An orally bioavailable pyrrolinone inhibitor of HIV-1 protease: Computational analysis and x-ray crystal structure of the enzyme complex

Full text of DOI:10.1021/jm970195u

2440
J . Med. Chem. 1997, 40, 2440-2444
An Or a lly Bioa va ila ble P yr r olin on e
In h ibitor of HIV-1 P r otea se:
Com p u ta tion a l An a lysis a n d X-r a y
Cr ysta l Str u ctu r e of th e En zym e
Com p lex
Amos B. Smith, III,* Ralph Hirschmann,*
Alexander Pasternak, Wenquing Yao, and
Paul A. Sprengeler
Department of Chemistry, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
M. Katharine Holloway,* Lawrence C. Kuo,*
Zhongguo Chen, Paul L. Darke, and
William A. Schleif
Departments of Molecular Design and Diversity, Molecular
Biology, and Virus and Cell Biology, Merck Research
Laboratories, West Point, Pennsylvania 19486
Received March 24, 1997
The importance of HIV protease inhibitors in the
treatment of AIDS is now well established.1 A very
promising current strategy employs a combination of
drugs to inhibit both the protease and the HIV reverse
transcriptase.2 The remarkable mutability of the HIV
retrovirus however remains a major obstacle to the
successful long-term management and cure of the
disease. More recently, the concept of a single-mecha-
nism cocktail (e.g., a mixture of protease inhibitors) has
attracted considerable interest.3,4 Assuming that the
single-mechanism approach proves viable, we suggest
that the best protection against the wild-type and
mutant strains may be obtained by maximizing struc-
tural diversity of the protease inhibitors.
We have described several potent inhibitors of the
HIV-1 protease (e.g., 1 and 2), based on the novel 3,5-
linked bispyrrolinone scaffold which we developed to
mimic the peptidal â-strand/â-sheet structural motif.5
The first-generation inhibitor 1 proved less effective
than its exact peptidal counterpart 3 in purified enzyme
inhibition assays (IC50) but displayed higher cellular
antiviral activity (CIC95). Importantly, the correspond-
ing CIC95/IC50 (C/I) ratios6 indicated that the bispyr-
rolinone entered infected lymphocytes more readily than
the analogous peptide. We have also confirmed that
polypyrrolinones are not cleaved by the proteolytic
enzyme R-chymotrypsin.7 Nonetheless, our most active
bispyrrolinone 2 (IC50 1.3 nM, CIC95 800 nM) was not
orally bioavailable in dogs,5c,g reflecting either nonab-
sorption from the gastrointestinal tract, first-pass me-
tabolism in the liver, or excretion into the bile. The
relatively high molecular weight of 735 may account for
the poor oral bioavailability of 2.8 We therefore sought
to design and synthesize monopyrrolinone inhibitors of
HIV-1 protease with molecular weights <600.
Design a n d In itia l Molecu la r Mod elin g. Replace-
ment of the C-terminal pyrrolinone moiety in 1 and 2
with a P2′ indanol, as employed in amide inhibitors
L-685,434 (7)9 and L-697,807 (8),10 generated the 3,5,5-
trisubstituted pyrrolin-4-one structures 4 (MW 569) and
6 (MW 583). Monopyrrolinones cannot form â-strand-
inducing intramolecular hydrogen bonds between a
pyrrolinone NH and the carbonyl of an adjacent het-
erocycle, as observed in our polypyrrolinones. However,
the potential does exist for a weaker H-bond between
the pyrrolinone NH and the transition-state-mimetic
secondary hydroxyl.
To investigate the influence of this variation in
hydrogen bonding on the solution conformation, a Monte
Carlo conformational search11,12 was performed on 6 and
the resultant stuctures were minimized with both
chloroform and water continuum-solvation models.13
The backbone conformations in the lowest-energy cal-
culated conformers closely resembled the X-ray stuc-
tures of peptidal inhibitors such as MVT-101 and J G-
365 cocrystallized with the HIV-1 protease.14 The major
difference between the chloroform and water models
proved to be a hydrogen bond in the former linking the
catalytic site and indanol hydroxyls; this interaction
caused a slight rotation of the indanol relative to the
pyrrolinone. The majority of the other preferred con-
formers for both solvents displayed the same overall
backbone shape, with minor changes in the P1 and P1′
ø1 angles and the indanol-pyrrolinone torsion angle.
S0022-2623(97)00195-7 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2441
Syn th esis a n d Biologica l Activity. Monopyrroli-
nones 4 and 6 were synthesized from lactone (-)-1315
via methodology we developed earlier (Scheme 1).5c-i
In contrast with the bispyrrolinone inhibitors,5d,g the
monopyrrolinones are less potent antiviral agents than
their amide counterparts in the cellular assay, with
CIC95 values of 800 and 100 nM for 4 and 6 compared
with 4009 and 3 nM10 for 7 and 8. Nevertheless, the
C/I ratios6 for 4 and 6 (67 and 50, respectively) are
improved vis-a`-vis 7 and 8 (1333 and 100), suggesting
that 4 and 6 are more readily transported into HIV-
infected lymphocytes.19 We previously attributed2c,g the
improvement in CIC95/IC50 ratios for the bispyrrolinones
to hydrogen bonding between adjacent rings, which
would decrease the desolvation energy required for
transport from the aqueous extracellular environment
into the lipophilic cell membrane.20,21 In the monopyr-
rolinones an intramolecular six-membered-ring H-bond
can link the pyrrolinone NH with the nearby transition-
state-mimetic hydroxyl group, but this interaction is
conformationally and energetically less favorable than
H-bonding between neighboring pyrrolinone rings, per-
haps accounting for the more modest improvements in
C/I ratios for the monopyrrolinones (relative to their
amide counterparts). The less polar nature of the
pyrrolinone enaminone functionality, compared with the
amide group in 7 and 8, may also contribute to the
improved C/I indices for 4 and 6.
Sch em e 1
Evaluation of the pharmacokinetic properties of 6 in
two dogs revealed an oral bioavailability of 13%, with a
t1/2 of 33-36 min, a clearance rate of 20.6 mL/min/kg, a
volume distribution of 0.81 L/kg, and an area under the
curve of 1.83 µM/h. Although the corresponding data
for amide 8 do not exist, 6 proved to be 5-fold less
bioavailable than the amide inhibitor Crixivan (18). Our
findings demonstrate that useful bioavailability can be
attained by replacing the peptide backbone with a
pyrrolinone scaffold.22 In addition, the results suggest
that high molecular weight may indeed be responsible
for the lack of oral bioavailability of bispyrrolinone 2.
In ter p r eta tion via Mod elin g a n d X-r a y Cr ysta l-
logr a p h y. We minimized complexes of pyrrolinones 4
and 6 and their peptidal counterparts (7 and 8) in the
active site of HIV-1 protease, using the MM2X force field
according to published protocols.23 The key question
was whether the pyrrolinone NHs participate in â-sheet-
like hydrogen bonding with the enzyme, as suggested
by the biological activity (vide supra). The energy-
minimized bound conformations of 4 and 6 proved to
be nearly identical. In each case, the pyrrolinone NH
was displaced relative to the P2′ amide NH in 7 or 8, as
expected; the angle between the pyrrolinone NH and
the Gly27 carbonyl would accommodate a weak hydrogen
bond at best. H-Bonding may also be disfavored by the
close contact between the Gly27 carbonyl and the
adjacent carbon in the pyrrolinone ring as well as the
distance (3.4 Å) between the indanol hydroxyl and the
backbone NH of Asp29. These unfavorable conforma-
tional changes and steric effects were reflected in the
interaction energies calculated for the pyrrolinone in-
hibitors and amides 7 and 8 (Table 1). A linear
correlation (R ) 0.988) was observed between the
calculated values of EInter and the experimental binding
affinities (IC50s); importantly, analogous correlations
have heretofore been observed23 for groups of molecules
that bind in similar fashion, suggesting that the pyr-
rolinones and amides have adopted essentially equiva-
lent bound conformations.
In the purified enzyme inhibition assay,16,6a IC50s of
11.9 and 2.0 nM for 4 and 6 demonstrated that monopyr-
rolinones can strongly inhibit the HIV-1 protease (Table
1). Comparison with the analogous amide-based inhibi-
tors 7 and 8 (0.4 and 0.03 nM) suggested a less favorable
fit for 4 and 6 in the enzyme active site. As we observed
for the related bispyrrolinones,5c,g replacement of the
N-terminal Boc functionality in 4 with the (3S)-hydrox-
ytetrahydrofuranyl carbamate in 6 led to improved
potency. Subsequently, three amide-based analogs of
7 were reported:17 L-687,965 (9, R-methyl), L-689,428
(10, N-methyl), and L-687,630 (11, γ-lactam). These
compounds were 18-, 465-, and 215-fold less active than
7, possibly because an H-bond to the carbonyl of Gly27
is important for high affinity. The decrease in activity
was much smaller for 4 (30-fold relative to 7) than for
10 and 11, suggesting that H-bonding of the pyrrolinone
NH group may partially compensate for the loss in
affinity caused by steric interactions.
Ta ble 1. Bioassay Data and Calculated Interaction Energies
for Pyrrolinones 1 and 4-6 and Related Amide-Based
Inhibitors
IC50 CIC95
(nM) (nM)
EInter
(kcal/mol)
inhibitor
type
C/I
150
1
pyrrolinone 10
1500
L-682,679 (3) amide
0.6 6000
10000
67
4
pyrrolinone 11.9 800
-120.0
-145.0
L-685,434 (7) amide
L-687,965 (9) amide
0.3 400
7.0
1333
L-689,428 (10) N-Me-amide 186
L-687,630 (11) γ-lactam 86
pyrrolinone 2.0 100
6
50
100
-134.1
-150.9
-134.3
L-697,807 (8) amide
0.03
3
5
pyrrolinone 6.0
12
amide
0.16
To explore this relationship further we modeled 5, a
p-hydroxy analog of 4. The calculated interaction
Crixivan (18)18 amide
0.36 25-100 69-277

2442 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Communications to the Editor
energy (EInter -134.3 kcal/mol) led to a predicted IC50
of 2.7 nM. The latter value was expected to be slightly
too low; an H-bond in the model, between the p-OH in
P1′ and a nitrogen of Arg8, is not likely to be significant
in solution because Arg8 lies exposed to solvent at the
end of the active-site cleft. The observed IC50 of 6.0 nM
agreed well with the calculation.
Ultimately the binding of 6 to the HIV-1 protease was
elucidated via X-ray crystallographic analysis of the
complex (Figure 1a). Single crystals, obtained at room
temperature via vapor diffusion in hanging drops,24
diffracted to 2.0 Å resolution. The space group was
P21212 (cell constants: a ) 58.26 Å, b ) 87.48 Å, and c
) 46.41 Å) with one molecule of the complex per
asymmetric unit. Following data collection and pro-
cessing, as described previously, the structure was
determined via the difference Fourier method, using the
Crixivan complex as the starting model.24 Data col-
lected at 2-6 Å resolution were refined with XPLOR,25
yielding an R factor of 0.183 with rms bond-length and
bond-angle deviations of 0.016 Å and 2.048°, respec-
tively. A single orientation was observed for 6 bound
to the protease.
Comparison of the crystal structures of enzyme-bound
Crixivan (18) and 6 (Figure 1c) revealed shifts of the
protease backbone in both the flap and Thr26-Asp30
regions to accommodate the pyrrolinone ring. As pre-
dicted, the pyrrolinone NH does not hydrogen bond to
Gly27. Surprisingly, this NH does H-bond to the car-
boxylate of Asp25, a catalytic residue of the protease
(N-O distance 3.209 Å), resulting in a 90° rotation of
the carboxylate relative to the Crixivan complex. In
addition, the distance between the indanol hydroxyl and
Asp29 (5.40 Å) not only is larger than the corresponding
values for similar amide-based inhibitors but also
exceeds the modeling prediction for 6. Not unexpect-
edly, we found that this space is occupied by a water
molecule which H-bonds to both the NH of Asp29 and
the indanol hydroxyl, compensating for the loss of the
Gly27 hydrogen bond. Recent work has revealed similar
binding of water to crystalline HIV-1 protease even in
the absence of an inhibitor.26 The entropically unfavor-
able inclusion of a water molecule in the active site may
contribute significantly (up to ca. 2 kcal/mol) to the
lower affinity of 6.27 The bound conformation of 6
differed only slightly from the calculated structures in
water and chloroform (Figure 1b), and a small rotation
(ca. 30°) of the scissile-mimetic bond yields identical
backbones (rms 0.164 Å). This preorganization probably
accounts for the relatively high affinity observed for the
monopyrrolinones despite the disadvantages noted above.
F igu r e 1. (a) X-ray crystal structure of monopyrrolinone 6
bound to HIV-1 protease.28 (b) Overlay of the lowest-energy
linear structures of 6 minimized in chloroform (green) and
water (blue) with the enzyme-cocrystallized conformation
(purple). (c) Overlay of the HIV-1 protease cocrystal structures
of 6 (purple; enzyme, blue) and Crixivan (18) (orange; enzyme,
yellow).28
Su m m a r y. Our search for novel scaffolds with
improved pharmacokinetic properties5c,g has led to
several potent pyrrolinone-based inhibitors of HIV-1
protease. These monopyrrolinones are completely stable
to proteases, and 6 proved to be orally bioavailable in
dogs. Cocrystallization of 6 with the enzyme and X-ray
analysis revealed an unexpected hydrogen bond between
Asp25 and the pyrrolinone NH as well as the incorpora-
tion of a water molecule linking the indane hydroxyl
conformation and H-bonding of the enzyme are discern-
ible both in the active site and in other regions.
We believe that if the concept of single-mechanism
cocktails3 of HIV-1 protease inhibitors is eventually
validated in the clinic, its benefits can be optimized by
maximizing the structural diversity of the protease
inhibitors comprising the cocktail. We are currently
synthesizing several pyrrolinones designed to provide
and the NH of Asp29
.
The protease complexes of
pyrrolinone 6 and Crixivan both crystallize in a single
orientation. Notwithstanding the overall similarity of
the latter structures, significant differences in the

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2443
rahedron Lett. 1993, 34, 63-66. (i) Smith, A. B., III; Keenan, T.
P.; Holcomb, R. C.; Sprengeler, P. A.; Guzman, M. C.; Wood, J .
L.; Carroll, P. J .; Hirschmann, R. Design, Synthesis, and Crystal
Structure of a Pyrrolinone-Based Peptidomimetic Possessing the
Conformation of a â-Strand: Potential Application to the Design
of Novel Inhibitors of Proteolytic Enzymes. J . Am. Chem. Soc.
1992, 114, 10672-10674.
higher affinity by preventing the entropically unfavor-
able binding of water in the active site while maintain-
ing the H-bond to Asp25
.
Ack n ow led gm en t. We are pleased to acknowledge
support of this investigation by the National Institutes
of Health (Institute of General Medical Sciences) through
Grant GM-41821. Additional funding was provided by
Bachem, Inc. (Torrance, CA), Merck Research Labora-
tories (West Point, PA), and Sankyo Co., Ltd. (Tokyo,
J apan). The authors wish to thank Dr. G. Furst, Dr.
P. Carroll, and Mr. J . Dykins, Directors of the Univer-
sity of Pennsylvania Spectroscopic Facilities, for as-
sistance in obtaining NMR spectra, small-molecule
X-ray crystal structures, and high-resolution mass
spectra, respectively. We also thank Dr. Donald Heefner,
Dr. J oel Huff (Merck Research Laboratories, West
Point), Mr. Charles A. Lesburg, and Dr. Christopher S.
Shiner for helpful suggestions and critical comments.
(6) (a) We employ C/I ratios rather than I/C ratios to quantify
transport because the former values are usually >1 and the
latter <1; cf., Thompson, W. J .; Fitzgerald, P. M. D.; Holloway,
M. K.; Emini, E. A.; Darke, P. L.; McKeever, B. M.; Schleif, W.
A.; Quintero, J . C.; Zugay, J . A.; Tucker, T. J .; Schwering, J . E.;
Homnick, C. F.; Nunberg, J .; Springer, J . P.; Huff, J . R. Synthesis
and Antiviral Activity of a Series of HIV-1 Protease Inhibitors
with Functionality Tethered to the P1 or P1′ Phenyl Substitu-
ents: X-Ray Crystal Structure Assisted Design. J . Med. Chem.
1992, 35, 1685-1701. (b) For a recent application of the I/C ratio,
see: Smith, R. A.; Coles, P. J .; Chen, J . J .; Robinson, V. J .;
MacDonald, I. D.; Carriere, J .; Krantz, A. Design, Synthesis, and
Activity of Conformationally-Constrained Macrocyclic Peptide-
Based Inhibitors of HIV Protease. Bioorg. Med. Chem. Lett. 1994,
4, 2217-2222.
(7) Upon treatment of our previously reported pentapyrrolinone i5e
with chymotrypsin in 70% MeOH/phosphate buffer containing
9-fluorenylmethanol as internal standard and the chymotrypsin
assay reagent N-benzoyltyrosine ethyl ester, a positive control,
no decrease was observed in the HPLC peak area for i relative
to the internal standard even after 20 h. In contrast, the peak
corresponding to N-benzoyltyrosine ethyl ester disappeared
almost completely after only 15 min, furnishing the expected
N-benzoyltyrosine and an intermediate, N-benzoyltyrosine meth-
yl ester, which was eventually converted to N-benzoyltyrosine.
Su p p or tin g In for m a tion Ava ila ble: Plots of the plasma
levels of 6 in dogs (po and iv) and of binding affinity vs
calculated interaction energy for the pyrrolinone- and amide-
based inhibitors; characterization data for 4-6, 13, 14, and
16 (5 pages). See any current masthead page for ordering
information.
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(19) We have initiated a collaboration with Prof. Ronald T. Borchardt
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(22) Interestingly, pyrrolinone-based analog ii of Crixivan proved to
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