Development of a new type of protease inhibitors, efficacious against FIV and HIV variants

Article abstract of DOI:10.1021/ja982893p

Based on the structural analysis of FIV protease and drug-resistant HIV proteases and molecular modeling, a new type of inhibitors with a small P3 residue has been developed. These inhibitors are effective against HIV and its drug-resistant mutants, as well as SIV and FIV. Modification of existing HIV protease inhibitors by reducing the size of the P3 residue has the same effect. This finding provides a new strategy for the development of HIV protease inhibitors effective against the wild-type and drug-resistant mutants. It further supports the use of FIV protease as a useful model for drug-resistant HIV proteases, which often have a more constricted binding region for the P3 group or the combined P3 and P1 groups.

Full text of DOI:10.1021/ja982893p

J. Am. Chem. Soc. 1999, 121, 1145-1155
1145
Development of a New Type of Protease Inhibitors, Efficacious
against FIV and HIV Variants
Taekyu Lee,† Van-Duc Le,† Dongyeol Lim,† Ying-Chuan Lin,‡ Garrett M. Morris,‡
Andrew L. Wong,† Arthur J. Olson,‡ John H. Elder,*,‡ and Chi-Huey Wong*,†
Contribution from the Department of Chemistry and the Skaggs Institute for Chemical Biology and
the Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
ReceiVed August 12, 1998
Abstract: Based on the structural analysis of FIV protease and drug-resistant HIV proteases and molecular
modeling, a new type of inhibitors with a small P3 residue has been developed. These inhibitors are effective
against HIV and its drug-resistant mutants, as well as SIV and FIV. Modification of existing HIV protease
inhibitors by reducing the size of the P3 residue has the same effect. This finding provides a new strategy for
the development of HIV protease inhibitors effective against the wild-type and drug-resistant mutants. It further
supports the use of FIV protease as a useful model for drug-resistant HIV proteases, which often have a more
constricted binding region for the P3 group or the combined P3 and P1 groups.
The aspartyl protease (PR) of human immunodeficiency virus
(HIV) has been the subject of extensive research for the
development of therapeutically useful inhibitors to control the
progression of human acquired immunodeficiency syndrome
(AIDS). Four competitive inhibitors1 of this enzyme have been
approved, and several others are in clinical trials. Despite the
high potency and high selectivity of these inhibitors used in
AIDS therapy, many drug-resistant variants of HIV have been
identified,2 including 45 distinct drug-resistant variants found
in the past 3 years. The drug-resistant mutants are generated
through incomplete suppression of the virus by inhibitors and
usually contain multiple substitutions in their proteases.2
Moreover, these mutant enzymes often exhibit cross-resistance
to many structurally distinct protease inhibitors.2 Therefore,
development of new broad-based protease inhibitors efficacious
against a wide spectrum of HIV variants may be necessary in
order to slow the development of drug resistance.
As a first step toward this goal, we have developed potent
inhibitors against feline immunodeficiency virus protease (FIV
PR), which has been shown to be a useful animal model for
drug-resistant mutant HIV PRs.3,4 FIV is a retrovirus which
causes an immunodeficiency syndrome in cats comparable to
AIDS in humans.5 Both HIV and FIV PRs are C2-symmetric
homodimeric enzymes, and they have almost superimposable
active-site structures that facilitate catalysis by an identical
mechanism.3,6 Similar to HIV PR, FIV PR also processes both
structural proteins of gag and the enzymes encoded by pol
during FIV replication.7 Furthermore, six mutated residues in
HIV PR causing drug resistance (K20I, V32I, I50V, N88D,
L90M, Q92K)2a,8 are found in the structurally aligned native
residues of FIV PR. Kinetic studies have also demonstrated that
various potent HIV PR inhibitors which interact with the binding
region from S4 to S4′ are less efficient inhibitors of FIV PR by
a factor of 100 or more,3,4 and good inhibitors of FIV PR are
often better inhibitors of the wild-type and drug-resistant HIV
PRs.4 In addition to the observation that FIV PR resembles many
known drug-resistant HIV PRs, the cat offers a potential animal
system to test the effectiveness of anti-lentiviral agents in ViVo
to speed up the drug development process.
† Department of Chemistry and the Skaggs Institute.
‡ Department of Molecular Biology.
(1) (a) Babine, R. E.; Bender, S. L. Chem. ReV. 1997, 97, 1359-1472.
(b) De Lucca, G. V.; Erickson-Viitanen, S.; Lam, P. Y. S. Drug DiscoVery
Today 1997, 2, 6-18. (c) Vacca, J. P.; Condra, J. H. Drug DiscoVery Today
1997, 2, 261-272. (d) Huff, J. R. J. Med. Chem. 1991, 34, 2305. (e)
Wlodawer, A.; Erickson, J. W. Annu. ReV. Biochem. 1993, 62, 543-585.
(2) (a) Schinazi, R. F.; Larder, B. A.; Mellors, J. W. Int. AntiViral News
1997, 5, 129-142. (b) Wilson, S. I.; Phylip, L. H.; Mills, J. S.; Gulnik, S.
V.; Erickson, J. W.; Dunn, B. M.; Kay, J. Biochim. Biophys. Acta 1997,
1339, 113-125. (c) Erickson, J. W.; Burt, S. K. Annu. ReV. Pharmacol.
Toxicol. 1996, 36, 545-571. (d) Gulnik, S. V.; Suvorov, L. I.; Liu, B.; Yu,
B.; Anderson, B.; Mitsuya, H.; Erickson, J. W. Biochemistry 1995, 34,
9282-9287. (e) Erickson, J. W. Nature Struct. Biol. 1995, 2, 523-529. (f)
Condra, J. H.; Schleif, W. A.; Blahy, O. M.; Gabryelski, L. J.; Grayyam,
D. J.; Quintero, J. C.; Rhodes, A.; Robbins, H. L.; Roth, E.; Shivaprakash,
M.; Titus, D.; Yang, T.; Teppler, H.; Squires, K. E.; Deutsch, P. J.; Emini,
E. A. Nature 1995, 374, 569-571. (g) Otto, M. J.; Garber, S.; Winslow,
D. L.; Reid, C. D.; Aldrich, P.; Jadhav, P. K.; Patterson, C. E.; Hodge, C.
N.; Cheng, Y.-S. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7543-7547.
(h) Wong, J. K.; Hezareh, M.; Gunthard, H. F.; Halvir, D. V.; Ignacio, C.
C.; Spina, C. A.; Richman, D. D. Science 1997, 278, 1291-1295 (i) Finzi,
D.; Hermankova, M.; Pierson, T.; Carruth, L. M.; Buck, C.; Chaisson, R.
E.; Quinn, T. C.; Chadwick, K.; Margolick, J.; Brookmeyer, R.; Gallant,
J.; Markowitz, M.; Ho, D. D.; Richman, D. D.; Siliciano, R. F. Science
1997, 278, 1295-1300.
(3) Slee, D. H.; Laslo, K. L.; Elder, J. H.; Ollmann, I. R.; Gustchina,
A.; Kervinen, K.; Zdanov, A.; Wlodawer, A.; Wong, C.-H. J. Am. Chem.
Soc. 1995, 117, 11867-11878.
(4) Lee, T.; Laco, G. S.; Torbett, B. E.; Fox, H. S.; Lerner, D. L.; Elder,
J. H.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 939-944.
(5) (a) Talbott, R. L.; Sparger, E. E.; Lovelace, K. M.; Fitch, W. M.;
Pedersen, N. C.; Luciw, P. A.; Elder, J. H. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 5743-5747. (b) Pedersen, N. C.; Ho, E. W.; Brown, M. L.;
Yamamoto, J. K. Science 1987, 235, 790-793.
(6) Wlodawer, A.; Gustchina, A.; Reshetnikova, L.; Lubkowski, J.;
Zdanov, A.; Hui, K. Y.; Angleton, E. L.; Farmerie, W. G.; Goodenow, M.
M.; Bhatt, D.; Zhang, L.; Dunn, B. M. Nat. Struct. Biol. 1995, 2, 480-
488.
(7) Kramer, R. A.; Schaber, M. D.; Skalka, A. M.; Ganguly, K.; Wong-
Staal, F.; Reddy, E. P. Science 1986, 231, 1580-1584.
(8) Mellors, J. W.; Larder, B. A.; Schinazi, R. F. Int. AntiViral News
1995, 3, 8-13.
10.1021/ja982893p CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/03/1999

1146 J. Am. Chem. Soc., Vol. 121, No. 6, 1999
Lee et al.
Table 1. Inhibition of FIV and HIV PRs by Small P3 Residue-Containing Inhibitors and Their Parent Compoundsa
HIV PRc
FIV PR,b
Ki (nM)
HIV (G48V),c
IC50 (nm)
HIV (V82F),c
IC50 (nM)
compd
1a
1b
1c
1d
Ki (nm)
IC50 (nM)
17000 ( 300d
41 ( 7d
nd
nd
ni
1.1 ( 0.2d
1.5 ( 0.3d
nd
3.8
5.3
20.5
158
277
14.9
60.8
64.4
nd
13.3
2a
60000e
2b
3a
3b
4a
4b
5a
7300 ( 300
499 ( 81
308 ( 70
ni
300000e
9400 ( 900
ni
212 ( 23
ni
2000e
10.3
5.4 ( 0.7
214e
131
86.1
5b
6b
7a
7b
46 ( 5
2.5 ( 0.4
3.0 ( 0.6
1.5 ( 0.2
11.3 ( 1.3
1.6 ( 0.6
7.8
5.0
4.0
68.7
34.5
26.1
44.4
24.0
13.3
3700 ( 600
2600 ( 300
133000 ( 38000
76000 ( 300
RO31-8959
4.4
106
26.5
a Ki and IC50 values were determined in duplicate using fluorescent substrate (see ref 4 for procedures; for HIV PR substrate, see Toth, M. V.;
Marshall, G. R. Int. J. Pept. Res. 1990, 36, 544). For a different assay condition, see ref 2b. nd, not determined. ni, No inhibition at 800 µM of
inhibitor. b Data were obtained at pH 5.25 at 37 °C in 0.1 M NaH2PO4, 0.1 M sodium citrate, 0.2 M NaCl, 0.1 mM DTT, 5% glycerol, and 5%
DMSO in volume. c Data were obtained at pH 5.25 at 37 °C in 0.1 M MES, 5% glycerol, and 5% DMSO in volume. d From ref 4. e From ref 3.
Results and Discussion
which define the S subsites are shown in Figure 2. In HIV PR,
Pro 81 and Val 82 are components for both S1 and S3 subsites
since they can affect binding of both P1 and P3 moieties,11
whereas Ile 98 and Gln 99 are the structurally aligned residues
for FIV PR.6 The X-ray structures also revealed that three
residues at S3 and S3′ subsites, Gly 48, Pro 81, and Val 82 of
HIV PR, were replaced with Ile 57, Ile 98, and Gln 99 in FIV
PR.6,11 As a result, the S3 and S3′ subsites of FIV PR are
sterically more congested than those in HIV PR, and these three
different residues may define the S3 and S3′ subsite specificities
of the enzymes.6 The results of these studies point to a new
direction for development of inhibitors effective against both
HIV PR and its drug-resistant variants.
Asymmetric Inhibitors with Small P3 Groups. The S3 and
S3′ subsite specificities of FIV PR and drug-resistant HIV PRs
with mutations affecting the S3 subsite were investigated further
to determine if there is a correlation between them. The new
inhibitors of HIV PR were synthesized with Ala at P3, Val or
Asn at P2, and various Phe-Pro isosteric cores for the P1-P1′
residues (Figure 3). The inhibitory activities of each compound
against FIV, HIV, and drug-resistant mutant HIV PRs were
determined as described previously,4 and the results are sum-
marized in Table 1.
Redesign of C2-Symmetric Inhibitors with small P3
Residues. Our initial work on the development of protease
inhibitors efficacious against both HIV and FIV was focused
on the systematic analysis of the S3 and S3′ subsite specificities
of the enzymes using a series of C2-symmetric inhibitors
containing (1S,2R,3R,4S)-1,4-diamino-1,4-dibenzyl-2,3-butane-
diol as a P1 and P1′ core and Val as P2 and P2′ residues.4 We
have demonstrated that FIV PR exhibited a strong preference
for small hydrophobic groups at the S3 and S3′ subsites, in
contrast to the high flexibility for the P3 and P3′ residues binding
to HIV PR.4 Kinetic studies have also indicated that the binding
preference observed in drug-resistant mutant HIV PRs is very
similar to that found in FIV PR.4 In addition, the most potent
FIV PR inhibitor, 1b, strongly inhibits FIV, HIV, and SIV
infections in tissue culture with virtually the same degree of
effectiveness.4
Each compound tested in this study is a competitive inhibitor
and is significantly more potent against HIV PR than FIV PR,
as expected. However, the new inhibitors 1b-6b and 7a showed
a remarkably different pattern of inhibitory activities compared
to their parent compounds 1a-5a, ABT-538, and RO31-8959.
Compounds 2a-4a, containing no P3 moieties, exhibited
marginal activities, with IC50 values in the range of 2-300 µM,
against HIV PR and did not show any significant inhibition of
FIV PR at 800 µM.3 Only the R-keto amide 5a showed
reasonable potency against HIV PR, with a Ki value of 214 nM.3
On the other hand, the modified inhibitors 2b-5b, which contain
a methyl group as the P3 residue, displayed 120-1000-fold
improved inhibitory activities against HIV PR and at least 3
orders of magnitude higher potency for FIV PR compared to
The X-ray structure of FIV PR complexed with inhibitor 1b
has been determined9 and has shown that the P1 and P3 side
chains are positioned very closely, consistent with previous
structural studies for HIV and FIV PRs that show that S1 and
S3 subsites are neighboring hydrophobic pockets that accom-
modate the corresponding P1 and P3 side chains.1e,4 Models of
1b bound to HIV and FIV PR (Figure 1) indicate that the S1
and S3 subsites in HIV PR constitute a much larger hydrophobic
pocket than the corresponding subsites found in FIV PR.
Because of its smaller size, FIV PR can only accommodate
inhibitors with a smaller size for P1 and P3 residues together,
and several drug-resistant HIV PRs are, indeed, found to have
smaller S3 and S1 subsites. Based on the X-ray structures of
HIV and FIV PRs complexed with inhibitors,1e,4 the residues
(10) Schno¨lzer, M.; Rackwitz, H.-R.; Gustchina, A.; Laco, G. S.;
Wlodawer, A.; Elder, J. H.; Kent, S. B. H. Virology 1996, 224, 268-275.
(11) Hosur, M. V.; Bhat, T. N.; Kempf, D. J.; Baldwin, E. T.; Liu, B.;
Gulnik, S.; Wideburg, N. E.; Norbeck, D. W.; Appelt, K.; Erickson, J. W.
J. Am. Chem. Soc. 1994, 116, 847-855.
(9) The X-ray structures of 1b complexed with HIV, FIV (3X), FIV
(V59I), and FIV (Q99V) PRs have been determined and will be published
separately: Li, M.; Morris, G.; Lee, T.; Laco, G. S.; Wong, C.-H.; Olson,
A.; Elder, J. H.; Wlodawer, A.; Gustchina, A. Submitted.

Protease Inhibitors with a Small P3 Residue
J. Am. Chem. Soc., Vol. 121, No. 6, 1999 1147
Figure 1. Models of HIV protease (top) and FIV protease (middle) complexed with 1b, indicating the small S3 site in FIV protease and the close
proximity of the P3 (CH3) and P1 (PhCH2) residues, a structural feature found in many drug-resistant HIV proteases. Bottom: a cut-away view of
the molecular surfaces of HIV (pink) and FIV PR (blue), in which the clipping plane is parallel to the plane of the two aspartates and cuts through
the P3 site.

1148 J. Am. Chem. Soc., Vol. 121, No. 6, 1999
Lee et al.
Figure 2. Amino acid side chains in the S subsites interacting with the inhibitors.
Figure 4. Proposed mechanism of inhibition by 5b. A water molecule
is added, with assistance of the enzyme, to the R-keto group to form a
gem-diol.
the hydroxyamide 4b is 57- and 44-fold more effective than its
diastereomer 3b against HIV and FIV PR, respectively. In
addition, the relative effectiveness of the P1-P1′ core structures
in 2b-5b against HIV PR is also consistent with the binding
pattern3 of their parent compounds 2a-5a. These results indicate
that introduction of Val and Ala as P2 and P3 residues can
improve the inhibition against both enzymes despite some
changes for the P1-P1′ core unit. In addition, extension of the
backbone of an inhibitor to contain an appropriate P3 moiety
is essential to exhibit high potency against FIV PR, which is
also consistent with our previous results4 with the C2-symmetric
inhibitor 1b.
Figure 3. Dissymmetric inhibitors with small P3 residues. Compounds
2b-6b and 7 contain a methyl group as the P3 residue.
The kinetic results of inhibitors 6b and 7a, which were
modified from existing drugs ABT-538 and RO31-8959
(Saquinavir), respectively, were even more promising. Com-
pound 7a was found to have Ki values of 1.5 nM and 2.6 µM
against HIV and FIV PR, respectively. The inhibitory activities
of the original drug RO31-8959, were also evaluated under the
same assay conditions, and it was found to have Ki values of
1.6 nM and 76 µM for HIV and FIV PR, respectively. These
results clearly indicated that 7a retained the original inhibitory
activity of RO31-8959 against HIV PR, which was already
optimized for the wild-type enzyme, but showed 29-fold
enhanced potency against FIV PR. Inhibitor 6b also exhibited
activities comparable to those of 7a, with Ki values of 3.0 nM
and 3.7 µM for HIV and FIV PR, respectively. However, 7b
was 7.5- and 51-fold less potent than 7a against HIV and FIV
PR, respectively. Changing the P2 Val residue of 7a to Asn
increased the hydrophilicity of the inhibitor, which could be a
major cause of lower activity found in 7b for both enzymes. In
their parent compounds. In particular, 5b was found to be a
slow binding inhibitor, with Ki values of 2.5 and 46 nM against
HIV and FIV PR, respectively. This level of potency against
FIV PR by the inhibitor with a molecular weight of only 649
was truly remarkable, considering that the smallest efficient
substrate for the enzyme is an eight-residue peptide, Ac-Pro-
Gln-Ala-Tyr∼Pro-Ile-Gln-Thr.10 Since the ketone moiety is not
hydrated in aqueous solution according to 13C NMR studies,
the increased inhibitory activity of 5b may occur via enzyme-
assisted hydration of the ketone moiety within the active site
to form a gem-diol as transition-state mimic, similar to the case
of a related R-keto amide inhibitor observed previously by X-ray
structure and 13C NMR analyses3 (Figure 4).
The relative inhibitory activities of inhibitors 2b-5b against
FIV PR are similar to the relative activities against HIV PR.
For example, 5b is a superior inhibitor of both enzymes, and

Protease Inhibitors with a Small P3 Residue
J. Am. Chem. Soc., Vol. 121, No. 6, 1999 1149
Table 2. Inhibition of FIV and HIV PRs by C2-Symmetric Diolsa
FIV PRb
FIV PRb
HIV PR,c
HIV PR,c
inhibitor (X)
Ki (nM)
IC50 (nM)
Ki (nM)
inhibitor (Y)
Ki (nM)
IC50 (nM)
Ki (nM)
8 (Ala-Cbz)
9 (Leu-Cbz)
10 (Phe-Cbz)
11 (Val-Cbz)
62 ( 9
235
11600
29300
18800
6.5 ( 1.3
0.87 ( 0.12
5.5 ( 0.8
nd
1b (Ala-Cbz)
9b (Leu-Cbz)
10b (Phe-Cbz)
12 (Ser-Cbz)
13 (Thr-Cbz)
14 (Abu-Cbz)
15 (Nva-Cbz)
16 (Nle-Cbz)
41 ( 7d
159 ( 15d
7000 ( 500d
32 ( 5
142 ( 25
nd
72
8200
92400
66
7250
95
1.5 ( 0.3d
1.4 ( 0.3d
2.6 ( 0.4d
0.58 ( 0.1
7.7 ( 1.9
nd
230 ( 34
487 ( 20
248 ( 47
nd
nd
6700
29400
nd
nd
a Ki values were determined in duplicate. nd, not determined. b Data were obtained at pH 5.25 at 37 °C in 0.1 M NaH2PO4, 0.1 M sodium citrate,
0.2 M NaCl, 0.1 mM DTT, 5% glycerol, and 5% DMSO in volume. c Data were obtained at pH 5.25 at 37 °C in 0.1 M MES, 5% glycerol, and
5% DMSO in volume. d From ref 4.
fact, 7b was more soluble in water than 7a and thus would
require higher desolvation energy to bind the hydrophobic active
sites of enzymes. It is interesting to note that desymmetrization
of 1b by changing one hydroxy group to a ketone (1c) or a
methylene (1d) reduces the HIV PR potency by 1.4- or 3.5-
fold, respectively. The above results suggest that inhibitors with
a small P3 residue are effective against both FIV PR and HIV
PR. These results are consistent with the results of molecular
modeling, which show HIV and FIV PR binding to Saquinavir
and its modified derivative with a small P3 residue (Figure 5).
with 1b,9 compounds (12, 13) containing a hydroxy group at
P3 and P3′ residues were synthesized with the hope that further
improvement in inhibition would be obtained, as favorable
H-bonding interactions of these polar side chains with the water
molecule may occur.
The inhibitory effects of each C2-symmetric inhibitor on HIV
and FIV PRs were determined, and the results are recorded in
Table 2. For comparison, the Ki values for inhibition of FIV
and HIV PR by the C2-symmetric inhibitors with a benzyl group
at P1 and P1′ are also included (1b, 9b, 10b).
Inhibition of Drug-Resistant Mutant HIV PRs. The modi-
fied inhibitors with dual efficacy against FIV and HIV PR (1b-
d, 4b, 5b, 6b, and 7a) were also tested against drug-resistant
mutant HIV PRs G48V and V82F. These mutant enzymes were
selected since Gly 48 and Val 82 are within the S3 and S3′
subsites6,11 and have been identified as some of the most
frequently mutated residues associated with development of drug
resistance.2 Against these mutant enzymes, all modified inhibi-
tors retained most of their original potency, and their relative
inhibitory activity was also directly proportional to the efficacy
against wild-type HIV PR. In particular, modification of the
FDA-approved drugs ABT-538 and RO31-8959 containing a
bulky P3 group to the ones with a methyl group at P3 (i.e., 6b
and 7a) showed significantly improved inhibitory activity against
the mutants, with only 3.2- and 6.5-fold higher IC50 values for
the V82F and G48V mutant variants, respectively, compared
to 6- and 27-fold higher IC50 values for the parent compounds.
It is noteworthy that V82F and G48V mutants are known to be
less efficient enzymes than the wild-type HIV PR.2b,d Therefore,
inhibitory activities of compounds tested against these mutant
enzymes are expected to be lower compared to wild-type HIV
PR. Compound 1b was also active in cell culture.12 The IC90
values of 1b against the wild-type HIV PR and the I84V and
82F/84V mutants are 0.1, 0.4, and 0.9 µM, respectively.
All the C2-symmetric diols tested in this study showed
competitive inhibition of both the feline and human lentivirus
PRs in every case, but with at least an order of magnitude higher
potency against HIV PR. Among the inhibitors with iso-butyl
groups at the P1-P1′ core (8-11), compound 8, with Ala at
P3 and P3′, is the best inhibitor of FIV. This maximum
inhibitory activity observed in 8 was reduced by increasing the
size of the side chain of the P3 and P3′ residues. In fact, the
measured Ki values of inhibitors 9, 10, and 11 were 3.7-, 7.9-,
and 4-fold higher, respectively, than that of 8. This specificity
for small hydrophobic groups at P3 and P3′ sites found among
the C2-symmetric inhibitors 8-10 with iso-butyl groups as the
P1-P1′ core against FIV PR was consistent with the observation
for the analogous inhibitors 1b, 9b, and 10b. In addition, 8 and
9 exhibited almost the same degree of potency against FIV PR
compared to the benzyl analogues 1b and 9b, respectively. This
result indicates that Leu can bind the S1 and S1′ subsites of the
enzyme as effectively as Phe. In the previous report,4 the
explanation for the observed preference for small P3 and P3′
moieties in FIV PR was that bulky groups at the P3 and P3′
positions of the inhibitor could provoke unfavorable interactions
at the sterically congested S3 and S3′ subsites of FIV PR. In
particular, 10b (Ki ) 7.0 µM) exhibited 170-fold lower potency
than 1b. This proposal is still valid for the lower inhibitory
activities of 9, 10, and 11 compared to that of 8 against FIV
PR. However, it is noteworthy that 10 showed 14-fold higher
inhibitory activity than its analogue 10b. This improved potency
against FIV PR observed in 10 cannot be explained by the above
proposal, since both inhibitors contain Phe as P3 and P3′
residues. Therefore, current observations also suggest that the
steric interaction between neighboring P1 and P3 side chains is
another crucial factor to be considered in the design of new
inhibitors. Indeed, 10, containing smaller P1 and P1′ side chains
than inhibitor 10b, can provide more room at S3 and S3′ binding
C2-Symmetric Inhibitors with an iso-Butyl-2,3-diol Core
as P1 and P1′. Since the side chains of P1 and P3 residues of
1b are positioned very close to each other in the S1 and S3
subsites of the enzyme, we have investigated the activities of
new C2-symmetric inhibitors (8-11) with an iso-butyl group
at P1 and P1′. In addition, since water molecules were identified
in the S3 and S3′ subsites of both HIV and FIV PR complexed
(12) Bacheler, L. T.; Paul, M.; Jadhar, P. K.; Otto, M.; Stone, B.; Miller,
J. AntiViral Chem. Chemother. 1994, 5, 111.

1150 J. Am. Chem. Soc., Vol. 121, No. 6, 1999
Lee et al.
Figure 5. Models of HIV protease (top) and FIV protease (middle) complexed with RO31-8959, and FIV PR complexed with the modified
inhibitor 7a (bottom). The P3 group of RO31-8959 is too big to fit the S3 subsite of FIV PR, whereas 7a, with methyl group as P3, residue shows
a good fit.

Protease Inhibitors with a Small P3 Residue
J. Am. Chem. Soc., Vol. 121, No. 6, 1999 1151
the binding specificity of the enzymes. We have also investi-
gated the effect of P2 residue in 1b on the inhibition of FIV
and HIV PR and found that Val is better than L-R-aminobutyric
acid (IC50 ) 41 and 1400 for HIV and FIV PR), norvaline
(IC50 ) 137 and >10 000 for HIV and FIV PR), and norleucine
(IC50 ) 9800 for HIV PR).
Finally, compounds containing a hydroxy group at P3 and
P3′ side chains (12 and 13) were also effective inhibitors of
both HIV and FIV PR. In particular, 12 displayed the highest
potency, with Ki values of 0.58 and 32 nM against HIV and
FIV PRs, respectively. It is noteworthy that 12 is more
hydrophilic than 1b by two additional hydroxy groups and would
require more desolvation energy in order to bind to the
hydrophobic active sites of enzymes. However, 12 exhibited a
3-fold higher potency against HIV PR and similar inhibition
against FIV PR, compared to 1b. These results indicate that
binding of 12 is significantly enhanced by the two hydroxyl
groups of the P3 and P3′ side chains, presumably by promoting
favorable electrostatic interactions with the crystallographic
water molecules at the S3 and S3′ subsites. The ability of 12 to
prevent infection of FIV in tissue culture was also examined. It
was, however, not as effective as 1b. It has been known that
introduction of hydrophilic functional groups, such as a hydroxyl
or carboxyl group, to the P2 and P2′ positions of C2-symmetric
inhibitors would cause a dramatic loss of potency against HIV
PR in tissue culture, though the inhibitory activity in vitro has
little change.15 Our ex ViVo assay results have confirmed a
similar activity loss in tissue culture by the presence of a
hydrophilic group at P3 and P3′. These results suggest that
increasing the overall polarity and hydrophilicity of compounds
may result in the reduction of efficacy in ViVo.
The above results indicate that the interaction of P1 and P3
residues in the active sites of HIV and FIV PR is a crucial factor
to be considered for maximizing the binding affinity of
inhibitors. Although it has been considered that Phe can provide
ideal binding at the S1 subsite of HIV PR,1 our observations
suggest that, with appropriate P3 and P3′ moieties, one can also
develop potent inhibitors with P1 and P1′ side chains smaller
than the benzyl group.
Figure 6. Effect of the size of the P3 side chain (based on the van der
Waals volume in Å3) on the inhibition of FIV and HIV proteases.
sites to accommodate bulky benzyl groups. This proposal was
further confirmed by the modeling of FIV PR complexed with
8. The structure reveals that the side chains of P1 and P3
residues are positioned closely, and the combined S1 and S3
pocket is not wide enough to accommodate two benzyl groups.
It is also expected that binding of P1 and P3 moieties can be
affected by each other, and an appropriate combination of P1
and P3 residues is essential for good binding. Figure 6 shows
the effects of the size of P3 side chain on the inhibition of FIV
and HIV proteases.
It has been determined that HIV PR exhibits a high degree
of flexibility in binding at the S3 and S3′ subsites.4 The results
from the inhibition studies of the diols 8-10 against HIV PR
showed very different patterns compared to their analogues 1b,
9b, and 10b. The Ki values of 8 and 10 were 4.3- and 2.0-fold
higher than those of 1b and 10b, respectively. This indicates
that Phe at P1 and P1′ is better than Leu for binding to HIV
PR. However, 9 is a more effective inhibitor than 9b and also
showed 7.5- and 6.3-fold higher potency against HIV PR
compared to 8 and 10, respectively. This significant improve-
ment was not observed from 1b, 9b, and 10b. These kinetic
results indicate that introduction of iso-butyl groups at S3 and
S3′ subsites can improve the potency of an inhibitor containing
a medium-size P1-P1′ core and suggest that the overall size
of the combined P1 and P3 residues will significantly affect
Synthesis
The key intermediates (1S,2R,3R,4S)-1,4-bis[(N-Cbz)amino]-
1,4-dibenzyl-2,3-diol and (1S,2R,3R,4S)-1,4-bis[(N-Cbz)amino]-
1,4-diisobutyl-2,3-diol (17) (Scheme 1) were prepared by the
stereoselective pinacol coupling of L-Cbz-phenylalanal and
L-Cbz-leucinal, respectively, using Pedersen’s procedure.16 The
minor diastereomeric impurities of the coupling reaction were
removed by flash column chromatography after protection of
the diol as an isopropylidene derivative (18). The Cbz groups
of 18 were removed to yield the diamine 19 by hydrogenation.
(13) (a) Kempf, D. J.; Sham, H. L.; Marsh, K. C.; Flentge, C. A.;
Betebenner, D.; Green, B. E.; McDonald, E.; Vasavanonda, S.; Saldivar,
A.; Wideburg, N, E.; Kati, W. M.; Ruiz, L.; Zhao, C.; Fino, L.; Patterson,
J.; Molla, A.; Plattner, J. J.; Norbeck, D. W. J. Med. Chem. 1998, 41, 602-
617. (b) Kempf, D. J.; Marsh, K. C.; Fino, L. C.; Bryant, P.; Craig-Kennard,
A.; Sham. H. L.; Zhao, C.; Vasavanond, S.; Kohlbrenner, W. E.; Wideburg,
N. E.; Saldivar, A.; Green, B. E.; Herrin, T.; Norbeck, D. W. Bioorg. Med.
Chem. 1994, 2, 847-858.
(14) Thompson, W. J.; Ghosh, A. K.; Holloway, M. K.; Lee, H. Y.;
Munson, P. M.; Schwering, J. E.; Wai, J.; Darke, P. L.; Zugay, J.; Emini,
E. A.; Schleif, W. A.; Huff, J. R.; Anderson, P. S. J. Am. Chem. Soc. 1993,
115, 801-803.
(15) Budt, K.-H.; Peyman, A.; Hansen, J.; Knolle, J.; Meichsner, C.;
Paessens A.; Ruppert, D.; Stowasser, B. Bioorg. Med. Chem. 1995, 3, 559-
571.
(16) Konradi, A. W.; Pedersen, S. F. J. Org. Chem. 1992, 57, 28-32.

1152 J. Am. Chem. Soc., Vol. 121, No. 6, 1999
Lee et al.
Scheme 3. Synthesis of the R-Ketoamides 5a and 5ba
Scheme 1a
a Conditions: (a) BH3, THF; (b) Swern oxidation (90%); (c)
NaHSO3, H2O; (d) KCN; (e) HCl (6 N in dioxane); (f) Cbz-Cl, NaOH,
H2O (52%, 4 steps); (g) HBTU, Et3N, CH3CN (73%); (h) Pd/C, H2,
EtOAc; (i) Cbz-Ala-Val-OH, HBTU, Et3N, CH3CN (65%, 2 steps); (j)
Dess-Martin (63%).
a Conditions: (a) 2,2-dimethoxypropane, p-TsOH (80%); (b) Pd/C,
H2, MeOH (99%); (c) HBTU, Cbz-Val, Et3N, CH3CN (89%); (d)
HBTU, Cbz-amino acids, Et3N, CH3CN; (e) p-TsOH, MeOH.
Scheme 4. Synthesis of the Modified Existing Drugs 6aa
and 7a,bb
Scheme 2. Synthesis of 2ba
a Conditions: (a) NMM, THF, i-BuOCOCl; (b)CH2N2, Et2O; (c) HCl
(85%, 3 steps); (d) NaBH4, EtOH (90% de, 81%); (e)NaOMe, MeOH
(96%); (f) MeOH, Et3N (90%); (g) Pd/C, H2, EtOAc; (h) CBz-Ala-
Val-OH, HBTU, Et3N,CH3CN (49%, 2 steps).
Amine 19 was directly coupled with Cbz-Val using HBTU17 to
give adduct 20. Four different P3 and P3′ residues were then
introduced to adduct 20 by applying the same deprotection and
coupling procedures described above to give 22-25. Finally,
the target inhibitors 8-11 were prepared by deprotection of the
isopropylidene group from the corresponding precursors (Scheme
1). Compounds 12-16 were obtained by applying the same
procedure described previously using (1S,2R,3R,4S)-1,4-bis[(N-
Cbz)amino]-1,4-dibenzyl-2,3-diol as a starting material.
The hydroxyethylamine inhibitor 2b was prepared by cou-
pling the proline derivative 27 to the epoxide 283,19 via reflux
in methanol, using triethylamine as shown in Scheme 2. The
synthesis of the core isostere 5a has been modified from the
method previously employed by our group. The R-hydroxy acid
29, prepared from known procedures,3,20 was coupled to the
proline derivative 27 to give the R-hydroxy amides 3a and 3b.
Hydrogenation of the diasteromers 3a and 3b followed by
HBTU-mediated coupling with Cbz-Ala-Val-OH gave 4a and
4b in 65% yield. Dess-Martin oxidation21 of the R-hydroxy
a Conditions: (a) PhB(OH)2, PhMe, reflux; (b) THF, rt (78%, 2
steps); (c) Cbz-Ala-Val-OH, HBTU, Et3N, CH3CN (73%). b Conditions:
(a) MeOH, Et3N (80%); (b) TFA, CH2Cl2; (c) HBTU, Et3N, CH3CN,
Cbz-Ala-Val-OH (76%) or Cbz-Ala-Asn-OH (78%).
amides 3a-4b gave the corresponding R-keto amides 5a and
5b in moderate yield (Scheme 3).
The synthesis of 6b began with the cyclic boronate 31
prepared by refluxing the diamine 30 with phenylboric acid.13
Condensation of the phenyl boronate 31 with carbonate 32
followed by HBTU-mediated coupling with Cbz-Ala-Val-OH
gave the desired product 6b (Scheme 4). The synthesis of the
modified Saquinavir derivatives 7a and 7b began with conden-
sation of the isoquinoline derivative 34 with epoxide 33 to give
the adduct 7 in 80% yield.14 Removal of the BOC group in 7
with TFA followed by HBTU coupling with Cbz-Ala-Val-OH
or Cbz-Ala-Asn-OH gave the modified inhibitors 7a and 7b,
respectively (Scheme 4).
(17) Dourtoglou, V., Gross, B. Synthesis 1984, 572-574.
(18) Biosym Technologies, San Diego, CA, 1998.
(19) Hung, R. R.; Straub, J. A.; Whitesides, G. M. J. Org. Chem. 1991,
56, 3849-3855.
Conclusion
In summary, manipulation of the backbone of HIV PR
inhibitors to create appropriate P3 residues can improve potency
against FIV, HIV, and mutant HIV PRs, regardless of the
structure of the P1-P1′ core units. The inhibitors with small
(20) Munoz, B.; Giam, C.-Z.; Wong. C.-H. Bioorg. Med. Chem. 1994,
2, 1085-1090.
(21) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7287.

Protease Inhibitors with a Small P3 Residue
J. Am. Chem. Soc., Vol. 121, No. 6, 1999 1153
a colorless oil. The crude amine, Cbz-Ala-Val-OH (12 mg, 0.039
mmol), HBTU (14 mg, 0.038 mmol), and Et3N (4.7 mg, 0.046 mmol)
in CH3CN (1 mL) gave 2b (12 mg, 49%) as a white solid: 1H NMR
(500 MHz, DMSO-d6, 60 °C) δ 0.76 (6H, d, J ) 6.7 Hz), 1.20 (3H, d,
J ) 7.1 Hz), 1.26 (9H, s), 1.64-1.70 (3H, br), 1.86-1.90 (1H, m),
1.98-2.00 (1H, m), 2.67 (1H, m), 2.69 (1H, m), 2.93 (1H, b), 2.96
(1H, br), 3.03 (3H, m), 3.50 (1H, br), 4.00-4.13 (3H, m), 5.04 (2H,
s), 7.10-7.50 (14H, m); 13C NMR (100 MHz, DMSO-d6, 60 °C) δ
18.0, 19.2, 28.3, 30.0, 30.8, 34.7, 38.2, 49.5, 50.1, 52.8, 55.5, 57.7,
59.4, 65.3, 67.8, 71.0, 125.7, 127.6, 127.7, 127.8, 128.3, 129.2, 137.0,
138.8, 155.6, 170.2, 170.3, 172.0; HRMS (FAB+) calcd for MCs+
C35H51N5O6Cs m/e 770.2894, found m/e 770.2922.
P3 and bulky P1 described in this study are effective against
FIV and HIV PR and exhibit high potency against drug-resistant
mutant HIV PRs. In addition, modification of some existing
HIV PR inhibitors to have a small P3 group also enhances their
inhibition of FIV PR and mutant HIV proteases without affecting
the activity against wild-type HIV PR. The development of
broad-based inhibitors described in this study contributes
significantly to our understanding of the specificity and resis-
tance development of the aspartyl protease and provides a new
strategy for the development of new antiviral pharmaceuticals.
Although the current results represent only an initial step toward
development of therapeutic agents efficacious against both native
and mutant HIV proteases, using FIV PR as a general model
for the drug-resistant mutant HIV PRs is clearly an effective
strategy. One may thus use cats as a drug-resistant animal model
to accelerate the drug development process.
Compounds 3b and 4b. A mixture of diastereomer 3a and 4a3 (85
mg, 0.170 mmol) was hydrogenated with 10% Pd/C (20 mg) in EtOAc
(3 mL) to give an amine as a colorless oil. The crude amine, Cbz-Ala-
Val-OH (55 mg, 0.170 mmol), HBTU (65 mg, 0.170 mmol), and Et3N
(20 mg, 0.20 mmol) in CH3CN (4 mL) gave separable mixture of 3b
and 4b (72 mg, 65%) as a white solid.
1
Compound 3b: H NMR (400 MHz, CD3OD, 20 °C) δ 0.82 (6H,
Experimental Section.
br s), 1.28-1.33 (12H, m), 1.80-2.05 (5H, m), 2.80-2.97 (4H, m),
3.36 (1H, br), 4.10-4.20 (2H, m), 4.25 (1H, br s), 4.44 (1H, br s),
5.09 (2H, s), 7.20 (1H, br), 7.24-7.55 (13H, m); 13C NMR (100 MHz,
CD3OD, 20 °C) δ 18.0, 18.3, 19.9, 25.5, 28.8, 30.7, 32.1, 38.8, 47.8,
52.0, 52.9, 60.1, 62.3, 67.7, 70.4, 127.7, 128.9, 129.0, 129.5, 129.6,
130.5, 139.4, 158.1, 172.0, 172.7, 173.8; HRMS (FAB+) calcd for
MCs+ C35H51N5O6Cs m/e 770.2894, found m/e 770.2922.
Chemical Synthesis. General Procedures. Analytical TLC was
performed on precoated plates (Merck, silica gel 60F-254). Silica gel
used for flash column chromatography was Mallinckrodt Type 60
(230-400 mesh). NMR (1H, 13C) spectra were recorded on either a
Bruker AMX-400 or an Am-500 MHz Fourier transform spectrometer.
Coupling constants (J) are reported in hertz (Hz), and chemical shifts
are reported in parts per million (δ) relative to tetramethylsilane (TMS,
0.0 ppm) with CDCl3, DMSO, or CD3OD as solvent.
1
Compound 4b: H NMR (400 MHz, DMSO-d6, 60 °C) δ 0.74-
0.77 (6H, m), 1.21-1.23 (3H, overlapping), 1.23 (9H, s), 1.81-1.91
(5H, br), 2.70 (1H, m), 2.71 (1H, m), 2.78 (1H, br), 2.89 (1H, br),
3.00 (1H, br), 3.36-3.41 (4H, m), 5.04 (2H, s), 7.16-7.44 (14H, m);
13C NMR (100 MHz, DMSO-d6, 60 °C) δ 17.1, 17.6, 18.7, 23.3, 28.1,
30.1, 36.8, 37.8, 45.7, 49.6, 50.0, 57.3, 60.1, 65.1, 69.4, 125.6, 127.1,
127.2, 127.6, 127.8, 128.6, 136.6, 138.2, 155.1, 169.9, 171.6; HRMS
(FAB+) calcd for MCs+ C35H51N5O6Cs m/e 770.2894, found m/e
770.2947.
All other reagents were commercial compounds of the highest purity
available and purchased from the Aldrich, Sigma, Nova Biochem, or
Bachem.
General Procedure for Coupling Reaction. To a solution of a free
amine (1.0 mol equiv) and carboxylic acid (1.0 mol equiv) in dry
CH3CN (0.1-0.15 M) was added HBTU (1.0 mol equiv) followed by
Et3N (1.0 mol equiv) at 20 °C under Ar atmosphere. The reaction
mixture was stirred for 15 min and then quenched by addition of brine
and extracted with EtOAc. The organic layer was then washed
sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, dried
over MgSO4, filtered, and concentrated in vacuo. The crude product
was purified by flash chromatography to give the desired product.
Compound 1c. Compound 1b4 (5.3 mg, 0.0058 mmol) was treated
with a mixture of freshly prepared dimethyldioxirane in acetone (0.047
M, 0.372 mL, 3 equiv) and CDCl3 (0.3 mL) at 25 °C. The reaction
was monitored with analytical HPLC. After 3 days of stirring, the
reaction mixture was concentrated and purified on a semipreparative
C18 column (HPLC yield 63%): 1H NMR (500 MHz, DMSO-d6, 20
°C) δ 0.65-0.72 (12H, m), 1.11 (3H, d, J ) 5.9 Hz), 1.14 (3H, d, J )
5.9 Hz), 1.75-1.85 (2H, m), 2.67-2.77 (2H, m), 2.82 (1H, dd, J )
4.1, 11.6 Hz), 3.02 (1H, bd, J ) 10.2 Hz), 4.05-4.13 (4H, m), 4.26
(1H, bd, J ) 5.9 Hz), 4.57-4.62 (1H, m), 4.95-5.03 (4H, m), 5.11
(1H, d, J ) 5.2 Hz), 7.10-7.15 (4H, m), 7.16-7.23 (6H, m), 7.26-
7.37 (9H, m), 7.43-7.47 (2H, m), 7.52 (1H, d, J ) 7.5 Hz), 7.54 (1H,
d, J ) 7.4 Hz), 7.72 (1H, d, J ) 7.4 Hz), 8.42 (1H, d, J ) 6.8 Hz);
HRMS (FAB+) calcd for MCs+ C50H62N6O10Cs m/e 1139.3582, found
m/e 1139.3538.
Compound 5b. Dess-Martin reagent (22 mg, 0.074 mmol) was
added to a solution of diasteromers 3b and 4b3 (16 mg, 0.025 mmol)
in CH2Cl2 (2 mL). The reaction mixture was stirred for 12 h and then
quenched by addition of brine and extracted with EtOAc. The organic
layer was then washed with saturated aqueous NaHCO3 and brine, dried
over MgSO4, filtered, and concentrated in vacuo. The crude product
was purified by flash chromatography to give 5b (10 mg, 63%) as a
mixture of isomers (1:1): 1H NMR (500 MHz, DMSO-d4, 60 °C) δ
0.76-0.85 (6H, m), 1.16, 1.18 (3H, d, J ) 7.0 Hz), 1.24, 1.26 (9H, s),
1.70-1.80 (3H, m), 1.82-2.08 (3H, m), 2.15-2.22 (1H, m), 2.60 (1H,
dd, J ) 10.5, 14.6 Hz), 2.96 (1H, dd, J ) 9.1, 14.2 Hz), 3.17 (1H, dd,
J ) 4.9, 14.2 Hz), 3.24-3.26 (1H, m), 3.38-3.65 (5H, m), 4.07-4.12
(3H, m), 4.19 (1H, t, J ) 6.6 Hz), 4.24 (2H, t, J ) 5.7 Hz), 4.62 (1H,
q, J ) 4.3 Hz), 4.95-4.99 (1H, m), 5.01 (2H, s), 5.26 (1H, m), 7.15-
7.45 (12H, m); 13C NMR (100 MHz, DMSO-d4, 60 °C) δ 17.6, 18.0,
19.1, 21.7, 24.4, 28.4, 29.1, 31.0, 32.4, 33.7, 34.2, 35.6, 47.4, 50.0,
50.2, 55.9, 56.7, 56.8, 57.1, 57.5, 59.8, 60.4, 65.3, 126.2, 126.5, 127.7,
127.7, 128.1, 128.2, 128.3, 128.4, 128.6, 128.9, 137.1, 137.2, 138.0,
140.5, 155.6, 162.0, 162.2, 169.8, 170.7, 171.0, 171.4, 172.1, 172.2,
196.0, 198.1; HRMS (FAB+) calcd for MCs+ C35H47N5O7Cs m/e
782.2530, found m/e 782.2558.
Compound 1d. (2S,3S,5S)-3-Hydroxy-2,5-bisamino-1,6-diphenyl-
hexane13b (15.7 mg, 0.055 mmol) was coupled to N-Cbz-Ala-Val-OH
(39 mg, 0.12 mmol) to give the product (39 mg, 79%) as a white
solid: 1H NMR (500 MHz, DMSO-d6, 20 °C) δ 0.56-0.61 (2H, m),
0.64-0.68 (2H, m), 0.72-0.76 (6H, m), 0.84-0.88 (2H, m), 1.17-
1.25 (12H, m), 1.49-1.53 (2H, m), 1.79-1.91 (2H, m), 2.55-2.60
(1H, m), 2.69-2.76 (2H, m), 3.60 (1H, m), 4.03-4.14 (6H, m), 4.47
(1H, m), 5.00 (4H, m), 7.08-7.43 (20H, m); 13C NMR (100 MHz,
DMSO-d6, 20 °C, major peaks) δ 17.9, 18.1, 18.2, 18.2, 19.2, 19.3,
30.7, 30.9, 37.4, 38.8, 46.8, 50.0, 53.2, 57.6, 57.7, 65.3, 68.2, 78.7,
79.2, 125.7, 125.8, 127.7, 127.74, 127.8, 127.83, 127.9, 128.0, 128.3,
129.0, 129.2, 137.0, 138.5, 139.2, 155.6, 169.9, 170.5, 172.0, 172.2,
172.4; HRMS (FAB+) calcd for MCs+ C50H64N6O9Cs m/e 1025.3789,
found m/e 1025.3748.
Compound 6b. The amine 6a13 (20 mg, 0.047 mmol), Cbz-Ala-
Val-OH (15 mg, 0.047 mmol), HBTU (18 mg, 0.047 mmol), and Et3N
(5.7 mg, 0.056 mmol) in CH3CN (1 mL) gave the title compound 6b
(25 mg, 73%) as a white solid: 1H NMR (400 MHz, DMSO-d6, 80
°C) δ 0.75-0.78 (6H, m), 1.21 (3H, d, J ) 7.1 Hz), 1.48-1.56 (2H,
m), 1.85-1.94 (1H, m), 2.62-2.79 (3H, m), 3.58 (1H, m), 3.79 (1H,
m), 4.05-4.08 (1H, m), 4.10-4.13 (2H, m), 5.04 (2H, d, J ) 2.5 Hz),
5.14 (2H, s), 7.08-7.34 (15H, m), 7.80 (1H, s), 8.98 (1H, s); 13C NMR
(100 MHz, DMSO-d6, 80 °C) δ 17.4, 18.7, 30.2, 37.0, 38.0, 46.9, 50.0,
52.9, 55.5, 57.0, 57.5, 65.1, 68.6, 125.3, 127.1, 127.2, 127.4, 127.4,
127.8, 128.5, 128.6, 128.7, 133.5, 136.9, 138.2, 138.8, 142.3, 154.4,
155.0, 169.2, 170.0, 171.5; HRMS (FAB+) calcd for MCs+ C39H47N5O7-
SCs m/e 862.2277, found m/e 862.2251.
Compound 2b. Compound 2a3 (18 mg, 0.039 mmol) was hydro-
genated with 10% Pd/C (15 mg) in EtOAc (2 mL) to give an amine as
Compound 7a. The amine14 (30 mg, 0.075 mmol) prepared from
the BOC derivative 7, Cbz-Ala-Val-OH (24 mg, 0.075 mmol), HBTU

1154 J. Am. Chem. Soc., Vol. 121, No. 6, 1999
Lee et al.
(28 mg, 0.075 mmol), and Et3N (9.1 mg, 0.09 mmol) in CH3CN (1
mL) gave the title compound 7a (40 mg, 76%) as a white solid: 1H
NMR (500 MHz, DMSO-d6, 80 °C) δ 0.73 (3H, d, J ) 6.8 Hz), 0.75
(3H, d, J ) 6.8 Hz), 1.19 (3H, d, J ) 7.08 Hz), 1.26 (9H, s), 1.31-
1.43 (4H, br), 1.48 (2H, br), 1.59 (1H, br), 1.63 (1H, br), 1.73 (1H,
br), 1.82-1.98 (3H, m), 2.05-2.13 (2H, m), 2.57-2.66 (3H, m), 2.93-
2.98 (2H, m), 4.06-4.20 (3H, m), 5.03 (2H, s), 7.09-7.39 (14H, m);
13C NMR (100 MHz, DMSO-d6, 80 °C) δ 16.9, 17.8, 18.2, 18.6, 19.3,
20.4, 24.8, 25.9, 28.4, 29.8, 30.6, 31.2, 32.4, 33.3, 35.7, 49.9, 52.1,
57.3, 58.0, 65.3, 69.8, 125.4, 127.6, 127.7, 127.8, 128.3, 129.3, 137.0,
140.2, 155.6, 170.1, 172.0, 172.7; HRMS (FAB+) calcd for MCs+
C40H59N5O6Cs m/e 838.3520, found m/e 838.3546.
NMR (500 MHz, DMSO-d6, 80 °C) δ 0.80-0.88 (12H, m), 1.15-
1.20 (1H, m), 1.22 (3H, d, J ) 7.0 Hz), 1.45-1.51 (1H, m), 1.53-
1.60 (1H, m), 2.04 (1H, q, J ) 6.2 Hz), 3.48 (1H, s), 4.06-4.15 (2H,
m), 4.24 (1H, dd, J ) 7.5, 2.5 Hz), 5.03 (2H, dd, J ) 16.5, 11.5 Hz),
7.23 (1H, d, J ) 9.0 Hz), 7.26-7.31 (1H, m), 7.33-7.40 (5H, m),
7.45 (1H, d, J ) 8.9 Hz); HRMS (FAB+) calcd for MCs+ C47H72N6O10-
Cs m/e 1013.4364, found m/e 1013.4324.
General Procedure for Deprotection. To a solution of 22 (25 mg,
0.030 mmol) in MeOH (1.5 mL) was added a catalytic amount of
p-TsOH. The reaction mixture was heated at 60 °C for 24 h and then
diluted with EtOAc (10 mL). The organic solution was washed with
saturated aqueous NaHCO3 (2 mL) and saturated aqueous NaCl (2 mL),
dried over MgSO4, filtered, and concentrated in vacuo to give free diol
21 (14 mg, 57%) as a white solid: 1H NMR (500 MHz, DMSO-d6, 80
°C) δ 0.82-0.88 (12H, m), 1.14-1.20 (1H, m), 1.24 (3H, d, J ) 7.1
Hz), 1.38-1.58 (2H, m), 1.53-1.60 (1H, m), 2.01 (1H, q, J ) 6.8
Hz), 2.86 (1H, s), 4.06-4.16 (3H, m), 5.03 (2H, s), 6.84-6.89 (2H,
m), 7.25-7.70 (1H, m), 7.31-7.74 (4H, m), 7.40 (1H, d, J ) 8.6 Hz);
13C NMR (125 MHz, DMSO-d6, 80 °C) δ 17.24, 17.5, 18.6, 21.5, 22.6,
23.6, 29.5, 41.5, 47.2, 49.8, 57.7, 65.0, 72.5, 126.9, 127.0, 127.6, 137.0,
154.54, 169.6, 171.6; HRMS (FAB+) calcd for MCs+ C44H68N6O10Cs
m/e 973.4051, found m/e 973.4028.
Compound 7b. The amine14 (49 mg, 0.122 mmol) prepared from
the BOC derivative 7, Cbz-Ala-Asn-OH (41 mg, 0.122 mmol), HBTU
(46 mg, 0.122 mmol), and Et3N (12.4 mg, 0.09 mmol) in DMF (1 mL)
gave the title compound 7a (42 mg, 78%) as a white solid: 1H NMR
(500 MHz, DMSO-d6, 60 °C) δ 1.22 (3H, d, J ) 7.1 Hz), 1.27 (9H, s),
1.31-1.85 (10H, m), 1.90 (2H, m), 2.22 (1H, br), 2.33 (1H, br), 2.49-
2.58 (3H, m), 2.71 (1H, dd, J ) 9.4, 14.3 Hz), 2.81 (1H, m), 2.91-
2.94 (1H, m), 3.18-3.25 (2H, m), 3.74 (1H, m), 4.08 (1H, q, J ) 7.3
Hz), 4.52 (1H, q, J ) 6.3 Hz), 5.03 (2H, s), 7.13-7.35 (10H, m); 13
C
NMR (100 MHz, DMSO-d6, 20 °C) δ 18.3, 20.2, 21.3, 25.2, 26.0,
28.3, 29.5, 30.5, 32.8, 34.7, 35.6, 37.5, 49.5, 50.1, 50.3, 58.3, 58.5,
61.2, 65.5, 68.0, 126.7, 127.8, 127.9, 128.4, 128.7, 129.6, 137.1, 137.4,
155.7, 158.0, 158.3, 172.1, 173.2; HRMS (FAB+) calcd for MH+
C39H57N6O7 m/e 721.4262, found m/e 721.4289.
The preparations of 9-16 were carried out using the general
procedures for coupling and deprotection.
Compound 9. In a similar manner, 21 (19 mg, 0.041 mmol) was
coupled to N-Cbz-Leu (23 mg, 0.086 mmol) to give 23 (23 mg, 58%)
as a white solid: 1H NMR (500 MHz, DMSO-d6, 80 °C) δ 0.75-0.88
(18H, m), 1.15-1.19 (1H, m), 1.28 (3H, s), 1.40-1.70 (5H, m), 1.95-
2.02 (1H, m), 3.49 (1H, s), 4.06-4.12 (2H, m), 4.20-4.28 (1H, m),
5.03 (2H, dd, J ) 17.1, 12.0 Hz), 7.12-7.20 (1H, br), 7.22-7.41 (6H,
m), 7.45 (1H, d, J ) 9.0 Hz); HRMS (FAB+) calcd for MCs+
C53H84N6O10Cs m/e 1097.5303, found m/e 1097.5351.
Compound 23 (20 mg, 0.021) was deprotected to give 9 (9.0 mg,
46%) as a white solid: 1H NMR (500 MHz, DMSO-d6, 80 °C) δ 0.80-
0.95 (18H, m), 1.12-1.19 (1H, m), 1.40-1.55 (4H, m), 1.60-1.68
(1H, m), 1.95-2.02 (1H, m), 3.17 (1H, s), 4.03-4.13 (3H, m), 5.03
(2H s), 7.00 (1H, d, J ) 9.5 Hz), 7.02-7.10 (1H, br), 7.25-7.35 (5H,
m), 7.49 (1H, d, J ) 9.0 Hz); 13C NMR (100 MHz, DMSO-d6, 25 °C)
δ 18.1, 19.4, 21.5, 21.9, 23.1, 23.6, 23.9, 24.2, 30.2, 40.7, 42.1, 46.9,
53.2, 57.7, 65.3, 73.2, 127.6, 127.8, 128.3, 137.1, 155.8, 170.2, 172.1;
HRMS (FAB+) calcd for MCs+ C50H80N6O10Cs m/e 1057.4990, found
m/e 1057.4954.
Compound 18. To a solution of 1,4-bis[(N-Cbz)amino]-1,4-diisobu-
tyl-2,3-diol (17) (450 mg, 0.90 mmol) in 2,2-dimethoxypropane (24
mL) was added a catalytic amount of p-TsOH. The reaction mixture
was heated at 60 °C for 5 h and cooled to 20 °C. The reaction mixture
was diluted with EtOAc (200 mL), and the resulting solution was
washed with saturated aqueous NaHCO3 and saturated aqueous NaCl,
dried over MgSO4, filtered, and concentrated in vacuo. The residue
was then purified by flash chromatography to give 2,3-protected
(1S,2R,3R,4S)-diastereomer 18 (467 g, 96%) as a white solid: 1H NMR
(400 MHz, DMSO-d6, 80 °C) δ 0.80-0.87 (6H, m), 1.11-1.20 (1H,
m), 1.27 (3H, s), 1.42-1.49 (1H, m), 1.53-1.61 (1H, m), 3.58 (1H,
s), 3.72-3.83 (1H, m), 5.00 (1H, d, J ) 12.8 Hz), 5.08 (1H, d, J )
12.8 Hz), 6.42 (1H, br s), 7.26-7.32 (5H, m); 13C NMR (100 MHz,
DMSO-d6, 80 °C) δ 21.1, 22.3, 23.8, 26.4, 41.0, 48.1, 64.8, 78.9, 126.8,
127.0, 127.6, 136.7, 153.4; HRMS (FAB+) calcd for MCs+ C31H44N2O6-
Cs m/e 673.2254, found m/e 673.2228.
Compound 20 (General Procedure for the Coupling Reaction).
Compound 18 (480 mg, 0.89 mmol) in EtOAc (30 mL) containing 10%
Pd/C (170 mg) was stirred under H2 (1atm) at 20 °C for 20 h. The
reaction mixture was filtered through Celite and then concentrated in
vacuo to give diamine 19 (226 mg, 93%) as a colorless oil, which was
used for the coupling reaction without purification.
Compound 10. Compound 21 (22 mg, 0.047 mmol) was coupled
to N-Cbz-Phe (30 mg, 0.098 mmol) to give 24 (30 mg, 63%) as a white
solid: 1H NMR (500 MHz, DMSO-d6, 80 °C) δ 0.76-0.92 (12H, m),
1.15-1.20 (1H, m), 1.29 (3H, s), 1.45-1.60 (2H, m), 1.98-2.10 (1H,
m), 2.78-2.85 (1H, m) 3.51 (1H, s), 4.08-4.15 (1H, br), 4.25-4.35
(2H, m), 4.95 (2H s), 7.12-7.35 (12H, m), 7.56 (1H, d, J ) 8.5 Hz);
HRMS (FAB+) calcd for MCs+ C59H80N6O10Cs m/e 1165.4990, found
m/e 1165.4936.
Compound 24 (20 mg, 0.019) was deprotected to give 10 (11 mg,
58%) as a white solid: 1H NMR (400 MHz, DMSO-d6, 80 °C) δ 0.83-
0.89 (12H, m), 1.18-1.25 (1H, m), 1.43-1.58 (2H, m), 2.02 (1H, q,
J ) 6.7 Hz), 2.82 (1H, dd, J ) 14.1, 9.8 Hz), 3.04 (1H, dd, J ) 14.2,
4.4 Hz), 3.23 (1H, s), 4.10-4.20 (2H, m), 4.32-4.38 (1H, m), 4.95
(2H, s), 6.95-7.00 (2H, m), 7.15-7.32 (10H, m), 7.54 (1H, d, J ) 8.5
Hz); 13C NMR (100 MHz, DMSO-d6, 25 °C) δ 17.3, 18.7, 21.5, 22.6,
23.6, 29.7, 37.0, 41.5, 47.3, 55.7, 57.7, 64.9, 72.5, 125.5, 126.7, 127.0,
127.3, 127.6, 128.5, 137.4, 139.1, 156.0, 169.6, 170.6; HRMS (FAB+)
calcd for MCs+ C56H76N6O10Cs m/e 1125.4677, found m/e 1125.4709.
Compound 11. Compound 21 (22 mg, 0.047 mmol) was coupled
to N-Cbz-Val (25 mg, 0.098 mmol) to give 25 (31 mg, 70%) as a white
solid: 1H NMR (500 MHz, DMSO-d6, 80 °C) δ 0.75-0.88 (18H, m),
1.12-1.19 (1H, m), 1.28 (3H, s), 1.45-1.60 (2H, m), 2.00 (2H, br s),
3.50 (1H, s), 3.92-3.99 (1H, m), 4.03-4.12 (1H, br s), 4.25-4.32
(1H, m), 4.98-5.08 (2H m), 6.89-6.95 (1H, br), 7.25-7.38 (6H, m),
7.48 (1H, d, J ) 8.9 Hz); HRMS (FAB+) calcd for MCs+ C51H80N6O10-
Cs m/e 1069.4990, found m/e 1069.4943.
To a solution of diamine 19 (194 mg, 0.71 mmol) and N-Cbz-L-
valine (377 mg, 1.50 mmol) in CH3CN (8 mL) was added HBTU (569
mg, 1.50 mmol) followed by Et3N (166 mg, 1.64 mmol). The reaction
mixture was stirred for 15 min at 20 °C under Ar and then quenched
by addition of brine (20 mL) and extracted with EtOAc (4 × 20 mL).
The organic layer was washed sequentially with 1 M HCl (5 mL),
saturated aqueous NaHCO3 (5 mL), and saturated aqueous NaCl (5
mL), dried over MgSO4, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography to give 20 (430 mg,
82%) as a white solid: 1H NMR (400 MHz, DMSO-d6, 25 °C) δ 0.78-
0.88 (12H, m), 1.05-1.15 (1H, m), 1.22 (3H, s), 1.47-1.63 (2H, m),
2.01 (1H, q, J ) 6.4 Hz), 3.42 (1H, s), 3.97 (1H, dd, J ) 9.0, 6.2),
4.02-4.10 (1H, m), 5.01 (2H, dd, J ) 17.3, 12.6 Hz), 7.27-7.41 (7H,
m); 13C NMR (100 MHz, DMSO-d6, 20 °C) δ 17.6, 19.3, 21.4, 23.9,
27.0, 30.1, 41.7, 44.5, 60.2, 65.4, 79.2, 107.3, 127.6, 127.7, 128.3, 136.8,
156.0, 171.1; HRMS (FAB+) calcd for MCs+ C41H62N4O8Cs m/e
871.3622, found m/e 871.3648.
Compound 8. Compound 20 (170 mg, 0.23 mmol) was hydroge-
nated with 10% Pd/C (50 mg) in EtOAc (8 mL) to give 21 (106 mg,
99%) as a colorless viscous oil. It was used for the coupling reaction
without purification.
Compound 21 (20 mg, 0.043 mmol) was coupled with N-Cbz-Ala
(20 mg, 0.089 mmol) to give 22 (28 mg, 75%) as a white solid: 1H
Compound 25 (24 mg, 0.025) was deprotected to give 11 (11 mg,
50%) as a white solid: 1H NMR (400 MHz, DMSO-d6, 80 °C) δ 0.80-
0.88 (18H, m), 1.13-1.22 (1H, m), 1.41-1.56 (2H, m), 1.95-2.04

Protease Inhibitors with a Small P3 Residue
J. Am. Chem. Soc., Vol. 121, No. 6, 1999 1155
(2H, m), 3.20 (1H, s), 3.94 (1H, dd, J ) 8.9, 6.6 Hz), 4.11 (1H, se, J
) 4.4 Hz), 4.17 (1H, dd, J ) 8.8, 6.6 Hz), 5.04 (2H, s), 6.74 (1H, d,
J ) 8.2 Hz), 6.94 (1H, d, J ) 9.3 Hz), 7.25-7.35 (5H, m), 7.43 (1H,
d, J ) 8.8 Hz); 13C NMR (100 MHz, DMSO-d6, 80 °C) δ 17.3, 18.5,
18.7, 21.5, 22.6, 23.6, 29.6, 29.7, 41.5, 47.3, 57.6, 60.0, 65.0, 72.5,
126.9, 127.0, 127.6, 137.4, 156.0, 169.7, 170.3; HRMS (FAB+) calcd
for MCs+ C48H76N6O10Cs m/e 1029.4677, found m/e 1029.4701.
Compound 12. (1S,2R,3R,4S)-1,4-Bis[(N-Cbz)amino]-1,4-dibenzyl-
2,3-diol derivative 26 was prepared by applying the procedure
described.4 Compound 26 (40 mg, 0.074 mmol) was coupled to N-Cbz-
Ser (38 mg, 0.16 mmol) to give the adduct (60 mg, 83%) as a white
solid: 1H NMR (500 MHz, DMSO-d6, 80 °C) δ 0.66 (3H, d, J ) 6.5),
0.72 (3H, d, J ) 6.0), 1.31 (3H, s), 1.90 (1H, d, J ) 6.2 Hz), 2.65-
2.80 (2H, m), 3.13-3.17 (1H, m), 3.58 (2H, s), 4.07-4.16 (2H, m),
4.23-4.31 (1H, m), 4.60-4.70 (1H, m), 5.05 (2H, s), 6.90-6.99 (1H,
br), 7.07-7.19 (6H, m), 7.25-7.35 (5H, m), 7.45-7.50 (1H, br); HRMS
(FAB+) calcd for MCs+ C53H68N6O12Cs m/e 1113.3990, found m/e
1113.3897.
The adduct (30 mg, 0.031 mmol) was then deprotected to give 12
(15 mg, 51%) as a white solid: 1H NMR (400 MHz, DMSO-d6, 80
°C) δ 0.70 (3H, d, J ) 6.5 Hz), 0.72 (3H, d, J ) 7.0 Hz), 1.91 (1H, se,
J ) 6.5), 2.68-2.82 (2H, m), 3.33 (1H, s), 3.60-3.65 (2H, m), 4.07
(1H, dd, J ) 9.0, 6.5 Hz), 4.16 (1H, q, J ) 8.0), 4.29 (1H, s), 4.37-
4.43 (1H, m), 4.63-4.70 (1H, m), 5.07 (2H, s), 6.82-6.92 (1H, br),
7.05-7.40 (12H, m); 13C NMR (100 MHz, DMSO-d6, 80 °C) δ 17.6,
19.3, 30.3, 38.5, 50.5, 57.0, 57.8, 61.8, 65.5, 73.2, 125.7, 127.8, 128.4,
129.1, 136.9, 139.0, 155.9, 169.9, 170.2; HRMS (FAB+) calcd for
MCs+ C50H64N6O12Cs m/e 1073.3637, found m/e 1073.3685.
) 8.3 Hz), 7.49 (1H, d, J ) 9.7 Hz), 7.51 (1H, d, J ) 9.3 Hz); 13C
NMR (100 MHz, DMSO-d6, 20 °C) δ 13.7, 17.8, 18.7, 19.3, 22.5,
30.7, 34.0, 38.5, 50.3, 54.5, 57.3, 65.3, 73.2, 125.6, 127.7, 127.8, 128.3,
129.0, 137.0, 138.9, 155.9, 170.3, 171.6; HRMS (FAB+) calcd for
MCs+ C54H72N6O10Cs m/e 1097.4364, found m/e 1097.4317.
Compound 16. Compound 26 (22 mg, 0.041 mmol) was coupled
to N-R-Cbz-L-norleucine (21.8 mg, 0.082 mmol). The adduct was then
deprotected to give 16 (20.5 mg, 50%) as a white solid: 1H NMR (500
MHz, DMSO-d6, 20 °C) δ 0.66 (3H, d, J ) 6.7 Hz), 0.70 (3H, d, J )
6.7 Hz), 0.83 (3H, t, J ) 6.8 Hz), 1.15-1.28 (4H, m), 1.40-1.48 (1H,
m), 1.49-1.57 (1H, m), 1.75-1.85 (1H, m), 2.61 (1H, dd, J ) 3.9,
13.8 Hz), 2.76 (1H, dd, J ) 9.9, 13.8 Hz), 3.25 (1H, s), 3.94-4.01
(1H, m), 4.10 (1H, dd, J ) 8.8, 6.6 Hz), 4.38-4.45 (1H, m), 4.63 (1H,
s), 5.01 (2H, s), 7.04-7.09 (1H, m), 7.10-7.17 (4H, m), 7.27-7.31
(1H, m), 7.32-7.37 (4H, m), 7.45 (1H, d, J ) 8.4 Hz), 7.48 (1H, d, J
) 9.6 Hz), 7.51 (1H, d, J ) 9.6 Hz); 13C NMR (100 MHz, DMSO-d6,
20 °C) δ 13.9, 17.8, 19.3, 21.9, 27.7, 30.7, 31.6, 38.4, 50.4, 54.8, 57.4,
65.3, 73.0, 125.6, 127.7, 127.8, 128.3, 129.0, 137.1, 138.9, 155.9, 170.3,
171.6; HRMS (FAB+) calcd for MCs+ C56H76N6O10Cs m/e 1125.4677,
found m/e 1125.4729.
Molecular Modeling
Models of the protease inhibitor 1b, complexed with HIV-1
protease and FIV protease, were built using the Brookhaven
Protein Data Bank entries 1HVI11 and 1FIV6 as starting points.
The entry 1HVI has a resolution of 1.8 Å and contains the C2-
symmetric Abbott inhibitor A77003 (R,S) co-crystallized with
the HIV-1 protease of strain HIVLAI expressed in E. coli. The
structure of A77003 is quite similar to that of 1b. The
stereochemistry of one of the “core” hydroxyls had to be
inverted, and the terminal pyrimidine groups were replaced by
the Cbz groups. The 1FIV structure has a resolution of 2.0 Å
and contains the inhibitor LP-149 co-crystallized with the feline
immunodeficiency virus protease from Felis catus and expressed
in E. coli.
Compound 13. Compound 26 (33 mg, 0.061 mmol) was coupled
to N-Cbz-Thr (33 mg, 0.13 mmol) to give the adduct (52 mg, 85%) as
a white solid: 1H NMR (500 MHz, DMSO-d6, 80 °C) δ 0.69 (3H, d,
J ) 7.0 Hz), 0.74 (3H, d, J ) 6.5 Hz), 1.03 (3H, d, J ) 6.5 Hz), 1.32
(3H, s), 1.93 (1H, se, J ) 6.0 Hz), 2.68-2.80 (2H, m), 3.60 (1H, s),
3.90-3.98 (1H, m), 4.02 (1H, dd, J ) 8.5, 5.0 Hz), 4.20 (1H, dd, J )
8.5, 6.0 Hz), 4.27-4.32 (1H, m), 4.56 (1H, d, J ) 5.5 Hz), 5.06 (2H,
s), 6.65-6.75 (1H, br), 7.10-7.19 (6H, m), 7.28-7.35 (5H, m), 7.44
(1H, d, J ) 9.0 Hz); HRMS (FAB+) calcd for MCs+ C55H72N6O12Cs
m/e 1141.4263, found m/e 1141.4316.
The adduct (35 mg, 0.037 mmol) was then deprotected to give 13
(20 mg, 56%) as a white solid: 1H NMR (500 MHz, DMSO-d6, 80
°C) δ 0.69 (3H, d, J ) 6.5 Hz), 0.72 (3H, d, J ) 7.0 Hz), 1.04 (3H, d,
J ) 6.5 Hz), 1.87-1.93 (1H, m), 2.68-2.80 (2H, m), 3.33 (1H, s),
3.90-4.15 (3H, m), 4.22-4.38 (2H, m), 5.06 (2H, s), 6.60-6.70 (1H,
br), 7.05-7.40 (11H, m), 7.50 (1H, d, J ) 8.0 Hz); 13C NMR (100
MHz, DMSO-d6, 20 °C) δ 17.6, 19.3, 19.7, 30.6, 38.5, 50.6, 57.5, 60.3,
65.5, 66.8, 73.1, 125.5, 127.7, 127.8, 128.0, 128.4, 129.0, 136.9, 138.9,
156.0, 169.7, 170.2; HRMS (FAB+) calcd for MCs+ C52H68N6O12Cs
m/e 1101.3950, found m/e 1101.3900.
Compound 14. Compound 26 (22 mg, 0.041 mmol) was coupled
to N-R-Cbz-2-aminobutyric acid (19.6 mg, 0.082 mmol). The adduct
was then deprotected to give 14 (16.4 mg, 43%) as a white solid: 1H
NMR (500 MHz, DMSO-d6, 20 °C) δ 0.66 (3H, d, J ) 5.5 Hz), 0.70
(3H, d, J ) 5.6 Hz), 0.79 (3H, t, J ) 6.2 Hz), 1.42-1.50 (1H, m),
1.52-1.58 (1H, m), 1.78-1.84 (1H, m), 2.57-2.65 (1H, m), 2.73-
2.82 (1H, m), 3.24 (1H, s), 3.90-3.95 (1H, m), 4.10 (1H, dd, J ) 7.4,
5.4 Hz), 4.42-4.49 (1H, m), 4.65 (1H, s), 5.01 (2H, s), 7.07 (1H, t, J
) 5.8 Hz), 7.11-7.18 (4H, m), 7.28-7.33 (1H, m), 7.32-7.37 (4H,
m), 7.41 (1H, d, J ) 6.8 Hz), 7.46 (1H, d, J ) 7.7 Hz), 7.52 (1H, d,
J ) 7.4 Hz); 13C NMR (100 MHz, DMSO-d6, 20 °C) δ 10.4, 17.8,
19.3, 25.2, 30.7, 38.5, 50.3, 56.1, 57.4, 65.3, 73.2, 125.6, 127.8, 128.3,
129.0, 137.0, 138.9, 155.9, 170.3, 171.4; HRMS (FAB+) calcd for
MCs+ C52H68N6O10Cs m/e 1069.4051, found m/e 1069.4004.
The model of 1b bound to HIV-1 protease was built first,
using InsightII 97.0’s Biopolymer18 and Discover modules and
the AMBER force field. Molecular mechanics minimization was
performed using the conjugate gradients minimizer until the
minimization converged, i.e., the derivative of the energy was
less than 0.001. Distance constraints were used in the early
stages of the modeling to ensure that the hydrogen bonds were
preserved between the inhibitor, water, and protease. Initially,
only the inhibitor model, five active-site water molecules, and
the residues of the active site that were in contact with the
inhibitor were allowed to move. These five waters consisted of
the water bound between the tips of the flaps, and four waters,
two in each subunit of the dimer, that bind inside a pocket
adjacent to the Arg-8 residue in HIV (Arg-13 in FIV). The
resulting model was minimized while allowing all atoms to
move. A similar procedure was applied to the modeling shown
in Figure 5, using HIV protease complexed with RO31-8959.
Acknowledgment. We thank Dr. M. C. Fitzgerald at Scripps
for providing HIV and FIV PR substrates, Dr. George Trainor
at Du Pont Pharmaceuticals for the inhibition analysis of 1b in
cell culture, and Dr. A. Wlodawer at NCI for providing the
coordinate of FIV protease complexed with 1b. Support from
Grants P01GM48870 (C.H.W. and A.J.O.) and R01AI40882
(J.H.E.) from the National Institutes of Health is gratefully
acknowledged. The contribution of reagents supplied by the
AIDS Reagents and Reference Program of the National Institutes
of Health is also gratefully acknowledged.
Compound 15. Compound 26 (22 mg, 0.041 mmol) was coupled
to N-R-Cbz-L-norvaline (20.6 mg, 0.082 mmol). The adduct was then
deprotected to give 15 (14 mg, 35%) as a white solid: 1H NMR (500
MHz, DMSO-d6, 20 °C) δ 0.67 (3H, d, J ) 6.7 Hz), 0.70 (3H, d, J )
6.7 Hz), 0.83 (3H, t, J ) 7.3 Hz), 1.15-1.34 (2H, m), 1.38-1.53 (2H,
m), 1.77-1.85 (1H, m), 2.57-2.64 (1H, m), 2.73-2.80 (1H, m), 3.25
(1H, s), 3.96-4.02 (1H, m), 4.10 (1H, dd, J ) 8.7, 6.6 Hz), 4.41-
4.47 (1H, m), 4.64 (1H, s), 5.01 (2H, s), 7.05-7.10 (1H, m), 7.11-
7.17 (4H, m), 7.26-7.31 (1H, m), 7.32-7.37 (4H, m), 7.44 (1H, d, J
JA982893P