Design and synthesis of spiro-cyclopentenyl and spiro-[1,3]-dithiolanyl substituted pyrrolidine-5,5-trans-lactams as inhibitors of hepatitis C virus NS3/4A protease

Article abstract of DOI:10.1016/S0960-894X(03)00274-9

Using the pyrrolidine-5,5-trans-lactam template, we have designed small, neutral, mechanism-based inhibitors of hepatitis C NS3/4A protease. Compound 2b, with a spiro-cyclobutyl P1 substituent and an isopropyl carbonyl substituent at the lactam nitrogen, has an IC₅₀ value in the replicon cell-based assay of 3 μM.

Full text of DOI:10.1016/S0960-894X(03)00274-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1657–1660
Design and Synthesis of Spiro-cyclopentenyl and Spiro-[1,3]-
dithiolanyl Substituted Pyrrolidine-5,5-trans-lactams as
Inhibitors of Hepatitis CVirus NS3/4A Protease
David M. Andrews,* Paul S. Jones, Gail Mills, S. Lucy Hind,
Martin J. Slater, Naimisha Trivedi and Katrina J. Wareing
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
Received 23 January 2003; revised 11 March 2003; accepted 11 March 2003
Abstract—Using the pyrrolidine-5,5-trans-lactam template, we have designed small, neutral, mechanism-based inhibitors of hepa-
titis C NS3/4A protease. Compound 2b, with a spiro-cyclobutyl P1 substituent and an isopropyl carbonyl substituent at the lactam
nitrogen, has an IC50 value in the replicon cell-based assay of 3 mM.
# 2003 Elsevier Science Ltd. All rights reserved.
Hepatitis C virus (HCV) infects chronically an esti-
mated 3% of the global human population,1 often
leading to cirrhosis, hepatocellular carcinoma and liver
failure in later life.2 Current therapies are based upon
interferon-a alone or in combination with ribavirin.
Although sustained response rates are markedly improved
using combination therapies, at least 50% of patients fail
to show a sustained response. Additionally, current
therapies have the disadvantage of frequent and severe
side effects.3 The development of new therapies to treat
HCV infection effectively is thus of paramount impor-
tance, and is currently an intensive area of research.4
hepatitis C virus NS3/4A protease enzyme based on the
a-ethyl,6 dimethyl and spirocycloalkyl7 pyrrolidine-5,5-
trans-lactam template (Fig. 1). The inhibitor 2a and its
close analogues are novel, potent in the replicon cell-
based surrogate assay and demonstrate moderate stabil-
ity in human plasma.
These compounds bear reactive functionality so they are
potentially vulnerable to metabolism. Although early
studies showed human plasma stability to be acceptable,
more recently we have shown that following intra-
venous administration to dogs, the compounds are
rapidly cleared in vivo.8 Thus, following a strategy
developed during the application of this template to
inhibition of HCMV protease,9 we attempted to find a
compound that would react with the viral enzyme after
binding to the active site, but would be sufficiently stable
to hydrolysis by plasma enzymes. Compounds 2a–e were
synthesized from the core template 3, with the aim of
either sterically hindering the approach of the hydrolytic
plasma enzymes to the lactam carbonyl, or reducing the
reactivity of the lactam carbonyl by making the sub-
stituent on the lactam nitrogen less electron withdrawing.
HCV is a small, enveloped virus, the genome of which is
a 9.5kb single-stranded RNA that encodes for a single
polyprotein of 3010–3030 amino acids. Mature non-
structural replicative proteins are released from this
polyprotein by the action of the viral proteases NS2 and
NS3. It has been established that introducing mutations
into the NS3 protease region of the HCV genome abol-
ishes infectivity,5 demonstrating that NS3 protease is
thus an essential viral function and should prove to be
an excellent target for the development of novel anti-
HCV agents.
The lactam 37 was deprotonated using lithium hexam-
ethyldisilazide and acylated at 0 ꢀC with a range of acyl- or
carbamoyl chlorides, or with methyl chlorothiol formate
in moderate to good yields. Acidolysis was followed by
diethyl ether trituration in a rapid synthesis fashion. The
poor yield of 5c was probably a consequence of higher
We recently reported the design and synthesis of a novel
class of mechanism-based inhibitors (1 and 2a) of the
*Corresponding author. Tel.: +44-1438-763644; fax: +44-1438-
763620; e-mail: da9978@gsk.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00274-9

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D.M.Andrews et al./ Bioorg.Med.Chem.Lett.13 (2003) 1657–1660
the preference for the methanethiol side chain of
cysteine at P1 could be driven by interaction of the SH
proton with the electron-rich p-cloud of Phe154.10 Since
we wished to retain the stability and synthetic accessi-
bility properties conferred by the spirocyclobutyl sub-
stituent, we chose to explore spirocyclopentenyl and
dithiolanyl substituents. In the case of the spiro-
cyclopentenyl, it was postulated that the olefin group
would be capable of p-p stacking with Phe154.11 In the
case of the diathiolanyl group, we postulated that it
could be possible for the sulfur lone-pair electrons to
interact with a ring proton of Phe154, the latter having
a partial positive character.12
Figure 1. Spirocyclobutyl pyrrolidine-5,5-trans-lactam template:
potential interactions with protease subsites.
Cyclopent-3-enylidene-ethoxymethoxy)-trimethylsilane
(7) was synthesized as previously described13 and sub-
ject to Lewis acid-mediated acyliminium coupling and
hydrolysis to form 8 (Scheme 2). Cyclization proceeded
under standard conditions to furnish 9 in excellent yield.
Previous work in our group had indicated that removal
of the Cbz protecting group under mild hydrogenolytic
conditions is difficult (e.g., over Lindlaar catalyst), so
the Cbz protecting group was removed using 4 equiva-
lents of TMS iodide in 60% yield and replaced by the
Boc protecting group to form 10. 10 was converted to
11a by methods analogous to those in Scheme 1.
than expected amine trifluoroacetate solubility in ether.
In similar fashion, 5d was obtained less pure than gen-
erally observed, due to carryover of an unidentified
impurity—this impacted on the isolated yield of 2d at
the next stage. HATU-mediated coupling of Boc-Val,
followed by column chromatography or Bond-Elut
solid phase cartridge extraction furnished 2a–e in gen-
erally good yields overall.
The compounds demonstrated a range of biochemical
potency against a fluorogenic assay system using full-
length NS3/4A protein (Table 1). The two most potent
compounds were 2a–b, both of which bear an sp3 center
adjacent to the carbonyl moiety. 2c (which has an sp2
center) is markedly less potent even than the close ana-
logue 2b. The ethyl groups of the urea 2d are almost
certainly too large to be accommodated in the S10
pocket, hence the compounds are poorly active. The
thiocarbamate 2e demonstrated interesting potency,
however, this group does not permit the application of
more small, branched groups, so we took the view that
it would be difficult to improve upon the modest
potency and stability of 2e, and the series was not pur-
sued. The most potent compounds (i.e., kobs/
([1,3]Dithiolan-2-ylidene-ethoxymethoxy)-trimethylsi-
lane (12)14 was subjected to the identical reaction
sequence, each step proceeding broadly similarly with
respect to reaction yield and compound quality. A
noteworthy exception was that the standard base
hydrolysis conditions used to remove the trifluoroacetyl
protecting group (to produce 13) unexpectedly caused
significant concomitant loss of the ethyl ester protecting
group—hence the low yield of 13. All other reactions
proceeded very similarly to the cyclopentenyl series.
The compounds were assayed biochemically and in the
cell-based replicon assay as shown in Table 2. The spiro-
cyclopentenyl compound 11a was at least an order of
I>100 MÀ1 À1) were tested in the replicon cell-based
s
assay. There was generally a good correlation between
biochemical assay potency and replicon potency,
although the isopropyl carbonyl analogue 2b was
slightly more potent in the cellular assay than would
have been predicted. (2a cf 2b and 2b cf 2e- all data is
for head-to-head IC50 determinations on compounds in
the same assay).
We next chose to re-examine the requirement for the
spirocyclobutyl P1 substituent. It has been noted that
Table 1. HCV NS3/4A isolated protease potency and replicon cel-
lular assay potency for compounds 2a–e
Compd
P10
HCV protease
kobs/I (MÀ1 sÀ1
ELISA replicon
IC50 (mM)
)
2a
2b
2c
2d
2e
CO–cyclopropyl
CO–CH(CH3)2
CO–C¼CH2(CH3)
CO–N(C2H5)2
CO–SMe
400
166
24
3.3
154
4.0
3.1
NT
NT
7.9
Scheme 1. Elaboration of core template. Reagents and conditions: (a)
LiHMDS; acyl chloride (4a–c), Et2NCOCl (4d) or MeSCOCl; THF,
0ꢀC; (b) TFA/DCM 1:1, diethyl ether trituration; (c) HATU, DIPEA,
RCO2H, MeCN or DMF.
NT, not tested.

D.M.Andrews et al./ Bioorg.Med.Chem.Lett.13 (2003) 1657–1660
1659
ꢀ
.
Scheme 2. Synthesis of cyclopentenyl and dithiolanyl pyrrolidine-5,5-trans-lactams. Reagents and conditions: (a) BF3 OEt2, DCM, À5 C;
(b) K2CO3, EtOH, 70 ꢀC; (c) tBuMgCl, THF, 5 ꢀC; (d) TMS-I (4 equiv), MeCN, 0 ꢀC; (e) Boc2O (1.3 equiv), THF; (f) LiHMDS; acyl chloride, THF,
0 ꢀC; (g) 4 M HCl in dioxane; (h) HATU, DIPEA, RCO2H, MeCN.
Table 2. HCV NS3/4A isolated protease potency and replicon cellular assay potency for compounds 11a–c (data for 2a and 2b reproduced for
comparison)
Compd
P1
P10
Calculated
logP15
HCV protease
kobs/I (MÀ1 sÀ1 16
ELISA replicon
IC50 (mM)17
Medium stability
(% turnover at 4 h)18
)
11a
16a
16b
2a
Cyclopentenyl
Dithiolanyl
Dithiolanyl
Cyclobutyl
Cyclobutyl
CO-cyclopropyl
CO-cyclopropyl
CO-isopropyl
CO-cyclopropyl
CO-isopropyl
2.9
3.2
3.7
2.4
3.0
19
621
362
400
166
NT
>100
17.0
4.0
NT
>50
>50
27
2b
3.1
5
NT, not tested.
magnitude less potent than any of the other compounds
examined and was not progressed to the cell-based assay.
Interestingly, although 2b was the least biochemically
potent of the remaining compounds, it displayed the
highest cellular potency. Conversely, the most potent
compound in the NS3/4A assay (16a) was essentially
inactive in the replicon assay. From the data presented,
it appears that the excellent biochemical potency con-
ferred by the cyclopropyl carbonyl lactam substituent
(2a cf 16a) translates less well to replicon potency than
is the case for the isopropyl carbonyl substituent (2b cf
16b). Clearly, in addition to biochemical potency, other
factors contribute to cellular potency. All the com-
pounds examined in the replicon assay were calculated
to be of similar lipophilicity (clogP 2.4–3.7) and are
broadly within the same molecular weight range (433–
485), leading us to suggest that cellular permeability was
unlikely to be the most important factor. In the absence
of any other factor to account for the trends seen in the
data, we speculated that, although the dithiolanyl moi-
ety confers excellent biochemical activity, those com-
pounds bearing this substituent are too inherently
unstable in cellular systems to be of utility.
This hypothesis is confirmed by inspection of the in
vitro stability behavior of the four compounds, also
shown in Table 2. Although 2b is of modest biochemical
potency, it is clearly the most stable compound of the
four examined- thus demonstrating the greatest cellular
activity. Evidently, in this series, a prerequisite for cel-
lular activity is that compounds possess both stability
and biochemical potency. It should be borne in mind
that there is a considerable difference in swept volume
for the isopropyl moiety compared to the cyclopropyl
moiety. The interatomic distance for the beta carbons is
calculated to be almost double for the isopropyl group
À2.54 A compared to 1.53 A for the cyclopropyl group.
The results presented are consistent with a hypothesis
that the smaller substituent is more readily accom-
modated in the S10 subsite of the NS3/4A protease, but
that the larger substituent reduces the rate of non-spe-
cific hydrolysis of the lactam ring by steric hindrance.

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D.M.Andrews et al./ Bioorg.Med.Chem.Lett.13 (2003) 1657–1660
Additionally, the larger isopropyl is less electronegative
than cyclopropyl, providing an electrostatic contribu-
tion to stability.19 The application of these observations
will be the subject of future communications.
8. Andrews, D. M.; Chaignot, H. M.; Coomber, B. A.;
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Acknowledgements
We thank Drs. Graham Baker, Sue Bethell and Mal-
colm Ellis for provision of NS3 protease protein and
initial assay systems; Derek Evans and Tracy Redfern
for provision of intermediates; Norman M. Gray and
Seb J. Carey for biochemical test data; Dr. Nigel Parry
and Liz Amphlett for replicon test results; Anne
Cheasty, Rebecca Fenwick and Neil Roughley for
medium stability and dog clearance data; and Richard
Upton for assistance with NMR interpretation.20
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17. Replicon assay details are described in ref 7(b) and in
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18. Each compound was incubated in Dulbecco’s Minimal
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a temperature of 37 ꢀC; an aliquot was withdrawn and depro-
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LC–MS on an API-300 using APCI source and single-ion
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1
20. NMR assignments: 2b: H NMR (CDCl3) d 5.20 (d, J=9
Hz, 1H, NH), 4.29 (dd, J=6.5, 9 Hz, 1H, NCH2CH2), 4.19 (t,
J=9 Hz, 1H, NHCH), 3.81 (td, J=6.5, 10 Hz, 1H,
NCH2CH2), 3.61 (m, 1H, COCH(CH3)2), 3.39 (td, J=5, 11
Hz, 1H, CH2CHN), 3.27 (d, J=11 Hz, 1H, NCHCH), 2.94
(m, 1H, CH2(CH2)2), 2.77 (m, 1H, NCH2CH2CH), 2.56 (m,
1H, CH2(CH2)2), 2.12–1.94 (m, 6H, NCH2CH2CH,
CHCH(CH3)2 and CH2(CH2)2), 1.44 (s, 9H (CH3)3), 1.17 and
1.15 (dꢁ2 J=6.5 Hz, CH(CH3)2), 1.03 and 0.97 (dꢁ2, J=6.5
Hz, 3H, COCH(CH3)2). 16b: 1H NMR (CDCl3) d 5.20 (d,
J=9 Hz, 1H, NH), 4.26 (dd, J=6.5, 9 Hz, 1H, NCH2CH2),
4.21 (t, J=9 Hz, 1H, NHCH), 3.87 (td, J=6.5, 10 Hz, 1H,
NCH2CH2), 3.78 (d, J=11 Hz, 1H, NCHCH), 3.66–3.54 (m,
4H, CH2CHN, SCH2CH2S and COCH(CH3)2), 3.43 (t, J=5.5
Hz, 2H, SCH2CH2S), 2.83 (m, 1H, NCH2CH2CH), 2.03–1.92
(m, 2H, NCH2CH2CH, CHCH(CH3)2), 1.42 (s, 9H (CH3)3),
1.19 and 1.17 (dꢁ2 J=6.5 Hz, CH(CH3)2), 1.01 and 0.94
(dꢁ2, J=6.5 Hz, 3H, COCH(CH3)2).
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