with 10% aqueous citric acid (2 X), 10% aqueous NaHCO 3 (2 X) and brine (2 X). The organic layer was dried (MgSO 4 ) and concentrated to give an oil. After SiO 2 chromatography of the oil using hexane-EtOAc (7:3) as the eluent, Boc-Asp(NEt 2 )-OBzl (1.55 g, 89%) was obtained as an oil. Under a N 2 atmosphere, a solution of the latter compound (1.55 g, 4.09 mmol) in MeOH (100 mL) was mixed with 5% Pd/C (155 mg). The mixture was shaken on a Parr apparatus under H 2 (50 psi) for 90 min. The mixture was filtered through a 45 µm membrane and the filtrate concentrated to give Boc-Asp(NEt 2 )-OH (1.159, 98%) as an oil. The structure of the product was confirmed by NMR. In the same manner, corresponding N-substituted asparagine analogs were obtained by replacing NHEt 2 .HCl with the appropriate amine or amine salt (e.g. pyrrolidine hydrochloride or piperidine hydrochloride). (b) Preparation of the Intermediate Boc-2(S)-Amino-4-oxo-undecanoic Acid Boc-Asp-OBzl (500 mg, 1.55 mmol) was dissolved in acetonitrile (10 mL) and N,N'-carbonyldiimidazole (277 mg, 1.71 mmol) was added to the solution. After 30 min, p-nitrobenzyl-magnesium malonate (860 mg, 1.71 mmol) was added and the mixture was stirred at room temperature (20-22°) for 1.5 h. The acetonitrile was evaporated. The residue was dissolved in EtOAc, washed with 1N aqueous HCl, water and then brine. The organic phase was dried (MgSO 4 ) and concentrated under reduced pressure. The resulting residue was purified by chromatography (SiO 2 , eluent: hexane-EtOAc) to give Boc-2(S)-amino-4-oxo-1,6-hexanedioic acid 1-benzyl ester 6-(4-nitrophenyl)methyl ester (600 mg, 80%). The latter compound (3.25 g, 6.5 mmol) was dissolved in DMF (40 mL). Cs 2 CO 3 (2.33 g, 7.14 mmol) and hexyl iodide (1.51g, 7.14 mmol) were added to the solution. The mixture was stirred at room temperature for 18 h. The solvent was evaporated. The residue was dissolved in EtOAc. The solution was washed with 1N aqueous HCl and H 2 O, dried (MgSO 4 ) and evaporated. The residue was purified by chromatography (SiO 2 , eluent = hexane-EtOAc) to give Boc-2(S)-amino-4-oxo-5-[(4-nitrophenyl)methoxycarbonyl]undecanoic acid benzyl ester (630 mg). A solution of the latter compound (630 mg) in MeOH (25 mL) was shaken on a Parr apparatus under an atmosphere of H 2 in the presence of 20% Pd(OH) 2 /C (70 mg) for 18 h. After filtration and concentration of the reaction mixture, the resulting residue was dissolved in EtOAc. The solution was stirred with 1N aqueous HCl for 10 min. The organic phase was separated, washed with H 2 O, dried (MgSO 4 ) and evaporated. The residue was purified by chromatography (SiO 2 , eluent: hexane-EtOAc) to give the title compound (160 mg). NMR and MS of the product were in agreement with the expected structure. (c) Preparation of the Intermediate Boc-2(S)-Amino-5-cyclopentyl-4-oxocyclopentanoic Acid Boc-2(S)-amino-4-oxo-1,6-hexanedioic acid 1-benzyl ester 6-(4-nitrophenyl)methyl ester (4.8 g, 9.6 mmol) was dissolved in DMF (100 mL). Na 2 CO 3 (4.07g, 38.4 mmol) and 1,4-diiodobutane (3.59 g, 11.6 mmol) were added to the solution. The mixture was stirred 18 h at room temperature and then heated at 50° for 3 h. Evaporation of the reaction mixture, extraction of the resulting residue with EtOAc, washing of the extract with 1N aqueous HCl and water, drying (MgSO 4 ) and evaporation of the extract gave a crude product. The crude product was purified by chromatography (SiO 2 , eluent: hexane-EtOAc) to give the corresponding benzyl ester of the title compound (4.3g). The benzyl ester of the latter compound was subjected to hydrogenolysis [5% Pd(OH) 2 /C in MeOH, 18 h] and worked up (see section (a) of this example) to give the title compound (140 mg). NMR and MS of the product were in agreement with the expected structure. Example 2 Preparation of (S)-α-Amino-1-carboxy-cycloalkylacetic Acid Intermediates These intermediates, which can be used to prepare peptides of formula 1 in which R 5 and R 6 are joined to form a lower cycloalkyl can be prepared according to the method of M. Bochenska and J.F. Biernat, Rocz. Chem., 50 , 1195 (1976); see Chem. Abstr., 86 , 43990r (1977). More specifically exemplified, (±)-Boc-Asp(cyPn)(OBzl)-OH was prepared as follows: To a solution of 1-bromocyclopentane-carboxylic acid ethyl ester [17.1 g, 77.3 mmol, described by D.N. Harpp et al., J. Org. Chem., 46 , 3420 (1975)] and freshly distilled ethyl isocyanoacetate (12.7 g, 122 mmol) in a mixture of dimethylsulfoxide and Et 2 O (1:1, 120 mL) was added sodium hydride (4.5 g, 60% dispersion in mineral oil, 122 mmol) in small portions over 5 h. The resulting red slurry was stirred at room temperature for 16 h after which time it was treated with a saturated aqueous solution of ammonium chloride (5 mL). The mixture was diluted with water (500 mL). The resulting mixture was extracted (2X) with EtOAc. The EtOAc layers were combined and washed with water (2X) and then with brine. Drying (MgSO 4 ), filtering and concentration of the extract afforded a dark red oil. This material was subjected to flash chromatography through a 5 x 25 cm column of silica gel [eluent: EtOAc-hexane (1:10)]. Concentration of the appropriate fractions provided α-cyano-1-carboxycyclopentaneacetic acid diethyl ester as a clear colorless viscous liquid (13 g, 66 %). The latter compound (13 g, 51 mmol) was mixed with 6 N aqueous HCl (60 mL) at 0°. After dissolution, the reaction mixture was heated in a oil bath at 120° for 24 h. After this time water was removed from the mixture using a dry ice rotory evaporator. The resulting white solid was dried under high vacuum for 18 h. The dried material was dissolved in a mixture of dioxane (50 mL) and 3N aqueous NaOH (52 mL). A solution of di(tertiarybutyl) dicarbonate (14.6 g, 67 mmol) in dioxane (25 mL) was added to the solution. The mixture was stirred at room temperature for 16 h. Additional 3N aqueous NaOH was added at intervals insuring a pH of about 10. The mixture was diluted with water (500 mL) and extracted (2X) with Et 2 O (200 mL). The aqueous phase was rendered acidic (pH = 3) with solid citric acid and extracted (2X) with EtOAc (300 mL). The combined EtOAc extracts were washed with water (3x) and brine. Drying, filtering and concentration of the extract afforded Boc-Asp(cyPn)-OH as a white solid (14 g, 96%). To a solution of the latter compound (7.2 g, 25 mmol) in dry DMF (50 mL) was added K 2 CO 3 (7.6 g, 55 mmol) and benzyl bromide (6.6 mL, 55 mmol). The reaction mixture was stirred at room temperature for about 7 h. Thereafter, the reaction mixture was poured into a mixture of water (500 mL) and EtOAc (350 mL). The organic phase was washed with water (2X) and brine. Drying, filtering and concentration of the extract provided a pale yellow viscous liquid. This material was subjected to flash chromatography through a 5 x 20 cm column of silica gel, eluting with hexane-EtOAc (12:1). Concentration of the appropriate fractions provided the dibenzyl derivative of Boc-Asp(cyPn)-OH as a low melting white solid (11 g, 94%). The dibenzyl product was dissolved in THF (100 mL) and an aqueous solution of LiOH (23.5 mL, 1N) was added. After 4 h, the reaction mixture was poured into water and extracted (3X) with Et 2 O. The aqueous phase was rendered acidic with 10% aqueous citric acid and extracted (2X) with EtOAc. The EtOAc layers were combined, dried (MgSO 4 ), filtered and concentrated to provide Boc-Asp(cyPn)(OBzl)-OH as a clear color less gum (7.3 g, 82%). Example 3 General Procedure for the Solid Phase Preparation of Peptides of Formula 1 A modified version of the solid phase method of R.B. Merrifield, J. Am. Chem. Soc., 85 , 2149 (1963) was used to prepare the peptides preferably using a BHA-photoresin such as [4-(2-chloropropionyl)phenoxy]acetamidomethylcopoly(styrene-1% divinyl-benzene) resin, see D. Bellof and M. Mutter, Chemia, 39 , 317 (1985). Protection of free carboxy groups and hydroxy groups was provided by the Bzl protective group. Typically, a Boc-amino acid, representing the C-terminal unit of the desired peptide, e.g. Boc-Leu-OH, was linked to the above noted BHA-photoresin by the potassium fluoride method of K. Horiki et al., Chem. Lett., 165 (1978), using 9 molar equivalents of KF and 3.6 molar equivalents of Boc-Leu-OH, for example in DMF at 70° C for 24 hours, to give [4-{2-(Boc-leucyl}-propionyl)phenoxy]acetamidomethyl-copoly(styrene-1% divinylbenzene) resin. The dried amino acid-solid support typically showed a leucine content of 0.6 to 0.8 mmol/g for the product, as determined by deprotection of an aliquot, followed by picric acid titration, B.F. Gisin, Anal. Chim. Acta, 58 , 248 (1972). The latter amino acid-solid support was used to build up the required sequence of units (i.e. amino acid residues, derived amino acid residues) of the desired peptide by solid phase methodology. Two molar equivalents (per mole of the amino-acid solid support) of the appropriate amino acid residues were coupled serially to the solid support system using BOP (2 molar equivalents), or BOP (2 molar equivalents)/HOBt (1 molar equivalent), in the presence of N-methylmorpholine (6 molar equivalents) in dry DMF. Completion of coupling was verified by a negative ninhydrin test, E. Kaiser et al., Anal Biochem., 34 , 595 (1979). Double coupling was used when necessary. Cleavage of the protected peptide from the solid support was accomplished by irradiation at 330 nm in EtOH/DMF (1:4) at 0° under an argon atmosphere for 6 to 18 h. Protective groups (Bzl), if present, were removed from the cleavage product by hydrogenolysis over 5% or 10% Pd/C or 20% Pd(OH) 2 /C by standard procedures. Purification of the final product was performed by reversed-phase HPLC to better than 95% homogeneity using 0.06% aqueous TFA/0.06% TFA in acetonitrile gradients. Example 4 Preparation of PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH (Example of a solution phase procedure for preparing a peptide as herein defined) To a solution of Boc-Asp(cyPn)(OBzl)-OH (5.3 g, 14 mmol, described in example 2) in dry CH 2 Cl 2 (50 mL), BOP (6.8 g, 16 mmol), NMM (4.6 mL, 42 mmol) and the (4 methyl-phenyl)sulfonic acid salt of LeuOBzl (6.6 g, 16 mmol) were added successively. The reaction mixture was stirred at room temperature for 5 h, after which time it was poured into a two component system of EtOAc (500 mL) and a saturated aqueous solution of NaHCO 3 (400 mL). The organic phase was washed with water and brine. Drying (MgSO 4 ), filtering and concentrating of the organic phase afforded a dark yellow oil. This material was purified by flash chromatography [SiO 2 , eluent: hexane-EtOAc (6:1)] to provide Boc-Asp(cyPn)(OBzl)-Leu-OBzl as a clear colorless gum (7 g, 86%, mixture of diastereoisomers). The latter compound (7 g, 12 mmol) was mixed with CH 2 Cl 2 (4 mL). TFA (6 mL) was added to the mixture and the resulting solution was stirred for 30 min at room temperature. Thereafter, the majority of the solvent was evaporated and the residue was poured into a two component system of EtOAc (200 mL) and a saturated aqueous solution of NaHCO 3 (400 mL). Drying (MgSO 4 ), filtering, and concentrating of the organic phase afforded the amine dipeptide H-Asp(cyPn)(OBzl)-Leu-OBzl as a clear colorless gum (mixture of diastereoisomers). The mixture was separated on a Waters LC-500 [2 columns of SiO 2 , eluent: hexane-EtOAc (1.5:1)]. The first diastereomer to elute (ca 2g, white solid) proved to provide the more active end products (peptides of formula 1). This observation proved general for all corresponding cycloalkyl aspartic acid derivatives made. For convenient storage of material, the pure amine dipeptide was treated briefly with 6N HCl/dioxane and concentrated to afford the hydrochloride salt as a white foam. The latter hydrochloride salt (400 mg, 0.8 mmol) was coupled with Boc-Asp(pyrrolidino)OH (250 mg, 0.87 mmol), following the same general procedure used above for preparing Boc-Asp(cyPn)(OBzl)-Leu-OBzl. The crude product was purified by flash chromatography [SiO 2 , eluent: hexane-EtOAc (1:1)] to provide Boc-Asn(pyrrolidino)-Asp(cyPn)(OBzl)-Leu-OBzl as a white foam (530 mg, 91%). The previous material (280 mg, 0.38 mmol) was treated with 6 N HCl/dioxane (4 mL) for 30 min at room temperature. The solvent was removed and the residue was pumped under high vacuum for 18 h. The resulting white foam was coupled to Boc-Tbg-OH (1.1 eq) in essentially the same manner as was done for the previous coupling. The crude product was purified by flash chromatography [SiO 2 , eluent: hexane-EtOAc (1:1)] to provide Boc-Tbg-Asp(pyrrolidino)-Asp(cyPn)(OBzl)-Leu-OBzl as a white foam (280 mg, 85%). The latter compound was coupled to Boc-N-Me-Val-OH in the same manner as was done for the previous coupling to provide Boc-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)(OBzl)-Leu-OBzl as a white foam (86% yield). The latter compound (88 mg, 0.09 mmol) was treated with 6 N HCl/dioxane (1.5 mL) for 20 min at room temperature. The solvent was removed and the residue was pumped under vacuum for 2 h. This material was dissolved in CH 2 Cl 2 (0.8 mL) and NMM (40 L, 0.36 mmol) was added, followed by a premixed solution of 3-phenylpropionic acid (27 mg, 0.18 mmol) and BOP (80 mg, 0.18 mmol) in CH 2 Cl 2 (0.35 mL). The reaction mixture was stirred at room temperature for 16 h, after which time it was poured into EtOAc (30 mL) and a saturated aqueous solution of NaHCO 3 (20 mL). The organic phase was washed with water and brine, dried (MgSO 4 ), filtered and concentrated to afford a yellow gum. This material was purified by flash chromatography [SiO 2 , eluent: EtOAc-hexane (2:1)] to provide PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)(OBzl)-Leu-OBzl (80 mg, 90%). The latter material (80 mg, 0.08 mmol) was dissolved in MeOH (2 mL). 20% Pd(OH) 2 /C (50 mg) and ammonium formate (50 mg) were added to the mixture. The mixture was stirred under an atmosphere of hydrogen for about 5 h. The reaction mixture was filtered through diatomaceous earth and the filtrate was concentrated. The residue was dissolved in water (20 mL), (a few drops of saturated aqueous solution of NaHCO 3 were added to insure basicity). The solution was washed (2X) with Et 2 O, acidified with solid citric acid and extracted (2X) with EtOAc. The combined EtOAc extracts were dried (MgSO 4 ), filtered and concentrated to afford the title compound of this example as a white solid (55 mg, 83%). The procedures of examples 3 or 4 can be used to prepare other peptides as defined herein. Commercially available Boc-amino acids were used. Unnatural amino acids were used in their Boc protected form; they were either commercially available, readily prepared from commercially available corresponding amino acids by reaction with di-tertiary-butyl carbonate, or prepared by standard methods. Note that N-alkylated Boc amino acids are commercially available, e.g. Boc-N-methylvaline, or they can be prepared by standard N-alkylation of corresponding Boc-amino acids. For example, Boc-N-Me-Asp(NEt 2 )-OH was obtained by reacting Boc-Asp(NEt 2 )-OH with 2.5 molar equivalents of methyl iodide and 2.1 molar equivalents of potassium hydride in THF at 0° for 18 h to give a mixture of Boc-N-Me-Asp(NEt 2 )-OH and its corresponding methyl ester. The mixture was esterified fully (diazomethane) and then saponified (NaOH/H 2 O/dioxane) to yield the desired compound. Example 5 Comparison of Acyclovir, the Peptide of Formula: PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-{(S)-NHCH[CH 2 C(CH 3 ) 3 ]CH 2 OH}, and the Combination of the Two Agents in Inhibiting HSV-2 Replication in Cell Culture . BHK-21/C13 cells (ATCC CCL 10) are incubated for two days in 150 cm 2 T-flasks (1.5 x 10 6 cells/flask) with alpha-MEM medium (Gibco Canada Inc., Burlington, Ontario, Canada) supplemented with 8% (v/v) fetal bovine serum (FBS, Gibco Canada Inc.). The cells are trypsinized and then transferred to fresh media in a 24 well plate to give 2.5 x 10 5 cells in 750 µL of media per well. The cells are incubated at 37°C for a period of 6 hours to allow them to adhere to the plate. Thereafter, the cells are washed once with 500 µL of alpha-MEM supplemented with 0.5% (v/v) FBS and then incubated with 750 µL of the same media (low serum) for 3 days. After this period of serum starvation, the low serum medium is removed and the cells are incubated in 500 µL of BBMT for 2 to 3 hours. [BBMT medium is described by P. Brazeau et al., Proc. Natl. Acad. Sci. USA, 79 , 7909 (1982).] Thereafter, the cells are infected with HSV-2 (multiplicity of infection = 0.02 PFU/cell) in 100 µL of BBMT medium. (Note: The HSV-2 used was strain HG-52, see Y. Langelier and G. Buttin, J. Gen. Virol., 57 , 21 (1981); the virus was stored at -80°C.) Following 1 hour of virus adsorption at 37°C, the media is removed and the cells are washed with BBMT (3 X 250 µL). The cells in each well are incubated with or without (control) appropriate concentrations of the test agent dissolved in 200 µL of BBMT medium. After 29 hours of incubation at 37°C, the infected cells are harvested by first freezing the plate at -80°C, followed by thawing. The cells in each well are scraped off the surface of the well with the help of the melting ice fragments. After complete thawing, the cell suspensions are collected and each well is rinsed with 150 µL of BBMT medium. The viral sample (suspension plus washing) is sonicated gently for 4 minutes at 4°C. Cell debris are removed by centrifugation (1000 times gravity for 10 minutes at 4°C). The supernatant is collected and stored at -80°C until determination of viral titer. Viral titration was performed by a modification of the colorimetric assay method of M. Langlois et al., Journal of Biological Standardization, 14 , 201 (1986). More specifically, in a similar manner as described above, BHK-21/C13 cells are trypsinized and transferred to fresh media in a 96 well microtiter plate to give 20,000 cells in 100 µL of media per well. The cells in the prepared plate are incubated at 37°C for 2 hours. During that time, the viral sample is thawed and sonicated gently for 15 seconds, and log dilutions of the sample are prepared (1/5 sequential: 50 µL of the sample plus 200 µL of BBMT medium, sequential dilutions being done with a multichannel pipette. On completion of the above 2 hour incubation of the BHK-21/C13 cells, the media is replaced with alpha-MEM medium supplemented with 3% (v/v) FBS. The cells are now ready to be infected with the various sample dilutions of virus. Aliquots (50 µL) of the various dilutions are transferred into the appropriate wells of the plate. The resulting infected cells are incubated for 2 days at 37°C. Then 50 µL of a 0.15% (v/v) solution of neutral red dye in Hank's Balanced Salt Solution (pH 7.3, Gibco Canada Inc.) is added to each well. The prepared plate is incubated for 45 minutes at 37°C. Medium from each well is then aspirated and the cells are washed once with 200 µL of Hank's Balanced Salt Solution. After the wash, the dye is released from the cells by the addition of 100 µL of a 1:1 mixture of 0.1 M Sorensen's citrate buffer (pH 4.2) and ethanol. [Sorensen's citrate buffer is prepared as follows: Firstly, a 0.1 M disodium citrate solution is prepared by dissolving citric acid monohydrate (21 g) in 1 N aqueous NaOH (200 mL) and adding sufficient filtered H 2 O to make 1 L. Secondly, the 0.1 M disodium citrate solution (61.2 mL) is mixed' with 0.1 N aqueous HCl (38.8 mL) and the pH of the resulting solution is adjusted to 4.2 if necessary.] The mixture in the wells is subjected to a gentle vortex action to ensure proper mixing. The plate wells are scanned by a spectrophotometer plate reader at 540 mM to assess the number of viable cells. In this manner, the percentage of virus growth inhibition can be determined for the various concentrations of the test agent, and the concentration of the test agent causing a 50% inhibition of virus replication, i.e. the IC 50 can be calculated. The following table is illustrative of the results obtained when acyclovir and the title peptide were evaluated according to the assay procedure of this example. COMPOUND RANGE OF SAMPLE CONCENTRATIONS EVALUATED IC 50 (µM) acyclovir 0.1 to 30 µM 1.8 peptide 18.75 to 300 µM 80 acyclovir +20 µM of peptide 0.005 to 30 µM 0.7 acyclovir +30 µM of peptide 0.005 to 30 µM 0.5 acyclovir +40 µM of peptide 0.005 to 30 µM 0.11 acyclovir +50 µM of peptide 0.005 to 30 µM 0.09 The synergism of the combination of acyclovir and the peptide can further be demonstrated by applying the isobole method to the above results, see J. Sühnel, Antiviral Research, 13 , 23 (1990) and references therein. The positive result obtained in the application of this method is illustrated graphically in the accompanying Figure 1. By following the procedure of this example, synergism was demonstrated also for combinations of acyclovir and PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, and of vidarabine and the title peptide. Examples of other specific peptides for use according to the present invention are: PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH (of example 4) [4-(Boc-NH)-Phe]CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH (4-NH 2 -Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-NH 2 (4-NH 2 -Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(N-Me-piperazino)-Asp(cyPn)-Leu-OH PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyBu)-Leu-OH PhCH 2 CH 2 CO-N-Me-Val-Tbg-NHCH(2-cyclopentyl-2-oxoethyl)-CO-Asp(cyPn)-Leu-OH Still other examples of peptides of formula 1 include: PhCH 2 CH 2 CO-N-Me-Val-Ile-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-NH 2 (4-MeO-Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH PhCH 2 CH 2 CO-N-Me-Tbg-Tbg-Asp(NEt 2 )-Asp(diMe)-Leu-OH Me 2 CHCH 2 CH 2 CO-N-Me-Tbg-Val-Asp(NMe 2 )-Asp(cyBu)-Leu-NH 2 (4HO-Phe)CH 2 CH 2 CO-N-Me-Val-Ile-Asp(piperazino)-Asp(cyBu)-Leu-OH

Claims: A pharmaceutical composition for treating herpes infections in a mammal comprising a pharmaceutically or veterinarily acceptable carrier, and an effective amount of the combination of an antiviral nucleoside analog, being selected from acyclovir, vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, brovavir, fiacitabine, penciclovir, famciclovir and rociclovir, or a therapeutically acceptable salt thereof, and a ribonucleotide reductase inhibiting peptide derivative being selected from: PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-{(S)-NHCH[CH 2 C(CH 3 ) 3 ]CH 2 OH}, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH, [4-(Boc-NH)-Phe]CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, (4-NH 2 -Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-NH 2 , (4-NH 2 -Phe)CH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-Leu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(N-Me-piperazino)-Asp(cyPn)-Leu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyPn)-γMeLeu-OH, PhCH 2 CH 2 CO-N-Me-Val-Tbg-Asp(pyrrolidino)-Asp(cyBu)-Leu-OH, and PhCH 2 CH 2 CO-N-Me-Val-Tbg-NHCH(2-cyclopentyl-2-oxoethyl)-CO-Asp(cyPn)-Leu-OH; or a therapeutically acceptable salt of any of these. A composition as claimed in claim 1 wherein the amount of the nucleoside analog, or a therapeutically acceptable salt thereof, is 0.01-1.0 % by weight of the composition, and the amount of the peptide or a therapeutically acceptable salt thereof, is 0.5-20 % by weight of the composition. A composition as claimed in claim 1 comprising 0.05-1.0% by weight of acyclovir and 1-10% by weight of the peptide as defined in claim 1. As cosmetic composition comprising a herpes viral prophylactic amount of a combination of acyclovir or a therapeutically acceptable salt thereof, and a ribonucleotide reductase inhibiting peptide as defined in claim 1, or a therapeutically acceptable salt thereof, and a physiologically acceptable carrier. A composition as claimed in any one of claims 1 to 4 in a form suitable for topical application. A composition as claimed in any one of claims 1 to 5 for treating herpes simplex virus type 1, or type 2 infections in a mammal.

Inventors: DEZIEL ROBERT, GUINDON YVAN, DEZIEL, ROBERT, GUINDON, YVAN, Déziel, Robert
Applicants: BOEHRINGER INGELHEIM CA LTD, BOEHRINGER INGELHEIM (CANADA) LTD.
Application year: 1991
Main classification: A61K38
IPC codes: A61Q19, A61K8, A61K31, A61K38, C07K7, A61P43, A61P31, C07K14