Herpes ribonucleotide reductase inhibitors

Disclosed herein are compounds of the formula ##STR1## wherein R.sup.1 is hydrogen or (1-4C) alkyl, R.sup.2 is (1-4C) alkyl or a therapeutically acceptable salt thereof. The compounds are useful for treating herpes infections.

FIELD OF INVENTION 
This invention relates to peptidomimetic compounds having antiviral 
properties and to means for using the compounds to treat viral infections. 
More specifically, the invention relates to peptidomimetic compounds 
exhibiting activity against herpes viruses, to pharmaceutical compositions 
comprising the compounds, and to methods of using the compounds to inhibit 
the replication of herpes virus and to treat herpes infections. 
BACKGROUND OF THE INVENTION 
Herpes viruses inflict a wide range of diseases against humans and animals. 
For instance, herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), are 
responsible for cold sores and genital lesions, respectively; varicella 
zoster virus (VZV) causes chicken pox and shingles; and the Epstein-Barr 
virus (EBV) causes infectious mononucleosis. 
Over the past two decades, a class of compounds known as the purine and 
pyrimidine nucleoside analogs has received the most attention by 
investigators in the search for new therapeutic agents for treatment of 
herpes virus infections. As a result, several nucleoside analogs have been 
developed as antiviral agents. The most successful to date is acyclovir 
which is the agent of choice for treating genital herpes simplex 
infections. 
Nevertheless, in spite of some significant advances, the need for 
effective, safe therapeutic agents for treating herpes viral infections 
continues to exist. For a review of current therapeutic agents in this 
area, see R. E. Boehme et al., Annual Reports in Medicinal Chemistry, 29, 
145 (1994). 
The present application discloses a group of compounds having activity 
against herpes simplex viruses. The selective action of these compounds 
against herpes viruses, combined with a wide margin of safety, renders the 
compounds as desirable agents for combating herpes infections. 
The following references disclose peptides or peptidomimetic compounds 
which have been associated with antiherpes activity: 
E. A. Cohen et al., U.S. Pat. No. 4,795,740, Jan. 3, 1989, 
R. Freidinger et al., U.S. Pat. No. 4,814,432, Mar. 21, 1989, 
P. Gaudreau et al., J. Med. Chem., 33, 723 (1990), 
J. Adams et al., European patent application 411,334, published Feb. 6, 
1991, 
R. L. Tolman et al., European patent application 412,595, published Feb. 
13, 1991, 
L. L. Chang et al., Bioorganic & Medicinal Chemistry Letters, 2, 1207 
(1992), 
P. L. Beaulieu et al., European patent application 560,267, published Sep. 
15, 1993, 
N. Moss et al., J. Med. Chem., 36, 3005 (1993), and R. Deziel and N. Moss, 
European patent application 618,226, published Oct. 5, 1994. 
The subject peptides of the previous reports can be distinguished from the 
peptides of the present application by characteristic structural and 
biological differences. 
Abbreviations and symbols used hereinafter are defined in "Details of the 
Invention" section of this application. 
SUMMARY OF THE INVENTION 
The compounds of this invention are represented by formula 1 
##STR2## 
wherein R.sup.1 is hydrogen or (1-4C)alkyl and R.sup.2 is (1-4C)alkyl; or 
a therapeutically acceptable salt thereof. 
A preferred group of the compounds of this invention are represented by 
formula 1 wherein R.sup.1 is hydrogen or methyl and R.sup.2 is methyl, 
ethyl, 1-methylethyl or propyl; or a therapeutically acceptable salt 
thereof. 
A more preferred group of the compounds are represented by formula 1 
wherein R.sup.1 is hydrogen and R.sup.2 is methyl, ethyl or 1-methylethyl; 
or a therapeutically acceptable salt thereof. 
Included within the scope of this invention is a pharmaceutical composition 
comprising an antiherpes virally effective amount of a compound of formula 
1, or a therapeutically acceptable salt thereof, and a pharmaceutically 
acceptable carrier. 
Also included within the scope of this invention is a cosmetic composition 
comprising a compound of formula 1, or a therapeutically acceptable salt 
thereof, and a physiologically acceptable carrier suitable for topical 
application. 
An important aspect of the invention involves a method of treating a herpes 
viral infection in a mammal by administering to the mammal an anti-herpes 
virally effective amount of the compound of formula 1, or a 
therapeutically acceptable salt thereof. 
Another important aspect involves a method of inhibiting the replication of 
herpes virus by contacting the virus with a herpes viral ribonucleotide 
reductase inhibiting amount of the compound of formula 1, or a 
therapeutically acceptable salt thereof. 
Still another aspect involves a method of treating a herpes viral infection 
in a mammal by administering thereto an antiherpes virally effective 
amount of a combination of the compound of formula 1, or a therapeutically 
acceptable salt thereof, and an antiviral nucleoside analog. A 
pharmaceutical composition comprising the combination is also within the 
scope of this invention. 
Processes for preparing the compounds of formula 1 are described 
hereinafter.

DETAILS OF THE INVENTION 
General 
Alternatively, formula 1 can be illustrated as: 
##STR3## 
wherein (N-Me)Val represents the amino acid residue of 
(S)-2-(methylamino)-3-methylbutanoic acid, Tbg represents the amino acid 
residue of (S)-2-amino-3,3-dimethylbutanoic acid, Me and Et represent the 
alkyl radicals methyl and ethyl, respectively, and Asp(cyPn) represents 
the amino acid residue of (S)-.alpha.-amino-1-carboxycyclo-pentaneacetic 
acid. 
The term "residue" with reference to an amino acid or amino acid derivative 
means a radical derived from the corresponding .alpha.-amino acid by 
eliminating the hydroxyl of the carboxy group and one hydrogen of the 
.alpha.-amino group. 
The term "(1-4C)alkyl" as used herein means an alkyl radical containing 
from one to four carbon atoms and includes methyl, ethyl, propyl, 
1-methylethyl, 1,1-dimethylethyl and butyl. 
The term "pharmaceutically acceptable carrier" as used herein means a 
non-toxic, generally inert vehicle for the active ingredient which does 
not adversely affect the ingredient. 
The term "physiologically acceptable carrier" as used herein means an 
acceptable cosmetic vehicle of one or more non-toxic excipients which do 
not react with or reduce the effectiveness of the active ingredient 
contained therein. 
The term "effective amount" means a predetermined antiviral amount of the 
antiviral agent, i.e. an amount of the agent sufficient to be effective 
against herpes virus in vivo. 
Process for Preparing the Compounds of Formula 1 
In general, the compounds of formula 1 are prepared by known methods using 
reaction conditions which are known to be suitable for the reactants. 
Description of the methods are found in standard textbooks such as "Annual 
Reports In Organic Synthesis--1994", P. M. Weintraub et al., Eds, Academic 
Press, Inc., San Diego, Calif., U.S.A., 1994 (and the preceding annual 
reports), "Vogel's Textbook Of Practical Organic Chemistry", B. S. Furniss 
et al., Eds, Longman Group Limited, Essex, UK, 1986, and "Comprehensive 
Organic Synthesis", B. M. Trost and I. Fleming, Eds, Pergaman Press, 
Oxford, UK, 1991, Volumes 1 to 8. 
An exception to the latter statement, however, is the unique stereospecific 
synthesis of a key intermediate for the preparation of the compounds of 
formula 1. This key intermediate is represented by formula 2 
EQU W.sup.1 -Tbg-CH.sub.2 --(R)--CH(CH.sub.2 C(O)CMe.sub.3)C(O)OW.sup.2(2) 
wherein W.sup.1 is an amino protective group, and W.sup.2 is a carboxyl 
protective group. In this instance, W.sup.2 is a protective group which 
can be selectively removed in the presence of the protective group 
W.sup.1. Preferably, W.sup.1 is tert-butyloxycarbonyl (Boc) or 
2,2,2-trichloroethoxycarbonyl and W.sup.2 is benzyl, 
(4-nitrophenyl)methyl, methyl or ethyl. 
The intermediate of formula 2 can be prepared by a stereospecific process 
illustrated in the following Scheme 1: 
##STR4## 
wherein W.sup.1 and W.sup.2 are as defined herein and Alk is methyl or 
ethyl. 
With reference to the preceding schematic representation, a starting 
material of formula W.sup.1 -Tbg-O-Alk (3) is reacted with the reagent 
LiCH.sub.2 P(O)(OCH.sub.3).sub.2 (4) (prepared from CH.sub.3 
P(O)(OCH.sub.3).sub.2 and butyllithium) to give a phosphonate of formula 
W.sup.1 -Tbg-CH.sub.2 P(O)(OCH.sub.3).sub.2 (5). Reaction of the latter 
phosphonate with a glyoxylyl ester of formula HC(O)C(O)OW.sup.2 (6) in the 
presence a suitable tertiary amine, preferably triethylamine or 
diisopropylethylamine, affords a .gamma.-keto-.alpha.,.beta.-unsaturated 
ester of formula W.sup.1 -Tbg-(E)-CH.dbd.CHC(O)OW.sup.2 (7). Reaction of 
the latter compound with the sodium enolate of a .beta.-ketoester of 
formula CH.sub.2 .dbd.CHCH.sub.2 OC(O)CH.sub.2 C(O)CMe.sub.3 (8) affords a 
Michael adduct of formula W.sup.1 -Tbg-CH.sub.2 --(R)--CH{CH 
(C(O)CMe.sub.3)-(C(O)OCH.sub.2 CH.dbd.CH.sub.2)}C(O)OW.sup.2 (9). 
Note (1): The .beta.-ketoester of formula 8, i.e. CH.sub.2 .dbd.CHCH.sub.2 
OC(O)CH.sub.2 C(O)CMe.sub.3, is prepared readily by reacting the lithium 
enolate of allyl acetate with trimethylacetyl chloride. 
Note (2): The sodium enolate of the .beta.-ketoester of formula 8 is 
generated in situ from the .beta.-ketoester in the presence of a 
catalytically effective amount of sodium hydride. 
Thereafter, reaction of the Michael adduct of formula 9 with 
tetrakistriphenylphosphine palladium(O) in the presence of a suitable 
secondary amine, preferably pyrrolidine or piperidine, similar to the 
method of R. Deziel, Tetrahedron Letters, 28, 4371 (1987), effects 
deallylation and subsequent decarboxylation of the allyl ester to give the 
key intermediate of formula 2. 
Noteworthy is the unexpected high stereo-selectivity obtained in the 
Michael addition reaction of the .gamma.-keto-.alpha.,.beta.-unsaturated 
ester of formula 7 with the sodium enolate of the .beta.-ketoester of 
formula 8 to give the Michael adduct of formula 9. The stereoselectivity 
of the Michael addition reaction is inferred by the fact that the 
intermediate of formula 2, derived directly from the Michael adduct, is 
obtained essentially as a single isomer. The diastereoisomeric purity of 
the intermediate of formula 2 can be demonstrated by nuclear magnetic 
resonance studies. The enantiomeric purity of the intermediate of formula 
2 can be assessed by removing the amino protective group (W.sup.1) and 
applying the method of J. A. Dale et al., J. Org. Chem., 34, 2543 (1969) 
to the resulting free amino derivative (see example 4 for more detail). 
Thereafter again, the carboxyl protective group (W.sup.2) of the key 
intermediate of formula 2 is selectively removed by standard methods, for 
example, by hydrogenolysis in the instance wherein W.sup.2 is benzyl, to 
give the corresponding free carboxylic acid derivative (see formula 14 in 
Scheme 2 below ) for incorporation into the process for preparing the 
compounds of formula 1 . 
In general, the incorporation of the preceding free carboxylic acid 
derivative into a process for the preparation of the compounds of formula 
1 can be envisaged as a sequence of chemical events wherein a carboxylic 
acid derivative (representing a first unit) is joined to two other units 
by forming amide bonds. 
In the following more detailed description of a convenient and practical 
process for preparing the compounds of formula 1, a certain order of the 
chemical events is followed. However, it will be appreciated that changes 
in the order of chemical events are not critical and therefore such 
changes are deemed to be within the scope of the present invention. 
Likewise, it should be appreciated that the intermediate of formula 2 
wherein protective group W.sup.1 can be selectively removed in the 
presence of protective group W.sup.2, allowing for a change in the order 
of the chemical events, also is deemed to be within the scope of the 
present invention. Accordingly, an important aspect of this invention 
includes a key intermediate of formula 2 in which W.sup.1 is an amino 
protective group for the amine at the N-terminus and W.sup.2 is a carboxyl 
protective group for the carboxyl at the C-terminus of the intermediate, 
with the proviso that the amino protective group W.sup.1 can be 
selectively removed in the presence of the carboxyl protective group 
W.sup.2 when the terminal amine is destined for the reaction to follow, or 
that, on the other hand, the carboxyl protective group W.sup.2 can be 
selectively removed in the presence of the amino protective group W.sup.1 
when the terminal carboxyl is destined for the reaction to follow. 
Examples of the intermediates of formula 2 wherein the amino protective 
group W.sup.1 can be selectively removed in the presence of the carboxyl 
protective group W.sup.2 include those in which W.sup.1 is 
tert-butyloxycarbonyl and W.sup.2 is benzyl, 2,2,2-trichloroethyl, methyl 
or ethyl. 
More particularly, with respect to an overall process, the compounds of 
formula 1 can be prepared by a convenient and practical process 
illustrated in the following Scheme 2. 
##STR5## 
In Scheme 2, W.sup.1 is as defined herein, W.sup.3 is a carboxyl 
protective group (preferably benzyl, tert-butyl or 2,2,2-trichloroethyl), 
R.sup.3 is azido for formula 12 and an amino for formula 13, R.sup.4 is 
W.sup.1 as defined herein for formula 15 and a hydrogen for formula 16, 
R.sup.5 is an amino protective group preferably tert-butyloxycarbonyl or 
2,2,2-trichloroethoxycarbonyl, for the compounds of formula 17 and 18, and 
a hydrogen for formula 19, and R.sup.1 and R.sup.2 are as defined herein. 
Referring to Scheme 2, a process for preparing compound of formula 1 
comprises: 
(a) coupling a carboxylic acid derivative of formula 10 with an amine of 
formula 11 to obtain an .alpha.-azidoamide of formula 12, 
(b) reducing the .alpha.-azidoamide of formula 12 to obtain a corresponding 
.alpha.-aminoamide of formula 13, 
(c) coupling the .alpha.-aminoamide of formula 13 with a carboxylic acid 
derivative of formula 14 to obtain a diprotected intermediate of formula 
15, 
(d) selectively deprotecting the diprotected intermediate of formula 15 to 
obtain a free N-terminal derivative of formula 16, 
(e) coupling the free N-terminal derivative of formula 16 with an 
N-protected valine of formula 17 to obtain a diprotected intermediate of 
formula 18, 
(f) selectively deprotecting the latter diprotected intermediate of formula 
18 to obtain a corresponding free N-terminal compound of formula 19, 
(g) reacting the free N-terminal compound of formula 19 with an acid 
chloride of formula 20 to obtain a corresponding protected carboxyl 
derivative of formula 21, and 
(h) deprotecting the latter derivative of formula 21 to obtain the 
corresponding compound of formula 1, and 
(i) if desired transforming the compound of formula 1 into a 
therapeutically acceptable salt thereof. 
The coupling steps (a), (c) and (e) and the deprotecting steps (d), (f) and 
(h) can be achieved by methods commonly used in peptide synthesis. 
More explicitly, the coupling step involves the dehydrative coupling of a 
free carboxyl of one reactant with the free amino group of the other 
reactant in the presence of coupling agent to form a linking amide bond. 
Description of such coupling agents are found in general textbooks on 
peptide chemistry, for example, M. Bodanszky, "Peptide Chemistry", 2nd rev 
ed, Springer-Verlag, Berlin, Germany, 1993. Examples of suitable coupling 
agents are N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in the 
presence of N,N'-dicyclohexylcarbodiimide or 
N-ethyl-N'-(3-dimethylamino)propyl!carbodiimide. A very practical and 
useful coupling agent is the commercially available (benzotriazol-1-yloxy) 
tri-(dimethylamino) phosphonium hexafluorophosphate, either by itself or 
in the presence of 1-hydroxybenzotriazole. Still another very practical 
and useful coupling agent is commercially available 
2-(1H-benzotriazol-1-yl)-N, N, N', N'-tetramethyluronium 
tetrafluoroborate. 
The coupling reaction is conducted in an inert solvent, e.g. 
dichloromethane or acetonitrile. An excess of a tertiary amine, e.g. 
diisopropylethylamine or N-methylmorpholine, is added to maintain the 
reaction mixture at a pH of about eight. The reaction temperature usually 
ranges between 0.degree. and 50 .degree. C. and the reaction time usually 
ranges between 15 minutes and 24 hours. 
In step (b), the azide group of the .alpha.-azidoamide of formula 12 is 
transformed into a corresponding amine of the .alpha.-aminoamide of 
formula 13 by a reducing agent capable of selectively reducing an azide to 
an amino group in the presence of an amido group and an ester group. This 
step can be accomplished conveniently and efficiently by the method of N. 
Maiti et al., Tetrahedron Letters, 27, 1423 (1986) using stannous chloride 
as the reducing agent and methanol as the reaction solvent. 
In step (g), the free N-terminal compound of formula 19 is reacted directly 
with 1 to 1.5 molar equivalents of the acid chloride of formula 20 to give 
the protected carboxyl derivative of formula 21. This step is based on the 
classical method for preparing amides whereby an acid chloride is reacted 
with the terminal amino group. The reaction proceeds readily in the 
presence of an (1.2 to 2.0 molar equivalents) excess of a suitable 
tertiary amine, for example N-methylmorpholine or diisopropylethylamine. 
The reaction is conducted in an inert solvent, such as dichloromethane or 
toluene, and at temperatures usually ranging from -20 .degree. C. to 20 
.degree. C. 
Furthermore, if desired, the compound of formula 1 can be obtained in the 
form of a therapeutically acceptable salt. Such salts can be considered as 
biological equivalents of the compounds of formula 1. Examples of such 
salts (of the carboxy group) are those formed by known methods with the 
sodium, potassium or calcium cation. 
The acid chlorides of formula 20 are known or can be prepared readily by 
known methods. For illustration, simple procedures for the preparation of 
certain acid chlorides of formula 20 are included in the examples. 
Antiherpes Activity 
The antiviral activity of the compounds of formula 1 can be demonstrated by 
biochemical, microbiological and biological procedures showing the 
inhibitory effect of the compounds on the replication of herpes simplex 
viruses, types 1 and 2 (HSV-1 and HSV-2), as well as acyclovir-resistant 
herpes simplex viruses. 
In the examples hereinafter, the inhibitory effect on herpes ribonucleotide 
reductase is noted for exemplary compounds of formula 1. Noteworthy, in 
the connection with this specific inhibition of herpes ribonucleotide 
reductase, is the relatively minimal effect or absence of such an effect 
of the compounds on cellular ribonucleotide reductase activity required 
for normal cell replication. 
A method for demonstrating the inhibitory effect of the compounds of 
formula 1 on viral replication is the cell culture technique; see, for 
example, T. Spector et al., Proc. Natl. Acad. Sci. U.S.A., 82, 4254 
(1985). 
The therapeutic effect of the compounds of formula 1 can be demonstrated in 
laboratory animals, for instance, by using an assay based on the murine 
model of herpes simplex virus-induced ocular disease for antiviral drug 
testing, described by C. R. Brandt et al., J. Virol. Meth., 36, 209 
(1992). 
When a compound of this invention, or one of its therapeutically acceptable 
acid addition salts, is employed as an antiviral agent, it is administered 
topically or systemically to warm-blooded animals, e.g. humans, pigs or 
horses, in a vehicle comprising one or more pharmaceutically acceptable 
carriers, the proportion of which is determined by the solubility and 
chemical nature of the compound, chosen route of administration and 
standard biological practice. For topical administration, the compound can 
be formulated in pharmaceutically accepted vehicles containing 0.1 to 5 
percent, preferably 0.5 to 5 percent, of the active agent. Such 
formulations can be in the form of a solution, cream or lotion. 
For systemic administration, the compound of formula 1 is administered by 
either intravenous, subcutaneous or intramuscular injection, in 
compositions with pharmaceutically acceptable vehicles or carriers. For 
administration by injection, it is preferred to use the compounds in 
solution in a sterile aqueous vehicle which may also contain other solutes 
such as buffers or preservatives as well as sufficient quantities of 
pharmaceutically acceptable salts or of glucose to make the solution 
isotonic. 
Suitable vehicles or carriers for the above noted formulations are 
described in standard pharmaceutical texts, e.g. in "Remington's 
Pharmaceutical Sciences", 18th ed, Mack Publishing Company, Easton, Pa., 
1990. 
The dosage of the compound will vary with the form of administration and 
the particular active agent chosen. Furthermore, it will vary with the 
particular host under treatment. Generally, treatment is initiated with 
small increments until the optimum effect under the circumstances is 
reached. In general, the compound is most desirably administered at a 
concentration level that will generally afford antivirally effective 
results without causing any harmful or deleterious side effects. 
With reference to topical application, the compound of formula 1 is 
administered cutaneously in a suitable topical formulation to the infected 
area of the body e.g. the skin or part of the oral or genital cavity, in 
an amount sufficient to cover the infected area. The treatment should be 
repeated, for example, every four to six hours until lesions heal. 
With reference to systemic administration, the compound of formula 1 is 
administered at a dosage of 10 mg to 150 mg per kilogram of body weight 
per day, although the aforementioned variations will occur. However, a 
dosage level that is in the range of from about 10 mg to 100 mg per 
kilogram of body weight per day is most desirably employed in order to 
achieve effective results. 
Another aspect of this invention comprises a cosmetic composition 
comprising a herpes viral prophylactic amount of the compound of formula 
1, or a therapeutically acceptable salt thereof, together with a 
physiologically acceptable cosmetic carrier. Additional components, for 
example, skin softeners, may be included in the formulation. The cosmetic 
formulation of this invention is used prophylactically to prevent the 
outbreak of herpetic lesions of the skin. The formulation can be applied 
nightly to susceptible areas of the skin. Generally, the cosmetic 
composition contains less of the compound than corresponding 
pharmaceutical compositions for topical application. A preferred range of 
the amount of the compound in the cosmetic composition is 0.5 to 5 percent 
by weight. 
Although the formulations disclosed hereinabove are indicated to be 
effective and relatively safe medications for treating herpes viral 
infections, the possible concurrent administration of these formulations 
with other antiviral medications or agents to obtain beneficial results is 
not excluded. Such other antiviral medications or agents include the 
antiviral nucleosides, for example, acyclovir, and antiviral surface 
active agents or antiviral interferons such as those disclosed by S. S. 
Asculai and F. Rapp in U.S. Pat. No. 4,507,281, Mar. 26, 1985. 
More specifically with respect to treating herpes viral infections by 
concurrent administration, it has been found that the antiherpes activity 
of an antiviral nucleoside analogs can be enhanced synergistically, 
without the concomitant enhancement of toxic effects, by combining the 
same with a compound of formula 1. Accordingly, there is provided herewith 
a pharmaceutical composition for treating herpes infections in a mammal 
comprising a pharmaceutically acceptable carrier, and an effective amount 
of the combination of an antiviral nucleoside analog or a therapeutically 
acceptable salt thereof, and a ribonucleotide reductase inhibiting 
compound of formula 1 or a therapeutically acceptable salt thereof. 
Also provided herein is a method of treating herpes viral infections in a 
mammal. The method comprises administering to the mammal an anti-herpes 
virally effective amount of a combination of a compound of formula 1 or a 
therapeutically acceptable salt thereof, and an antiviral nucleoside 
analog or a therapeutically acceptable salt thereof. 
The antiviral nucleoside analog employed in the combination is one which is 
enzymatically convertible (in vivo) to a viral DNA polymerase inhibitor 
of, and/or an alternative substrate for, a herpes DNA polymerase. The 
antiviral nucleoside analog can be selected from known nucleoside analogs. 
Preferred nucleoside analogs of the invention include acyclovir and its 
analogs; for example, the compounds of formula 22 
##STR6## 
wherein R.sup.6 is hydrogen, hydroxy or amino, or a therapeutically 
acceptable salt thereof. (Formula 22 wherein R.sup.6 is hydroxy represents 
acyclovir.) 
Other preferred antiviral nucleoside analogs for use according to the 
present invention include penciclovir, famciclovir and valacyclovir. 
An example of a therapeutically acceptable salt of the nucleoside analogs 
is the sodium salt. 
The term "synergistic effect" when used in relation to the antiviral or 
antiherpes activity of the above defined combination of the nucleoside 
analog and the compound of formula 1 means an antiviral or antiherpes 
effect which is greater than the predictive additive effect of the two 
individual components of the combination. 
When utilizing the combination of this invention for treating herpes 
infections, the combination is administered to warm blooded animals, e.g. 
humans, pigs or horses, in a vehicle comprising one or more 
pharmaceutically acceptable carriers, the proportion of which is 
determined by the solubility and chemical nature of the nucleoside analog 
and the compound of formula 1, chosen route of administration, standard 
biological practice, and by the relative amounts of the two active 
ingredients to provide a synergistic antiviral effect. The combination may 
be administered topically. For example, the two active agents (i.e. the 
antiviral nucleoside analog and the compound of formula 1, or their 
therapeutically acceptable salts) can be formulated in the form of 
solutions, emulsions, creams, or lotions in pharmaceutically acceptable 
vehicles. Such formulation can contain 0.01 to 1.0 percent by weight of 
the nucleoside analog, or a therapeutically acceptable salt thereof, and 
about 0.05 to 1 percent by weight of the compound of formula 1, or a 
therapeutically acceptable salt thereof. 
In any event, the two active agents are present in the pharmaceutical 
composition in amounts to provide a synergistic antiherpes effect. 
The following examples illustrate further this invention. Temperatures are 
given in degrees Celsius. Solution percentages express a weight to volume 
relationship, and solution ratios express a volume to volume relationship, 
unless stated otherwise. Nuclear magnetic resonance (NMR) spectra were 
recorded on a Bruker 400 MHz spectrometer; the chemical shifts (.delta.) 
are reported in parts per million. Abbreviations used in the examples 
include Boc: tert-butyloxycarbonyl; Bzl: benzyl; DMSO: dimethyl-sulfoxide; 
Et: ethyl; EtOH: ethanol; EtOAc: ethyl acetate; Et.sub.2 O: diethyl ether; 
HPLC: high performance liquid chromatography; Me: methyl; MeOH: methanol; 
Pr: propyl; TLC: thin layer chromatography; THF: tetrahydrofuran. 
EXAMPLE 1 
General Procedure for Coupling Reactions 
{See also R. Knorr et al., Tetrahedron Letters, 30, 1927 (1989).} 
The first reactant, i.e. a free amine (or its hydrochloride salt), is 
dissolved in CH.sub.2 Cl.sub.2 or CH.sub.3 CN and the solution is cooled 
to 4.degree.. Under a nitrogen atmosphere, four equivalents of 
N-methylmorpholine are added to the stirred solution. After 20 min, one 
equivalent of the second reactant, i.e. a free carboxylic acid, and 1.05 
equivalents of the coupling agent are added. (Practical and efficient 
coupling reagents for this purpose are (benzotriazol-1-yloxy) 
tris-(dimethylamino) phosphonium hexafluorophosphate or preferably 
2-(1H-benzotriazol-1-yl)-N, N,N',N'-tetramethyluronium tetrafluoroborate. 
The reaction is monitored by TLC. After completion of the reaction, the 
solvent is evaporated under reduced pressure. The residue is dissolved in 
EtOAc. The solution is washed successively with 1 N aqueous citric acid, 
10% aqueous Na.sub.2 CO.sub.3 and brine. The organic phase is dried 
(MgSO.sub.4), filtered and concentrated under reduced pressure. The 
residue is purified on silica gel (SiO.sub.2) according to Still's flash 
chromatography technique {W. C. Still et al., J. Org. Chem., 43, 2923 
(1978)}. 
EXAMPLE 2 
Preparation of 1(R)-Ethyl-2,2-dimethylpropylamine Hydrochloride 
(NH2--(R)--CH(Et)CMe.sub.3.HCl). 
To a cooled solution (0.degree.) of 4,4-dimethyl-3-pentanone (106 g, 0.928 
mol) and (R)-.alpha.-methylbenzylamine (111 g, 0.916 mol) in benzene (1 
L), a solution of TiCl.sub.4 (50.5 mL, 0.461 mol) in benzene (200 mL) was 
added at a rate that kept the temperature of the mixture below 10.degree.. 
Thereafter, the mixture was stirred mechanically for 3 h at 40.degree., 
cooled to room temperature (20.degree.-22.degree.) and filtered through 
diatomaceous earth. The diatomaceous earth was washed with Et.sub.2 O. The 
combined filtrate and wash was concentrated. The residue was dissolved in 
dry MeOH (2 L). The solution was cooled to 0.degree. and NaBH.sub.4 (20 g, 
0.53 mol) was added portionwise while maintaining the temperature of the 
mixture below 5.degree.. The methanol was evaporated. The residue was 
dissolved in Et.sub.2 O. The solution was washed with brine, dried 
(MgSO.sub.4) and concentrated to give a reddish oil (a 18:1 mixture of 
diastereoisomers as indicated by NMR). The oil was purified by flash 
chromatography (SiO.sub.2, eluent: EtOAc/hexane, 7:93) to afford 
N-(1(R)-phenylethyl)-1(R)-ethyl-2,2-dimethylpropylamine as a liquid (110 
g, 54% yield). This material was dissolved in hexane (1.5 L). 1N HCl in 
E.sub.2 O (550 mL) was added to the solution over a period of 15 min. The 
resulting white solid was collected on a filter and then washed with 
hexane to provide N-(1(R)-phenylethyl)-1(R)-ethyl-2,2-dimethylpropylamine 
hydrochloride (125 g, 97% yield). .sup.1 H NMR(CDCl.sub.3) 
.delta.7.79-7.74 (m, 2H), 7.48-7.30 (m, 3H), 4.49-4.31 (m, 1H), 2.44-2.36 
(m, 1H), 2.23 (d, J=6.5 Hz, 3H), 1.95-1.54 (m, 2H), 1.14 (s, 9H), 0.55 (t, 
J=7.5 Hz, 3H). 
A solution of the latter compound (41.5 g) in MeOH (120 mL) was mixed with 
10% Pd/C (w/w) (4.2 g) and the mixture was shaken under 50 psi of hydrogen 
in a Parr hydrogenator at room temperature for 48 h. The mixture was 
filtered through diatomaceous earth and the filtrate was concentrated to 
give the desired NH.sub.2 --(R)--CH(Et)CMe.sub.3 in the form of its 
hydrochloric acid addition salt, as a white solid (25 g, 100% yield). 
.sup.1 H NMR(CDCl.sub.3) .delta. 8.40-8.10 (broad s, 3H), 2.85-2.70 (m, 
1H), 1.90-1.58 (m, 2H), 1.22 (t, J=7 Hz, 3H), 1.10 (s, 9H). 
EXAMPLE 3 
Preparation of the Intermediate H-Asp(cyPn)(Bzl)--NH--(R)--CH (Et)CMe.sub.3 
(the compound of formula 13 wherein R.sup.4 is NH.sub.2 and W.sup.3 is 
Bzl) 
(a) (S)-.alpha.-Azido-1-{(phenylmethoxy)carbonyl}cyclo-pentaneacetic acid 
(the compound of formula 10 wherein W.sup.3 is Bzl): This compound was 
prepared from 2-oxospiro4.4!nonane-1,3-dione, described by M. N. 
Aboul-Enein et al., Pharm. Acta Helv., 55, 50 (1980), according to the 
asymmetric azidation method utilizing the Evan's auxiliary; see Evans et 
al., J. Amer. Chem. Soc., 112, 4011 (1990). 
More explicitly, a 1.6M hexane solution of butyllithium (469 mL, 750 mmol) 
was added dropwise under an argon atmosphere to a solution of the chiral 
auxiliary, 4(S)-(1-methylethyl)-2-oxazolidinone, (96.8 g, 750 mmol) 
{described by L. N. Pridgen and J. Prol., J. Org, Chem, 54, 3231 (1989)} 
in dry THF at -40.degree.. The mixture was stirred at -40.degree. for 30 
min and then cooled to -78.degree.. 2-Oxospiro4.4!nonane1,3-dione was 
added dropwise to the cooled mixture. The mixture was stirred at 0.degree. 
for 1 h. Thereafter, a 20% aqueous solution of citric acid (600 mL) was 
added to the mixture. The organic phase was separated and the aqueous 
phase was extracted with EtOAc. The combined organic phases were washed 
with brine, dried (MgSO.sub.4) and concentrated under reduced pressure to 
give 
3-2-(1-carboxycyclopentyl)-1-oxoethyl)}-4(S)-(1-methylethyl)-2-oxazolidin 
one as a pink solid (300 g). 
The latter solid (ca 750 mmol) was dissolved in CH.sub.3 CN (1 L). Benzyl 
bromide (89.2 mL, 750 mmol) and 1,8-diazabicyclo5.4.0!undec-7-ene (112 
mL, 750 mmol) were added to the solution. The mixture was stirred under 
argon for 16 h. The volatiles were removed under reduced pressure. The 
residue was dissolved in H.sub.2 O/EtOAc. The organic phase was separated, 
washed with a 10% aqueous solution of citric acid and brine, dried 
(MgSO.sub.4) and concentrated under reduced pressure to give an oil. 
Crystallization of the oil from hexane/EtOAc gave the corresponding benzyl 
ester as a white solid (204 g, 73% yield). 
A solution of the latter compound (70 g, 190 mmol) in dry THF (200 mL) was 
cooled to -78.degree.. A 0.66M THF solution of potassium 
bis(trimethylsilyl)amide (286 mL, 190 mmol) containing 6% cumene was added 
over a period of 15 min to the cooled solution. The mixture was stirred at 
-78.degree. for 45 min. A solution of 2,4,6-triisopropylbenzenesulfonyl 
azide (67 g, 220 mmol) in dry THF (100 mL) was added in one portion to the 
cold mixture, followed two minutes later by the addition of glacial acetic 
acid (50 mL, 860 mmol). The mixture was warmed and stirred at 
35.degree.-45.degree. for 1 h. The volatiles were removed under reduced 
pressure. The yellow residue was triturated with hexane/EtOH (4:1, 1.7 L). 
The resulting white solid was collected on a filter. The filtrate was 
mixed with SiO.sub.2 (230-240 mesh). Volatiles were removed under reduced 
pressure and the residual solid was dried at 35.degree. under reduced 
pressure to remove cumene. The residual solid then was placed on a column 
of SiO.sub.2. Elution of the column with hexane-EtOAc (9:1) and 
concentration of the eluent gave 
3-{{2(S)-azido-1-oxo-2-{(1-{(phenylmethoxy)carbonyl}cyclopentyl}-ethyl}-4( 
S)-(1-methylethyl)-2-oxazolidinone (66 g, 86% yield). 
A solution of the latter compound (13.4 g, 32.4 mmol) in THF/H.sub.2 O 
(3:1, 608 mL) was cooled to 0.degree.. Hydrogen peroxide/H.sub.2 O (3:7, 
16.3 mL, 141 mmol of H.sub.2 O.sub.2) was added to the cooled solution, 
followed by the addition of LiOH.H.sub.2 O (2.86 g, 68.2 mmol). The 
mixture was stirred at 0.degree. for 45 min and then quenched with a 10% 
aqueous solution of sodium sulfite (400 mL). After NaHCO.sub.3 (1.93 g) 
had been added, the mixture was concentrated under reduced pressure. The 
chiral auxiliary was recovered by continuous extraction (aqueous 
NaHCO.sub.3 /chloroform) for 20 h. Thereafter, the aqueous phase was 
cooled to 0.degree. rendered acidic by the addition of concentrated HCl 
and then extracted with EtOAc. The extract was washed with brine, dried 
(MgSO.sub.4) and concentrated under reduced pressure to give 
(S)-.alpha.-azido-1-{(phenylmethoxy)carbonyl}cyclopentaneacetic acid as a 
white solid (8.2 g, 84% yield). .sup.1 H NMR (CDCl.sub.3) .delta. 
7.40-7.28 (m, 5H), 5.12 (s, 2H), 4.55 (s, 1H), 2.30-2.20 (m, 1H), 
2.05-1.95 (m, 2H), 0 1.8-1.6 (m, 5H). 
(b) The title compound of this example: By following the coupling procedure 
of example 1 and using the hydrogen chloride salt of NH.sub.2 
--(R)--CH(Et)CMe.sub.3 of example 2 as the first reactant and 
(S)-.alpha.-azido-1-{(phenylmethoxy)carbonyl}cyclo-pentaneacetic acid of 
section (a) of this example as the second reactant, 
N-{1(R)-ethyl-(2,2-dimethylpropyl) }-(S)-.alpha.-azido-1-{(phenylmethoxy) 
carbonyl}cyclopentaneacetamide was obtained. Reduction of the latter 
compound with tin(II) chloride in MeOH according to the method of N. Maiti 
et al., Tetrahedron Letters, 27, 1423 (1986), followed by purification by 
chromatography (SiO.sub.2, hexane-Et.sub.2 O, 1:1), gave the title 
compound of this example. .sup.1 H NMR (CDCl.sub.3) .delta. 7.36-7.27 (m, 
5H), 7.08 (d, J=10.5 Hz, 1H), 5.17 (d, J=12.3 Hz, 1H), 5.09 (d, J=12.3 Hz, 
1H), 3.72 (s, 1H), 3.56 (ddd, J=10.5, 10.5, 2.5 Hz, 1H), 2.23-1.15 (m, 
2H), 1.87-1.80 (m, 1H), 1.76-1.57 (m, 8H), 1.17-1.03 (m, 1H), 0.88 (s, 9H) 
and 0.86 (t, J=7.3 Hz, 3H). 
EXAMPLE 4 
Preparation of the Intermediate Boc-Tbg-CH.sub.2 --(R)--CH (CH.sub.2 
C(O)CMe.sub.3)C(O)OBzl (the compound of formula 2 wherein W.sup.1 is Boc 
and W.sup.2 is Bzl) 
(a) Boc-Tbg-OMe (the compound of formula 3 wherein W.sup.1 is Boc): A 
solution of Boc-Tbg-OH (68 g, 0.30 mol) in dry CH.sub.3 CN (0.5 L) was 
cooled to 0.degree.. 1,8-Diazabicyclo5.4.0!undec-7-ene (54 mL, 0.36 mol) 
was added over a period of 10 min to the cooled solution, followed by the 
addition of CH.sub.3 I (37 mL, 0.60 mol). The reaction mixture was stirred 
at room temperature (20.degree.-22.degree.) for 4 h and then concentrated 
under reduced pressure. The residue was partitioned between EtOAc and 
H.sub.2 O. The organic phase was washed with H.sub.2 O, an aqueous 
saturated solution of NaHCO.sub.3 (2 X), and brine. Thereafter, the 
organic phase was dried (MgSO.sub.4) and concentrated to afford a clear 
viscous liquid. This material was distilled bulb to bulb (oil pump vacuum, 
air bath temperature at 110.degree.) to provide the desired product as a 
colorless oil (65 g, 88% yield). .sup.1 H NMR (CDCl.sub.3) .delta. 5.10 
(broad d, J=9.0 Hz, 1H), 4.10 (d, J=9.0 Hz, 1H), 3.72 (s, 3H), 1.44 (s, 
9H), 0.96 (s, 9H). 
(b) Boc-Tbg-CH.sub.2 -P(O)(OMe).sub.2 (the compound of formula 5 wherein 
W.sup.1 is Boc): At -78.degree. under a nitrogen atmosphere, a 5 L flask 
equipped with a mechanical stirrer, an addition funnel with jacket and a 
thermometer was charged with a solution of BuLi in hexane (3.60 mol, 361 
mL of a 10N solution). A cold (-78.degree.) solution of freshly distilled 
dimethyl methylphosphonate (391 mL, 3.60 mol) in dry THF (1 L) was added 
dropwise via the addition funnel over a 1 h period. The mixture was 
stirred at -78.degree. for 30 min. A cold (-78.degree.) solution of 
Boc-Tbg-OMe (111 g, 0.452 mol) in THF (0.5 L) was added dropwise over a 20 
min period. The reaction was stirred at -78.degree. for 45 min, and then 
allowed to warm to about -30.degree. over a 30 min period. Following the 
sequential addition of glacial acetic acid (0.25 L) and H.sub.2 O (0.3 L), 
the mixture was extracted with EtOAc (1 L). The organic layer was washed 
with H.sub.2 O, a 10% aqueous solution of NaHCO.sub.3 and brine, dried 
(MgSO.sub.4) and concentrated. The resulting solid was triturated with 
hexane to give the desired phosphonate as a white powder with mp 
84.degree.-86.degree. (144 g, 95% yield). .sup.1 H NMR (CDCl.sub.3) 
.delta. 5.23 (broad d, J=9.0 Hz, 1H), 4.25 (d, J=9.0 Hz, 1H), 3.80 (d, 
J=11.4 Hz, 6H), 3.30 (dd, J=22.0, 14.6 Hz, 1H), 3.12 (dd, J =22.0, 14.6 
Hz, 1H), 1.44 (s, 9H), 1.00 (s, 9H). 
The phosphonate is used in section (d) of this example. 
(c) HC(O)C(O)OBzl (the compound of formula 6 wherein W.sup.2 is Bzl): Solid 
H.sub.5 IO.sub.6 (49.3 g, 0.216 mol) was added portionwise to a solution 
of dibenzyl L-tartrate (70 g, 0.21 mol) in Et.sub.20 (900 mL). The mixture 
was stirred for 2.5 h at room temperature and then filtered. The filtrate 
was dried (MgSO.sub.4) and concentrated. The residual syrup was dissolved 
in hexane-E.sub.2 O (2:3). The resulting milky solution was filtered 
through a pad of diatomaceous earth. The pad was washed with 
hexane-Et.sub.2 O (2:5). The combined filtrate and washing were 
concentrated to yield benzyl-glyoxylate as an oil (69.9 g, .about.90% 
yield). H.sup.1 NMR (CDCl.sub.3) showed a mixture of aldehyde and hydrate 
form. Characteristic chemical shifts: .delta. 9.25 (s), 7.87-7.21 (m, 5H), 
5.47-5.03 (m), 4.56 (broad s). 
(d) The .gamma.-keto-.alpha.,.beta.-unsaturated ester 
Boc-Tbg-(E)-CH.dbd.CHC (O)OBzl (the compound of formula 7 wherein W.sup.1 
is Boc and W.sup.2 is Bzl): A solution of Boc-Tbg-CH.sub.2 --P(O) 
(OMe).sub.2 (121 g, 0.359 mol), described in section (b) of this example, 
and triethylamine (0.10 L, 0.72 mol) in CH.sub.3 CN (0.7 L) was stirred 
under nitrogen for 10 min at room temperature. Thereafter, a solution of 
HC(O)C(O)OBzl (121 g, .about.0.36 mol) in CH.sub.3 CN (0.15 L) was added 
over 30 min. The mixture was stirred for 24 h and then concentrated. The 
residue was dissolved in E.sub.2 O -hexane (2:1, 0.8 L). The solution was 
washed with a 10% aqueous solution of citric acid, a saturated solution of 
NaHCO.sub.3 and brine, dried (MgSO.sub.4) and concentrated. The resulting 
orange oil was passed through a silica gel pad (12.times.10 cm) using 
EtOAc-hexane (3:20) as the eluent. Concentration of the eluate gave the 
desired .gamma.-keto-.alpha.,.beta.-unsaturated ester as a yellow oil (112 
g, 83% yield). .sup.1 H NMR (CDCl.sub.3) .delta. 7.42-7.32 (m, 5H), 7.23 
(d, J=15.9 Hz, 1H), 6.80 (d, J=15.9 Hz, 1H), 5.25 (s, 2H), 5.21 (broad d, 
J=8.9 Hz, 1H), 4.43 (d, J=8.9 Hz, 1H), 1.42 (s, 9H), 0.96 (s, 9H). 
The .gamma.-keto-.alpha.,.beta.-unsaturated ester is used in section (f) of 
this example. 
(e) CH.sub.2 .dbd.CHCH.sub.2 OC(O)CH.sub.2 C(O)CMe.sub.3 (the compound of 
formula 8): A solution of lithium bis(trimethylsilyl)amide in THF (1N, 0.8 
L) was cooled to -78.degree.. A solution of allyl acetate (39 mL, 0.36 
mol) in THF (40 mL) was added dropwise to the cooled solution. The mixture 
was stirred at -78.degree. for 1 h. Thereafter, a solution of 
trimethyl-acetyl chloride (47 mL, 0.38 mol) was added dropwise and the 
resulting mixture was stirred for 25 min at -78.degree.. Hexane (0.3 L) 
and an aqueous solution of HCl (3N, 0.6 L) were added to the mixture. The 
organic phase was separated and washed with a saturated aqueous solution 
of sodium bicarbonate, brine and water. The organic phase was dried 
(MgSO4), and concentrated to afford an orange oil. Distillation (bulb to 
bulb, air bath temperature of 60.degree., 0.25 Tor.) of the crude product 
gave desired ester as a colorless oil (62 g, 92% yield). .sup.1 NMR 
(CDCl.sub.3) .delta. 6.02-5.87 (m, 1H), 5.35 (broad d, J=17.2 Hz, 1H), 
5.25 (broad d, J=9.5 Hz, 1H), 4.63 (broad d, J=5.6 Hz, 2H), 3.59 (s, 2H), 
1.19 (s, 9H). 
(f) The Michael adduct, i.e. Boc-Tbg-CH.sub.2 --(R)--CH{CH 
(C(O)CMe.sub.3)(C(O)OCH.sub.2 CH.dbd.CH.sub.2)}C(O)OBzl (the compound of 
formula 9 wherein W.sup.1 is Boc and W.sup.2 is Bzl): Solid NaH (2.7 g of 
a 60% oil dispersion, 0.07 mol) was added over a 15 min period to a 
solution of CH.sub.2 .dbd.CHCH.sub.2 OC(O)CH.sub.2 C(O)CMe.sub.3 (83.2 g, 
0.452 mol) in THF (0.8 L). The reaction mixture was stirred at room 
temperature under an atmosphere of argon until all the solid dissolved (30 
min). The homogeneous solution was cooled to -60.degree. (solution 
temperature) and a solution of Boc-Tbg-(E)-CH.dbd.CHC (O)OBzl (170 g, 0.45 
mol), described in section (d) of this example, in THF (0.5 L) was added 
slowly over a period of 45 min. Thereafter, the reaction mixture was 
stirred at -60.degree. for 5 h. A 10% aqueous solution of citric acid was 
added and the mixture was allowed to warm to room temperature. The mixture 
was extracted with Et.sub.2 O. The organic phase was washed with a 5% 
aqueous solution of sodium bicarbonate and brine, dried (MgSO.sub.4) and 
concentrated to afford an orange oil (250 g) which was used without 
further purification in the next reaction. 
(g) Boc-Tbg-CH.sub.2 --(R)--CH (CH.sub.2 C (O) CMe.sub.3) C (O)--OBzl: 
Pyrrolidine (56 mL, 0.54 mol) was added to a stirred solution of 
tetrakistriphenylphosphine palladium (O) (2.60 g, 2.25 mmol, 0.5% molar) 
in CH.sub.2 Cl.sub.12 (250 mL) and CH.sub.3 CN (250 mL) at 0.degree. under 
an atmosphere of argon. The mixture was allowed to warm to room 
temperature. A solution of the Michael adduct from the preceding section 
(250 g, 0.45 mol) in CH.sub.2 Cl.sub.2 --CH.sub.3 CN (200 mL:200 mL) was 
added to the mixture. After 3 h, the mixture was concentrated to yield an 
orange oil. The crude oil was dissolved in a mixture of Et.sub.2 O-hexane 
(1:1, 1 L). The solution was washed with a 10% aqueous solution of citric 
acid, 10% aqueous solution of sodium bicarbonate, and brine, dried 
(MgSO.sub.4) and concentrated to give the title compound of this example 
as an orange oil (203 g, &gt;90% yield). This material was used without 
further purification in example 5. A small sample was purified by 
SiO.sub.2 chromatography. Elution with hexane-EtOAc (9:1) gave the pure 
title compound as a colorless oil. .alpha.!.sub.D.sup.25 +11.5 (c =1.3, 
CHCl.sub.3); .sup.1 H NMR (CDCl.sub.3) .delta. 7.38-7.28 (m, 5H), 5.10 (s, 
2H), 5.07 (broad d, J=9.2 Hz, 1H), 4.08 (d, J=9.2 Hz, 1H), 3.38-3.31 (m, 
1H), 3.09 (dd, J=18.8, 6.0 Hz, 1H), 2.94 (dd, J=18.4 6.1 Hz, 1H), 2.82 
(dd, J=18.4, 6.1 Hz, 1H), 2.77 (dd, J=18.8, 6.0 Hz, 1H), 1.42 (s, 9H), 
1.10 (s, 9H), 0.95 (s, 9H). The diastereoisomeric purity was assessed to 
be &gt;35:1 by NMR; see P. L. Beaulieu et al., European patent application 
560 267, published Sep. 15, 1993. In order to assess the enantiomeric 
purity of the title compound, the Boc protective group (W.sup.1) was 
removed with 4N HCl in dioxane and the resulting amine was converted to a 
Mosher amide (see J. A. Dale et al., vide supra). By comparing results 
from a product prepared by the procedure of this example with results 
obtained with a racemic mixture of the title compound, the enantiomeric 
excess for said product was determined to be &gt;96% by NMR and &gt;99% by 
chiral column chromatography. The latter determination was performed by 
normal phase HPLC on a Chiracel.RTM. OD column from Daicel Chemical 
Industries Limited, Tokyo, Japan (U.S. distributor: Chiral Technologies 
Inc., Exton Pa, U.S.A.). EtOH-hexane (1:19) was the eluent and UV 
detection at 215 nmwas employed. 
EXAMPLE 5 
Preparation of the Intermediate Boc-Tbg-CH.sub.2 --(R)--CH(CH.sub.2 C 
(O)CMe.sub.3)C(O)OH (the compound of formula 14 wherein W.sup.1 is Boc) 
To a solution of the title compound of example 4 (171 g, 0.36 mol) in EtOH 
(1.4 L) was added 10% Pd/C (10 g). The resultant mixture was stirred 
vigorously under one atmosphere of hydrogen for 5 h. Thereafter, the 
reaction mixture was subjected to filtration through diatomaceous earth. 
The filtrate was concentrated under reduced pressure. The residue was 
dissolved in a saturated aqueous solution of Na.sub.2 CO.sub.3. The 
aqueous solution was washed with hexane-Et.sub.2 O (8:2), rendered acidic 
with citric acid and extracted with EtOAc. The extract was dried 
(MgSO.sub.4) and concentrated. The orange residue was dissolved in 
Et.sub.2 O and the resulting solution was passed through a silica gel pad 
(12.times.12 cm). Concentration gave the title compound of this example as 
a solid with mp 62.degree.-65.degree. (117 g, 84% yield). .sup.1 H NMR 
(CDCl.sub.3) .delta. 5.18 (d, J=8.8 Hz, 1H), 4.09 (d, J=8.8 Hz, 1H), 
3.35-3.29 (m, 1H), 3.09 (dd, J=18.8, 6.3 Hz), 2.94 (dd, J=18.4, 6.3 Hz, 
1H), 2.83 (dd, J=18.4, 6.3 Hz, 1H), 2.78 (dd, 18.8, 6.3 Hz, 1H), 1.43 (s, 
9H), 1.14 (s, 9H), 0.96 (s, 9H). 
EXAMPLE 6 
Preparation of the Intermediate Boc-Tbg-CH.sub.2 --(R)--CH(CH.sub.2 C 
(O)CMe.sub.3)C(O)-Asp(cyPn)(Bzl)--NH--(R)--CH(Et)CMe.sub.3 (the compound 
of formula 15 wherein R.sup.5 is Boc and W.sup.3 is Bzl) 
By following the coupling procedure of example 1 and using the title 
compound of example 3 as the first reactant and the title compound of 
example 5 as the second reactant, the title compound of this example is 
obtained. .sup.1 H NMR (CDCl.sub.3) .delta. 7.43-7.26 (m, 6H), 6.76 (d, 
J=10.0 Hz, 1H), 5.16 (s, 2H), 5.06 (d, J=8.9 Hz, 1H), 4.62 (d, J=8.9 Hz, 
1H), 4.07 (d, J=8.9 Hz, 1H), 3.60 (ddd, J=10.0, 10.0, 2.5 Hz, 1H), 
3.18-2.83 (m, 3H), 2.70 (dd, J=16.9, 4.1 Hz, 1H), 2.68-2.54 (m, 1H), 
1.90-1.52 (m, 9H), 1.42 (s, 9H), 1.11 (s, 9H), 0.94 (s, 9H), 0.88 (s, 9H), 
0.78 (t, J=7.3 Hz, 3H). 
EXAMPLE 7 
Preparation of the Intermediate Boc-(N-Me) Val-Tbg-CH.sub.2 
--(R)--CH(CH.sub.2 C(O)CMe.sub.3)C(O)-Asp(cyPn)-(Bzl)--NH--(R)--CH 
(Et)CMe.sub.3 (the compound of formula 18 wherein R.sup.5 is Boc and 
W.sup.3 is Bzl) 
The title compound of example 6 (18.00 g, 0 24.8 mmol) was dissolved in 4M 
HCl.sub.1/ dioxane (125 mL). The mixture was stirred at room temperature 
for 45 min and then concentrated under reduced pressure to give 
H-Tbg-CH.sub.2 --(R)--CH(CH.sub.2 C(O)CMe.sub.3)--C 
(O)Asp(cyPn)(Bzl)-NH--(R)--CH(Et)CMe.sub.3 in the form of its hydrochloric 
acid addition salt. 
The latter salt was dissolved in CH.sub.2 Cl.sub.12 (300 mL). The solution 
was washed successively with 10% aqueous Na.sub.2 CO.sub.3 and brine. The 
organic phase was concentrated to give the corresponding free base as a 
clear oil (.about.17 g). The clear oil was dissolved in CH.sub.2 Cl.sub.2 
(200 mL). N-Methylmorpholine (7 mL, 70 mmol) and Boc-(N-Me)Val-OH (6.93 g, 
30 mmol) were added to the solution. At this point, the free base was 
coupled with Boc-(N-Me)Val-OH according to the procedure of example 1 to 
give the title compound (18.8 g, 89% yield). .sup.1 H NMR (CDCl.sub.3) 
.delta. 7.40-7.29 (m, 6H), 6.83 (d, J=8.5 Hz, 1H), 6.77 (d, J=10 Hz, 1H), 
5.17 (s, 2H), 4.62 (d, J=9.5 Hz, 1H), 4.55 (d, J=10 Hz, 1H), 4.28 (d, J=8 
Hz, 1H), 3.64-3.56 (m, 1H), 2.97 (s, 3H), 3.05-2.50 (m, 7H), 2.33-2.23 (m, 
1H), 1.91-1.56 (m, 15H), 1.34-1.14 (m, 7H), 1.11 (s, 9H), 1.05 (d, J=7 Hz, 
3H), 0.95 (d, J=8.5 Hz, 3H), 0.90 (s, 9H), 0.77 (t, J=7 Hz, 3H). 
EXAMPLE 8 
Preparation of Some Representative Intermediates for the Elaboration of the 
N-Terminus of the Compound of Formula 1 
(a) .alpha.(R)-Methylcyclohexanepropionic acid chloride: Under argon, a 
1.6M hexane solution of burylithium (100 mL, 160 mmol) was added to a 
cooled solution (-30.degree. to -40.degree.) of 
4(S)-(1-methylethyl)-2-oxazolidinone (20.7 g, 160 mmol), see L. N. Pridgen 
et al., J. Org. Chem., 54, 3231 (1989), in dry THF (200 mL). After 15 min, 
the mixture was cooled to -78.degree. and propionyl chloride (14.2 mL, 163 
mmol) was added. After 5 min at -78.degree., the reaction mixture was 
allowed to warm to 0.degree.. The mixture then was treated with a 
saturated aqueous solution of NaHCO.sub.3 (500 mL). The resultant mixture 
was extracted with EtOAc (2 X). The combined organic extracts were dried 
(MgSO.sub.4) and concentrated to afford a yellow liquid. This material was 
purified by flash chromatography SiO.sub.2, eluent: EtOAc-hexane (1:10 to 
1:4)! to provide (4S)-(1-methylethyl)-3-(1-oxopropyl)-2-oxazolidinone as a 
clear liquid (10.9 g, 74% yield). .sup.1 H NMR (400 MHz, CDCl.sub.3) 
.delta. 4.46-4.41 (m, 1H), 4.29-4.19 (m, 2H), 3.03-2.86 (m, 2H), 2.43-2.33 
(m, 1H), 1.17 (t, J=7.3 Hz, 3H), 0.92 (d, J=7 Hz, 3H), 0.88 (d, J=7 Hz, 
3H). 
A solution of lithium hexamethyl-disilazane (LiHMDS, 1.0M in THF, 120 mL, 
120 mmol) was added to dry THF (300 mL). The resultant solution was cooled 
to 0.degree.. Meanwhile, a solution of 
(4S)-(1-methylethyl)-3-(1-oxopropyl)-2-oxazolidinone (20.9 g, 113 mmol) in 
dry THF (200 mL) was cooled to 0.degree., and then cannulated into the 
LiHMDS solution. After 30 min at 0.degree., benzyl bromide (13.4 mL, 113 
mmol) was added. The resultant mixture was stirred at 0.degree. for 2 h 
and then allowed to warm to room temperature. The mixture was treated with 
10% aqueous citric acid and then extracted with EtOAc (2 X). The combined 
organic extracts were washed with brine, dried (MgSO.sub.4) and 
concentrated to provide a yellow oil mixed with a solid. This material was 
purified by flash chromatography SiO.sub.2, eluent: EtOAc-hexane (1:10 to 
1:3)! to provide 
4(S)-(1-methylethyl)-3-(2(R)-methyl-1-oxo-3-phenylpropyl)-2-oxazolidinone 
as a clear pale yellow liquid (26.8 g, 86% yield). .sup.1 H NMR 
(CDCl.sub.3) .delta. 7.29-7.24 (m, 4H), 7.22-7.16 (m, 1H), 4.45-4.41 (m, 
1H), 4.26-4.13 (m, 3H), 3.13 (dd, J=13, 7.5 Hz, 1H), 2.64 (dd, J=13, 7.5 
Hz, 1H), 2.22-2.12 (m, 1H), 1.16 (d, J=6.5 Hz, 3H), 0.84 (d, J=7 Hz, 3H), 
0.61 (d, J=7 Hz, 3H). 
To a cooled solution (0.degree.) of the latter oxazolidine derivative (26.7 
g, 97.0 mmol) in THF (9500 mL) and H.sub.2 O (1.5 L) was added a 30% 
aqueous solution of hydrogen peroxide (55 mL, 0.5 mol), followed by the 
addition of a solution of LiOH.H.sub.2 O (8.67 g, 200 mmol) in H.sub.2 O 
(15 mL). The resultant mixture was vigorously stirred for 1 h at 
0.degree.. A solution of Na.sub.2 SO.sub.3 (100 g) in H.sub.2 O (700 mL) 
and solid NaHCO.sub.3 (20 g) were added sequentially. After 5 min, the THF 
was removed under reduce pressure. The residual aqueous solution was 
washed with CH.sub.2 Cl.sub.12 (3 X). The aqueous phase was rendered 
acidic with 10% aqueous HCl.sub.1 and extracted with Et.sub.2 O (3 X). The 
combined Et.sub.2 O extracts were washed with brine, dried (MgSO.sub.4) 
and concentrated to afford .alpha.(R)-methylbenzenepropionic acid as a 
clear liquid (12.8 g, 81% yield). .sup.1 H NMR (CDCl.sub.3) .delta. 
7.33-7.19 (m, 5H), 3.10 (dd, J=13.5, 6.5, 1H), 2.83-2.74 (m, 1H), 2.79 
(dd, J=13.5, 8 Hz, 1H), 1.20 (d, J=7 Hz, 3H). 
A mixture of .alpha.(R)-methylbenzenepropionic acid (3.0 g, 18 mmol) and 5% 
rhodium on alumina (800 mg) in methanol (100 mL) was shaken under 40 
p.s.i. of H.sub.2 on a Parr hydrogenation apparatus. After 15 h, the 
mixture was filtered through diatomaceous earth and concentrated to afford 
.alpha.(R)-methylcyclohexanepropionic acid as a clear liquid (2.4 g, 77%). 
.sup.1 H NMR (CDCl.sub.3) .delta. 2.62-2.53 (m, 1H), 1.79-1.59 (m, 6H), 
1.38-1.16 (m, 5H), 1.17 (d, J=7 Hz, 3H), 0.95-0.83 (m, 2H). 
To a solution of .alpha.(R)-methylcyclohexane-propionic acid (2.4 g, 14 
mmol) in dry CH.sub.2 Cl.sub.12 (30 mL) was added DMF (1 drop) and oxalyl 
chloride (2 g, 15 mmol). After 2 h at room temperature, the mixture was 
concentrated. The residue was dissolved in Et.sub.2 O (10 mL). This 
solution was filtered. The filtrate was concentrated to provide 
.alpha.(R)-methylcyclohexanepropionic acid chloride as a clear yellow 
liquid (2.6 g, 98% yield). .sup.1 H NMR (400 MHz, CDCl.sub.3) 3.02-2.91 
(m, 1H), 1.80-1.63 (m, 6H), 1.39-1.10 (m, 5H), 1.28 (d, J=7 Hz, 3H), 
0.99-0.85 (m, 2H). This material was used without further purification in 
the coupling reaction described in the following example. 
(b) .alpha.(S)-(1-Methylethyl)cyclohexanepropionic acid chloride: By 
following procedure (a) of this example but replacing propionyl chloride 
with 3-methylbutanoyl chloride, .alpha.(S)-(1-methylethyl)-cyclohexane 
propionic acid is obtained. .sup.1 H NMR (CDCl.sub.3) .delta. 2.27 (ddd, 
J=10.5, 7, 3.5 Hz, 1H), 1.91-1.80 (m, 2H), 1.73-1.55 (m, 5H), 1.35-1.08 
(m, 5H), 0.98-0.77 (m, 2H), 0.97 (d, J=7 Hz, 3H), 0.96 (d, J=7 Hz, 3H). 
Thereafter, the latter compound was converted to its corresponding acid 
chloride in the same manner as described in section (a) of this example. 
(c) .alpha.(R),.beta.(R)-Dimethylcyclohexanepropionic acid: Oxalyl chloride 
(2.9 mL, 33.3 mmol) and then 2 drops of dimethylformamide were added to a 
solution of .beta.(R)-methylbenzenepropionic acid (4.0 mL, 26.1 mmol). The 
mixture was stirred at room temperature for 2h, and then evaporated to 
dryness under reduced pressure to give the corresponding acid chloride 
(i.e. first reactant), which was used hereinafter. 
In a separate preparation, a solution of 
4(R)-(1-methylethyl)-2-oxazolidinone (3.06 g, 23.7 mmol) in dry THF (30 
mL) was cooled to -50.degree.. Under argon, a 1.6M hexane solution of 
butyllithium (14.8 mL, 23.7 mmol) was added dropwise to the cooled 
solution of the oxazolidinone derivative. After 15 min at -78.degree., a 
solution of the first reactant in dry THF (10 mL) was added. The reaction 
mixture was stirred at -70.degree. for 30 minutes and then allowed to warm 
to room temperature over a 45 min period. The mixture was quenched with 
excess 10% aqueous NH.sub.4 Cl. Thereafter, the THF was removed under 
reduced pressure and the resulting concentrate was dissolved in EtOAc. The 
solution was washed with 5% aqueous NaHCO.sub.3 (2 X) and brine (2 X), 
dried (MgSO.sub.4) and concentrated to give an oil. The oil was purified 
by flash chromatography SiO .sub.2, hexane-EtOAc (43:7)! to yield 
(4R)-(1-methylethyl)-3-(3(R)-methyl-1-oxo-3-phenylpropyl)- 2-oxazolidinone 
(6.1 g, 93% yield). 
A solution of potassium hexamethyldisilazane (KHMDS, 0.692M in THF, 23.3 
mL, 16.1 mmol) was added to dry THF (50 mL) and the mixture was cooled to 
-78.degree. A solution of the preceding oxazolidine derivative (4.03 g, 
14.7 mmol) in dry THF (40 mL) was cooled to -78.degree.. The latter 
solution was then cannulated into the KHMDS solution. The mixture was 
stirred at -78.degree. for 1 h. Methyl iodide (1.75 mL, 28.1 mmol) was 
added. After being stirred at -78.degree. for 2.5 h more, the reaction 
mixture was warmed to room temperature. The mixture was quenched with 10% 
aqueous citric acid. After the THF was removed under reduced pressure, the 
resulting concentrate was dissolved in EtOAc. The solution was washed with 
10% aqueous citric acid (2 X), 5% aqueous NaHSO.sub.3 and brine, dried 
(MgSO.sub.4) and concentrated to dryness. The residue was purified by 
flash chromatography SiO.sub.2, eluent: hexane-EtOAc (42:8)! to yield 
4(R)-(1-methylethyl)-3-(2(R),3(R)-dimethyl-1-oxo-3-phenylpropyl)-2-oxazoli 
donone (2.84 g, 67% yield). 
Reaction of the latter oxazolidinone derivative (2.80 g, 9.69 mmol) with 
30% aqueous hydrogen peroxide (5.5 mL, 48.5 mmol) in the presence of 
LiOH.H.sub.2 O (0.81 g, 19.3 mmol), followed by reduction of the resulting 
.alpha.(R),.beta.(R)-dimethyl-benzenepropionic acid with 5% rhodium on 
alumina (1.76 g) in MeOH, according to the procedure of section (a) of 
this example, afforded .alpha.(R),.beta.(R)-dimethylcyclohexanepropionic 
acid (1.74 g, 96% yield from the latter oxazolidinone derivative). .sup.1 
H NMR (CDCl.sub.3) 2.63 (qd, J=6.5, 6.5 Hz, 1H), 1.80-1.71 (m, 4H), 
1.68-1.63 (m, 2H), 1.29-0.91 (m, 6H), 1.08 (d, J=7 Hz, 3H), 0.84 (d, J=6.5 
Hz, 3H). Thereafter, the latter compound was converted to its 
corresponding acid chloride in the same manner as described in section (a) 
of this example. 
EXAMPLE 9 
Preparation of (3-Cyclohexyl-2(R)-methyl-1-oxopropyl)-(N--Me) 
Val-Tbg-CH.sub.2 --(R)--CH(CH.sub.2 C(O)CMe.sub.3)--C 
(O)-Asp(cyPn)-NH--(R)--CH(Et)CMe.sub.3 (the compound of formula 1 wherein 
R.sup.1 =H and R.sup.2 =Me). 
The title compound of example 7 (18.8 g, 21.98 mmol) was dissolved in 4M 
HCl.sub.1/ dioxane (200 mL). The solution was stirred at room temperature 
for 7 h and then concentrated. The resulting residue was dissolved in 
CH.sub.2 Cl.sub.12 (350 mL). The solution was washed with 10% aqueous 
Na.sub.2 CO.sub.3 and then brine. Concentration of the solution provided 
H--(N--CH.sub.3)Val-Tbg-CH.sub.2 --(R)--CH(CH.sub.2 C(O)--CMe.sub.3) 
C(O)-Asp(cyPn)-NH--(R)--CH(Et)CMe.sub.3 (.about.17 The latter compound was 
dissolved in CH.sub.2 Cl.sub.2 (200 mL). After the addition of 
N-methylmorpholine (2.5 mL, 25 mmol) and 
.alpha.(R)-methylcyclohexane-propionic acid chloride, the mixture was 
stirred at room temperature for 1.5 h. Thereafter, the mixture was washed 
with 10% aqueous Na.sub.2 CO.sub.3, 10% aqueous citric acid and brine, 
dried (MgSO.sub.4) and concentrated to give the corresponding protected 
carbonyl derivative of the title compound of this example (12.5 g, 63% 
yield), i.e. the compound of formula 21 wherein R.sup.1 is hydrogen, 
R.sup.2 is methyl and W.sup.3 is Bzl. 
The latter derivative (12.2 g, 13.5 mmol) was subjected to hydrogenolysis 
10% Pd(OH).sub.2 /C (1.3 g), 1 atmosphere of H.sub.2, MeOH (150 mL), 1 
h!. Thereafter, charcoal was added to the reaction mixture and the 
resulting suspension was filtered through a glass microfiber filter and 
diatomaceous earth. The filtrate was concentrated under reduced pressure 
to yield the title compound as a fine white powder (10.7 g, 97% yield). Mp 
115.degree.-116.degree.; .sup.1 H NMR (d.sub.6 -DMSO) .delta. 8.31 (d, J=7 
Hz, 0.25H), 8.23 (broad, J=10 Hz, 1H), 7.86 (d, J=8.5 Hz, 0.75H), 6.93 
(overlap, d, J=10 Hz, 1H), 4.91 (overlap, d, J=10 Hz, 1H), 4.70 (d, J=10 
Hz, 0.75H), 4.22 (d, J=10.5 Hz, 0.25H), 4.16 (overlap, d, J=8.5 Hz, 1H), 
3.45-3.37 (m, 1H), 3.24-3.15 (m, 1H), 2.96 (s, 2.25H), 2.88 (s, 0.75H), 
2.88-2.50 (m, 5H), 2.19-2.00 (m, 2H), 1.70-1.44 (m, 14H), 1.44-0.62 (m, 
47H, characteristic singlets at 1.04, 0.88 and 0.87); FAB MS (m/z): 817.6 
(M+H).sup.+. 
By following the procedure of this example but replacing 
.alpha.(R)-methylcyclohexanepropionic acid chloride with 
.alpha.(S)-(1-methylethyl)-cyclohexanepropionic acid chloride, then 
{3-cyclohexyl-2(S)-(1-methylethyl)-1-oxopropyl}-(N-Me) Val-Tbg-CH.sub.2 
--(R)--CH(CH.sub.2 C(O)CMe.sub.3)C(O)-Asp(cypn)-NH--(R)--CH (Et)CMe.sub.3 
was obtained. .sup.1 H NMR (d.sub.6 -DMSO) .delta. 8.23 (d, J=10 Hz, 1H), 
8.01 (d, J=8 Hz, 1H), 6.93 (d, J=10 Hz, 1H), 4.92 (d, J=10 Hz, 1H), 4.80 
(d, J=11 Hz, 1H), 4.13 (d, J=8.5 Hz, 1H), 3.40-3.37 (m, 1H), 3.23-3.16 (m, 
1H), 3.00 (s, 3H), 2.84-2.56 (m, 4H), 2.53-2.48 (m, 1H, overlap with Me's 
of DMSO), 2.18-2.08 (m, 1H), 2.06-2.02 (m, 1H), 1.73-1.42 (m, 15H), 
1.39-1.28 (m, 1H), 1.24-1.00 (m, 4H), 1.04 (s, 9H), 0.92-0.77 (m, 15H), 
0.90 (s, 9H), 0.87 (s, 9H), 0.64 (t, J=7 Hz, 3H); FAB MS (m/z): 845 
(M+H).sup.+. 
Again, by following the procedure of this example but replacing 
.alpha.(R)-methylcyclohexane-propionic acid chloride with .alpha.(R), 
.beta.(R)-dimethyl-cyclohexanepropionic acid chloride, then 
(3-cyclohexyl-2 (R), 3 (R)-dimethyl-1-oxopropyl)-(N-Me) Val-Tbg-CH.sub.2 
--(R)--CH (CH.sub.2 C(O) CMe.sub.3)C(O)-Asp (cyPn)-NH--(R)--CH (Et) 
CMe.sub.3 was obtained. .sup.1 H NMR (d6-DMSO) .delta. 8.23 (d, J=9.5 Hz, 
0.9H), 8.16 (d, J=5 Hz, 0.1H), 7.84 (d, J=8.5 Hz, 1H), 6.98 (broad, 0.1H), 
6.94 (d, J=10 Hz, 0.9H), 4.96 (broad, 0.1H), 4.91 (d, J=10 Hz, 0.9H), 4.70 
(d, J=11.5 Hz, 0.9H), 4.23 (d, J=10 Hz, 0.1H), 4.16 (d, J=8.5 Hz, 0.9H), 
3.96 (d, J=5 Hz, 0.1 H), 3.42-3.37 (m, 1H), 3.24-3.16 (m, 1H), 2.96 (s, 
2.7H), 2.90 (s, 0.3H), 2.83-2.50 (m, 5H)2.19-2.02 (m, 2H), 1.73-1.35 (m, 
15H), 1.20-0.96 (m, 6H), 1.04 (s, 9H), 0.91-0.87 (m, 6H), 0.89 (s, 9H), 
0.87 (s, 9H), 0.79 (d, J=7 Hz, 3H), 0.77 (d, J=7 Hz, 3H), 0.65 (t, J=7 Hz, 
3H); FAB MS (m/z): 831 (M+H).sup.+. 
EXAMPLE 10 
Inhibition of Herpes Simplex Virus (HSV-1) Ribonucleotide Reductase 
a) Preparation of Enzyme 
HSV-1 ribonucleotide reductase (partially purified) was obtained from 
quiescent BHK-21/Cl13 cells infected with strain F HSV-1 virus at 10 
plaque forming units/cell as described by E. A. Cohen et al., J. Gen. 
Virol., 66, 733 (1985). 
b) Assay 
The assay described by P. Gaudreau et al., J. Biol, Chem., 262, 12413 
(1987), is used to evaluate the capability of the compounds of formula 1 
to inhibit HSV-1 ribonucleotide reductase activity. The assay results are 
expressed as the concentration of the compound producing 50% of the 
maximal inhibition (IC.sub.50) of enzyme activity. The number of units of 
the enzyme preparation used in each assay was constant, based on the 
specific activity of the enzyme preparation. The results are relative to 
the activity obtained in control experiments without the test compound and 
represent the means of four assays that varied less than 10% with each 
other. 
The following TABLE I illustrates the assay results obtained for 
exemplified compounds of formula 1. 
TABLE I 
______________________________________ 
Compound of the Formula 
##STR7## 
wherein R.sup.1 and R.sup.2 are 
IC.sub.50 
as designated herein below 
.mu.M 
______________________________________ 
R.sup.1 =H and R.sup.2 =Me 
0.147 
R.sup.1 =H and R.sup.2 =CHMe.sub.2 
0.123 
R.sup.1 and R.sup.2 =Me 
0.191 
______________________________________ 
EXAMPLE 11 
Inhibition of Herpes Simplex Virus (HSV-1) Replication in Cell Culture 
Assay 
BHK-21 cells clone 13 (ATCC CCL10) were incubated for two days in 850 
cm.sup.2 roller bottles (2.times.10.sup.7 cells/bottle) with alpha-MEM 
medium (Gibco Canada Inc., Burlington, Ontario, Canada) supplemented with 
8% (v/v) fetal bovine serum (FBS, Gibco Canada, Inc.). The cells were 
trypsinized and then transferred to fresh media in a 96-well microtiter 
plate at a density of 50,000 cells per well in 100 .mu.L. The cells were 
incubated at 37.degree. for a period of 6 hours to allow adhesion to the 
plate. The cells then were washed once with 100 .mu.L of alpha-MEM 
supplemented with 0.5% FBS (v/v) and incubated with 100 .mu.L of the same 
media for 3 days. After this period of serum starvation, the low serum 
media was removed. The cells were washed once with 100 .mu.L BBMT and 
incubated for two hours in 100 .mu.L of the same media. {Note: BBMT medium 
is described by P. Brazeau et al., Proc. Natl. Acad. Sci. U.S.A., 79, 7909 
(1980).} 
Thereafter, the cells were infected with HSV-1 strain F or KOS 
(multiplicity of infection =0.05 PFU/cell) in 50 .mu.L of BBMT medium. 
Following one hour of virus absorption at 37.degree., the media was 
removed and the cells were washed with BBMT (2.times.100 .mu.L). The cells 
were incubated with or without 100 .mu.L of the appropriate concentration 
of test reagent in BBMT medium. After 24 hours of incubation at 
37.degree., the extent of viral replication was determined by an ELISA 
assay; for instance, the following assay that detects the late 
glycoprotein C of HSV-1. 
Cells were fixed in a microtiter plate with 100 .mu.L of 0.063% 
glutaraldehyde in phosphate buffered saline for 30 minutes at room 
temperature. The microtiter plate was then washed once with casein 
blocking solution and blocked with 200 .mu.L of the same solution for one 
hour at room temperature. Thereafter, 100 .mu.L of mAB Cll recognizing 
HSV-1 gC envelope protein see E. Trybala et al., Journal of General 
Virology, 75, 743 (1994)! was added to each well for two hours at room 
temperature. The plate was washed three times with phosphate buffered 
saline containing 0.05% polyoxyethylene (20) sorbitan monooleate. The 
cells were and incubated with 100 .mu.L of sheep anti-mouse IgC 
horseradish peroxidase for one hour at room temperature in the dark. 
The plate then was washed three times with 200 .mu.L of the above-noted 
phosphate buffer saline preparation, and then once with 0.1M sodium 
citrate (pH 4.5). Thereafter, 100 .mu.L of orthophenylenediamine 
dihydrochloride (OPD, Gibco, Canada Inc.) was added to each well. The 
plate was agitated on a microplate shaker for 30 minutes in the dark. 
Color development was monitored at 450 nm using a microplate 
spectrophotometer. 
SAS was used to calculate % inhibition of viral replication and to generate 
ECl.sub.50 values. 
Results 
The following TABLE II provides examples of the results obtained when 
compounds of formula 1 were evaluated according to the cell culture assay 
(HSV-1 strain F) of this example. 
TABLE II 
______________________________________ 
Compound of the formula 
##STR8## 
wherein R.sup.1 and R.sup.2 are 
EC.sub.50 
as designated herein below 
.mu.M 
______________________________________ 
R.sup.1 =H and R.sup.2 =Me 
0.4 
R.sup.1 =H and R.sup.2 =CHMe.sub.2 
0.2 
R.sup.1 and R.sup.2 =Me 
0.2 
______________________________________ 
EXAMPLE 12 
Synergistic Combinations 
The synergistic action between the title compound of example 9 and 
acyclovir (ACV) against HSV-1 was demonstrated by evaluating the two 
agents, each alone and then in various combinations in the cell culture 
assay, using strain KOS of HSV-1 and applying the isobole method to the 
results obtained in these studies; see J. Suhnel, J. Antiviral Research, 
13, 23 (1990) for a description of the isobole method. The results are 
illustrated in accompanying FIG. 1. 
More explicitly with reference to the isobole method, this method requires 
experimental data generated for the two test compounds, each alone and in 
different combinations. In this way selected concentrations of the title 
compound of example 9 (EC.sub.5, EC.sub.10, EC.sub.20 and EC.sub.30) were 
added to a given concentration of ACV and the EC.sub.50 's were evaluated 
as described previously. For these experiments, the EC.sub.5, EC.sub.10, 
EC.sub.20 and EC.sub.30 of the title compound of example 9 (i.e. the test 
compound) were derived from inhibition curves previously obtained. An 
isobologram is generated using for the Y axis a value termed FEC.sub.60 
(ACV) (which is the ratio of the concentration of ACV required to inhibit 
HSV replication by 60% in the presence of a fixed concentration of the 
test compound to the concentration required in the absence of the test 
compound). This is plotted against a term representing the ratio of the 
fixed concentration of the test compound to the concentration of the test 
compound that reduced inhibition of HSV replication in the absence of ACV 
(the X axis). 
Equations 
##EQU1## 
The following TABLE III is illustrative of results obtained when 
combinations of ACV and the title compound of example 9 (TC) were 
evaluated for their antiherpes activity against HSV-1. The virus strain 
and the multiplicity of infections (MOI) employed were HSV-1 KOS strain 
(MOI =0.05 PFU/cell). 
TABLE III 
______________________________________ 
SYNERGISTIC STUDIES OF ACYCLOVIR (ACV) 
AND THE TITLE COMPOUND OF EXAMPLE 9 (TC) 
AGAINST HSV-1 
EC.sub.50 
COMPOUNDS (.mu.M).sup.1 
______________________________________ 
Compound Alone 
ACV.sup.2 6.95 
TC 0.473 
Synergistic Studies 
ACV + 0.05 .mu.M of TC 
6.3 
ACV + 0.1 .mu.M of TC 
3.2 
ACV + 0.15 .mu.M of TC 
1.79 
ACV + 0.2 .mu.M of TC 
1.79 
ACV + 0.3 .mu.M of TC 
1.0 
ACV + 0.4 .mu.M of TC 
0.5 
______________________________________ 
.sup.1 Stock solutions of the title compound of example 9 were filtered 
through a 0.22 .mu.M membrane and then the concentration of the compound 
in the filtered solution was determined by HPLC. 
.sup.2 Acyclovir was obtained from Burroughs Wellcome Inc., Kirkland, 
Quebec, Canada. 
Note: In the preceding studies of TABLE III, the inhibition of the HSV 
replication was observed at concentrations significantly below the 
cytotoxic levels for the test compounds as determined by the cytotoxicity 
assay of F. Denizot and R. Lang, J. Immunol. Methods, 89, 271 (1986). 
The results of TABLES III show that, on combining the title compound of 
example 9 with acyclovir, a proportional lowering of the EC.sub.50 of 
acyclovir is effected as the ratio of the concentrations of the title 
compound of example 9 is increased. Hence, these synergistic studies 
demonstrate that the compounds of formula 1 are able to potentiate the 
antiherpes activity of acyclovir against HSV-1.