CA2095408A1 - Method of combating acyclovir-resistant herpes simplex viral infections - Google Patents

Abstract

Method of Combating Acyclovir-resistant Herpes Simplex Viral Infections Abstract of the Disclosure Disclosed herein is a method for treating acyclovir-resistant herpes infections in a mammal.
The method comprises administering a peptide derivative or a combination of the peptide derivative and an antiviral nucleoside analog to the infected mammal. The peptide derivative used for the method is represented by the formula A-B-D-CH2CH{CH2C(O)R1}C(O)-NHCH{CR2(R3)COOH}C(O)-E wherein A
is a terminal group, for example an optionally substituted phenylalkanoyl, and B is a N-methyl amino acid residue; or A and B together form a saturated alkylaminocarbonyl; D is an amino acid residue; R2 is, for example, an alkyl, cycloalkyl, or a monosubstituted or a disubstituted amino; R2 is, for example, hydrogen or alkyl and R3 is alkyl, or R2 is hydrogen and R3 is phenylalkyl, or R2 and R3 are joined to from a cycloalkyl; and E is a terminal unit, for example, an alkylamino or a monovalent amino acid radical such as NHCH(alkyl)C(O)OH.

Description

2 0 9 ~ ~ 0 8 ..
~ethod of CoFbating Acyclovir-resistant ~erpes Si~plex Viral I~fections ~ield ~e eb- Y~venti~n :~
This invention concern~ a method for treating acyclovir-resistant herpes infections in a mammal.
The method comprises admini~tering a peptide ~ -derivative or a combination of the peptide derivative and an antiviral nucleo~ide analog.

Backqround of the In~ention Acyclovir i~ the most widely used drug for treating herpes infections. However, the frequency of reports of acyclovir-resi~tant herpes infections has been increasing in recent years;
for example, see P.~. Chatis et al., N. Engl. J.
~ed., 320, 297 (1989) and S. Safrin, Res. Virol., 143, 125 (1992). Lately, this type of resistant infection i~ being observed much more often in patients with severe human immunodeficiency virus (~IV) infections. A~ a result of ~heir immunocompromized condition, ~he latter patients are very suceptible to infection~ by herpe~
simplex virus (HSV~, type 1 and especially type 2, infections.

The biochemical feature~ which characterize acyclovir-re3istant HSV isolates have received considerable attention; 3ee the review concerning acyclovir re~istant HSY by C.S. Crumpacker, J. ~n.
Acad. Dermatol., 18, 190 (1988). Briefly, the resistant strains display functional alterations in one or both of the virally encoded enzymes, thymidine kinase (TK) or DNA polymerase (POL).

2~4~8 In ~he instance where cells, infected with a wild type HSV, are exposed to acyclovir, viral T~
catalyses the monophosphorylation of acyclovir to a much greater extent than cellular enzyme~. The S resulting monophosphate sub~equently is trans formed by cellular enzymes to triphosphorylated acyclovir. The latter triphosphate interacts more readily with viral DNA polymerase than with cellulax DNA polymerases. Consequently, it is able to reduce viral DNA ~ynthesi~ by becoming an alternate substrate for the viral enzyme, and by acting a~ a chain terminator.

Two resistance mechanisms involving viral thymidine kina~e have been described: (1) the selection of thymidi~e kinase-deficient mutants that induce very little enzyme activity after infection, and (b) the selection of mutants posse~sing a thymidine kinase of altered sub~trate ~pecificity that is able to phosphorylate thymidine but not acyclovir.

A third resistance mechanism involving D~A
polymerase is the result of the selection of mutants encoding an altered ~nz~me which i5 resistant to inactivation by acyclovir triphosphate.

Based on the resi~tance mechanism, acyclovir resistant HSV isolate~ can be classified as thymidine deficient (TKD) strains, thymidine altered 1TKA) strain~ or DNA polymi~rase altered (POLA) strains.

Although some success ha~ been reported for the treatment o~ acyclovir-resistant ~SV

3 209~

infection~ with the antiviral agent foscarnet, the acyclovir-resistant type of infection i~ being encountered with increasing frequency. It is now considered to ~e a wide spread problem in AIDS
patient~; see K.S. ~rlich et al., N. Engl. J.
Med., 320, 293 (1989) and SO Safrin, J. Acquired Immun. Defic. Syndr., 5 (Suppl. 1), S29 (1992).
Hence, there is a great need for means to manage this manifestation.
The present invention provides an effective, relatively afe method for the treatment of acyclovir-resistant herpes infections.

The method involve3 the u~e of peptide derivatives described by P.L. Beaulieu, R. Déziel, N. Moss and R. Plante in copending patent application PCT/CA/93/0095, filed March 12, 1993 directed to the use of the compounds for treating herpes infections. It i~ known, however, that antiviral agents effective against herpe~
infections are not necessarily effectivP again~t acyclovir-re3i3tant herpe~ simplex virus; for example see J.J. O'Brien and D.~. C~mpoli-Richards, Drugs 37, 233 (1989), pp 252-253. It wa~ therefore surprising and rewarding to find that the present peptide derivativss were effective against acyclovir-resi~tant herpes ~implex virus.
Summary of the Invention 'rhe pre~ent invention provide~ a method for treating acyclovir-re~istant herpes simplex viral in~ection~ in a mammal~ The method compri3e~
admini~tering to the mammal an anti-acyclovir-4 2 ~ û 8 re~istant herpe~ effec~ive amount of a peptide derivative of formula 1 : ~ ;

A-B-D-cH2cH{cH2c(o)Rl}clo)-NHcH{cR2(R3)cooH}c(o)-E
~; 1 wherein A is phenylacetyl, phenylpropionyl, ~4-aminophenyl)propionyl, (4--fluorophenyl)propionyl, (4-hydroxyphenyl)propionyl, (4-methoxyphenyl)~
propionyl, 2-(phenylmethyl)-3-phenylpropionyl, 2~
10 {(4-fluorophenyl)methyl}-3-(4-fluorophenyl~pro- :
pionyl, 2-{~4-methoxyphenyl)methyl}~3-(4-methoxy-phenyl)propionyl or benzyl~minocarbonyl; B is (N-Me)-Val or ~N-Me)-Ile; or A and B taken together form a saturated alkylaminocarbonyl selected from the group of bukylaminocarbonyl, 1-methylethylaminocarbonyl, l-methylpropylamino-carbonyl, 1-ethylpropylaminocarbonyl, 1,1-dimethetylbutylaminocarbonyl, l-ethylbutylamino-carbonyl, 1-propylbutylaminocarbonyl, 1-ethylpent-ylaminocarbonyl, l-butylpentylaminocarbonyl, 1-ethylbutylaminocarbonyl, 2-ethylpentylaminocar~on-yl, l-methyl-l-propylbutylaminocarbonyl, 1-ethyl-1-propylbutylaminocarbonyl, 1,1-dipropylbutyl-aminocarbonyl, ~1-propylcyclopentyl)aminocarbonyl and (1-propylcyclohexyl)aminocarbonyl; D i~ Val, Ile or Tbg; Rl i 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, l-methylcyclopentyl, NR4R5 wherein R4 is hydrogen or lower alkyl and R5 is lower alkyl, or R4 and R5 together with khe nitrogen atom to which they are attached form a pyrrolidino, piperidino, morpholino or 4-methylpiperazino; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, 1,1-dimethylethyl, propyl, 2-propenyl or benzyl, and the carbon atom beariny R2 and R3 ~ V .,~ : - ' ' : ' 2 09 ~

has the (R)-configuration, or R2 and R3 each independently i~ methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or S cyclohexyl; and E is NHR6 wherein R6 is 2-methylpropyl, 2,2-dimethylpropyl, l(R),2,~-trimethylpropyl, 1,1,2,2-tetramethylpropyl, l(R~-ethyl-2,2-dimethylpropyl, 2-(R~S)-methylbutyl, 2,2-dilmathylbutyl, 3,3-dimethylbutyl, l(R)~2,2-trimethylbutyl, l~R),3,3-trimethylbutyl, 2-ethylbutyl, 2,2-diethylbutyl, 2-ethyl-l(R)-methylbutyl, 2-ethyl-2-methylbutyl, l(R)-ethyl-3,3-dimethylbutyl, 2,2-dimethylpentyl, cis- or trans-2-methylcyclohexyl, 2,2-dimethylcyclohexyl or cyclohexylmethyl; or E is NHCH(R7)-Z wherein the carbon atom bearing R7 ha~ the (S)-configuration/ R7 is l,l-dimethylethyl, 1-methylpropyl, 2-methylprspyl, 2,2-dimethylpropyl or cyclohexylmethyl and Z is C~2OH, C(O)OH, C~O)NH2 or C(o)oR3 wherein R8 is methyl, ethyl or propyl; or a therapeutically acceptable salt thereof.

A preferred method of this invention for treating acyclovir-re~i~tant herpe~ simplex infections comprises adminiistering a peptide of formula 1 wherein A is phenylpropionyl, 2-(phenylmethyl)-3-phenylpropionyl or benzylami~o-carbonyl; B is (N-Me~Val; D is Tbg; R~
methylethyl, l,l-dimethylethyl, l-methylpropyl, l,l-dimethylp.ropyl, 2,2-dimethylpropyl, cyclobut-yl, cyclopintyl, cyclohexyl or 1-methylcyclopentyl; R2 is hydrogen and R3 is methyl, ethyl, l-methylethyl, propyl or benzyl, and the carbon atom bearing ~2 and R3 has the (R)-configuration, or R2 and ~3 each independently is '-. . i- ' . ~ . : .

6 2~9~
.
methyl or ethyl, or ~2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and ~
NHR6 wherein R6 is 2,2-dimethylpropyl, l(R~,2,2-S trimethylpxopyl, l(R)-ethyl-2,2-dimethylpropyl, 2,2-dimethylbutyl or llR)-ethyl-3,3-dimethylbutyl or E is NHCH(R7)-Z wherein the car~on atom bearing R7 ha~ the (S)-configuration, R7 i~ 2,2-dimethylpropyl and Z i~ CH2OH, C(O)OH, C(O)NH2 or C(O)OR8 wherein R8 i5 methyl, ethy:L or propyl; or a therapeutically acceptable salt thereof.

Another prsferred method for treating acyclovir-resistant herpes simplex infections co~prises administering a peptide derivative of formula 1 wher~in A and B together form a saturated alkylaminocarbonyl selected from the group consisting of l-ethylpropylaminocarbonyl, 1-ethylbutylaminocarbonyl, 1-propylbutylaminocarbon-yl, 2-ethylpentylaminocarbonyl, 1-methyl-1-propylbutylaminocarbonyl, l-~thyl-l propylbutyl-aminocarbonyl, 1,1-dipropylbutylaminocarbonyl and (1-propylcyclopentyl)aminocarbonyl; and D, R1, R2, R3 and E are as defined in the last in~tance, or a therapeutically acceptable salt thereof.

Another aspect of this i~vention involve3 a method of treating`an acyclovir-re3istant herples viral infection in a mammal by administering thereto an anti-acyclovir-re~istant herpes effective amount of a combination of the peptide derivatlve o~ formula 1, or a therapeutically acceptable ~alt thereof, and an antivir,al nucleo~ide analog, or a therapPutically acceptable ~alt thereofO

;~: ' ' ' . "".'.'.~

7 209~8 The antiviral nucleo3ide analog employed in the combination is one which is enzymatically convertible (in vivo) to a viral DNA polymera~e inhibitor of, and/or an alternative substrate for, a herpe~ DNA polymerase. The antiviral nucleo~ide analog can be ~elected from known nucleoside analog~ Preferred nucleo~ide analogs of the invention include acyclovir and its analogs, for example, the compound~ of formula 2 R

wherein R9 i~ hydrogen, hydroxy or amino, or a therapeutically acceptable salt thereof. (Formula lS 2 wherein R9 is hydroxy represents acyclovir.) :
Other preferred antiviral nucleo~ide analog~
for use according to the present invention include vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, brovavir, fiacitabine, penciclovir, famciclovir and rociclovir.

Description of the Drawin~

Figures 1 and 2 are ~raphic representations of re~ult~ obtained by applying the isobole method for demonstrating ~ynergy in a ~tudy involving the activiky of combination~ of acyclovir and peptide derivativP of formula 1 against acyclovir-resistant herpe~ ~implex viru~es.

"~

8 2~4~8 , - `
Details of the_Invention G~NERAL

Alternatively, formula 1 can be illustrated as:
~ COR

A-B-D` ~ NH ~

o R2 COOH
R

The term "residue" with reference to an amino acid or amino acid derivative mean3 a radical derived from the corresponding a-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the a~amino group.

In general t the abbreviation~ used herein for designating the amino acids and the protective group~ are based on recommendations of the IUPAC-IUB Co~mision of Biochemical Nomenclatur~, ~ee European Journal of Biochemistry 1~, 9 ~1984).
For in~tance, Val, Ile, A3p, and Leu represent th xe~idue~ of L-valine, L-i~oleucin~, L-aspartic acid and L-leucine, respectively.

The a~ymmetric carbon atoms re~iding in the principal linear axis (i.e. the backbone~ of the peptide derivatives of formula 1/ exclusive of the terminal groups A and Z (of E) but including the carbon atom bearing "R7" when E i~ NHCH(R7)-Z a~
defined herein, have an S configuration~ An exception occur3l however, for the carbon atom 9 2 0 ~ 8 , bearing the CH2C(O)R1 ~ide chain wherein Rl is a lower alkyl or lower cycloalkyl as defined h~rein.
For the latter exception, the carbon atom has the R configuration.

Asymmetric carbon atoms residing in the side chain of an amino acid or derived amino acid re~idue, in the terminal group A, and in the terminal group E when E represent~ N~R6 as defined herein, may have the S or configuration.

The symhols "~e", "Et"~ "Pr" and "Bu"
represent the alkyl radicals m~thyl, ethyl, propyl and butyl, re~pectively.
The symbols "MeEt2C" and "EtPr2C" for example repre~ent the radical3 l-ethyl-1-methylpropyl and l-ethyl-l-propylbutyl, re~pectively.
;
The symbol "Tbg" repr~sents the amino acid re~idue of (S)-2-amino-3,3-dimethylbutanoic acid.
"~MeLeu" represent~ the amino acid residue of (S)-2-amino-4,4-dimethylpentanoic acid. The symbol "yMeLeucinol" represents (5)-2-~mino-4,4-dimethylpenkanol with one hydrogen remo~ed from the a-amino group.

Other symbols used herein are- (N-Me)Val for the residue of ts) 3 methyl-2-(methylamino)-butanoic acld; (N-Me)Ile for khe residue of (S)-3-methyl-2-(methylamino)pentanoic acid; (N-Me3)Tbg for the residue of (S)-~-(methylamino)-3,3-dimethyl butanoic acid; Asp(cyBu~ for the residue o~ (5)-a-amino-1-carboxycyclobutaneacekic acid;
and Asp(cyPn) for the re idue of (S)-a-amino-1-carboxycyclopentaneacetic acid.

"'` . ', ' ~ ~ , ' `
~:'' '' . ., ` ~ I :' ;''.'''' ' 2 0 9 5 ~ 0 8 .

The term "lower alkyl" a~ used herein, either alone or in combination with another radical, means straight chain alkyl radicals containing one to six carbon atoms and branched chain alkyl S radicals containing three to ~ix carbon atom~ and includes methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl r 2-methylpropyl and 1,l-dimlethylethyl.

The term "1-(lower alkyl)-(lower cycloalkyl)"
as used herein means a lower cycloalkyl radical bearing a lower alkyl 3ub~tituent at position 1;
for example, 1-ethylcyclopropyl, l-propylcyclo-pentyl and l-propylcyclohexyl.
The term "lower cycloalkyl" a~ used herein, either alone or in combination with another radical, mean~ saturated cyclic hydrocarbon radicals containing from ~hree to 9iX carbon atom3 and includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term "pharmaceutically acceptable carrier" as use herein means a non-toxic, generally inert vehicle for the acti~e ingredient which does not adversely affect the ingredient.

The term "phy~iologically 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 ingredient contained thereinO

The term "effective amount" mean~ a predetermined antiviral amount o-f the antiviral .~.: . ., ','. ~, ~ ~ !

~1 209~08 agent, i.e. an amount of the agent ~ufficient to be effective again~t the viral organisms in vivo.

The term "coupling agent" as used herein mean~ an agen~ capable of effecting the dehydrative coupling of an amino acid or peptide free carboxy group with a free amino group of another amino acid or peptide to form an amide bond between the reactant~. Similarly, ~uch agents can effect the coupling of an acid and an alcohol to form corre~ponding e~ters. The agents promote or facilitate the dehydrative coupling by activating the carboxy group. Descriptions of such coupling agents and activated groups are included in general text books of peptide chemi~try; for in~tance, E. Schroder and K.L.
Lubke, "The Peptides", VolO 1, Academic Press, ~ew York, N~Yo ~ 1965~ pp 2-128, and K.D. Kopple, "Peptides and Amino acid~", W.A. Benjamin, Inc., New York, N.Y., 1966, pp 33-51. Examples of coupling agents are diphenylphosphoryl azide, l,l/-carbonyldiimidazol~, dicyclohexyl-carbodiimide, N-hydroxy~uccinimide, or l-hydroxy-benzotriazole ln the presence of dicycl~-hexylcarbodiimide. A very practical and useful coupling agent i5 (benzotriazol-l-yloxy~tri~-(dimethylamino)phosphonium hexafluorophosphate, described by B~ Ca~tro et al., Tetrahedron Letters, 1219 (lg75~, see also D. Hudson, J. Org.
Chem., 53, 617 (1988)~ either by itself or in the presence of l-hydroxybenzotriazole~ Still another very practical and useful coupling age~t is the commercially available 2-(lH-benzotriazol-l-yl)-N, N, N/, N/-tetramethyluronium tetrafluoroborate.

.~ ~
,;.:~:

1~ 2D~D8 The antiviral nucleo~ide analog~, and their therapeutically acceptable salts, for use according to the present invention are a well known class of compounds. As noted abovs, the S members of this clas are characterized by the manner in which they mediate an antiviral effect again3t herpes viruses, i.e. by in vivo inhibition of viral DNA poly~erase. Important members of this cla~s are acyclovir and its analogs which are described by H.J. Schaeffer in US patent 4,19~,574, issued April 22, 1980; see al o H.J. Schaeffer et al., Nature (London), 272, 583 (1978) and T.A. Krenitsk et al., Proc. Natl. Acad.
Sci. USA, 81, 3209 (1984). The compound of formula 2 wherein R9 is hydroxy is "acyclovir", also known by its chemical name, 9-[(2-hydroxy~
ethoxy~methyl3guanine. The compound of formula 2 wherein R9 i~ hydrogen has the names 6-deoxyacyclovir and 2-amino-9-[(2-hydroxyethoxy)methyl]adenine; and the compound offormula 2 wherein R9 i5 amino has the chemical name, 2,6-diamino-9-[(2 hydroxyetho~y)-methyl]purine.

Is to be under3tood that the csmpound of formula 2 in which R9 is hydroxy can exist in it~
tautomeric form, i.e. 2-amino-1,9-dihydro-9-1(2-hydroxyethoxy)methyl~-6H-purin-6-one, and that the compound can be a mixture of the two tautomeric forms, the percentage of each tautomer in the mixture being dependent on the physical environment of the compoundO Tautomeric forms also are possible for the other antiviral nucleoside analog~ having an enolizable carbonyl.

13 2 09 5~ 08 " .
Other antiviral nucleosides contemplated for u~e according to the pre~ent invention include vidarabine (9-~-D-arabinofuranosyladenine mono-hydrate), see R.J. Whitley et al., N. Engl. J.
S ~ed., 307, 971 (1982); idoxudine (2/-deoxy-5-iodouridine), ~ee W.H. Pru30ff, Biochim. Biophys.
Acta, 32, 295 (1959~; trifluridine ~2/-deoxy-5-~trifluoromethyl)uridine], see C. Eleidelberger, US
patent 3,201,387, i~sued Augu~t 17, 1965, 10 ganciclovir 9-[(1,3-dihydroxy-2-propoxy)methyl]-guanine, see J.P. Verheyden and J.C. Martin~ US
paten~ 4,355,032, i~sued October 19, 1982;
edoxudine (5-ethyl-2/-deoxyuridine), 3ee K.K.
Gauris US patent 3,553,192, is~ued January 5, 1971; brovavir [~E)-5-~2-hromovinyl)-2/-deoxyuridine], see Y. ~enoît et al., Eur. J.
Pediatric~, 143, 198 ~1985); fiacitabine (2/-fluoro-deoxy-5-iodouridine), see B. Leyland-Jones et al., J. Infect. Di~., 154, 430 (1986), penciclovir (9-[4-hydroxy-3 (hydroxy-methyl)-butyl~guanine, ~ee S.E. Fowler Qt al., Br. J.
Clin. Pharmacol., 28, 236P ~1989~; famci~lovir (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-diisopropoxy-2-propoxy~methyl]adenine, ~ee E. Winklemann et al., Arzneim.-Forsch., 38, 1545 (~98~).

~ of E~m~

The peptide derivatives of formula 1 can ~e prepared by processes which incorporate therein methods commonly u3ed in peptide synthe3i~ ~uch a~
the cla~ical solution coupling of amino acid 14 2095~
, , residue3 andJor peptide fras~ent~. Such methods are de~cribed, for example, by E. Schroder and Ko Lubke, cited above, in the textbook serie3, "The Peptides: ~nalysis, Synthesis, ~iology", E. Gro~s et al., Ed~., Academic Pres~, New York, N.Y., 1979-1987, Volume~ 1 to 8, and by JoM~ Stewart and J.D. Young in "Solid Pha~e Peptide ~ynthesis", 2nd ed., Pierce Chem. Co., Rockford, IL, USA, 1984.

~ common feature of the aforementioned processes for the peptide derivatives is the protection of the reactive ~ide chain groups of the various amino acid residue~ or derived amino acid residues (or, if required, non-peptidic fragments of the peptide derivative) with suitable protective group~ which will preven~ a chemical reaction from occurring at that site until the protective group is ultimately removed. Also common i5 the protection of an a-amino group on an amino acid or a fragment while that entity react~
at the carboxy group, followed by the selective removal of the a-amino prokective group to allow ~ub3equent reaction to taXe place at that location. AnothPr common feature is khe initial protection of the C-terminal carboxyl of the amino acid residue or peptide fragment, if pre~ent, which i~ to become the C-terminal function of khe peptide derivative, with a ~uitable protective group which will prevent a chemical reaction fro:m occurring at that 3ike until the protecti~e group i~ removed after the desired ~equence of the peptide derivative has been a~sembled.

A key intermediate for the pepkide~ of formula 1 i~ the intermediate of formula 2 209S~08 W-D-CH2CH{CH2C(O)Rl}C(O)OH

wherein W is an a-aminoprotective group, e.g.
tert-butyloxycarbonyl ~Boc), benzyloxycarbonyl (Z) S or fluoren-9-ylmethoxycarbonyl (Fmoc), and D and R1 are as defined herein. The c~mpound can be prepared by a Michael addition of an allyl ester of the formula W-D-C~2C(O)OC~2CH=CH2 to a fumaroyl derivative of the formula (lower alkyl)-OC(O)CH=C~C(O)R1 to give the Michael adduct offormula W-D-CH{C(~)OCH2CH=CH2~CH{CH2C~O~Rl}C(030-(lower alkyl).

Thereafter, treatment of the latter compound lS with tetrakistriphenylphosphine palladium and triphenylphosphine in the presence of pyrrolidine, according to the m~thod of R. Dziel, Tetrahedron Letters, 28, 4371 (1987), effects hydroly~is and subsequent decarboxylation of the allyl ester to give the corresponding alkyl e~ter of the key intermediate. ~ydrolysi~ of the latter e~ter in the presence of a base, e.g. sodium hydroxidP or lithium hydroxide, give~ the key intermediate as a diastereoi30meric mixtureO
Alternatively~ the key intermediate of formula 2 wherein W and D are a3 defined in the last instance and Rl i~ NR4R5 wherein R4 and R5 each i~ lower alkyl or R4 and R5 together with the nitrogen atom to which they are attachsd form a pyrrolidino, piperid.ino, morpholino or 4-methylpiperazino can be prepared a~ ~ollow~: The previou~ly mentioned allyl e~ter of formula W-D-CH2C(O)OC~2C~=CH2 wherein W and D are a~ defined herein i~ reacted according to the condition-~ of the Michael reaction with a fumaroyl derivatiYe of 16 2~5~ i~8 formula BzlOC ( O ) CH=CHC ( O ) OBzl { ( E ) -2-butenedioc acid dibenzyl ester} to give the corresponding Michael adduct of formula W-D-(RS)-CH{CH{C(O)OBzl}CH2C(O)OBzl~C(O)-OCH2CH=CH2.
Treatment of the latter adduct with tetraki~tri-phenylpho~phine palladium and triphenylpho3phine in the presence of pyrrolidin~ (Déziel, vide ~upra) ~ives the corre~ponding dibenzyl e~ter of formula W-D-CH2-(R,S)-CH{CH2C(O)O~zl}C(O)OBZl.
Subsequent hydrogenolysi~ in the presence of palladium hydroxide on carbon gives the corre~ponding dicarboxylic acid which i~
transformed into the Gorrs~ponding anhydride by heating with excess acetic anhydride. Reaction of the anhydride with the appropriate secondary amine in the presence of pyridine gives a mixture of regioisomer~ with a preponderance of the de~ired i30mer of formula W-D-C~2-(RS~-CH{CH2C(o~NR4R5}-C(O)-OH in which W,D and ~R4R5 are as defined herein. The latter product i~ converted to its benzyl e~tsr and the resulting mixture of regioi~omers i5 separated by high performance liquid chromatography to give the de~ired isomer as the preponderant product. Subse~uent hydrogena-tion of the lat~er product in the presence of palladium hydroxide on carbon yield~
the key intermediate of formula 2 wherein W and D
are as defined herein and R1 is N~4R5 as defined hexein.
In general, therefore, a peptide of formula 1 can be prepared by the stepwise coupling, in the order of the ~equence of the peptide, of the appropriate amino acid or derived amino a~id residue~, and non-peptidic fragment~ of the peptide (such a~ the key intermadia-tes), which if required are ~uitably protected, and eliminating :. :. ~ . , :: ~ , .
:: : : .., ~ .: :: . - : :
": . : ~ ~ ; `, :

17 ~D~

all protecting group~, if present, at the completion of th~ stepwise coupling to obtain the peptide of formula 1. More specific processes are illustrated in the example~ hereinafterO
s The peptide of formula 1 of this invention can be obtained in the form of a therapeutically acceptable salt. In the instance where a particular peptide has a re3idue which functions as a base, examples of such salts of the base are those with organic acids, e.gO acetic, lactic, succinic, methanesulfonic or p-toluenesulfonic acid, as well as polymeric acid~ such as tannic acid or carboxymethyl cellulose, and also salt3 with inorganic acids such as hydrohalic acids, e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid. If desiredt a particular acid addition salt is converted into another acid addition salt, such as a non-toxic, pharma-ceutically accepta~le salt, by treatment with theappropriate ion exchange re~in in the manner described by R.A. ~oissonnas et al., Helv. Chim.
Acta, 43, 1849 11960).

In the instance where a particular peptide has one or more freP carboxy group~, examples of such salts of the carboxy group are those with the sodium, potassium or calcium cations, or with organic base , for example, triethyl~mine or N-m~thylmorpholine.

~ntL~hexpe5 Activity The antiviral activity of the peptide derivatives of formula 1 can be demonstrated b~y biochemical, microbiological and bioloyical 18 2 0 9 ~ 08 procedures ~howing the inhibitory effect of the compound~ on the replication of the acyclovir-resi~tant herpes simplex viruses, type~ 1 and 2 (HSV-l and HSV-2).
S ~.
A method for demonstrating the inhibitory effect of the p~ptide derivatives of formula 1 on viral ~plication i~ the cell culture technique;
~ee, for example, T. Spector et al., Proc. Natl.
10Acad. Sci. USA, 82, 4254 (1985).

The therapeutic effect of the peptide derivatives can be demon~trated in laboratory animal~, for instance, by u~ing an as~ay baaed on the murine model of herpe~ ~implex virus-induced ocular di~ease for antiviral drug te~ting, described by C.R. Brandt et al., J0 Virol. Meth., 36, 209 (1992).

20~hen a peptide derivative of this invention, or on~ of its therapeutically acceptable 3alt~
employed as an antiviral agent, it is admini~tered topically or systemically to warm-blooded animal3, e.g. humans, pigs or hor~e~, in a vehicle 25comprising one or more pharmaceu~ically acceptable carrier~, the proportion of which i5 determined by the ~olubility and chemical nature of the peptide derivative, cho~en `route of admini~tration and ~tandard biological practice. For topical 30administration, the peptide derivative can be formulated in pharmaceutically accepted vehicle~
containing 0.1 to 5 percent, preferably 0.5 to 2 percent, of the active agent. Such formulation~
can be in the form of a solution, cream or lotion.

19 2~

For ~y~temic admini~tration, the peptide derivative of formula 1 is administered orally or by either intravenous, ~ubcutaneous or intramus-cular injection, in composition~ with pharmaceuti-cally acceptable vehicles or carriers. Foradmini~tration by injection, it i~ preferred to u~e the peptide in solution in a ~terile aqueous vehicle which may al~o contain other ~olute such as buffer~ or pre~ervative~ a~ well a~ sfficient quantitie3 of pharmaceutically acceptable salt~ or of gluco~e to make the solution isotonic.

Suitable vehicle~ or carrier~ for the above noted formulations are de~cribed in standard pharmaceutical text~, e.g. in "Remington's Pharmaceutical Sciences", 18th ed, Mack Publi~hing Comp~ny, Easton, Penn., 1990.

The dosage of the peptide derivative will vary with the form of admini~tration and the particular active agent chosen. Furthermore, it will vary with ~he particular host under treatment. Generally, treatment is initiated with ~mall increment~ until the optimum effeck under the circumstance~ is reached. In general, the peptide derivati~e i~ most desirably administer!ed at a concentration level that will generally afford antivirally effective re~ultx without cau~ing any harmful or deleteriou~ ~ide effect3.
With reference to topical application, the peptide derivative is administered directly in a suita~le topical formulation ~o the infected area of the body e.g. the ~kin, the eye, or part of the oral or genital cavity, in an amount sufficien-t ko cover the infected area. The treatment ~hould be ... ,, ~ .. . ........... . . .

:,'~'' .. . ` :' . ' , 20 20~0~

repeated, for example, every four to six hours until lesion~ heal.

With reference to systemic admini~tration, S the peptide derivative of formula 1 is admini~tered at a dosage of 1.0 mg to 10 mg per kilogram of body weight per day, although the aforementioned variations will occur. However, a do~age level that i in the range of from about 1.0 mg to 5 mg pex kilogram of body weight p~r day is most de irably employed in order to achieve effective re~ult~

Although the formulation disclo~ed hereinabove are indicated to be effective and relatively safe medications for treating herpes viral infections, the pos~ible concurrent administration of these formulations with other antiviral medication~ or agents to obtain beneficial results is not excluded. Such antiviral medication or agents include the previously noted antiviral nucleosides, antiviral ~urface active agent~ or antiviral interferons such as those disclosed by S.S. A~culai and F.
Rapp in U.S. patent 4,507,2B1, ~arch 2S, 1985.

More specifically with respect to treaking acyclovir-re~istant herpes viral infection~ by concurrent administration, it has bsen found that the antiherpe~ activity of an antiviral nucleoside analogs can be enhanced ~ynergi~tically, without the concomitant enhancement of toxic effects, by combining the same with a peptide derivative of formula 1. Accordingly, there is pro~ided herewith a pharmaceutical composition for treating acyclovir-resi~tant herpe~ infections in a mammal ,. ~, .. :
,".,. : ;
,:: . : : .
: .:, :

21 2 ~ 8 comprising a pharmaceutically or veterinarily acceptable carrier, and an effective amount of the combination of an antiviral nucleo3ide analog or a therapeutically acceptable salt thereof, and a S peptide derivative of formula 1 or a therapeutically acceptable salt thereof.

The term "~ynergistic effectl' when used in relation to the antiviral or antiherpes activity of the above defined combination of the nucleoside analog and peptide derivative of formula 1 means an antiviral or antiherpes effect which i3 greater than the predictive additive effect of the two individual components of the combination.
When utilizing the combination of this invention ~or tr~ating herpes acyclovir-re~istant infection~, the combination is administered to warm blooded animals, e.g. human~, pig~ or hor~e~, 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 derivative of formula 1, chosen route of administration, ~tandard biological practic~, and by the relative ~mounts of the two active ingredients to provide a synergistic antiviral effect. Tn a prPferred ~:
manner, the combination is administered topically.
For example, the two active agents ~i.e. the antiviral nucleoside analog and the pPptide derivative of formula 1, or their therap~utically accepta~le salts) can be formulated in the form of ~olution~, emul~ions, creams, or lotion~ in pharmaceutically acceptable vehicles. Such formulation can contain 0.01 to 1.0 percent by 22 2095~08 weight of the nucleoside analog, or a therapeutically acceptable alt thereof, and about 0.05 to 1 percent by weight of the peptide derivative of formula 1, or a therapeutically acceptable salt thereof.

In any event, the two active agent~ are pre~ent in the pharmaceutical composition in amounts to provide a ~ynergistic antiherpe~
effect.

~ he following examples illustrate further this invention. Temperatures are given in degrees Celsius. Solution percentages or ratios express a volume to volume relationship, unless ~tated otherwise. Nuclear magnetic resonance spectxa were recorded on a Bruker 200 M~z or 400 MHz spectrometer (a 400 MHz spectrum being noted :in the preamble); the chemical shifts (~) axe reproted in parts per million. Abbreviations u~ed in the examples include Boc: tert-butyloxycarbonyl; Bu: butyl; Bzl: benzyl; DMF:
dimethylformamide; Et: ethyl; E~OH: ethanol;
EtOAc. ethyl acetate; Et2O: diethyl ether; Me:
methyl; MeOHO methanol; Pr: propyl; T~C: thin layer chromatography; THF: tetrahydrofuran.
'~"
~. :

~

{See also R. Rnorr et al., Tetrahedron Letter~, 30, 1927 (19B9).}

The fir~t reactant, iOe. a free ~mine (or it~
hydrochloride salt), is di~solved in CH2C12 or ,.,, ~ . - - - ~ , . .

~ r~ ~

23 2~9~

acetonitrile and the 301ution i5 cooled to 4 .
Under a nitrogen atmosphere, four equivalents of N-methylmorpholine i~ added to the ~tirred ~olution. After 20 min., one equivalent of the second reactant, i.e. a free carboxylic acid, and 1.05 equivalent of the coupling agent are added.
~Practical and efficient coupling reagents for thi~ purpose are ~benzotriazol-1-yloxy)tris-(dimethylamino)pho~phonium hexafluorophosphat~ or preferably 2-(lH-benzotriazol-1-yl)-N,~,Nt,N/-tetramethyluronium tetrafluoroborate. The reactio~ i3 monitered by TLC. After completion of the reaction, the CH2C12 ~or acetonitrile) i~
Pvaporated under reduced pre~sure. The residue i~
di~solved in EtOAc. The solution i~ washed ~uccessively with lN aqueous citric acid, 10~
aqueous Na2C03 and brine. The oxganic phase is dried (MgS04), filt~red and concentrated to dryness under reduced pressure. The residue i~
purified on silica gel (SiO2) according to Still' 9 flash chromatography technique {W C. Still et alO, J. Org. Chem., 43~ 2923 ~1978)}.

Example 2 Pre~aration of the ~ 1-CMe31CH20Bzl (a) (S)-a-Azido-1-{~phenylmethoxy)carbonyl}-cyclopentaneacetic acid: This compound was prepared from 2-oxo~piro[4.4]nonane-1,3-cllone, de~cribed by M.M~ Aboul-Enein et al., Pharm. Acta Helv., S5, 50 (1980), according to the a~ymmetric azidation method utilizing the Evan' 3 auxiliary, see D.A. Evans et al., J. Amer. Chem. Soc., 112, 4011 (1990).

: . . . ~ . . ~ :

24 2~9540~

More explicitly, a 1.5 M hexane solution of butyllithium (469 ml, 750 mmol) was added dropwis~
under an argon atmosphere to a solution of the chiral auxiliary, 4(S)~ methylethyl)-2-S oxazolidinone, {96.8 g, 750 mmol, descxibed by h.
N. Pridgen and J. Prol., J. Org. Chem., 54, 3231 (1989)} in dry THF at -40 The mixture was stirred at -40 for 30 min and then cooled to 78 . 2-Oxospiro[4~4]nonane-1,3-dione was added 10 dropwise to the cooled mixture. The mixture then was stirred at 0 for 1 h. Thereafter, a 20%
(w/v) aqueous solution of citric acid (600 mL) was added to the mixture. The organic pha~e was separated and the aqueous phase was extracted wikh 15 EtOAc. The combined organic phases were washled with brine, dried (Mg50~) and concentrated und~er reduced pre~sure to give 3-{2~ carboxy-cyclopentyl)-1-oxoethyl~}-4(S)-(1-methylethyl)-2-oxazolidinone as a pink ~olid (300 g).
2~
The latter solid (ca 750 mmol) was dissolved in acetonitrile ~ enzyl bromide (128.3 g, 89.2 mL, 750 mmol) and ~,8-diazabicyclor5.4.0]-undec-7-ene (114 g, 112 mL, 750 mmol) were added ~5 to the 501ution. The mixture was skirred under argon for 16 h. The volatiles were removed und~r reduced pres~ure. The residue wa~ di3solved in H2O/EtOAc. The organic pha~e wa~ separated, washed with a 10% ~w/v) aqueous solution of citric acid~
30 brine, dried (MgSO4) and concentrated to drynes~
under reducad pre~sure to give an oi]L.
Crystallization of the oil from hexane/EkOAc ga~
khe corresponding ben2yl ester as a white solid (~4 g, 73%).
t n. . : .. . . ~ . .. . . .

.` -' . - .: ; ~ ..
, ' ,. ' A ~olution of the latter compound (70 g, 187 mmol) in dry THF (200 mh) was cooled to -78 . A
0.66 M THF solution of potas~ium 1,1,1,3,3,3-hexamethyldisilazane (286 mL, 189 mmol) containing 6% (w/v) cumene wa~ added over a period of 15 min to the cooled solution. The mixture wa-~ stirred at -78 for 45 min. A solution of 2,4,6-triisopropyl-benzenesulfonyl azide (67 g, 215 mmol) in dry TH~ 0 mL) wa~ addad in one portion to the cold mixture, ~ollowed two minute~ later by the addition of glacial acetic acid (50 mL, ~60 mmol). The mixture was warmed and tirred at 35-45 for 1 h. The volatiles were removed under reduced pressure. The yellow residue wa~
triturated with hexane/EtOH (4:1, 1.7 L). The re~ulting white solid was collected on a filtex.
The filtrate was mixed with SiO2 (230-240 m~sh).
Volatile~ were removed under reduced pressure and the residual solid was dried a~ 35 under reduced pressure to remove cumene. The residual ~olid then wa~ plac~d on a column of SiO2. Elution of residual solid and SiO2 with hexane-EtOAc, 9:1 and concentration of the eluent gave 3-{~S3-azido-1-oxo-2-{1-{(phenylmethoxy)carbonyl~cyclopen-tyl}-ethyl}-4(S)-(1-methylethyl)-2-oxazolidinonQ [66 g, 86%~.

A solution of the latter compound ~13 7 42 g, 32.4 mmol) in THF/~20 (3:1, 608 mL) wa~ cooled to 0 . Hydrogen peroxids/H20 (3:7, 16.3 mL, 518 mmol of H202) was added to the cooled ~olution, followed by the addition of LiO~H20 (2.86 g, S8.~
mmol). The mixture wa~ ~tirred at O for 45 min and then quenched wlth a 10% (w/v) aqueous solution of sodium ~ulfite (400 mL)~ Afker NaHC03 (1.93 g) had been added, the mixtl~re was ~, .. ,.. ~ .. . , . ~ , ~; ,.. ,. . -: . ~

26 2 ~

concentrated under reduced pressure. The chiral auxiliary was recovered by continuous extraction (aqueous Na~CO3/chloroform) for 20 h. Thereafter, the aqueou~ phase wa~ cooled to 0 rendered acidic by the addi~ion of concentrated HCl and then extracted with EtOAc. The extract wa washed with brine, dried (MgSO4) and concentrated under reduced pres~ure to give the de~ired compound a~ a white solid (8.2 g, 84~). The lH ~MR (CDC13) of the compound ~howed: ~ 1.6-1.8 (m, 5H~, 1.95-2.05 ~m, 2H), 2.20-2.30 (m, lH), 4.55 (s,lH), 5.12 (s,2H) and 7.4 (m,5~), The compound i~ u~ed in section (c) of this example.
(b) NH2-(S)-CH(CH2CMe3)CH2OBzl: H-yMeLeu-O~ was reduced with LiBH4/Me3SiCl according to the method of A. Giannis and K. Sandhoff, ~ngew. Chem. Int.
Ed. Engl., 28, 218 (1989) to give the aminoalcohol N~2-(S)~ H2CMe3)CH2OH. A mixture of the latter compound (812 mg, 6.2 mmol), triethylamine (659 mg, 6.51 mmol~ and di-tert-butyl dicarbonate (1.42 g, 6.51 mmol) in dry THF (15 m~ was stirred under a nitrogen atmosphere at 4 for 15 min and then at room temperature for 4 h. The THF wa~ evaporated under reduced pre~ure. The re~idue was dis301ved in EtOAc. The ~olution wa~ wa~hed with 10%
aqueou~ citric acid, 5% aqueous NaHCO3 and brin~e.
The organic pha~e was dried ~Mg5O4~ and concentrated to dryna~ under reduced pressur~e~
The residue was purified by flash chromatography (SiO2, elu0nt: hexane-~tOAc, 2,1) to give Boc-NH-(S)-CH(CH2CMe3)-CH~OH (1.23 g, 86%).

Tetrabutyl~mmonium bi~ulfate (106 mg) a~d 50%
aqueou~ NaOH (3 mL) were added ~ucce3si~ely to a 27 2 ~9 ~

~olution of Boc-NH-(S)-CH(CH2CMe3)CH2OH (1.23 g, 5.35 mmol) in benzyl chloride (13 mL). The resulting mixture wa~ ~tirred at 35-40 for 90 min, diluted with EtOAc, and washed wi~h H20 and brine. The organic phase was dried (MgSO4) and concentrated to dryne~s under reduced pressure.
The residue wa3 dissolved in hexane. The solution was poured onto a column of SiO2. The column wa~
eluted with hexane to remove benzyl chloride~ and then with hexane-EtO~c (2:1) to give Boc-NH-(S)-CH(CH2CMe3)CH2O~zl. The lH NMR (CDCl3) of the latter compound ~howed ~ 0.95 (~,9H), 1.42 (~
9H), 1.30-1.55 (m, 2~, 3.42 td, J = 4 Hz~ 2 3.88 (broad, lH), 4.54 (m, 3H), 7.23-7.4 (m, 5 The latter compound (1.28 g, 3.99 mmol) wa~
di~olved in 6 N ~ICl/dioxane (10 mL). The solution was ~tirred under a nitrogen atmosphere at 4 for 45 min. Evaporation of the ~olvent gave the hydrogen chloride ~alt of the desired compound (1~05 g)~ The compound i~ used without further purification in the next ~ection of thi~ example.
(c) The title compound of thi~ example: By following the coupling procedure of example 1 and u~ing the hydrogen chloride 3alt of NH2-(S)~
CH(CH2CMe3)CH2OBzl of the preceding ~ection a~ the fir~t reactant and (S)-a-azido-1-{tphenylmethOXy) carbonyl}cyclopentaneacetic acid of ~ection (a) of this example a3 t~e ~econd reactant, ~J-~(5~-:L-benzyloxymethyl-3,3-dimethylbutyl~(5)-a-azido-1 {(phenylmethoxy)carbvnyl} cyclopentaneacetamide wa~ obtained. Reduction of the latter compound with tin(II) chloride in MeOH according to the method of N. Maiti et al., Tetrahedron ~etter~ t ~, 1423 (1986) gave the title compou~d of thi3 example. The lH NMR (CDCl3~ of the compound ~howed ~ 0.98 (~, 9H~, 1,22-2.25 (m, 12H), 3.4 (d, J = 4 :.. ~ .~ . ~ . .

~!.': . ' 2 ~ 8 . i Hz, 2H), 3.64 (s, lH3, 4.18 (broad m, lH~, 4.52 (s, 2H), 5.12 (s, 2H), 7.18 (d, J = 7 Hz, lH), 7.22-7.38 (broad m, 10H).

S j~me~

Preparation of the Ip~aL-~i~t-e~ oç=~eg=--2-( CH~CH~CIOI~M_3~C~O~oH
. ' 10 (a) Magnesium salt of monoallyl malonate: A
solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (100 g; 0.69 mol) and allyl alcohol (47 mL, 0.69 mol in benzene (800 mL) was heated at reflux for 24 h. The solvent was evaporated under reduced pressure. Distillation of the residue under reduced pressure qave monoallyl malonate (71 g, 71%, bp 123-127 /2.7 mm Hg). The latter ester (71 g, 0.48 mol) was dls301ved in dry THF (300 mL).
Magnesium ethoxide (28.5 g, 0,245 mol) waq added to the olution. The mixture was stirred under argon for 4 h at room temperature (20-22~. The solvent was evaporated under reduced pressure and the residue was triturat~d with Et2O to give a tan ~olid. The solid was ground into fine particles and dried under reduced pressure to give the t desired magnesium salt (56 g, 73%). The ~alt i~
used in the next section o~ this e~ample.
(b) Boc-Tbg-Cff2C(O)OCH2=CH~: 1,1-Carbonyldiimidazole (12.6 g, 78 mmoll was added to a solution of Boc Tbg-O~ (15 g, 64 mmol) in dry acetonitrile (150 mL). The mixture was stirred under argon for 2 h at room temperature. The magnesium salt of monoallyl malonate (24 gl 78 mmol) and 4-(N/N-dimethylamino~pyridine (100 mg) were added to the mixture. The mixture wa~ heated at reflux for 1 h and then stirred at room 5 ~ ~ 8 temperature for 18 h. Thereafter, the mixture wa~
concentrated under reduced pre~sure. The re~idue was dis~olved in EtOAc ~300 mL). The solution wa~
washed with 10% aqueous citric acid (2 x 130 mL) S and brine (2 x 100 mL), dried (MgSO4) and evaporated to dryness. The re~idue wa~ purified by fla~h chromatography ~ SiO2, eluent: hexane-EtOAc, S:l) to give the de~ired allyl ester (19.4 g, 96%) as a brown oil which crystallized on ~tanding. lH NMR (CDCl3),note that this compound exi~ts as a mixture of keto-enol tautomer~ in a 3:1 ratio in chloroform, ~ 0.96 (s, 9~, enol form), 1.04 (5, 9H), 1.46 (5, 9H), 3.65 (~, 2H), 3O90 (d, J = 8.5 Hz, 1~, ~nol form), 4.20 (d, J =
7.5 Hz, lH), 4.65 (m, 2H), 5.10 (broad d, J = 7.5 Hz, lH), 5.20-5.40 (m, 2H), 5.80-6.05 (m, lH), 12 (~, lH, enol form). The allyl e~ter is used in section (d) of this example.
(c3 (E)-5,5-Dimethyl 4-oxo-2-hexenoic acid ethyl ester: Thi~ ethyl ester was prepared according to the method of S. Manfredini et al., Tetrahedron hetters, 29, 3997 (1988). The oily crude product was purified by flash chromatography (SiO2, eluent: hexane) to give the desired ethyl ester as a yellow oil. lH NMR (CDCl3) ~ 1.21 (~, 9H~, 1.33 (t, J = 7.5 Hz~ 3H3, 4.28 (q, J = 7.5 Hz, 2H~, 6.78 (d, J = 15.5 Hz, lH), 7.51 (d, J = 15.5 Elz, lH~. The ethyl e~ter is used in the next ~ection of thi~ example.
Id) The title compound of thi~ example: Boc-Tbg-CH2C(O)OCH2=CH2 (0.67 g, 2.1 mmol), described :in section (b) of this example, wa~ dissolved :in anhydrous TRF (25 mL) under an argon atmosphere.
Sodium hydride (60% oil disper~ion, 0.095 g, 2.4 mmol) was added to the solution. The mixture wa~
stirred at room temperature for 30 min. (E)-5/5-.. ^ ~, ~ ~ , :: .
,,. ~ ~ :

30 2 ~ 8 dimethyl-4-oxo-2-hexenoic acid ethyl ester (0.435 g, 2.36 mmol), described in section (c) of thi~
example, was added to the mixture. The reaction mixture was stirred until the reaction was S complete as judged by TLC (about 6 h).
Thereafter, the mixture was quenched with 10%
aqueou~ citric acid. THF wa~ removed under reduced pressure and the resulting concentrate wa~
extracted with EtOAc (3 x 25 mL). The extract wa~
washed with H2O, dried (MgSO4) and concentrated under reduced pressure. The re~idue was purified by flash chromatography (SiO2, eluent: hexane-EtOAc, 9:1) to give the corresponding Michael reaction adduct (1.06 g, 100%).
The Michael adduct was transformed to Boc-Tbg-cH2-(Rs)-cH(cH2c(o)cM83)c(o)oH ~ follow5:
Tetraki~triphenylphosphine palladium(0) (0.20 g, 0.18 mmol) and triphenylphosphine (0.060 g, 0.23 mmol) were dissolved in ~H2C12 ~10 mL~ under an argon atmo~phere. Acetonitrile (20 m~) wa~ added and the solution was cooled to 0 . Pyrrolidine (O.28 mL, 2.7 mmol) and then the Michael adduct (1.06 g~ 2.1 mmol) were added to the ~ollltion.
The mixture was allowed to çome to room temperature over 1 h and then stirred for 20 h.
Thereafter, the reaction m~xture wa~ heated at reflux for 1 h under argon to complete the reaction. The solvent was evapora~ed and the 3~ re~idue wa~ purified by flash chromatography (SiO2, eluent: hexane-EtOAc, 9:1) to give Boc-Tbg-C~2-(RS)-CH(CH2C(O)CMe3)C(O)OE~ (0.77 g, 83~).
Thereafter, the latter compound (0.77 g, 1.7 mmol) wa~ dis~olved in ethyleneglycol dimethyl ether -H2O (1:1, 10 mL). Lithium hydroxide monohydrate (0.31 g, 7.4 mmol) was added to the ~olution. The 31 2~95~08 mixture was stirred at room temperature for 4 h, rendered aridic with 10% aqueous citric acid (20 mL) and extracted with EtOA~ (3x25 mL). The extract wa~ dried (MgSO4) and concentrated under S reduced pre3sure to give the title compound of thiq example as a tan solid (O.69 g, ~0% yield overall from Boc-Tbg-CH2C(O~O-CH2CH=C~2~. The product was a 55:45 mixture of diaqtereoi~omers (~hown by NMR). lH NMR (CDC13) ~ 0.97 (s, 9H, minor isomer), 0.99 (s, 9H, major isomer), 1.14 (g, 18~), 1.48 (~, 18H), 2.68-3.12 (m, 8H), 3.30 (m, 2H), 4.08 (d, J = 9Hz, lH, minor i~omer), 4.10 (d, J = 9Hz, lH, major i.q~mer), 5.11 (d, J = 9Hz, 2~).
~xam~le 4 Preparation of the Intermediate H-Tb~=
_~CH~CH2C~O!CMe3)C~OL-A~p~cyPn~B~ NH-LSl-CH(C~12C~O~Me3~_20Bzl By following the coupling procedure of example 1 and using the title compound of exampl 2~3.42 g, 7.13 mmol~ as the first reactant and the title compound of e~ample 3 (2.50 g, 6.48 mmol3 as the ~econd reactant, flash chromatography (SiO2, eluent~ hexane-EtOAc, 4:1) of the cruda product gave the corresponding N-Boc derivative of the title compound (4~64 g, 84%; ~f = 0.21, hexane-EtOAc, 7:3). A ~qolution of the latker derivati~e (4.64 g, 5.44 mmol) in 6 N ~Cl/dioxane (50 mL) was stirred at room temperature for 1 h and then concentrated under reduced pressure. The re~idlle was dissolved in Et20. The latter solution was washed with saturated aqueous solution of NaEI~03 and brine, dried (MgS04) and concentrated to give ,'1~.' ~ . . ': ~ :

32 2 ~ 0 8 a yellow oil consisting of two diastereoisomers.
The two isomer~ were separated by flash chromatography (SiO2, eluent: hexan~-EtO~c-MeO~, 5:4.5:0.5). The de~ired isomer (i.~. the more polar; Rf = 0.18, EtOAc-hexane-MeOH, 7:3:0.5) was obtained as a colourless oil (2.52 g, 52%). The isomer, the title compound of this example, is u~ed without further purification in the ne~t example.

Preparation of Boc-(N-Me ! va l ~q~ ! R ! ~
5 ~ 2C(~? ~ e3)CJQ)-ASptcyPn~_LBzl)~NH~
5~ 2 _ 3 ! C~Q~l By following the coupling procedure of example 1 and using the title compound of example 4(4.45 g, 5.95 mmol) as the fir~t reactant and N-methylvaline ~4.41 g, 17.9 mmol) as the second react nt, flash chromatography (SiO2, eluent, hexane-EtOAc, 7:3) of the crude product gave the title compound of this example (4.02 g, 72%
yi~ld). lH NMR (CDC13) ~ 0.87 (d, 3 = 7 Hz, 6H), 0.90 (s, 18H), 1.10 ~5, 9H), 1.48 (s, 9~, 1.5 -2.0 (m, 10H), 2.30 (m, lH), 2.5 - 3.1 ~m, 5H), 2.80 (s! 3H), 3.30 (m, 2H3, 4.0 (d, J = 12.5 Hz, lH), 4.20 (m, lH~, 4.32 (d, J = 8.5 Hz, lH), 4.49 (d, J = 4.5 Hz, 2H), 4.64 (d, J ~ 11 Hz, lH), 5.18 (d, J = 5 Hz, 2H), 6.78 (broad d, J = 8.5 Hz, lH), 7.11 (broad d, J = 8.5 Hz, lH), 7.18 (broad d, J = 11 Hz, lH), 7.2-7.45 (ml 10H).

.

,..:-:,:: .

~, :
P,r,eparation _of P-hC-H-2~2 ~ -(R)-CH~cH2cto~cMe3!c~o)-A~p~cypnl-yMeLaucin S
A ~olution of the title compound of example 5 ~4.02 g, 4~25 mmol) in 6N ~Cl/dioxane (30 mL~ was stirred at room temperature for 1 h and then concentrated under reduced pre sure to give the free N-terminal amino derivative of the title compound of example 5 in the form of its hydrochoride 3alt. Thereafter, by following the coupling procedure of example 1 and using the latter amino derivative as the fir3t reactant a]nd benzenepropionic acid (2.00 g, 13.3 mmol) as the second reactant, the purification of the crude product by flash chromatography ~SiO2, eluent:
hexane-EtOAc, 3:2) gave PhCH2CH2C(O)-N-Me-Val-Tbg-CH2-(R)-cH(cH2c(o)cMe3)c(o)-Asp-(cypn)(~Bzl)-~H-(S)-CH(CH2CMe3)CH20Bzl a3 a white foam (4.09 g, 94%; Rf = 0.35, he~ane-EtOAc, 1:1).

The latter compound (4.00 g, 4.03 mmol~ wa~
~ubjected to hydrogenolysi3 {20~ Pd~OH)2/C (200 mg), 1 atmosphere of H2, EtOH, 5 h}. After completion of the reaction, the cataly~t wa~
removed from the reaction mixtur~ by filtration through a 45 ~m membrane. The filtrate was concentrated under raduced pre3sure to give a clear oil. The oil was dissolved in Et2O (100 mL). The solution was evaporated to dryness under reduced pressure. Tha dissol~ing and evaporakiny process wa~ repaated whereby a white solid was obtained. The solid wa~ trikurated with hexane, filtered and dried under reduced pr~ssure to gi~e the title compound (3.12 g, ~5%) lH NMR (d6-DMSO), .,, ~ ~ , , .
, ,, ~ . 1 ~;

34 2~9~8 400 MHz; note: the compound exii3ts in DMSO as a 50:50 mixture of two rotamers ~ O.71-0.92 (m, 24H), 1.05 (~, 4.5H), 1.06 (s, 4.5H), 1.20-1.78 (m, 10H), 1.93-2~16 (m, 2H), 2.48-2.83 I~, 6H), 2.84 (8, 1.5H~, 2.92 ~8, 1.5~), 2.g6-3.06 ~m, lH), 3rlO~3~23 (m, 2H), 3~72-3~81 (m, lH), 4.09-4.14 (m, lH), 4.22 (d, 8 Hz, 0.5H), 4.54-4.62 (broad m, lH), 4.~3-4.81 (m, 1.5H), 7.12-7.29 ~m, 6H), 7.94 (d, 3 = 10 Hz, lH), 8.04 (d, J = 8 Hz, 0.5H), 8.32 (d, J = 8.5 Hz, 0~5H).

By following the procedure of example 6 but replacing benzenepropionic acid with 2-(phenyl methyl)-3-phenylpropionic acid (dibenzylacetic acid), ~phcH2)~cHc(o)-(N-Me)val-Tbg-cH2-(R
CH(cH2c~o)-cMe3)c(o)-A~p(cypn~-~MeLeucinGl i~
obtained.

rr~ "_~L~ CHNHC ~ -Tbq-cH2-~RL~cHr~25 C 3~5Lblu~L!~D~ y~

l-Ethylpropyl isocyanate (28 mg, 0.24g mmol) wa~ added to a solution of the title compound of example 4 (23 mg, 0.030 mmol) and triethylamine (6 mg, 0.057 mmol) in anhydrou~ C~2C12. Tha reaction mixture wa~ stirxed under an aryon atmosphere at 0 for 1 h and then at room temperature for 1~ h.
TLC (EtOAc-hexane, 1 1) indicated the completion of the reaction, The ~olvent was evaporated ~nder reduced pre sure. The residue wa~ purified by flash chromatography ( SiO2, eluent: hexane-EtOAc, 6:4) to give the corresponding dibenzyl derivative of the title compound of thi~ example (16 mg)1 1H
NMR (400 MHz, CDC13) ~ 0.9 (t, J = 7 Hz, 3H), 3.92 (t, J = 7 Hz, 3H), 0.94 (s, 9H), 0.95 (~, 9H), 35 2~9~

1.10 (~, 9H), 1.25-1.90 (m, 14H), 2.52 ~m, lH), 2.68 (m, lH), 2.81 (m, lH~, 2.94-3.07 (m, 2H), 3.27 (dd, J = 7.2, 9 Hzl lH), 3.36 (dd, J = 5.5, 9 Hz, lH), 3.46 (m, lH), 4.07 (d, J = 9 Hz, lH~, 4.23 (m, lH), 4.28 (d, J = 9 Hz, lH), 4.48 (dd, J
= 10 Hz, 2~), 4.66 (d, J = 10 Hz, lH); 4.74 (d, J
= 9 Hz, lH), 5.17 (dd, J = 14 H~, 2~), 7.10 (d, J
= 8.5 ~z, lH), 7.2-7,45 ~m, llH3.

The latter dibenzyl derivative wa~ ~ubjected to hydrogenolysi~ tlO~ palladi~m on carbon, atmosphere, EtOH) to give the title compound.
Mass spectrum: 703 (M + Na)+.

Exampl~_8 Preparation of Other Representative Intermediate~
for the Elaboration of the C-Terminu~ of Peptide~
of Formula 1.
(a) N~2-(R~-CH(Et)CMe3: To a cooled solution (0) of 4,4-dimethyl-3-pentanone (lOÇ g/ 0.92 mmol) and (R)-a-methylbenzylamine ~111 g, 0~92 mmol) in benzene (1 L~, a ~olution of TiC14 150.5 mL~ 0.46 mmol) in benzene (200 mL) was added at a rate that kept the temperature of the mixture below 10 . Thereafter~ the mixture wa~ ~tirred mechanically for 3 h at 40 I cooled to ro~m temperature and filtered through diatomaceous earth. The diatomaceou~ earth wa~ washed wlth Et20. The combined filtrate and wash was concentrated. The residue wa~ di~olved in dry MeO~ (2 L). The ~olution wa~ cooled to 0 and NaBH4 (20 g, O.53 mmol) was added portionwi~e while maintaining the temperature of the mixtuxe below 5 . The methanol wa~ evaporated. The ~6 2 ~
.-residue was dissolved in Et20. The solution wa~washed with brine, dried (MgS04 ) and evaporated o dryne~i~; to give a reddish oil (a 18 :1 mixture of diastereoi~omers as indicated by NMR). The oil S was purified by flash chrsmatography ~SiO2, eluent: EtOActhexane, 7:93) to afford N-(l(R)-phenylethyl ) -1 ( R ) -ethyl-2, 2 -dimethylpropyl-amine as a liquid (110 g, 54%). Thi~ material wa~
dissolved in hexane l l . 5 L) . 6~1 HCl in dioxane (90 mL~ was added to the ~olution over a period of 15 min. ~he re~ulting white solid wa collected on a f ilter and then washed with hexane to provide N-(l(R)-phenylethyl)-l(R)-ethyl-2,2-dim0thylpropyl hydrochloride (125 g, 97~ H ~MR(CDCl3) a 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, lH), 4.31-4.49 (m, lH), 7.30-7.48 (m, 3H), 7.74-7.79 (m, 2H ) .

A solution of the la~ter compound (4~.5 g) in MeOH (120 mL) was mixed with 10% (w/w) Pd/C and the mixture was ~haken undier 50 psi of hydrogen on a Parr hydrogenator at room temperature f or 4 8 h .
The mixture wa~ filtered and ~he filtrate wa~
concentrated to give the desired NH2-~R)-CH(Et)C~
Me3 in the form of its hydrochloric acid addition 3alt, as a white solid (25 g, 190%) 5 lH
~JMR(CDC13) ~ 1-10 (~, 9H), 1.22 (t, J = 7 Hz, 2H), 1.58-1.90 (m, 2H), 2.70-2.85 (m, 1~), 8710-8.40 (broad ~, 3H).

In the same manner but replacing 4,4-dimethyl-3-pentanone with 3,3-dimethyl-2-butanone in the preceding proced1lre, NH~ (R)-C~I(Me~CMe3.HCl i9 obtained.

., . . . ~, . . . .

.;,-. i: ~
... ,: . :- .

37 2 U ~ 8 Example 9 Preparation of Other Repre~entative Intermediates for ~laborating the N-Terminuq of Peptide S Derivative3 of Formula 1 According to the Procedure of ~xample 7.

(a) 1-Propylbutyl i~ocyanate: Thi~ intermediate was prepared from commercially available 4~
aminoheptane by the procedure of V.S, Golde~midt and M. Wick, Liebigs Ann. Chem., 5?5, 217 (1952~.

(b) 1-~thyl-1-(2-propenyl3-3-butenyl i30cyanate:
A solution of propionitrile (14.5 g, 264 mmol) in dry Et20 (40 m~) was added dropwise to 1.0 M allyl magnesium bromide/Et20 (880 mL). The reaction mixture was mechanically ~tirred at reflux fox 2 h, a~ter which time it was cooled to 0 . A
saturated aqueous ~olution of NH4Cl (320 mL) was added cautiously to the cooled reaction mixture.
The organic pha~e was separated, dried (MgSO4), cooled to 0 and then mixed at the same temparature with 1 M HCl/Et2O (200 mL). The re~ulting solid wa~ collected and dried under reduc~d pres~ure (ca 27 g). The latter material dis~olved in CH2C12 (200 mL). The solution was wa3hed with a 10% (w/v) aqueous ~olution of Na2CO3 (2 X) and then brine, dried (MgSO4) and concentrated to dryne ~ to afford a yellow oi:L.
The oil wa~ distilled (82-85 /20 Torr] to give 1-ethyl-1-(2-propenyl)-3-butenylamin~ a~ a colorless liquid (11.6 g, 34%); lH NMR(CDCl3), 400 MHz) 0.89 (t, J = 7Hz, 3H), 1-39 !q, J ~ 7 Hz, 2H), 2.11 ~d, J = 7 Hz, 2H3, 5.06-5,14 (m, 4H), 5.80-5.89 (m, 2H).

38 2~ 8 The latter compound wa~ converted to l-ethyl-1-(2-propenyl)-3-butenyl isocyanate by the procedure of V.S, ~oldesmidt and M. Wick, supra~

S The first step of the process of preceding section b, i.e. the preparation of l-ethyl 1-(2-propenyl)-3-butenylamine, i~ based on a general method described by G. Alvernhe and A. Laurent, Tetrahed.ron Lett " 1057 (1973). The overall process, with the appropriate choice of reactants, can b~ used to prepare other requisite isocyanate intermediate~ for the eventual preparation of peptide derivatives having unsaturation at the N-terminus, iOe. an un~aturated alkylaminocarbonyl such a~ 1-methyl-1-(2-propenyl)-3-butenyl. Note, however, that when the requisite isocyanate intermediates are applied according to the procedure of example 7, then the ultimate product will be the correRponding peptide derivative of formula 1 in which the N-terminus is ~aturated.
Moreover, the latter procedure represent~ a practical proces~ for preparing such corresponding peptide~; for example, Pr2CHNHC(O)-Tbg-CH2-~R)-CH(CH2C~O~CMe3)C10)-A~3p(cyPn)~NH-(R~-CH(Et)CMe3, ~FAB/mass ~pectrum (m~z): 693 [M + H~ he title peptide derivative noted in example 10, hereinafter.

Thu~, by u~ing the appropriate intermediates, the ~erial coupling and the deprotection procedure~ of examples 1 to 7 can be u~ed to prepare other compound3 of formula 1, ~uch a~
those exemplified in the table of the following example~ In some cases, precipitation of the final product does not afford pure material~ In those instances, the product can be purified by 3g 2~5~08 semi preparative HPLC on a C-18 rever~ed~pha~e column using a gradient of acetonitrile and water, each containing 0.06% TFA. To thi~ end, the crude product was di3solved in 0.1 ~ aqueous NH40~ and the pH of the solution was brought back to about 7 u~ing 0.1 M aqueou3 AcOH, prior to purification.
When applicable, dia~tereoi~omeric mixtures were ~eparated in this fashion.

Some examples o~ other compound of formula 1 that can be prepared thus are (PhCH2)2CHC(0~-~N-Me)Val-Tbg-CH2-(R)-CHtCH2C(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3 and (phc~2)2cHc(o)-(N-Me)val-Tbg-cH
(R)-CH(CH2C(O)CMe3)C(O)-A~;p~cyPn)-NH-(R)- .'~
CH(Me)CMe3. :
~ ~' Still some other examples of compound~ of formula 1 are:

pr2cHNHc(o)-Tbg-cH2-(R)-cH~cH2c(o)cMe3)c(o)-Asp~cyPn)-NHCH2CMe3, FAB/mas~ ~pectrum (m/z): 665 [M + ~]+;

Etpr2c~Hc(o)-Tbg-cH~-(R)-c~(cH2c(o)cMe3)c(o) A~p(cyPn)-NH-(R) CH(Et)CMe3, F~B/mas3 spectrum (m/z): 722 [~ ~ H3+; and EtPr2CNHC(0) Tbg-C~2-(R)-CH(CH2C~O)CMe3)C(0~-A~p(cyPn)-NHCH2CMe3, FABtma~s ~pectrum (m/z)- 693 [M + H]+-~m~

Study Showing The Effect Of The Peptide Derivative, Pr2c~lNHc(o)~Tbg-cH2-(R)-cH(cH2c(o)-Me3)~C(0)-A~p(cyPn~-NH-(R)-CH(Et)CMe3, And 2 ~

Combina~ion Thereof With Acyclovir AgainRt A Wild Type HSV-l Clinical Isolate And Two Acyclovir Resistant HSV 1 Clinical Isolate ~;

Viruses. The clinical isolates have been described by S.L. S~ck~ et al., Annals of Internal Medicine, 111, 893 (1989). They were obtained from a 25-year~old woman with acute myelogenous ;~
leukemia in first relapse from an allogeneic bone marrow transplant. The initial cultures for herpe~ ~implex virus from day 8 after the bone graft were found to be po~itive (isolate 294, defined a~ wild type HSV-l). Acyclovir treatment was then initiated, and viral cultures were still positive on day 36 (isolate 615 representing re~istant HSV-1). One sub-isolate 515.9 (ACVrTR
clinical isolate) was characterized to be acyclovir-re~i~tant, forcarnet-sen~itive and to have a dimini~hed thymidine kinase ~TX) activity.
615.9 was ~ubsequently confirmed to be a TK
deficient mutant. In contrast, subisolate 615.8 (~CVrPOL clinical isolate~ was characterized to be acyclovir and forcarnet re~istant and had thymidine kinase activitie~ near to that ob~erved in the pretreatment isolate 294. 615.8 wa~
subsequently confirmed to be a DNA polymera~e mutant.

Assay:
BHK-21/C13 cells (ATCC CCL 10~ are incubat~d for two days in 150 cm2 T-fla~k~ (1.5 x 106 cells/flask) with alpha-MEM medium (Gibco Canada Inc~, ~urlington, Ontario, Canada) ~upplemented with 8% (v/v) fetal bovine serum (FBS, Gibco Canada Inc.). The cells are trypsinized and then 41 2 ~ Q ~
, tran~ferred to fresh media in a 24 well plate to give 2.5 x 105 cells in 750 ~L of media per well.
The cell~ are incubated at 37 for a period of 6 h to allow them to adhere to the plate. Thereafter, the cells are washed once with 500 ~L of alpha-MEM
supplemented with 0.5~ (v/v) FBS and then incubaked with 750 ~h of the sc~me media (low ~erum~ for 3 day~. After thi~ period of serum ~tarvation, the low serum medium i~ removed and -the cells are incubated in 500 ~L 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 ~L of ~BMT medium. (Note: The HSV-2 used wa~ strain HG-52, see Y. Langelier and G. Buttin, J. Gen. Virol~, 57, 21 (1981~; the viru~ wa ~tored at -80 ~) Following 1 h of virus adsorption at 37 ~ the media i3 removed and the cell~ are washed with BB~T (3 X 250 ~L). The cell~ in each well ar~ incubated with or without (control) appropriate concentration~ of the test agent dissolved in 200 ~h of BBMT medium. Aft~r 29 h of incubation at ~7 , the infected cell are harvested by fir~t freezing the plate ak 80 , followed by thawi~g The cell in each well are scraped off the surface of the well with the help of the melting ice fragments. After complete thawing, the cell suspension~ are collected and each we:Ll i~ rinsed with 150 ~L of BB~T m~dium.
The viral ~ample ~suspen~ion plu~ wa~hing) i~
sonicated gently ~or 4 min ak 4 . Cell debri3 are removed by centrifugation (10~0 time3 gravity for 10 minute~ at 4 ~. The upernatant is collecked and stored at -80 until determination of viral titer.

, '. ~" ~' ' :: . ' ' . ' ' 42 2 ~
~ ~.
; ~
Viral titration wa~, performed by a modification of the colorimetric asc7ay method of M. Langlois et al., Journal of Biological S Standardization, 14, 201 (1986).

More specifically, in a ~imilar manner as described above, BHK-21/C13 cells are tryp~inized and transferred to fresh media in a 96 well microtiter plate to give 20,000 cells in 100 ~L of media per well. The cells in the prepared plate are incubated at 37 for 2 h. 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 ~L of the sample plus 200 ~, of BB~T medium, sequential dilutions being done with a multichannel pipette.

On completion of the above 2 hour incubation of the BHR-21/C13 cell~ the media i5 replaced with alpha-MEM medium supplemented with 3~ (v/v) FBSo The cells are now ready to be infected with the various sample dilutions of viru~. Aliquots (50 ~L) of the variou3 dilution~ are transferred into the appropriate wells of the plate. l'he resulting infectsd cell~ are incubated for 2 dayc, at 37 . Then 50 ~L of a 0.15~ ~v/v) solution of neutral red dye in Hank's Balanced Salt Solution tpH 7.3, Gibco Canada Ins.) is added to each well.
The prepared plate i~ incubated ~or 45 min at 37 .
Medium from each well is then aspirated and the cells are wa~,hed once with 200 ~L of Hank's Balanced Salt Solution. After the wash, the dye is released from the cells by the addition o~ 100 ~L 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 follow~- 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 H2O to make 1 L. Secondly, the 0.1 M
disodium citrate ~olution (61.2 ~3 is mixed with O.1 N aqueous HCl (38.8 mL) and the pH of the re~ult~ng solution i~ adjusted to 4.2 if nece~sary.] The mixture in the wells is ~ubjected to a gentle vortex action to ensure proper mixing.
The plate well~ are ~canned by a ~pectrophotometer plate reader at 540 nm to a5se55 the number of viable cell~. In thi~ manner, the percentage of viru~ growth inhibition can be determined for the various concentration~ of the test agent, and the concentration of the test agent causing a 50~
inhibition of virus replication, i.e. the IC50 can be calculated.

The approach taken was to evaluate acyclovir and the peptide derivative, each alone and then in variou~ combinations in the prPceding a~say.
Thu~, the co~parative effectiveness of the two compounds against the isolate~ was demonstrated.
Furthermore, a synergistic effect bekween the two compounds was evaluated and confirmed by applying the isobole method to the reRults obtained in the~e studies; s~e J. S~hnel, JO Antiviral Research, 13, 23 l1990~ for a description of the isobole method.

More explicitly with raference to the i~obole method, thi~ method requires experimental data generated for the two test co~pounds, each alone and in different dose combination~ at equi-effective levels. In thi~ way Relected concentrations of the title peptide derivative (ICs, IC1o, IC20 and IC30) were added in combination with variou~ do~e~ of acyclovir and the IC50'~ were evaluated. For thi~ experiment, S the ICs~ IClor IC20 and IC30 of the title peptide derivative a~ well a~ the do~es of acyclovir were derived from curve previou~ly obtained. An isobologram i~ generated using a value termed FIC60~acyclovir) ~which is the ratio of the concentration of acyclovir requixed to inhibit ~SV
replication by 60% in the presence of a fi~ed concentration of the title peptide derivative to the concentration required in the absence of the peptide derivative). This is plotted against a term repre~enting the ratio of the fixed concentration of the peptide derivative to the concentration of the peptide derivative that reduced 60% inhibition of HSV replication in the absence of acyclovirO
equations.

X axis :
= [the fixed concentration of the peptide ~ _ IC60 of the peptide derivative alone Y axis :
FIC60(acyclovir) = IC60(acyclovir + X IAM of thP
peptide derivative IC60(acyclovir alone) Reslllt_:

3S TABLE I lists the re~ults obtained when acyclovir and the title peptide of this example 45 2'~

were a~sayed alone in the preceding cell culture a~ay with the wild type ~ISV-1 clinical isolate and the two acyclovir-resi~tant HSV-1 clinical ~:
isolates.
STABLE I ~ :-. _ _ _ . .
Isolates Isolate Isolate Isolate ¦Compounds 294 615.8 615.9 IC ~(~M) ICs~(~M) ICs~(~M) 5 ~ ~r ~ ~
Acyclovir 1.1 19 55 Title Peptide 2.0 2O0 6.2 Derivati~e . . .

The result~ from TABLE I demonstrate that compounds of formula 1 are active againRt wild type HSV-1, and that they e~hibit similar efficacy again~t acyclovir-resi~tnat HSV~ uch as POL-mutants and TK-deficient mutants; whereas acyclovir lo~es much of its efficacy (being above 20 to 40 times lass effective) against the m~tant strain~.

~ he following TABLE5 II, III and IV are illustrative of the r~ult~ obtained when combinations of acyclovir and the title peptide derivative of this example were evaluated according to the preceding cell culture assay with the wild type HSV-1 clinical isolate and the two acyclovir-re~istant HSV-l clinical i~olates.

v~J~

462 ~

TAHL~ II
ANTIVIRAL ACTIVITY
SYNERGY STUDY
_ Isolate 294 (Pretreatment c Linical isolate) Fixed concentration of Acyclovir IC50 (1l the Title Peptide in combination with DerivativeTitle Peptide Derivative (~q) (~) 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 ~.13 _ TA~L~ III
¦ ANTIVIRAL ACTIVITY
SYNERGY STUDY
Isolate 615.8 (ACVrPOL treal ,ment i501ate ) Fixed concentration ofAcyclovir IC50 ~2) the Title Peptidein combination with Derivative Title Peptide Derivative ~ ).... . ~ =~= I
0.3 15.0 0.5 13.0 1.4 1~7 1.6 2.~
--~-- =e _ _ 47 2 ~9 ~ 08 TABLE IV
ANTIVIRAL ACTIVITY
SY~RGY STUDY
Isolate 615.9 (ACVrTR treatment i olate) ¦
_ ,.
Fixed concentration ofAcycloYir IC50 ~2) the Title Peptidein combination with ¦
DerivativeTitle Peptide Derivati~e¦
(lll.Mo) _ (llM) . :

2.0 17.0 6.0 0.9 7.0 ~ 0.02 _ _ ~.0 < 0.02 _ Notes re~pecting the preceding four ta~les:
o IC50 values are not corrected for 301ubility.
All stock ~olutions were ultracentrifuged at 100,000 x g for 30 minutes at 4 prior to use.
The ~olubility of the title peptide deriva~ive at 20~M is 85%.
o The cytotoxicity of the title peptide derivatiYe is 89 ~M (value not corrected).
tl) IC50 obtained from a dose response curvP of acyclovir ranging from 7.6 x 10-4 to 1.5 x 1 ~M in the presence of the corresponding fixled concentration of the title peptide derivative as shown in the col~nn to the left.
~2) IC50 obtained from a dose response curve of acyclovir ranging from 1.5 x 10-4 to 1.5 ~ 10~
~M in the presence of the corre~ponding fixed conc~ntration of the title peptide derivative as shown in the column to the left.

.,:..... ~:
;.,. . . ~ . :
,:.,.,.. , . ~ , :,. .. .. . .

,..... ,,: ~ , :

48 ~ 0~

TABLES II, III and IV demonstate that the peptides of formula 1 are able to potentiate the activity of acyclovir against wild type HSV-1 and again~t acyclovir-resistant H~V-1 mutants, when the two agents are used in c~mhin~tion. The re~ults ~how a proportional lowering of the IC50 of acyclovir as the ratio of the concentration3 of the peptide to acyclovir i9 increased.

Accompanying Figure 1 i3 a graphic illu~tration of the positive results (synergism) obtained in the application of the isobole m~thod in a study with acyclovir and the title peptide of this example using the subisolate 615.8 (ACVrPOL
clinical isolate).

Similarly, accompanying Figure 2 is a graphic illustration of the positive results in a study with acyclovir and the peptide of this example using the subisolate 615.9 ~ACVrTK).

Claims (12)

1. The use of a peptide derivative for treating acyclovir-resistant herpes simplex viral infections in a mammal wherein the peptide derivative is a compound of formula 1 A-B-D-CH2CH{CH2C(O)R1}C(O)-NHCH{CR2(R3)COOH}C(O)-E

wherein A is phenylacetyl, phenylpropionyl, (4-aminophenyl)propionyl, (4-fluorophenyl)propionyl, (4-hydroxyphenyl)propionyl, (4-methoxyphenyl)-propionyl, 2-(phenylmethyl)-3-phenylpropionyl, 2-{(4-fluorophenyl)methyl}-3-(4-fluorophenyl)pro-pionyl, 2-{(4-methoxyphenyl)methyl}-3-(4-methoxy-phenyl)propionyl or benzylaminocarbonyl; B is (N-Me)-Val or (N-Me)Ile; or A and B taken together form a saturated alkylaminocarbonyl selected from the group of butylaminocarbonyl, 1-methylethylaminocarbonyl, 1-methylpropylamino-carbonyl, 1-ethylpropylaminocarbonyl, 1,1-dimethethylbutylaminocarbonyl, 1-ethylbutylamino-carbonyl, 1-propylbutylaminocarbonyl, 1-ethylpent-ylaminocarbonyl, 1-butylpentylaminocarbonyl, 1-ethylbutylaminocarbonyl, 2-ethylpentylaminocarbon-yl, 1-methyl-1-propylbutyaminocarbonyl, 1-ethyl-1-propylbutylaminocarbonyl, 1,1-dipropylbutyl-aminocarbonyl, (1-propylcyclopentyl)aminocarbonyl and (1-propylcyclohexyl)aminocarbonyl; D is Val, Ile or Tbg; R1 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl,

2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl, NR4R5 wherein R4 is hydrogen or lower alkyl and R5 is lower alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino, morpholino or 4-methylpiperazino; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, 1,1-dimethylethyl, propyl, 2-propenyl or benzyl, and the carbon atom bearing R2 and R3 has the (R)-configuration, or R2 and R3 each independently is methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR6 wherein R6 is 2-methylpropyl, 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-dimethylbutyl, 3,3-dimethylbutyl, 1(R),2,2-trimethylbutyl, 1(R),3,3-trimethylbutyl, 2-ethylbutyl, 2,2-diethylbutyl, 2-ethyl-1-(R)-methylbutyl, 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 NHCH(R7)-Z wherein the carbon atom being R7 has the (S)-configuration, R7 is 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or cyclohexylmethyl and Z is CH2OH, C(O)OH, C(O)NH2 or C(O)OR8 wherein R8 is methyl, ethyl or propyl; or a therapeutically acceptable salt thereof.
2. A use of a peptide derivative as claimed in claim 1 wherein the peptide derivative is a compound of formula 1 wherein A is phenylpropionyl, 2-(phenylmethyl)-3-phenylpropion-yl or benzylaminocarbonyl; B is (N-Me)Val; D is Tbg; R1 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimetylpropyl, 2,2-dimethyl-propyl, cyclobutyl, cyclopentyl, cyclohexyl or 1-methylcyclopentyl; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, propyl or benzyl, and the carbon atom bearing R2 and R3 has the (R)-configuration, or R2 and R3 each independently is methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR6 wherein R6 is 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 NHCH(R7)-Z wherein the carbon atom bearing R7 has the (S)-configuration, R7 is 2,2-dimethylpropyl and Z is CH2OH, C(O)OH, C(O)NH2 or C(O)OR8 wherein R8 is methyl, ethyl, or propyl; or a therapeutically acceptable salt thereof.
3. A use of a peptide derivative as claimed in claim 1 wherein the peptide derivative is a compound of formula 1 wherein A and B together form a saturated alkylaminocarbonyl selected from the group of 1-ethylpropylaminocarbonyl, 1-ethyl-butylaminocarbonyl, 1-propylbutylaminocarbonyl, 2-ethylpentylaminocarbonyl, 1-methyl-1-propylbutyl-aminocarbonyl, 1-ethyl-1-propylbutylaminocarbonyl, 1,1-dipropylbutylaminocarbonyl and (1-propylcyclo-pentyl)aminocarbonyl; D is Tbg; R1 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobut-yl, cyclopentyl, cyclohexyl or 1-methylcyclo-pentyl; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, propyl or benzyl, and the carbon atom bearing R2 and R3 has the (R)-configuration, or R2 and R3 each independently is methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR6 wherein R6 is 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 NHCH(R7)-Z
wherein the carbon atom bearing R7 has the (S)-configuration, R7 is 2,2-dimethylpropyl and Z is CH2OH, C(O)OH, C(O)NH2 OR C(O)OR8 wherein R8 is methyl, ethyl or propyl; or a therapeutically acceptable salt thereof.

4. A use of a peptide derivative as claimed in claim 1 wherein the peptide derivative is selected from the group consisting of:
PhCH2CH2C(O)-(N-Me)Val-Tbg-CH2-(R)-CH(CH2C(O)-CMe3)C(O)-Asp(cyPn).gamma.MeLeucinol, Et2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-.gamma.MeLeucinol, Pr2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NH-(R)-CH(Et)CMe3, Pr2CHNHC(O)-Tbg-CH2-(R)-CH(CHC(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3, EtPr2CNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NH-(R)-CH(Et)CMe3, and EtPr2CNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3.

5. A use of a combination of the peptide derivative as defined in claim 1, or a therapeutically acceptable salt thereof, and an antiviral nucleoside analog, or a therapeutically acceptable salt thereof.

6. A use of a combination as claimed in claim 5 wherein the peptide derivative is a compound of formula 1 wherein A is phenylpropionyl, 2-(phenylmethyl)-3-phenylpropionyl or benzylamino-carbonyl; B is (N-Me)Val; D is Tbg; R1 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobut-yl, cyclopentyl, cyclohexyl or 1-methylcyclo-pentyl; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, propyl or benzyl, and the carbon atom bearing R2 and R3 has the (R)-configuration, or R2 and R3 each independently is methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR6 wherein R5 is 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 NHCH(R7)-Z
wherein the carbon atom bearing R7 has the (S)-configuration, R7 is 2,2-dimethylpropyl and Z is CH2OH, C(O)OH, C(O)NH2 or C(O)OR8 wherein R8 is methyl, ethyl or propyl; or a therapeutically acceptable salt thereof.

7. A use of a combination as claimed in claim 5 wherein formula 1 wherein A and B together form a saturated alkylaminocarbonyl selected from the group of 1-ethylpropylaminocarbonyl, 1-ethyl-butylaminocarbonyl, 1-propylbutylaminocarbonyl, 2-ethylpentylaminocarbonyl, 1-methyl-1-propylbutyl-aminocarbonyl, 1-ethyl-1-propylaminocarbonyl, 1,1-dipropylbutylaminocarbonyl and (1-propylcyclo-pentyl)aminocarbonyl; D is Tbg; R1 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobut-yl, cyclopentyl, cyclohexyl or 1-methylcyclo-pentyl; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, propyl or benzyl, and the carbon atom bearing R2 and R3 has the (R)-configuration, or R2 and R3 each independently is methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR6 wherein R6 is 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 NHCH(R7)-Z
wherein the carbon atom bearing R7 has the (S)-configuration, R7 is 2,2-dimethylpropyl and Z is CH2OH, C(O)OH, C(O)NH2 or C(O)OR8 wherein R8 is methyl, ethyl or propyl; or a therapeutically acceptable salt thereof.

8. A use of a combination as claimed in claim 5 wherein the group consisting of:

PhCH2CH2C(O)-(N-Me)Val-Tbg-CH2-(R)-CH(CH2C(O)-CMe3)C(O)-Asp(cyPn)-.gamma.MeLeucinol, Et2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-.gamma.MeLeucinol, Pr2CHNHC(o)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NH-(R)-CH(Et)CMe3, Pr2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3, EtPr2CNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NH-(R)-CH(Et)CMe3, and EtPr2CNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3.

9. The use of a combination as claimed in claim 5 wherein the nucleoside analog is the compound of formula 2 wherein R9 is hydrogen hydroxy or amino, or a therapeutically acceptable salts thereof.

10. The use of a combination as claimed in claim 5 wherein the nucleoside analog is selected from the group consisting of vidarabine, idoxuridine, trifluridine, ganciclovir, edoxudine, broavir, fiacitabine, penciclovir, famciclovir and rociclovir.

11. The use of a combination as claimed in claim 5 wherein the peptide derivative is selected from the group consisting of:

PhCH2CH2C(O)-(N-Me)Val-Tbg-CH2-(R)-CH(CH2C(O)-CMe3)C(O)-Asp(cyPn)-.gamma.MeLeucinol, Et2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-.gamma.MeLeucinol, Pr2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NH-(R)-CH(Et)CMe3, Pr2CHNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3, EtPr2CNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NH-(R)-CH(Et)CMe3, EtPr2CNHC(O)-Tbg-CH2-(R)-CH(CH2C(O)CMe3)C(O)-Asp(cyPn)-NHCH2CMe3; and the nucleoside analog is acyclovir.

12. A pharmaceutical composition for the treat-ment of acyclovir-resistant herpes simplex viral infections, comprising as active ingredient a peptide derivative of formula 1 A-B-D-CH2CH{CH2C(O)R1}C(O)-NHCH{CR2(R3)COOH}C(O)-E
wherein A is phenylacetyl, phenylpropionyl, (4-aminophenyl)propionyl, (4-fluorophenyl)propionyl, (4-hydroxyphenyl)propionyl, (4-methoxyphenyl)-propionyl, 2-(phenylmethyl)-3-phenylpropionyl, 2-{(4-fluorophenyl)methyl}-3-(4-fluorophenyl)pro-pionyl, 2-{(4-methoxyphenyl)methyl}-3-(4-methoxy-phenyl)propionyl or benzylaminocarbonyl; B is (N-Me)-Val or (N-Me)-Ile; or A and B taken together form a saturated alkylaminocarbonyl selected from the group of butylaminocarbonyl, 1-methylethylaminocarbonyl, 1-methylpropylamino-carbonyl, 1-ethylpropylaminocarbonyl, 1,1-dimethetylbutylaminocarbonyl, 1-ethylbutylamino-carbonyl, 1-propylbutylaminocarbonyl, 1-ethylpent-ylaminocarbonyl, 1-butylpentylaminocarbonyl, 1-ethylbutylaminocarbonyl, 2-ethylpentylaminocarbon-yl, 1-methyl-1-propylbutylaminocarbonyl, 1-ethyl-1-propylbutylaminocarbonyl, 1,1-dipropylbutyl-aminocarbonyl, (1-propylcyclopentyl)aminocarbonyl and (1-propylcyclohexyl)aminocarbonyl; D is Val, Ile or Tbg; R1 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-methylcyclopentyl, NR4R5 wherein R4 is hydrogen or lower alkyl and R5 is lower alkyl, or R4 and R5 together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino, morpholino or 4-methylpiperazino; R2 is hydrogen and R3 is methyl, ethyl, 1-methylethyl, 1,1-dimethylethyl, propyl, 2-propenyl or benzyl, and the carbon atom bearing R2 and R3 has the (R)-configuration, or R2 and R3 each independently is methyl or ethyl, or R2 and R3 together with the carbon atom to which they are attached form a cyclobutyl, cyclopentyl or cyclohexyl; and E is NHR6 wherein R6 is 2-methylpropyl, 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-dimethylbutyl, 3,3-dimethylbutyl, 1(R),2,2-trimethylbutyl, 1(R),3,3-trimethylbutyl, 2-ethylbutyl, 2,2-diethylbutyl, 2-ethyl-1(R)-methylbutyl, 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 NHCH(R7)-Z wherein the carbon atom bearing R7 has the (S)-configuration, R7 is 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or cyclohexylmethyl and Z is CH2OH, C(O)OH, C(O)NH2 or C(O)OR8 wherein R8 is methyl, ethyl or propyl; or a therapeutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.