Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase with improved in vivo antiviral activity

Article abstract of DOI:10.1021/jm960324r

We have been investigating the potential of a new class of antiviral compounds. These peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus (HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replica

Full text of DOI:10.1021/jm960324r

J . Med. Chem. 1996, 39, 4173-4180
4173
P ep tid om im etic In h ibitor s of Her p es Sim p lex Vir u s Ribon u cleotid e Red u cta se
w ith Im p r oved in Vivo An tivir a l Activity
Neil Moss,*,† Pierre Beaulieu, J ean-Simon Duceppe, J ean-Marie Ferland, Michel Garneau, J ean Gauthier,
Elise Ghiro, Sylvie Goulet, Ingrid Guse, J orge J aramillo, Montse Llinas-Brunet, EÄ ric Malenfant,
Raymond Plante, Martin Poirier, Francois Soucy, Dominik Wernic, Christiane Yoakim, and Robert De´ziel
Bio-Me´ga/ Boehringer Ingelheim Research Inc., 2100 Cunard, Laval, Que´bec, Canada H7S 2G5
Received April 30, 1996X
We have been investigating the potential of a new class of antiviral compounds. These
peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus
(HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replication of viral DNA.
The compounds disclosed in this paper build on our previously published work. Structure-
activity studies reveal beneficial modifications that result in improved antiviral potency in
cell culture in a murine ocular model of HSV-induced keratitis. These modifications include
a stereochemically defined (2,6-dimethylcyclohexyl)amino N-terminus, two ketomethylene amide
bond isosteres, and a (1-ethylneopentyl)amino C-terminus. These three modifications led to
the preparation of BILD 1351, our most potent antiherpetic agent containing a ureido
N-terminus. Incorporation of the C-terminal modification into our inhibitor series based on a
(phenylpropionyl)valine N-terminus provided BILD 1357, a significantly more potent antiviral
compound than our previously published best compound, BILD 1263.
Ta ble 1
In tr od u ction
We have been pursuing a new class of antiviral
compounds for their potential utility as inhibitors of
herpes simplex virus (HSV) replication. These com-
pounds prevent association of the two subunits of HSV
ribonucleotide reductase (RR), an enzyme that catalyzes
the conversion of ribonucleotide diphosphates into the
corresponding deoxyribonucleotides, thereby preparing
the DNA building blocks required for HSV genomic
replication.1 Our subunit association inhibitors are
based on the C-terminal amino acid sequence of the
HSV RR small subunit (R2). This sequence binds to the
RR large subunit (R1) and consequently permits subunit
association and subsequent catalytic activity.2 Our
inhibitors, which mimic this C-terminal sequence, com-
pete with R2 for binding to R1. This mechanism of
inhibiting HSV RR is attractive because compounds
based on the HSV R2 C-terminal sequence do not inhibit
host cell RR.3 Despite the fact that herpes and human
RRs function by the same mechanism and have similar
structural features, the critical R2 C-terminal regions
ing efforts to identify new inhibitors of HSV RR that
are more potent antiherpetics in vitro and in vivo.
Our structure-activity studies are based on the five
C-terminal amino acids of HSV R2 (Val-Val-Asn-Asp-
Leu), since this sequence contains the most important
functionalities for binding to HSV R1.7 Table 1 depicts
three classes of RR inhibitors that we have previously
published. The distinction between these three classes
lies at the inhibitor N-terminus. Our first structure-
activity disclosure, compound 1 being a representative
example, highlighted the role and relative importance
of the various amino acid side-chain functionalities for
potency in an HSV RR enzyme assay.8 However, none
of the compounds in this paper proved efficacious
against HSV in cell culture. Progress toward efficacy
in HSV cell culture was obtained with the inhibitor
series represented by compound 2.9 A binding interac-
tion between the phenyl ring in 2 and R1 accounted for
the 30-fold increase in binding assay potency over
of these enzymes have little sequence homology. Con-
sequently mimics of either enzyme's R2 C-terminus do
not cross-inhibit the other ribonucleotide reductase.4
Given that our inhibitors mimic an amino acid
sequence, these compounds have thus far maintained
a degree of peptidic character. The suitability of pep-
tide- and peptidomimetic-based compounds as effective
drugs remains an important question.5 As an important
first step, we recently demonstrated that inhibitors of
HSV RR subunit association could not only prevent HSV
replication in cell culture but also reduce the severity
of HSV-induced keratitis in a murine ocular model.6
This work provided the first illustration that peptido-
mimetic compounds can prevent enzyme subunit as-
sociation in vivo. In this report, we detail our continu-
† Current address: Boehringer Ingelheim Pharmaceuticals, Inc.,
R&D Center, 175 Briar Ridge Rd., Ridgefield, CT 06877.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(96)00324-X CCC: $12.00 © 1996 American Chemical Society

4174 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Moss et al.
Ta ble 2
compound 1. Incorporation of various beneficial modi-
fications into compound 2 eventually led to the discovery
of BILD 1263 (Table 7), a potent inhibitor of HSV RR
subunit association (IC50 ) 0.2 nM) that also inhibited
the replication of HSV types 1 and 2 in cell culture (EC50
) 3 and 4 µM). It was this molecule that became the
first RR subunit association inhibitor to reduce the
severity of HSV induced keratitis in a murine ocular
model.6
More recently, we revealed that a minor modification
to our first published inhibitor series, represented by
compound 1, could increase binding potency to the same
level as the inhibitor series represented by compound
2. Insertion of an NH group near the N-terminus of
compound 1 improved binding potency over 50-fold (cf.
compounds 1 and 3).10 Evidence led us to conclude that
this increase in binding potency was due to a hydrogen-
bonding interaction between R1 and the NH group. The
inhibitor series represented by 3 also provided a point
of reference for a series of conformationally restricted
inhibitors designed to probe the bioactive conformation
of these peptide-based compounds. However, this pub-
lication did not describe the antiviral properties of these
new ureido-based inhibitors. Herein, we reveal modi-
fications to the inhibitor series represented by com-
pound 3 that have proven beneficial for in vitro and in
vivo antiviral potency. Although the molecules about
to be discussed do not represent an exhaustive list of
modifications investigated, they do include the most
important ones identified so far. This paper provides a
summary of the structure-activity studies and the
thought process that led to the discovery of these
inhibitors.
Before discussing the new results, it is important to
point out a protocol that has often proven necessary to
help assess the effect of new inhibitor modifications.
Binding assay potency has proven to be an important
criterion for good potency in HSV cell culture.9 The
radioligand binding assay used to determine the potency
of our inhibitors against HSV RR is reliable for com-
pounds with IC50s of around 1 nM or higher.9,11 How-
ever, virtually all compounds that have good cell culture
potency are believed to have true IC50s substantially
lower that 1 nM. This makes assessment of the effect
of a new modification on binding potency difficult if we
use our most potent molecules as a point of reference
for further structure-activity studies. Consequently,
we frequently investigate new structural modifications
in a less potent series first. Modifications that improve
binding potency or alter physicochemical properties
without significantly lowering binding potency are then
incorporated into our most potent inhibitors for evalu-
ation in HSV cell culture. This protocol is used in this
paper to illustrate the effects of the new modifications
about to be discussed.
a larger lipophilic group improved binding potency 2-fold
(cf. compounds 3 and 4). The importance of the N-
terminal lipophilic group in inhibitors 3 and 4 for good
binding potency, combined with the assumed inherent
flexibility of these alkyl chains, raised the potential that
locking of these chains in a favorable orientation for
binding to R1 might improve inhibitor potency. How-
ever, it was not immediately obvious how to achieve
this.
Through screening of various readily available alkyl-
amines at the inhibitor N-terminus, we noticed the
similar potencies of inhibitors containing either a cy-
clohexylamino or isopropylamino group (cf. compounds
5 and 6, Table 2). On the basis of the assumption that
the N-termini of 5 and 6 bound to R1 similarly, the
equivalent potencies of these inhibitors implied that
much of the cyclohexyl ring in 6 did not interact with
R1. This suggested the use of the cyclohexyl ring as a
semirigid scaffold for orienting alkyl groups in specific
directions. Adding two methyl groups to the cyclohex-
ane ring as shown in compound 7 improved binding
potency 10-fold. Moreover, changing the stereochem-
istry of one of the methyl groups, as shown for the
compound 8 mixture, further improved the binding
potency to the level of compound 3. Since these results
showed that both equatorial or axial methyl groups on
the cyclohexane ring could improve binding potency, the
possible benefit of a lipophilic group that could occupy
both equatorial and both axial positions became an
obvious extrapolation. One N-terminal group that
achieved this general arrangement of alkyl functionality
Resu lts a n d Discu ssion
Modification of th e N-Ter m in al Lipoph ilic Gr ou p.
The presence of the 1-ethylpropyl N-terminus in com-
pound 3 is very important for good binding potency.
Replacement of this group with an isopropyl moiety
lowers potency approximately 40 times (compound 5,
Table 2), and further truncation to a methyl group
results in a 2800-fold loss of binding potency.10 Con-
versely, replacement of the 1-ethylpropyl group in 3 with

Peptidomimetic Inhibitors of HSV RR
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4175
Ta ble 3
this in mind, we sought to further reduce hydrophilicity
at the inhibitor C-terminus by removing the hydroxy-
methyl group. The binding potency of the resultant
inhibitor, compound 12, drops a further 15-fold, and
unfortunately, molecules containing the (3,3-dimethyl-
butyl)amino C-terminus do not look promising in cell
culture.
We previously showed that replacement of a leucine
residue at the inhibitor C-terminus with an alanine
lowered inhibitory potency over 2000-fold.8 This result
demonstrated the importance of the C-terminal lipo-
philic group and suggested that this region of the
inhibitor might be participating in a relatively precise
binding interaction with R1. Therefore, we were some-
what surprised to discover that a neopentylamine
C-terminus produced an inhibitor more potent than the
corresponding (3,3-dimethylbutyl)amino analogue (cf.
compounds 12 and 14). Also surprising was the neces-
sity of all three methyl groups of the neopentylamine
for good binding potency. Unlike compound 12 where
removal of one methyl group lowered binding potency
only 3.5 times,12 removal of one methyl group from
compound 14 lowered binding potency almost 50-fold
(cf. compounds 12 with 13 and 14 with 15). These
results indicated the potential existence of multiple
binding modes for the C-terminal lipophilic groups of
various inhibitors. More significantly, inhibitors con-
taining the neopentylamine C-terminus consistently had
similar cell culture potencies to those containing the
synthetically more complex γ-methylleucinol (see com-
pounds 27 and 28, Table 6 for one example).
Despite the appealing simplicity of the neopentyl-
amine C-terminus, we also explored the potential of
more elaborate neopentylamine derivatives. One of the
most beneficial modifications resulted from the intro-
duction of a stereochemically defined methyl or ethyl
group R to the C-terminal nitrogen. Substituted neo-
pentylamines possessing an (R)-alkyl group, compounds
16 and 17, were at least two times more potent than
the parent compound 14, while the corresponding S-
analogues 18 and 19 were approximately 50-fold less
potent. Although the binding potency increases associ-
ated with the addition of an (R)-alkyl group were not
large, the resultant improved cell culture potencies
maintained our interest in these neopentylamine de-
rivatives (see compounds 27 and 29, Table 6 for one
example).
was the (dimethylcyclohexyl)amino group shown in
compound 9. The two methyl groups on the cyclohexane
ring forced the cyclohexane ring to adopt the conforma-
tion shown in Table 2. NMR coupling constants pro-
vided evidence for the predominance of this conforma-
tion in solution (coupling constant of 10, 3, and 3 Hz
for the hydrogen adjacent to the N-terminal nitrogen).
Importantly, compound 9 turned out to be at least 10-
fold more potent than isomers 7 and 8 and more potent
than either compounds 3 or 4.
The lipophilic N-terminal group in compound 9 con-
stitutes the most potent substitution identified at this
position so far. More significantly, this particular
(dimethylcyclohexyl)amino group also provided inhibi-
tors with improved in vivo potency. This point will be
further elaborated later.
Mod ifica tion of th e In h ibitor C-Ter m in u s. Our
previous structure-activity studies show that replace-
ment of the C-terminal carboxylic acid with a hy-
droxymethyl group lowers binding potency 7-10-fold.8,9
Compounds 10 and 11 in Table 3 further illustrate this
observation. However, inhibitors containing a hydroxy-
methyl C-terminus are up to 10 times more potent in
cell culture than the corresponding carboxylic acid
analogue.9 These results demonstrate the important
relationship between inhibitor physicochemical proper-
ties and cell culture potency. Polar functionality such
as carboxylic acids may impede the ability of inhibitors
to reach efficacious concentrations inside cells. With
Th e Bis-Ketom eth ylen e Isoster e. The advent and
incorporation of amide bond isosteres into peptide and
peptidomimetic compounds is an area of active inves-
tigation. Removal of an amide bond can potentially
increase metabolic stability and change physicochemical
properties which can favorably affect cellular uptake.
With the knowledge that amide bonds provide sites for
hydrogen bonding to water, we hoped that removal of
heteroatoms associated with an amide bond might
decrease the desolvation energy required for passive
diffusion through the cell’s lipid bilayer and thus
improve cell culture potency. Before testing this hy-
pothesis, we first investigated amide linkages that could
be modified with minimal effect on binding assay
potency.
One amide bond that could be successfully modified
is located between the pyrrolidine-modified asparagine
and tert-leucine residues (see Table 4). We previously

4176 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Moss et al.
Ta ble 4
Ta ble 5
demonstrated that the pyrrolidine-modified asparagine
backbone NH was likely not involved in a binding
interaction with R1 since it could be alkylated with no
adverse effect on inhibitor binding potency.10 This led
us to replace this NH with a CH2 (a ketomethylene
isostere, cf. compounds 20 and 21, Table 4). We were
pleased to discover that this modification had little effect
on inhibitor binding potency.
Further attempts to improve the cell culture potency
of the ureido-based inhibitors led to the disappointing
discovery that replacement of the amide bond between
the tert-leucine and the pyrrolidine-modified asparagine
residues with a ketomethylene isostere did not, as
predicted, improve potency (cf. compounds 24 and 25,
Table 5). Nevertheless, modifying the side-chain amide,
i.e. replacing the pyrrolidine for a tert-butyl-group,
improved tissue culture potency approximately 2-fold
(cf. compounds 24 and 26). Somewhat surprisingly,
unlike compound 25, incorporation of the backbone
ketomethylene isostere into compound 26 improved cell
culture potency a further 2-fold. The resultant com-
pound, BILD 1351, has a cell culture potency compa-
rable to that of the larger BILD 1263. The increase in
cell culture potency obtained by replacing the pyrroli-
dine-modified asparagine (compound 24) with the bis-
ketomethylene derivative (BILD 1351) was consistently
observed when we incorporated this modification into
other ureido-based inhibitors.
The side-chain amide of the pyrrolidine-modified
asparagine residue could be modified in a similar
manner. We previously demonstrated that dialkylation
of the asparagine side-chain nitrogen (pyrrolidine found
to be the best example) improved inhibitor binding
potency 100-fold over the unsubstituted nitrogen.8,9
Attempts to alter the shape of this group, with the
optimistic intention of increasing binding potency, led
to the preparation of the corresponding tert-butyl de-
rivative (compounds 22 and 23). Although the com-
pounds shown in Table 4 showed that replacement of
the pyrrolidine moiety with a tert-butyl group had
virtually no affect on binding potency, we had removed
one amide bond, and more significantly, inhibitors con-
taining a tert-butyl group proved less hydrophilic than
the corresponding inhibitor containing a pyrrolidine.13
In h ibitor Cell Cu ltu r e P oten cy. Incorporation of
the new N- and C-terminal modifications just discussed
into inhibitors containing cyclopentylaspartic acid, a
modification previously shown to improve both binding
and cell culture potencies,9 led to the preparation of
compound 24 (Table 5). Unlike the inhibitors shown
in Tables 1-4, compound 24 inhibited the replication
of HSV-1 and -2 in cell culture below 1000 µM (EC50 28
and 40 µM). This molecule demonstrated that inhibitors
containing a ureido N-terminus could inhibit HSV
replication in cell culture. Previously, only inhibitors
based on the larger (phenylpropionyl)valine N-terminus,
e.g. BILD 1263, showed efficacy in cell culture.
Table 6 uses BILD 1351 as a frame of reference to
illustrate the effect on cell culture potency of the
previously discussed N- and C-terminal modifications.
A comparison between BILD 1351 and compounds 27
and 28 shows the (1-ethylneopentyl)amino C-terminus
in BILD 1351 to be superior to either the γ-methylleu-
cinol (28) or the unsubstituted neopentylamine (27)
C-termini. However, a comparison between BILD 1351
and compound 29 shows that the (dimethylcyclohexyl)-
amino N-terminus in BILD 1351 provides no benefit in
terms of cell culture potency over the (3-propylbutyl)-
amino N-terminus. Nevertheless, we maintained a
strong interest in the (dimethylcyclohexyl)amino N-
terminus after discovering that BILD 1351 appeared to
be superior to compound 29 in vivo (see next section).

Peptidomimetic Inhibitors of HSV RR
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4177
Ta ble 6
F igu r e 1. Effect of BILD1351 on HSV-1-induced ketatitis.
F igu r e 2. Effect of BILD1357 on HSV-1-induced ketatitis.
Ta ble 7
cell culture potency. Unfortunately, unlike the ureido-
based inhibitor series, replacing the pyrrolidine-modi-
fied asparagine residue in BILD 1357 with the bis-
ketomethylene derivative did not improve cell culture
potency (see compound 30). The reason for the latter
observation was not immediately clear. Nevertheless,
BILD 1357 and compound 30 constituted the most
potent inhibitors of HSV RR subunit association made
to date.
In h ib it or P ot en cy in a Mu r in e Mod el of HSV-
In d u ced Ker a titis. The potential utility of BILD 1263
as an antiherpetic drug has been highlighted.6 In
addition to cell culture potency comparable to the most
widely used antiherpetic drug, acyclovir, BILD 1263 also
strongly potentiated the antiviral activity of acyclovir
and was effective against acyclovir resistant strains of
HSV. Most significantly, BILD 1263 reduced the sever-
ity of HSV-1-induced keratitis in a murine ocular model
when administered as a 5% ophthalmic cream.14 We
have used this ocular model as a screen for evaluating
the relative potency of our newer RR inhibitors, con-
centrating on testing the most potent inhibitors of HSV
replication in cell culture.
BILD 1351 and 1357 proved to be significantly more
potent in this model than BILD 1263. As can be seen
from Figures 1 and 2, both BILD 1351 and 1357 reduced
the severity of HSV-1-induced keratitis in a dose de-
pendent manner. In both cases, disease could be almost
completely suppressed (ED90) when the RR inhibitor
was administered as a 0.3% cream formulation. We
previously showed that BILD 1263 could only come close
to this level of disease suppression when administered
as a 5% cream formulation.
Considering the improvements in cell culture potency
obtained from ureido based inhibitors containing the bis-
ketomethylene derivative and the (1-ethylneopentyl)-
amino C-terminus, we investigated the effect of these
modifications on the antiviral properties of the inhibitor
series represented by BILD 1263 (Table 7). As observed
previously in the ureido-based inhibitor series, replace-
ment of the C-terminal γ-methylleucinol (BILD 1263)
with a 1-ethylneopentylamine (BILD 1357) improved
As mentioned earlier, even though BILD 1351 and
compound 29 had similar cell culture potencies, BILD
1351 proved superior in the mouse ocular model.

4178 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Moss et al.
Sch em e 1
Compound 29 only managed to achieve the efficacy level
of BILD 1351 when administered as a 5-10% cream
formulation (data not shown).
Compound 30 was also shown to be a potent inhibitor
of HSV replication in cell culture. However, when this
compound was applied as a 1% cream formulation in
the mouse ocular model, significant hair loss (alopecia)
was observed around the eye. Even at higher concen-
trations of BILD 1351 and 1357, no toxic effect was
observed. On this basis compound 30 was not investi-
gated further.
BILD 1351 and 1357 constitute the most potent RR
subunit association inhibitors found so far in this in vivo
model of HSV disease. These results constitute further
support for the potential of these peptidomimetic com-
pounds as effective antiherpetic agents. Further phar-
macological evaluation of these compounds has com-
menced.
Con clu sion
The compounds disclosed in this paper further high-
light the potential of peptidomimetic inhibitors of HSV
RR subunit association as potential antiherpetic agents.
Structure-activity studies show that inhibitors based
on a ureido N-terminus can be as efficacious in vivo as
the larger (phenylpropionyl)valine based N-terminus.
Beneficial modifications that result in improved anti-
viral potency include a stereochemically defined (2,6-
dimethylcyclohexyl)amino N-terminus, a bis-ketometh-
ylene derivative as a replacement for the pyrrolidine
modified asparagine residue, and a (1-ethylneopentyl)-
amino C-terminus. These three modifications led to the
preparation of BILD 1351, our most potent antiherpetic
agent containing a ureido N-terminus. The new C-
terminal modification allowed the preparation of BILD
1357, a significantly more potent antiviral than our
previously published compound, BILD 1263.
oxobutanoic acid (pyrrolidine modified asparagine moiety)8,
N-Boc-L-γ-methylleucine,8 and the appropriately protected
precursor of cyclopentylaspartic acid9 have been described in
our earlier structure-activity papers. The various stere-
ochemically defined 2,6-dimethylcyclohexylamines were pre-
pared from 2,6-dimethylphenol according to the procedures in
refs 17 and 18. The enantioselective synthesis of 2(R)- or 2(S)-
ethyl/methyl-3,3-dimethylpropylamine has been published.19
The tert-butyl ketone amino acid residue found in inhibitors
22 and 26 was prepared according to published procedure.20
The preparation of the ketomethylene dipeptide isosteres
found in compound 25 and compounds 27-30 and BILD 1351
was based on a diastereoselective synthesis of ketomethylene
dipeptide isosteres of the type AAΨ[COCH2]Asp (see Scheme
1).21 The tert-butyl keto ester 32 was prepared as follows. To
a solution of lithium bis(trimethylsilyl)amide (1 N, 800 mL)
in THF at -78 °C was added dropwise a solution of allyl
acetate (39 mL, 0.36 mol) in THF (40 mL). This mixture was
stirred at -78 °C under an atmosphere of nitrogen for 1 h,
after which time trimethylacetyl chloride (47 mL, 0.38 mol)
was added dropwise. After 30 min at -78 °C, hexane (300
mL) followed by 3 N aqueous HCl (600 mL) was added. The
organic phase was washed with saturated aqueous NaHCO3
and brine, dried (MgSO4), filtered, and concentrated. This
material was distilled bulb to bulb (60 °C air bath, 0.25 Torr)
to provide keto ester 32 as a clear colorless liquid (62 g, 92%
yield). 1H NMR (400 MHz, CDCl3) δ 6.02-5.87 (m, 1 H), 5.35
(br d, J ) 17 Hz, 1 H), 5.25 (br d, J ) 9.5 Hz, 1 H), 4.63 (br d,
J ) 5.5 Hz, 2 H), 3.59 (s, 2 H), 1.19 (s, 9 H).
Exp er im en ta l Section
Ribon u cleotid e Red u cta se Bin d in g Assa y. The inhibi-
tory effect of our compounds in an HSV ribonucleotide reduc-
tase radioligand binding assay was measured according to a
published protocol.11 The reported IC50 values are the mean
of at least four separate determinations, and the standard
deviation from the mean is also reported.
HSV Cell Cu ltu r e Assa y. The potencies of our inhibitors
against HSV in cell culture were determined by using serum-
starved baby hamster kidney (BHK) cells in a colorimetric viral
yield assay.15 The reported EC50 values are the mean of at
least three separate determinations, and the standard devia-
tion from the mean is also reported. To confirm that the
observed antiviral effect was not a consequence of cytotoxicity,
the cytotoxic effect of all inhibitors was determined by a
tetrazolium salt (MTT) metabolic assay.16 Typically, selectivity
indices (EC50 versus CC50) of greater than 15 were observed.
BILD 1351, BILD 1357, and compound 29 had selectivity
indices of 15-20 while compound 30 showed a lower index of
5-10. All compounds referred to in this paper proved stable
under the assay conditions.
Mou se Ocu lar Model of HSV-In du ced Ker atitis.14 Com-
pounds were formulated in a mixture of Eucerin anhydrous,
water, and light mineral oil (70:15:15). The desired RR
inhibitor was ground with warm mineral oil (37 °C) and then
thoroughly mixed with the mixture of Eucerin and water.
To a solution of keto ester 32 (83.2 g, 0.452 mol) in THF
(0.8 L) was added NaH (2.7 g of a 60% oil dispersion, 0.07 mol)
over a 15 min period. The reaction mixture was stirred at
room temperature under an atmosphere of nitrogen, and the
Ma ter ia ls. Common amines and N-Boc-L-amino acids,
N-Boc-L-tert-leucine, N-Boc-L-γ-methylleucine, and L-leucine
O-benzyl ester pTsOH salt, were obtained from commercial
sources. The preparations of Boc-2(S)-amino-4-pyrrolidino-4-
resultant homogeneous solution was cooled to -60 °C. A
solution of Michael acceptor 3121 (170 g, 0.45 mol) in THF (0.5
L) was added over a period of 45 min, and the reaction mixture
was stirred at -60 °C for 5 h. A 10% aqueous solution of citric

Peptidomimetic Inhibitors of HSV RR
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4179
acid (500 mL) was added, and the mixture was allowed to
warm to room temperature. The mixture was extracted with
Et2O (1 L). The organic phase was washed with saturated
aqueous NaHCO3 and brine, dried (MgSO4), filtered and
concentrated to afford crude 33 as an orange oil (250 g). This
material was used without further purification in the next
reaction. To a solution of tetrakis(triphenylphosphine)-
palladium(0) (2.60 g, 2.25 mmol, 0.5% molar) in CH2Cl2 (250
mL) and CH3CN (250 mL) at 0 °C was added pyrrolidine (56
mL, 0.54 mol). The mixture was allowed to warm to room
temperature, and a solution of 33 (250 g, 0.45 mol) in CH2Cl2
(200 mL) and CH3CN (200 mL) was added. After 3 h, the
mixture was concentrated to yield an orange oil. This material
was dissolved in Et2O-hexane (1:1, 1 L). The resultant
solution was washed with a 10% aqueous citric acid, 10%
aqueous NaHCO3, and brine, dried (MgSO4), filtered, and
concentrated to give an orange oil (203 g). This material could
be used in the next reaction without further purification or it
could be purified by silica gel flash chromatography (elution
with hexane-EtOAc, 9:1) to give compound 35 as a colorless
oil. The diastereoisomeric purity was assessed to be >35:1
by NMR: 1H NMR (400 MHz, CDCl3) δ 7.38-7.28 (m, 5 H),
5.10 (s, 2 H), 5.07 (br d, J ) 9 Hz, 1 H), 4.08 (d, J ) 9 Hz, 1
H), 3.38-3.31 (m, 1 H), 3.09 (dd, J ) 19, 6 Hz, 1 H), 2.94 (dd,
J ) 18.5, 6 Hz, 1 H), 2.82 (dd, J ) 18.5, 6 Hz, 1 H), 2.77 (dd,
J ) 19, 6 Hz, 1 H), 1.42 (s, 9 H), 1.10 (s, 9 H), 0.95 (s, 9 H).
To a solution of crude compound 35 (171 g, ∼0.36 mol) in
EtOH (1.4 L) was added 10% Pd/C (10 g). The resultant
mixture was stirred vigorously under 1 atm of hydrogen for 5
h. Thereafter, the reaction mixture was filtered through
Celite. The filtrate was concentrated under reduced pressure,
and the residue was dissolved in saturated aqueous Na2CO3.
The aqueous solution was washed with hexane-Et2O (8:2),
rendered acidic with citric acid and extracted with EtOAc. The
organic phase was dried (MgSO4) and concentrated. The
resultant orange residue was dissolved in Et2O and the
solution was passed through a silica gel pad (12 × 12 cm).
Concentration provided compound 38 as a white solid (117 g,
The various N-terminal alkylureido functionalities were
introduced as follows. To a solution of the appropriate peptide
hydrochloride salt (0.5 mmol) in dry dichloromethane (5 mL)
was added N-methylmorpholine (1.5 mmol) and the relevant22
isocyanate (1 mmol, usually neat). The reaction mixture was
stirred at room temperature for 16 h, after which time the
volatiles were removed and the residue was partitioned
between EtOAc and 0.1 M aqueous HCl. The organic phase
was washed with 5% aqueous sodium bicarbonate and brine,
dried (MgSO4), filtered, and concentrated. The resultant crude
product was purified by silica gel flash chromatography to
provide the desired ureido derivatives usually as white foams
(64-76% yield).
The last reaction involved in inhibitor preparation involved
removal of the benzyl ester protective group(s) by catalytic
hydrogenolysis (10 mol % of 10% Pd/C in methanol under 1
atm of H2 for 3 h). The resultant inhibitor was often obtained
in greater than 95% purity (HPLC and NMR), but when
necessary it was purified by preparative HPLC on a C18
reverse-phase column (Vydac, 15 µm particle size) eluting with
0.06% TFA in water-0.06% TFA in acetonitrile gradients.
In h ibitor Ch a r a cter iza tion a n d P u r ity. All inhibitors
showed satisfactory 1H NMR spectra (400 MHz), FAB mass
spectra (M+ + H) and/or (M+ + Na), and HPLC purity in two
solvent systems (>95%). All inhibitors not containing the
ketomethylene isostere fragment showed satisfactory amino
acid analysis including peptide recovery. Satisfactory elemen-
tal analysis was obtained for BILD 1351 and BILD 1357. An
X-ray structure of compound BILD 1351 was obtained, provid-
ing proof of the assigned stereochemistries for all asymmetric
centers.
Ack n ow led gm en t. We are grateful to M. Liuzzi and
E. Scouten for determining the IC50s of our inhibitors
in the binding assay. We are also grateful to C.
Bousquet and N. Dansereau for determining EC50s of
our inhibitors in cell culture. We are also grateful to
L. Tong for the X-ray structure of BILD 1351.
1
84% yield): mp 62-65 °C; H NMR (CDCl3) δ 5.18 (d, J ) 9
Hz, 1 H), 4.09 (d, J ) 9 Hz, 1 H), 3.35-3.29 (m, 1 H), 3.09 (dd,
J ) 19, 6.5 Hz, 1 H), 2.94 (dd, J ) 18.5, 6.5 Hz, 1 H), 2.83 (dd,
J ) 18.5, 6.5 Hz, 1 H), 2.78 (dd, 19, 6.5 Hz, 1 H), 1.43 (s, 9 H),
1.14 (s, 9 H), 0.96 (s, 9 H).
Su p p or tin g In for m a tion Ava ila ble: Full tabulation of
1H NMR, FAB mass spectra, amino acid analysis, elemental
analysis, HPLC purity data for new inhibitors and coordinates
for the X-ray structure of BILD 1351 (21 pages). Ordering
information is given on any current masthead page.
Compound 36 could be prepared as described above for
compound 35 by using diallyl malonate. Decarboxylation
following deallylation of the diallyl malonate derivative 34
required heating of the crude diacid in xylene. Compound 36
was obtained as a clear gum: 1H NMR (400 MHz, DMSO-d6)
δ 7.38-7.21 (m, 5 H), 7.27 (d, J ) 8 Hz, 1 H), 5.06 (s, 2 H),
3.79 (d, J ) 8 Hz, 1 H), 3.14-3.08 (m, 1 H), 2.92 (dd, J ) 18.5,
6 Hz, 1 H), 2.79 (dd, J ) 18.5, 6 Hz, 1 H), 2.54 (dd, J ) 17, 7
Hz, 1 H), 2.44 (dd, J ) 17, 6 Hz, 1 H), 1.38 (s, 9 H), 0.89 (s, 9
H). Compound 36 could be coupled with pyrrolidine according
to the general coupling procedure in the inhibitor synthesis
section. Compound 37 was obtained as a clear gum: 1H NMR
(400 MHz, CDCl3) δ 7.29-7.22 (m, 5 H), 5.08 (d, J ) 8.5 Hz,
1 H), 5.05 (s, 2 H), 4.01 (d, J ) 8.5 Hz, 1 H), 3.40-3.20 (m, 5
H), 3.10 (dd, J ) 18.5, 7 Hz, 1 H), 2.88 (dd, J ) 18.5, 5 Hz, 1
H), 2.63 (dd, J ) 16, 5.5 Hz, 1 H), 2.48 (dd, J ) 16, 7 Hz, 1 H),
1.87-1.72 (m, 4 H), 1.35 (s, 9 H), 0.88 (s, 9 H). Compound 39
was obtained as a clear gum from 37 using the hydrogenolysis
procedure described for compound 38: 1H NMR (400 MHz,
DMSO-d6) δ 7.22 (d, J ) 8.5 Hz, 1 H), 3.80 (d, J ) 8.5 Hz, 1
H), 3.38-3.21 (m, 4 H), 3.04-2.98 (m, 1 H), 2.82 (dd, J ) 18.5,
6 Hz, 1 H), 2.72 (dd, J ) 18.5, 6 Hz, 1 H), 2.52 (dd, J ) 16.5,
7 Hz, 1 H), 2.38 (dd, J ) 16.5, 6.5 Hz, 1 H), 1.88-1.72 (m, 4
H), 1.39 (s, 9 H), 0.91 (s, 9 H).
Refer en ces
(1) Cory, J . G. Role of ribonucleotide reductase in cell division. In
Inhibitors of Ribonucleoside Diphosphate Reductase Activity;
Cory J . G., Cory, A. H., Eds.; Pergamon Press Inc.: New York,
1989; pp 1-17.
(2) Filatov, D.; Ingemarson, R.; Graslund, A.; Thelander, L. The role
of herpes simplex virus ribonucleotide reductase small subunit
carboxyl terminus in subunit interaction and formation of iron-
tyrosyl center structure. J . Biol. Chem. 1992, 267, 15816.
(3) (a) Dutia, B. M.; Frame, M. C.; Subak-Sharpe, J . H.; Clarke, W.
N.; Marsden, H. S. Specific inhibition of herpes virus ribonucle-
otide reductase by synthetic peptides. Nature 1986, 321, 439-
441. (b) Cohen, E. A.; Gaudreau, P.; Brazeau, P.; Langelier, Y.
Specific inhibition of herpes simplex virus ribonucleotide reduc-
tase activity by
a nonapeptide derived from the carboxyl
terminus of subunit 2. Nature 1986, 321, 441-443.
(4) Cosentino, G.; Lavalle´e, P.; Rakhit, S.; Plante, R.; Gaudette, Y.;
Lawetz, C.; Whitehead, P. W.; Duceppe, J .-S.; Le´pine-Frenette,
C.; Dansereau, N.; Guilbault, C.; Langelier, Y.; Gaudreau, P.;
Thelander, L.; Guindon, Y. Specific inhibition of ribonucleotide
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(5) Peptide-based drug design; Taylor, M. D., Amidon, G. L., Eds.;
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(6) Liuzzi, M.; De´ziel, R.; Moss, N.; Beaulieu, P.; Bonneau, A.-M.;
Bousquet, C.; Chafouleas, J . G.; Garneau, M.; J aramillo, J .;
Krogsrud, R. L.; Lagace´, L.; McCollum, R. S.; Nawoot, S.;
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In h ibitor Syn th esis. All inhibitors were prepared by
sequentially coupling N-Boc-amino acid derivatives (azide
derivative for cyclopentylaspartic acid, see ref 9) from C- to
N-terminus by using benzotriazol-1-yl 1,1,3,3-tetramethyl-
uronium tetrafluoroborate (TBTU) as the coupling agent.
Removal of the N-Boc protective group was effected with 4 N
HCl in dioxane (azide derivative of cyclopentylaspartic acid
reduced to amine with SnCl2 in methanol). Representative
procedures can be found in reference 9.
(7) Gaudreau, P.; Paradis, H.; Langelier, Y.; Brazeau, P. Synthesis
and inhibitory potency of peptides corresponding to the subunit
2 C-terminal region of herpes virus ribonucleotide reductase. J .
Med. Chem. 1990, 33, 723-730.

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M.; Beaulieu, P.; DiMaio, J .; Duceppe, J .-S.; Ferland, J .-M.;
Gauthier, J .; Ghiro, E.; Goulet, S.; Grenier, L.; Lavalle´e, P.;
Le´pine-Frenette, C.; Plante, R.; Rakhit, S.; Soucy, F.; Wernic,
(15) Langlois, M.; Allard, J . P.; Nugier, F.; Aymard, M. A rapid and
automated colorimetric assay for evaluating the sensitivity of
herpes simplex strains to antiviral drugs. J . Biol. Standardiza-
tion 1986, 14, 201-211.
(16) Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth
and survival. Modifications to the tetrazolium dye procedure
giving improved sensitivity and reliability. J . Immunol. Methods
1986, 89, 271-277.
(17) Borowitz, I. J .; Williams, G. J .; Gross, L.; Beller, H.; Kurland,
D.; Suciu, N.; Bandurco, V.; Rigby, R. D. G. The synthesis of
2-methyl-7-ketoundecanolide, 9-ketodecanolide, and 2,4,6-tri-
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(18) Bellucci, G.; Macchia, F.; Poggianti, M. Configurazione, confor-
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1
ribonucleotide reductase by substituted tetrapeptide derivatives.
J . Med. Chem. 1993, 36, 3005-3009.
(9) Moss, N.; Beaulieu, P.; Duceppe, J .-S.; Ferland, J .-M.; Gauthier,
J .; Ghiro, E.; Goulet, S.; Grenier, L.; Llinas-Brunet, M.; Plante,
R.; Wernic, D.; De´ziel, R. Peptidomimetic inhibitors of HSV
ribonucleotide reductase: a new class of antiviral agents. J . Med.
Chem. 1995, 38, 2723-2730.
(10) Moss, N.; Plante, R.; Beaulieu, P.; Duceppe, J .-S.; Ferland, J .-
M.; Gauthier, J .; Ghiro, E.; Goulet, S.; Guse, I.; Llinas-Brunet,
M.; Plamondon, L.; Wernic, D.; De´ziel, R. Ureido based pepti-
domimetic inhibitors of herpes simplex virus ribonucleotide
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J . Med. Chem. 1996, 39, 2178-2187.
(11) Krogsrud, R. L.; Welchner, E.; Scouten, E.; Liuzzi, M. A solid-
phase assay for the binding of peptidic subunit association
inhibitors to the herpes simplex virus ribonucleotide reductase
large subunit. Anal. Biochem. 1993, 213, 386-390.
(12) This observation has also been made for γ-methylleucine versus
leucine (ref 9) and γ-methylleucinol versus leucinol (ref 8).
(13) An approximately one log unit increase in log K′w was observed
between inhibitors 24 and 26 and 25 and BILD 1351 (HPLC
solvent: 60-75% MeOH-aqueous Na2HPO4, 20 mM, pH 7.4).
(14) Brandt, C. R.; Spencer, B.; Imesch, P.; Garneau, M.; De´ziel, R.
Evaluation of a peptidomimetic ribonucleotide reductase inhibi-
tor in a murine model of HSV-1 ocular disease. Antimicrob.
Agents Chemother. 1996, 40, 1078-1084.
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e basicita delle 2,6-dimetilcicloesilammine e delle
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(19) Moss, N.; Gauthier, J .; Ferland, J .-M. The enantioselective
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type AAΨ[COCH2]Asp. J . Org. Chem. 1996, 61, 2901-2903.
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J M960324R