Design and synthesis of pyrrolidine-5,5′-trans-lactams (5-oxo-hexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 4. Antiviral activity and plasma stability

Article abstract of DOI:10.1021/jm030810w

A series of chiral, (S)-proline-α-methylpyrrolidine-5,5-trans-lactam serine protease inhibitors has been developed as antivirals of human cytomegalovirus (HCMV). The SAR of the functionality on the proline nitrogen has shown that derivatives of para-subst

Full text of DOI:10.1021/jm030810w

4428
J . Med. Chem. 2003, 46, 4428-4449
Design a n d Syn th esis of P yr r olid in e-5,5′-tr a n s-La cta m s
(5-Oxo-h exa h yd r op yr r olo[3,2-b]p yr r oles) a s Novel Mech a n ism -Ba sed In h ibitor s
of Hu m a n Cytom ega lovir u s P r otea se. 4. An tivir a l Activity a n d P la sm a Sta bility
Alan D. Borthwick,*,† Dave E. Davies,† Peter F. Ertl,‡ Anne M. Exall,† Terry M. Haley,§ Graham J . Hart,|
Deborah L. J ackson,† Nigel R. Parry, Angela Patikis,# Naimisha Trivedi,† Gordon G. Weingarten,† and
J ames M. Woolven§
Department of Medicinal Chemistry CVU UK, Department of Molecular Immunology, Department of Biomolecular Structure,
Department of Enzyme Pharmacology, and Department of Virology, GlaxoSmithKline Research and Development, Medicines
Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, United Kingdom
Received February 13, 2003
A series of chiral, (S)-proline-R-methylpyrrolidine-5,5-trans-lactam serine protease inhibitors
has been developed as antivirals of human cytomegalovirus (HCMV). The SAR of the
functionality on the proline nitrogen has shown that derivatives of para-substituted phenyl
ureas > para-substituted phenyl sulfonamides > para-substituted phenyl carboxamide for
activity against HCMV δAla protease, producing para-substituted phenyl ureas with single
figure nM potency (Ki) against the viral enzyme. The SAR of the functionality on the lactam
nitrogen has defined the steric and electronic requirements for high human plasma stability
while retaining good activity against HCMV protease. The combination of high potency against
HCMV δAla protease and high human plasma stability has produced compounds with
significant in vitro antiviral activity against human cytomegalovirus with the 6-hydroxymethyl
benzothiazole derivative 72 being equivalent in potency to ganciclovir. The parent benzothiazole
56 had good pharmacokinetics in dogs with 29% bioavailability and good brain and ocular
penetration in guinea pigs.
In tr od u ction
with enzymatic degradation of the acylated HCMV
protease showed that these inhibitors acylate HCMV
protease at the active site serine (Ser 132) in a time-
dependent and reversible manner.9 SAR in this series
of R-methylpyrrolidine-5,5′-trans-lactam has defined the
size, the relative stereochemisty of the substituent
adjacent to the lactam carbonyl, and its chirality.
Optimization of the acyl function on the lactam nitrogen
has extended the substitution pattern on the lactam
nitrogen for activity against HCMV δAla protease to
CO-cyclopropyl > COMe > CO2Me > SO2Me > CON-
HMe. Optimization of the functionality on the pyrroli-
dine nitrogen gave the highly potent dansyl-S-proline
derivatives 1 and 2, with Ki’s in the low nanomolar
range against HCMV δAla protease, which are highly
selective over the mammalian enzymes elastase, throm-
bin and acetylcholine esterase.9 However, they were not
stable to human plasma.14 We now report on work to
define further the requirements of the substituent on
the pyrrolidine nitrogen that accesses the S4 pocket, and
on work to exploit the functionality on the lactam
nitrogen of this template to give stability in human
plasma and whole cell antiviral activity. This has given
plasma stable inhibitors with low nanomolar potency
against the HCMV δAla protease and antiviral activity
equivalent to ganciclovir in whole cells.
Human herpes viruses cause a range of diseases:
HSV-1 (cold sores), HSV-2 (genital herpes), VZV (chicken
pox, shingles), and HCMV (retinitis, pneumonitis). The
current treatment of these diseases uses nucleoside
(acyclovir, ganciclovir) and phosphate (PFA) substrate
analogues. Because of the toxicity associated with PFA
and ganciclovir, together with the emergence of mutants
resistant to acyclovir, there is a need for a new class of
antiherpes compounds based on a novel mechanism.
Human herpes viruses encode a serine protease,
which is essential for viral replication.1 Recent X-ray
structures of the serine proteases of HCMV, HSV-1,
HSV-2, and VZV revealed that these enzymes belong
to a novel class of serine proteases where the active site
is composed of the His, His, Ser triad.3-6 Substrate
cleavage sites across all the herpes virus family are
unique and highly conserved, and these enzymes have
become attractive molecular targets for the design of
novel antiviral drugs.1,2 We recently reported on the
design and synthesis of a novel class of mechanism
based inhibitors of human cytomegalovirus protease,7-9,14
based on the R-methylpyrrolidine-5,5′-trans-lactam tem-
plate incorporating the natural substrate requirements
of the consensus sequence of HCMV protease (Scheme
1). Mechanism of action studies using ESI-MS together
Ch em istr y
We reported previously9 that a dansyl-S-proline group
on the pyrrolidine nitrogen and a cyclopropyl- or methyl-
carbonyl function on the lactam nitrogen was optimal
for maximum potency in the novel chiral pyrrolidine-
5,5′-trans-lactam inhibitors (1 and 2) of HCMV protease
(Scheme 1). To investigate further the potency require-
* Corresponding author. Telephone: +44 (0)1438 763422. E.mail:
alan.d.borthwick@gsk.com. Fax: + 44 1438 768483.
† Department of Medicinal Chemistry CVU UK.
‡ Department of Molecular Immunology.
§ Department of Biomolecular Structure.
| Department of Enzyme Pharmacology.
Department of Virology.
Department of Drug Metabolism.
#
10.1021/jm030810w CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4429
Sch em e 1
Sch em e 2a
a
Reagents and conditions: TFA, 1 h, RT; (b) LHMDS (1.4eq)/THF, -78 °C then cyclopropylCOCl; (c) 10% Pd/C (Degussa, 50% H2O),
i
isoPrOH, 2 h, RT, then HCl (1.1eq) Et2O; (d) Cbz-(S)-proline, TBTU, HOBT, Pr2EtN, DMF, RT; (e) Boc anhydride (1.3eq)/ 10% Pd/C
(Degussa, 50% H2O), isoPrOH, 2 h, RT.
ments of substituents on the pyrrolidine nitrogen and
the carbonyl function on the lactam nitrogen, the chiral
intermediates 8 and 12 were prepared as outlined in
Scheme 2.
Deprotection of 3 with TFA followed by acylation of
4 with cyclopropyl carbonyl chloride gave the chiral
(cyclopropyl) trans-lactam 5 in 65% overall yield. Hy-
drogenolysis of 5 with hydrogen in the presence of
palladium gave the amine 6 in 74% yield, which was
coupled with Cbz-(S)-proline using O-benzotriazol-1-yl-
N,N,N′,N′,-tetramethyluronium tetrafluoroborate/1-hy-
droxybenzotriazole (TBTU/HOBT) in DMF at room
temperature to give the protected prolyl trans-lactam
7. This, on further hydrogenolysis, gave the amine 8 in
87% overall yield. Similarly hydrogenolysis of 3 with
hydrogen in the presence of palladium gave 9 in 93%
yield, which was coupled with Cbz-(S)-proline using
TBTU/HOBT at room temperature to give protected
prolyl trans-lactam 10 in 88% yield. Deprotection of 10
with trifluoroacetic acid gave the lactam 11 in 98% yield,
which on hydrogenolysis in the presence of di-tert-butyl
dicarbonate gave the Boc-protected prolyl trans-lactam
12 in 90% yield.
Exploration of the substituents on the proline nitro-
gen has been done with a variety of linkers, namely,
sulfonamide, amide, and urea (Scheme 3). The sulfona-

4430 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
Sch em e 3a
a
i
Reagents and conditions: (a) RSO2Cl, (1.1-1.5eq), Et3N, MeCN, RT; (b) RCO2H, TBTU, HOBT, Pr2EtN, DMF, RT; (c) PhNMeCOCl,
Et3N, MeCN, RT; (d) RNdCdO, Et3N, MeCN, RT.
mides 1 and 14-23 were prepared by reacting the
proline 8 with the corresponding sulfonyl chloride at RT
in the presence of triethylamine. Similarly 13 was
prepared from the ethyl analogue of 8.13 Sulfonyl
chlorides that were not commercially available were
prepared by diazotisation of the corresponding aniline
in the presence of SO2 and CuCl,10 or by reaction of the
sulfonic acid with triphosgene.11 The reaction of proline
8 with the corresponding aromatic acids in the presence
of TBTU/HOBT at room temperature gave the amides
24-29. The ureas 30-39 were prepared by reacting 8
with the corresponding isocyanides, while the N-me-
thylated urea 40 was prepared in 79% yield by reacting
8 with N-methyl-N-phenylcarbamoyl chloride in the
presence of triethylamine.
lactam 4 with (c-2, c-3-dimethylcyclopropyl-r-1-carboxy-
lic) anhydride gave the dimethylcyclopropanyl deriva-
tive 41 in 96% yield. Deprotection of 41 with hydrogen
in the presence of 10% of palladium on carbon gave the
amine 42 in 92% yield, which was then coupled with
Cbz-(S)-proline to give the protected proline 43 in 70%
yield. Deprotection of 43 with hydrogen in the presence
of 10% of palladium on carbon gave the amine 44 in
96% yield, which was reacted with 4-(isopropyl)phenyl
isocyanate in the presence of triethylamine to give the
urea 45 in 47% yield. A similar sequence of reactions
starting from 4 and (t-2, t-3-dimethylcyclopropyl-r-1-
carboxylic) anhydride gave the t-2, t-3-dimethylcyclo-
propyl-r-1-carbonyl derivative urea 46.15 Similarly reac-
tion of the Boc protected lactam 12 with tetramethyl-
cyclopropanecarboxylic anhydride gave 47 in 68% yield,
which was carried through to the urea 51 via the proline
49 in a manner similar to 45 (Scheme 4). Reaction of
the lactam 12 with 4-nitrobromobenzene under Cu
The cyclopropyl derivatives (Scheme 4) were prepared
by acylating the anions of the lactam 4 and lactam 12
with cyclopropyl carbonyl chloride or the corresponding
mixed anhydride. Acylation of the anion of the chiral

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4431
Sch em e 4a
a
Reagents and conditions: (a) LHMDS (1.4eq)/THF, -78 °C then (diMecyclopropylCO)(Me3CO)O; (b) 10% Pd/C (Degussa, 50% H2O),
i
isoPrOH, 2 h, RT, then HCl (1.1eq) Et2O; (c) Cbz-(S)-proline, TBTU, HOBT, Pr2EtN, DMF, RT; (d) 4-Me2CHC6H4NdCdO, Et3N, MeCN,
RT; (e) LHMDS (1.4eq)/THF, -78 °C then (tetraMecyclopropylCO)(Me3CO)O or CuCl, K2CO3, TDA-1, pNO2PhBr, pXylene; (f) TFA, 1 h,
RT;
catalysis using modified Goldberg conditions16 (CuCl/
TDA-1/K2CO3/xylene/∆) gave the N-aryl lactam 48 in
65% yield. Similarly 53 and 54 were prepared (Scheme
5) in 37% and 47% yield, respectively, by reacting 12
with 2-bromothiazole and 2-bromobenzothiazole. Depro-
tection of 48 with trifluoroacetic acid gave the amine
50 in quantitative yield, which reacted with 4-(isopro-
pyl)phenyl isocyanate to give the urea 52 in 49% yield.
Similarly deprotection of 53 and 54 with trifluoroacetic
acid gave 55 and 56 in 87% and 85% yield, respectively.
The substituted benzothiazole derivatives 58-71 were
prepared by reacting lactam 12 with the corresponding
substituted bromobenzothiazole 57 under Cu catalysis
using modified Goldberg conditions.16 Deprotection of
58-71 with trifluoroacetic acid and reaction of the
resulting prolines with 4 -(isopropyl)phenyl isocyanate

4432 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
Sch em e 5a
a
Reagents and conditions: (a) CuCl, 2-bromoheterocycle, TDA-1, K2CO3, xylene, ∆; (b) TFA, then 4-Me2CH-C6H4NCO, Et3N, MeCN,
RT.
in the presence of triethylamine gave the corresponding
ureas 72-85 (Scheme 5).
of either ring from the unsubstituted 1-naphthyl deriva-
tive 15 (Ki ) 60nM) to give the phenyl derivative 18 or
the benzyl analogue 17 caused a large loss of potency.
However, modeling indicated that the space occupied
by the 5-NMe2 on the naphthalene ring could be
occupied by 4-substitution of a monocyclic phenyl ring.
It was found that additional binding could be gained
by having a medium-sized, hydrophobic group at this
position, illustrated by the greater potency of the
4-isopropyl 20 over the unsubstituted phenyl derivative
18 (Table 1). Also the 4-isopropoxy 21 (Ki ) 39nM) was
equivalent in terms of potency to the 2-naphthalene
derivative 16 (Ki ) 40nM). However, moving the iso-
propoxy group to the 3-position 22 caused a large loss
in activity, while the 4-propoxy 23 (Ki ) 33nM) il-
lustrates that for high potency, it is not essential to have
the fused bicyclic ring that is present in the lead
dansylproline derivative 1. Hence, increasing the size
of the 4-substitutent increased potency in the order H
< Me2CH < Me2CHO ≡ MeCH2CH2O.
Resu lts a n d Discu ssion s
F u n ction a liza tion a t th e P yr r olid in e Nitr ogen .
Previously we had shown that the conformationally
restricted, chiral, dansyl-S-proline R-methyl-5, 5′-trans-
lactam 1 (Ki ) 20nM) (Table 1) had high potency against
human cytomegalovirus (HCMV) protease.9 To reduce
the overall molecular weight, the first part of our lead
exploration focused on whether the bicyclic ring and the
dimethylamino functionality of the dansyl group are
essential for activity. Removal of the 5-NMe2 group to
give the unsubstituted 1-naphthyl derivative 15 (Ki )
60nM) caused a loss in potency, and no improvement
was obtained with the unsubstituted 2-naphthyl deriva-
tive 16 (Ki ) 40nM). Moving the NMe2 group to the
6-position 14 caused a larger loss in potency, indicating
that this group is not tolerated an this position. Removal

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4433
Ta ble 1. Pyrrolidine-5, 5′-trans-Lactam Sulfonamide Inhibitors
Ta ble 3. Pyrrolidine-5,5′-trans-Lactam Urea Inhibitors of
of HCMV Protease
HCMV Protease
HCMV proteasea
HCMV proteasea
IC50 (µM)17
Ki (nM)17
compd
R
IC50 (µM)17
Ki (nM)17
compd
R
1
5-NMe2-naphth-1-yl
6-NMe2-naphth-1-yl
naphth-1-yl
0.34
1.1
0.37
<0.19
2.1
20
30
31
32
33
34
35
36
37
38
39
4-Me2CH-Ph-
4-MeCH2CH2O-Ph-
4-CF3-Ph-
0.20
0.21
0.23
0.18
0.26
0.65
0.47
0.59
0.65
1.4
2
9
15
18
38
14
15
16
17
18
19
20
21
22
23
60
40
naphth-2-yl
4-Me2CHO-Ph-
4-Cl-Ph-
PhCH2-
Ph-
1.8
naphth-1-yl
2-Me2CH-Ph-
3-CF3-Ph-
3-Cl-Ph-
Ph-
4-Cl-Ph-
0.84
0.46
0.40
65
4-Me2CH-Ph-
4-Me2CHO-Ph-
3-Me2CHO-Ph-
4-MeCH2CH2O-Ph-
99
39
0.23
33
HCMV pNA assay.9
a
HCMV pNA assay.9
a
Removing the second ring in the 1-naphthyl deriva-
tive 35 to give the phenyl derivative 39 resulted in a
loss in potency; however, compounds with increased
potency relative to the 1-naphthyl derivative 35 could
be obtained by increasing the size of the 4-substituent
in the phenyl series as shown by moving from the chloro
derivative 34 (Ki ) 38nM) to the trifluoromethyl deriva-
tive 32 (Ki ) 15nM) to the n-propoxy derivative 31 (Ki
) 9nM) to the isopropyl derivative 30 (Ki ) 2nM).
Moving the chloro substituent in 34 and trifluoromethyl
substituent in 32 to the 3 positions in 38 and 37,
respectively, caused a loss of activity, as did moving the
isopropyl substituent in 30 to the 2 position in 36.
Ta ble 2. Pyrrolidine-5,5′-trans-Lactam Carboxamide Inhibitors
of HCMV Protease
HCMV proteasea
compd
R
IC50 (µM)17
Ki (nM)17
24
5-Cl-3-Me-
0.19
2
benzothien-2-yl
4-Ph-Ph-
25
26
27
28
29
0.30
0.39
0.54
0.89
1.4
21
79
4-Me2CH-Ph-
4-MeCH2CH2O-Ph-
naphth-1-yl
Compared to the dansylproline series, the 4-substi-
tuted phenyl ureas have two advantages, reduced mo-
lecular weight and increased solubility. The SAR es-
tablished for inhibition of HCMV protease have shown
that in the substituted proline trans-lactams, the 4-sub-
stituted phenyl ureas > 4-substituted phenyl sulfona-
mides > 4-substituted phenyl carboxamides in terms of
potency (Table 4).
4-Cl-Ph-
a
HCMV pNA assay.9
Having achieved potency with monocyclic sulfona-
mides, we investigated changing the linker to carboxa-
mide. In general the monocyclic carboxamide (Table 2)
were less active than the monocyclic sulfonamides. This
is possibly due to a shorter linker between the proline
and aromatic rings preventing access of the latter into
the hydrophobic binding pocket thought to be necessary
for activity. This can be seen by comparing the 4-chlo-
rophenyl, the naphth-1-yl, and 4-propoxyphenyl deriva-
tives 29, 28, and 27 of the carboxamide (Table 2) with
the corresponding derivatives in the sulfonamide series
19, 15, and 23 (Table 1), the exception being the
isopropyl derivatives 20 and 26, which have similar
potency. The lower activity of the carboxamide could be
overcome by increasing the lipophilic bulk around the
aromatic group, as illustrated to the larger 5-chloro-3-
methyl-benzothienyl-2-carboxamide derivative 24.
Generally, the urea series looked the most promising,
with 30 showing the highest potency against the enzyme
(IC50 ) 0.2µM, Ki ) 2nM) (Table 3). It is noteworthy
that when 30 was assayed with 0 min as well as the
normal 15 min preincubation time the IC50 values in
the pNA assay were closely similar (0.24µM and 0.20µM
respectively), indicating rapid binding and inhibition of
the enzyme. This has been confirmed by ESI-MS studies
showing that the compound rapidly acylates HCMV
protease, giving 90% of the acyl-enzyme complex after
5 min, and remaining 58% acylated after 24 h (Figure
1). This stability of the acyl-enzyme complex is higher
than that of other cyclopropylcarbonyl trans-lactams.14
Interestingly, methyl substitution on the urea nitrogen
in 39 (IC50 ) 1.4µM) to give derivative 40 (Scheme 3)
leads to a complete loss of activity (IC50 ) >20µM). This
could be due to an altered conformation of the rigid urea
linker, or the methyl group could make an unfavorable
contact with the enzyme.
We further investigated extending the length of the
linker by changing from carboxamide and sulfonamide
to urea in the monocyclic series in an effort to increase
potency. In general the monocyclic ureas (Table 3) were
more active than the carboxamide and sulfonamides,
possibly due to the longer linker between the proline
and aromatic rings allowing further access of the latter
into the hydrophobic binding pocket.
P la sm a Sta bility. The most potent monocyclic urea
is the 4-isopropyl-phenyl urea 30, however it is unstable
in human plasma (t1/2 < 1 h). Recently we have achieved

4434 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
F igu r e 1. MS characterization of the acylation of HCMV δAla protease by R-methyl-pyrrolidine-5,5′-trans-lactams.
Ta ble 4. Variation in Potency of Pyrrolidine-5,5′-trans-Lactam
Inhibitors of HCMV Protease with Aryl to Proline Ring Linkage
and the other was to make the lactam carbonyl less
reactive by making the lactam nitrogen substituent less
electron withdrawing. Substitution of the cyclopropyl
ring with methyl groups increased the plasma stability
of these acyl derivatives in the order cyclopropyl car-
bonyl < t-2, t-3-dimethylcyclopropyl-r-1-carbonyl < c-2,
c-3-dimethylcyclopropyl-r-1-carbonyl < 2,2,3,3-tetram-
ethylcyclopropyl carbonyl. However, although the t-2,
t-3-dimethylcyclopropyl-r-1-carbonyl derivative 46 is
slightly more stable (t1/2 ) 1.5 h) than 30, it is signifi-
cantly less active, whereas the tetramethylcyclopropyl
derivative 51, which is >16 times more stable, is
considerably less active (>100 fold). A better balance is
achieved with the c-2, c-3-dimethylcyclopropyl-r-1-car-
bonyl derivative 45 that is > 6 times more stable than
the cyclopropyl derivative 30 while retaining good
a
HCMV pNA assay.9
potency. Replacing the cyclopropyl carbonyl substituent
success14 in increasing stability to human plasma, while
retaining potency against HCMV protease, in the dan-
sylproline series of R-methyl pyrrolidine-5,5′-trans-
lactams. This strategy has been applied to the 4-subti-
tuted phenyl urea series (Table 5). Two approaches were
pursued, one was to sterically hinder the approach of
the hydrolytic plasma enzymes to the lactam carbonyl,
with an electron withdrawing aryl or heterocyclic ring
dramatically increased stability to human plasma (t1/2
>24 h) in this series (Table 5). However, even though
the 2-thiazole 55 is more potent than the 4-nitrophenyl
derivative 52, both are significantly less active than 30.
In contrast, the benzothiazole 56 had comparable po-
tency to 30 with a Ki ) 10 nM. Although the thiazole

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4435
Ta ble 5. Human Plasma Stability, Inhibition of HCMV Protease and HCMV Antiviral Activity of Chiral Pyrrolidine-5,
5′-trans-Lactam Analogues
a
b
d
Tested in fresh human plasma 100 µM14
.
HCMV pNA assay.9 c HCMV Elisa assay (see experimental). Vero cell cytotoxicity assay.12
Ta ble 6. Selectivity of Pyrrolidine-5,5′-trans-Lactam Inhibitors
for HCMV Protease Compared to Mammalian Serine Proteases
over during a 24h incubation period. By contrast, com-
pounds with greater plasma stability (e.g., 45, 51, and
56) generate acyl-enzymes that show no turnover in
24h.
acetylcholine
esterase
thrombin
elastase
IC50 (µM) IC50 (µM)17 Ki (nM)17
HCMV proteasea
IC50 µM
IC50 µM
Most compounds studied acylated the enzyme rapidly
under the conditions used; acylation of the enzyme was
normally complete within 5 min. However, the tetram-
ethylated cyclopropylcarbonyl 51 acylated the enzyme
much more slowly. Less than 5% of the enzyme was
acylated within 30 min, and even after 4h 25% was still
unacylated, although by 24 h the enzyme was fully
acylated. The reason for the slow acylation of HCMV
protease by 51 has not been investigated further, but it
may well be due to the presence of the four methyl
groups sterically hindering the approach of the com-
pound to the active site serine residue.
56
45
>200
>200
>100
>100
>10
>10
0.18
0.30
10
16
a
HCMV pNA assay.9
and the benzothiazole groups have similar electron with-
drawing properties, the greater potency of benzothiazole
56 compared to thiazole 55 is probably due to the
greater hydrophobic contact of the former making a
better fit in the S1’pocket (see Modeling section).
Selectivity a n d Mech a n ism of Action . The 4-iso-
propylphenylurea-(S)-proline-trans-lactams 56 and 45
show selectivity for the viral HCMV protease (Table 6)
over acetylcholine esterase and the mammalian pro-
teases elastase and thrombin by at least 2 orders of
magnitude, showing no significant activity against these
enzymes at the concentrations tested.
MS An a lysis of th e Acyla tion of HCMV p r otea se.
Comparison of the plasma stability’s of the compounds
(Table 5) with the rates at which the compounds acylate
the enzyme and the rates at which the acyl-enzyme
complexes are deacylated (Figure 1) enables the follow-
ing general conclusions to be drawn.
An tivir a l Activity. Because HCMV cellular assays
require 7 days to complete, plasma unstable compounds
did not initially show antiviral activity. Studies have
since shown that a modified ELISA technique(see
experimental), delaying the single addition of com-
pounds to 48 h post infection (hpi), is suitable for the
titration of both stable and unstable trans-lactams for
activity against HCMV. Parallel comparisons with
plaque reduction assays12 yield similar IC50 values for
stable trans-lactams, but not for the plasma labile
analogue 1. The technique has the additional advantage
of providing an exposure time of the cells to the drug
equal to the Vero cytotoxicity assay.12 Of the five plasma
Compounds that have poor plasma stability (e.g., 30)
produce an acyl-enzyme that shows significant turn-

4436 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
Ta ble 7. Human Plasma Stability, Inhibition of HCMV Protease and HCMV Antiviral Activity of Chiral
Pyrrolidine-5,5′-trans-Lactam Benzothiazole Analogues
HCMV proteaseb
human plasma
IC50
Ki
HCMVc strain
AD169 IC50 (µM)
cytotoxicityd
CCID50 (µM)
compd
R
stability,a t1/2 (hr)
(µM)17
(nM)17
56
76
78
73
74
80
79
83
81
84
82
77
72
85
75
H
>24
>24
0.18
1.1
10
5
17.4 pl
17.1
5.2
>500
126
4-MeO-7-Me
4-Cl
4-MeO
5-Cl
5-SO2CHF2
5-MeO
0.29
0.24
0.46
0.21
0.23
0.36
0.39
0.35
0.46
0.22
0.31
0.29
0.87
41
38
27
10
22
17
25
7.4
20
43
>500
89
49
266
2.2 pl
2.4 pl
12.3
8.7
5.1
5.3
3.2
0.52
3.6
>500
>500
298
6-F
6-NO2
>24
24
9
11
>24
>20
8
128
6-CO2Et
6-OCF3
6-MeO
6-CH2OH
7-aza
>500
>500
>500
125
<31
>500
40
21
117
6- MeO-7-aza
9
31 pl
ganciclovir
-
-
0.72 pl
>500
a
b
Tested in fresh human plasma 100 µM.14 HCMV pNA assay.9 c HCMV Elisa assay (see experimental). pl ) IC50 in antiviral plaque
reduction assay.12 Vero cell cytotoxicity assay.12
d
stable compounds 45, 51, 52, 55, and 56, (Table 5) only
45 and 56 had antiviral activity, showing that low nM
potency against HCMV protease and sufficient plasma
stability are required to obtain low µM activity against
the virus in whole cells (Table 7). A variety of substi-
tuted benzothiazoles was prepared in an attempt to
increase potency against the protease and antiviral
activity. In general, potency against the HCMV protease
is greater with electron-withdrawing groups and less
with electron-donating groups; however, most deriva-
tives were less active than the unsubstituted parent 56.
Also replacing the benzothiazole in 56 by 7-pyridothia-
zole to give 85 had no significant effect on protease
activity. The best HCMV protease inhibitors were the
6-CO2Et derivative 84 (Ki ) 7.4nM) and 5-SO2CHF2
derivative 80(Ki ) 10 nM), which were similar to 56.
Modeling suggests that this is because these benzothia-
zoles make no extra interactions in the “canyon” at S1′
(see Modeling section). The disubstituted 4-OMe-7-Me
benzothiazole 76 was 5-fold less active than the 4-OMe
analogue 73, indicating that the 7-Me substitution
prevents the efficient binding of the benzothiazole ring
in the canyon at S1′. Also, the 6-OMe analogue 75 of
the 7-aza derivative 85 was 5-fold less active than the
parent. In general potency against the protease does not
translate to the in vitro HCMV antiviral assay, possibly
because the extent of penetration of both the cellular
and nuclear membranes to reach the target protease
varies for each compound. Substitution on the ben-
zothiazole ring at the 4, 5, 6, or 7 positions with electron-
donating or -withdrawing groups had only a small effect
on anti-viral potency (IC50 ) 2.2-19 µM) compared to
the unsubstituted parent. Only four analogues, 5-OMe
79, 5-SO2CHF2 80, 6-OMe 77, and 6-CH2OH 72, were
more potent as antivirals than the parent 56. Also the
analogues 4-OMe 73 and 7-pyridyl 85 were toxic to Vero
cells. The 6-CH2OH analogue 72 designed to interact
with Asn 62 at S1′ has no increase in potency against
the protease but possess submicromolar activity (IC50
) 0.5µM) against the virus and is equivalent in potency
to ganciclovir.
Mod elin g. The crystal structure of HCMV protease
was obtained recently and used to model the conforma-
tionally restricted, chiral, dansyl-(S)-proline-R-methyl-
5,5-trans-lactam 1 into the active site groove of the
enzyme.9 Similarly we have modeled our antiviral,
plasma stable, 4-isopropylphenylurea-(S)-proline ben-
zothiazole analogue 56 into the active site groove of
HCMV protease, in what could be considered as an
initial binding complex (Figure 2).
Both 1 and 56 can make a lot of similar interactions
with HCMV protease when modeled into the active site.
In both, the C-1 trans-lactam carbonyl is situated in the
oxyanion pocket formed by the backbone amide NH of
arginine 165 and arginine 166, and it is in position for
attack by the hydroxyl of serine 132, which is part of
the active-site catalytic triad formed by serine 132,
histidine 63, and histidine 157. The lactam substituent
extends into the prime sites of the enzyme. The model
shows that R-Me in the (S)-configuration is easily
accommodated in the S1 pocket, as expected from the
substrate requirements of a conserved alanine at this
position for this enzyme. However, different length,
rigidity, hybridization, and directional aspects of sub-
stituents on the proline and lactam nitrogens of 1 and
56 account for the greater potency of the latter and are
seen in their different interactions with HCMV protease.
The urea carbonyl oxygen in 56 is capable of forming a

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4437
F igu r e 2. HCMV protease active site and inhibitor 56.
Ta ble 8. Pharmacokinetics of 56 in Dog and Guinea Pig
Cmax
(ng/mL)
Tmax
(h)
AUC0-t
AUC0-inf
t1/2
(h)
CL
Vdss
(L/kg)
56
(ng mL-1 h-1
)
(ng mL-1 h-1
)
(mL min-1 kg-1
)
F (%)
dog
guinea pig
5480
32000
0.08
0.08
9660
2210
9750
2230
1.5
1
8
60
0.8
1.6
29
-
hydrogen bond to the backbone NH of Ser135, which is
performed by one of the sulfonamide oxygen’s present
in 1. The trans-lactam of 56 fits better into the very
shallow S2 pocket compared with the same region of
the trans-lactam moiety in 1. The (S)-proline carbonyl
oxygen in 56 is almost within hydrogen bonding dis-
tance of Arg166 (2.95Angstrom), whereas the same
carbonyl oxygen in 1 is almost 3.8 Å away. The 4-iso-
propylphenyl ring of 56 fits further into the P4 pocket
than the 5-dimethylaminoaryl ring of the dansyl group
in 1. Similarly the benzothiazole of 56 fits further into
the P1′ pocket than the cyclopropylcarbonyl of 1.
Inspection of our inhibitor 56 at S1 indicated that a
group larger than methyl at this position may be
tolerated. However preparation of the dansylproline
trans-lactam 1313 (IC50 g 100 µM) with ethyl at this
position resulted in >300-fold loss of potency compared
to the corresponding methyl analogue 1 (IC50 ) 0.35
µM). Longer preincubation times, up to 24 h, did not
result in significant improvement on the inhibition of
HCMV protease by 13 (3% at 30 min to 42% at 24 h at
100 µM concentration). Substitution of the ureas NH
in the phenylurea 39 (IC50 ) 1.4 µM) with NMe 40 (IC50
g 20 µM) leads to a complete loss of activity. This could
be due to an altered conformation of the rigid urea
linker, or the methyl group could make an unfavorable
contact with the enzyme. Modeling suggests that al-
though no untoward interaction is seen with the protein
in noncovalent docking, as 40 approaches closer to the
covalent mode the urea N-Me substituent would be
likely to cause a severe steric clash in the region of
Glu31.
The energy minimized semi-rigid trans-lactam proline
urea 56 docked into the active site of the enzyme fits
round the “nose” of the enzyme. This enables the proline
ring, via its rigid urea linker, to position the para-
substituted aromatic ring into the S4 pocket, which is
occupied by tyrosine at P4 in the natural substrate. Part
of the proline ring occupies the S3 pocket.
The benzothiazole function extends into the S′ prime
sites of the enzyme. The benzothiazole makes a better
hydrophobic interaction in the S1 pocket than the
smaller thiazole, which accounts for its greater potency
(Table 5). However, the disubstituted 4-OMe-7-Me ben-
zothiazole 76 was 5-fold less active than the 4-OMe
analogue 73. Modeling suggests that although no un-
toward interaction is seen between 76 and the protease
in the noncovalent docking, as 76 approaches closer to
the covalent mode the substitution at the 7-position
would cause sterically undesirable clashes with the
protease, since the molecule lies much deeper in the P1′
pocket. A variety of substitutions on the benzothiazole
ring at the 4, 5, 6, or 7 positions had little positive effect
on protease activity. Even the 6-CH2OH analogue 72
designed to interact with Asn 62 at S1′ had no increase
in potency against the protease. Modeling suggests that
this is because these benzothiazoles make no effective
extra interactions in the active site at S1′.
P h a r m a cok in etics. The pharmacokinetics of the
benzothiazole 56 was determined in the dog after
intravenous administration. It had a low plasma clear-
ance (8 mL min-1 kg-1), a moderate volume of distribu-
tion (0.8 L/kg), a half-life of 1.5 h, and a reasonable
bioavailability after oral dosing (29%) (Table 8). The

4438 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
Ta ble 9. Plasmas Stability, HCMV Protease and Antiviral Activity of 56 and 72, and Pharmacokinetics of 56 in Dog
dog
human plasma
HCMV proteaseb
HCMVcstrain
AD169 IC50 (µM)
cytotoxicityd
CCID50 (µM)
Vdss
(L/kg)
CL
F
(%)
compd
stability,a t1/2 (hr)
Ki (nM)17
t1/2 (hrs)
(mL min-1 kg-1
8
)
56
72
>24
>24
10
16
5
0.5
490
125
1.5
0.8
29
a
b
d
Tested in fresh human plasma 100 µM14
.
HCMV pNA assay.9 c HCMV Elisa assay (see experimental). Vero cell cytotoxicity assay.12
pharmacokinetics was also determined in the guinea pig
after intravenous administration (Table 8). Further-
more, since the compound is a potential treatment for
infection of the CNS and the retinitis caused by HCMV,
the ability of 56 to access the brain and the eye was
also investigated in this study. Although 56 had a
reasonable half-life in the guinea-pig (1 h) and a
moderate volume of distribution (1.6 L/kg), it had a high
plasma clearance (60 mL/min/kg). Despite this high
clearance, drug levels were measurable in both the brain
and the vitreous humor. These declined in a biexponen-
tial manner with a half-life of approximately 0.5 h in
both tissues. The plasma:brain ratio was 1:5, while the
plasma:vitreous humor ratio was 1:2, indicating that the
drug gains access to both tissues.
active site groove of the enzyme enabling us to direct
and rationalize the SAR in this series. Developing the
SAR in the substituted benzothiazoles produced the
6-hydroxymethyl benzothiazole 72, which has an in vitro
antiviral potency equivalent to ganciclovir. Both plasma
stable, benzothiazole trans-lactams 56 and 72 are potent
inhibitors of HCMV protease in the low nM range and
have low micromolar potency against the HCMV virus
and a good therapeutic index (g100) (Table 9), and are
(>100) selective for the viral protease (HCMV) over the
mammalian proteases elastase and thrombin and also
acetylcholine esterase. Also 56 has a good PK profile in
the dog with an oral bioavailability of 29% and good
CNS and ocular penetration in the guinea pig.
Gen er a l P r oced u r es
Con clu sion s
Melting points were obtained using an Electrothermal
digital melting point apparatus and are uncorrected. All
purifications by flash chromatography were performed using
Kieselgel 60, Merck 9385 silica gel. Preparative plate chro-
matography was performed using Whatman PK6F silica gel
60A plates eluting with ethyl acetate-cyclohexane mixtures.
Monitoring of reactions by TLC used Merck 60 F254 silica gel
glass backed plates (5 × 10 cm), eluted with mixtures of ethyl
acetate and cyclohexane, and visualized by UV light, followed
by heating with aqueous phosphomolybdic acid. Analytical
HPLC were run on a Hewlett-Packard 1090 HPLC instrument,
equipped with an Intersil M column ODS2. Standard condi-
tions were eluent system A (H2O, 0.1% H3PO4), B (95% MeCN/
H2O, 0.1% H3PO4): gradient 0% B 2 min, 0-100% B 40 min,
100% B 10 min; flow rate ) 1 mL/min, λ ) 215 nm). Retention
times (tr) are given in minutes. LCMS were run on a Hewlett-
Packard 1050 coupled with a Micromass Platform II equipped
with a Supelco ABZplus column. Standard conditions were
eluent system A (H2O, 0.1% formic acid, 10 mmol ammonium
acetate) B (MeCN, 0.05% formic acid): gradient 1 100% A 0.7
min, 100% A-100% B 3.5 min, 100% B 3.5 min, 100% to 0%
B 0.3 min; flow rate ) 1 mL/min). Gradient 2 100%A 0.7 min,
100% A-100% B 4.2 min, 100% B 1.1 min, 100% to 0% B 0.2
min; flow rate ) 1 mL/min). Gradient 3 100% A 3 min, 100%
A-100% B 20 min, 100% B 5 min, 100% to 0% B 2 min; flow
rate ) 1 mL/min). All NMR spectra were run on a Bruker 250
MHz instrument generally as solutions in CDCl3 unless
otherwise stated. IR spectra were recorded on a Bio-rad FTS7
spectrometer from thin films on NaCl plates, a KBr mix or
solutions in the solvent specified. Mass spectra were run by
an electrospray Hewlett-Packard 5989B instrument. CD spec-
tra were recorded in acetonitrile on a J asco J -720A spectropo-
larimeter. Final organic solutions were dried over MgSO4
The chiral, dansyl-(S)-proline-(SRS)-R-methyl-5,5′-
trans-lactam 1 was defined 9 previously as the required
template for nanomolar potency against HCMV δAla
protease. We have now developed the SAR for the
substituent on the (S)-proline and have shown that a
single 4-substituted phenyl ring is sufficient for good
potency against HCMV δAla protease, and the potency
increases in the order CO < SO2 < NHCO for the linker
from this 4-substituted phenyl ring to the (S)-proline.
The optimal (S)-proline substituent was found to be the
4-isopropylphenylurea. Plasma stability has been opti-
mized in the 4-isopropylphenylurea-(S)-proline series,
while retaining low nanomolar potency against HCMV
δAla protease, by modifying the substituents on the
lactam nitrogen to give the c-2, c-3-dimethylcyclopropyl-
r-1-carbonyl derivative 45 and the benzothiazole deriva-
tive 56. ESI/MS studies have revealed that the plasma
stable trans-lactams 45 and 56 rapidly acylate HCMV
protease and the enzyme remained fully acylated after
24 h. SAR studies have shown that plasma stability and
low nM potency against HCMV δAla protease are
required to give antiviral activity in whole cells. Both
45 and 56 have low nM potency against the viral
protease and micromolar antiviral activity against
HCMV. The crystal structure of HCMV protease was
obtained and used to model the conformationally re-
stricted, chiral, 4-isopropylphenylurea-(S)-proline-(SRS)-
R-methyl-5,5′-trans-lactam benzothiazole 56 into the

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4439
before filtration and evaporation using a Buchi Rotavapor.
Ambient temperature was 20 °C. All solvents used were Fisons
analytical reagents except for pentane (Aldrich Chemical Co.)
and anhydrous THF (Fluka sureseal). All other reagents were
usually obtained from Aldrich, Fluka or Lancaster. Elemental
microanalyses were determined by the Microanalytical Labo-
ratory, GlaxoSmithKline Stevenage.
(broad s, 2H, NH2+ ), 4.08-3.80 (m, 3H, NCH2CH2, NCHCH2),
3.38-3.29 (m, 1H, NCHCHMe), 3.14-3.04 (m, 1H, CHMe),
2.88-2.77 (m, 2H, NCH2CHH, COCHCHMeCHMe), 2.01-1.88
(m, 1H, NCH2CHH), 1.73-1.56 (m, 2H, COCHCHMeCHMe),
1.46 (d, J ) 7.5 Hz, 3H, CHMe), 1.23-1.13 (m, 6H, COCH-
CHMeCHMe). HPLC: 99.7% (tR 11.78 min); LCMS m/z 237
(MH+) single component 100%, gradient 2 (tR 1.93 min)
gradient 3 (tR 8.44min); HRMS calcd for C13H21N2O2 (MH+)
237.1603, found 237.1609.
((3S,3a R,6a S)-1-[(cis-2,3-Dim eth ylcyclop r op yl)-cis-ca r -
bon yl]-3-m eth yl-4-[(2S)-p yr r olid in -2-ylca r bon yl]h exa h y-
d r op yr r olo[3,2-b]p yr r ol-2(1H)-on e h yd r och lor id e (44).
Compound 43 was deprotected with hydrogen in the presence
of Pd /C as described for 6 to give 44 (96%) as a white solid:
1H NMR (CDCl3) δ 11.20 and 8.00 (2 broad s, 2H, NH2+ ), 4.80-
4.68 (m, 1H, NCHCO), 4.21 (t, J ) 9.5 Hz, 1H, NCHHCH2-
CH2), 3.85-3.40 (m, 5H, NCHHCH2CH2, NCH2CH2, NCHCH2,
NCHCHMe), 3.28-3.13 (m, 1H, CHMe), 2.88-2.69 (m, 2H,
NCH2CHH, COCHCHMeCHMe), 2.25-1.53 (m, 7H, NCH2-
CHH, NCH2CH2CH2, COCHCHMeCHMe); 1.25-1.09 (m, 9H,
CHMe, COCHCHMeCHMe); MS (thermospray) m/z 334 (MH+).
Anal. (C18H27N3O3‚HCl) C, H, N.
ter t-Bu tyl (3S,3a R,6a S)-4-({(2S)-1-[(Ben zyloxy)ca r bo-
n yl]p yr r olid in -2-yl}ca r bon yl)-3-m eth yl-2-oxoh exa h yd r o-
p yr r olo[3,2-b]p yr r ole-1(2H)-ca r boxyla te (10). To a stirred
suspension of Cbz-(S)-proline (8 g, 32.1 mmol), TBTU (10.3 g,
32.1 mmol), and HOBT‚H2O (4.5 g, 32.1 mmol) in anhydrous
acetonitrile (65 mL) was added diisopropylethylamine (10.6
mL, 61 mmol), and the resulting solution was stirred for 20
min. The hydrochloride salt 9 (7.45 g, 27 mmol) was added,
followed by acetonitrile (35 mL) and anhydrous dimethylfor-
mamide (30 mL) to give a clear solution which was stirred at
20 °C for 24 h. The solution was poured into ethyl acetate (500
mL), and the solution was washed with water (500 mL). The
aqueous phase was back-extracted with ethyl acetate (300 mL),
and the combined organic phases were washed sequentially
with 2M HCl (3 × 200 mL), water (2 × 200 mL), and saturated
brine (200 mL), then dried and evaporated to give a gum (18.5
g). This gum was purified by flash column chromatography
eluting with cyclohexanes-ethyl acetate (2:1 to 3:1) to yield
10 (11.26 g, 88.5%) as a white foam: 1H NMR (CDCl3 shows
rotameric forms) δ 7.40-7.28 (m, 5H, C6H5), 5.22-4.93 (m, 2H,
PhCH2), 4.49-4.20 and 3.83-3.45 and 3.32-2.97 and 2.64-
2.51 (4m, 9H, NCHCO, NCH2CH2CH2, NCH2CH2, NCHCHMe,
NCHCH2, NCHCHH, CHMe), 2.32-1.76 (m, 5H, NCHCHH
+ NCH2CH2CH2), 1.54 (s, 9H, But), 1.13 and 1.06 and 1.86 (d,
3H, J ) 7 Hz, CHMe); HPLC 100% (tR 26.4 min); LCMS m/z
472 (MH+) gradient 1 (tR 3.01 min); TLC Rf 0.31 (cyclohex-
anes-ethyl acetate, 4:1); HRMS calcd for C25H34N3O6 (MH+)
472.2448, found 472.2446.
Ben zyl (3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ole-1(2H )-ca r b ox-
yla te (5). To a solution of 49 (922 mg, 1 equiv, 2.75 mmol) in
dry tetrahydrofuran (7 mL) at -78 °C under nitrogen was
added 1M LHMDS solution in tetrahydrofuran (3.3 mL, 1.2
equiv, 3.3 mmol), keeping the temperature below -70 °C. The
solution was kept at -78 °C for 10 min, then at 0 °C for 10
min, and recooled to -78 °C; cyclopropanecarbonyl chloride
(0.75 mL, 3 equiv, 8.25 mmol) was added, and the reaction
mixture was then stirred at -78 °C for 1 h. The reaction
mixture was quenched with saturated aqueous ammonium
chloride (25 mL) and then allowed to warm to room temper-
ature. Water was added (20 mL), and then the aqueous phase
was extracted with ethyl acetate (100 mL), and the combined
organic phase was washed with water (30 mL) and brine (30
mL), dried, and evaporated to give a yellow oil. This oil was
purified by flash column chromatography eluting with cyclo-
hexanes-ethyl acetate (4:1) to yield 5 (334 mg, 35%) as a pale
yellow gum: IR (KBr) νmax 1747, 1713, 1704, 1693, 1681 cm
-1; 1H NMR (CDCl3) δ 7.36 (s, 5H, C6H5), 5.13 (ABq, J ) 12.5
Hz, 2H, PhCH2), 3.93-3.60 (m, 3H, NCH2CH2, NCHCH2),
3.55-3.44 (m, 1H, NCHCHMe), 3.30-2.80 (m, 2H, CHMe,
COCH(CH2)2), 2.75-2.62 (m, 1H, NCH2CHH), 2.05-1.85 (m,
1H, NCH2CHH), 1.30-0.85 (m, 7H, COCH(CH2)2), and CHMe);
MS (thermospray) m/z 343 (MH)+. Anal. (C19H22N2O4)
C, H, N.
(3S,3aR,6aS)-1-(Cyclopr opylcar bon yl)-3-m eth ylh exah y-
d r op yr r olo[3,2-b]p yr r ol-2(1H)-on e h yd r och lor id e (6). A
solution of 5 (330 mg, 0.96 mmol) in 2-propanol (30 mL) was
added to the palladium catalyst (119 mg, 10% Pd/C, Degussa
type, E101, NE/W, 50% H2O) under nitrogen and the resulting
mixture stirred vigorously under an atmosphere of hydrogen
for 2.75 h. The catalyst was filtered off under an atmosphere
of nitrogen, and a 1 M solution of hydrogen chloride in diethyl
ether (1 mL, 1 equiv, 1 mmol) was added to the filtrate.
Evaporation of the solvent gave 6 (175 mg, 74%) as a colorless
gum: 1H NMR (CDCl3) δ 4.10-3.79 (m, 2H, NCHHCH2
+
NCHCH2), 3.37 (dd, J ) 11.6 Hz, 6.7 Hz, 1H, NCHHCH2),
3.22-3.05 (m, 1H, NCHCHMe), 2.95-2.75 (m, 2H, CHMe,
COCH(CH2)2), 2.07-1.87 (m, 1H, NCHCHH), 1.67-1.56 (m,
1H, NCHCHH), 1.27-0.88 (m, 7H, CHMe, COCH(CH2)2); MS
(thermospray) m/z 209 (MH+), 417 (2MH+). Anal. (C11H16N2O2)
C, H, N.
The following compounds were similarly prepared.
The following compounds were similarly prepared.
ter t-Bu tyl (3S,3a R,6a S)-3-Meth yl-2-oxoh exa h yd r op yr -
r olo[3,2-b]p yr r ole-1(2H)-ca r boxyla te h yd r och lor id e (9).
Compound 39 was deprotected with hydrogen in the presence
of Pd /C as described for 6 to yield 9 (93%) as a white solid:
1H NMR (d6-DMSO) δ 9.44 (br s, 1H, NH), 3.93-3.29 (m, 4H,
NCH2CH2, NCHCH2, NCHCHMe), 2.91-2.75 (m, 1H, CHMe),
2.46-2.30 (m, 1H, NCH2CHH), 2.12-1.92 (m, 1H, NCH2CHH),
1.46 (s, 9H, t-Bu), 1.20 (d, J ) 7.3 Hz) and 1.04 (d, J ) 6.1 Hz)
(3H, CHCH3); LCMS m/z 241 (MH+) single component 100%,
gradient 1 (tR 1.66 min); MS (Thermospray) m/z 481 (2MH+),
241 (MH+), 141 (M-Boc+). HPLC: 95% (tR 8.5 min). Anal.
(C12H20N2O3.HCl‚0.25C3H8O) C, H, N.
((3S,3aR,6aS)-1-(Cyclopr opylcar bon yl)-3-m eth yl-4-[(2S)-
p yr r olid in -2-ylca r bon yl]h exa h yd r op yr r olo[3,2-b]p yr r ol-
2(1H)-on e h yd r och lor id e (8). Compound 7 was deprotected
with hydrogen in the presence of Pd/C as described for 6 to
give 8 (94%) as a white foam, identical to that prepared
previously.9
Ben zyl (2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-
6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]-
ca r bon yl}p yr r olid in e-1-ca r boxyla te. (7). Compound 6 was
reacted with Cbz-(S)-proline as described for 10 to give 7 (87%)
as a white amorphous solid, identical to that prepared previ-
ously.9
Ben zyl (2S)-2-{[(3a R,6R,6a S)-4-{[cis-2,3-Dim eth ylcyclo-
p r op yl]-cis- ca r bon yl}-6-m eth yl-5-oxoh exa h yd r op yr r olo-
[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyl-
a t e (43). Compound 42 was reacted with Cbz- (S)-proline
as described for 10 to give 43 (77%) as a white foam: 1H
NMR (DMSO heated at 120° to get the rotomers to coalesce)
δ 7.40-7.23 (m, 5H, C6H5), 5.08 (d, J ) 13 Hz, 1H) and 5.02
(d, J ) 13 Hz, 1H, PhCH2), 3.95-3.30 (m, 6H, NCH2CH2,
NCH2CH2CH2, NCHCHMe and NCHCH2), 3.38 (m, 1H, NCH-
CO), 3.03 (m, 1H, CHMe), 2.70 (t, J ) 9 Hz, 1H, COCHCHMe),
2.28-2.15 and 2.00-1.75 (2m, 1H and 5H, NCH2CH2 and
NCH2CH2CH2), 1.58-1.47 (m, 2H, CHCHMeCHMe), 1.15 and
1.12 (2d, J ) 7.5 Hz, 2 × 3H, CHCHMeCHMe), 1.06 (br m,
3H, Me); MS (thermospray) m/z 468 (MH+); Circular dichroism
(CH3CN) λmax199.8 nm, dE 3.89, E22341, λmax221.6 nm, dE
-17.90, E11748, λmax242.6 nm, dE 16.00, E1661; LCMS m/z
468 (MH+) single component 95%, gradient 2 (tR 3.30 min)
(3S,3a R,6a S)-1-[(cis-2,3-Dim eth ylcyclop r op yl)-cis-ca r -
b on yl]-3-m et h ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H )-
on e h yd r och lor id e (42). Compound 41 was deprotected with
hydrogen in the presence of Pd /C as described for 6 to give
42 (98%) as a white solid: 1H NMR (CDCl3) δ 10.28-9.80

4440 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
gradient 3 (tR 15.08min); HRMS calcd for C26H34N3O5 (MH+)
468.2498, found 468.2489.
P r ep a r a tion of 1, 14, a n d 16-23. Using essentially the
same procedure as for the preparation of 15, the following
compounds listed in Scheme 3 were prepared from 8.
Ben zyl (2S)-2-{[(3a S,6S,6a R)-6-Met h yl-5-oxoh exa h y-
d r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-
1-ca r boxyla te (11). To a solution of 10 (11.2 g, 23.8 mmol)
in dichloromethane (60 mL) was added trifluoroacetic acid (20
mL). The solution was stirred in a cold water bath for 45 min
and then concentrated in vacuo. Toluene (100 mL) was added
and evaporated off and the resulting gum dissolved in ethyl
acetate (250 mL). The solution was washed sequentially with
saturated aqueous sodium bicarbonate solution (2 × 100 mL),
water (2 × 100 mL), and saturated brine (100 mL), then dried
and evaporated to a foam. The aqueous washes were combined
and extracted with ethyl acetate (3 × 300 mL), and the organic
extracts were combined, washed with saturated brine (50 mL),
then dried and evaporated to give a crisp foam. The batches
of foam were combined, dissolved in ethyl acetate (50 mL), and
evaporated to give 11 (8.68 g, 98%) as a white solid: 1H NMR
(CDCl3 shows rotameric forms) δ 7.39-7.28 (m, 5H, C6H5),
6.14-5.92 (2m, 1H, NH), 5.22-4.92 (m, 2H, PhCH2), 4.48-
3.98 (2m, 2H, NCHCO, NCHHCH2), 3.82-2.84 (2m, 6H, NCH2-
CH2CH2, NCHHCH2, NCHCHMe, NCHCH2, CHMe), 2.34-
1.77 (m, 6H, NCH2CH2CH2, NCHCH2), 1.48-0.78 (4m, 3H,
CHMe); HPLC 100% (tR 18.2 min); TLC Rf 0.12 (ethyl acetate-
toluene- acetic acid, 25:5:1); LCMS m/z 372 (MH+) single
component 99%, gradient 2 (tR 2.46 min), gradient 3 (tR
10.92min); HRMS calcd for C20H26N3O4 (MH+) 372.1923, found
372.1928.
(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-[((2S)-1-{[6-
(d im et h yla m in o)-1-n a p h t h yl]su lfon yl}p yr r olid in -2-yl)-
car bon yl]-3-m eth ylh exah ydr opyr r olo[3,2-b]pyr r ol-2(1H)-
on e (14). Compound 8 was reacted with [6-(dimethylamino)-
naphth-1-yl]sulfonyl chloride as described for 15 to give 14
(64%) as a green/yellow solid: IR (KBr) νmax 1747.6, 1684.9,
1
1665.5, 1618.9 cm-1; H NMR (CDCl3) δ 8.64 (d, J ) 9.3 Hz,
1H, arylH), 7.94 (d, J ) 7 Hz, 1H, arylH), 7.85 (d, J ) 9.3 Hz,
1H, arylH), 7.44-7.27 (m, 2H, arylH), 6.96-6.89 (m, 1H,
arylH), 4.67 (dd, J ) 4.8 Hz, J ) 7.8 Hz, 1H, NCHCO),
4.31-4.10 (m, 1H, NCHHCH2CH2), 3.88-3.25 (m, 6H,
NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2, CHMe),
3.14-3.06 (m, 6H, NMe2), 3.02-2.88 (m, 1H, COCHCH2CH2),
2.83-2.68 (m, 1H, NCH2CHH), 2.22-1.77 (m, 5H,
NCH2CH2CH2, NCH2CH2CH2 and NCH2CHH), 1.31-0.94
(m, 7H, CHMe, COCHCH2CH2); MS (thermospray) m/z 539
(MH+); HPLC 99% (tR 27.20 min). Anal. (C28H34N4O5S)
C, H, N.
(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-[((2S)-1-{[5-
(d im et h yla m in o)-1-n a p h t h yl]su lfon yl}p yr r olid in -2-yl)-
car bon yl]-3-m eth ylh exah ydr opyr r olo[3,2-b]pyr r ol-2(1H)-
on e (1). Compound 8 was reacted with 5-(dimethylamino)-
naphth-1-yl]sulfonyl chloride as described for 15 to give 1 as
a
green/yellow foam (80%), identical with that prepared
previously.9
(3S,3aR,6aS)-1-(Cyclopr opylcar bon yl)-3-m eth yl-4-{[(2S)-
1-(2-n a p h t h y ls u lfo n y l)p y r r o li d i n -2-y l]c a r b o n y l}-
h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H )-on e (16). Com-
pound 8 was reacted with 2-naphthylsulfonyl chloride as
described for 15 to give 16 (59%) as a white solid: IR (KBr)
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-6-Meth yl-5-oxoh exa h y-
d r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-
1-ca r boxyla te (12). A solution of 11 (8.0 g, 21.5 mmol) and
di-tert-butyl dicarbonate (6.0 g, 27.5 g) in 2-propanol (350 mL)
was hydrogenated in the presence of the palladium catalyst
(2 g, 10% Pd/C, Degussa type, E101, NE/W, 50% H2O) for 5 h.
The catalyst was filtered off and the filtrate evaporated to a
white foam (6.5 g). Crystallization from toluene (35 mL) and
cyclohexane (40 mL) gave 12 (5.6 g, 77%) as a white solid: 1H
NMR (CDCl3) δ 6.09-5.90 (2m, 1H, CONH), 4.45-3.97 (2m,
2H, NCHCO, NCHHCH2), 3.81-3.36 (m, 5H, NCH2CH2CH2,
NCHHCH2, NCHCHMe, NCHCH2), 3.20-2.78 (2m, 1H, CHMe),
2.37-1.75 (m, 6H, NCH2CH2CH2, NCHCH2), 1.50-1.05 (3m,
12H, But, CHMe); LCMS m/z 338 (MH+) single component
99.8%, gradient 1 (tR 2.25min); HPLC 100% (tR 16.77 min);
TLC Rf 0.12 (ethyl acetate- toluene- acetic acid, 25:5:1). Anal.
(C17H27N3O4) C, H, N.
1
νmax 1753.8, 1673.9, 1658.1 cm-1; H NMR (CDCl3) δ 8.46 (s,
1H, arylH), 8.01-7.87 (m, 4H, arylH), 7.69-7.59 (m, 2H,
arylH), 4.65-4.60 (dd, J ) 4.9 Hz, J ) 7.9 Hz, 1H, NCHCO),
4.32 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.94-3.38 (m, 5H,
NCHHCH2CH2, NCH2CH2, NCHCHMe and NCHCH2), 3.30
(m, 1H, CHMe), 2.95 (m, 1H, COCHCH2CH2), 2.81 (m, 1H,
NCH2CHH), 2.20-1.72 (m, 5H, NCH2CH2CH2, NCH2CH2CH2
and NCH2CHH), 1.28-0.95 (m, 7H, CHMe and COCHCH2CH2);
MS (thermospray) m/z 496 (MH+). HPLC: 98% (tR 27.18 min).
Anal. (C26H29N3O5S) C, H, N, S.
(3S,3a R,6a S)-4-{[(2S)-1-(Ben zylsu lfon yl)p yr r olid in -2-
yl]ca r b on yl}-1-(cyclop r op ylca r bon yl)-3-m et h ylh exa h y-
d r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (17). Compound 8 was
reacted with benzylsulfonyl chloride as described for 15 to
give 17 (58%) as a white solid: IR (KBr) νmax 1747.46 (m),
(3S,3aR,6aS)-1-(Cyclopr opylcar bon yl)-3-m eth yl-4-{[(2S)-
1-(1-n a p h t h y ls u lfo n y l)p y r r o li d i n -2-y l]c a r b o n y l}-
h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (15). To a so-
lution of 8 (30.7 mg, 89.8 µmol, 1 equiv) in dry MeCN (2 mL)
was added triethylamine (29 µL, 207 µmol, 2.3 equiv). To this
solution was added a solution of 1-naphthylsulfonyl chloride
(28.2 mg, 105 µmol, 1.2 equiv) in dry MeCN (1 mL). The
reaction mixture was stirred at room temperature for 3 h
before 2-propanol (10 µL) was added to quench unreacted
sulfonyl chloride. The mixture was evaporated to dryness,
redissolved in dichloromethane (15 mL), and washed with
water (15 mL), sat. NaHCO3 solution (15 mL) and water (15
mL) and then dried, and the solvent was evaporated to give a
white solid. This solid was purified by flash column chroma-
tography eluting with cyclohexanes-ethyl acetate to give 15
(29 mg, 65%) as a white solid: IR (KBr) νmax 1747.8, 1682.4,
1668.2 cm-1; 1H NMR (CDCl3) δ 8.77 (d, J ) 8.8 Hz, 1H, arylH),
8.30 (d, J ) 7 Hz, 1H, arylH), 8.05 (d, J ) 7 Hz, 1H, arylH),
7.92 (d, J ) 7.9 Hz, 1H, arylH), 7.72-7.50 (m, 3H, arylH), 4.74
(dd, J ) 7.9 Hz, J ) 5.1 Hz, 1H, NCHCO), 4.254 (t, J ) 10.2
Hz, 1H, NCHHCH2CH2),3.88-3.36 (m, 5H, NCHHCH2CH2,
NCH2CH2, NCHCHMe, NCHCH2), 3.34-3.22 (quintet, J ) 7.4
Hz, 1H, CHMe), 3.00-2.87 (m, 1H, COCHCH2CH2), 2.83-2.69
(m, 1H, NCH2CHH), 2.30-1.83 (m, 5H, NCH2CH2CH2,
NCH2CH2CH2 and NCH2CHH), 1.30-0.92 (m, 7H, CHMe,
COCHCH2CH2); MS (thermospray) m/z 496 (MH+); HPLC
100% (tR 27.79 min). Anal. (C26H29N3O5S) C, H, N, S.
1
1667.65 (m), 1650.87 (s) cm-1; H NMR (CDCl3) δ 7.68-7.49
and 7.44-7.32 (2m, 5H, arylH), 4.51-4.25 (m, 3H, NCHCO,
PhCH2SO2), 4.12-4.01 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2),
3.77-3.28 (m, 5H, NCHHCH2CH2, NCH2CH2, NCHCHMe and
NCHCH2), 3.15-3.04 (m, 1H, CHMe), 3.00-2.88 (m, 1H,
COCHCH2CH2), 2.80-2.67 (m, 1H, NCH2CHH), 2.29-1.80 (m,
5H, NCH2CH2CH2, NCH2CHH), 1.20-0.95 (m, 7H, CHMe and
COCHCH2CH2); MS (thermospray) m/z 460 (MH+). HPLC:
96% (tR 25.05 min). Anal. (C23H29N3O5S) C, H, N, S.
(3S ,3a R ,6a S )-1-(Cyclop r op ylca r b on yl)-3-m e t h yl-4-
{[(2S )1-(p h e n y ls u lfo n y l)p y r r o lid in -2-y l]c a r b o n y l}-
h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (18). Compound
8 was reacted with phenylsulfonyl chloride as described for
15 to give 18 (80%) as a white solid: 1H NMR (CDCl3) δ 7.93-
7.85 (m, 2H, arylH), 7.60-7.49 (m, 3H, arylH), 4.57-4.52 (dd,
J ) 4.5 Hz, J ) 7.6 Hz, 1H, NCHCO), 4.28 (t, J ) 9.5 Hz, 1H,
NCHHCH2CH2), 3.90-3.64, 3.57-3.38 (m, 5H, NCHHCH2CH2,
NCH2CH2, NCHCHMe and NCHCH2), 3.32 (m, 1H, CHMe),
2.94 (m, 1H, COCHCH2CH2), 2.79 (m, 1H, NCH2CHH), 2.20-
1.75 (m, 5H, NCH2CH2CH2, NCH2CH2CH2 and NCH2CHH),
1.25-0.95 (m, 7H, CHMe and COCHCH2CH2); LCMS m/z 446
(MH+) single component 99%, gradient 1 (tR 4.12 min).
HPLC: 100% (tR 24.31 min). Anal. (C22H27N3O5S) C, H, N, S.
(3S,3a R,6a S)-4-({(2S)-1-[(4-Ch lor op h en yl)su lfon yl]p yr -
r olid in -2-yl}ca r bon yl)-1-(cyclop r op ylca r bon yl)-3-m eth -
ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (19). Com-

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4441
pound 8 was reacted with 4-chlorophenylsulfonyl chloride as
described for 15 to give 19 (49%) as a white solid: 1H NMR
(CDCl3) δ 7.94-7.81 (m, 2H, arylH), 7.54-7.45 (m, 2H, arylH),
4.61-4.56 (dd, J ) 4.7 Hz, J ) 7.0 Hz, 1H, NCHCO), 4.26 (t,
J ) 9.8 Hz, 1H, NCHHCH2CH2), 3.91-3.64 and 3.64-3.26
(2m, 6H, NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2
and CHMe), 2.94 (m, 1H, COCHCH2CH2), 2.79 (m, 1H,
NCH2CHH), 2.26-1.80 (m, 5H, NCH2CH2CH2, NCH2CH2CH2
and NCH2CHH), 1.28-0.95 (m, 7H, CHMe and COCHCH2CH2);
LCMS m/z 480 (MH+) single component 99% gradient 3 (tR
14.44 min). HPLC: 100% (tR 28.26 min); HRMS calcd for
4-MeCH2CH2OPh); MS (thermospray) m/z 504 (MH+). HPLC:
100% (tR 28.86 min). Anal. (C25H33N3O6S) C, H, N, S.
(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-[((2S)-1-{[5-
(d im et h yla m in o)-1-n a p h t h yl]su lfon yl}p yr r olid in -2-yl)-
ca r bon yl]-3-eth ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-
on e (13). Compound 13 was prepared from ethyl analogue of
3 as described for the methyl isomer 1 (Schemes 2 and 3) 13 as
a white solid: 1H NMR (CDCl3) δ 8.54 (d, J ) 8.0 Hz, 1H,
dansyl-2H), 8.42 (d, J ) 8.5 Hz, 1H, dansyl-4H), 8.28 (dd, J )
7.3 Hz, 1.2 Hz, 1H, dansyl-8H), 7.60-7.48 (m, 2H, dansyl-3H,
dansyl-7H), 7.18 (d, J ) 7.3 Hz, 1H, dansyl-6H), 4.75 (dd, J )
7.9 Hz, 4.9 Hz, 1H, NCHCO), 4.27 (t, J ) 9.5 Hz, 1H,
NCHHCH2CH2), 3.84-3.36 (m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCHEt, NCHCH2), 3.10-2.85 (m, 8H, CHEt, NMe2,
COCHCH2CH2), 2.78-2.67 (m, 1H, NCH2CHH), 2.29-1.82 (m,
5H, NCH2CH2CH2, NCH2CHH), 1.31-1.22 (m, 2H, CHCH2-
Me), 1.28-0.87 (7H, m, CHCH2Me, COCHCH2CH2); MS (ther-
mospray) m/z 553 (MH+); LCMS m/z 553 (MH+) single com-
ponent 99% gradient 1 (tR 4.72 min). Anal. (C29H36N4O5S) C,
H, N, S.
C
22H27ClN3O5S (MH+) 480.1360, found 480.1353.
(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-({(2S)-1-[(4-
isop r op ylp h en yl)su lfon yl]p yr r olid in -2-yl}ca r b on yl)-3-
m et h ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H )-on e (20).
Compound 8 was reacted with 4- isopropyphenylsulfonyl
chloride as described for 15 to give 20 (44%) as a white solid:
1H NMR (CDCl3) δ 7.84-7.77 (m, 2H, arylH), 7.40-7.32 (m,
2H, arylH), 4.56-4.51 (dd, J ) 4.9 Hz, J ) 7.9 Hz, 1H,
NCHCO), 4.28 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.91-3.64
and 3.57-3.26 (2m, 6H, NCHHCH2CH2, NCH2CH2, NCH-
CHMe, NCHCH2 and CHMe), 3.05-2.88 (m, 2H, COCHCH2-
CH2 and arylCHMe2), 2.78 (m, 1H, NCH2CHH), 2.17-1.72 (m,
5H, NCH2CH2CH2, NCH2CH2CH2 and NCH2CHH), 1.27 (d,
J ) 7.3 Hz, 6H, arylCHMe2), 1.20-0.94 (m, 7H, CHMe and
COCHCH2CH2); LCMS m/z 488 (MH+) single component 99.3%
gradient 1 (tR 4.57 min), gradient 3 (tR 15.22 min). HPLC:
100% (tR 29.52 min). HRMS calcd for C25H34N3O5S (MH+)
488.2219, found 488.2202.
(3S ,3a R ,6a S )-1-(Cyclop r op ylca r b on yl)-3-m e t h yl-4-
{[(2 S )-1 -(1 -n a p h t h o y l )p y r r o l i d i n -2 -y l ]c a r b o n y l}-
h exah ydr opyr r olo[3,2-b]pyr r ol-2(1H)-on e (28). To a stirred
solution of 1-naphthoic acid (22 mg, 0.125 mmol) in dry
dimethylformamide (2 mL) at room temperature were added
solutions of HOBT (16 mg, 0.115 mmol) in DMF (0.5 mL) and
TBTU (39 mg, 0.122 mmol) in acetonitrile (0.5 mL). After
stirring at room temperature for 20 min, a solution of 8 (0.036
mg, 0.105 mmol) in dry DMF (0.5 mL) was added followed by
diisopropylethylamine (0.037 mL, 0.21 mmol). After stirring
at room temperature for 18 h, the mixture was diluted with
dichloromethane (15 mL) and water (15 mL), the organic and
aqueous layers were separated, and the latter was extracted
with dichloromethane (2 × 10 mL). The combined organic
extracts were washed with 2N HCl (15 mL), water (15 mL),
saturated NaHCO3 solution (15 mL), and again with water
(15 mL), dried over MgSO4, filtered, and evaporated to leave
an orange oil, which was purified by preparative plate chro-
matography eluting with ethyl acetate-cyclohexane(1:1) to give
28 (25 mg, 51%) as an off-white, glassy solid: IR (KBr) νmax
1746.5, 1687.8, 1660.1, 1650.5, 1631.2 cm-1; 1H NMR (CDCl3)
δ 8.11-8.04 (d, J ) 7.9 Hz, 1H, arylH), 7.92-7.83 (m, 2H,
arylH), 7.63-7.45 (m, 4H, arylH), 4.87-4.78 (dd, J ) 5.5 Hz,
J ) 8.0 Hz, 1H, NCHCO), 4.67 (t, J ) 9.5 Hz, 1H, NCHHCH2-
CH2), 3.94-3.66, 3.52-3.36 and 3.27-3.17 (3m, 6H, NCHHCH2-
CH2, NCH2CH2, NCHCHMe, NCHCH2 and CHMe), 2.96 (m,
1H, COCHCH2CH2), 2.80 (m, 1H, NCH2CHH), 2.39-1.78 (m,
5H, NCH2CH2CH2, NCH2CH2CH2 and NCH2CHH), 1.28-
0.93 (m, 7H, CHMe and COCHCH2CH2); MS (thermospray)
m/z 460 (MH+); 478 (MNH4+); HPLC 98% (tR 25.45 min). Anal.
(C27H29N3O4) C, H, N.
(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-({(2S)-1-[(4-
isop r op oxyp h en yl) su lfon yl]p yr r olid in -2-yl}ca r b on yl)-
3-m eth ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (21).
Compound 8 was reacted with 4- isopropoxyphenylsulfonyl
chloride as described for 15 to give 21 (43%) as a beige solid:
1H NMR (CDCl3) δ 7.83-7.77 (m, 2H, arylH), 6.97-6.91 (m,
2H, arylH), 4.67-4.58 (m, 1H, arylOCHMe2), 4.55-4.48 (dd,
J ) 4.9 Hz, J ) 7.9 Hz, 1H, NCHCO), 4.28 (t, J ) 9.5 Hz, 1H,
NCHHCH2CH2), 3.91-3.65, 3.59-3.27 (m, 6H, NCHHCH2CH2,
NCH2CH2, NCHCHMe, NCHCH2 and CHMe), 2.99-2.89 (m,
H, COCHCH2CH2), 2.78 (m, 1H, NCH2CHH), 2.20-1.74 (m,
5H, NCH2CH2CH2, NCH2CH2CH2 and NCH2CHH), 1.37 (d,
J ) 6.1 Hz, 6H, arylOCHMe2), 1.20-0.94 (m, 7H, CHMe and
COCHCH2CH2); MS (thermospray) m/z 504 (MH+). HPLC:
94% (tR 28.36 min). Anal. (C25H33N3O6S) C, H, N, S.
(3S,3a R,6a S)-1-(Cyclop r op ylca r b on yl)-4-({(2S)-1-[(3-
isop r op oxyp h en yl) su lfon yl]p yr r olid in -2-yl}ca r b on yl)-
3-m eth ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)- (22). Com-
pound 8 was reacted with 3-isopropoxyphenylsulfonyl chloride
as described for 15 to give 22 (43%) as a white solid: IR (KBr)
νmax 1753.9 (m), 1673.9 (m), 1660.9 (s) cm-1; 1H NMR (CDCl3)
δ 7.44-7.36 and 7.11-7.03 (2m, 4H, arylH), 4.67-4.50 (m, 2H,
arylOCHMe2, NCHCO), 4.34-4.23 (t, J ) 9.2 Hz, 1H,
NCHHCH2CH2), 3.91-3.23 (m, 6H, NCHHCH2CH2, NCH2CH2,
NCHCHMe, NCHCH2, CHMe), 3.00-2.83 (m, 1H, COCHCH2-
CH2), 2.84-2.72 (m, 1H, NCH2CHH), 2.20-1.73 (m, 5H,
NCH2CH2CH2, NCH2CHH), 1.86 and 1.85 (2d, J ) 6.1 Hz, 6H,
arylOCHMe2), 1.20-0.95 (m, 7H, CHMe and COCHCH2CH2);
MS (thermospray) m/z 504 (MH+), 436 (MH-COcyclopropyl+).
HPLC: 100% (tR 28.32 min). Anal. (C25H33N3O6S) C, H, N, S.
The following compounds were similarly prepared.
(3S ,3a R ,6a S )-4-({(2S )-1-[(5-Ch lor o-3-m e t h yl-1-b e n -
zoth ien -2-yl)ca r bon yl]p yr r olid in -2-yl}ca r bon yl)-1-(cyclo-
pr opylcar bon yl)-3-m eth ylh exah ydr opyr r olo[3,2-b]pyr r ol-
2(1H)-on e (24). Compound 8 was reacted with (5-chloro-3-
methyl-1-benzothien-2-yl)carbonyl chloride as described for 28
to give 24 (78%) as a white solid: 1H NMR (CDCl3) δ 7.76-
7.70 and 7.39-7.34 (2m, 3H, aryl), 4.78-4.70 (m, 1H,
NCHCO), 4.56 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.90-3.30
(m, 6H, NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2,
CHMe), 3.00-2.89 (m, 1H, COCHCH2CH2); 2.85-2.73
(m, 1H, NCH2CHH), 2.49 (s, 3H, arylMe), 2.41-1.86 (m, 5H,
NCH2CH2CH2, NCH2CHH), 1.22-0.93 (m, 7H, CHMe,
COCHCH2CH2); MS (thermospray) m/z 514 (MH+); HPLC
98.51% (tR ) 29.54 min). LCMS m/z 514 (MH+) single
(3S,3a R ,6a S)-1-(Cyclop r op ylca r b on yl)-3-m e t h yl-4-
({(2S )-1-[(4-p r op oxyp h e n yl)su lfon yl]p yr r olid in -2-yl}-
ca r bon yl)h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (23).
Compound 8 was reacted with 4-propoxyphenylsulfonyl chlo-
ride as described for 15 to give 23 (85%) as a beige solid: 1H
NMR (CDCl3) δ 7.85-7.78 (m, 2H, arylH), 7.02-6.94 (m, 2H,
arylH), 4.56-4.44 (dd, J ) 4.9 Hz, J ) 7.3 Hz, 1H, NCHCO),
4.56-4.44 (dd, J ) 4.9 Hz, J ) 7.3 Hz, 1H, NCHCO), 4.28 (t,
J ) 9.8 Hz, 1H, NCHHCH2CH2), 3.97 (m, 2H, 4-MeCH2CH2-
OPh), 3.92-3.62, 3.62-3.4, 3.47-3.26 (3m, 6H, NCHHCH2-
CH2, NCH2CH2, NCHCHMe, NCHCH2 and CHMe), 2.94 (m,
H, COCHCH2CH2), 2.78 (m, 1H, NCH2CHH), 2.21-1.68 (m,
7H, NCH2CH2CH2, NCH2CH2CH2, NCH2CHH and 4-MeCH2-
CH2OPh), 1.30-0.93 (m, 9H, CHMe, COCHCH2CH2 and
component 99.5% gradient 3 (tR 15.40 min). Anal. (C26H28
-
ClN3O4S) C, H, N.
(3S,3a R,6a S)-4-{[(2S)-1-(1,1′-Bip h en yl-4-ylca r b on yl)-
p yr r olid in -2-yl]ca r bon yl}-1-(cyclop r op ylca r bon yl)-3-m e-
th ylh exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (25). Com-
pound 8 was reacted with 4-phenylbenzoyl chloride as described
for 28 to give 25 (54%) as a white solid: 1H NMR (CDCl3) δ

4442 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
7.69-7.58 and 7.50-7.33 (2m, 9H, arylH), 4.78-4.70 (m, 1H,
NCHCO), 4.61 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.91-3.58
(m, 5H, NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2),
3.41-3.28 (m, 1H, CHMe), 3.00-2.89 (m, 1H, COCHCH2CH2),
2.84-2.71 (m, 1H, NCH2CHH), 2.35-1.81 (m, 5H,
NCH2CH2CH2, NCH2CHH), 1.22-0.93 (m, 7H, CHMe,
COCHCH2CH2); MS (thermospray) m/z 486 (MH+); LCMS m/z
508 (MNa+) single component 99.8% gradient 2 (tR 3.10 min),
gradient 3 (tR 14.52 min); HRMS calcd for C29H32N3O4 (MH+)
486.2393, found 486.2397.
(3S,3a R,6a S)-1-(Cyclop r op ylca r bon yl)-4-{[(2S)-1-(4-iso-
pr opylben zoyl)pyr r olidin -2-yl]car bon yl}-3-m eth ylh exah y-
d r op yr r olo[3,2-b]p yr r ol-2(1H)- (26). Compound 8 was re-
acted with 4-isopropylbenzoyl chloride as described for 28 to
give 26 (18%) as a white solid: 1H NMR (CDCl3) δ 7.53-7.48
and 7.26-7.21 (2m, 4H, arylH), 4.75-4.68 (m, 1H, NCHCO),
4.60 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.88-3.55 (m, 5H,
NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2), 3.40-3.27
(m, 1H, CHMe), 3.02-2.70 (m, 3H, COCHCH2CH2, NCH2CHH,
arylCHMe2), 2.35-1.81 (m, 5H, NCH2CH2CH2, NCH2CHH),
1.28-0.95 (m, 13H, arylCHMe2, CHMe, COCHCH2CH2); MS
(thermospray) m/z 452 (MH+); LCMS m/z 474 (MNa+) single
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-(4-isop r op ylp h en yl)p yr r olid in e-1-ca r b oxa m id e
(30). To a solution of proline 8 (0.03 g, 0.09 mmol) in dry
acetonitrile (2 mL) was added dry triethylamine (0.03 mL, 0.22
mmol) followed by 4-(isopropyl)phenyl isocyanate (0.022 g,
0.135 mmol). The reaction mixture was left standing at room-
temperature overnight, then quenched with 2-propanol (0.1
mL). It was then evaporated to dryness and purified using
preparative plate chromatography eluting with ethyl acetate-
cyclohexane (2:1) to give 30 (77%) as an amorphous solid: 1H
NMR (CDCl3) δ 7.26-7.21 and 7.16-7.11 (2m, 4H, arylH), 6.19
(s, 1H, CONH), 4.65 (dd, J ) 7.6 Hz, 3.7 Hz, 1H, NCHCO),
4.47 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.88-3.44 (m, 5H,
NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2), 3.37-3.24
(m, 1H, CHMe), 2.99-2.66 (m, 3H, COCHCH2CH2, NCH2CHH,
arylCHMe2), 2.45-1.85 (m, 5H, NCH2CH2CH2, NCH2CHH),
1.28-0.95 (m, 13H, arylCHMe2, CHMe, COCHCH2CH2); MS
(thermospray) m/z 467 (MH+); HPLC 100% (tR 27.843min);
LCMS m/z 467 (MH+) single component 99.6% gradient 1 (tR
4.28min); HRMS calcd for C26H34N4O4 (MH+) 467.265831,
found 467.264975.
The following compounds were similarly prepared.
component 99.4% gradient 3 (tR 14.18 min); HPLC 100% (tR
)
26.50 min). HRMS calcd for C26H34N3O4 (MH+) 452.2549, found
452.2548.
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-(4-pr opoxyph en yl)pyr r olidin e-1-car boxam ide (31).
Compound 8 was reacted with 4-propoxyphenyl isocyanate as
described for 30 to give 31 (53%) as a white solid: 1H NMR
(CDCl3): δ 7.24-7.18 and 6.85-6.79 (2m, 4H, arylH), 6.11 (s,
1H, CONH), 4.63 (dd, J ) 8.0 Hz, 4.0 Hz, 1H, NCHCO), 4.45
(t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.91-3.45 (m, 7H,
arylOCH2CH2Me, NCHHCH2CH2, NCH2CH2, NCHCHMe,
NCHCH2), 3.36-3.23 (m, 1H, CHMe), 2.98-2.88 (m, 1H,
COCHCH2CH2), 2.78-2.66 (m, 1H, NCH2CHH), 2.40-1.70 (m,
7H, arylOCH2CH2Me, NCH2CH2CH2, NCH2CHH), 1.19-0.95
(m, 10H, arylOCH2CH2Me, CHMe, COCHCH2CH2); MS (ther-
mospray) m/z 483 (MH+). Anal. (C26H34N4O5) C, H, N.
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-[4-(tr iflu or om eth yl)p h en yl]p yr r olid in e-1-ca r box-
amide (32).Compound8wasreactedwith 4-(trifluoromethyl)phenyl
isocyanate as described for 30 to give 32 (90%) as a white
powder: 1H NMR (CDCl3): δ 7.55-7.43 (m, 4H, arylH), 6.42
(s, 1H, CONH), 4.65 (dd, J ) 8.0 Hz, 4.0 Hz, 1H, NCHCO),
4.41 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.86-3.47 (m, 5H,
NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2), 3.36-3.23
(m, 1H, CHMe), 2.99-2.89 (m, 1H, COCHCH2CH2), 2.82-2.70
(m, 1H, NCH2CHH), 2.42-1.86 (m, 5H, NCH2CH2CH2, NCH2-
CHH), 1.19-0.95 (m, 7H, CHMe, COCHCH2CH2); MS (ther-
mospray) m/z 493 (MH+); LCMS m/z 493 (MH+) single com-
ponent 99.7% gradient 1 (tR 4.65min), gradient 3 (tR 14.39min);
HRMS calcd for C24H28F3N4O4 (MH+) 493.2063, found 493.2047.
(2S )-2-{[(3a S ,6S ,6a R )-4-(C y c lo p r o p y lc a r b o n y l)-6-
m eth -yl-5-oxoh exah ydr opyr r olo[3,2-b]pyr r ol-1(2H)-yl]car -
b on yl}-N-(4-isop r op oxyp h e n yl)p yr r olid in e -1-ca r b ox-
a m id e (33). Compound 8 was reacted with 4-(isopropoxy)-
phenyl isocyanate as described for 30 to give 33 (12%) as a
white powder: 1H NMR (CDCl3): δ 7.24-7.17 and 6.85-6.78
(2m, 4H, arylH), 6.11 (s, 1H, CONH), 4.63 (dd, J ) 7.3 Hz, 4.3
Hz, 1H, NCHCO), 4.51-4.40 (m, 2H, arylOCHMe2, NCHHCH2-
CH2), 3.84-3.44 (m, 5H, NCHHCH2CH2, NCH2CH2, NCH-
CHMe, NCHCH2), 3.36-3.23 (m, 1H, CHMe), 2.98-2.88 (m,
1H, COCHCH2CH2), 2.77-2.66 (m, 1H, NCH2CHH), 2.40-1.87
(m, 5H, NCH2CH2CH2, NCH2CHH), 1.19-0.95 (m, 13H, ary-
lOCHMe2, CHMe, COCHCH2CH2); MS (thermospray) m/z
483 (MH+); HPLC 100%, (tR 25.36min). Anal. (C26H34N4O5)
C, H, N.
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-(4-ch lor op h en yl)p yr r olid in e-1-ca r boxa m id e (34).
Compound 8 was reacted with 4-chlorophenyl isocyanate as
described for 30 to give 34 (66%) as a white powder: 1H NMR
(CDCl3): δ 7.32-7.20 (m, 4H, arylH), 6.24 (s, 1H, CONH), 4.63
(dd, J ) 8.0 Hz, 4.3 Hz, 1H, NCHCO), 4.41 (t, J ) 9.5 Hz, 1H,
(3S,3aR,6aS)-1-(Cyclopr opylcar bon yl)-3-m eth yl-4-{[(2S)-
1 -(4 -p r o p o x y b e n z o y l )p y r r o l i d i n -2 -y l ]c a r b o n y l }-
h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (27). Compound
8 was reacted with 4-propoxybenzoyl chloride as described for
28 to give 27 (59%) as a white solid: 1H NMR (CDCl3) δ 7.58-
7.53 and 6.91-6.86 (2m, 4H, arylH), 4.71 (t, J ) 7.0 Hz, 1H,
NCHCO), 4.60 (t, J ) 9.2 Hz, 1H, NCHHCH2CH2), 3.94
(t, J ) 6.4 Hz, 2H, arylOCH2CH2Me), 3.89-3.55 (m, 5H,
NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2), 3.39-3.26
(m, 1H, CHMe), 3.00-2.89 (m, 1H, COCHCH2CH2), 2.82-2.71
(m, 1H, NCH2CHH), 2.35-1.74 (m, 7H, arylOCH2CH2Me,
NCH2CH2CH2, NCH2CHH), 1.26-0.95 (m, 10H, arylOCH2-
CH2Me, CHMe, COCHCH2CH2); MS (thermospray) m/z 468
(MH+); HPLC 100% (tR ) 26.02 min); LCMS m/z 468 (MH+)
single component 99.6% gradient 3 (tR 13.92 min); HRMS calcd
for C26H34N3O5 (MH+) 468.2498, found 468.2482.
(3S,3a R,6a S)-4-{[(2S)-1-(4-Ch lor oben zoyl)p yr r olid in -2-
yl]ca r bon yl}-1-(cyclop r op ylca r bon yl)-3-m et h ylh exa h y-
d r op yr r olo[3,2-b]p yr r ol-2(1H)-on e (29). Compound 8 was
reacted with 4-chlorobenzoyl chloride as described for 28 to
give 29 (87%) as a white solid: 1H NMR (CDCl3) δ 7.56-7.50
(m, 2H, arylH), 7.41-7.30 (m, 2H, arylH), 4.70 (t, J ) 7.0 Hz,
1H, NCHCO), 4.55 (t, J ) 9.8 Hz, 1H, NCHHCH2CH2), 3.90-
3.46, 3.39-3.27 (2m, 6H, NCHHCH2CH2, NCH2CH2, NCH-
CHMe, NCHCH2 and CHMe), 2.94 (m, 1H, COCHCH2CH2),
2.78 (m, 1H, NCH2CHH), 2.37-1.78 (m, 5H, NCH2CH2CH2 and
NCH2CH2CH2 and NCH2CHH), 1.31-0.95 (m, 7H, CHMe and
COCHCH2CH2); MS (thermospray) m/z 444 (MH+); LCMS m/z
444 (MH+) single component 99.7% gradient 3 (tR 13.23 min);
HPLC 100% (tR ) 24.01min); HRMS calcd for C23H27ClN3O4
(MH+) 444.1690, found 444.1689.
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-m eth yl-N-p h en ylp yr r olid in e-1-ca r boxa m id e (40).
To a solution of 8 (20 mg, 0.06 mmol) in acetonitrile (2 mL)
was added triethylamine (0.02 mL, 0.15 mmol, 2.5 equiv)
followed by N-methyl-N-phenylcarbamoyl chloride (15 mg, 0.15
mmol, 1.5 equiv). The resulting solution was left at room
temperature for 16 h, quenched with 2-propanol (0.1 mL) and
then evaporated to dryness. The resulting residue was purified
by preparative TLC eluting with ethyl acetate/cyclohexane (2:
1) to give 40 (22 mg, 79%) as a white solid: 1H NMR (CDCl3)
δ 7.41-7.08 (m, 5H, arylH), 4.60-4.40 (m, 2H, NCHCO,
NCHHCH2CH2), 3.84-3.60 (m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCHMe, NCHCH2), 3.42-3.30 (m, 1H, CHMe), 3.24 (s, 3H,
arylNMeCO), 3.00-2.66 (2m, 2H, COCHCH2CH2, NCH2CHH),
2.17-1.55 (m, 5H, NCH2CH2CH2, NCH2CHH), 1.30-0.95 (m,
7H, CHMe, COCHCH2CH2); MS (thermospray) m/z 439 (MH+).
Anal. (C24H30N4O4) C, H, N.

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4443
NCHHCH2CH2), 3.85-3.43 (m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCHMe, NCHCH2), 3.36-3.23 (m, 1H, CHMe), 2.99-2.89
(m, 1H, COCHCH2CH2), 2.80-2.69 (m, 1H, NCH2CHH), 2.40-
1.88 (m, 5H, NCH2CH2CH2, NCH2CHH), 1.19-0.95 (m, 7H,
CHMe, COCHCH2CH2); MS (thermospray) m/z 459 (MH+);
LCMS m/z 459 (MH+) single component 99.7% gradient 3 (tR
13.64 min); HPLC 99.65% (tR 24.99min). HRMS calcd for
4.3 Hz, 1H, NCHCO), 4.45 (t, J ) 9.5 Hz, 1H, NCHHCH2CH2),
3.85-3.45 (m, 5H, NCHHCH2CH2, NCH2CH2, NCHCHMe,
NCHCH2), 3.36-3.24 (m, 1H, CHMe), 2.99-2.87 (m, 1H,
COCHCH2CH2), 2.79-2.68 (m, 1H, NCH2CHH), 2.40-1.90 (m,
5H, NCH2CH2CH2, NCH2CHH), 1.27-0.95 (m, 7H, CHMe,
COCHCH2CH2); MS (thermospray) m/z 425 (MH+); HPLC
98.62% (tR 22.08min). Anal. (C23H28N4O4) C, H, N.
(2S)-2-{[(3a S,6S,6a R)-4-{[-cis-2,3-Dim et h ylcyclop r o-
p yl] -cis-ca r bon yl}-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-
b]p yr r ol-1(2H )-yl]ca r b on yl}-N-(4-isop r op ylp h en yl)p yr -
r olid in e-1-ca r boxa m id e (45). Compound 44 was reacted
with 4-(isopropyl)phenyl isocyanate as described for 30 to give
45 (47%) as a white solid: 1H NMR (CDCl3) δ 7.29-7.19 and
7.18-7.08 (pseudo-ABq, J ) 8.5 Hz, 4H, arylH), 6.20 (bs,1H,
NH), 4.68-4.60 (m, 1H, NCHCO), 4.55-4.38 (m, 1H,
NCHHCH2CH2), 3.84-3.34 (m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCH2, NCHCHMe), 3.32-3.18 (m, 1H, CHMe), 2.94-2.69
(m, 3H, NCH2CHH, arylCHMe2, NCOCHCHMeCHMe), 2.40-
1.87 (m, 5H, NCH2CH2CH2, NCH2CHH), 1.60 (m, 2H, NCO-
CHCHMeCHMe), 1.20 (m, 15H, 5× CH3); MS (thermospray)
m/z 495 (MH+), 334 (M-4-(isopropyl)phenyl isocyanate
group+); LCMS m/z 495 (MH+) single component 99.5% gradi-
ent 1 (tR 4.50 min); HRMS calcd for C28H38N4O4 (MH+)
495.297131, found 495.297733; HPLC 100% (tR 28.71min).
C
23H28ClN4O4 (MH+) 459.1799, found 459.1787
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-(1-n a ph th yl)p yr r olidin e-1-ca r boxa m id e (35). Com-
pound 8 was reacted with 1-naphthyl isocyanate as described
for 30 to give 35 (74%) as a white solid: 1H NMR (CDCl3): δ
7.91-7.41 (m, 7H, arylH), 6.57 (s, 1H, CONH), 4.70 (dd, J )
8.0 Hz, 4.3 Hz, 1H, NCHCO), 4.42 (t, J ) 9.5 Hz, 1H,
NCHHCH2CH2), 3.89-3.50 (m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCHMe, NCHCH2), 3.36-3.25 (m, 1H, CHMe), 2.98-2.87
(m, 1H, COCHCH2CH2), 2.76-2.65 (m, 1H, NCH2CHH), 2.48-
1.94 (m, 5H, NCH2CH2CH2, NCH2CHH), 1.29-0.95 (m, 7H,
CHMe, COCHCH2CH2); MS (thermospray) m/z 475 (MH+);
LCMS m/z 475 (MH+) single component 99.5% gradient 3 (tR
13.31 min); HPLC 99% (tR 24.7 min). HRMS calcd for
C
27H31N4O4 (MH+) 475.2345, found 475.2331.
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
(2S)-2-{[(3a S,6S,6a R)-6-Met h yl-5-oxo-4-[(2,2,3,3-t et r a -
m eth ylcyclopr opyl)car bon yl]h exah ydr opyr r olo[3,2-b]pyr -
r ol-1(2H)-yl]car bon yl}-N-(4-isopr opylph en yl)pyr r olidin e-1-
ca r boxa m id e (51). Compound 49 was reacted with 4-(iso-
propyl)phenyl isocyanate as described for 30 to give 51 (37%)
as a white solid: 1H NMR (CDCl3) δ 7.26-7.21 and 7.15-7.10-
(2m, 4H, arylH), 6.19 (s, 1H, CONH), 4.97-4.61 (m, 1H,
NCHCO), 4.44 (t, J ) 9.4 Hz, 1H, NCHHCH2CH2), 3.83-3.45
(m, 5H, NCHHCH2CH2, NCH2CH2, NCHCH2, NCHCHMe),
3.29-3.16 (m, 1H, CHMe), 2.91-2.67 (m, 2H, NCH2CHH,
arylCHMe2),2.40-1.87(m,6H,COCHCMe2CMe2,NCH2CH2CH2,
NCH2CHH), 1.30-1.10 (m, 21H, arylCHMe2, CHMe,
COCHCMe2CMe2); LCMS m/z 523 (MH+) single component
98% gradient 1 (tR 5.48min). Anal. (C30H42N4O4) C, H, N.
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-(2-isop r op ylp h en yl)p yr r olid in e-1-ca r b oxa m id e
(36). Compound 8 was reacted with 2-(isopropyl)phenyl iso-
cyanate as described for 30 to give 36 (73%) as a white solid:
1H NMR (CDCl3): δ 7.61 (dd, J ) 7.9 Hz, 1.8 Hz, 1H, aryl-
6H), 7.25-7.06 (m, 3H, aryl-3,4,5H), 6.13 (s, 1H, CONH), 4.65
(dd, J ) 7.9 Hz, 4.3 Hz, 1H, NCHCO), 4.46 (t, J ) 9.5 Hz, 1H,
NCHHCH2CH2), 3.84-3.48 (m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCHMe, NCHCH2), 3.37-3.23 (m, 1H, CHMe), 3.11-2.86
(m, 2H, arylCHMe2, COCHCH2CH2), 2.75-2.64 (m, 1H,
NCH2CHH), 2.45-1.89 (m, 5H, NCH2CH2CH2, NCH2CHH),
1.28-0.95 (m, 13H, arylCHMe2, CHMe, COCHCH2CH2); MS
(thermospray) m/z 467 (MH+); HPLC 97.69% (tR 26.13min).
Anal. (C25H34N4O4) C, H, N.
(2S)-2-{[(3a S,6S,6a R)-6-Meth yl-4-(4-n itr op h en yl)-5-oxo-
h exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}-N-(4-
isop r op ylp h en yl)p yr r olid in e-1-ca r boxa m id e (52). Com-
pound 50 was reacted with 4-(isopropyl)phenyl isocyanate as
described for 30 to give 52 (49%) as a pale yellow foam: 1H
NMR (CDCl3) δ 8.30-8.23 and 7.62-7.55 (2m, 4H, 4-O2N-
arylH), 7.26-7.21 and 7.16-7.11 (2m, 4H, 4-iPr-arylH), 4.71-
4.60 (m, 2H, NCHCO, NCHHCH2CH2), 4.07-3.29 (m, 6H,
NCHHCH2CH2, NCH2CH2, NCHCH2, NCHCHMe, CHMe),
2.91-2.77 (m, 1H, NCH2CHH), 2.67-2.54 (m, 1H, arylCHMe2),
2.45-1.90 (m, 5H, NCH2CH2CH2, NCH2CHH), 1.24-1.13 (m,
9H, arylCHMe2, CHMe); MS (thermospray) m/z 520 (MH+);
HPLC 100% (tR 29.4 min). Anal. (C28H33N5O5) C, H, N.
Ben zyl (3a S,6S,6a R)-4-{[-cis-2,3-Dim eth ylcyclop r op yl]
-cis-ca r b on yl}-6-m et h yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ole-1(2H)-ca r boxyla te (41). To a solution of (c-2, c-3-
dimethylcyclopropyl-r-1-carboxylic) acid (678 mg, 5.95 mmol)
in anhydrous tetrahydrofuran (20 mL), stirred under nitrogen
at -13 °C, was added methanesulfonyl chloride (250 µL, 3.23
mmol) followed by a solution of triethylamine (1.5 mL, 10.8
mmol) in anhydrous tetrahydrofuran (10 mL) dropwise over
15 min at -12 to -14 °C. The resulting suspension was stirred
below -11 °C for 1h and then allowed to warm to 20 °C over
2.5 h. Solid was filtered off and washed with ether, and the
combined filtrates were evaporated. The residue was parti-
tioned between ether (2 × 50 mL) and ice-cold saturated
aqueous sodium bicarbonate solution (25 mL). The combined
organic phases were washed with water (20 mL) and saturated
brine (20 mL), dried, and evaporated to give (c-2, c-3-dimeth-
ylcyclopropyl-r-1-carboxylic) anhydride (543 mg, 87%) as an
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-[3-(tr iflu or om eth yl)p h en yl]p yr r olid in e-1-ca r box-
a m id e (37). Compound 8 was reacted with 3-(trifluoro)phenyl
isocyanate as described for 30 to give 37 as a white solid
(95%): 1H NMR (CDCl3): δ 7.72-7.21 (m, 4H, arylH), 6.41 (s,
1H, CONH), 4.64 (dd, J ) 7.9 Hz, 4.3 Hz, 1H, NCHCO), 4.41
(t, J ) 9.5 Hz, 1H, NCHHCH2CH2), 3.85-3.49 (m, 5H,
NCHHCH2CH2, NCH2CH2, NCHCHMe, NCHCH2), 3.36-3.24
(m, 1H, CHMe), 2.99-2.87 (m, 1H, COCHCH2CH2), 2.82-2.70
(m, 1H, NCH2CHH), 2.40-1.90 (m, 5H, NCH2CH2CH2, NCH2-
CHH), 1.27-0.95 (m, 7H, CHMe, COCHCH2CH2); MS (ther-
mospray) m/z 493 (MH+); HPLC 83.35% (tR 27.19min). Anal.
(C24H27F3N4O4) C, H, N.
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-(3-ch lor op h en yl)p yr r olid in e-1-ca r boxa m id e (38).
Compound 8 was reacted with 3-chlorophenyl isocyanate as
described for 30 to give 38 (62%) as a white solid: 1H NMR
(CDCl3): δ 7.51-7.49 (m, 1H, aryl-2H), 7.20-7.16 (m, 2H, aryl-
4H, -6H), 7.02-6.97 (m, 1H, aryl-5H), 6.27 (s, 1H, CONH),
4.63 (dd, J ) 7.9 Hz, 4.3 Hz, 1H, NCHCO), 4.41 (t, J ) 9.5
Hz, 1H, NCHHCH2CH2), 3.85-3.43 (m, 5H, NCHHCH2CH2,
NCH2CH2, NCHCHMe, NCHCH2), 3.36-3.24 (m, 1H, CHMe),
2.99-2.86 (m, 1H, COCHCH2CH2), 2.81-2.70 (m, 1H,
NCH2CHH), 2.40-1.90 (m, 5H, NCH2CH2CH2, NCH2CHH),
1.24-0.95 (m, 7H, CHMe, COCHCH2CH2); MS (thermospray)
m/z 459 (MH+); HPLC 98.97% (tR 25.52min). Anal. (C23H27
-
ClN4O4) C, H, N.
oil which crystallized: IR (KBr) νmax 1788, 1732, 1024 cm-1
;
(2S)-2-{[(3a S,6S,6a R)-4-(Cyclop r op ylca r bon yl)-6-m eth -
yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b o-
n yl}-N-p h en ylp yr r olid in e-1-ca r boxa m id e (39). Compound
8 was reacted with phenyl isocyanate as described for 30 to
give 39 (80%) as a white solid: 1H NMR (CDCl3): δ 7.37-
6.99 (m, 5H, arylH), 6.24 (s, 1H, CONH), 4.65 (dd, J ) 7.9 Hz,
1H NMR (CHCl3) δ 1.68 (dd, J ) 9.0 Hz, 7.5 Hz, 2H,
COCHCHMeCHMe x 2), 1.61-1.54 (m, 4H, COCHCHMeCHMe
x 2), 1.24-1.21 (m, 12H, COCHCHMeCHMe x 2); MS (ther-
mospray) m/z 228 (MNH4+). To a solution of the trans-lactam
4 (600 mg, 2.19 mmol) in anhydrous tetrahydrofuran (8 mL)

4444 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
stirred under nitrogen at -78 °C was slowly added 1M
LHMDS solution in tetrahydrofuran (2.4 mL, 2.4 mmol). The
yellow solution was stirred for a further 20 min at -78 °C,
and then a solution of (c-2, c-3-dimethylcyclopropyl-r-1-car-
boxylic) anhydride (510 mg, 2.42 mmol) in anhydrous tetrahy-
drofuran (7 mL) was slowly added. The resulting solution was
stirred for 1 h under nitrogen at -78 °C and then poured into
saturated aqueous ammonium chloride solution (50 mL). The
solution was extracted with ethyl acetate (2 × 50 mL), and
the combined organic phases were washed sequentially with
saturated aqueous sodium bicarbonate solution (20 mL), water
(20 mL) and saturated brine (20 mL), dried, and evaporated
to give an oil which crystallized (1.09 g). The crude product
was purified by column chromatography eluting with cyclo-
hexane-dichloromethane to give 41 (785 mg, 96%) as a white
foam: IR (KBr) νmax 1746, 1713, 1682 cm-1; 1H NMR (CHCl3)
δ 7.40-7.31 (m, 5H, arylH), 5.16 and 5.09 (Abq, J ) 12.5 Hz,
2H, PhCH2O), 3.92-3.63 (m, 3H, NCH2CH2, NCHCH2), 3.46
(dd, J ) 11.0 Hz, 7.0 Hz, 1H, NCHCHMe), 3.25-3.11 and
3.04-2.90 (2 broad s, 1H, CHMe), 2.87-2.78 (m, 1H, COCH-
CHMeCHMe), 2.75-2.67 (m, 1H, NCH2CHH), 1.98-1.85 (m,
1H, NCH2CHH), 1.69-1.53 (m, 2H, COCHCHMeCHMe),
1.30-1.06 (m, 9H, CHMe, COCHCHMeCHMe); LCMS m/z 371
(MH+) single component 100% gradient 2 (tR 3.53 min)
gradient 3 (tR 16.38); HRMS calcd for C21H27N2O4 (MH+)
371.1971, found 371.1962.
(3S,3a R,6a S)-3-Meth yl-1-(4-n itr op h en yl)-4-[(2S)-p yr r o-
lid in -2-ylca r bon yl]h exa h yd r op yr r olo[3,2-b]p yr r ol-2(1H)-
on e t r iflu or oa cet a t e (50). Compound 48 was deprotected
with trifluoroacetic acid as described for 49 to give 50 (99%)
as a yellow glass: 1H NMR (CDCl3) δ 12.40-12.05 (broad s,
exch., 0.5H, NH), 8.33-8.24 and 7.62-7.53 (2m, 4H, arylH),
4.94-4.84 (m, 1H, NCHCO), 4.28-3.30 (m, 7H, NCH2CH2CH2,
NCH2CH2, NCHCH2, NCHCHMe, CHMe), 2.80-2.60 (m, 1H,
NCH2CHH), 2.37-1.90 (m, 5H, NCH2CH2CH2, NCH2CHH),
1.34-1.13 (m, 3H, CHMe); MS (thermospray) m/z 359 (MH+).
Anal. (C18H22N4O4‚C2HF3O2) C, H, N.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-4-(1,3-Ben zoth ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (54). A mixture of
Boc-proline translactam 12 (40.4 mg, 0.12 mmol, 1 equiv),
copper (1) chloride (12 mg, 0.12 mmol, 1 equiv), potassium
carbonate (27.5 mg, 0.2 mmol, 1.66 equiv), TDA-1 (11.1µL, 0.03
mmol, 0.29 equiv), and 2-bromobenzothiazole (45 mg, 0.21
mmol, 1.75 equiv) in p-xylene (25 mL) was stirred and refluxed
under nitrogen. After 4.5 h the mixture was left to stand at
room temperature overnight before it was reheated and stirred
at reflux for a further 5.25 h and then allowed to cool to room
temperature overnight. Then the mixture was filtered and the
residue washed with ethyl acetate (10 mL). The filtrate and
washings were combined and washed with 1M hydrochloric
acid (10 mL), water (7.5 mL), and brine (7.5 mL), dried, and
evaporated to leave a yellow solid. The crude material was
purified by preparative plate chromatography eluting with
ethyl acetate-cyclohexane (1:1) to give 54 (26.2 mg, 46%) as a
white solid: NMR (CDCl3) δ 7.80 (t, J ) 6.7 Hz, 2H, ben-
zothiazolyl-4H and -7H) 7.44 and 7.31 (2t, J ) 7.5 Hz, 2H,
benzothiazolyl-5H and -6H), 4.44 (m, 1H, NCHCO), 4.15, 3.85
and 3.53 (3m, 6H, NCHCHMe, NCH2CH2CH2, NCH2CH2 and
NCHCH2),3.45 (m, 1H, CHMe), 3.17 and 2.37 (2m, 2H,
NCH2CH2), 2.16 and 1.91 (2m, 4H, NCH2CH2CH2, NCH2-
CH2CH2), 1.44 (d, J ) 6.1 Hz, (rotamers) 9H, But), 1.20 (m,
3H, CHMe); MS (thermospray) m/z 471 (MH+), 371 (M-Boc+);
HPLC 100% (tR 31.44min); HRMS calcd for C24H30N4O4S
(MH+) 471.206603, found 471.206283.
(2S)-2-{[(3a S,6S,6a R)-4-{[-cis-2,3-Dim et h ylcyclop r o-
p yl] -tr a n s-ca r bon yl}-6-m eth yl-5-oxoh exa h yd r op yr r olo-
[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}-N-(4-isop r op ylp h en yl)-
p yr r olid in e-1-ca r boxa m id e (46). Compound 4 was reacted
with the anhydride prepared from (t-2, t-3-dimethylcyclopro-
pyl-r-1-carboxylic) acid15 and the intermediate converted through
as described for the cis, cis-dimethyl isomer 45 to give 46 (67%)
as a white solid: 1H NMR (CDCl3) δ 7.24 and 7.11 (pseudo-
Abq, J ) 8.5 Hz, 4H, arylH), 6.21 (s, 1H, CONH), 4.64 (dd, J
) 8.0 Hz, 4.3 Hz, 1H, NCHCO), 4.45 (t, J ) 8.0 Hz, 1H,
NCHHCH2CH2), 3.83-3.45(m, 5H, NCHHCH2CH2, NCH2CH2,
NCHCH2, NCHCHMe), 3.32-3.20 (m, 1H, CHMe), 2.92-2.65
(m, 2H, NCH2CHH, arylCHMe2), 2.42-1.89 (m, 6H, COCH-
CHMeCHMe, NCH2CH2CH2, NCH2CHH), 1.74-1.53 (m, 2H,
COCHCHMeCHMe), 1.25-1.10 (m, 15H, arylCHMe2, CHMe,
COCHCHMeCHMe). Anal. (C28H38N4O4) C, H, N.
The following compounds were similarly prepared.
ter t-Bu t yl (2S)-2-{[(3a S,6S,6a R)-6-Met h yl-4-(4-n it r o-
p h en yl)-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]-
ca r b on yl}p yr r olid in e-1-ca r b oxyla t e (48). Compound 12
was reacted with 4-nitrobromobenzene as described for 54 to
give 48 (65%) as a yellow foam: 1H NMR (CDCl3) δ 8.30-8.22
and 7.64-7.56 (2m, 4H, arylH), 4.53-3.10 (m, 8H, NCHCO,
NCH2CH2CH2, NCH2CH2, NCHCH2, NCHCHMe, CHMe),
2.75-2.55 (m, 1H, NCH2CHH), 2.30-1.81 (m, 5H, NCH2CHH,
NCH2CH2CH2), 1.49-1.40 (m, 9H, But), 1.20-1.13 (m, 3H,
CHMe); MS (thermospray) m/z 459 (MH+), 359 (M-Boc+);
LCMS m/z 459 (MH+) single component 100% gradient 2 (tR
3.12 min) gradient 3 (tR 14.04min); HPLC 100% (tR 26.7min);
HRMS calcd for C23H30N4O6 (MNa+) 481.2063, found 481.2071.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-6-Meth yl-5-oxo-4-(1,3-
t h ia zol-2-yl)h exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]-
ca r bon yl}p yr r olid in e-1-ca r boxyla te (53). Compound 12
was reacted with 2-bromothiazole as described for 54 to give
53 (37%) as a white solid: 1H NMR (CDCl3) δ 7.44 (d, J ) 3.7
Hz, 1H, SCHCHN), 7.02 (m, 1H, SCH), 4.42 (m, 1H, NCHCO),
4.22-3.38 (m, 7H, NCHCHMe, CHMe, NCH2CH2CH2, NCH2-
CH2 and NCHCH2), 3.01 (m, 1H, NCH2CHH), 2.43-1.82 (m,
5H, NCH2CH2CH2, NCH2CH2CH2, NCH2CHH), 1.46 (d, J )
6.7 Hz, 9H, But), 1.17 (dd, J ) 7.3 Hz, 3.7 Hz, 3H, CHMe); MS
(thermospray) m/z 421 (MH+); LCMS m/z 421 (MH+) single
component 98% gradient 2 (tR 2.91 min), gradient 3 (tR 13.13
min); HRMS calcd for C20H29N4O4S (MH+) 421.1910, found
421.1905; HPLC 100% (tR 24.06 min).
ter t-Bu t yl
(2S)-2-{[(3a S,6S,6a R)-6-m et h yl-5-oxo-4-
[(2,2,3,3-t et r a m et h ylcyclop r op yl)ca r b on yl]h exa h yd r o-
pyrrolo[3,2-b]pyrrol-1(2H)-yl]carbonyl}pyrrolidine-1-carbox-
yla te (47). Derived from 12 and the mixed anhydride prepared
from 2,2,3,3-tetramethylcyclopropanecarboxylic acid and tri-
methylacetyl chloride as described for 41 to give 47 (68%) as
a white solid. 1H NMR (CDCl3) shows complex overlapping
signals due to rotameric forms: δ 4.47-3.20 (m, 8H, NCHCO,
NCH2CH2CH2, NCH2CH2, NCHCH2, NCHCHMe, CHMe),
3.08-2.63 (m, 1H, NCH2CHH), 2.38-1.75 (m, 6H, COCHCMe2-
CMe2, NCH2CHH, NCH2CH2CH2), 1.49-1.09 (m, 24H, But,
CHMe, COCHCMe2CMe2); MS (thermospray) m/z 462 (MH+),
362 (M-Boc+); TLC Rf 0.36 (cyclohexane-ethyl acetate, 1:1);
LCMS m/z 462 (MH+) single component 99% gradient 1 (tR
4.88 min), gradient 2 (tR 3.46 min), gradient 3 (tR 16.17 min);
HRMS calcd for C25H40N3O5 (MH+) 462.2968, found 462.2974.
(3S,3aR,6aS)-3-Meth yl-4-[(2S)-pyr r olidin -2-ylcar bon yl]-
1-[(2,2,3,3-t e t r a m e t h y lc y c lo p r o p y l)c a r b o n y l]h e x a -
h ydr opyr r olo[3,2-b]pyr r ol-2(1H)-on e tr iflu or oacetate (49).
To a solution of 47 (75 mgs, 0.16 mmol) in dichloromethane (3
mL) was added trifluoroacetic acid (1 mL), and the mixture
was left at 21° C for 1.5 h. The mixture was evaporated to
dryness, toluene (2 × 5 mL) was added, and the mixture
evaporated and then left under high vacuum for 24 h at room
temperature to give 49 (76 mg, 99%) as a pale yellow foam:
1H NMR (CDCl3)
δ
6.20-5.70 (broad s, 2H, NH2+),
ter t-Bu tyl (2S)-2-{[(3aS,6S,6aR)-4-[6-({[ter t-Bu tyl(diph e-
n yl)silyl]oxy} m eth yl)-1,3-ben zoth ia zol-2-yl]-6-m eth yl-5-
oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b on yl}-
4.60-4.40 (m, 2H, NCHCO, NCHHCH2CH2), 4.35-3.20 (m,
6H, NCHHCH2CH2, NCH2CH2, NCHCH2, NCHCHMe,
CHMe), 2.50-2.00 (m, 7H, NCH2CH2, NCH2CH2CH2,
p yr r olid in e-1-ca r boxyla te (58). Prepared in
a manner
COCHCMe2CMe2),
1.30-1.05
(m,
15H,
CHMe,
similar to 54 by reacting 12 with 2-bromo-6-(tert-butyl-
diphenylsilanyloxymethyl)-benzothiazole12 to give 58 (57%
yield) as a pale yellow foam:1H NMR (CDCl3) δ 7.81-7.63 (m,
COCHCMe2CMe2); MS (thermospray) m/z 362 (MH+), 380
(MNH4+). Anal. (C20H31N3O3‚C2HF3O2) C, H, N.

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4445
4H, arylH), 7.49-7.31 (m, 9H, arylH), 4.85 (s, 2H, arylCH2),
4.52-4.04 (m, 3H, NCHCO, NCHHCH2CH2, NCHCHMe),
3.98-3.07 (m, 6H, NCHHCH2CH2, NCH2CH2, NCHCH2, CHMe,
NCH2CHH), 2.49-1.80 (m, 5H, NCH2CHH, NCH2CH2CH2),
1.44 (2s, 9H, t-BuO2C), 1.29-1.16 (m, 3H, Me), 1.10 (s, 9H,
t-BuSiO); MS (thermospray) m/z 739 (MH+), 639 ((MH-Boc+);
LCMS m/z 739 (MH+) single component 99.5% gradient 1 (tR
4.51 min); HPLC 100% (tR 43.6min). Anal. (C41H50N4O5SSi) C,
H, N, S.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-4-(4-Meth oxy-1,3-ben -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (59).
Prepared in a manner similar to 54 by reacting 12 with
2-bromo-4-methoxy-benzothiazole12 to give 59 (61% yield) as
a white solid:1H NMR (CDCl3) δ 7.41 (d, J ) 7.9 Hz, 1H,
benzothiazolyl-7H), 7.27 (m, 1H, benzothiazolyl-6H), 6.90 (d,
J ) 7.9 Hz, 1H, benzothiazolyl-5H), 4.43 (m, 1H, NCHCO),
4.02 (s, 3H, OCH3), 4.29-4.07, 3.95-3.69 and 3.68-3.36
(3m, 7H, NCHCHMe, NCH2CH2CH2, CHMe, NCH2CH2 and
NCHCH2), 3.19 and 2.39 (2m, 2H, NCH2CH2), 2.20 and 1.90
(2m, 4H, NCH2CH2CH2, NCH2CH2CH2), 1.45 (d, J ) 7.3 Hz,
(rotamers) 9H, But), 1.18 (m, 3H, CHMe); MS (thermospray)
m/z 501 (MH+), 401 (MH-Boc+); HPLC 98.5% (tR 29.5min).
Anal. (C25H32N4O5S‚0.15 CHCl3) C, H, N.
ter t-Bu t yl (2S)-2-{[(3a S,6S,6a R)-4-(5-Ch lor o-1,3-b en -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (60).
Prepared in a manner similar to 54 by reacting 12 with
2-bromo-5-chloro-benzothiazole12 to give 60 (16% yield) as a
white solid: 1H NMR (CDCl3) δ 7.78 (d, J ) 1.2 Hz, 1H,
benzothiazolyl-4H), 7.72 (d, J ) 8.5 Hz, 1H, benzothiazolyl-
7H), 7.28 (m, 1H, benzothiazolyl-6H), 4.44 (m, 2H, NCHCO,
NCHHCH2), 4.13 (m, 1H, NCHCH2), 3.99-3.70 and 3.68-3.36
(2m, 5H, NCHCHMe, NCH2CH2CH2, CHMe, NCHHCH2) 3.12
and 2.37 (2m, 2H, NCH2CH2), 2.30-2.00 and 2.00-1.81 (2m,
4H, NCH2CH2CH2), 1.45 (d, J ) 5.5 Hz, (rotamers) 9H, But),
1.20 (m, 3H, CHMe); MS (thermospray) m/z 505 (MH+), 405
(MH-Boc+). Anal. (C24H29ClN4O4S) C, H, N, S.
benzothiazolyl-4H), 7.28 (d, J ) 2.5 Hz, 1H, benzothiazolyl-
7H), 7.03 (dd, J ) 8.7 Hz, 2.5 Hz, 1H, benzothiazolyl-5H), 4.44
(m, 1H, NCHCO), 3.87 (s, 3H, OCH3), 4.24-4.01, 3.98-3.69
and 3.67-3.36 (3m, 7H, NCHCHMe, NCH2CH2CH2, CHMe,
NCH2CH2 and NCHCH2), 3.13 and 2.35 (2m, 2H, NCH2CH2),
2.27-2.00 and 2.00-1.80 (2m, 4H, NCH2CH2CH2, NCH2-
CH2CH2), 1.44 (d, J ) 6.1 Hz, (rotamers) 9H, But), 1.19 (m,
3H, CHMe); MS (thermospray) m/z 501 (MH+), 401 (MH-
Boc+); LCMS m/z 501 (MH+) single component 100% gradient
2 (tR 3.38 min), gradient 3 (tR 15.44 min); HRMS calcd for
C
25H33N4O5S (MH+) 501.2172, found 501.2165.
ter t-Bu t yl (2S)-2-{[(3a S,6S,6a R)-4-(4-Ch lor o-1,3-b en -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (64).
Prepared in a manner similar to 54 by reacting 12 with
2-bromo-4-chloro-benzothiazole12 to give 64 (55% yield) as a
white solid: 1H NMR (CDCl3) δ 7.71 and 7.46 (2d, J ) 8 Hz,
2H, benzothiazolyl-5H and -7H), 7.23 (m, 1H, benzothiazolyl-
6H), 4.54-4.07 (m, 3H, NCHCO, NCH2CH2), 4.01-3.70
and 3.69-3.36 (2m, 5H, NCHCHMe, NCH2CH2CH2, CHMe,
NCHCH2), 3.21 and 2.40 (2m, 2H, NCH2CH2), 2.32-2.01 and
1.98-1.78 (2m, 4H, NCH2CH2CH2, NCH2CH2CH2), 1.46 (d, J
) 6.1 Hz, (rotamers) 9H, But), 1.20 (m, 3H, CHMe); MS
(thermospray) m/z 505 (MH+), 405 (MH-Boc+). Anal. (C24H29
ClN4O4S) C, H, N, S.
-
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-4-(5-Meth oxy-1,3-ben -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (65).
Prepared in a manner similar to 54 by reacting 12 with
2-bromo-5- methoxy-benzothiazole12 to give 65 (37% yield) as
an off-white foam: 1H NMR (CDCl3) δ 7.66 (d, J ) 8 Hz, 1H,
benzothiazolyl-7H), 7.30 (d, J ) 2 Hz, 1H, benzothiazolyl-4H),
6.95 (dd, J ) 8.5 Hz, 2.5 Hz, 1H, benzothiazolyl-6H), 4.48 (m,
1H, NCHCO), 4.42 (m, 1H, NCHHCH2), 4.13 (m, 1H, NCHCH2),
3.88 (s, 3H, OCH3), 3.96-3.69 and 3.65-3.36 (2m, 5H, NCH-
CHMe, NCH2CH2CH2, CHMe and NCHHCH2), 3.14 and 2.38
(2m, 2H, NCH2CH2), 2.30-2.01 and 1.98-1.80 (2m, 4H,
NCH2CH2CH2, NCH2CH2CH2), 1.45 (d, J ) 6 Hz, (rotamers)
9H, But), 1.20 (m, 3H, CHMe); MS (thermospray) m/z 501
(MH+), 401 (MH-Boc+); LCMS m/z 501 (MH+) single compo-
nent 98% gradient 2 (tR 3.39 min) gradient 3 (tR 15.44 min);
HRMS calcd for C25H33N4O5S (MH+) 501.2172, found 501.2162.
ter t-Bu t yl
(2S)-2-{[(3a S,6S,6a R)-4-(5-Met h oxy[1,3]-
th ia zolo[5,4-b]p yr id in -2-yl)-6-m eth yl-5-oxoh exa h yd r op y-
r r olo[3,2-b]p yr r ol-1(2H)-yl]ca r b on yl}p yr r olid in e-1-ca r -
boxyla te (61). Prepared in a manner similar to 54 by reacting
12 with 2-bromo-6- methoxy-7aza-benzothiazole12 to give 61
(80% yield) as a glassy solid:1H NMR (CDCl3) (rotamers) δ 7.78
(d, J ) 8.5 Hz, 1H, azabenzothiazolyl-4H); 6.80 (d, J ) 8.5
Hz, 1H, azabenzothiazolyl-5H); 4.50-4.33 (m, 2H, NCHCO,
NCHHCH2); 4.20-4.01 (m, 1H, NCHCH2); 4.00 (s, 3H, ArOMe);
3.97-3.02 (m, 6H, NCHHCH2, NCHCHMe, NCH2CH2CH2,
CHMe,NCH2CHH),2.42-1.81(m,5H,NCH2CHH,NCH2CH2CH2),
1.47-1.41 and 1.24-1.16 (2m, 12H, But, CHMe). MS (ther-
mospray) m/z 502 (MH+). Anal. (C24H31N5O5S) C, H, N, S.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-4-(4-Meth oxy-7-m eth -
yl-1,3-ben zoth iazol-2-yl)-6-m eth yl-5-oxoh exah ydr opyr r olo-
[3,2-b]p yr r ol-1(2H )-yl]ca r b on yl}p yr r olid in e-1-ca r b oxy-
la te (62). Prepared in a manner similar to 54 by reacting 12
with 2-bromo-4-methoxy-7-methyl-benzothiazole12 to give 62
(53% yield) as a yellow solid: 1H NMR (CDCl3) δ 7.05 (d, J )
7 Hz, 1H) and 6.83 (d, J ) 8 Hz, 1H benzothiazolyl-5H and
-6H), 4.47 (m, 1H, NCHCO), 4.39 (m, 1H, NCHHCH2), 4.18
(m, 1H, NCHCH2), 4.00 (s, 3H, OCH3 ), 3.94-3.71 and 3.67-
3.36 (2m, 5H, NCHCHMe, NCH2CH2CH2, CHMe, NCHHCH2),
3.20 (m, 1H, NCH2CHH), 2.48 (s, 3H, arylCH3), 2.50-2.02 and
1.97-1.80 (2m, 5H, NCH2CHH, NCH2CH2CH2, NCH2CH2CH2),
1.45 (d, J ) 7.3 Hz, (rotamers) 9H, But), 1.19 (m, 3H, CHMe);
MS (thermospray) m/z 515 (MH+), 415 (MH-Boc+); LCMS m/z
515 (MH+) single component 100% gradient 2 (tR 3.42 min),
gradient 3 (tR 15.83 min); HRMS calcd for C26H35N4O5S (MH+)
515.2328, found 515.2345.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-4-{5-[(Diflu or om eth yl)-
su lfon yl]-1,3-b en zot h ia zol-2-yl}-6-m et h yl-5-oxoh exa h y-
d r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-
1-ca r boxyla te (66). Prepared in a manner similar to 54 by
reacting 12 with 2-bromo-5-(difluoromethyl)sulfonyl-benzothi-
azole12 to give 66 (27.5% yield) as a cream solid: 1H NMR
(CDCl3) δ 8.40 (d, J ) 1.2 Hz, 1H, benzothiazolyl-4H), 8.07 (d,
J ) 8 Hz, 1H, benzothiazolyl-7H), 7.87 (m, 1H, benzothiazolyl-
6H), 6.24 (t, J ) 54 Hz, 1H, CF2H), 4.45 (m, 2H, NCHCO and
NCHHCH2), 4.31-4.05, 4.01-3.73 and 3.67-3.34 (3m, 6H,
NCHCHMe, NCH2CH2CH2, CHMe, NCHHCH2 and NCHCH2),
3.14 (m, 1H, NCH2CHH), 2.51-2.02 and 1.97-1.78 (2m, 5H,
NCH2CHH, NCH2CH2CH2, NCH2CH2CH2), 1.45 (d, J ) 5 Hz,
(rotamers) 9H, But), 1.20 (m, 3H, CHMe); MS (thermospray)
m/z 585 (MH+), 485 (MH-Boc+). Anal. (C25H30F2N4O6S2) C,
H, N.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-6-Meth yl-4-(6-n itr o-1,3-
ben zoth ia zol-2-yl)-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-
1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (67). Pre-
pared in a manner similar to 54 by reacting 12 with 2-bromo-
6-nitro-benzothiazole12 to give 67 (13% yield) as a yellow
solid: 1H NMR (CDCl3) δ 8.72 (d, J ) 2.5 Hz, 1H, benzothia-
zolyl-7H), 8.30 (dd, J ) 9 Hz, 2 Hz, 1H, benzothiazolyl-5H),
7.82 (d, J ) 8.5 Hz, 1H, benzothiazolyl-4H), 4.53-4.37 and
4.29-4.06 (2m, 3H, NCHCO, NCHHCH2 and NCHCH2), 4.04-
3.73 and 3.69-3.36 (2m, 5H, NCHCHMe, NCH2CH2CH2,
CHMe, NCHHCH2), 3.14 and 2.42 (2m, 2H, NCH2CH2), 2.32-
2.02 and 1.99-1.83 (2m, 4H, NCH2CH2CH2, NCH2CH2CH2),
1.45 (d, J ) 5 Hz, (rotamers) 9H, But), 1.24 (m, 3H, CHMe);
MS (thermospray) m/z 516 (MH+), 416 (MH-Boc+). Anal.
(C24H29N5O6S) C, H, N, S.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-4-(6-Meth oxy-1,3-ben -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (63).
Prepared in a manner similar to 54 by reacting 12 with
2-bromo-6-methoxy-benzothiazole12 to give 63 (58% yield) as
a white solid: 1H NMR (CDCl3) δ 7.68 (d, J ) 8.6 Hz, 1H,

4446 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
ter t-Bu t yl (2S)-2-{[(3a S,6S,6a R)-6-Met h yl-5-oxo-4-[6-
(tr iflu or om eth oxy)-1,3-ben zoth ia zol-2-yl]h exa h yd r op yr -
rrolo[3,2-b]pyrrol-1(2H)-yl]carbonyl}pyrrolidine-1-carboxylate
(68). Prepared in a manner similar to 54 by reacting 12 with
2-bromo-6-trifluoromethoxy-benzothiazole12 to give 68 (53%
yield) as a white solid: 1H NMR (CDCl3) δ 7.77 (d, J ) 8.5
Hz, 1H, benzothiazolyl-4H), 7.67 (m, 1H benzothiazolyl-7H),
7.30 (m, 1H, benzothiazolyl-5H), 4.54-4.01 (m, 3H, NCHCO,
NCH2CH2), 3.98-3.70 and 3.69-3.35 (2m, 5H, NCHCHMe,
NCH2CH2CH2, CHMe, and NCHCH2), 3.13 (m, 1H, NCH2CHH),
2.48-1.79 (m, 5H, NCH2NHH, NCH2CH2CH2, NCH2CH2CH2),
1.46 (d, J ) 6.1 Hz, (rotamers) 9H, But), 1.20 (m, 3H, CHMe);
MS (thermospray) m/z 555 (MH+), 455 (MH-Boc+); LCMS m/z
555 (MH+) single component 100% gradient 2 (tR 3.70 min),
gradient 3 (tR 17.11 min); HRMS calcd for C25H30F3N4O5S
(MH+) 555.1889, found 555.1902.
4 Hz, 2H, benzothiazolyl-4H and -7H); 7.44 (m, 1H, ben-
zothiazolyl-5H); 7.36-7.22 (m, 3H, benzothiazolyl-6H, aryl);
7.14 (d, J ) 8 Hz, 2H, aryl); 6.23 (s, 1H, NH); 4.70 (dd, J ) 8
Hz, 4 Hz, 1H, NCHCO); 4.62 (t, J ) 10 Hz, 1H, NCHHCH2);
4.13 (m, 1H, NCHCH2); 3.99-3.64 (m, 3H, NCHCHMe,
NCHHCH2CH2, NCHHCH2); 3.54 (m, 1H, NCHHCH2CH2);
3.45 (m, 1H, CHMe); 3.14 (m, 1H, NCH2CHH); 2.86 (m, 1H,
CHMe2); 2.48-1.92 (m, 5H, NCH2CHH, NCH2CH2CH2), 1.21
(d, J ) 7 Hz, 9H, 3 x Me); MS (thermospray) m/z 532 (MH+),
371 (M-[4-(isopropyl)phenyl isocyanate group]+); Circular dichro-
ism (CH3CN) λmax198.0 nm, dE -11.0, E42843, λmax212.4 nm,
dE 8.47, E34234, λmax228.0 nm, dE -4.01, E25880, λmax239.0
nm, dE 2.94, E25077, λmax248.2 nm, dE 0.47, E23038, λmax
-
257.2 nm, dE 4.78, E11031; LCMS m/z 532 (MH+) single
component 99% gradient 2 (tR 3.61 min), gradient 3 (tR 16.55
min); HRMS calcd for C29H34N5O3S (MH+) 532.2382, found
532.2393.
ter t-Bu t yl (2S)-2-{[(3a S,6S,6a R)-4-(6-F lu or o-1,3-b en -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H)-yl]ca r bon yl}p yr r olid in e-1-ca r boxyla te (69).
Prepared in a manner similar to 54 by reacting 12 with
2-bromo-6-fluoro-benzothiazole12 to give 69 (45% yield) as an
off-white foam: 1H NMR (CDCl3) δ 7.71 (dd, J ) 8.5 Hz, 4.5
Hz, 1H, benzothiazolyl-4H), 7.46 (dd, J ) 8 Hz, 2.5 Hz, 1H,
benzothiazolyl-7H), 7.16 (m, 1H, benzothiazolyl-5H), 4.52-4.36
(m, 2H, NCHCO, NCHHCH2), 4.14 (m, 1H, NCHCH2), 3.97-
3.70, 3.66-3.36 (2m, 5H, NCHCHMe, NCH2CH2CH2, CHMe,
NCHHCH2), 3.12 and 2.38 (2m, 2H, NCH2CH2), 2.34-2.04 and
1.98-1.80 (2m, 4H, NCH2CH2CH2, NCH2CH2CH2), 1.45 (d, J
) 6 Hz, (rotamers) 9H, But), 1.20 (m, 3H, CHMe); MS
The following compounds were similarly prepared.
(2S)-2-{[(3a S,6S,6a R)-6-Met h yl-5-oxo-4-(1,3-t h ia zol-2-
yl) h exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}-N-
(4-isop r op ylp h en yl) p yr r olid in e-1-ca r boxa m id e (55). Pre-
pared in a manner similar to 56 from 53 to give 55 (96% yield)
1
as a white solid: H NMR (CDCl3) δ 7.45 (d, 1H, J ) 3.5 Hz,
thiazolyl-4H), 7.25-7.22 (m, 2H, aryl -2H, -6H), 7.17-7.12 (m,
2H, aryl -3H, -5H), 7.01 (d, J ) 3.5 Hz, 1H, thiazolyl-5H), 6.19
(s, exch., 1H, CONH), 4.67 (dd, J ) 8.0 Hz, 4.5 Hz, 1H,
NCHCO), 4.58 (t, J ) 9.5 Hz, 1H, NCHHCH2), 4.09-4.00 (m,
1H, NCHCH2), 3.93-3.85 (m, 1H, NCHHCH2), 3.78-3.73 (m,
1H, NCHHCH2CH2), 3.72-3.65 (m, 1H, NCHHCH2CH2), 3.52
(dd, J ) 14.5 Hz, 7.5 Hz, 1H, NCHCHMe), 3.40 (quintet, J )
7.5 Hz, 1H, CHMe), 3.02-2.95 (m, 1H, NCH2CHH), 2.84
(septet, J ) 7.0 Hz, 1H, arylCHMe2), 2.41-2.17 (m, 3H,
NCH2CHH, NCH2CHHCH2, NCH2CH2CHH), 2.13-2.03 (m,
1H, NCH2CHHCH2), 2.03-1.94 (m, 1H, NCH2CH2CHH), 1.20
(d, J ) 7.0 Hz, 6H, arylCHMe2), 1.17 (d, J ) 7.5 Hz, 3H,
CHMe); MS (thermospray) m/z 482 (MH+). Anal. (C25H31N5O3S)
C, H, N.
(thermospray) m/z 489 (MH+), 389 (MH-Boc+). Anal. (C24H29
FN4O4S) C, H, N, S.
-
Eth yl 2-[(3S,3a R,6a S)-4-{[(2S)-1-(ter t-Bu toxyca r bon yl)-
p yr r olid in -2-yl]ca r bon yl}-3-m eth yl-2-oxoh exa h yd r op yr -
r olo[3,2-b]pyr r ol-1(2H)-yl]-1,3-ben zoth iazole-6-car boxylate
(70). Prepared in a manner similar to 54 by reacting 12 with
2-bromo-6-ethoxycarbonylbenzothiazole12 to give 70 (38% yield)
as a white solid: 1H NMR (CDCl3) δ 8.53 (d, J ) 1.8 Hz, 1H,
benzothiazolyl-7H); 8.12 (dd, J ) 8.6 Hz, 1.8 Hz, 1H, ben-
zothiazolyl-5H); 7.80 (d, J ) 8.6 Hz, 1H, benzothiazolyl-4H);
4.50-4.36 (m, 3H, NCHCO, CO2CH2Me); 4.30-3.07 (m, 8H,
NCH2CH2CH2, NCH2CH2, NCHCH2, NCHCHMe, CHMe,
NCH2CHH); 2.49-1.84 (m, 5H, NCH2CHH, NCH2CH2CH2);
1.50-1.38 and 1.27-1.17 (2m, 15H, But, CO2CH2Me, CHMe);
MS (thermospray) m/z 543 (MH+); LCMS m/z 543 (MH+) single
component 100% gradient 1 (tR 4.78 min), gradient 2 (tR 3.58
min), gradient 3 (tR 14.63 min); HRMS calcd for C27H35N4O6S
(MH+) 543.2277, found 543.2283.
ter t-Bu tyl (2S)-2-{[(3a S,6S,6a R)-6-Meth yl-5-oxo-4-[1,3]-
th ia zolo[5,4-b]p yr id in -2-ylh exa h yd r op yr r olo[3,2-b]p yr -
r ol-1(2H)-yl]car bon yl}pyr r olidin e-1-car boxylate (71). Pre-
pared in a manner similar to 54 by reacting 12 with 2-bromo-
7-aza-benzothiazole12 to give 71 (28% yield) as a white solid:
1H NMR (CDCl3) shows rotameric forms: δ 8.50-8.45 (m, 1H,
pyridyl-2H), 7.98 (dd, J ) 8.3 Hz, 1.5 Hz, 1H, pyridyl-4H), 7.36
(dd, J ) 8.3 Hz, 4.9 Hz, 1H, pyridyl-3H), 4.49-4.00 (2m, 3H,
NCHCO, NCHHCH2, NCHCH2), 4.99-3.70 (m, 2H, NCH-
HCH2, NCHCHMe), 3.61-3.05 (2m, 4H, NCH2CH2CH2, CHMe,
NCH2CHH), 2.47-1.80 (3m, 5H, NCH2CHH, NCH2CH2CH2),
1.48-1.40 (m, 9H, But) 1.26-1.16 (m, 3H, CHMe); MS (ther-
mospray) m/z 472 (MH+), 372 (MH-Boc+). Anal. (C23H29N5O4S)
C, H, N, S.
(2S)-2-{[(3aS,6S,6aR)-4-(1,3-Ben zoth iazol-2-yl)-6-m eth yl-
5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H )-yl]ca r b on -
yl}-N-(4-isopr opylph en yl)pyr r olidin e-1-car boxam ide (56).
To 54 (64 mg, 136 µmol) was added trifluoroacetic acid (315
µL, 4.09 mmol) at room temperature. After 10 min the solution
was azeotroped with toluene (1 mL × 2) to leave a yellow gum
(78 mg), which was dissolved in acetonitrile (2 mL) and
4-(isopropyl)phenyl isocyanate (30 µL, 188 µmol, 1.37 equiv)
added followed by triethylamine (47.5 µL, 341 µmol, 2.5 equiv).
The mixture was left to stand at room temperature for 4 h
before it was directly purified using preparative plate chro-
matography eluting with ethyl acetate to give 56 (62 mg, 85%)
as an off-white solid: 1H NMR (CDCl3) δ 7.80 (dd, J ) 8 Hz,
(2S)-2-{[(3a S,6S,6a R)-4-(4-Meth oxy-1,3-ben zoth ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isopr opylp h en yl)pyr r olidin e-1-ca r box-
a m id e (73). Prepared in a manner similar to 56 from 59 to
1
give 73 (90% yield) as a white solid: H NMR (CDCl3) δ 7.40
(d, J ) 7 Hz, 1H, benzothiazolyl-7H); 7.25 (m, 3H, benzothia-
zolyl-6H, aryl); 7.13 (d, J ) 8.5 Hz, 2H, aryl); 6.89 (d, J ) 8
Hz, 1H, benzothiazolyl-5H); 6.20 (s, 1H, NH); 4.68 (m, 1H,
NCHCO); 4.58 (m, 1H, NCHHCH2); 4.19 (m, 1H, NCHCH2);
4.02 (s, 3H, OMe), 3.98-3.62 (m, 3H, NCHCHMe, NCHHCH2-
CH2, NCHHCH2); 3.58-3.34 (m, 2H, NCHHCH2CH2, CHMe);
3.16 (m, 1H, NCH2CHH); 2.85 (m, 1H, CHMe2); 2.53-1.91 (m,
5H, NCH2CHH, NCH2CH2CH2), 1.20 (m, 9H, CHMe2, Me); MS
(thermospray) m/z 562 (MH+), 401 (M-[4-(isopropyl)phenyl
isocyanate group]+). Anal. (C30H35N5O4S‚0.05 EtOAc) C, H, N.
(2S)-2-{[(3a S,6S,6a R)-4-(5-Ch lor o-1,3-b en zot h ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isopr opylp h en yl)pyr r olidin e-1-ca r box-
a m id e (74). Prepared in a manner similar to 56 from 60 to
give 74 (74% yield) as a white solid: 1H NMR (CDCl3) δ 7.78
(d, J ) 2.5 Hz, 1H, benzothiazoyl-4H), 7.71 (d, J ) 8.5 Hz,
1H, benzothiazolyl-7H), 7.27 (m, 3H, benzothiazolyl-6H, aryl),
7.12 (d, J ) 8.5 Hz, 2H, aryl), 6.33 (s, 1H, NH), 4.67 (dd, J )
8 Hz, 4 Hz, 1H, NCHCO), 4.59 (t, J ) 10 Hz, 1H, NCHHCH2),
4.10 (m, 1H, NCHCH2), 3.91 (m, 1H, NCHHCH2), 3.74 (dd, J
) 11 Hz, 8 Hz, 1H, NCHCHMe), 3.67 (m, 1H, NCHHCH2CH2),
3.58-3.36 (m, 2H, CHMe, NCHHCH2CH2), 3.07 (m,1H, NCH2-
CHH), 2.84 (m, 1H, CHMe2), 2.44-1.70 (m, 5H, NCH2CHH,
NCH2CH2CH2), 1.19 (d, J ) 7 Hz, 9H, CHMe2, Me): MS
(thermospray) m/z 566 (MH+), 405 (M-[4-(isopropyl)phenyl
isocyanate group] +); LCMS m/z 566 (MH+) single component
98.0% gradient 1 (tR 4.92 min); HPLC 100% (tR 35.58 min).
Anal. (C29H32ClN5O3S‚0.15CHCl3) C, H, N.
(2S)-2-{[(3a S,6S,6a R)-4-(5-Meth oxy[1,3]th ia zolo[5,4-b]-
pyr id in -2-yl)-6-m eth yl-5-oxoh exa h ydr opyr r olo[3,2-b]p yr -
r ol-1(2H)-yl]car bon yl}-N-(4-isopr opylph en yl)pyr r olidin e-
1-ca r boxa m id e (75). Prepared in a manner similar to 56 from

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4447
61 to give 75 (95% yield) as a white solid: 1H NMR (CDCl3) δ
7.89 (d, J ) 8.5 Hz, 1H, pyridothiazolyl-4H), 7.28-7.21 and
7.17-7.10 (2m, 4H, arylH), 6.80 (d, J ) 8.5 Hz, 1H, pyridot-
hiazolyl-5H), 6.19 (s, 1H, CONH), 4.72-4.51 (m, 2H, NCHCO,
NCHHCH2CH2), 4.15-3.35 (m, 9H, NCHHCH2CH2, heteroary-
lOMe, NCH2CH2, NCHCH2, NCHCHMe, arylCHMe2), 3.14-
3.00 (m, 1H, CHMe), 2.91-2.78 (m, 1H, NCH2CHH), 2.45-
1.90 (m, 5H, NCH2CHH, NCH2CH2CH2), 1.25-1.15 (m, 9H,
arylCHMe2, CHMe); MS (thermospray) m/z 563 (MH+); LCMS
m/z 563 (MH+) single component 100% gradient 2 (tR 3.49 min),
gradient 3 (tR 16.30min); HRMS calcd for C29H35N6O4S (MH+)
563.2441, found 563.2425.
J ) 8.5 Hz, 2H, aryl), 6.95 (dd, J ) 9 Hz, 2.5 Hz, benzothia-
zolyl-6H), 6.27 (s, 1H, NH), 4.68 (dd, J ) 7.5 Hz, 4 Hz, 1H,
NCHCO), 4.60 (t, J ) 9.5 Hz, 1H, NCHHCH2), 4.09 (m, 1H,
NCHCH2), 3.92 (m, 1H, NCHHCH2), 3.88 (s, 3H, OCH3), 3.76
(dd, J ) 11 Hz, 7 Hz, 1H, NCHCHMe), 3.68 (m, 1H, NCHHCH2-
CH2), 3.52 (m, 1H, NCHHCH2CH2), 3.43 (m, 1H, CHMe), 3.11
(m, 1H, NCH2CHH), 2.84 (m, 1H, CHMe2), 2.46-1.92 (m, 5H,
NCH2CHH, NCH2CH2CH2), 1.20 (m, 9H, CHMe2, Me); MS
(thermospray) m/z 562 (MH+), 401 (M-[4-(isopropyl)phenyl
isocyanate group] +); LCMS m/z 562 (MH+) single component
99.7% gradient 2 (tR 3.52 min), gradient 3 (tR 16.47 min);
HRMS calcd for C30H36N5O4S (MH+) 562.2488, found 562.2162.
(2S)-2-{[(3a S,6S,6a R)-4-(4-Met h oxy-7-m et h yl-1,3-b en -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H )-yl]ca r b on yl}-N-(4-isop r op ylp h en yl)p yr r o-
lid in e-1-ca r boxa m id e (76). Prepared in a manner similar
to 56 from 62 to give 76 (91% yield) as a cream solid: 1H NMR
(CDCl3) δ 7.25 (d, J ) 9 Hz, 2H, aryl), 7.13 (d, J ) 8.5 Hz, 2H,
aryl), 7.04 (d, J ) 8 Hz, 1H, benzothiazolyl-5H), 6.82 (d, J )
8 Hz, 1H, benzothiazolyl-6H), 6.22 (s, 1H, NH), 4.68 (dd, J )
7 Hz, 4 Hz, 1H, NCHCO), 4.58 (t, J ) 9.5 Hz, 1H, NCHHCH2),
4.19 (m, 1H, NCHCH2), 4.00 (s, 3H, OCH3), 3.89 (m, 1H,
NCHHCH2), 3.78 (dd, J ) 11 Hz, 7 Hz, 1H, NCHCHMe), 3.68
(m, 1H, NCHHCH2CH2), 3.52 (m, 1H, NCHHCH2CH2), 3.44
(m, 1H, CHMe,), 3.15 (m,1H, NCH2CHH), 2.84 (m, 1H,
CHMe2), 2.47 (s, 3H, arylCH3), 2.46-1.91 (m, 5H, NCH2CHH,
NCH2CH2CH2), 1.20 (m, 9H, CHMe2, Me); MS (thermospray)
m/z 576 (MH+), 415 (M-[4-(isopropyl)phenyl isocyanate
group] +); LCMS m/z 576 (MH+) single component 99.7%
gradient 2 (tR 3.55 min), gradient 3 (tR 16.59 min); HRMS calcd
for C31H38N5O4S (MH+) 576.2645, found 576.2626.
(2S)-2-{[(3a S,6S,6a R)-4-{5-[(Diflu or om eth yl)su lfon yl]-
1,3-ben zoth ia zol-2-yl}-6-m eth yl-5-oxoh exa h yd r op yr r olo-
[3,2-b]p yr r ol-1(2H)-yl]ca r bon yl}-N-(4-isop r op ylp h en yl)-
p yr r olid in e-1-ca r boxa m id e (80). Prepared in a manner
similar to 56 from 66 to give 80 (55% yield) as a cream solid:
1H NMR (CDCl3) δ 8.40 (d, J ) 1 Hz, 1H, benzothiazolyl-4H),
8.06 (d, J ) 8 Hz, 1H, benzothiazolyl-7H), 7.86 (dd, J ) 8 Hz,
1.5 Hz, 1H, benzothiazoyl-6H), 7.24 (d, J ) 9 Hz, 2H, aryl),
7.13 (d, J ) 8.5 Hz, 2H, aryl), 6.27 (s, 1H, NH), 6.23 (t, J ) 53
Hz, 1H, CHF2), 4.66 (m, 2H, NCHCO, NCHHCH2), 4.12 (m,
1H, NCHCH2), 3.95 (m, 1H, NCHHCH2), 3.80 (dd, J ) 11 Hz,
7 Hz, 1H, NCHCHMe), 3.68 (m, 1H, NCHHCH2CH2), 3.50 (m,
2H, NCHHCH2CH2, CHMe), 3.01 (m, 1H, NCH2CHH), 2.84
(m, 1H, CHMe2), 2.49-1.92 (m, 5H, NCH2CHH, NCH2CH2CH2),
1.20 (m, 9H, CHMe2, Me); MS (thermospray) m/z 646 (MH+),
485 (M-[4-(isopropyl)phenyl isocyanate group] +); LCMS m/z
646 (MH+) single component 99.5% gradient 2 (tR 3.53 min),
gradient 3 (tR 16.32 min); HRMS calcd for C30H34F2N5O5S2
(MH+) 646.1969, found 646.1976.
(2S)-2-{[(3a S,6S,6a R)-4-(6-Meth oxy-1,3-ben zoth ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isop r op ylp h en yl)p yr r olidin e-1-ca r box-
a m id e (77). Prepared in a manner similar to 56 from 63 to
give 77 (85% yield) as a white solid: 1H NMR (CDCl3) δ 7.67
(d, J ) 8.5 Hz, 1H, benzothiazolyl-4H); 7.25 (m, 3H, benzothia-
zole-7H, aryl); 7.13 (d, J ) 8.5 Hz, 2H, aryl); 7.02 (dd, J ) 9
Hz, 3 Hz, 1H, benzothiazolyl-5H); 6.21 (s, 1H, NH); 4.68 (m,
1H, NCHCO); 4.59 (t, J ) 9.5 Hz, 1H, NCHHCH2); 4.10 (m,
1H, NCHCH2); 3.87 (s, 3H, OMe), 3.88-3.64 (m, 3H,
NCHCHMe, NCHHCH2CH2, NCHHCH2); 3.58-3.36 (m, 2H,
NCHHCH2CH2, CHMe); 3.09 (m, 1H, NCH2CHH); 2.84 (m, 1H,
CHMe2); 2.45-1.83 (m, 5H, NCH2CHH, NCH2CH2CH2), 1.20
(m, 9H, CHMe2, Me); MS (thermospray) m/z 562 (MH+), 401
(M-[4-(isopropyl)phenyl isocyanate group] +); LCMS m/z 562
(MH+) single component 99.5% gradient 2 (tR 3.52 min),
gradient 3 (tR 16.52 min); HRMS calcd for C30H36N5O4S (MH+)
562.2488, found 562.2477.
(2S)-2-{[(3a S,6S,6a R)-6-Meth yl-4-(6-n itr o-1,3-ben zoth i-
a zol-2-yl)-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-yl]-
ca r bon yl}-N-(4-isop r op ylp h en yl)p yr r olid in e-1-ca r boxa -
m id e (81). Prepared in a manner similar to 56 from 67 to
give 81 (76% yield) as a yellow solid: 1H NMR (CDCl3) δ 8.74
(d, J ) 2.5 Hz, 1H, benzothiazolyl-7H), 8.31 (dd, J ) 9 Hz, 2
Hz, 1H, benzothiazolyl-5H), 7.83 (d, J ) 9 Hz, 1H, benzothia-
zolyl-4H), 7.24 (d, J ) 8.5 Hz, 2H, aryl), 7.13 (d, J ) 8.5 Hz,
2H, aryl), 6.28 (s, 1H, NH), 4.65 (m, 2H, NCHCO, NCHHCH2),
4.13 (m, 1H, NCHCH2), 3.94 (m, 1H, NCHHCH2), 3.79 (dd, J
) 11 Hz, 7 Hz, 1H, NCHCHMe), 3.69 (m, 1H, NCHHCH2CH2),
3.59-3.41 (m, 2H, CHMe, NCHHCH2CH2), 3.11 (m, 1H, NCH2-
CHH), 2.84 (m, 1H, CHMe2), 2.49-1.92 (m, 5H, NCH2CHH,
NCH2CH2CH2), 1.20 (m, 9H, CHMe2, Me); MS (thermospray)
m/z 577 (MH+), 416 (M-[4-(isopropyl)phenyl isocyanate
group] +); LCMS m/z 577 (MH+) single component 99.8%
gradient 2 (tR 3.60 min), gradient 3 (tR 16.74 min); HRMS calcd
for C29H33N6O5S (MH+) 577.2233, found 577.2222.
(2S)-2-{[(3a S,6S,6a R)-4-(4-Ch lor o-1,3-b en zot h ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isop r op ylp h en yl)p yr r olidin e-1-ca r box-
a m id e (78). Prepared in a manner similar to 56 from 64 to
give 78 (93% yield) as a pale yellow solid: 1H NMR (CDCl3) δ
7.70 and 7.45 (2dd, J ) 1.2 Hz, 8 Hz, 2H, benzothiazolyl-5H
and -7H), 7.25 (m, 3H, benzothiazolyl-6H, aryl), 7.13 (d, J )
8.5 Hz, 2H, aryl), 6.21 (s, 1H, NH), 4.69 (dd, J ) 7 Hz, 4 Hz,
1H, NCHCO), 4.59 (t, J ) 9.5 Hz, 1H, NCHHCH2), 4.16 (m,
1H, NCHCH2), 3.94 (m, 1H, NCHHCH2), 3.80 (dd, J ) 11 Hz,
7 Hz, 1H, NCHCHMe), 3.69 (m, 1H, NCHHCH2CH2), 3.59-
3.39 (m, 2H, CHMe, NCHHCH2CH2), 3.19 (m, 1H, NCH2CHH),
2.85(m,1H,CHMe2),2.51-1.86(m,5H,NCH2CHH,NCH2CH2CH2),
1.22 (m, 9H, CHMe2, Me); MS (thermospray) m/z 566 (MH+),
405 (M-[4-(isopropyl)phenyl isocyanate group]+); LCMS m/z
566 (MH+) single component 99.8% gradient 2 (tR 3.69 min),
gradient 3 (tR 17.36 min); HRMS calcd for C29H33ClN5O3S
(MH+) 566.1993, found 566.1979.
(2S )-2-{[(3a S ,6S ,6a R )-6-Me t h y l-5-o xo -4-[6-(t r iflu o -
r om eth oxy)-1,3-ben zoth ia zol-2-yl]h exa h yd r op yr r olo[3,2-
b]p yr r ol-1(2H )-yl]ca r b on yl}-N-(4-isop r op ylp h en yl)p yr -
r olid in e-1-ca r boxa m id e (82). Prepared in a manner similar
to 56 from 68 to give 82 (81% yield) as a white solid: 1H NMR
(CDCl3) δ 7.78 (d, J ) 8.5 Hz, 1H, benzothiazolyl-4H), 7.67
(br, 1H, benzothiazolyl-7H), 7.27 (m, 3H, benzothiazolyl-5H,
aryl), 7.13 (d, J ) 8.5 Hz, 2H, aryl), 6.20 (s, 1H, NH), 4.74-
4.58 (m, 2H, NCHCO, NCHHCH2), 4.12 (m, 1H, NCHHCH2),
3.93, 3.78 and 3.69 (3m, 3H, NCH2CH2CH2, NCHCH2), 3.53
(m, 1H, NCHCHMe), 3.45 (m, 1H, CHMe), 3.10 (m, 1H, NCH2-
CHH), 2.85 (m, 1H, CHMe2), 2.47-1.91 (m, 5H, NCH2CHH,
NCH2CH2CH2), 1.22 (m, 9H, CHMe2, Me); MS (thermospray)
m/z 616 (MH+), 455 (M-[4-(isopropyl)phenyl isocyanate
group] +); LCMS m/z 616 (MH+) single component 99.7%
gradient 2 (tR 3.74 min), gradient 3 (tR 17.74 min); HRMS calcd
for C30H33F3N5O4S2 (MH+) 616.2205, found 616.2194.
(2S)-2-{[(3a S,6S,6a R)-4-(6-F lu or o-1,3-b en zot h ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isopr opylp h en yl)pyr r olidin e-1-ca r box-
a m id e (83). Prepared in a manner similar to 56 from 69 to
give 83 (89% yield) as a white solid: 1H NMR (CDCl3) δ 7.72
(dd, J ) 9 Hz, 4.5 Hz, 1H, benzothiazolyl-4H), 7.49 (dd, J )
8.5 Hz, 2.5 Hz, 1H, benzothiazolyl-7H), 7.24 (d, J ) 9.5 Hz,
2H, aryl), 7.15 (m, 3H, aryl, benzothiazolyl-5H), 6.23 (s, 1H,
(2S)-2-{[(3a S,6S,6a R)-4-(5-Meth oxy-1,3-ben zoth ia zol-2-
yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isop r op ylp h en yl)p yr r olidin e-1-ca r box-
a m id e (79). Prepared in a manner similar to 56 from 65 to
give 79 (65% yield) as a cream solid: 1H NMR (CDCl3) δ 7.66
(d, J ) 8.5 Hz, 1H, benzothiazolyl-7H), 7.31 (d, J ) 2.5 Hz,
1H, benzothiazolyl-4H), 7.25 (d, J ) 8.5 Hz, 2H, aryl), 7.13 (d,

4448 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Borthwick et al.
NH), 4.68 (dd, J ) 8 Hz, 4 Hz, 1H, NCHCO), 4.61 (t, J ) 10
Hz, 1H, NCHHCH2), 4.10 (m, 1H, NCHCH2), 3.91 (m, 1H,
NCHHCH2), 3.77(dd, J ) 11 Hz, 7 Hz, 1H, NCHCHMe), 3.69
(m, 1H, NCHHCH2CH2), 3.52 (m, 1H, NCHHCH2CH2), 3.43
(m, 1H, CHMe), 3.09 (m, 1H, NCH2CHH), 2.84 (m, 1H,
CHMe2), 2.47-1.92 (m, 5H, NCH2CHH, NCH2CH2CH2), 1.20
(m, 9H, CHMe2, Me); MS (thermospray) m/z 550 (MH+), 389
(M-[4-(isopropyl)phenyl isocyanate group] +); LCMS m/z 550
(MH+) single component 99.4% gradient 2 (tR 3.54 min),
gradient 3 (tR 16.71 min); HRMS calcd for C29H33FN5O3S
(MH+) 550.2288, found 550.2278.
(MH+) single component 98.8% gradient 1 (tR 3.16 min); HPLC
94% (tR 27.4 min). Anal. (C30H35N5O4S) C, H, N, S.
An tivir a l Assa y. (Enzyme Linked ImmunoSorbant Assay
(ELISA) for HCMV protease inhibitors). Trypsin stripped
MRC5 cells were suspended to a concentration of 105 cells per
ml in assay medium (Gibco DMEM supplemented with 5%
fetal calf serum and antibiotics). This cell suspension was
dispensed into 96-well microplates at 100 µL per well. Three
columns of wells were allocated for each compound to be tested.
Two of these columns received 50 µL per well of human
cytomegalovirus (HCMV strain AD169), diluted to give an
infectivity ratio of 0.01 plaque-forming unit per cell, and the
third column received 50 µL of medium. Then the plates were
incubated at 37°C in a 5% CO2 atmosphere for 48 h. Test
compounds were formulated to a concentration of 40mM in
DMSO to provide a stock solution. Twofold dilution series in
medium were prepared from this stock at 4 times the required
final concentrations. The compound dilutions were added to
the assay plates at 50 µL per well using three wells (two
infected and one uninfected) per dilution. Virus and cell
controls received 50 µL of medium. The plates were then re-
incubated for a further 5 days.
(2S)-2-{[(3a S,6S,6a R)-4-(6-E t h ylca r b oxyla t e-1,3-b en -
zoth ia zol-2-yl)-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H )-yl]ca r b on yl}-N-(4-isop r op ylp h en yl)p yr r o-
lid in e-1-ca r boxa m id e (84). Prepared in a manner similar
to 56 from 70 to give 84 (62% yield) as a cream solid: 1H NMR
(CDCl3) δ 8.53 (d, J ) 1.8 Hz, 1H, benzothiazolyl-7H); 8.12
(dd, J ) 1.8 Hz, J ) 8.6 Hz, 1H, benzothiazolyl-5H); 7.81 (d,
J ) 8.6 Hz, 1H, benzothiazolyl-4H); 7.24-7.13 (ABq, J ) 8.5
Hz, 4H, arylH); 6.20 (s, 1H, CONH); 4.72-4.57 (m, 2H,
NCHCO, NCHCH2); 4.41 (q, J ) 7.1 Hz, 2H, CO2CH2Me);
4.20-4.05 (m, 1H, NCHCH2); 4.00-3.39 (m, 5H, NCHHCH2-
CH2, NCHHCH2, NCHCHMe and CHMe); 3.19-3.06 (m, 1H,
NCH2CHH); 2.91-2.78 (m, 1H, CHMe2); 2.45-1.94 (m, 5H,
NCH2CHH, NCH2CH2CH2); 1.43 (t, J ) 7.0 Hz, 3H, CO2-
CH2Me); 1.21-1.17 (m, 9H, CHMe, CHMe2); LCMS m/z 604.5
(MH+) single component 98.% gradient 1 (tR 4.96 min). Anal.
(C32H37N5O5S) C, H, N, S.
Growth medium was tipped from the plates and the cell
sheets were washed once by gentle immersion in phosphate
buffered saline (PBS). The wash was removed and the cells
fixed by the addition of 100 µL per well of 1:1 mix of acetone
and methanol for 3 min. Following a further wash in PBS,
the plates were blocked with 100 µL per well ELISA diluent
(PBS + 0.05% v/v Tween 20 + 2% w/v skimmed milk powder)
at 37 °C for 30 min. The plates were then washed once with
PBS + 0.05% Tween 20, and 50 µL of murine monoclonal
antibody (HCMV MAb 34 binding to the viral gB protein,
Biogenesis Ltd.), diluted 1:760, was added to each well. After
incubation at 37°C for 2 h, the plates were washed three times
in PBS/Tween, blotted dry and 50 µL of rabbit, anti-mouse
IgG antibody conjugated to horseradish peroxidase (DAKO),
preadsorbed with uninfected MRC5 cells and diluted 1:1500,
was added to each well. The plates were incubated for another
hour and then washed thoroughly five times and dried.
Substrate solution, orthophenylene diamine (OPD)/peroxide
in urea buffer (Sigmafast kit), was added at 50 µL per well,
and color allowed to develop at room temperature. The reaction
was stopped by the addition of 25 µL of 12.5% sulfuric acid to
each well, and the plates were read spectrophotometrically at
a wavelength of 490 nm.
(2S)-2-{[(3a S,6S,6a R)-6-Met h yl-5-oxo-4-[1,3]t h ia zolo-
[5,4-b]p yr id in -2-ylh exa h yd r op yr r olo[3,2-b]p yr r ol-1(2H)-
yl]ca r bon yl}-N-(4-isop r op ylp h en yl)p yr r olid in e-1-ca r box-
a m id e (85). Prepared in a manner similar to 56 from 71 to
give 85 (75% yield) as a white solid: 1H NMR (400 MHz,
CDCl3): δ 8.47 (dd, J ) 4.8 Hz, 1.5 Hz, 1H, pyridyl-2H), 8.01
(dd, J ) 8.3 Hz, 1.5 Hz, 1H, pyridyl-4H), 7.37 (dd, J ) 8.3 Hz,
4.8 Hz, 1H, pyridyl-3H), 7.26-7.22 (m, 2H, aryl-2H, 6H), 7.16-
7.12 (m, 2H, aryl-3H, 5H), 6.19 (s, exch., 1H, CONH), 4.67 (dd,
J
) 8 Hz, 4 Hz, 1H, NCHCO), 4.63 (t, J ) 9.5 Hz, 1H,
NCHHCH2), 4.15-4.07 (m, 1H, NCHCH2), 3.97-3.89 (m, 1H,
NCHHCH2), 3.83-3.77 (m, 1H, NCHCHMe), 3.72-3.66
(m, 1H, NCHHCH2CH2), 3.56-3.50 (m, 1H, NCHHCH2CH2),
3.46 (quintet, J ) 7.3 Hz, 1H, CHMe), 3.14-3.07 (m, 1H,
NCH2CHH), 2.85 (septet, J ) 7 Hz, 1H, arylCHMe2), 2.45-
2.32 (m, 2H, NCH2CHH, NCH2CHHCH2), 2.29-2.19 (m, 1H,
NCH2CH2CHH), 2.15-2.05 (m, 1H, NCH2CHHCH2), 2.03-
1.95 (m, 1H, NCH2CH2CHH), 1.21 (d, J ) 7 Hz, 3H, CHMe),
1.20 (d, J ) 7 Hz, 6H, arylCHMe2); LCMS m/z 533 (MH+)
single component 99.7% gradient 2 (tR 3.23 min), gradient 3
(tR 15.03 min); HRMS calcd for C28H33N6O3S (MH+) 533.2335,
found 533.2325.
The mean color development of duplicate infected wells at
each compound concentration was calculated as a percentage
of the mean adsorption of untreated, infected controls after
both values had been adjusted for nonspecific background.
These percentage inhibition values were plotted against
compound concentration and the 50% inhibitory concentration
(IC50) derived by regression analysis.
(2S)-2-{[(3a S,6S,6a R)-4-[6-(H yd r oxym et h yl)-1,3-b en -
zoth ia zol-2-yl]-6-m eth yl-5-oxoh exa h yd r op yr r olo[3,2-b]-
p yr r ol-1(2H )-yl]ca r b on yl}-N-(4-isop r op ylp h en yl)p yr r o-
lid in e-1-ca r boxa m id e (72). To a solution of 58 (0.125 g, 0.169
mmol) in dry 1,4-dioxane (1 mL) was added a 4.0M solution
of HCl in 1,4-dioxane (0.5 mL). The reaction mixture was
allowed to stir at room temperature under an atmosphere of
nitrogen for 68 h. The reaction mixture was then evaporated
to dryness and azeotroped with toluene (×3) to give a cream
residue, which was used in the next step without further
purification. The crude mixture was treated in a manner
similar to that used to prepare 56 to give 72 (25% yield) as an
off-white solid: IR (KBr) νmax 1729.8, 1684.8, 1669.6, 1654.3,
On completion of the ELISA stage, plates were washed with
water and stained with 20% v/v carbol fuchsin for 30 min, then
washed again and dried. The uninfected columns of cells for
each compound were examined microscopically. In-assay cy-
totoxicity was recorded as the lowest concentration of com-
pound that produced any visible effect on the cell monolayers.
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1518.3 cm-1 1H NMR (CDCl3) δ 7.82 (d, J ) 1.8 Hz, 1H,
;
benzothiazolyl-7H);); 7.6 (d, J ) 8.6 Hz, 1H, benzothiazolyl-
4H); 7.43 (dd, J ) 1.8 Hz, J ) 8.6 Hz, 1H, benzothiazolyl-5H);
7.26-7.14 (ABq, J ) 8.8 Hz, 4H, arylH); 6.23 (s, 1H, NH), 4.79
(s, 2H, CH2OH), 4.68 (dd, J ) 3.8 Hz, J ) 7.5 Hz, 1H, NCHCO);
4.59 (t, J ) 9.4 Hz, 1H, NCHHCH2); 4.12-4.00 (m, 1H,
NCHCH2); 3.96-3.82 (m, 1H, NCHHCH2); 3.75-3.63 (m, 1H,
NCHCHMe); 3.59-3.30 (m, 3H, NCH2CH2CH2, CHMe); 3.13-
3.01 (m, 1H, NCH2CHH); 2.91-2.78 (m, 1H,CHMe2); 2.43-
1.90 (m, 5H, NCH2CH2CH2, NCH2CHH); 1.20 (d, J ) 7 Hz,
6H, CHMe2); 1.16 (d, J ) 7.5 Hz, 3H, CHMe); LCMS m/z 562

Inhibitors of Human Cytomegalovirus Protease
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