Note: Descriptions are shown in the official language in which they were submitted.
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
H2 N N N
CH2 OCH2 0
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
28
. 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).
.
,..:-:,:: .
33
~, :
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
44
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
0 7 7 0 --
0 5 3
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).
