Design, synthesis, and biological evaluation of monopyrrolinone-based HIV-1 protease inhibitors possessing augmented P2′ side chains

Article abstract of DOI:10.1016/j.bmcl.2005.11.011

A series of monopyrrolinone-based HIV-1 protease inhibitors possessing rationally designed P2′ side chains have been synthesized and evaluated for activity against wild-type HIV-1 protease. The most potent inhibitor displays subnanomolar potency in vitro

Full text of DOI:10.1016/j.bmcl.2005.11.011

Bioorganic & Medicinal Chemistry Letters 16 (2006) 859–863
Design, synthesis, and biological evaluation
of monopyrrolinone-based HIV-1 protease inhibitors
possessing augmented P20 side chains
Amos B. Smith, III,a,* Adam K. Charnley,a Hironori Harada,a Jason J. Beiger,a
Louis-David Cantin,a Craig S. Kenesky,a Ralph Hirschmann,a Sanjeev Munshi,b
David B. Olsen,b Mark W. Stahlhut,b William A. Schleifb and Lawrence C. Kuob
aDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
bMerck Research Laboratories, West Point, PA 19486, USA
Received 17 October 2005; accepted 3 November 2005
Available online 18 November 2005
Abstract—A series of monopyrrolinone-based HIV-1 protease inhibitors possessing rationally designed P20 side chains have been
synthesized and evaluated for activity against wild-type HIV-1 protease. The most potent inhibitor displays subnanomolar potency
in vitro for the wild-type HIV-1 protease. Additionally, the monopyrrolinone inhibitors retain potency in cellular assays against
clinically significant mutant forms of the virus. X-ray structures of these inhibitors bound in the wild-type enzyme reveal important
insights into the observed biological activity.
Ó 2005 Elsevier Ltd. All rights reserved.
Despite significant advances in the treatment of AIDS,
in particular the advent of HAART (highly active
anti-retroviral therapy), the development of new HIV
therapeutics remains essential to combat this epidemic.1
The remarkable ability of the virus to circumvent cur-
rent clinical agents via mutation clearly highlights the
need for improved therapeutics. In fact, development
of a cocktail of highly potent, structurally diverse inhib-
itors of HIV-1 encoded enzymes may provide the best
opportunity to thwart viral resistance.2
Aspartic acid proteases including the HIV-1 protease
most often bind their substrate in an extended b-strand
conformation, and thus, we initiated a program to
develop potent HIV-1 protease inhibitors based on our
pyrrolinone b-strand peptidomimetic scaffold. Our early
work directed at pyrrolinone-based HIV-1 protease
inhibitors resulted in a series of bispyrrolinones which
displayed both potent (nM) activity, and improved cell
permeability relative to peptidal inhibitors, yet lacked
oral bioavailability in dogs.5 Reasoning that the lack
of oral bioavailability was due, at least in part, to their
high molecular weight, we subsequently designed and
synthesized a monopyrrolinone-based HIV-1 protease
inhibitor (ꢀ)-1 (Fig. 2), which proved to be both potent
(nM) and to possess 13% oral bioavailability in dogs.6
X-ray structure determination of (ꢀ)-1 cocrystallized
A principal goal of the Smith/Hirschmann collaboration
at Penn has been the development of the pyrrolinone
scaffold as a privileged peptidomimetic. The homochiral
3,5-linked pyrrolinone scaffold (Fig. 1) was first intro-
duced in 1992 and has been demonstrated to be a highly
successful b-strand/b-sheet mimic,3 while the heterochi-
ral 3,5-linked pyrrolinone (Fig. 1) scaffold has recently
been shown to hold considerable potential as a b-turn
mimic.4
O
O
O
O
HN
HN
HN
HN
Heterochiral (LDL)
HN
HN
O
O
Homochiral (DDD)
Keywords: Pyrrolinone; HIV-1; Protease inhibitors.
*
Figure 1. Homochiral
polypyrrolinones.
(DDD)
and
heterochiral
(LDL)
Corresponding author. Tel.: +1 215 898 4860; fax: +1 215 898
5129; e-mail: smithab@sas.upenn.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.011

860
A. B. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 859–863
Ph
OH
Ph
HN
H
O
N
P2'
O
2
O
O
O
O
O
P2'
H N
O
2
OH
NH
O
NH
NH
O
(–)-1
(–)-2
(+)-3
4
5
Figure 2. Monopyrrolinone-based HIV-1 protease inhibitors.
with the wild-type HIV-1 protease revealed the presence
of a water molecule bridging the indanol hydroxyl on
the P20 side chain and the Asp29 NH in the S2 pocket.6
Based on X-ray crystallographic information, molecular
modeling led to the design of inhibitors with modified
P20 side chains [cf. (ꢀ)-2 and (+)-3] holding the promise
of improved potency via displacement of the adventi-
tious water molecule. Unfortunately, these early at-
tempts proved unsuccessful.6b
(Schemes 1 and 2, respectively). The synthesis of (+)-6
began with allyl indanone (ꢀ)-8, which was readily
available in enantiopure form via the Johnson sulfoxide
resolution method.8 Ozonolysis of the olefin followed in
turn by chemoselective reduction of the aldehyde and
protection as the triisopropylsilyl ether (TIPS) provided
ketone (ꢀ)-9. Attachment of the side chain onto ketone
(ꢀ)-9 was achieved via Reformatsky addition, followed
by elimination of the resulting alcohol9 and hydrogena-
tion to afford ester (ꢀ)-10 as a mixture (10:1) of diaste-
reomers favoring the desired cis-product. Ester (ꢀ)-10
was then converted to the primary amide in two steps,
followed by removal of the silyl-protecting group and
oxidation of the resulting alcohol to furnish aldehyde
(+)-6.
In continuation of our efforts to prepare highly potent
pyrrolinone-based HIV-1 protease inhibitors with im-
proved P20 side chains, we report here the design, syn-
thesis, and biological evaluation of a third-generation
series of monopyrrolinone-based inhibitors (4 and 5),
including the X-ray analysis of the inhibitors cocrystal-
lized with wild-type HIV-1 protease.
For the preparation of aldehyde (ꢀ)-7, we turned to the
rhodium-mediated nitrene C–H insertion chemistry
developed by Du Bois et al.10 Here we envisioned 7 to
arise from carbamate 13 (Scheme 2) via a late-stage
C–H insertion. In the event, (ꢀ)-13 was prepared from
known lactone (ꢀ)-11, the latter readily available from
the synthesis of (ꢀ)-1. Initially, the C–H insertion pro-
ceeded only in modest yield with [Rh2(OAc)4] and
[Rh2(esp)2]; better results (ca. 50%) were obtained with
[Rh2(O2CPh3)4].
Molecular modeling suggested that P20 side chains
incorporating the benzylic acetamide and cyclic carba-
mate substituted indane skeletons (4 and 5, respectively)
would improve the hydrogen-bonding interactions in the
S20 pocket, particularly with Asp29. In addition, the
cyclic carbamate could be anticipated to gain additional
potency via preorganization of the side chain, thereby
reducing the entropy penalty associated with binding.7
From the synthetic perspective, incorporation of the P20
side chains into our pyrrolinone synthetic protocol
called for preparation of aldehydes (+)-6 and (ꢀ)-7
With aldehydes (+)-6 and (ꢀ)-7 in hand, application of
our pyrrolinone cyclization protocol involving imine
formation followed by metalloenamine-mediated
1. Zn, BrCH2CO2Et
TIPSO
1. O3, PPh3 (78%)
O
O
(EtO)3B-THF (58%)
2. Li(t-BuO)3AlH
(74%)
3. TIPSCl, imidazole
(quant.)
2. Martin Sulfurane (99%)
3. HCO2NH4, Pd/C
MeOH
(–)-8
(–)-9
(86% 10:1 cis/trans)
1. aq. NaOH
MeOH (98%)
2. CDI, NH4OAc (95%)
TIPSO
O
CO Et
2
CONH
2
3. TBAF (quant.)
4. SO3•Pyr, DMSO
(92%)
(–)-10
(+)-6
Scheme 1. Preparation of aldehyde (+)-6.

A. B. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 859–863
861
of the resultant pyrrolinones, followed by treatment
with succinimidyl carbonate (+)-16,11 provided the pro-
spective inhibitors (+)-15 and (+)-17 in good yield for
the two steps. In addition to the targeted compounds,
analogues incorporating t-butyl carbamate and t-butyl
acetamide P2 substituents were prepared in a similar
fashion [e.g. (+)-18 to (ꢀ)-21].
O
1. Cl3C(O)NCO
2. K2CO3
1. DIBAL
2. Ph3PCH3Br
O
OH
(quant., 2 steps)
3. O3, PPh3 (57%)
KOt-Bu
(90% 2 steps)
(–)-11
(–)-12
O
O
O
Rh cat.
PhI(OAc)2, MgO
DCM, 40 oC
The monopyrrolinone inhibitors were evaluated for
activity against wild-type HIV-1 protease in isolated
enzyme12 and cellular13 assays (Table 1). Pleasingly,
the prospective inhibitors, with the exception of those
incorporating a t-butyl acetamide at P2 [e.g. (+)-19
and (ꢀ)-21], displayed potent activity in the HIV-1
protease enzyme assay. Analogue (ꢀ)-5 exhibits subn-
anomolar activity, and as such represents an order of
magnitude improvement in potency relative to our first-
and second-generation pyrrolinone-based inhibitors.
O
Rh cat.
[Rh2(OAc)4]
[Rh2(esp)2]
Yield
33%
36%
O
N
H N
2
O
(–)-13
(–)-7
H
[Rh2(O2CCPh3)4] 50%
Scheme 2. Preparation of aldehyde (ꢀ)-7.
cyclization,3b proceeded with both aldehydes, albeit the
yields were modest (Scheme 3). That pyrrolinone forma-
tion proved viable in the presence of amide and carba-
mate hydrogens was however gratifying, and in the
case of amide (+)-6, represents the first demonstration
of such functional compatibility. Global deprotection
Activity in the cellular assays, however, did not reflect
the improved in vitro potency, resulting in significant
but somewhat diminished CIC95 values, with higher
O
CONH
2
Ph
HN
Ph
H N
2
O
a.
O
NH
O
2
BocN
Ph
OCH
BocN
Ph
(+)-6
PhMe (-H2O)
3
O
O
(+)-14
b. KHMDS, THF
(34%)
(+)-15
Ph
HN
H N
2
O
OH
1. TFA, DCM
H
(+)-15
R
N
2. Reagent, Et3N
O
O
O
Ph
O
O
N
(67%)
O
O
O
O
O
(+)-16
R =
Boc2O (59%)
O
t-BuCH2COCl (21%)
(+)-4
(+)-18
(+)-19
O
O
Ph
HN
Ph
O
O
O
NH
O
2
N
NH
(–)-7
O
BocN
Ph
H
OCH
BocN
Ph
3
Benzene
KHMDS, THF
(42%)
O
O
(+)-14
(+)-17
O
Ph
O
OH
HN
H
1. 1N HCl/MeOH
NH
R
N
(+)-17
2. Reagent, (i-Pr)2NEt
O
O
O
O
Ph
O
O
N
(63%)
O
O
O
O
(+)-16
R =
Boc2O (88%)
t-BuCH2COCl, HOBt
(64%)
O
(–)-5
(–)-20
(–)-21
Scheme 3. Preparation of monopyrrolinone-based protease inhibitors.

862
A. B. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 859–863
Table 1. Bioassay data for monopyrrolinone-based HIV-1 protease
inhibitors
X-ray structures of the third-generation inhibitors (+)-4
and (ꢀ)-5 complexed with wild-type HIV-1 protease
were obtained. Analysis of the cocrystal structure of
(+)-4 indicated that, as predicted, the P20 substituent
had displaced the undesired6b water molecule (Fig. 3),
despite the fact that we did not obtain an increase in
potency.15 Gratifyingly, the cocrystal structure of (ꢀ)-
5, our most potent inhibitor (IC50 0.44 nM), revealed
that we had again succeeded in displacing the water mol-
ecule, reestablishing important interactions in the S20
pocket (Fig. 4).16
Inhibitor
IC50 (nM)
CIC95 (nM)
C/I ratio
(ꢀ)-1
2
2
100–250
250
781
50–125
125
137
(ꢀ)-2
(+)-3
(+)-4
5.7
2.4
781
400
325
909
(ꢀ)-5
0.44
20.5
>250
2.5
(+)-18
(+)-19
(ꢀ)-20
(ꢀ)-21
Indinavir
>400
12,500
200
80
32.5
69–277
96
3125
25–100
0.36
Comparison of the P20 side chains of (ꢀ)-5 and (+)-4
indicates that the indane ring and the cyclic carbamate
of (ꢀ)-5 are superimposable onto the indane ring and
benzylic acetamide, respectively, of (+)-4. In the two
X-ray structures, the B-factors associated with these
moieties are indistinguishable within experimental error.
For these reasons, the increase in binding affinity
(ꢁ1 kcal/mol) of (ꢀ)-5 over that of (+)-4 can be attrib-
uted to a difference in ground-state energetics between
the two compounds in the bulk solvent, likely a result
of reducing the rotational entropy arising from the ben-
zylic acetamide of (+)-4 (i.e., preorganization).7
Experimental error for IC50 is 5%, based on standard errors of the
mean, and for CIC95, one-half to 2-fold of the value shown, based on
serial dilutions of compound concentration.
CIC95/IC50 (C/I) ratios relative to our previous inhibi-
tors [ca. (ꢀ)-1] as well as indinavir.6b
Having demonstrated both potent in vitro and signifi-
cant in vivo activity against wild-type HIV-1, we next
tested the most potent third- generation inhibitors
against three clinically significant mutant virus strains.14
Pleasingly, the third- generation pyrrolinone-based
inhibitors maintained good inhibitory activity across
the panel of clinically significant mutants. Additionally,
by comparing wild-type CIC95 values, the pyrrolinone-
based inhibitors displayed minimal effects from protein
binding [in the presence of either 10% fetal bovine serum
(Table 2) or 50% normal human serum (Table 3)]. These
results are quite encouraging and support our belief that
the binding conformation of the pyrrolinone scaffold
may be sufficiently unique, so as to overcome established
mutations.
The design, synthesis, biological evaluation, and X-ray
structural analyses of a series of third-generation mono-
pyrrolinone HIV-1 protease inhibitors have been
achieved. These studies culminated in the preparation
Table 2. Bioassay data against different viral isolates in the presence of
10% normal human serum (NHS) or 10% fetal bovine serum (FBS)
Inhibitor
CIC95 (nM)
1002-60C
Wild type
Mutants
4X
H9IIIB
(FBS)
NL4-3
(FBS)
1026-60C
(ꢀ)-1
156
1250
625
156
50
78
312
625
156
50
1250
1250
1250
2500
400
625
625
156
312
312
625
400
(+)-4
(ꢀ)-5
Figure 3. X-ray cocrystal structure of (+)-4 with HIV-1 protease.
156
(ꢀ)-20
Indinavir
2500
100
Table 3. Bioassay data against different viral isolates in the presence of
50% normal human serum (NHS)
Inhibitor
CIC95 (nM)
Wild type
H9IIIB NL4-3
Mutant
4X
1002-60C
1026-60C
(ꢀ)-1
156
1,250
625
156
625
625
625
50
1250
2,500
1250
1250
1250
312
312
625
(+)-4
(ꢀ)-5
312
(ꢀ)-20
Indinavir
312
25
10,000
400
5000
100
1250
400
Experimental error for CIC95, one-half to 2-fold of the value shown,
based on serial dilutions of compound concentration.
Figure 4. X-ray cocrystal structure of (ꢀ)-5 with HIV-1 protease.

A. B. Smith et al. / Bioorg. Med. Chem. Lett. 16 (2006) 859–863
863
Sprengeler, P. A.; Darke, P. L.; Emini, E. A.; Holloway,
M. K.; Schleif, W. A. J. Med. Chem. 1994, 37, 215; (b)
Smith, A. B., III; Hirschmann, R.; Pasternak, A.;
Guzman, M. C.; Yokoyama, A.; Sprengeler, P. A.; Darke,
P. L.; Emini, E. A.; Schleif, W. A. J. Am. Chem. Soc. 1995,
117, 11113.
of (ꢀ)-5 displaying subnanomolar activity in vitro. The
combination of potent biological activities and cocrystal
structure analysis validated our hypothesis that rational
modification of the P20 side chain of these monopyrroli-
none-based HIV-1 protease inhibitors would lead to an
improvement in in vitro potency. Importantly, the third-
generation monopyrrolinone-based inhibitors main-
tained their in vivo activity against several clinically sig-
nificant HIV-1 mutants, further illustrating the potential
value of the pyrrolinone inhibitor scaffold.
6. (a) Smith, A. B., III; Hirschmann, R.; Pasternak, A.; Yao,
W.; Sprengeler, P. A.; Holloway, M. K.; Kuo, L. C.; Chen,
Z.; Darke, P. L.; Schleif, W. A. J. Med. Chem. 1997, 40,
2440; (b) Smith, A. B., III; Cantin, L.-D.; Pasternak, A.;
Guise-Zawacki, L.; Yao, W.; Charnley, A. K.; Barbosa, J.;
Sprengeler, P. A.; Hirschmann, R.; Munshi, S.; Olen, D.
B.; Schleif, W. A.; Kuo, L. C. J. Med. Chem. 2003, 46,
1831. In our previous report (b), we mistakenly referred to
the indanol as a P2 side-chain, while according to
convention, it should be labeled P20.
7. (a) Cram, D. J. Angew. Chem. Int. Ed. Engl. 1986, 25,
1039(b) Andrews, P. In The Practice of Medicinal Chem-
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Acknowledgments
Financial support was provided by the National Insti-
tutes of Health (National Institute of Allergy and Infec-
tious Diseases) through Grant No. AI-42010.
Additional support was provided by Merck Research
Laboratories, the University of Pennsylvania, and grad-
uate fellowships from Bristol-Myers Squibb and the
University of Pennsylvania to A.K.C. The authors also
thank Professor Justin Du Bois (Stanford University)
for providing samples of the rhodium catalysts.
9. Martin, J. C.; Arhart, R. J. J. Am. Chem. Soc. 1971, 93,
4327.
10. (a) Espino, C. G.; Du Bois, J. Angew. Chem. Int. Ed. 2001,
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3. (a) Smith, A. B., III; Keenan, T. P.; Holcomb, R. C.;
Sprengeler, P. A.; Guzman, M. C.; Wood, J. L.; Carroll, P.
J.; Hirschmann, R. J. Am. Chem. Soc. 1992, 114, 10672;
(b) Smith, A. B., III; Guzman, M. C.; Sprengeler, P. A.;
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J.; Hirschmann, R. J. Am. Chem. Soc. 1994, 116, 9947; (c)
Smith, A. B., III; Benowitz, A. B.; Sprengeler, P. A.;
Barbosa, J.; Guzman, M. C.; Hirschmann, R.; Schweiger,
E. J.; Bolin, D. R.; Nagy, Z.; Campbell, R. M.; Cox, D.
C.; Olson, G. L. J. Am. Chem. Soc. 1999, 121, 9286; (d)
Lee, K. H.; Olson, G. L.; Bolin, D. R.; Benowitz, A. B.;
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15. This structure has been deposited with the Protein Data
Bank with Accession No. 2BBB.
5. (a) Smith, A. B., III; Hirschmann, R.; Pasternak, A.;
Akaishi, R.; Guzman, M. C.; Jones, D. R.; Keenan, T. P.;
16. This structure has been deposited with the Protein Data
Bank with Accession No. 2BB9.