HUT73779A - Method of combating acyclovir-resistant herpes simplex viral infections - Google Patents

Description

A method for controlling acyclovir-resistant Herpes simplex virus infections

BIO-MEGA / BOEHRINGER INGELHEIM RESEARCH INC., QUEBEC, CA

Date of filing: 29.4.1994

Priority 03/03/1993 (2,095,408) CA.

International Application Number: PCT / CA94 / 00242

International Publication Number: WO 94/25046

The present invention relates to a method for combating acyclovir-resistant herpes infections in a mammal by a peptide derivative of formula (I) wherein

The various substituted arylalkanoyl or N-benzylcarbamoyl groups,

B (N-Me) Val or (N-Me) Ile or

A and B together form different N- (saturated alkyl) carbamoyl groups,

D Val, Ile or TBg,

R ^{1 is C} 3 -C 6 branched or cyclic alkyl or -NR ⁴ R ^{3 in} which R ⁴ and R ^{5 are} hydrogen or lower alkyl, or R ⁴ and R ³ together with the nitrogen atom form one to six membered ring amines .

R ^{2 is} hydrogen,

R ^{3 is a C} 1-7 straight or branched, saturated or unsaturated alkyl or benzyl group, or

R ² and R ³ together with the carbon atom to which they are attached form a C 4 -C 6 saturated cycloalkyl group,

E is -NHR ⁸ wherein R ^{8 is C} 4 -C ⁸ branched or cyclic saturated alkyl, or

E is -NHCH (R ⁷ ) -Z in which

R ^{7 is C} 4 -C ⁷ alkyl,

Z is hydroxymethyl, carboxy, carbamoyl or -C (O) OR ⁸ wherein

R ^{8 is} a combination of a C 1-3 alkyl group and a nucleoside analog.

The peptide derivatives of formula (I) or a therapeutically acceptable salt thereof and a suitable nucleoside analogue or a therapeutically acceptable salt thereof potentiate one another and the combination preparations prepared therefrom are effective against various acyclovir-resistant herpes simplex viruses.

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Patent Office

H-1062 Budapest. Andrássy ut113. Phone: 34-24-950, Fax '. 34-24-3J3

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313 5 <95 PUBLISHING MANUAL

A method for controlling acyclovir-resistant Herpes simplex virus infections

BIO-MEGA / BOEHRINGER INGELHEIM RESEARCH INC., QUEBEC, CA

inventors:

CHAFOULEAS, James, Gus, QUEBEC, CA

Róbert DEZIEL, QUEBEC, CA.

Date of filing: 29.4.1994

Priority 03/03/1993 (2,095,408) CA.

International Application Number: PCT / CA94 / 00242

International Publication Number: WO 94/25046 · · · · · · · · ·

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The present invention relates to a method for treating acyclovir-resistant herpes infections in mammals. The process involves the use of a combination of a peptide derivative or a peptide derivative and an antiviral nucleoside analog.

Acyclovir is the most widely used drug for the treatment of herpes infections. However, the incidence of acyclovir-resistant herpes infections has increased in recent years; for example, two publications refer to P.A. Chatis et al., N. Eng. J. Med. 320, 297 (1989) and S. Safrin, Res. Virol., 143, 125 (1992).

This type of resistant infection has recently been very frequently studied in patients with severe human immunodeficiency virus (HIV) infection. Due to their impaired immune system, these latter patients are highly susceptible to infections caused by herpes simplex virus type 1, and especially herpes simplex virus type 2 (HSV).

Considerable attention has been paid to the biochemical characteristics that characterize acyclovir-resistant HSV isolates; this is illustrated, for example, by the following summary publication on acyclovir-resistant HSV: C.S. Crumpacker, J. Am. Acad. Dermatol. , 18, 190 (1988). Essentially, resistant strains show functional changes in one or two enzymes encoded by the virus, namely thymidine kinase (TK) and DNA polymerase (POL).

By way of example, when cells infected with wild-type HSV are exposed to acyclovir, viral TK catalyses acyclovir monophosphorylation to a much greater extent than cellular enzymes. The monophosphate formed is then converted by the cellular enzymes to triphosphorylated acyclovir. The latter triphosphate reacts much more readily with viral DNA polymerase than with cellular DNA polymerase. Consequently, it is able to reduce viral DNA synthesis as it represents a variable substrate for viral enzymes and acts as a chain terminator.

Two mechanisms of resistance to viral thymidine kinase have been described:

(1) selection of mutants deficient in thymidine kinase, which results in only very low enzyme activity being induced after infection; and (b) selection of mutants with altered substrate specificity that are capable of phosphorylating thymidine but not aciclovir.

The third mechanism of resistance to DNA polymerase is the selection of mutants that encode an altered enzyme and, consequently, are resistant to acyclovir triphosphate inactivation.

Based on the above resistance mechanisms, acyclovir-resistant HSV isolates can be classified as thymidine deficient (TK ^D ) strains, altered thymidine kinase (TKII) producing strains, or altered DNA polymerase (POL ^A ) producing strains.

Although acyclovir-resistant HSV infection has been successfully treated with antiviral foscarnet in a number of cases, cases of acyclovir-resistant infections are becoming more common. This is a particularly widespread problem for AIDS patients; see

for example, K.S. Erlich et al., N.

S. Safrin, J. Acquired Immun.

S29 (1992). It is therefore significant

Eng. J-Med. 320, 293 (1989) and

Defic, Syndr., 5, (Suppl. 1), there is a need for these problems to be a relatively safe method of treating herpes infections.

R. Déziel, N. Moss, and R. (filed March 1993, pepper for use in compounds against herpes infections not necessarily against simplex virus;

Campoli-Richards, Drugs, 37, to resolve.

The invention is effective in providing acyclovir resistance

The procedure is described in P.L. International Patent Application No. PCT / CA / 93/0095, issued by Beaulieu Plante, discloses the use of tide derivatives for the treatment of herpes infections. However, it is known that an antiviral drug effective against acyclovir-resistant herpes can be found, for example, in J.J. O'Brien and D.M.

233 (1989), p. 252-253. Therefore, it has been surprising and successful to observe that the peptide derivatives of the invention are effective against acyclovir-resistant herpes simplex virus.

The present invention relates to a method for treating acyclovir-resistant herpes simplex virus infections in mammals. The method comprises treating a mammal with an effective amount of a peptide derivative of formula (I) or a therapeutically acceptable salt thereof, against acyclovir-resistant herpes, wherein:

A is phenylacetyl, phenylpropionyl, (4-aminophenyl) propionyl, (4-fluorophenyl) propionyl, (4-hydroxyphenyl) propionyl, (4-methoxy) phenyl) -propionyl, 2-benz-5 - ........

zyl-3-phenylpropionyl, 2- (4-fluorobenzyl) -3- (4-fluorophenyl) propionyl, 2- (4-methoxybenzyl) -3- (4-methoxyphenyl) ) -propionyl or N-benzylcarbamoyl,

B is (N-Me) -Val or (N-Me) -le, or

A and B together form a saturated N-alkylcarbamoyl group selected from the group consisting of N-butylcarbamoyl, N- (2-propyl) -carbamoyl, N-isobutylcarbamoyl, N- (1-ethyl- propyl) -carbamoyl, N- (1,1-dimethyl-butyl) -carbamoyl, N- (1-ethyl-butyl) -carbamoyl, N- (1-propyl-butyl) -carbamoyl, N- ( 1-ethyl-pentyl) -carbamoyl, N- (1-butyl-pentyl) -carbamoyl, N- (1-ethyl-butyl) -carbamoyl, N- (2-ethyl-pentyl) -carbamoyl, N - (1-methyl-1-propyl-butyl) -carbamoyl, N- (1-ethyl-1-propyl-butyl) -carbamoyl, N- (1,1-dipropyl-butyl) -carbamoyl, N- (1-propylcyclopentyl) carbamoyl and N- (1-propylcyclohexyl) carbamoyl,

D is Val, lle or Tbg,

R ¹ is isopropyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl or is a group of the formula -NR ⁴ r 5 in which

R ⁴ is hydrogen or lower alkyl,

^R5 is lower alkyl, or

R ⁴ and R ² together with the nitrogen atom to which they are attached form pyrrolidino, piperidino, morpholino or 4-methylpiperazino,

R ² is hydrogen, and

R ³ is methyl, ethyl, isopropyl, tert-butyl, propyl, allyl or benzyl and the carbon atom to which R ² and R ^{3 are} attached is in the (R) configuration, or

R ² and R ³ are independently methyl or ethyl, or

R ² and R ³ together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl group,

E is a group of the formula -NHR ^{6 in} which

R 4 is isobutyl, neopentyl, 1 (R), 2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1 (R) -ethyl-2,2-dimethylpropyl -, 2- (R, S) -methyl-butyl, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 1 (R), 2,2-trimethyl-butyl, 1 (R ), 3,3-trimethyl-butyl, 2-ethyl-butyl, 2,2-diethyl-butyl, 2-ethyl-1 (R) -methyl-butyl, 2-ethyl-2-methyl-butyl -, 1 (R) -ethyl-3,3-dimethyl-butyl, 2,2-dimethyl-pentyl, cis or trans-2-methyl-cyclohexyl, 2,2-dimethyl-cyclohexyl or cyclohexyl- or methyl

E is a group of the formula -NHCH (R ⁷ ) -Z in which the carbon atom bearing R ⁷ is of the (S) -configuration;

R ⁷ is tert-butyl, sec-butyl, neopentyl or cyclohexylmethyl, and

Z is hydroxymethyl, carboxy, carbamoyl, or a group -C (O) OR (R)

t

R ⁸ is methyl, ethyl or propyl.

A preferred method of the present invention is the treatment of acyclovir-resistant herpes simplex infections by administering the peptides of formula (I) or a therapeutically acceptable salt thereof wherein:

A is phenylpropionyl, 2-benzyl-3-phenylpropionyl or N-benzylcarbamoyl,

B is (N-Me) -Val,

D is Tbg,

R 1 is isopropyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl or 1-methylcyclopentyl group,

R ² is hydrogen,

R ³ is methyl, ethyl, isopropyl, propyl or benzyl and has the carbon atom of (R) configuration bearing the substituents R ² and R ³ , or

R ² and R ³ are independently methyl or ethyl, or

R ² and R ³ together with the carbon to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl group,

E is a group of formula NHR ^{6 in} which

R ⁸ is 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethyl-propyl, 2,2-dimethyl-butyl- or 1 (R) -ethyl-3,3-dimethyl-butyl, or

E is -NHCH (R ⁷ ) -Z,

__ 'γ in which the carbon atom bearing the R substituent is in the (S) configuration, and

R ⁷ is 2,2-dimethylpropyl, and

Z is hydroxymethyl, carboxy, carbamoyl or a group -C (O) OR ⁸ wherein

R ⁸ is methyl, ethyl or propyl.

Another preferred method of treating acyclovir-resistant herpes simplex infections is the administration of a peptide derivative of formula (I) or a therapeutically acceptable salt thereof, wherein:

A and B together form a saturated N-alkylcarbamoyl group selected from the group consisting of N- (1-ethylpropyl) carbamoyl, N- (1-ethylbutyl) carbamoyl, N- (1-propyl) -butyl) -carbamoyl, N- (2-ethyl-pentyl) -carbamoyl, N- (1-methyl-1-propyl-butyl) -carbamoyl, N- (1-ethyl-1-propyl-butyl) -carbamoyl, N- (1,1-dipropyl-butyl) -carbamoyl and N- (1-propyl-cyclopentyl) -carbamoyl,

D, r 1, R ² , R ³ and E are as defined above.

Another embodiment of the present invention is the treatment of acyclovir-resistant herpes viral infections in mammals by combining an effective amount of a combination of acyclovir-resistant herpes infections, a combination of a peptide derivative of formula (1) or a therapeutically acceptable salt thereof and an antiviral is formed from its acceptable salt.

Among the antiviral nucleoside analogs, they are suitable

Such a combination may be enzymatically (in vivo) converted to a viral DNA polymerase inhibitor and / or a modified substrate for the herpesvirus DNA polymerase enzyme. The antiviral nucleoside analogs may be selected from known nucleoside analogs. Preferred nucleoside analogues according to the invention are acyclovir and its analogs, for example, compounds of formula (2)

R ⁹ is hydrogen, hydroxy or amino, or therapeutically acceptable salts of these compounds. [Formula 2 represents the formula for acyclovir when R ⁹ is hydroxy.]

Other preferred antiviral nucleoside analogues for use in the present invention include vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudin, brovavir, fiacitabine, penciclovir, famciclovir and rociclovir.

Figures 1 and 2 graphically illustrate the results obtained by the isobol method for demonstrating synergism in an experiment investigating the efficacy of a combination of acyclovir and a peptide derivative of formula (1) against acyclovir-resistant herpes simplex viruses.

The term "residue" as used in reference to an amino acid or an amino acid derivative means a residue obtained when the hydroxy group of the carboxy group and one of the hydrogen atoms of the amino group at the α-position are removed.

Generally, the abbreviations we use to denote amino acids and protecting groups are based on the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [see

Journal of Biochemistry 138: 9 (1984)]. For example, Val, Ile, Asp and Leu represent residues of L-valine, L-isoleucine, L-aspartic acid and L-leucine.

Asymmetric carbon atoms in the main linear axis of the peptide derivatives of formula (I) (i.e., in the peptide backbone), except for the Z terminal groups in A and E, but including the carbon bearing R ⁷ , when E is NHCH ( R ⁷ ) -Z are all of the (S) configuration. Except, however, is a carbon atom bearing a -CH ₂ C (O) R ¹ side chain, wherein R ¹ is lower alkyl and lower cycloalkyl, as defined above. With the latter exception, the carbon atom has the (R) configuration.

Asymmetric carbon atoms in the side chain of an amino acid or amino acid derivative, in the terminal group A and in the terminal group E, when E is the group -NHR ⁶ as defined above, may have the configuration (S) or (R).

The symbols Me, Et, Pr and Bu represent the methyl, ethyl, propyl and butyl groups.

The MeEt2C and EtPr ₂ C symbols include, for example, 1-ethyl-1-methylpropyl and 1-ethyl-1-propyl butyl.

The Tbg symbol represents the amino acid residue of (S) -2-amino-3,3-dimethylbutanoic acid. ^ MeLeu represents the amino acid residue of (S) -2-amino-4,4-dimethylpentanoic acid. The MeLeucinol symbol denotes the residue of (S) -2-amino-4,4-dimethylpentanol in the α-amino group less than one hydrogen atom.

Other symbols used herein include: (N-Me) Val the residue of (S) -3-methyl-2- (methylamino) butanoic acid; (N-Me) Ile is the residue of (S) -3-methyl-2- (methylamino) pentanoic acid; (N-Me ₃ ) Tbg is the residue of (S) -2- (methylamino) -3,3-dimethylbutanoic acid; Asp (cyBu) is the residue of (S) -α-amino-1-carboxycyclobutane acetic acid; and finally, Asp (cyPn) represents the residue of (S) -α-amino-1-carboxycyclopentane-acetic acid.

The term "lower alkyl" as used herein, alone or in combination with other radicals, means straight-chained alkyl radicals having from 1 to 6 carbon atoms and branched alkyl radicals having from 3 to 6 carbon atoms and includes the following groups: methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl and 1,1-dimethylethyl (tert-butyl).

As used herein, the term 1- (lower alkyl) - (lower cycloalkyl) means a lower cycloalkyl radical substituted at position 1 with a lower alkyl group; for example, 1-ethylcyclopropyl, 1-propylcyclopentyl and 1-propylcyclohexyl.

The term lower cycloalkyl, alone or in combination with other radicals, means a saturated cyclic hydrocarbon radical containing from 3 to 6 carbon atoms; this includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl radicals.

• ·

The term pharmaceutically acceptable carrier, as used herein, is a non-toxic, generally inert carrier for active ingredients that does not exert a deleterious (adverse) effect on the active ingredient.

The term physiologically acceptable carrier, as used herein, is an acceptable cosmetic carrier of one or more non-toxic excipients which does not react with the active ingredient in admixture or diminish its efficacy.

The term effective amount refers to a predetermined amount of an antiviral agent having a predetermined antiviral effect, i.e., an amount of the agent that is sufficiently effective against viruses in vivo.

The term "coupling agent" as used herein refers to an agent (reagent) capable of dehydrating the free carboxyl group of an amino acid or peptide to a free amino group of another amino acid or peptide, thereby forming an amide bond between the two reactants. Similarly, such an agent is capable of promoting the coupling of an acid and an alcohol to form the corresponding ester. The agent accelerates or promotes dehydrative coupling by activating the carboxy group. Such linking agents and activated moieties are generally described in the peptide chemistry manuals; for example, E. Schroeder and K.L. Lübke, The Peptides, Vol. 1, Academic Press, New York, N.Y., 1965, pp. 2-128, and K.D. Kopple, Peptides and Amino Acids, W.A. Benjamin, Inc. New York, N.Y., 1966, pp. 33-51. Such coupling agents include, for example, diphenyl-? -? -? -? -? -? -? -? 1-carbonyldiimidazole, dicyclohexylcarbodiimide, N-hydroxysuccinimide, or 1-hydroxybenzotriazole in the presence of dicyclohexylcarbodiimide. A preferred practical coupling agent is (benzthiazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate, described by B. Castro et al., Tetrahedron Letters, 1219 (1975). ) and D. Hudson, J. Org. Chem., 53, 617 (1988), which may be used alone or in the presence of 1-hydroxybenzotriazole. Another very useful and useful coupling agent is the commercially available 2- (1H-benzotriazol-1-yl) -Ν, Ν, Ν ', N'-tetramethyluronium tetrafluoroborate.

Antiviral nucleoside analogs and their therapeutically acceptable salts form a class of compounds well known for use in the present invention. As noted above, members of this class of compounds exhibit antiviral activity against herpes viruses, i.e., inhibit viral DNA polymerase in vivo. An important member of this class of compounds is acyclovir and analogues thereof, disclosed in U.S. Patent 4,199,574 (filed April 22, 1980) to HJ Schaeffer; see also HJ Schaeffer et al., Naturre (London), 272, 583 (1978) and TA Krenitsk et al., Proc. Natl. Acad. Sci. USA, 81, 3209 (1984). The compound of formula (2) wherein R ⁹ is hydroxy is acyclovir having the chemical name 9 - [(2-hydroxyethoxy) methyl] -guanine. When R ^{9 in the} formula (2) is hydrogen, it is called 6-deoxyacyclovir, more specifically: 214 · »·

amino-9 - [(2-hydroxyethoxy) methyl] adenine; and the chemical name of the compound for which, in formula (2), R ⁹ is amino, 2,6-diamino-9 - [(2-hydroxyethoxy) methyl] purine.

It is understood that when R ⁹ is hydroxy in the formula (2), this compound may exist in its tautomeric form with the chemical name 2-amino-1,9-dihydro-9 - [(2-hydroxy). ethoxy) methyl] -6H-purin-6-one, and the percentage of each tautomer in the mixture of the two tautomeric forms depends on the physical environment of the compound. Tautomeric forms may also exist for any other nucleoside analogue having an enolizable carbonyl group.

Other antiviral nucleoside analogs for use in the present invention include vidarabine [9-β-D-arabinofuranosyl-adenine-water (1/1)], see R.J. Whitley et al., N. Engl. J. Med., 307, 971 (1982); idoxudine (2'-deoxy-5-iodo-uridine), see W.H. Prusoff, Biochim. Biophys. Acta, 32, 295 (1959); trifluridine [2'-deoxy-5- (trifluoromethyl) uridine], see U.S. Patent No. 3,201,387 to C. Heidelberger (filed August 17, 1965); ganciclovir {9 - [(1,3-dihydroxy-2-propoxy) -methyl] -guanine}, see J.P. Verheyden and J.C. Martin, U.S. Patent No. 4,355,032, filed October 19, 1982; edoxudine (5-ethyl-2'-deoxyuridine), see K.K. Gauri, U.S. Patent 3,553,192, filed January 5, 1971; brovavir [(E) -5- (2-bromo-vinyl) -2'-deoxy-uridine], see Y.

15 Benőit et al., Eur. J. Pediatrics, 143: 198 (1985); fiacitabine (2'-fluoro-deoxy-5-iodo-uridine), 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- (acetoxymethyl) butyl] adenine}, see R.A.V. Hodge et al. , Antimicrob. Agents Chemotherap. , 33, 1765 (1989); and rociclovir [9 - {[1,3-bis (2-propoxy) -2-propoxy] methyl} adenine], see E. Winklemann et al. , Arzneim. -Forsch. , 38, 1545 (1988).

The peptide derivatives of formula (1) may be prepared by conventional methods for peptide synthesis, such as by coupling amino acid residues and / or peptide fragments in solution. Such methods are described, for example, by E. Schroeder and K. Lübke in their previously cited book, and by E. Gross et al. In The Peptides: Analysis, Synthesis, New York, NY, 1979-1987, Volumes 1-8. and JM Stewart and J.D. Young in their Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, IL, USA, 1984.

A common feature of the above methods for the preparation of peptide derivatives is the protection of reactive side-chain groups present in amino acid residues or modified amino acid residues (or, where appropriate, in non-peptide fragments of peptide derivatives); they prevent undesired chemical reactions at the sites mentioned until they are removed. Typical examples include α-amino groups

protection of amino acids or other fragments until the carboxy group is reacted and then selectively removes the protecting group on the αί-amino group, thereby allowing it to react with a suitable reagent. Another common feature of the above methods is the initial protection of the C-terminal carboxy group of amino acid residues or peptide fragments, if present, with an appropriate protecting group that prevents chemical reactions at this site until the desired peptide sequence is formed, and after deprotection, this will be the C-terminal function of the peptide derivative.

A key intermediate of the peptides of formula (1) is represented by the following formula: wd-ch ₂ ch {ch ₂ c (O) R ¹ } C (O) OH wherein W is a protecting group for α-amino groups, such as tert-butoxy; -carbonyl (Boc), benzyloxycarbonyl (Z) or (9-fluorenyl) methoxycarbonyl (Fmoc), while D and R ¹ are as previously defined. The compound of the above formula may be prepared by: a

The allyl ester of formula WD-CH ₂ C (O) och ₂ ch = ch _{2 is} subjected to a Michael addition reaction with the fumaroyl derivative of the formula (lower alkyl) -O (O) CH = CHC (O) R ¹ . a wd-ch {c (o) och ₂ ch = ch ₂ } ch {ch ₂ c (0) R ¹ } C (0) 0

- (lower alkyl) • Michael adduct of the formula 0 · * ·· * · · · · ···.

This compound is then treated with tetrakis triphenylphosphine palladium and triphenylphosphine in the presence of pyrrolidine as described by R. Deziel (Tetrahedron Letteres, 2.8, 4371 (1987)), at which time the allyl ester is hydrolyzed and decarboxylated. the corresponding alkyl ester of the key intermediate is formed. Hydrolysis of the latter ester in the presence of a base such as sodium hydroxide or lithium hydroxide provides the key intermediate, which is a mixture of diastereomers.

Alternatively, the above key intermediate of the formula wherein W and D are as defined above, and R ¹ is -NR ⁴ R ⁵ , wherein R ⁴ and each is lower alkyl, or R ⁴ and R ⁵ together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino, morpholino or 4-methylpiperazino group, may be prepared as follows: wd-ch ₂ c (o) ch ₂ ch = ch _The allyl ester of formula ₂ , wherein W and D are as previously defined, is reacted under the conditions of the Michael reaction

BzlOC (0) CH = CHC (0) OBzl, a fumaroyl derivative, i.e. dibenzyl (E) -2-butenoate, then

WD- (R, S) -CH {CH [C (0) OBzl] CH ₂ C (0) 0Bzl} C (0) OCH ₂ CH = CH ₂ Michael adduct of formula is obtained.

This adduct is treated with tetrakis triphenylphosphine palladium and triphenylphosphine in the presence of pyrrolidine (see Dieliel supra), at which time

WD-CH ₂ - (R, S) -CH (CH ₂ C (O) 0Bzl} C (O) OBzl) is obtained by hydrogenation in the presence of palladium hydroxide on activated carbon to give the corresponding dicarboxylic acid. This anhydride is reacted with the appropriate secondary amine in the presence of pyridine to give a mixture of regioisomers, which is predominantly of the desired isomer, having the general formula:

WD-CH ₂ - (R, S) -CH- {CH ₂ C (O) NR ⁴ R ⁵ } -C (O) -OH wherein W, D and NR ⁴ R ² are as previously defined. This product is converted to the dibenzyl ester which is separated by high performance liquid chromatography to give the predominant isomer. Subsequently, hydrogenolysis in the presence of palladium hydroxide on activated carbon affords the desired key intermediate wherein W and D are as defined above, and R ¹ is -NR ⁴ R ⁴ which is also defined above.

Therefore, generally speaking, the peptides of formula (1) may be prepared by stepwise linkage according to the peptide sequence by linking the appropriate amino acid or amino acid derivative, or, where appropriate, a non-peptide-like fragment of the peptide (such as the key interferer). , then all protecting groups present will be removed to complete ··· ♦ ·· «· ·

• ··· • · · ··· «··« step by step! coupling to give the peptide of formula (1). More specific methods are illustrated in the following examples.

The peptides of formula (I) may also be prepared in the form of therapeutically acceptable salts. In the case where a peptide functions as a base, salts of the base include those with organic acids such as acetic acid, lactic acid, succinic acid, methanesulfonic acid or p-toluenesulfonic acid, and polymeric acids such as tannic acid or (carboxylic acid). ) cellulose, as well as inorganic acids such as hydrohalic acids such as hydrochloric acid, sulfuric acid or phosphoric acid. If necessary, an acid addition salt can be converted to another acid addition salt, for example, a non-toxic pharmaceutically acceptable salt, if it is a compound of R.A. As described by Boissonnas et al. Chim. Acta 43, 1849 (1960)] with the appropriate ion exchange resin.

When a typical peptide contains one or more free carboxy groups, the salts formed are typically those formed by the sodium, potassium or calcium cations or by organic bases such as triethylamine or 4-methylmorpholine.

The antiviral efficacy of the peptide derivatives of formula (I) can be demonstrated by biochemical, microbiological and biological methods that demonstrate the inhibitory effect of the compounds on the replication of acyclovir-resistant herpes simplex virus types 1 and 2 (HSV-1 and HSV-2).

The inhibitory effect of the peptide derivatives of formula (1) on viral replication can be demonstrated by the cell culture technique, see, for example, T. Spector et al.

Proc. Natl. Acad. Sci. USA, 82, 4254 (1985).

The therapeutic efficacy of the peptide derivatives can also be demonstrated in laboratory animals, for example, by using a test method based on a rat model of herpes simplex virus-induced ocular disease, which is based on C.R. Brandt et al., J. Virol. Meth. , 1992, 3.6, 209].

When used as an antiviral agent, a peptide derivative of the invention or one of its therapeutically acceptable salts may be administered topically or to pigs or horses in the form of a composition comprising one or more pharmaceutically acceptable carriers in proportion to the solubility, chemical nature, dosage regimen and standard biological practice. For topical use, the peptide derivative may be formulated in a pharmaceutically acceptable carrier which comprises the active ingredient.

In an amount of 0.1-5%, preferably 0.5-2%. The dosage form may be a solution, a cream or a lotion.

Systemic Application The peptide derivative may be administered orally, for example, in the form of a compound of formula (I) or intravenously, subcutaneously or intramuscularly, in a formulation with a pharmaceutically acceptable carrier or vehicle. For injection, the peptide is preferably dissolved in a sterile aqueous medium containing additional solutes, e.g.

for example, buffers, preservatives, and a sufficient amount of a pharmaceutically acceptable salt or glucose to provide isotonic solution.

Suitable carriers and excipients for use in the above pharmaceutical formulations are described in standard pharmaceutical manuals such as

Remington's

Pharmaceutical Sciences, 18th ed, Mac Publishing Company,

Easton, Penn., 1990.

The dosage of the peptide derivative will depend on the route of administration and the particular nature of the active ingredient selected. It also depends on the nature of the virus host being treated. Usually, small doses are started and continued until the optimum effect is achieved under the circumstances. Generally speaking, it is desirable to administer the peptide derivative at a dosage level that is generally sufficient to achieve the desired antiviral effect without causing any dangerous or deleterious side effects.

With regard to topical application, it is noted that the peptide derivative is applied directly to the infected area of the body, such as the skin, eyes, mouth or genital cavities, in an amount suitable for covering the infected area. The treatment may be repeated, for example, every four or six hours, until the abnormalities have healed.

For systemic use, the peptide derivative of formula (1) is administered in a dose of 1.0 to 10 mg / kg body weight / day.

however, the variations mentioned above may occur here. However, the most suitable dosage level for effective healing is 1.0-5 mg / kg body weight / day.

Although the drug formulations described above are effective and relatively safe drug treatments for herpes viral infections, the concomitant use of other antiviral drugs or agents cannot be excluded for good results. Such antiviral drugs or agents may be the aforementioned antiviral nucleosides, antiviral surfactants, or antiviral interferons as described in S.S. Asculai and F. Rapp, U.S. Patent 4,507,281, filed March 26, 1985.

More particularly, during the concomitant treatment of acyclovir-resistant herpes viral infections, it has been found that the anti-herpes activity of an antiviral analogue of a nucleoside can be enhanced synergistically when combined with a peptide derivative of the general formula (1). Accordingly, there is provided a pharmaceutical composition for the treatment of acyclovir-resistant herpes infections in a mammal comprising a pharmaceutically or veterinarily acceptable carrier, an antiviral nucleoside analog or a therapeutically acceptable salt thereof, and a pharmaceutically acceptable peptide derivative thereof. salt combination.

The synergistic effect of the combination of a nucleoside analogue as defined above and a peptide derivative of formula (I) as defined above means that the antiviral and herpes effect of the combination is greater than the sum of the expected effects of the two individual components of the combination.

When used in the treatment of acyclovir-resistant herpes infections, the combination of the invention is administered to warm-blooded animals, such as humans, pigs or horses, as a composition comprising one or more pharmaceutically acceptable excipients in which the nucleoside analog and ) is determined by the chemical nature and solubility of the peptide derivative of the general formula (I), synergistic antiviral activity can be achieved by appropriate choice of dosage, standard biological practice and correct determination of the relative amounts of the two active ingredients. The combination is preferably administered topically. For example, the two active ingredients (i.e., the antiviral nucleoside analogue and the peptide derivative of formula (I), or a therapeutically acceptable salt thereof) may be formulated with a pharmaceutically acceptable carrier, and may be solutions, emulsions, creams or lotions. These preparations contain from 0.01% to 1.0% by weight of the nucleoside analogue or a therapeutically acceptable salt thereof, and from a peptide derivative of the formula (I) or a therapeutically acceptable salt thereof by weight.

- 24 may contain from 0.05% to 1%.

In each case, the two active ingredients are present in the pharmaceutical composition in a ratio that provides a synergistic antiherpes effect.

The following examples illustrate the invention. Temperatures are given in degrees Celsius. Unless otherwise indicated, the percentage composition of solutions is by volume. Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker 200 MHz or 400 MHz spectrometer (the 400 MHz spectrum is indicated separately); chemical shift values are given in parts per million (ppm). The following abbreviations are used in the examples: Boc = tert-butoxycarbonyl; Bu = butyl; Bzl = benzyl; DMF = N, N-dimethylformamide; Et = ethyl; EtOH = ethanol; EtOAc = ethyl acetate; Et ₂ O = diethyl ether; Me = methyl; MeOH = methanol; Pr = propyl; TLC = thin layer chromatography; THF = tetrahydrofuran.

Example 1:

General procedure for coupling reactions [See R. Knorr et al. , Tetrahedros Letters, 30 0, 1927 (1989).]

The first reagent, the free amine (or its hydrochloride salt) is dissolved in dichloromethane or acetonitrile and cooled to 4 ° C. Four equivalents of 4-methylmorpholine were added under nitrogen with stirring. After 20 minutes, one equivalent of the second reactant, the free carboxylic acid, and 1.05 equivalents of coupling agent are added. [Useful and effective coupling reagents for this purpose are: (benzthiazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate or better 2- (1H-benzothiazol-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate.] The reaction is followed by thin layer chromatography. After completion of the reaction, methylene chloride (or acetonitrile) is distilled off under reduced pressure. The residue was dissolved in ethyl acetate and the solution was washed successively with 0.33 M aqueous citric acid, 10% w / w aqueous sodium carbonate, and saturated aqueous sodium chloride. The organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure to remove solvent. The residue was purified on silica gel (SiO ₂ ) by Still flash chromatography [WC Still et al., J. Org. Chem., 43, 2923 (1978)].

Example 2

Preparation of H-Asp (cyPn) (Bzl) -NH- (S) -CH (CH 2 CMe ^) C 1 -B 2 O 2 Intermediate (a) (S) -α-Azido-1 - [(benzyloxy) carbonyl] ] -cyclopentane-acetic acid

The compound was prepared according to M.N. Aboul-Enein et al., Pharm. Acta Helv. , 55, 50 (1980)] was prepared from 2-oxo-spiro [4.4] nonane-1,3-dione by the asymmetric azide addition method and the Evans chiral auxiliary reagent [see D.A. Evans et al., J. Amer. Chem. Soc., 1990, 112, 4011 (1990).

More specifically, a solution of butyllithium in 1.6 M hexane (469 mL, 750 mmol) was added dropwise under argon to the chiral auxiliary, 4 (S) - (2-propyl) -3-oxazolidine.

non-anhydrous tetrahydrofuran [96.8 g, 750 mmol; described by L.N. Pridgen and J. Prol, J. Org. Chem., 54, 3231 (1989)] at -40 ° C. The mixture was stirred at -40 ° C for 30 minutes, then cooled to -78 ° C. Add 2-oxo-spiro [4.4] nonane-1,3-dione dropwise to the cooled solution. The mixture was then stirred at 0 ° C for one hour. Subsequently, 600 ml of a 20% (w / v) aqueous citric acid solution are added. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The product, 3- [2- (1-carboxycyclopentyl) -1-acetyl] -4 (S) - [1- (2-propyl)] - 2-oxazolidinone, is obtained as a pink solid. Quantity: 300 g.

This solid (about 750 mmol) was dissolved in one liter of acetonitrile. To the solution was added benzyl bromide (89.2 mL, 128.3 g, 750 mmol) and 1,8-diazabicyclo [5.4.0] undec-7-ene (114 g, 112 mL, 750 mmol). The mixture was stirred under argon for 16 hours. The volatiles were removed under reduced pressure. The residue was taken up in water / ethyl acetate. The organic layer was separated, washed with 10% (w / v) aqueous citric acid solution, saturated aqueous sodium chloride solution, dried over magnesium sulfate and the solvent was distilled off under reduced pressure. The residual oil was crystallized from hexane / ethyl acetate to give the corresponding benzyl ester as a white solid. Yield: 204 g, theoretical 73

-the.

A solution of the latter compound (200 g, 187 mmol) in anhydrous tetrahydrofuran (200 ml) was cooled to -78 ° C. To the cooled solution was added a solution of potassium 1,1,1,3,3,3-hexamethyldisilazane in 0.66 M tetrahydrofuran (286 minutes) over a period of 15 minutes.

189 mmol) containing 6% (w / v) cumene.

A solution of 2,4,6-triisopropylbenzenesulfonyl azide (216 mmol) in dry tetrahydrofuran (100 mL) was added all at once to the cold mixture and acetic acid (50 mL, 860 mmol) was added two minutes later. The mixture was heated and stirred at 35-45 ° C for one hour. The volatiles were removed under reduced pressure. The yellow residue was triturated with 1.7 L of hexane / ethanol (4: 1). The resulting white solid was filtered. The filtrate was mixed with silica (230-240 mesh). The volatiles were removed under reduced pressure and the residual solids held under reduced pressure at 35 ° C to remove the cumene. The decolourised solid was applied to a silica gel column. The mixture of solid residue and silica was eluted with 9: 1 hexane: ethyl acetate. This is obtained by evaporation of the eluent

-2- [1- (benzyloxy) carbonyl] cyclopentyl} ethyl] -4 (S) -1- (2-propyl) -2-oxazolidinone. Yield: 66 g, theoretical 86

A solution of the latter compound (13.42 g, 32.4 mmol) in water (3: 1) was cooled to 0 ° C in 608 mL of tetrahydro. To the cooled solution was added a mixture of 16.3 mL (518 mmol hydrogen peroxide), followed by * ··· ····

Lithium hydroxide water (2.86 g, 68.2 mmol) (1: 1) was added. The reaction mixture was stirred at 0 ° C for 45 min and quenched with 400 mL of 10% w / v aqueous sodium sulfite. To the mixture was added sodium bicarbonate (1.93 g) and the mixture was concentrated under reduced pressure. The chiral auxiliary is obtained by continuous extraction (aqueous sodium bicarbonate solution / chloroform) for 20 hours. The aqueous phase is then cooled to 0 ° C and acidified by the addition of concentrated aqueous hydrochloric acid and extracted with ethyl acetate. The extract was washed with a saturated aqueous sodium chloride solution, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The title compound is a white solid (8.2 g, 84% of theory).

¹ H NMR (CDCl 3) δ 1.6-1.8 (m, 5H), 1.95-2.15 (m, 2H), 2.20-2.30 (m, 1H), 4.55 (s, 1H), 5.12 (s, 2H) and 7.4 (m, 5H).

The compound is used in part (c) of the present example.

(b) <RTIgt; <-methyl-leucinol-benzyl-ether

Reduction of H 1 -MeLeu-OH with lithium (tetrahydroborate (III)) / trimethylchlorosilane reagent according to A. Giannis and K. Sandhoff [Angew. Chem. Int. Ed. Engl., 28,

218 (1989)] yields Y-methylleucinol. A mixture of this compound (812 mg, 6.2 mmol) in triethylamine (659 mg, 6.51 mmol) and di-tert-butyl dicarbonate (1.42 g, 6.51 mmol) in dry tetrahydrofuran (15 mL) was heated at 4 ° C under nitrogen. stirring for 15 minutes and then stirring at room temperature for another 4 hours. Tetrahydrofuran under reduced pressure * · · ·

29 was distilled off and the residue was dissolved in ethyl acetate. The solution was washed with 10% aqueous citric acid solution, 5% aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution. The organic phase is dried over magnesium sulfate and the solvent is distilled off under reduced pressure. The residue was purified by flash chromatography (silica gel; eluent: hexane-ethyl acetate 2: 1) to give Boc-Y-methylleucinol. Yield: 1.23 g, 86% of theory.

To a solution of 1.23 g (5.35 mmol) of Boc-4-methyl-leucinol in 13 ml of benzyl chloride were successively 106 mg of (tetrabutylammonium) bisulfate and 3 ml of 50% aqueous sodium hydroxide solution. We are added. The resulting mixture was stirred at 35-40 ° C for 90 minutes, then diluted with ethyl acetate, and the solution was washed with water and a saturated aqueous solution of sodium chloride. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure to remove solvent. The residue was dissolved in hexane and the solution was poured onto a silica gel column. The column was eluted with hexane to remove benzyl chloride and eluted with hexane / ethyl acetate (2: 1) to yield Boc-Y-methyl-leucinol-benzyl ether.

^{Σ Η-NMR} (CDC1 _3): δ 0.95 (s, 9H), 1.42 (s, 9H), 1.30-1.55 (m, 2H), 3.42 (d, 2H , J = 4Hz), 3.88 (broad, 1H), 4.54 (m, 3H), 7.23-7.4 (m, 5H).

A solution of the latter compound (1.28 g, 3.99 mmol) in 10 mL of 6M hydrogen chloride in dioxane was added. The solution was stirred under a nitrogen atmosphere at 4 ° C for 45 minutes. After distilling off the solvent, the expected compound is hydrochloride ···· ····

30 salts which are used in the next step without further purification.

(c) H-Asp (cyPn) (Bzl) -NH- (S) -CH (CH ₂ CMe ₃ ) CH ₂ OBzl

Following the coupling procedure described in Example 1, using the first reagent Y-methyl leucinol benzyl ether hydrochloride described in (b) and the second reagent using (S) -α-azido-1- as described in (a). [(Benzyloxy) carbonyl] -cyclopentane-acetic acid was prepared to yield N - [(S) -1- (benzyloxy) methyl-3,3-dimethylbutyl] - (S) -α-aza- 1 - [(benzyloxy) carbonyl] cyclopentane-acetamide. Reduction of this compound with tin (II) chloride in methanol following the method of N. Maiti et al. (Tetrahedron Letters, 27, 1423 (1986)) affords the title compound.

¹ H NMR (CDCl ₃ ): δ 0.98 (s, 9H), 1.22-2.25 (m, 12H), 3.4 (d, J = 4Hz, 2H), 3, 64 (s, 1H), 4.18 (bm, 1H), 4.52 (s, 2H), 5.12 (s, 2H), 7.18 (d, J = 7Hz, 1H), 7 , 22-7.38 (br m, 10H).

Example 3:

Boc-Tbg-CH ₂ - (R, S) -CH [CH ₂ C (O) CMe 3] C (O) OH (a) Monoallyl magnesium malonate

A solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (100 g, 0.69 mol), allyl alcohol (47 ml, 0.69 mol) and benzene (800 ml) was heated at reflux for 24 hours. The solvent was distilled off under reduced pressure. The residue was also distilled under reduced pressure to give 71 g of monoallyl malonate; boiling point: 123-127 ° C / 360 Pa; yield theoretical ···· ····

- 71% of the 31 values. The resulting monoester (71 g, 0.48 mol) was dissolved in anhydrous tetrahydrofuran (300 ml), magnesium ethanolate (28.5 g, 0.245 mol) was added and the mixture was stirred under argon for 4 hours at room temperature (20-22 ° C). The solvent was distilled off under reduced pressure and the residue was triturated with diethyl ether to give a yellow-brown solid. It is finely pulverized and dried under reduced pressure. The desired amount of magnesium salt was 56 g, 73% of theory. This salt is used in the next reaction step.

(b) Boc-Tbg-CH ₂ C (O) OCH = CH ₂

1,1-Carbonyldiimidazole (12.6 g, 78 mmol) was added to a solution of Boc-Tbg-OH (15 g, 64 mmol) in dry acetonitrile (150 mL). The mixture was stirred under argon at room temperature for 2 hours. Monoallyl magnesium malonate (24 g, 78 mmol) and 4- (N, N-dimethylamino) pyridine (100 mg) were added and the mixture was heated under reflux for 1 hour and then stirred at room temperature for 18 hours. The solvent was then distilled off under reduced pressure. The residue was dissolved in ethyl acetate (300 mL), washed with 10% aqueous citric acid (2 x 100 mL), brine (2 x 100 mL), dried over magnesium sulfate and the solvent was distilled off under reduced pressure. The residue was purified by flash chromatography (silica gel; eluent: hexane / ethyl acetate = 9: 1). The desired allyl ester is obtained as a brown oil which crystallizes on standing. Yield: 19.4 g, 96% of theory.

1 H NMR (CDCl 3): note that the compound is a 3: 1 mixture of keto-enol tautomers in chloroform, δ 0.96 (s,

9H, enol form), 1.04 (s, 9H), 1.46 (s, 9H), 3.65 (s, 2H),

3.90 (d, J = 8.5 Hz, 1H, enol form), 4.20 (d, J = 7.5 Hz,

1H), 4.65 (m, 2H), 5.10 (broad d, J = 7.5 Hz, 1H), 5.20-5.40 (m, 2H), 5.80-6.05 ( m, 1H), 12 (s, 1H, enol form). It's ally

ester is used in part (d) of the example.

(c) Ethyl (E) -5,5-dimethyl-4-oxo-2-hexenoate

The ethyl ester was prepared by the method of S. Manfredini et al., Tetrahedron Letteres, 29, 3997 (1988). The crude oily product was purified by flash chromatography (silica gel; eluent: hexane) to give the desired ethyl ester as a yellow oil.

¹ H NMR (CDCl 3) δ 1.21 (s, 9H), 1.33 (t, J = 15.5 Hz, 1H), 4.28 (q, J = 7.5 Hz, 2H) ), 6.78 (d, J = 15.5 Hz, 1H), 7.51 (d, J = 15.5 Hz, 1H).

The ethyl ester is used in the following section of this example.

(d) Boc-Tbg-CHo- (R, S) -CH [CH ₂ C (O) CMe ₃ ] C (O) OH

From Boc-Tbg-CH ₂ C (O) OCH = CH ₂ as described in Example b)

0.67 g (2.1 mmol) was dissolved in 25 mL of anhydrous tetrahydrofuran under argon. Ag (C), 095 g ^ ^v sodium Joldathoz

hydride (60% oily dispersion, 2.4 mmol) was added and the mixture was stirred for 30 minutes at room temperature. Then, 0.435 g (2.36 mmol) of ethyl (E) -5,5-dimethyl-4-oxo-2-hexeno33 described in part (c) of the example is added. Stir the reaction mixture until the reaction is complete; the reaction is followed by TLC, which requires about 6 hours. The reaction was quenched with 10% aqueous citric acid solution, the tetrahydrofuran was removed under reduced pressure, and the resulting concentrate was extracted with ethyl acetate (3 x 25 mL). The extract was washed with water, dried over magnesium sulfate and the solvent was distilled off under reduced pressure. The residue was purified by flash chromatography (silica gel; eluent: hexane: ethyl acetate = 9: 1) to give the corresponding Michael adduct. Yield: 1.06 g, 100% of theory.

The Michael adduct was converted to 0.20 g (0.18 g) of Boc-Tbg-CH ₂ - (R, S) -CH [CH ₂ C (O) CMe ₃ ] -C (O) OH. tetrakis (triphenylphosphine) palladium (O) and triphenylphosphine (0.060 g, 0.23 mmol) were dissolved in dichloromethane (10 mL) under argon. To the solution was added acetonitrile (20 mL) and cooled to 0 ° C. Pyrrolidine (0.28 mL, 2.7 mmol) and Michael adduct (1.06 g, 2.1 mmol) were then added. The mixture was allowed to warm to room temperature over an hour and then stirred for an additional 20 hours. The reaction mixture was then heated and refluxed for one hour under argon to complete the reaction. The solvent was distilled off and the residue was purified by flash chromatography (silica gel; eluent: hexane / ethyl acetate = 9: 1) to give Boc-Tbg-CH ₂ - (R, S) -CH [CH ₂ C (O) CMe. ₃ ] C (O) OEt; Yield 0.77 g, 83% of theory. This compound (0.77 g, 1.7 mmol) was dissolved in 10 mL of a 1: 1 mixture of ethylene-34-glycol dimethyl ether and water, and 0.31 g (7.4 mmol) of lithium hydroxide was added. water (1/1) and the mixture was stirred at room temperature for 4 hours. Aqueous citric acid (20 mL) was added and the mixture was extracted with ethyl acetate (3 x 25 mL). The extract was dried over magnesium sulfate and the solvent was distilled off under reduced pressure. The title compound is a yellow-brown solid. Yield: 0.69 g, 80% based on Boc-Tbg-CH ₂ C (O) O-CH ₂ CH = CH ₂ . The product, as shown by ¹ H NMR, is a 55:45 mixture of diastereomers.

1 H NMR (CDCl ₃ ): δ 0.97 (s, 9H, minor isomer), 0.99 (s, 9H, major isomer), 1.14 (s,

18H), 1.48 (s, 18H), 2.68-3.12 (m, 8H), 3.30 (m, 2H), 4.08 (d, J = 9Hz, 1H, minor isomer). , 4.10 (d, J = 9Hz, 1H, larger isomer), 5.11 (d, J = 9Hz, 2H9).

Example 4:

H-Tb <? - CH ₂ CH [CH ₂ C (O) CMe ₃ l C (O) -Asp (cyPn) (Bzl) -NH- (S) -CH [CH ₂ C (O) Me ₃ 1 CH ₂ OBzl

Following the coupling procedure described in Example 1, using the final product of Example 2 (3.42 g, 7.13 mmol) as the first reactant and the final product of Example 3 (2.50 g, 6.48 mmol) as the second reactant. and purification of the crude final product by flash chromatography (silica gel; eluent: hexane: ethyl acetate = 4: 1) gave the title compound (Boc, 4.64 g, yield: 84% of theory, Rf). = 0.21; Hexane / ethyl acetate 7: 3). 4.64 g (5.44 mmol) of the latter derivative were dissolved in 50 ml of 6 M hydrochloric acid in dioxane.

After stirring for one hour at room temperature, the solvent was distilled off under reduced pressure. The residue was dissolved in diethyl ether, washed with a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution, and the separated organic layer was dried over magnesium sulfate. The yellow oil remaining after distilling off the solvent is a mixture of the two diastereomers. The two isomers were separated by flash chromatography (silica gel; eluent: hexane / ethyl acetate / methanol = 5: 4.5: 0.5). The desired (i.e., more polar) isomer (Rf = 0.18; ethyl acetate / hexane / methanol = 7: 3: 0.5) is a colorless oil. Yield: 2.52 g (52% of theory). This isomer, the title compound, was used in the next step without further purification.

Example 5:

Boc- (N-Me) Val-Tbg-CH ₂ (R) ICH -CH ₂ C (0) _CMe3 1C (0) -Asp (cyPn) (Bzl) -NH- (S) -CH [CH ₂ CMe ₃ 1CHnOBzl

Following the coupling procedure described in Example 1, using the title compound of Example 4 (4.45 g, 5.95 mmol) as the first reactant and N-methylvaline (4.41 g, 17.9 mmol) as the second reactant. , the crude product was purified by flash chromatography (silica gel; eluent: hexane: ethyl acetate = 7: 3) to give the title compound of this example (4.02 g, m.p.

72% (corresponding to yield) ¹ H NMR (CDCl ₃ ): δ 0.87 (d, J = 7Hz, 6H), 0.90 (S, 18H) , 1.10 (s, 9H), 1.48 (s, 9H), 1.5-2, 0 (m, 10H), 2.30 (M, 1H), 2.5-3.1 (m, 5H), 2.80 (s, 3H) , 3.30 (m, 2H), 4.0 (D, J =

12.5 Hz, 1H), 4.20 (m, 1H), 4.32 (d, J = 8.5 Hz, 1H), 4.49 (d,

J = 4.5 Hz, 2H), 4.64 (d, J = 11Hz, 1H) , 5.18 (d, J = 5 Hz, 2 H) , 6.78 (wide d, J = 8, 5 Hz, 1H) , 7, 11 (wide d. J = 8.5 Hz, 1H), 7.18 (broad d, J = 11 Hz, 1H), 7.2-7.45 (M, 10H).

Example 6:

(3-Phenylpropionyl) - (N-Me) Val-Tbq-CH ₂ - (R) -CH [CHqC (O) CMe ₃ JC (O) -Asp (cyPn) -N-Me-Leucinol

The title compound of Example 5 (4.02 g, 4.25 mmol) was dissolved in 30 mL of 6M hydrogen chloride in dioxane and the resulting solution was stirred at room temperature for one hour. The solvent was then distilled off under reduced pressure to give the free N-terminal amino derivative of the title compound of Example 5 as its hydrochloride salt. Subsequently, following the coupling procedure in Example 1, using this amino derivative as the first reactant and 3-phenylpropionic acid (2.00 g, 13.3 mmol) as the second reactant, the crude product was purified by flash chromatography (silica gel; eluent: hexane / ethyl acetate 3: 2) to give (3-phenylpropionyl) - (N-Me) Val-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) (Bzl) -NH- (S) -CH (CH ₂ CMe ₃ ) CH ₂ OBzl as a white foam. Yield: 4.00 g, 94% of theory (Rf = 0.35; hexane: ethyl acetate = 1: 1).

The latter compound (4.00 g, 4.03 mmol) was subjected to hydrogenation [20% Pd (OH) ₂ / C; 200 mg; hydrogen gas pressure 101.3 kPa; ethanol; 5 hours]. After completion of the reaction, the catalyst was removed from the reaction mixture by filtration through a 45 μτη pore size membrane. The filtrate was concentrated under reduced pressure to give a light oil. The oil was dissolved in diethyl ether (100 mL) and the solvent was distilled off under reduced pressure. Dissolution and evaporation of the solvent were repeated until a white solid was obtained. This solid was triturated with hexane, filtered and dried under reduced pressure. 3.12 g (95% of theory) of the title compound are obtained.

¹ H NMR (dg-DMSO), 400 MHz; Note: the compound

In DMSO, the two rotamers are present as a 50:50 mixture: δ

0.71-0.92 (m, 24H), 1.05 (s, 4.5H), 1.06 (s, 4.5H), 1.20-1.78 (m, 10H), 1, 93-2.16 (m, 2H), 2.48-2.83 (m, 6H), 2.84 (s,

1.5H), 2.92 (s, 1.5H), 2.96-3.06 (m, 1H), 3.10-3.23 (m, 2H),

3.72-3.81 (m, 1H), 4, 09-4.14 (m, 1H)

4.22 (d, 8 Hz, 0.5H),

4.54-4.62 (br m, 1H), 4.73-4.81 (m, 1.5H), 7.12-7.29 (m,

6H),

7.94 (d, J

Hz, 1H), 8.04 (d, J)

Hz, 0.5H), (d, J = 8.5 Hz, 0.5H).

Following the procedures outlined in Example 6 The procedure but replacing 3-phenylpropionic acid using 2-benzyl-3-phenylpropionic acid (dibenzylamino-acetic acid) was prepared from (dibenzyl-acetyl) - (N-Me) _Val-Tgb-CH2 - (R) -CH [CH ₂ C (O) -CMe ₃ JC (O) -Asp (cyPn) -Y-Me-leucinol.

Example 7:

N- (1-Ethyl-propyl) -carbamoyl-Tbq-CH ₂ - (R) -CH ₂ CH (C) -CMe ₃ 1 CO-Asp (cyPn) -Me-leucinol

To a solution of the title compound of Example 4 (23 mg, 0.030 mmol) and 6 mg (0.057 mmol) of triethylamine in anhydrous methylene chloride was added (1-ethyl) propyl isocyanate (28 mg, 0.248 mmol) and the reaction mixture was stirred at room temperature. argon gas atmosphere38 • hu · · · · · ··················································•

The reaction mixture was stirred at 0 ° C for 1 hour and then at room temperature for 18 hours. The reaction was followed by thin layer chromatography (ethyl acetate / hexane 1: 1) to ensure the reaction was complete. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica gel, eluent: hexane: ethyl acetate = 6: 4) to give the dibenzyl derivative of the title compound.

Yield: 16 mg.

⁴ Η-NMR (CDCl3, 400 MHz): 0.9 (t, J = 7 Hz, 3H), 0.92 (t, J = 7 Hz, 3H), 0.94 (s, 9H), 0.95 (s, 9H), 1.10 (s, 9H), 1.25-1.90 (m, 14H), 2.52 (m, 1H), 2.68 (m, 1H), 2.85 (m, 1H), 2.94-3.07 (m, 2 H) , 3.27 (dd, J = 7.2, 9Hz, 1H) , 3.36 (dd, J = 5.5, 9Hz, 1H), 3.46 (m, 1H), 4.07 (d, J = 9 Hz, 1H), 4.23 (m, 1H), 4.28 (d, J = 9Hz, 1H) , 4.48 (dd, J = 10 Hz, 2H), 4.66

(D,

1H),

4.74 (d,

J

J = 9 Hz,

1H), 5.17 (dd,

J = 10 Hz,

Hz,

2H), 7.10 (d, J = 8.5 Hz,

1H), 7.2-7.45 (m, 11H).

This dibenzyl derivative was subjected to hydrogenation (10 palladium on charcoal; hydrogen pressure 101.3 kPa;

ethanol) to give the title compound.

MS:

(M + Na) ⁺ = 703.

Example 8:

Preparation of Other Typical Intermediates for the Formation of the C-Terminal Part of Peptides of Formula 1 (a) 3 (R) -Amino-4,4-dimethylpentane

To a solution of 106 g (0.92 mol) of 4,4-dimethyl-3-pentanone and 111 g (0.92 mol) of (R) -g-methylbenzylamine in 1 liter of benzene cooled to 0 ° C. , 5 mL (0.46 mol) of titanium tetrachloride

··· • ·

A solution of benzene (200 mL) was added at a rate such that the temperature of the mixture did not exceed 10 ° C. The mixture was stirred at 40 ° C for 1 hour, cooled to room temperature and filtered through diatomaceous earth. Diatomaceous earth is diethyl

-ether. The filtrate and the washings were combined and the solvents were evaporated. The residue is dissolved in 2 liters of anhydrous methanol, the solution is cooled to 0 ° C and 20 g (0.53 mol) of sodium tetrahydroborate (III) are added portionwise while maintaining the temperature below 5 ° C. left. The methanol was then distilled off and the residue was dissolved in diethyl ether. The solution was saturated aqueous sodium

washed with brine and dried over magnesium sulfate. Distillation of the diethyl ether left a reddish oil, which was shown to be an 18: 1 mixture of diastereomers by ¹ H NMR. The oil was purified by flash chromatography (silica gel; eluent: ethyl acetate / hexane 7:93) to give N- [1 (R) -phenylethyl] -1 (R) -ethyl-2,2-dimethyl-propyl- as a liquid. Yield: 110 g (54% of theory). This material was dissolved in 1.5 L of hexane and 90 mL of 6M hydrogen chloride in dioxane was added over 15 minutes. The resulting white solid was filtered, washed with hexane to give N- [1 (R) -phenylethyl] -1 (R) -ethyl-2,2

dimethylpropylamine hydrochloride is obtained. Yield: 125 g (97% of theory).

¹ H NMR Spectrum (DCD ₁₃ ): 0.55 (t, J = 7.5 Hz, 3H), 1.14 (s, 9H), 1.54-1.95 (m, 2H), 2 , 23 (d, J = 6.5 Hz, 3H), 2.36-2.44 (m, 1H), 4.31-4.49 (m, 1H), 7.30-7.48 (m , 3H), 7.74-7.79 (m, · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

41.5 g of the latter compound are dissolved in 120 ml of methanol, 10% (w / w) palladium on activated carbon is added and the mixture is hydrogenated in a Parr hydrogenator at room temperature under a hydrogen pressure of 344.7 kPa for 48 hours. across. The mixture was filtered and the filtrate concentrated to give the desired 3 (R) -amino-4,4-dimethylpropylamine hydrochloride as a white solid. Yield: 25 g, quantitative.

1 H NMR (CDCl ₃ ): δ 1.10 (s, 9H), 1.22 (t, J = 7Hz, 2H), 1.58-1.90 (m, 2H), 2, 70-2.85 (m, 1H), 8.10-8.40 (broad s, 3H).

Proceeding as above, but replacing 4,4-dimethyl-3-pentanone with 3,3-dimethyl-2-butanone gives 2 (R) -amino-3,3-dimethyl-butane hydrochloride.

Example 9:

Preparation of Other Typical Intermediates for the Formation of the N-Terminal Part of the Peptides of Formula (I) Using the Procedure of Example 7 (a) (1-Propyl-butyl) -isocyanate

This intermediate was prepared from commercially available 4-aminoheptane according to V.S. Goldesmidt and M. Wick, see Liebigs Ann. Chem., 575, 217 (1952)].

(b) 1-Allyl-1-ethyl-3-butenyl isocyanate

To a solution of allyl magnesium bromide (1.0 mol) in diethyl ether (880 ml) was added dropwise a solution of propionitrile (14.5 g, 264 mmol) in diethyl ether (40 ml). The reaction mixture

stir mechanically and reflux for 2 hours, then cool to 0 ° C. 320 ml of saturated aqueous ammonium chloride solution was carefully added. The organic phase was separated, dried over magnesium sulfate, cooled to 0 ° C, and treated with 200 ml of diethyl ether containing 1M hydrogen chloride at the same temperature. The resulting solid was suction filtered and dried under reduced pressure. Yield 27 g. This material was dissolved in methylene chloride (200 mL), washed with 10% (w / v) aqueous sodium carbonate (twice), then with saturated aqueous sodium chloride, dried over magnesium sulfate, and the solvent was evaporated. evaporated. A yellow oil is obtained which is distilled under reduced pressure (b.p. 82-85 ° C, pressure: 2666 Pa) to give 1-allyl-1-ethyl-3-butenylamine as a colorless oil. Yield:

11.6 g, 34% of theory.

⁴ H NMR (CDCl ₃ , 400 MHz): δ 0.89 (t, J = 7 Hz, 3H),

1.39 (q, J = 7Hz, 2H), 2.11 (d, J = 7Hz, 2H), 5.06-5.14 (m, 4H), 5.80-5.89 (m , 2 H).

The latter compound was converted to 1-ethyl-1- (2-propenyl) -3-butenyl isocyanate according to V.S. Goldesmidt and M. Wick (see above for reference).

The process described in part (b) of the example is the first, i.e. the preparation of 1-allyl-1-ethyl-3-butenylamine

The step of the general method described by Alvernhe and A. Laurent, G.

(Tetrahedron Lett., 1057 (1973)). The entire process, including proper selection of reactants, can be used at the N-terminal unsaturation.

also for the preparation of isocyanate intermediates for the preparation of carrier peptide derivatives; such unsaturation may be, for example, an N- (unsaturated alkyl) carbamoyl such as 1-allyl-1-methyl-3-butenyl. It is noted, however, that when the required isocyanate intermediate is used according to the procedure described in Example 7, a peptide derivative of formula (1) is obtained which has an N-terminus saturated. In addition, the latter process is of practical importance for the preparation of peptides such as Pr ₂ CHNHC (O) -Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe] C (O) -Asp ( cyPn) -NH- (R) -CH (Et) CMe ₃ (Mass Spectrum (FAB) (m / z): (M + H) ⁺ = 693) which is the title peptide derivative of Example 10 below.

Therefore, by taking the appropriate intermediates and using the methods of 1-7. By employing serial coupling and deprotection of the Examples of the present invention, other compounds of formula (1) may be prepared, as shown in the table below. However, in some cases, precipitation of the final product does not result in pure material. In such cases, the product may be purified by semi-preparative HPLC on a C-18 reverse phase column, eluting with a gradient of acetonitrile / water containing 0.06% trifluoroacetic acid. For this purpose, the crude product was dissolved in 0.1 M aqueous ammonium hydroxide solution and the pH of the solution was restored with 0.1 M aqueous acetic acid solution.

In the case of purification, diastereomeric mixtures can also be separated in this way.

Some examples of these are: • ·

- 43 compounds which can be prepared as follows:

(PhCH ₂ ) ₂ CHC (O) - (N-Me) Val-Tbg-CH ₂ - (R) -CH [-CH ₂ C (O) CMe ₃ JC (O) -Asp (cyPn) -NHCH ₂ CMe and (PhCH ₂ ) CHC (O) -N-Me) Val-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ JC (O) -Asp (cyPn) -NH- (R ) -CH (Me) CMe ₃ .

Some other examples of compounds of formula (1) are:

Pr ₂ CHNHC (O) -Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NHCH ₂ CMe ₃ ,

Mass Spectrum (FAB, m / z): (M + H) ⁺ = 66;

EtPr ₂ CNHC (O) -Tbg-CH ₂ (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH- (R) -CH (Et) CMe ₃ ,

Mass Spectrum (FAB, m / z): (M + H) ⁺ = 722; and

EtPrCNHC (O) -Tbg-CH ₂ (R) -CH [CH ₂ C (O) CMe ₃ JC (O) -Asp (cyPn) -NHCH ₂ CMe ₃ , Mass spectrum (FAB, m / z): (M +) H) ⁺ = 693.

Example 10:

Assay for Pr ₂ CHNHC (O) -Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe, 1C (O) -Asp (cyPn) -NH- (R) -CH (et) CMe ₃ for the detection of the action of a clinical isolate against wild-type HSV-1 and two clinical isolates of acyclovir-resistant HSV-1 virus strain and its combination with aciclovir

Viruses: Clinical isolates are described by SLSacks et al., Annals of Medicine, 1989, 111, 893. They were isolated from a 25-year-old young woman suffering from myeloid leukemia following a first relapse of a foreign bone marrow transplant. Initial culture of herpes simplex virus was found positive 8 days after bone marrow transplantation (identified as wild type HSV-1 isolate 294). Thereafter, aciclovir treatment was initiated and virus culture was positive at day 36 (isolate 615, defined as resistant to HSV-1). Subsequent isolate 615.9 (clinical isolate ACV ^r TK) was found to be acyclovir resistant, foscarnet sensitive, and having reduced thymidine kinase (TK) activity. Later, the TK-deficient mutant of isolate 615.9 was confirmed. In contrast, the following isolate 615.8 (clinical isolate ACV ^r POL) was found to be acyclovir and foscarnet resistant, and thymidine kinase activity was nearly identical to that of isolate 294 before treatment. The DNA polymerase mutant of isolate 615.8 was later confirmed.

tests:

BHK-21 / C13 cells (ATCC CCL 10) were incubated for 2 days in 150 ml T-flasks (1.5 x 10 ⁶ cells / flask) in alpha-MEM medium (Gibco Canada Inc., Burlington, Ontario, Canada). It was supplemented with 8% v / v calf serum (FBS, Gibco Canada Inc.). Cells were treated with trypsin and placed in fresh medium in a 24-well plate containing 2.5 x 10 4 cells / well in 750 μΐ medium. The cells were incubated at 37 ° C for 6 hours, allowing the cells to adhere to the plate. Cells were then washed once with 500 μΐ of alpha-MEM medium supplemented with 0.5% v / v bovine serum and incubated with 750 μΐ of the same medium (low serum content) for 3 days. After this period of serum starvation, low serum medium was removed and the cells were incubated with 500 μΐ BBMT for 2-3 hours. [BBMT medium was described by P.Brazeau et al; see Proc. Natl. Acad. Sci. USA, 79, 7909 (1982)]. Cells were then infected with HSV-2 virus (infection rate 0.02 PFU per cell) in 100 μΐ BBMT medium. (Note: The HSV-2 virus strain used was HG-52, see Y. Langelier and G. Buttin, J. Gen. Virol., 57, 21 (1981); the virus was stored at -80 ° C) . The virus was allowed to adsorb at 37 ° C for one hour, then the medium was removed and the cells were washed with 3 x 250 μΐ BBMT. Cells from each titration site were incubated with 200 μΐ BBMT medium with or without the appropriate concentration of test substance (control). After incubation at 37 ° C for 29 hours, infected cells were harvested; the plate was first frozen at -80 ° C and then allowed to thaw. Cells from each titration site were scraped from the wall of the titration well with the melting ice scrap. After complete thawing, the cell suspension was harvested and each titration site was rinsed with 150 μΐ BBMT each. The virus-containing sample (cell suspension plus wash) was subjected to mild ultrasound at 4 ° C for 4 minutes. Cell debris was removed by centrifugation (1000 g at 10 ° C for 10 minutes). The supernatant was collected and stored at -80 ° C until the virus titer was determined.

Virus titration was performed by a modified colorimetric method of M. Langlois et al., 1986, Journal of Biological Standardization, 14, 201.

In more detail, in the same manner as above, • · ·

BHK-21 / C13 cells were treated with trypsin, and cells were plated in fresh medium in a 96-well plate, and 20,000 cells per titration were placed in 100 μΐ medium. The cells in the prepared plate were incubated at 37 ° C for 2 hours. During this time, the viral sample was thawed and subjected to mild ultrasound treatment for 15 seconds, then a logarithmic dilution series of the sample (1/5 serial dilution: 50 μΐ sample plus 200 μΐ BBMT medium) was serially diluted using a multichannel pipette.

At the end of the 2 hour incubation as described above, BHK-21 / C13 cells were replaced with alpha-MEM medium containing 3% v / v bovine serum by volume. The cells are now ready to be infected with various dilutions of the virus sample. A 50 μΐ aliquot of each dilution is transferred to the appropriate titration site of the plate. The infected cells were incubated at 37 ° C for 2 days. Hank's equilibrated saline (pH 7.3; Gibco Canada Inc.) containing 50 μΐ of a 0.15% neutral red solution was then added to each titration site. The prepared plate was incubated at 37 ° C for 45 minutes. Media were aspirated from each titration site and cells were washed once with 200 μΐ Hank's balanced saline. After washing with 100 μΐ 0.1 M Sörensen's citrate buffer (pH 4.2) and ethanol 1: 1, the dye was liberated from the cells. [Sörensen's citrate buffer was prepared by first preparing a 0.1 M disodium citrate solution; 21 g of citric acid-water (1: 1) are dissolved in 200 ml of 1 M aqueous sodium hydroxide.

and make up to one liter with filtered water. Second, 61.2 mL of 0.1 M disodium citrate solution was mixed with 38.8 mL of 0.1 M aqueous hydrochloric acid and the pH of the resulting solution was adjusted to 4.2 if necessary.] exposed to a slight Vortex mixing to ensure proper mixing. The titration sites on the plate are read with a scanning spectrophotometer at 540 nm to determine the number of viable cells. In this way, the percent inhibition of virus growth as a function of concentration of test compound can be determined and the concentration of test compound that causes 50% inhibition of viral replication, i.e., IC 50, can be calculated.

For the evaluation of acyclovir and the peptide derivative, the approach chosen was to investigate them separately and in combination in different combinations according to the method previously described. We also investigated the synergistic effect between the two compounds by evaluating the results obtained using the isobola method; for a description of the isobol method see J. Sühnel, J. Antiviral Research, 13, 23 (1990).

Illustrating the essence of the isobol method: This method requires experimental data for the two test compounds when evaluated individually and in different dose combinations of equal efficacy. Accordingly, the title peptide derivative selected concentration bolts (IC ^ q, IC _10, IC ₃₀ and IC ₂ q) are administered in combination with various doses of acyclovir and determining the

IC50 values. For this purpose, the IC ₅ , ^{Ι (2} ₁₀ , IC ₂ q and IC ₂ q) of the title peptide derivative and the doses of acyclovir were determined from the curves recorded in previous experiments. The FIC g ₀ (acyclovir) values for one isobologram were determined. one is the concentration of acyclovir which, in the presence of a constant concentration of the title peptide derivative, reduces the HSV virus replication by 60% compared to the concentration of aciclovir that exerts the same effect in the absence of the peptide derivative. The FICg ₀ (acyclovir) values are plotted against the values obtained when the constant concentration of the peptide derivative is divided by the concentration of the peptide derivative which inhibits the replication of the HSV virus by 60% without acyclovir.

The equations:

the constant concentration of the added peptide derivative

X axis: ----------------------------------------------- --- the ICgg value of the single acting peptide derivative

Y axis:

IC ₆₀ (X μovir value of acyclovir + peptide derivative)

FICg ₀ (acyclovir) = ----------------------------------------- (acyclovir alone) • ··· · ···

Results :

Table 1 shows the results obtained when the efficacy of acyclovir and the title peptide derivative of this example on the wild-type clinical isolate of HSV-1 and on two clinical isolates of HSV-1 resistant to acyclovir were examined. -módszerrel.

The results of Table 1 demonstrate that the compounds of formula (1) are effective against the wild type HSV-1 and show similar efficacy against acyclovir-resistant HSV-1 strains, both with the POL mutants and with TK. deficient against mutants; however, acyclovir loses much of its potency (20-40 times less active) against mutant strains.

···· ·· * · • «

Table 1

isolates 294 615.8 s z amps 615.9 compounds isolate isolate isolate IC ₅₀ (μΜ) IC ₅₀ (μΜ) IC ₅₀ (μΜ) acyclovir 1.1 19 55 The title peptide- derivatives 2.0 2.0 6.2

The following Tables 2, 3, and 4 contain the results obtained by testing the combinations of acyclovir with the title peptide derivative according to the cell culture method described above with the clinical isolate of the wild type HSV-1 and the two acyclovir-resistant HSV- 1 clinical isolate.

. spreadsheet

Antiviral activity

Studying synergism

294 isolate (Clinical isolate treatment)

Values for the constant concentration of the title peptide derivative (μΜ) Aciclovir IC ^ q (D values in combination with the title peptide derivative (μΜ) 0.3 0.85 0.5 0.70 0.7 0.35 0.9 0.30 1.0 0.30 1.2 0.22 1.4 0.10 1.6 0.13

- 52 3. spreadsheet

Antiviral activity

Studying synergism

Isolate 615.8 (Treatment of ACV ^r POL isolate)

The title peptide derivative constant concentration (μΜ) Acyclovir IC5Q ^ ² ^ value in combination with the title peptide derivative (μΜ) 0.3 15.0 0.5 13.0 0.7 7.0 0.9 8.0 1.0 5.3 1.2 5.0 1.4 1.7 1.6 2.4

β

Table 4

Antiviral activity

Studying synergism

Isolate 615.9 (Treatment of ACV ^r TK isolate)

The title peptide derivative Aciclovir IC ₅₀ constant value (μΜ) in combination with the title peptide derivative (μΜ)

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

33.0

17.0

5.5

5.6

2.0

0.9

0.02

0.02

Notes to the previous four tables:

• IC ₅₀ values were not corrected for solubility.

• All stock solutions were ultracentrifuged at 100,000 g at 4 ° C for 30 minutes prior to use.

The solubility of the title peptide derivative was 20% at 20 μΜ. · · · · · · · · · · · · · ·

• The cellular toxicity (cytotoxicity) of the title peptide derivative was 89 μΜ (uncorrected).

(1) Its value was determined from the dose-response curve;

the dose of acyclovir varied between 7.6 χ 10 ^-4 and 1.5 χ 10 ¹ μΜ in the presence of constant concentration of the title peptide derivative, shown in the left column.

(2) The IC ₅₀ was determined from the dose-response curve; the concentration of acyclovir varied between 1.5 χ 10 ^-4 and 1.5 x 10 Μ μΜ in the presence of constant concentration of the title peptide derivative, shown in the left column.

Tables 2, 3 and 4 show that peptides of formula (1) are capable of potentiating the effect of acyclovir against wild type HSV-1 and acyclovir-resistant HSV-1 mutants when both combination. The results also show that increasing the peptide concentration relative to the acyclovir concentration decreases the IC 50 of acyclovir.

Fig. 1 attached to this description graphically illustrates the positive results (synergism) obtained using the isobol method in the experiment when tested for the efficacy of aciclovir and the title peptide of this example on isolate 615.8 (ACV ^r POL clinical isolate). .

Similarly, the attached Figure 2 graphically illustrates the positive results when tested for the efficacy of acyclovir and the title peptide of this example against isolate 615.9 (ACV _r TK).

• · · · ····· · ·

- 56 PATENT CLAIMS

Claims (13)

PATENT CLAIMS Use of a peptide derivative in the manufacture of a medicament for the treatment of acyclovir-resistant herpes simplex viral infections in a mammal, wherein the peptide derivative is a compound of formula (I): wherein: A is phenylacetyl, phenylpropionyl, (4-aminophenyl) propionyl, (4-fluorophenyl) propionyl, (4-hydroxyphenyl) propionyl, (4-methoxy) phenyl) propionyl, 2-benzyl-3-phenylpropionyl, 2- (4-fluorobenzyl) -3- (4-fluorophenyl) propionyl, 2- (4-methoxybenzyl) - 3- (4-methoxyphenyl) propionyl or N-benzylcarbamoyl; B is (N-Me) -Val or (N-Me) -Ile; obsession A and B together form an N- (saturated alkyl) carbamoyl group selected from the group consisting of N-butylcarbamoyl, N-isopropylcarbamoyl, N-isobutylcarbamoyl, N- (1-ethylpropyl) ) -carbamoyl, N- (1,1-dimethyl-butyl) -carbamoyl, N- (1-ethyl-butyl) -carbamoyl, N- (1-propyl-butyl) -carbamoyl, N- (1) -ethyl-pentyl) -carbamoyl, N- (1-butyl-pentyl) -carbamoyl, N- (1-ethyl-butyl) -carbamoyl, N- (2-ethyl-pentyl) -carbamoyl, N- (1-methyl-1-propyl-butyl) -carbamoyl, N- (1-ethyl-1-propyl-butyl) -carbamoyl, N- (1,1-dipropyl-butyl) -carbamoyl, N- ( 1-propylcyclopentyl) carbamoyl and és- (1-propylcyclohexyl) carbamoyl; D is Val, Ile or Tbg; R ¹ is 2-propyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methyl -cyclopentyl or a group -NR ⁴ r 5 in which R ⁴ is hydrogen or lower alkyl, and ^R5 is lower alkyl, or R ⁴ and R ^{3 taken} together with the nitrogen atom to which they are attached form pyrrolidino, piperidino, morpholino or 4-methylpiperazino; R ² is hydrogen, and R ³ is methyl, ethylisopropyl, tert-butyl, propyl, allyl or benzyl and the carbon atom to which R ² and R ^{3 are} attached has the (R) configuration; obsession R ² and R ³ are independently methyl or ethyl, or R ² and R ^{3 taken} together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; E is a group of the formula -NHR ^{6 in} which R 1 is isobutyl, 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1 (R) -ethyl-2 , 2-dimethylpropyl, 2- (R, S) -methyl-butyl, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 1 (R), 2,2-trimethyl- butyl, 1 (R), 3,3-trimethyl-butyl, 2-ethyl-butyl, 2,2-diethyl-butyl, 2-ethyl-1 (R) -methyl-butyl, 2-ethyl-2-methyl-butyl, 1 (R) -ethyl-3,3-dimethyl-butyl, 2,2-dimethylpentyl, cis or trans-2-methylcyclohexyl, 2,2-dimethylcyclohexyl or cyclohexylmethyl, or E is a group of the formula -NHCH (R ⁷ ) -Z in which the carbon atom bearing the R ⁷ group is of the (S) -configuration; R ⁷ is tert-butyl, sec-butyl, 2,2-dimethylpropyl or cyclohexylmethyl, and Z is hydroxymethyl, carboxy, carbamoyl, or a group -C (O) O R ⁸ wherein R ⁸ is methyl, ethyl or propyl or a therapeutically acceptable salt thereof. Use of a peptide derivative or a therapeutically acceptable salt thereof according to claim 1, wherein the peptide derivative is a compound of formula (I) A is phenylpropionyl, 2-benzyl-3-phenylpropionyl or N-benzylcarbamoyl; B is (N-Me) Val; D is Tbg; R ¹ represents isopropyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl or 1-methylcyclopentyl group ; R ² is hydrogen; R ² is methyl, ethyl, isopropyl, propyl, or benzyl and has the same carbon atom in the (R) configuration of the substituents R ² and R ² , or R ² and R ² each independently represent methyl or ethyl, or R ² and R ^{3 * * *} , taken together with the carbon to which they are attached, form a cyclobutyl, cyclopentyl, or cyclohexyl group; E is a group represented by the formula -NHR 4, wherein 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethylpropyl -, 2,2-dimethylbutyl or 1 (R) -ethyl-3,3-dimethylbutyl, or E is a group of the formula -NHCH (R ⁷ ) -Z in which R has the (S) -configuration of the carbon bearing the substituent, and R ⁷ is 2,2-dimethylpropyl, and Z is hydroxymethyl, carboxy, carbamoyl, or a group -C (O) OR ⁸ wherein R ⁸ is methyl, ethyl or propyl. The use of a peptide derivative or a therapeutically acceptable salt thereof according to claim 1, wherein the peptide derivative is a compound of formula (I): wherein: A and B together form an N- (saturated alkyl) carbamoyl group selected from the group consisting of N- (1-ethylpropyl) carbamoyl, N- (1-ethylbutyl) carbamoyl, N - (1-propyl-butyl) -carbamoyl, N- (2-ethyl-pentyl) -carbamoyl, N- (1-methyl-1-propyl-butyl) -carbamoyl, N- (1-ethyl-propyl) butyl) carbamoyl, N- (1,1, dipropylbutyl) carbamoyl and N- (1-propylcyclopentyl) carbamoyl; D is Tbg; R ¹ is isopropyl, tert-butyl, isobutyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl or 1-methylcyclopentyl; R ² is hydrogen, and R ² is methyl, ethyl, isopropyl, propyl or benzyl and the carbon atom to which the R ² and R ² substituents are attached has the (R) configuration, or R ² and R ² are independently methyl or ethyl, or R ² and R ^{2 taken} together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; and E is a group of the formula -NHR ^{8 in} which R ⁸ is 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethyl-propyl, 2,2-dimethyl-butyl- or 1 (R) -ethyl-3,3-dimethyl-butyl, or E is a group of the formula -NHCH (R ⁷ ) -Z in which the carbon atom bearing R ⁷ is of the (S) -configuration; R ⁷ is 2,2-dimethylpropyl, and Z is hydroxymethyl, carboxy, carbamoyl or a group -C (O) OR ⁸ wherein R ⁸ is methyl, ethyl or propyl. ··· · ····· · · Use of a peptide derivative selected from the group consisting of: phenylpropionyl (N-Me) Val-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) Asp (cyPn) -Y-Me-leucinol, N- (3-pentyl) -carbamoyl-Tbg-CH ₂ (R) -CH [CH ₂ C (O) CMe ₃ JC (O) -Asp (cyPn) -N-Me-leucinol, N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH- (R) -CH (Et) CMe ₃ , N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ , N- [4- (4-Ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ JC (O) -Asp (cyPn) -NH- (R) -CH (Et) CMe ₃ , and N- [4- (4-ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ . Use of a combination of a peptide derivative or a therapeutically acceptable salt thereof and an antiviral nucleoside analogue or a therapeutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the treatment of acyclovir-resistant herpes simplex virus infections in mammals. The use of a combination according to claim 5, wherein the peptide derivative is a compound of formula (1) or a therapeutically acceptable salt thereof, wherein: A is phenylpropionyl, 2-benzyl-3-phenylpropionyl or N-benzylcarbamoyl, B is (N-Me) Val; D is Tbg; R ¹ represents isopropyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl or 1-methylcyclopentyl group ; R ² is hydrogen, and R ³ is a methyl, ethyl, isopropyl, propyl or benzyl group and has the carbon (R) configuration bearing both R ² and R ³ ; or R ² and R ³ are independently methyl or ethyl, or R ² and R ^{3 taken} together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; E is a group of the formula -NHR ^{8 in} which R ⁸ is 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethyl-propyl, 2,2-dimethyl-butyl- or 1 (R) -ethyl-3,3-dimethyl-butyl, or E is a group of the formula -NHCH (R ⁷ ) -Z in which the carbon atom bearing R ⁷ is of the (S) -configuration; R ⁷ is 2,2-dimethylpropyl, and Z is hydroxymethyl, carboxy, carbamoyl, or a group -C (O) OR ⁸ wherein R ⁸ is methyl, ethyl or propyl. The use of a combination according to claim 5, wherein the peptide derivative is a compound of formula (1):. .. or a therapeutically acceptable salt thereof, wherein A and B together form an N- (saturated alkyl) carbamoyl group selected from the group consisting of N- (1-ethylpropyl) carbamoyl, N- (1-ethylbutyl) carbamoyl, N - (1-propyl-butyl) -carbamoyl, N- (2-ethyl-pentyl) -carbamoyl, N- (1-methyl-1-propyl-butyl) -carbamoyl, N- (1-ethyl-1) -propylbutyl) carbamoyl, N- (1,1-dipropylbutyl) carbamoyl and N- (1-propylcyclopentyl) carbamoyl; D is Tbg; r! isopropyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl or 1-methylcyclopentyl; R ² is hydrogen, and R ² is methyl, ethyl, isopropyl, propyl or benzyl and has the carbon (R) -configuration for both R ² and R ² substituents, or R ² and R ² are each independently methyl or ethyl, or R ² and R ^{2 taken} together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; and E is a group represented by the formula -NHR 1, in which R ⁶ is 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1 (R) -ethyl-2,2-dimethyl-propyl, 2,2-dimethyl-butyl- or 1 (R) -ethyl-3,3-dimethyl-butyl, or • · has the formula -NHCH (R ⁷ ) -Z in which R ^{7 has} the (S) -configuration, and ^R7 is 2,2-dimethylpropyl group, and ···· ···· • · «·· group, a carbon group is hydroxymethyl, carboxyl, carbamoyl, or C (O) ^OR8 radical of formula wherein R ⁸ is methyl, ethyl or propyl. The use of a combination according to claim 5, wherein the peptide derivative is selected from the group consisting of phenylpropionyl (N-Me) Val-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C ( 0) Asp (cyPn) - V-Me-leucinol, N- (3-pentylcarbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -Y-Me-leucinol), N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH- (R) -CH (Et) CMe ₃ , N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ , N- [4- (4-ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH- (R ) -CH (Et) CMe ₃ , and N- [4- (4-ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH-CH ₂ -CMe ₃ . The use of a combination according to claim 5, wherein the nucleoside analog is a compound of formula (2), or a therapeutically acceptable salt thereof, wherein: R.9 is hydrogen, hydroxy or amino. The use of a combination according to claim 5, wherein the nucleoside analogue is selected from the group consisting of vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, brovavir, fiacitabine, penciclovir, famciclovir and rociclovir. Use of a combination according to claim 5, wherein the nucleoside analogue is acyclovir and the peptide derivative is selected from the group consisting of: phenyl-propionyl (N-Me) Val-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -Me-leucinol, N- (3-pentyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -Me-leucinol, N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH- (R) -CH (Et) CMe ₃ , N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ JC (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ , N- [4- (4-ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ) C (O) -Asp (cyPn) -NH- (R ) -CH (Et) CMe ₃ and N- [4- (4-ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ . 12. A pharmaceutical composition for the treatment of acyclovir-resistant herpes simplex virus infections, which comprises as an active ingredient a peptide derivative of the formula (I) or a therapeutically acceptable salt thereof - ··· ·· ben A is phenylacetyl, phenylpropionyl, (4-aminophenyl) propionyl, (4-fluorophenylpropionyl, (4-hydroxyphenyl) propionyl, (4-methoxyphenyl) ) -propionyl, 2-benzyl-3-phenylpropionyl, 2- (4-fluorobenzyl) -3- (4-fluorophenyl) propionyl, 2- (4-methoxybenzyl) -3 - (4-methoxyphenyl) propionyl or N-benzylcarbamoyl; B is (N-Me) Val or (N-Me) -Ile; obsession A and B together form an N- (saturated alkyl) carbamoyl group selected from the group consisting of N-butylcarbamoyl, N- (2-propyl) -carbamoyl, N-isobutylcarbamoyl, N- (1-ethyl-propyl) -carbamoyl, N- (1,1-dimethyl-butyl) -carbamoyl, N- (1-ethyl-butyl-carbamoyl, N- (1-propyl-butyl) -carbamoyl- , N- (1-ethyl-pentyl) -carbamoyl, N- (1-butyl-pentyl) -carbamoyl, N- (1-ethyl-butyl) -carbamoyl, N- (2-ethyl-pentyl) - carbamoyl, N- (1-methyl-1-propyl-butyl) -carbamoyl, N- (1-ethyl-1-propyl) -carbamoyl, N- (1,1-dipropyl-butyl) -carbamoyl, N- (1-propylcyclopentyl) carbamoyl and N- (1-propylcyclohexyl) carbamoyl; D is Val, Ile or Tbg; R ¹ is isopropyl, tert-butyl, sec-butyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl or is a group of the formula NR ⁴ R ^ in which R ⁴ is hydrogen or lower alkyl; ^R5 is lower alkyl, or R and R taken together with the nitrogen atom to which they are attached form pyrrolid67, ηο, piperidino, morpholino or 4-methylpiperazino; R ² is hydrogen, and R ² is methyl, ethyl, isopropyl, tert-butyl, propyl, allyl or benzyl and has the same carbon atom in the (R) configuration of the substituents R ² and R ² , or R ² and R ² are each independently methyl or ethyl, or R ² and R ^{2 taken} together with the carbon atom to which they are attached form cyclobutyl, cyclopentyl or cyclohexyl; and E is a group represented by the formula -NHR 1, wherein isobutyl, 2,2-dimethylpropyl, 1 (R), 2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl -, 1 (R) -ethyl-2,2-dimethylpropyl, 2- (R, S) -methylbutyl, 2,2-dimethyl-butyl, 3,3-dimethyl-butyl, 1 (R), 2,2-trimethyl-butyl, 1 (R), 3,3-trimethyl-butyl, 2-ethyl- butyl, 2,2-diethyl-butyl, 2-ethyl-1 (R) -methyl-butyl, 2-ethyl-2-methylbutyl, 1 (R) -ethyl-3,3-dimethylbutyl, 2,2-dimethylpentyl, cis or trans-2-methylcyclohexyl, 2,2-dimethylcyclohexyl or cyclohexylmethyl, or E is a group of the formula -NHCH (R ⁷ ) -Z in which the carbon atom bearing R ⁷ is of the (S) -configuration, and R ⁷ is tert-butyl, sec-butyl, 2,2-dimethylpropyl-pyl or cyclohexylmethyl; and Z is hydroxymethyl, carboxy, carbamoyl or a -C (O) OR ⁸ group in which R ⁸ is methyl, ethyl or propyl and contains a pharmaceutically acceptable carrier. 13. A 12. The pharmaceutical composition for the treatment of acyclovir resistant according to claim 1, wherein the active ingredient is a combination of claim 12 or a therapeutically acceptable salt thereof and an active nucleoside analogue or a therapeutically acceptable antiviral salt thereof. 14. A pharmaceutical composition according to claim 13, wherein the peptide derivative is selected from the group consisting of: phenyl-propionyl (N-Me) Val-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -Y-Me-leucinol, N- (3-pentyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe] C (O) -Asp (cyPn) -N-Me-leucinol. N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe] C (O) -Asp (cyPn) -NH- (R) -CH (Et) CMe ₃ , N- (4-heptyl) -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ , N- [4- (4-ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₃ ] C (O) -Asp (cyPn) -NH- (R ) -CH (Et) CMe ₃ and> * ··· ··· ♦ [- [4- (4-Ethyl) -heptyl] -carbamoyl-Tbg-CH ₂ - (R) -CH [CH ₂ C (O) CMe ₂ ] C (O) -Asp (cyPn) -NH-CH ₂ CMe ₃ , and the nucleoside analogue acyclovir. The agent shall: