Combination of non-natural D-amino acid derivatives and allophenylnorstatine-dimethylthioproline scaffold in HIV protease inhibitors have high efficacy in mutant HIV

Article abstract of DOI:10.1021/jm701555p

Several non-natural D-amino acid derivatives were introduced as P2/P3 residues in allophenylnorstatine-containing (Apns; (2S,3S)-3-amino-2-hydroxy-4- phenylbutyric acid) HIV protease inhibitors. The synthetic analogues exhibited potent inhibitory activity

Full text of DOI:10.1021/jm701555p

2992
J. Med. Chem. 2008, 51, 2992–3004
Combination of Non-natural D-Amino Acid Derivatives and
Allophenylnorstatine-Dimethylthioproline Scaffold in HIV Protease Inhibitors Have High
Efficacy in Mutant HIV
Shingo Nakatani,† Koushi Hidaka,† Ei’ichi Ami,† Koichiro Nakahara,‡ Akihiko Sato,‡ Jeffrey-Tri Nguyen,† Yoshio Hamada,†
Yasuko Hori,† Nobuyuki Ohnishi,† Akinori Nagai,† Tooru Kimura,† Yoshio Hayashi,† and Yoshiaki Kiso*,†
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program, Kyoto Pharmaceutical
UniVersity, Yamashina-ku, Kyoto 607-8412, Japan, DiscoVery Research Laboratories, Shionogi Co. Ltd., Settsu, Osaka 566-0022 Japan
ReceiVed December 12, 2007
Several non-natural D-amino acid derivatives were introduced as P2/P3 residues in allophenylnorstatine-
containing (Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) HIV protease inhibitors. The synthetic
analogues exhibited potent inhibitory activity against HIV-1 protease enzyme and HIV-1 replication in MT-4
cells. Structure–activity relationships revealed that D-cysteine or serine derivatives contributed to highly
potent anti-HIV activities. Interestingly, anti-HIV activity of all the D-amino acid-introduced inhibitors was
remarkably enhanced in their anti-HIV activities against a Nelfinavir-resistant clone, which has a D30N
mutation in the protease, over that of the wild-type strain. HIV inhibitory activity of several analogues was
moderately affected by an inclusion of R1-acid glycoprotein in the test medium.
Introduction
We have previously described the design and synthesis of
highly potent peptidomimetic HIV protease inhibitors, 1
(KNI-27211,12) and 2 (KNI-764,13–15 also known as JE-2147,
AG-1776, SM-319777), which contain allophenylnorstatine
(Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) with
a hydroxymethylcarbonyl (HMC) isostere as a transition-state
mimic as the P1 residue.16–18 Considering that HIV-1 protease
prefers for the cleavage site specific sequences Phe-Pro and Tyr-
Pro, (R)-1,3-thiazolidine-4-carboxylic acid (Thz) and (R)-5,5-
dimethyl-1,3-thiazolidine-4-carboxylic acid (Dmt) serve as
alternatives for the P1′ proline residue (Figure 1). X-ray and
NMR analyses of an Apns-containing inhibitor-protease com-
plex revealed hydrogen bond interactions between the HMC
and the two catalytic Asps, that is, HMC is an ideal transition-
state mimic.11a,19,20 The concept resulted in the development
of 1 and 2 as clinical candidates. On the basis of the
structure–activity relationships of HIV-1 protease inhibitors, we
identified a preliminary series of compounds21,22 that exhibited
extremely potent enzyme-inhibitory activity, lower cytotoxicity,
and high antiviral activities against both wild-type and mutant
proteases. In the present study, we describe our continuing
efforts to generate novel HIV protease inhibitors containing
various P2/P3 non-natural D-amino acid analogues, leading to
highly potent compounds whose potencies surpass those of
clinically used drugs against wild-type and drug-resistant HIVs,
while not being significantly affected by undesired protein
binding. Of the 32 compounds described in this present work,
12 preliminary compounds that have been briefly reported22 are
elaborated in full detail in relation to a much larger data sample
size so as to obtain a more accurate and deeper interpretation
of structure–activity relationships. Moreover, specific binding
to serum protein, particularly R1-acid glycoprotein (AGP), is
known to drastically reduce the potency of clinically used drugs.
Concerned by the detrimental effect of AGP-binding, in the
current work, we have carried out the evaluation of some
interesting compounds in the presence and absence of AGP.
Continuous pandemic of human immunodeficiency virus
(HIVa) infection, particularly a recent striking increase in Asia,
Eastern Europe, and Africa, is still recognized as a serious
problem that causes acquired immune deficiency syndrome
(AIDS) and increases mortality.1 To treat HIV infection,
therapies involving more than two drugs from reverse tran-
scriptase inhibitors, protease inhibitors, and a fusion inhibitor,
called HAART (highly active antiretroviral therapy), form an
effective options to reduce plasma HIV mRNA to undetectable
level.2 Despite the success of such chemotherapy, treatment is
life-long while monitoring that HIV resistance to these clinical
drugs do not develop. HIV type-1 (HIV-1) protease is a
homodimeric aspartic protease that processes its own polypro-
teins, Pr55 gag and Pr160 gag-pol, which contain all the
structural and catalytic proteins required for the formation of
mature and infectious virions.3 Mutations in the HIV-1 protease
region is also known to cause resistance to clinically used HIV
protease inhibitors.4 Tipranavir5 and Darunavir6 have recently
been approved by the FDA for use in salvage therapies against
the emergence of HIV mutants that are resistant to available
drugs. Multimutations via natural polymorphism in other HIV
subtypes, however, could develop corresponding resistances in
the future.7,8 Consequently, the development of structurally new
and highly potent HIV protease inhibitors with different
resistance profiles is still of interest.9,10
* To whom correspondence should be addressed. Phone: +81-75-595-
4635. Fax: +81-75-591-9900. E-mail: kiso@mb.kyoto-phu.ac.jp.
†
Department of Medicinal Chemistry, Center for Frontier Research in
Medicinal Science, 21st Century COE Program, Kyoto Pharmaceutical
University.
‡
Discovery Research Laboratories, Shionogi Co. Ltd.
a Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired
immune deficiency syndrome; HAART, highly active antiretroviral therapy;
Apns, allophenylnorstatine; HMC, hydroxymethylcarbonyl; Thz, (R)-1,3-
thiazolidine-4-carboxylic acid; Dmt, (R)-5,5-dimethyl-1,3-thiazolidine-4-
carboxylic acid; AGP, R1-acid glycoprotein; Boc, tert-butyloxycarbonyl;
Hph, homophenylalanine; EDC, 1-ethyl-3-(3,3-dimethylamino-propyl)car-
bodiimide; HOBt, 1-hydroxybenzotriazole; RTV, Ritonavir; NFV, Nelfi-
navir; rmsd, root-mean-square deviations; SI, selectivity index; MD,
molecular dynamics.
Design Rationale. Information on the interactions between
compound 1 and HIV-1 protease based on X-ray crystal-
lographic studies of the complex19 and molecular modeling23
10.1021/jm701555p CCC: $40.75
2008 American Chemical Society
Published on Web 04/22/2008

D-Amino Acid Containing HIV Protease Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2993
Figure 1. Structures of clinically tested Apns-containing HIV protease inhibitors.
Figure 2. Incorporation of D-amino acid derivative into Apns-Dmt scaffold.
suggest a feasible replacement of the L-amino acid residue with
different moieties at the P2/P3 positions to increase interactions
with the enzyme. A P2 methylthioalanine is not preferred
according to a pharmacokinetic study with 112 because the sulfur
atom is oxidized in physiological conditions, resulting in a short
plasma half-life. In 1994, Eli Lilly group reported an incorpora-
tion of non-natural D-amino acids at the P2/P3 positions of HIV
protease inhibitors with an hydroxyethylene isostere.24 The
D-amino acid residue was suggested to bind as the side chain
points toward the S3 pocket of the protease and the substituted
R-amino group occupies the S2 pocket (Figure 2). Eli Lilly group
named this idea as the “D-amino acid concept” and optimized
the non-natural D-amino acid residue to exhibit adequate antiviral
potency for oral availability in rat.25,26 Later, Fujii and co-
workers reported an application of the D-amino acid concept to
hydroxyethylamine isosteres.27 Among their compounds, two
sulfoxide analogues maintained their potencies against multidrug
resistant HIV-1 strains. These reports prompted us to disclose
our independent approach, which is the incorporation of various
D-amino acid derivatives as the P2/P3 residues to the Apns-Dmt
scaffold. D-Amino acid-containing inhibitors may sustain their
plasma concentration longer than our previous lead compounds
because of their stability. In our design, compound 2 was used
as a template to replace its P2 hydroxylmethylbenzoyl moiety
with a D-amino acid residue. The amino group of the D-amino
acid residue was substituted with a mesyl or acetyl group, which
are effective to mimic the L-asparagine residue in the Eli Lilly’s
report. A virtual compound containing D-N-mesyl-S-naphthyl-
cysteine (Figure 2) suggested a preferred binding orientation
in the active site of HIV protease by a molecular modeling
simulation as the S-naphthylthiomethyl side chain points to the
S3 pocket and the N-mesylamino group is oriented in the S2
pocket. We diversified the side chain and N-substituent of the
D-amino acid residue at three specific positions in order to
optimize, within our Apns-Dmt scaffold, for enzyme–inhibitory
and antiviral activity.
Chemistry. We synthesized D-cysteine analogues following
a precedent study of Eli Lilly’s group. The D-cysteine derivatives

2994 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Nakatani et al.
Scheme 1. Reagents: (i) Ph3P, DEAD, -78 °C (ii) R1-SNa, THF; (iii) m-CPBA (2 equiv), CH2Cl2
Scheme 2. Reagents: (a) Boc-D-Hph-OH or Boc-D-Ser(Bzl)-OH or 5a-h or 6a-h, EDC, HOBt, Et3N, DMF; (b) 4N HCl-dioxane; (c)
Ac2O or MsCl, NMM, CH2Cl2
were prepared by a ring-opening reaction of Vederas’ N-Boc-
D-serine ꢀ-lactone28 (4), starting from N-Boc-D-serine (3), with
a variety of mercaptans in a single step (5a, 5b, 5d-h) (Scheme
1). For R1 p-methoxybenzyl analogues, commercially available
N-Boc-D-(S-p-methoxybenzyl)cysteine (5c) was used. Additional
oxidation of D-N-Boc-cysteine analogues using m-chloroper-
benzoic acid provided the corresponding sulfones (6a-h). The
incorporation of D-amino acid derivatives is shown in Scheme
2. As a first attempt to apply the “D-amino acid concept” to our
Apns-Dmt skeleton, we chose commercially available optically
pure N-Boc protected D-homophenylalanine (Boc-D-Hph-OH).
An amine intermediate 7, H-Apns-Dmt-(2-methyl)benzylamide,
was prepared as previously reported.14 The D-amino acid was
coupled with 7 using the conventional EDC-HOBt method to
afford Boc-protected intermediate. After deprotection of the Boc
group, the mesyl and acetyl groups were attached to the amino
group to afford the N-substituted analogues 8 and 9, respectively.
We introduced another commercially available N-Boc protected
D-O-benzylserine, leading to the corresponding N-substituted
analogues 10, 11 using the same procedure as described above.
Each sulfoxide and sulfone was condensed with amine 7,
followed by the removal of Boc-group and the treatment of
mesyl chloride or acetic anhydride to give compounds 12–39.
Apns-Dmt scaffold. Compounds 8 and 9, which have a mesyl
and an acetyl, respectively, possessed an R1 benzyl without
an X moiety. These compounds exhibited nanomolar HIV-1
protease inhibition (about 50% at 1 nM inhibitor, Table 1)
with a slight difference between the R2 groups. This result
evidenced the compatibility of D-amino acid alteration to our
scaffold. Incorporation of an oxygen atom at the X position
in D-serine derivatives increased protease inhibitory activity
(10 and 11). Compounds 12 and 13, with a replacement of
the oxygen to sulfur atom in the D-cysteine structure,
exhibited improved potencies. In the case of O or S at the X
position, the acetyl moiety was slightly better for inhibitory
potency than the mesyl group. We also tested the oxidated
cysteine derivative, i.e., sulfonyl (X ) SO2) analogues. The
resultant mesyl form 14 was more potent than the acetyl form
15 with moderate activity. Among these R1 benzyl analogues,
a combination of an X sulfur with an R2 acetyl (compound
13) exhibited the best result for enzyme inhibition. Inhibitory
activity against HIV-1 IIIB of these R1 benzyl analogues
(8–15) was evaluated in MT-4 cells. EC50 values ranged from
15 to 219 nM. The contribution of X structure to EC50 value
seemed to be higher than that of enzyme-inhibitory activity.
The anti-HIV activity of X sulfonyl derivatives 14 and 15
was dramatically attenuated compared to other X moieties
with R1 benzyl analogues. As a result, the combination of
an X sulfur with an R2 acetyl was preferred for anti-HIV
activity as well as protease inhibition.
Results and Discussion
The D-homophenylalanine analogue was the first attempt
to confirm the function of the “D-amino acid concept” to our

D-Amino Acid Containing HIV Protease Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2995
Table 1. HIV-1 Protease Inhibitory Activity and Anti-HIV-1 Activity
compound
structure
HIV-1 PR inhibition
anti-HIV activity (HIV-1IIIB) MT-4/MTT
R1
X
R2
% at 1 nM
IC50, nM
EC50, nM
CC50, nM
SI
190
>610
850
>1400
580
1000
>120
>240
520
8 (KNI-1827)
9 (KNI-1880)
10 (KNI-1876)
11 (KNI-1878)
12 (KNI-1831)
13 (KNI-1976)
14 (KNI-1992)
15 (KNI-1991)
16 (KNI-1987)
17 (KNI-1881)
18 (KNI-2015)
19 (KNI-2016)
20 (KNI-1932)
21 (KNI-1931)
22 (KNI-1986)
23 (KNI-1990)
24 (KNI-1910)
25 (KNI-1909)
26 (KNI-1993)
27 (KNI-1960)
28 (KNI-1966)
29 (KNI-1964)
30 (KNI-1933)
31 (KNI-1961)
32 (KNI-1947)
33 (KNI-1946)
34 (KNI-1969)
35 (KNI-1965)
36 (KNI-1935)
37 (KNI-1934)
38 (KNI-1938)
39 (KNI-1937)
1
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
55
45
59
64
66
81
66
47
58
76
52
53
77
87
66
59
95
80
94
79
74
68
97
97
67
53
98
96
28
27
91
85
40
90
29
59
51
20
21
17
11000
>31000
17000
>30000
9800
15000
>27000
>28000
8900
O
O
S
S
SO2
SO2
S
0.56
0.39
15
219
117
17
12
125
82
9.3
13
203
102
nt
p-methylbenzyl
p-methylbenzyl
p-methylbenzyl
p-methylbenzyl
p-methoxybenzyl
p-methoxybenzyl
p-methoxybenzyl
p-methoxybenzyl
p-chlorophenyl
p-chlorophenyl
p-fluorophenyl
p-fluorophenyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
2-quinolinyl
S
8800
730
SO2
SO2
S
>26000
>28000
10000
17000
>26000
>27000
nt
18000
>27000
>28000
4400
>210
>340
1100
1300
>130
>260
nt
0.26
0.18
S
SO2
SO2
SO2
SO2
SO2
SO2
S
0.11
0.22
47
380
212
105
6.4
17
61
40
13
18
81
28
169
151
473
94
22
36
>130
>270
690
470
310
400
470
460
230
0.27
S
8000
SO2
SO2
S
0.083
0.090
19000
16000
6100
S
8300
SO2
SO2
S
S
SO2
SO2
0.090
0.086
19000
16000
8500
570
50
64
2-quinolinyl
2-quinolinyl
2-quinolinyl
9700
>26000
>27000
>30000
17000
11000
>55
>290
>1400
470
Ritonavir
Nelfinavir
16
690
We focused on the sulfur and sulfonyl for the X structure
because of their relatively stronger enzyme-inhibitory activity
and synthetic prospects. Derivatives 16–23 with a methyl or
methoxy substitution at the p-position of the R1 benzyl moiety
were evaluated. The methoxy substituent was better for HIV
protease inhibition than the methyl substituent in the case of
the X sulfur (compounds 16, 17 < compounds 20, 21). Anti-
HIV activities of these compounds were similarly potent;
compound 21 exhibited an EC50 value of 9.3 nM. On the
other hand, compounds 18, 19, 22, and 23 with an X sulfonyl
moiety were less potent against not only the protease but
also the virus.
Replacements of the R1 benzyl group with p-chlorophenyl
and p-fluorophenyl groups resulted in improved enzyme inhibi-
tion (24–27), especially compounds 24 and 26, with a R2 mesyl
group exhibiting extremely potent enzyme inhibition (>90%
inhibition at 1nM). The anti-HIV activities of 25–27 were as
low as the R1 benzyl derivatives with an X SO2 moiety because
of the disadvantage of the sulfonyl group observed in the first
modification. Compound 25 with an R1 p-chlorophenyl and a
R2 acetyl was relatively potent (47 nM) against the virus among
the X sulfonyl derivatives.
These results from the R1 halophenyl derivatives encouraged
us to introduce planar moieties such as a naphthyl group.
Compounds 28–35 possess 1-naphthyl or 2-naphthyl as R1. In
this series, it is clear that compounds with an X sulfonyl were
more potent than that of a sulfur in enzyme inhibition (30, 31,
34, and 35 with 96–98%). The IC50 values of these sulfonyl
moiety ranged from 80 to 90 pM. Unfortunately, we observed
a decrease in anti-HIV activity, which is similar to that observed
with our previous compounds, that is, an X sulfur was better
than a sulfonyl in antiviral assay. The difference between the
R2 mesyl and acetyl was ambiguous against enzyme inhibition
but rather obvious against antiviral activity. Compound 28 was
the most potent against HIV in the D-amino acid series with an
EC50 value of 6.4 nM, exceeding the potency of Ritonavir (RTV)
and Nelfinavir (NFV) despite its moderate potency against the
protease. R1 quinolinyl derivatives, with a minute replacement
of a nitrogen atom in the 2-naphthyl group, dramatically
attenuated potency (36–39). The decreased activity of these
compounds indicated that an improper substitution of a polar
nitrogen atom disrupted the hydrophobic interactions with the
enzyme as evidenced by a 2-naphthyl group. Equation 1 shows

2996 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Nakatani et al.
tion from the original sample as the validation data and the
remaining observations as the training data, and the process was
repeated such that each observation in the sample was used once
as the validation data. The equation is valid because the root-
mean-square deviations (rmsd) of the coefficients and intercept
are relatively small (0.597 ( 0.055, 0.649 ( 0.053, 0.789 (
0.017, -2.855 ( 0.035), and the coefficient of determination
did not greatly vary during cross-validation (r2 ) 0.85-0.88).
Overall, each descriptor contributed almost equally to the
equation (Enz, 29%; R1, 32%; X, 39%).
The cyototoxicity of these D-amino acid series were suf-
ficiently low and shown at the µM level (Table 1). Consequently,
the selectivity index (SI) values of the D-amino acid derivatives
were high. This result elucidated that our selective inhibitor
design with an Apns-Dmt scaffold is based on the Phe-Pro
residues of the virus-specific cleavage substrate. Exceptions are
analogues with 2-quinolinyl structures, possessing relatively high
toxicity. Concerning high anti-HIV activity and SI value
(>1000), compounds 11, 13, 20, and 21 would be potential
candidates for further pharmacokinetic study.
Figure 3. Chart showing agreement between predicted and observed
anti-HIV activity: log(EC50) values for compounds 8–23 and 25–39.
Predicted values were calculated from eq 1.
RTV and NFV are widely used in clinical therapy. Recently,
RTV has been used only for boosting as a CYP3A4 inhibitor
to maintain plasma levels of other protease inhibitors.2 The
potency of RTV and NFV is reduced in plasma because of
specific binding to serum protein,29 especially AGP. The plasma
level of AGP is reported to be increased in some HIV infected
patients,30 suggesting that a low plasma concentration of the
free drugs would bind the protease and, therefore, the resistance
of HIV to these drugs may easily occur. We selected some
compounds with interesting potencies against wild-type HIV-1
to test against drug resistant mutants. The susceptibility of these
D-amino acid derivatives against HIV-1 molecular clones of the
NL-432 strain, which have resistance to each or both of RTV
and NFV, is summarized in Table 2. The D-amino acid-
containing compounds exhibited fairly potent activities against
the wild-type NL-432 strain as tested to HIV-1 IIIB. The RTV-
resistant clone is 2.8-fold less sensitive to RTV, while NFV
maintained its activity (1.1-fold). Compound 1 distinctly at-
tenuated its activity against the RTV-resistant strain (5.6-fold).
On the other hand, compounds with D-amino acid derivatives
generally exhibited some reduction as much as 4-fold in anti-
HIV activity smaller than that of 1. The most potent compound
against RTV-resistant strain was 28 with an EC50 value of 17
nM, reducing its activity 1.8-fold from the wild-type strain.
Among the D-amino acid-containing series, compound 21
maintained its activity (1.1-fold). On the other hand, an increase
in potency was observed in the case of compound 25 with a
relatively low activity. At the three mutation sites, N37S and
G57R are polymorphisms derived from the wild-type protease
of HIV-1 IIIB, therefore I84V is a critical change in the enzyme
binding sites. These results suggest that the binding of the
D-amino acid moiety is less dependent on the I84V mutation
on the protease than the binding of the P2/P3 residue in 1.
a quantitative structure–activity relationship correlating HIV
protease inhibition and structural features with cellular antiviral
EC50.
(
)
( )
-log EC ) 0.597 Enz + 0.649 R + 0.789 X -
(
)
(
)
1
50
2.855 n ) 31, r2 ) 0.86, F ) 56, p < 0.001 (1)
where Enz: normalized log (% HIV protease inhibition at 1
nM), ranging from 0 to 1; R1: benzyl ) 0.531, p-methoxy-
benzyl ) 0.707, p-methylbenzyl ) 0.639, p-chlorophenyl )
0.693, p-fluorophenyl ) 0.226, 1-naphthyl ) 1.000, 2-naph-
thyl ) 0.963, 2-quinolinyl ) 0.000, ranging from 0 to 1; X:
null ) 0.450, O ) 0.964, S ) 1.000, SO2 ) 0.000, ranging
from 0 to 1.
In our preliminary report, we derived a well-fitted, normal-
ized, multiple collinear, quantitative SAR equation in which HIV
protease inhibition and the nature of the X moiety were
parametrized to correlate with antiviral activity (EC50).22 The
R1 and R2 moieties were excluded as descriptors because they
did not greatly contribute to the overall equation. Although, at
the time, we only considered R1 as p-methoxybenzyl, 1-naph-
thyl, and 2-naphthyl moieties (20–23 and 28–35), the current
series of compounds has a larger complexity of R1 moieties to
such an extent that the R1 has become involved as a descriptor
(eq 1). Each descriptor, namely Enz, R1, and X, is a normalized
percentile value from 0 to 1, where 1 is the most potent and 0
is the least potent based on EC50 values. To be exact, to award
scores to the R1 and X descriptors, groups of compounds, where
only one portion of the structure was changed (either R1, X, or
R2), were ranked as normalized percentiles from 0 to 1 based
on their EC50 values. To compare between the groups, the
average of the respective normalized percentiles represented the
score of the moiety being examined. The scores suggest that
compounds containing an R1 1-naphthyl or 2-naphthyl moiety
and an X oxygen or sulfur atom are expected to exhibit potent
antiviral activity. On the other hand, compounds possessing an
R1 2-quinolinyl or p-fluorophenyl moiety and an X SO2 moiety
are expected to exhibit low antiviral activity. The final equation
is well fitted (r2 ) 0.86, Figure 3) and statistically significant
(p < 0.001). A form of K-fold cross-validation, namely leave-
one-out cross validation, was performed using a single observa-
NFV drastically attenuated its anti-HIV activity (12-fold)
against the NFV-resistant clone, while RTV was more sensitive
(0.20-fold). The D30N mutation in the protease of the resistant
strain is one of the main reasons for the decrease because a
hydrogen bond interaction between NFV and Asp30 of the
protease, observed in the crystal structure, may not be main-
tained in the Asn30 mutation. Compound 1 also diminished its
potency against NFV-resistant clone (2.3-fold), although all of
the D-amino acid derivatives surprisingly exhibited higher
activity against the mutant than wild-type HIV (from 0.04- to
0.42-fold). These results proposed improvements in hydrophobic

D-Amino Acid Containing HIV Protease Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2997
Table 2. Inhibitory Activity against Drug-Resistant HIV-1 Strains
structure
EC50, nM (fold resistance)
compound
R1
X
R2
WT (NL-432)
RTV resistanta
NFV resistantb
18 (0.23)
2.3
RTV and NFV resistantc
9
10
11
12
13
16
17
20
21
25
28
29
32
33
1
benzyl
benzyl
benzyl
benzyl
benzyl
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ms
Ac
Ac
Ms
Ac
Ms
Ac
78
22
23
20
18
17
22
16
17
88
9.3
20
17
17
36
80
25
101
90
77
63
40
58
41
20
18
44
17
70
31
65
201
220
28
(1.3)
(4.1)
(3.3)
(3.2)
(2.2)
(3.4)
(1.9)
(1.3)
(1.1)
(0.50)
(1.8)
(3.5)
(1.8)
(3.8)
(5.6)
(2.8)
(1.1)
132
92
44
55
21
43
27
23
14
19
13
52
23
32
237
162
67
(1.7)
(4.2)
(1.9)
(2.8)
(1.2)
(2.5)
(1.2)
(1.4)
(0.82)
(0.22)
(1.4)
(2.6)
(1.4)
(1.9)
(6.6)
(2.0)
(2.7)
O
O
S
S
S
S
S
S
SO2
S
(0.10)
(0.28)
(0.09)
(0.33)
(0.09)
(0.16)
(0.04)
(0.14)
(0.11)
(0.07)
(0.32)
(0.05)
(0.42)
(2.3)
6.5
1.8
5.9
1.5
3.6
0.67
2.4
9.6
0.67
6.3
0.80
7.2
p-methylbenzyl
p-methylbenzyl
p-methoxybenzyl
p-methoxybenzyl
p-chlorophenyl
1-naphthyl
1-naphthyl
2-naphthyl
2-naphthyl
S
S
S
84
Ritonavir
Nelfinavir
16
308
(0.20)
(12)
a Ritonavir-resistant clone from NL-432 strain established by Shionogi Co. Ltd.: N37S, G57R, and I84V. b Nelfinavir-resistant clone from NL-432 strain
established by Shionogi Co. Ltd.: D30N, N37S, and G57R. c Ritonavir- and Nelfinavir-resistant clone from NL-432 strain established by Shionogi Co. Ltd.:
L19V, V32I, M46L, G57R, L63P, and I85V.
Figure 4. Structures of compounds 21 and 25.
interactions of the D-amino acid moiety with Asn30 of the
protease similar to that of RTV.
and the side chain of D-amino acid derivative was directed to
the S3 pocket while the N-acetylamino moiety was directed to
the S2 pocket. The common pose of 21 during the molecular
dynamics (MD) simulations surrounded by explicit water was
similar to that of 1 (Figure 5a). In the S2 pocket, the carbonyl
of the N-acetyl group could form hydrogen bond interactions
with the two amide NH groups of Asp29 and Asp30 (Figure
5b). Additionally, the NH of the N-acetyl moiety could interact
with the carbonyl of Gly48 from the flap region. This hydrogen
bond network seemed to be a common feature among the
D-amino acid containing analogues. Contrary to the hydrogen
bond network, compound 1’s hydrophobic side chain of
methylthioalanine was tightly packed in the pocket space. On
the other hand, the side chain of the D-amino acid residue of
compound 21, that is p-methoxybenzylthiomethyl, fitted in the
S3 pocket. The thioether of the side chain was flexibly bent to
point to the S3 pocket, letting the p-methoxybenzyl moiety
contact favorably with the side chains of Arg8′, Pro81′, and
Val82′ (Figure 5b).
The I84V mutation enlarges the space of the binding pockets
with a volume of two methyl groups from I84V and I84′V than
that of the wild-type enzyme. The acetylamino moiety of
compound 21, however, did not contact with the side chain of
Ile84 in the S2 pocket. Figure 5c shows one of the poses of 21
bound to the Val84 protease during an MD simulation. The rmsd
from 21 in wild-type protease was 0.556 Å. The acetylamino
moiety showed a slight contact with Val84 similar to the wild-
type protease, keeping the aforementioned hydrogen bond
network with amide NHs and CO of the protease backbone.
Furthermore, slight rotations at the P3 position kept the
hydrophobic interactions seen in the wild-type protease. Of
course, there are hydrophobic contacts within the S2′ pocket in
which the rotation of the methylene moiety in P2′ 2-methyl-
The RTV/NFV-resistant clone was less sensitive to both RTV
and NFV, with 2.0- and 2.7-fold decreases, respectively. This
clone possesses multiple mutations of L19V, V32I, S37N,
M46L, L63P, and I85V compared to the original protease
sequence of HIV-1 IIIB containing N37S and G57R. Among
these mutations, V32I is thought to directly change the shape
of the protease pockets. The activity of 1 was reduced by 6.6-
fold. The results of the D-amino acid containing analogues
against the RTV/NFV-resistant clone were similar to the results
against the RTV-resistant clone. Interestingly, compound 21
maintained its activity (0.82-fold), similar to the RTV-resistant
clone. In the series, compound 28 was the most potent with a
decreased activity (1.4-fold). On the other hand, the activity of
compound 25 was increased to a similar extent as the com-
pound’s potency against the RTV-resistant HIV strain. Com-
pound 25 exhibited higher potencies against the three tested
mutant proteases than the wild type protease. We believe that
because the active sites of the mutant proteases are slightly
different from that of the wild protease as a result of different
amino acid sequences, certain functional groups could be better
accommodated by the active subsites of the mutant proteases
than that of the wild protease.
The high activity sustenance of compound 21 (Figure 4)
against all three HIV-1 mutants with different protease se-
quences remains to be a matter to discuss. To speculate the
binding mode of compound 21 with HIV-1 protease, we
constructed a molecular model using an X-ray crystal structure
of compound 1 complex (PDB, 1hpx). On the basis of the design
rationale of the D-amino acid containing Apns derivatives, the
HMC of 21 ideally interacted with the two catalytic aspartic
acids of wild-type HIV protease similar to that observed in 1,19,20

2998 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Nakatani et al.
Figure 5. MD simulated poses of compound 21 (green stick) bound to HIV-1 proteases with a water soak. (a) Superimposition of compound 1
(purple stick, PDB 1hpx) in the wild-type protease. Flap regions of the protease are hidden. (b) Close-up view of the substituted D-cysteine residue
of 21 with a green meshed molecular surface. (c) Overlay of four poses of 21 in the four proteases (wild-type, I84V (red), D30N (blue), V32I
(yellow)). Mutated residues in each protease are shown with contact surfaces to 21 (meshed surface for wild-type). (d) Overlay of four poses of 25
in the four proteases with a close up view of S2 and S3 sites.
benzyl compensated for the change in I84V by a similar manner
to an X-ray observation of compound 2 in V32F/I84V pro-
tease.31 These observations suggest the reason why compound
21 could keep its activity against the RTV-resistant HIV-1 clone
while compound 1 could not.
in the S2′ pocket was inverted to accept the P2′ moiety from
the inhibitor. Finally, the conformations of compound 21 in the
wild-type protease were kept, even in the V32I protease.
Compound 25 was the only one to exhibit improvements in
activity against all tested mutant HIVs. We constructed various
molecular models of compound 25 bound to these mutated
proteases (Figure 5d). The poses were similar to that of
compound 21 except for the side chain of the D-amino acid
residue. We observed two additional bridging water molecules
between the inhibitor and the protease. One interacted between
one of the sulfonyl oxygens and Gly27 NH or Asp29 carboxy-
late, while the other one was observed between another SO and
Gly48 CO or Asp/Asn30. These stable interactions with water
and the sulfonyl derivative could form a basis for the improve-
ments in activity of compound 25.
We compared the potency of D-amino acid containing
inhibitors in the absence or presence of AGP at the same
concentration as normal human serum (Table 3). The addition
of AGP worsened the EC50 values of RTV and NFV to 18- and
29-fold, respectively. The potency of compound 1 also decreased
24-fold. The potency shifts of the D-amino acid containing
analogues varied from 4- to 87-fold. A large shift in potency
was observed in the case of compound 28, which exhibited the
highest activity among the series in the absence of AGP. On
the other hand, compounds 12, 17, and 21 were relatively potent
with moderate attenuation by the AGP addition (4.9-, 7.0-, 6.8-
fold, respectively). These potency shifts are lower than that of
the 12.7-fold of Lopinavir.29 Lopinavir in combination with
RTV is recommended for protease inhibitor-based regimens in
antiretroviral naïve patients. As seen in compounds 12, 17, and
21, a low AGP binding may increase the plasma concentration
A mutation of Asp30 to Asn in the protease sequence
represents the NFV-resistant strain. In this case, compound 21
became more sensitive to Asn than Asp. Molecular modeling
experiments revealed a movement of the neutral amide side
chain of Asn to contact with the hydrophobic acetylamino
moiety of D-amino acid residue in the S2 pocket, although the
anionic carboxylate of Asp is directed toward the solvent,
avoiding contact with the nonpolar acetyl group in the wild-
type protease (Figure 5c). This additional hydrophobic contact
between Asn and the acetyl moiety suggested an increased
affinity in compound 21 while keeping the specific interactions
of the D-amino acid residue described above (rmsd, 0.584 Å).
The acetyl and mesyl moiety of other D-amino acid containing
compounds can similarly contact with the side chain of Asn30,
exhibiting an increased potency compared to that of the wild-
type enzyme. In the S2′ pocket, the phenyl of the P2′ 2-meth-
ylbenzyl group interacted with the methylene side chain of the
Asp or Asn residue.
In the case of the V32I mutation, as seen in the RTV/NFV-
resistant clone, the space of the protease binding sites seemed
to be relatively smaller. One of the poses of compound 21
slightly contacted with Val or Ile of the S2 pocket during a
simulation (Figure 5c). As observed in the I84V mutation, the
hydrogen bond network between the D-amino acid residue and
protease backbone of the S2 pocket was kept, adopting the side
chain in the S3 pocket (rmsd, 0.398 Å). The side chain of Ile32′

D-Amino Acid Containing HIV Protease Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2999
Table 3. Anti-HIV-1 Activity in the Presence of AGP
8.1 Hz, 2H), 7.09 (d, J ) 7.9 Hz 2H), 6.44 (d, J ) 7.0 Hz, 1H),
3.99–3.86 (m, 1H), 3.73–3.64 (m, 2H), 2.82 (dd, J ) 13.4, 4.2 Hz,
1H), 2.66 (dd, J ) 13.4 Hz, 7.3 Hz, 1H), 2.26 (s, 3H), 1.39 (s,
3H). MS (ES-) m/z: 340 for [M - H]-.
EC50, nM
compound
10% FCSa
10% FCS + AGPb
potency shift
9
10
11
12
13
16
17
20
21
25
28
29
32
33
1
25
20
20
18
16
14
12
9.4
13
27
4.0
14
8.0
14
22
24
21
155
90
278
88
96
110
84
94
89
1268
347
401
88
6.2
4.5
14
4.9
6.0
7.9
7.0
10
6.8
47
87
29
11
The compound 5b (500 mg, 1.54 mmol) was dissolved in CHCl3
(20 mL), and then m-chloroperoxybenzoic acid (590 mg, 3.42
mmol) was added at 0 °C. The mixture was stirred at room
temperature for 10 min. After the reaction mixture was concentrated
in vacuo, purification of the residue by silica gel column chroma-
tography (CHCl3-MeOH) gave 480 mg of compound 6b; yield 76%
from thiol. 1H NMR (DMSO-d6) δ (ppm): 7.75–7.63 (m, 1H), 7.27
(d, J ) 8.1 Hz, 2H), 7.19 (d, J ) 8.1 Hz, 2H), 4.43 (s, 2H),
4.41–4.25 (m, 1H), 4.14 (dd, J ) 5.6 Hz, 1.9 Hz, 1H), 3.49 (dd, J
) 14.6 Hz, 3.4 Hz, 1H), 2.31 (s, 3H), 1.38 (s, 9H); MS (ES-) m/z:
396 for [M - H]-.
(S)-2-(tert-Butoxycarbonyl)amino-3-(benzylsulfonyl)propano-
ic acid (6a). Compound 6a was prepared from benzylmercaptan
in a manner similar to that described for compound 6b; yield 23%;
mp 176–179 °C. 1H NMR (DMSO-d6) δ (ppm): 7.49–7.27 (m, 5H),
7.15 (br, 1H), 4.50 (s, 2H), 4.36 (br, 1H), 3.54 (d, J ) 3.1 Hz,
1H), 3.50 (d, J ) 2.8 Hz, 1H), 1.38 (s, 9H); MS (ES-) m/z: 342 for
[M - H]-.
95
6.8
24
18
529
422
606
Ritonavir
Nelfinavir
29
a FCS, fetal calf serum. b AGP, R1-acid glycoprotein.
of a free drug, reducing its dose and its undesired side effects
such as lipodystrophy.
(S)-2-(tert-Butoxycarbonyl)amino-3-(4-methoxybenzylsulfo-
nyl)propanoic acid (6c). Compound 6c was prepared from (S)-2-
(tert-butoxycarbonylamino)-3-(4-methoxybenzylthio)propanoic acid
in a manner similar to that described for compound 6b; yield 94%.
1H NMR (DMSO-d6) δ (ppm): 7.76–7.63 (m, 1H), 7.30 (d, 2H, J
) 8.8 Hz), 6.95 (d, 2H, J ) 8.6 Hz), 4.47–4.28 (m, 3H), 4.13 (dd,
1H, J ) 5.6 Hz, 1.9 Hz), 3.76 (s, 1H), 3.48 (dd, 1H, J ) 14.4 Hz,
3.2 Hz), 1.38 (s, 9H); MS (ES-) m/z: 356 for [M - H]-.
(S)-2-(tert-Butoxycarbonyl)amino-3-(4-clorophenylsulfonyl)-
propanoic acid (6e). Compound 6e was prepared from 4-chlo-
robenzenethiol in a manner similar to that described for compound
Conclusion
We synthesized a series of D-amino acid containing HIV
protease inhibitors in order to apply the “D-amino acid concept”
into an allophenynorstatine-based structure. Evaluation of these
analogues suggested the usefulness of D-amino acid residues
for high potency against not only the wild-type but also resistant
HIV, especially the D30N mutation, in the protease. Several
derivatives had promising results against all resistant HIVs and
prospective profiles of cytotoxicity and AGP binding. These
results suggest the potentials of the D-amino acid derived
structure in the development of the next generation of HIV
protease inhibitors.
1
6b; yield 33%; mp 128–136 °C. H NMR (DMSO-d6) δ (ppm):
7.85 (d, J ) 8.4 Hz, 2H), 7.71 (d, J ) 8.3 Hz, 2H), 6.94 (d, J )
7.2 Hz, 1H), 4.28–4.18 (m, 1H), 3.75–3.70 (m, 2H), 1.29 (s, 9H);
MS (ES-) m/z: 362 for [M - H]-.
(S)-2-(tert-Butoxycarbonyl)amino-3-(naphthalene-1-yl-sulfo-
nyl)propanoic acid (6f). Compound 6f was prepared from 1-thion-
aphthol in a manner similar to that described for compound 6b;
Experimental Section
1
yield 82%; mp 133–142 °C. H NMR (DMSO-d6) δ (ppm): 8.58
(d, J ) 8.3 Hz, 1H), 8.33 (d, J ) 8.2 Hz, 1H), 8.16 (t, J ) 8.0 Hz,
2H), 7.84–7.58 (m, 3H), 6.88 (br, 1H), 4.27 (br, 2H), 3.95–3.78
(m, 2H), 1.23 (s, 9H). MS (ES-) m/z: 378 for [M - H]-.
Commercial reagents and solvents were used without further
purification. Column chromatography was performed on Merck
Silica Gel 60 (70–230 mesh). Melting points were measured on a
Yanagimoto micromelting apparatus without corrections. Analytical
1H NMR spectra were recorded on a JEOL JNM-AL 300 FT NMR
system with TMS as an internal standard. ES mass spectra were
obtained from a Finnigan SSQ 7000 spectrometer. High-resolution
FAB and EI mass spectra data were obtained on a JEOL JMS-SX
102A QQ and a JEOL JMS-GC MATE, respectively. Preparative
HPLC was carried out on a C18 reverse phase column (20 mm ×
250 mm; YMC Pack ODS SH343-5) with a binary solvent system:
a linear gradient of CH3CN in 0.1% aqueous TFA with a flow rate
of 5.0 mL/min and detection at 230 nm. Analytical HPLC was
performed using a C18 reverse-phase column (20 mm × 250 mm;
YMC Pack ODS SH343–5) with binary solvent systems: (A) linier
gradient of CH3CN 40–100% in 0.1% aqueous TFA in 15 min,
(B) liniar gradient of CH3CN 20–80% in 0.1% aqueous TFA in 30
min at a flow rate of 0.9 mL/min, detected at 230 nm.
(S)-2-(tert-Butoxycarbonyl)amino-3-(4-methylbenzylthio)pro-
panoic acid (5b) and (S)-2-(tert-Butoxycarbonyl)amino-3-(4-
methylbenzylsulfonyl)propanoic acid (6b). To a solution of (p-
methylphenyl)methanethiol (0.72 g, 5.80 mmol) in THF (30 mL)
was added NaH (0.2 g, 0.49 mmol, 60% oil dispersion), and the
mixture was stirred at room temperature for 10 min. A solution of
N-tert-Boc-D-serine ꢀ-lactone (4) (1.14 g, 5.78 mmol) in THF (20
mL) was added dropwise over 10 min, and the mixture was
additionally stirred at room temperature for 10 min. The reaction
mixture was concentrated in vacuo and then purified by silica gel
column chromatography (CHCl3-MeOH) to give 1.59 g of com-
pound 5b; yield 88%. 1H NMR (DMSO-d6) δ (ppm): 7.18 (d, J )
(S)-2-(tert-Butoxycarbonyl)amino-3-(naphthalene-2-yl-sulfo-
nyl)propanoic acid (6g). Compound 6g was prepared from
2-naphthalenethiol in a manner similar to that described for
1
compound 6b; yield 30%; mp 107–113 °C. H NMR (DMSO-d6)
δ(ppm): 8.52 (s, 1H), 8.16 (t, J ) 8.1 Hz, 2H), 8.08 (d, J ) 7.9
Hz, 1H), 7.85 (d, J ) 9.3 Hz, 1H), 7.81–7.59 (m, 2H), 6.83 (d, J
) 7.9 Hz, 1H), 4.33–4.22 (m, 1H), 3.85–3.67 (m, 2H), 1.11 (s,
9H). MS (ES-) m/z: 378 for [M - H]-.
(S)-2-(tert-Butoxycarbonyl)amino-3-(quinoline-2-yl-sulfonyl)-
propanoic acid (6h). Compound 6h was prepared from 2-quino-
linethiol in a manner similar to that described for compound 6b;
1
yield 57%; mp 115–123 °C. H NMR (DMSO-d6) δ (ppm): 8.76
(d, J ) 8.6 Hz, 1H), 8.19 (d, J ) 9.4 Hz, 2H), 8.07 (d, J ) 8.4 Hz,
1H), 7.96 (d, J ) 7.7 Hz, 1H), 7.91–7.79 (m, 1H), 6.96 (d, J ) 7.3
Hz, 1H), 4.42–4.31 (m, 1H), 4.10–3.93 (m, 2H), 1.11 (s, 9H). MS
(ES-) m/z: 379 for [M - H]-.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(quin-
oline-2-yl-thio)-2-(methylsulfonylamino)propanoyl)amino-4-phe-
nylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (36):
General Method for N-Mesyl Derivatives. N-Boc-S-quinolinyl-
D-cysteine 6h (400 mg, 1.15 mmol) was dissolved in DMF (4 mL).
The solution was cooled to 0 °C, and hydrocloride of allophenyl-
norstatine unit 7 (549 mg, 1.15 mmol), HOBt ·H2O (193 mg, 1.26
mmol), EDC ·HCl(242 mg, 1.26 mmol), and triethylamine (240 µL,
1.72 mmol) were added. The mixture was stirred at room temper-
ature for overnight, concentrated in vacuo, and diluted with EtOAc.
The mixture was washed with 10% citric acid, 5% sodium

3000 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Nakatani et al.
bicarbonate and brine, and then dried (Na2SO4) and concentrated
in vacuo. The silica gel column chromatography gave 594 mg, yield
67%, of the Boc-protected intermediate. To the protected intermedi-
ate (400 mg, 0.52 mmol) were added anisole (85 µL, 0.78 mmol)
and 4 N HCl/dioxane (2.6 mL), and the mixture was stirred at room
temperature for 1 h. After the reaction mixture was concentrated
in vacuo, ether was added. The precipitate was filtered and dried
to give the hydrochloride salt (368 mg, quantitative). The amine
(100 mg, 0.14 mmol) in CH2Cl2 (2 mL) was treated with
N-methylmorpholine (78.1 µL, 0.71 mmol) and methanesulfonyl
chloride (43.8 µL, 0.57 mmol) at 0 °C, and the mixture was stirred
at room temperature for 2 h. After removal of the solvent in vacuo,
the residue was dissolved in EtOAc, washed sequentially with 10%
citric acid, 5% sodium bicarbonate, and brine, dried over Na2SO4,
and concentrated in vacuo to give 62 mg, yield 59%. Purification
of the product by preparative TLC (CHCl3-MeOH) and preparative
HPLC gave the title compound; yield 20% from 8. 1H NMR
(DMSO-d6) δ (ppm): 8.53 (d, J ) 8.1 Hz, 1H), 8.42 (d, J ) 5.7
Hz, 1H), 8.18 (d, J ) 8.6 Hz, 1H), 7.93 (d, J ) 8.6 Hz, 2H), 7.76
(d, J ) 8.2 Hz, 1H), 7.52 (d, J ) 7.4 Hz, 1H), 7.38–7.02 (m, 10H),
5.31 (d, J ) 7.7 Hz, 1H), 5.08 (d, J ) 9.2 Hz, 1H), 4.97 (d, J )
9.2 Hz, 1H), 4.52 (s, 1H), 4.50 (dd, J ) 7.1 Hz, 2.4 Hz, 1H), 4.41
(dd, J ) 15.4 Hz, 6.4 Hz, 1H), 4.32–4.16 (m, 3H), 3.22–3.15 (m,
2H), 2.87 (s, 3H), 2.80–2.57 (m, 2H), 2.27 (s, 3H), 1.51 (s, 3H),
1.37 (s, 3H). HRMS (FAB+) calcd for C37H44N5O6S3 [M + H]+,
750.2454; found, m/z 750.2448.
1.36 (s, 3H). HRMS (FAB+) calcd for C36H45N4O5S [M + H]+,
645.3111; found, m/z 645.3120.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2R)-3-benzyloxy-2-(me-
thylsulfonylamino)propanoyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (10). Compound 10
was prepared from compound 7 in a manner similar to that described
for compound 36. 1H NMR (DMSO-d6) δ (ppm): 8.48 (d, J ) 8.6
Hz, 1H), 8.41 (t, J ) 5.6 Hz, 1H), 7.37–7.08 (m, 15H), 5.06 (d, J
) 8.8 Hz, 1H), 4.98 (d, J ) 9.2 Hz, 1H), 4.52 (s, 1H), 4.48–4.11
(m, 7H), 3.24 (d, J ) 5.9 Hz, 2H), 2.82 (s, 3H), 2.75–2.63 (m,
2H), 2.27 (s, 3H), 1.51 (s, 3H), 1.36 (s, 3H). HRMS (EI+) calcd
for C35H44N4O7S2 [M]+, 696.2651; found, m/z 696.2635.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2R)-2-acetylamino-3-
(benzyloxy)propanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (11). Compound 11 was
prepared from compound 7 in a manner similar to that described
for compound 37. 1H NMR (DMSO-d6) δ (ppm): 8.41 (t, J ) 5.4
Hz, 1H), 8.36 (d, J ) 8.7 Hz, 1H), 7.97 (d, J ) 8.6 Hz, 1H),
7.35–7.08 (m, 14H), 5.08 (d, J ) 9.5 Hz, 1H), 4.94 (d, J ) 9.5 Hz,
1H), 4.66–4.57 (m, 1H), 4.51 (s, 1H), 4.47–4.38 (m, 2H), 4.27–4.09
(m, 4H), 3.27–3.18 (m, 2H), 2.73–2.62 (m, 2H), 2.27 (s, 3H), 1.84
(s, 3H), 1.50 (s, 3H), 1.35 (s, 3H). HR-MS (FAB+) calcd for
C36H45N4O6S [M + H]+, 661.3060; found, m/z 661.3069.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-3-benzylthio-2-(me-
thylsulfonylamino)propanoyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (12). Compound 12
was prepared from compound 7 in a manner similar to that described
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(quinoline-2-yl-thio)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (37): Gen-
eral Method for N-Acetyl Derivatives. To a solution of the amine
intermediate mentioned above, hydrochloride salt of H-D-Cys(naph-
thalene-2-yl)-Apns-Dmt-NH-Bzl(2-Me) (100 mg, 0.14 mmol) in
CH2Cl2 (2 mL) and N-methylmorpholine (31 µL, 0.28 mmol) was
added, followed by acetic anhydride (41 µL, 0.42 mmol), slowly
under stirring at 0 °C. The resulting solution was stirred at room
temperature for 1 h. After removal of the solvent in vacuo, the
residue was dissolved in EtOAc, washed sequentially with 10%
citric acid, 5% sodium bicarbonate, and brine, dried over Na2SO4,
and concentrated in vacuo to give 54 mg, yield 54%. The crude
product was purified by preparative TLC (CHCl3-MeOH) and
1
for compound 36. H NMR (DMSO-d6) δ (ppm): 8.48-8.37 (m,
2H), 7.47 (d, J ) 9.7 Hz, 1H), 7.37–7.02 (m, 14H), 5.29 (d, J )
7.4 Hz, 1H), 5.07 (d, J ) 9.0 Hz, 1H), 4.91 (d, J ) 9.0 Hz, 1H),
4.52 (s, 1H), 4.49–4.34 (m, 2H), 4.22 (d, J ) 4.8 Hz, 1H), 4.17 (d,
J ) 4.6 Hz, 1H), 3.94 (d, J ) 5.7 Hz, 1H), 3.77 (s, 2H), 3.17 (d,
J ) 5.1 Hz, 1H), 2.80–2.56 (m, 3H), 2.39 (s, 3H), 2.26 (s, 3H),
1.49 (s, 3H), 1.36 (s, 3H). HRMS (FAB+) calcd for
C35H44N4O6S3Na [M + Na]+, 735.2321; found, m/z 735.2327.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(benzylthio)propanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (13). Compound 13 was
prepared from compound 7 in a manner similar to that described
for compound 37. 1H NMR (DMSO-d6) δ (ppm): 8.36 (d, J ) 5.5
Hz, 1H), 8.05 (d, J ) 8.4 Hz, 1H), 8.01 (d, J ) 8.4 Hz, 1H),
7.35–7.06 (m, 14H), 5.33 (d, J ) 7.1 Hz, 1H), 5.01 (d, J ) 9.0 Hz,
1H), 4.91 (d, J ) 9.2 Hz, 1H), 4.49 (s, 1H), 4.47–4.34 (m, 3H),
4.20 (d, J ) 4.8 Hz, 1H), 4.15 (d, J ) 4.4 Hz, 1H), 3.71 (s, 1H),
3.17 (d, J ) 5.1 Hz, 1H), 2.80–2.60 (m, 3H), 2.26 (s, 3H), 1.82 (s,
3H), 1.49 (s, 3H), 1.34 (s, 3H). HRMS (FAB+) calcd for
C36H45N4O5S2 [M + H]+, 677.2831; found, m/z 677.2827.
1
HPLC to give compound 37; yield 44%. H NMR (DMSO-d6) δ
(ppm): 8.42 (t, J ) 5.7 Hz, 1H), 8.33 (d, J ) 9.0 Hz, 1H), 8.16 (d,
J ) 8.4 Hz, 1H), 8.15 (d, J ) 8.6 Hz, 1H), 7.90 (d, J ) 8.8 Hz,
1H), 7.74 (t, J ) 7.6 Hz, 1H), 7.51 (d, J ) 7.5 Hz, 1H), 7.35–7.06
(m, 10H), 5.32 (d, J ) 7.3 Hz, 1H), 5.07 (d, J ) 9.0 Hz, 1H), 4.95
(d, J ) 9.2 Hz, 1H), 4.70–4.62 (m, 6H), 4.52 (s, 1H), 4.50–4.35
(m, 2H), 4.22 (d, J ) 5.0 Hz, 1H), 4.17 (d, J ) 4.4 Hz, 1H), 3.47
(dd, J ) 13.1 Hz, 4.7 Hz, 1H), 3.05 (dd, J ) 13.4 Hz, 9.3 Hz, 1H),
2.82–2.63 (m, 2H), 2.27 (s, 3H), 1.79 (s, 3H), 1.51 (s, 3H), 1.36
(s, 3H). HRMS (FAB+) calcd for C38H44N5O5S2 [M + H]+,
714.2784; found, m/z 714.2789.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-3-benzylsulfonyl-
2-(methylsulfonylamino)propanoyl)amino-2-hydroxy-4-phenylb-
utanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (14). Com-
pound 14 was prepared from compound 7 in a manner similar to
1
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2R)-2-meth-
ylsulfonylamino-4-phenylbutanoyl)amino-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (8). Compound 8
was prepared from compound 7 in a manner similar to that described
for compound 36. 1H NMR (DMSO-d6) δ (ppm): 8.51 (d, J ) 8.4
Hz, 1H), 8.42 (t, J ) 6.0 Hz, 1H), 7.37–6.98 (m, 14H), 5.14 (d, J
) 9.4 Hz, 1H), 4.97 (d, J ) 9.5 Hz, 1H), 4.53 (s, 1H), 4.48–4.39
(m, 2H), 4.27–4.16 (m, 2H), 3.91–3.81 (m, 1H), 2.78 (s, 3H),
2.73–2.65 (m, 2H), 2.27 (s, 3H), 2.21–2.12 (m, 2H), 1.60–1.47 (m,
5H), 1.37 (s, 3H). HRMS (FAB+) calcd for C35H45N4O6S2 [M +
H]+, 681.2781; found, m/z 681.2793.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2R)-2-acetylamino-4-
phenylbutanoyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (9). Compound 9 was prepared
from compound 7 in a manner similar to that described for
compound 37. 1H NMR (DMSO-d6) δ (ppm): 8.39 (t, J ) 5.4 Hz,
1H), 8.32 (d, J ) 8.4 Hz, 1H), 7.94 (d, J ) 8.3 Hz, 1H), 7.33–6.95
(m, 14H), 5.13 (d, J ) 9.2 Hz, 1H), 4.95 (d, J ) 9.4 Hz, 1H), 4.52
(s, 1H), 4.47–4.34 (m, 3H), 4.22–4.16 (m, 2H), 2.73–2.63 (m, 2H),
2.27 (s, 3H), 2.23–2.12 (m, 2H), 1.84 (s, 3H), 1.63–1.43 (m, 5H),
that described for compound 36. H NMR (DMSO-d6) δ (ppm):
8.59 (d, J ) 8.4 Hz, 1H), 8.42 (t, J ) 4.8 Hz, 1H), 7.74 (br, 1H),
7.47–7.03 (m, 14H), 5.42 (d, J ) 7.0 Hz, 1H), 5.01 (d, J ) 9.2 Hz,
1H), 4.97 (d, J ) 9.3 Hz, 1H), 4.52 (s, 1H), 4.48 (dd, J ) 7.4 Hz,
3.0 Hz, 1H), 4.45–4.34 (m, 4H), 4.23 (d, J ) 4.6 Hz, 1H), 4.18 (d,
J ) 5.0 Hz, 1H), 3.16–3.23 (m, 1H), 3.02–2.97 (m, 1H), 2.88 (s,
3H), 2.80–2.60 (m, 2H), 2.26 (s, 3H), 1.51 (s, 3H), 1.36 (s, 3H).
HRMS (FAB+) calcd for C35H44N4O8S3Na [M + Na]+, 767.2219;
found, m/z 767.2214.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(benzylsulfonyl)propanoyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (15). Compound 15
was prepared from compound 7 in a manner similar to that described
for compound 37. 1H NMR (DMSO-d6) δ (ppm): 8.44 (t, J ) 5.4
Hz, 1H), 8.27 (d, J ) 9.1 Hz, 1H), 8.24 (d, J ) 8.8 Hz, 1H),
7.42–7.03 (m, 14H), 5.39 (d, J ) 7.5 Hz, 1H), 5.01 (d, J ) 8.8 Hz,
1H), 4.92 (d, J ) 9.0 Hz, 1H), 4.89–4.78 (m, 1H), 4.50 (s, 1H),
4.47–4.38 (m, 2H), 4.34 (d, J ) 8.4 Hz, 2H), 4.18 (d, J ) 5.4 Hz,
1H), 4.13 (d, J ) 4.4 Hz, 1H), 3.24–3.13 (m, 1H), 2.94 (dd, J )
14.7 Hz, 9.6 Hz, 1H), 2.86–2.62 (m, 2H), 2.25 (s, 3H), 1.87 (s,

D-Amino Acid Containing HIV Protease Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3001
3H), 1.49 (s, 3H), 1,35 (s, 3H). HRMS (FAB+) calcd for
C36H44N4O7S2Na [M + Na]+, 731.2549; found, m/z 731.2562.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(4-me-
thylbenzylthio)-2-(methylsulfonylamino)propanoyl)amino-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(16). Compound 16 was prepared from compound 7 in a manner
(s, 1H), 4.46–4.38 (m, 2H), 4.22–4.13 (m, 2H), 3.73 (s, 3H), 3.54
(d, J ) 2.7 Hz, 2H), 2.72–2.62 (m, 2H), 2.35–2.27 (m, 4H), 2.14
(dd, J ) 13.5 Hz, 9.3 Hz, 1H), 1.84 (s, 3H), 1.50 (s, 3H), 1.35 (s,
3H). HRMS (FAB+) calcd for C37H47N4O6S2 [M + H]+, 707.2937;
found, m/z 707.2949.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(4-
methoxybenzylsulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (22). Compound 22 was prepared from compound 7 in a
manner similar to that described for compound 36. 1H NMR
(DMSO-d6) δ (ppm): 9.28 (d, J ) 9.3 Hz, 1H), 8.63 (d, J ) 8.6
Hz, 2H), 8.56 (d, J ) 8.8 Hz, 1H), 8.40 (m, 1H), 7.32–7.12 (m,
13H), 5.02–4.98 (m, 2H), 4.51–4.17 (m, 7H), 3.76 (s, 3H),
3.00–2.26 (m, 4H), 2.88 (s, 3H), 2.27 (s, 3H), 1.51 (s, 3H), 1.36
(w, 3H). HR-MS (FAB+) calcd for C36H47N4O9S3 [M + H]+,
775.2505; found, m/z 775.2499.
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.66 (d, J ) 7.5 Hz, 1H), 8.44(t, J ) 6.0 Hz, 1H), 7.43
(d, J ) 9.2 Hz, 1H), 7.33–7.09 (m, 13H), 5.09 (d, J ) 8.8 Hz,
1H), 4.96 (d, J ) 9.2 Hz, 1H), 4.53 (s, 1H), 4.49–4.39 (m, 2H),
4.23–4.02 (m, 3H), 3.62 (d, J ) 13.2 Hz, 1H), 3.53 (d, J ) 13.2
Hz, 1H), 2.84 (s, 3H), 2.73–2.65 (m, 2H), 2.32–2.18 (m, 8H,
overlapped with DMSO), 1.51 (s, 3H), 1.32 (s, 3H). HRMS (FAB+)
calcd for C36H46N4O6S3Na [M + Na]+, 749.2477; found, m/z
749.2487.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-(4-
methylbenzylthio)propanoyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (17). Compound 17
was prepared from compound 7 in a manner similar to that described
for compound 37. 1H NMR (DMSO-d6) δ (ppm): 8.58 (d, J ) 9.2
Hz, 1H), 8.42 (br, 1H), 7.98 (d, J ) 9.7 Hz, 1H), 7.30–7.09 (m,
13H), 5.09 (d, J ) 8.8 Hz, 1H), 4.94 (d, J ) 9.2 Hz, 1H), 4.68–4.54
(m, 1H), 4.52 (s, 1H), 4.46–4.38 (m, 2H), 4.24–4.09 (m, 2H), 3.55
(br, 2H), 2.73–2.27 (m, 2H), 2.37–2.10 (m, 8H, overlapped with
DMSO), 1.81 (s, 3H), 1.50 (s, 3H), 1.32 (s, 3H). HRMS (FAB+)
calcd for C37H47N4O5S2 [M + H]+, 691.2988; found, m/z 691.2979.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(4-me-
thylbenzylsulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (18). Compound 18 was prepared from compound 7 in a manner
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-(4-
methoxybenzylsulfonyl)propanoyl)amino-2-hydroxy-4-phenylb-
utanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23). Com-
pound 23 was prepared from compound 7 in a manner similar to
1
that described for compound 37. H NMR (DMSO-d6) δ (ppm):
8.44 (t, J ) 5.1 Hz, 1H), 8.27–8.22 (m, 2H), 7.29–7.13 (m, 11H),
6.91 (d, J ) 8.4 Hz, 2H), 5.01 (d, J ) 9.3 Hz, 1H), 4.92 (d, J )
8.7 Hz, 1H), 4.86–4.78 (m, 1H), 4.50 (s, 1H), 4.42–4.11 (m, 4H),
3.74 (s, 3H), 3.15–3.06 (m, 1H), 2.93 (dd, J ) 14.0 Hz, 9.2 Hz,
1H), 2.84–2.64 (m, 2H), 2.25 (s, 3H), 1.86 (s, 3H), 1.49 (s, 3H),
1.34 (s, 3H). HRMS (FAB+) calcd for C37H47N4O8S2 [M + H]+,
739.2835; found m/z 739.2823.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(4-
chlorophenylsulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (24). Compound 24 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.57 (d, J ) 8.6 Hz, 1H), 8.41 (t, J ) 5.4 Hz, 1H), 7.72
(d, J ) 9.4 Hz, 1H), 7.32–7.10 (m, 13H), 5.01 (d, J ) 9.0 Hz,
1H), 4.97 (d, J ) 9.0 Hz, 1H), 4.52 (s, 1H), 4.47 (d, J ) 3.0 Hz,
1H), 4.43–4.34 (m, 4H), 4.20 (dd, J ) 15.0 Hz, 4.8 Hz, 2H),
3.06–2.91 (m, 2H), 2.87 (s, 3H), 2.82–2.61 (m, 2H), 2.31 (s, 3H),
2.26 (s, 3H), 1.51 (s, 3H), 1.36 (s, 3H). HRMS (FAB+) calcd for
C36H46N4O8S3Na [M + Na]+, 781.2375; found, m/z 781.2380.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-(4-
methylbenzylsulfonyl)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (19). Com-
pound 19 was prepared from compound 7 in a manner similar to
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.58 (d, J ) 8.3 Hz, 1H), 8.40 (t, J ) 4.9 Hz, 1H), 7.81
(d, J ) 8.8 Hz, 2H), 7.70 (d, J ) 8.6 Hz, 2H), 7.49–7.05 (m, 10H),
5.37 (d, J ) 7.5 Hz, 1H), 4.98 (d, J ) 9.0 Hz, 1H), 4.93 (d, J )
9.2 Hz, 1H), 4.50 (s, 1H), 4.48–4.34 (m, 2H), 4.26 (d, J ) 7.5 Hz,
1H), 4.22 (d, J ) 5.1 Hz, 1H), 4.17 (d, J ) 5.0 Hz, 1H), 3.17–3.01
(m, 2H), 2.76 (s, 3H), 2.74–2.57 (m, 2H), 2.26 (s, 3H), 1.49 (s,
3H), 1.35 (s, 3H). HRMS (FAB+) calcd for C34H41ClN4O8S3Na
[M + Na]+, 787.1673; found, m/z 787.1678.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-(4-
chloropheylsulfonyl)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (25). Com-
pound 25 was prepared from compound 7 in a manner similar
to that described for compound 37. 1H NMR (DMSO-d6) δ
(ppm): 8.44 (t, J ) 5.5 Hz, 1H), 8.18 (d, J ) 9.2 Hz, 1H), 7.97
(d, J ) 9.0 Hz, 1H), 7.74 (d, J ) 9.0 Hz, 2H), 7.70 (d, J ) 9.0
Hz, 2H), 7.32–7.04 (m, 9H), 5.37 (d, J ) 7.4 Hz, 1H), 4.97 (d,
J ) 9.2 Hz, 1H), 4.89 (d, J ) 9.0 Hz, 1H), 4.69–4.54 (m, 1H),
4.48 (s, 1H), 4.43–4.33 (m, 2H), 4.19 (d, J ) 4.9 Hz, 1H), 4.14
(d, J ) 4.6 Hz, 1H), 3.22–3.16 (m, 2H), 2.80–2.57 (m, 2H),
2.25 (s, 3H), 1.64 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H). HR-MS
(FAB+) calcd for C35H41ClN4O7S2 Na [M + Na]+, 751.2003;
found, m/z 751.2009.
1
that described for compound 37. H NMR (DMSO-d6) δ (ppm):
8.43 (t, J ) 5.4 Hz, 1H), 8.25 (d, J ) 9.0 Hz, 1H), 8.21 (d, J )
10.8 Hz, 1H), 7.31–7.11 (m, 13H), 5.01 (d, J ) 8.8 Hz, 1H), 4.92
(d, J ) 9.0 Hz, 1H), 4.90–4.80 (m, 1H), 4.50 (s, 1H), 4.48–4.23
(m, 4H), 4.15 (dd, J ) 14.7 Hz, 4.8 Hz, 2H), 3.17–3.12 (m, 1H),
2.99–2.91 (m, 1H), 2.85–2.58 (m, 2H), 2.28 (s, 3H), 2.25 (s, 3H),
1.86 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H). HRMS (FAB+) calcd for
C37H47N4O7S2 [M + H]+, 723.2886; found, m/z 723.2878.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(4-
methoxybenzylthio)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (20). Compound 20 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.65 (d, J ) 8.8 Hz, 1H), 8.43 (t, 1H, J ) 5.1 Hz, 1H),
7.44 (d, J ) 9.5 Hz, 1H), 7.33–7.10 (m, 11H), 6.86 (d, J ) 8.6 Hz,
2H), 5.10 (d, J ) 9.2 Hz, 1H), 4.96 (d, J ) 9.4 Hz, 1H), 4.53 (s,
1H), 4.49 (br, 1H), 4.42 (dd, J ) 15.2 Hz, 5.9 Hz, 1H), 4.28–4.16
(m, 2H), 4.09–4.00 (m, 1H), 3.73 (s, 3H), 3.63–3.50 (m, 2H), 2.84
(s, 3H), 2.73–2.10 (m, 2H), 2.33–2.19 (m, 5H), 1.51 (s, 3H), 1.36
(s, 3H). HRMS (FAB+) calcd for C36H47N4O7S3 [M + H]+,
743.2607; found, m/z 743.2610.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(4-
fluorophenylsulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (26). Compound 26 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.58 (d, J ) 9.2 Hz, 1H), 8.41 (t, J ) 5.5 Hz, 1H), 7.89
(d, J ) 5.3 Hz, 1H), 7.86 (d, J ) 5.1 Hz, 1H), 7.47 (t, 2H, J ) 8.7
Hz 2H), 7.33–7.18 (m, 3H), 7.17–7.01 (m, 7H), 5.37 (d, J ) 7.1
Hz, 1H), 4.98 (d, J ) 9.5 Hz 1H), 4.93 (d, J ) 9.0 Hz, 1H), 4.50
(s, 1H), 4.48–4.32 (m, 2H), 4.27 (dd, J ) 9.6 Hz, 3.2 Hz, 1H),
4.21 (d, J ) 4.6 Hz 1H), 4.17 (d, J ) 4.4 Hz, 1H), 3.17 (dd, J )
9.9, 4.8 Hz 1H), 2.99 (dd, J ) 14.8 Hz, 3.8 Hz, 1H), 2.76 (s, 3H),
2.74–2.57 (m, 2H), 2.26 (s, 3H), 1.49 (s, 3H), 1.35 (s, 3H). HRMS
(FAB+) calcd for C34H42FN4O8S3 [M + H]+, 749.2149; found,
m/z 749.2140.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-(4-
methoxybenzylthio)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (21). Com-
pound 21 was prepared from compound 7 in a manner similar to
1
that described for compound 37. H NMR (DMSO-d6) δ (ppm):
8.48 (d, J ) 8.1 Hz, 1H), 8.42 (t, J ) 5.4 Hz, 1H), 7.98 (d, J ) 8.7
Hz, 1H), 7.33–7.09 (m, 11H), 6.86 (d, J ) 8.4 Hz, 2H), 5.09 (d, J
) 9.0 Hz, 1H), 4.94 (d, J ) 9.3 Hz, 1H), 4.64–4.38 (m, 1H), 4.52

3002 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Nakatani et al.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-(4-
fluoropheylsulfonyl)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (27). Com-
pound 27 was prepared from compound 7 in a manner similar to
7.98–7.80 (m, 3H), 7.72 (s, 1H), 7.63 (br, 1H), 7.59–7.44 (m, 2H),
7.40–7.27 (m, 4H), 7.25–7.00 (m, 6H), 5.29 (d, J ) 7.4 Hz, 1H),
5.09 (d, J ) 9.2 Hz, 1H), 4.97 (d, J ) 9.2 Hz, 1H), 4.52 (s, 1H),
4.50 (dd, J ) 7.2 Hz, 2.7 Hz, 1H), 4.41 (dd, J ) 14.8 Hz, 6.4 Hz,
1H), 4.14–4.05 (m, 1H), 2.95–2.86 (m, 2H), 2.83 (s, 3H), 2.79–2.57
(m, 2H), 2.27 (s, 3H), 1.51 (s, 3H), 1.37 (s, 3H). HR-MS (FAB+)
calcd for C38H45N4O6S3 [M + H]+, 749.2501; found, m/z 749.2507.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(naphthalene-2-yl-thio)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (33). Com-
pound 33 was prepared from compound 7 in a manner similar to
1
that described for compound 37. H NMR (DMSO-d6) δ (ppm):
8.44 (t, J ) 5.4 Hz, 1H), 8.18 (d, J ) 8.8 Hz, 1H), 7.98 (d, J ) 9.0
Hz, 1H), 7.82 (d, J ) 5.3 Hz, 1H), 7.79 (d, J ) 5.1 Hz, 1H), 7.47
(t, J ) 8.8 Hz, 2H), 7.34–7.03 (m, 9H), 5.38 (d, J ) 7.5 Hz, 1H),
4.97 (d, J ) 9.2 Hz, 1H), 4.89 (d, J ) 9.2 Hz, 1H), 4.70–4.53 (m,
1H), 4.48 (s, 1H), 4.44–4.31 (m, 2H), 4.20–4.09 (m, 2H), 3.22–3.15
(m, 2H), 2.80–2.58 (m, 2H), 2.25 (s, 3H), 1.65 (s, 3H), 1.49 (s,
3H), 1.34 (s, 3H). HRMS (FAB+) calcd for C35H42FN4O7S2 [M +
H]+, 713.2479; found, m/z 713.2487.
1
that described for compound 37. H NMR (DMSO-d6) δ (ppm):
8.54 (d, J ) 8.4 Hz, 1H), 8.41 (d, J ) 5.5 Hz, 1H), 8.15 (d, J )
8.4 Hz, 1H), 7.95–7.83 (m, 16H), 7.73 (s, 1H), 7.58–7.42 (m, 2H),
7.39–7.25 (m, 4H), 7.24–7.02 (m, 6H), 5.26 (d, J ) 7.3 Hz, 1H),
5.08 (d, J ) 9.2 Hz, 1H), 4.94 (d, J ) 8.8 Hz, 1H), 4.66–4.54 (m,
1H), 4.51 (s, 1H), 4.50–4.35 (m, 2H), 4.21 (d, J ) 4.8 Hz, 1H),
4.16 (d, J ) 4.4 Hz, 1H), 2.94 (dd, J ) 13.2 Hz, 4.8 Hz, 1H), 2.82
(dd, J ) 13.0 Hz, 9.3 Hz, 1H), 2.77–2.61 (m, H), 2.27 (s, 3H),
1.81 (s, 3H), 1.51 (s, 3H), 1.36 (s, 3H). HRMS (FAB+) calcd for
C39H45N4O5S2 [M + H]+, 713.2831; found, m/z 713.2823.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(naph-
thalene-1-yl-thio)-2-(methylsulfonylamino)propanoyl)amino-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(28). Compound 28 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.66 (d, J ) 8.4 Hz, 1H), 8.45–8.38 (m, 1H), 8.16 (d, J
) 9.3 Hz, 1H), 7.97 (d, J ) 6.9 Hz, 1H), 7.84 (d, J ) 7.8 Hz, 1H),
7.66–6.99 (m, 14H), 5.08 (d, J ) 9.4 Hz, 1H), 4.97 (d, J ) 9.0 Hz,
1H), 4.52 (s, 1H), 4.49 (d, J ) 2.4 Hz, 1H), 4.44–4.08 (m, 4H),
2.84 (d, J ) 7.5 Hz, 2H), 2.81 (s, 3H), 2.73–2.65 (m, 2H), 2.27 (s,
3H), 1.51 (s, 3H), 1.36 (s, 3H). HR-MS (FAB+) calcd for
C38H45N4O6S3 [M + H]+, 749.2501; found, m/z 749.2515.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(naphthalene-1-yl-thio)propanoyl)amino-2-hydroxy-4-phenylbu-
tanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (29). Com-
pound 29 was prepared from compound 7 in a manner similar to that
described for compound 37. 1H NMR (DMSO-d6) δ (ppm): 8.46–8.38
(m, 2H), 8.14 (d, J ) 8.4 Hz, 1H), 7.96 (d, J ) 9.3 Hz, 1H), 7.83 (d,
J ) 8.4 Hz, 1H), 7.60–7.01 (m, 14H), 5.07 (d, J ) 8.6 Hz, 1H), 4.94
(d, J ) 9.7 Hz, 1H), 4.62–4.53 (m, 1H), 4.51 (s, 1H), 4.45–4.37 (m,
2H), 4.22–4.09 (m, 2H), 2.94–2.63 (m, 5H), 2.26 (s, 3H), 1.82 (s, 3H),
1.50 (s, 3H), 1.36 (s, 3H). HRMS (FAB+) calcd for C39H45N4O5S2
[M + H]+, 713.2831; found, m/z 713.2838.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(naph-
thalene-2-yl-sulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (34). Compound 34 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.58–7.86 (m, 7H), 7.82–6.93 (m, 12H), 4.97 (d, J ) 8.6
Hz, 1H), 4.91 (d, J ) 8.8 Hz, 1H), 4.49 (s, 1H), 4.43–4.16 (m,
5H), 2.76 (s, 3H), 2.73–2.64 (m, 4H, overlapped with DMSO), 2.25
(s, 3H), 1.49 (s, 3H), 1.34 (s, 3H). HRMS (FAB+) calcd for
C38H45N4O8S3 [M + H]+, 781.2400; found, m/z 781.2394.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(naphthalene-2-yl-sulfonyl)propanoyl)amino-2-hydroxy-4-phe-
nylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (35).
Compound 35 was prepared from compound 7 in a manner similar
to that described for compound 37. 1H NMR (DMSO-d6) δ (ppm):
8.50–8.34 (m, 2H), 8.22–8.04 (m, 2H), 7.94 (d, J ) 8.8 Hz, 1H),
7.81–7.65 (m, 3H), 7.32–6.95 (m, 9H), 5.36 (d, J ) 7.3 Hz, 1H),
4.95 (d, J ) 9.0 Hz, 1H), 4.87 (d, J ) 9.5 Hz, 1H), 4.73–4.60 (m,
1H), 4.46 (s, 1H), 4.41–4.29 (m, 2H), 4.19–4.07 (m, 2H), 3.51–3.16
(m, 2H, overlapped with DMSO), 2.79–2.57 (m, 2H), 2.22 (s, 3H),
1.47 (s, 6H), 1.32 (s, 3H). HR-MS (FAB+) calcd for C39H45N4O7S2
[M + H]+, 745.2730; found, m/z 745.2726.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(naph-
thalene-1-yl-sulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (30). Compound 30 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6) δ
(ppm): 8.64–8.58 (m, 2H), 8.41- 8.36 (m, 2H), 8.20 (d, J ) 8.1 Hz,
1H), 8.11 (d, J ) 7.5 Hz, 1H), 7.84–7.68 (m, 3H), 7.58 (br, 1H),
7.30–7.27 (m, 1H), 7.19–6.96 (m, 5H), 6.77–6.57 (m, 3H), 5.33 (d, J
) 7.3 Hz, 1H), 4.95 (d, J ) 9.0 Hz, 1H), 4.91 (d, J ) 9.5 Hz, 1H),
4.52 (dd, J ) 9.7 Hz, 3.5 Hz, 1H), 4.49 (s, 1H), 4.46–4.30 (m, 2H),
4.20 (d, J ) 5.0 Hz, 1H), 4.15 (d, J ) 4.8 Hz, 1H), 3.17 (d, J ) 5.3
Hz, 1H), 3.05 (dd, J ) 14.1 Hz, 3.5 Hz, 1H), 2.81 (s, 3H), 2.24 (s,
3H), 1.48 (s, 3H), 1.34 (s, 3H). HRMS (FAB+) calcd for
C38H45N4O8S3 [M + H]+, 781.2400; found, m/z 781.2396.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(quin-
oline-2-yl-sulfonyl)-2-(methylsulfonylamino)propanoyl)amino-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxam-
ide (38). Compound 38 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.72 (d, J ) 8.6 Hz, 1H), 8.57 (d, J ) 8.8 Hz, 1H), 8.40
(t, J ) 5.6 Hz, 1H), 8.18 (d, J ) 8.8 Hz, 2H), 7.97 (t, J ) 7.6 Hz,
2H), 7.83 (t, J ) 7.6 Hz, 1H), 7.38–6.95 (m, 10H), 5.36 (d, J )
7.5 Hz, 1H), 4.94 (d, J ) 9.2 Hz, 1H), 4.89 (d, J ) 9.2 Hz, 1H),
4.49 (s, 1H), 4.47–4.33 (m, 3H), 4.22 (d, J ) 4.7 Hz, 1H), 4.17 (d,
J ) 4.8 Hz, 1H), 3.61–3.49 (m, 1H), 3.17 (d, J ) 5.1 Hz, 1H),
2.81–2.57 (m, 5H), 2.25 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H). HRMS
(FAB+) calcd for C37H43N5O8S3Na [M + Na]+, 804.2171; found,
m/z 804.2177.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(naphthalene-1-yl-sulfonyl)propanoyl)amino-2-hydroxy-4-phe-
nylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (31).
Compound 31 was prepared from compound 7 in a manner similar
to that described for compound 37. 1H NMR (DMSO-d6) δ (ppm):
8.53 (d, J ) 8.1 Hz, 1H), 8.39 (t, J ) 5.3 Hz, 1H), 8.34 (d, J ) 8.4
Hz, 1H), 8.18 (t, J ) 7.2 Hz, 2H), 8.05 (dd, J ) 7.5 Hz, 1.1 Hz,
1H), 7.99–7.91 (m, 1H), 7.83–7.63 (m, 3H), 7.31–7.21 (m, 1H),
7.13–6.97 (m, 5H), 6.95–6.80 (m, 3H), 5.33 (d, J ) 7.5 Hz, 1H),
4.93 (d, J ) 9.2 Hz, 1H), 4.87 (d, J ) 9.0 Hz, 1H), 4.80 (dd, J )
15.4 Hz, 7.0 Hz, 1H), 4.45 (s, 1H), 4.42–4.27 (m, 2H), 4.17–4.05
(m, 2H), 3.61–3.44 (m, 1H, overlapped with H2O), 3.17 (d, J )
5.1 Hz, 2H), 2.77–2.57 (m, 2H), 2.22 (s, 3H), 1.56 (s, 3H), 1.47 (s,
3H), 1.31 (s, 3H). HRMS (FAB+) calcd for C39H45N4O7S2 [M +
H]+, 745.2729; found, m/z 745.2723.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-((2S)-2-acetylamino-3-
(quinoline-2-yl-sulfonyl)propanoyl)amino-2-hydroxy-4-phenylb-
utanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (39). Com-
pound 39 was prepared from compound 7 in a manner similar to
1
that described for compound 37. H NMR (DMSO-d6) δ (ppm):
8.73 (d, J ) 8.4 Hz, 1H), 8.44 (t, J ) 5.3 Hz, 1H), 8.22–8.07 (m,
3H), 8.00 (d, J ) 9.3 Hz, 1H), 7.94 (d, J ) 8.6 Hz, 1H), 7.83 (t,
J ) 7.5 Hz, 1H), 7.34–6.99 (m, 10H), 5.36 (d, J ) 7.7 Hz, 1H),
4.94 (d, J ) 8.8 Hz, 1H), 4.86 (d, J ) 9.4 Hz, 1H), 4.82–4.70 (m,
1H), 4.47 (s, 1H), 4.32–4.44 (m, 2H), 4.20 (d, J ) 4.6 Hz, 1H),
4.15 (d, J ) 5.0 Hz, 1H), 3.56 (d, J ) 6.0 Hz, 1H), 3.17 (d, J )
5.1 Hz, 1H), 2.82–2.56 (m, 2H), 2.22 (s, 3H), 1.48 (s, 6H), 1.33 (s,
3H). HRMS (FAB+) calcd for C38H44N5O7S2 [M + H]+, 746.2682;
found, m/z 746.2687.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-((2S)-3-(naph-
thalene-2-yl-thio)-2-(methylsulfonylamino)propanoyl)amino-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(32). Compound 32 was prepared from compound 7 in a manner
1
similar to that described for compound 36. H NMR (DMSO-d6)
δ (ppm): 8.72 (d, J ) 8.6 Hz, 1H), 8.43 (t, J ) 5.2 Hz, 1H),

D-Amino Acid Containing HIV Protease Inhibitor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3003
HIV-1 Protease Inhibition. In the HIV protease assay, 25 µL
of 200 mM 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH
buffer (pH 5.5), containing 2 mM dithiothreinol (DDT), 2 mM
EDTA-2Na, and 1 M NaCl, was mixed with 5 µL of inhibitor (10
nM) dissolved in DMSO and 10 µL of HIV-1 protease (2 µg/mL)
in 50 mM AcOH (pH 5.5), containing 1 mM EDTA-2Na, 25 mM
NaCl, 0.2% mercaptoethanol, 0.2% Nonidet P-40, and 15%
glycerol. The mixture of was preincubated for 5 min at 37 °C, and
the enzymatic reaction was initiated by addition of 10 µL of a 75
mM substrate solution (H-Lys-Ala-Arg-Val-Tyr*Phe(p-NO2)-Glu-
Ala-Nle-NH2, synthesized by a conventional solid-phase method).
After incubation for 60 min at 37 °C, the reaction was terminated
by addition of 75 µL of 1N HCl. The C-terminal cleavage fragment
(H-Phe(p-NO2)-Glu-Ala-Nle-NH2) was separated by reverse-phase
HPLC on a C18 column with linear gradient of 0.1% aqueous TFA
to CH3CN, detected by monitoring the fluorescence intensity.
Percent inhibition was obtained compared to intensity without
inhibitor.
Antiviral Assays. The anti-HIV activity was assayed by
determining the level of inhibition of virus-induced cytopathic effect
in the cells as previously described.32 HIV-1 IIIB, kindly provided
by Prof. Harada, was used for anti-HIV assay. MT-4 cells were
added to each well of a 96-well flat-bottom plate containing serial
2-fold dilution of test compounds in duplicates. HIV-1 was added
to each well. Plates were incubated for 4 day in a CO2 incubator at
37 °C. After treatment with 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), the optical density of plates
were measured (560 nm) and percent cytopathic effect reduction
was calculated, then EC50 values were estimated by fitting the data
to a median-effect equation. Cytotoxicity was determined by
incubation in the absence of the virus. Antiviral assay in the
presence of R1-acid glycoprotein (AGP) was performed using
similar method with or without 1 mg/mL of AGP.
Supporting Information Available: Results of HPLC analysis
of final compounds. This is available free of charge via the Internet
at http://pubs.acs.org.
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Models. Initial conformation of inhibitor was constructed based
on an X-ray crystal structure of compound 1 with wild-type HIV-1
protease (PDB, 1HPX) using the Molecular Operating Environment
modeling package (MOE 2006.08, Chemical Computing Group,
Inc., Montreal, Canada) with a MMFF94x force field. A carboxyl
group of an Asp25 close to the HMC carbonyl group was
protonated. The binding area was immersed in a 20 Å sphere of
TIP3P water, centered on an oxygen atom of structural water
between inhibitor and the enzyme flap. The inhibitor, contacted
residues and flap region of the enzyme, and surrounded water were
energy-minimized while the other atoms were fixed. MD simula-
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equilibration with 1.5 fs time step. An additional 50 ps of MD
simulation was performed for data collection. Two residues in the
protease dimer were mutated from 1HPX to model each mutant
protease of the three drug-resistant HIV-1 clones, and energy-
minimizations and MD simulations were performed as described
above.
Acknowledgment. This research was supported in parts by
the Frontier Research Program and the 21st Century COE
Program from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan, the Japan Health
Sciences Foundation, and Japan Society for Promotion of
Science’s Post-Doctoral Fellowship for Foreign Researchers.
We gratefully acknowledge the assistance of T. Ito and H.
Tsukamoto for determining enzyme inhibition.

3004 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
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