Structure-activity relationships of novel HIV-1 protease inhibitors containing the 3-amino-2-chlorobenzoyl-allophenylnorstatine structure

Article abstract of DOI:10.1016/j.bmc.2007.10.062

A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted allophenylnorstatine (Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) were designed and synthesized. From the structure-activity relationship of this series of compounds, SM-309515 was found to have potent antiviral activity against wild-type and resistant HIV-1s and to possess a desirable pharmacokinetic profile in dogs.

Full text of DOI:10.1016/j.bmc.2007.10.062

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1299–1308
Structure–activity relationships of novel HIV-1 protease
inhibitors containing the 3-amino-2-chlorobenzoyl-
allophenylnorstatine structure
Tsutomu Mimoto,* Satoshi Nojima, Keisuke Terashima, Haruo Takaku,
Makoto Shintani and Hideya Hayashi
Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd., Enoki 33-94, Suita, Osaka 564-0053, Japan
Received 26 September 2007; revised 17 October 2007; accepted 18 October 2007
Available online 23 October 2007
Abstract—A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted allophenyl-
norstatine (Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) were designed and synthesized. From the structure–activity
relationship of this series of compounds, SM-309515 was found to have potent antiviral activity against wild-type and resistant
HIV-1s and to possess a desirable pharmacokinetic profile in dogs.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
which is known to sustain high plasma drug levels by
inhibiting cytochrome P450 (CYP) 3A4-mediated drug
metabolism.2
Human immunodeficiency virus (HIV) protease is essen-
tial for viral replication and is the main target for ap-
proved antiviral drugs. Combination chemotherapy
with reverse transcriptase inhibitors and protease inhib-
itors (PI) has proven to be highly effective in suppressing
viral replication to undetectable levels.1 Despite such
remarkable achievement, the use of current antiretrovi-
ral regimens continues to be limited by complexity, tol-
erability, drug resistance, and cross-resistance. In
addition, non-adherence to such regimens can lead to re-
duced effectiveness and increased drug resistance.
Although newer drugs offer improvements over existing
agents by having simpler dosing schedules, better toler-
ability, and/or improved antiviral activity, one of the
most desirable properties of any novel protease inhibitor
is that it can be dosed once daily. Atazanavir,2 the first
once-daily protease inhibitor, has strong anti-HIV effi-
cacy and favorable pharmacokinetic profile. However,
when used for salvage therapy in patients with a degree
of resistance, atazanavir is combined with ritonavir,
We have previously reported that JE-2147 (1, Fig. 1)
represents a class of transition-state mimetic dipeptide
HIV protease inhibitors containing allophenylnorstatine
(Apns:
(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric
acid) with a hydroxymethylcarbonyl (HMC) isostere as
the active moiety.3 JE-2147 completely suppresses
HIV-1 and HIV-2 strains as well as clinical HIV-1 vari-
ants that are highly resistant to marketed protease inhib-
itors.4 Although the strong anti-HIV efficacy and safety
of JE-2147 were proven in a pilot clinical study, the
pharmacokinetic profile of JE-2147 is still not satisfy-
ing.5 In vitro studies have shown that the main metabo-
lites of JE-2147 produced by human hepatocytes are the
phenol glucuronide M1 and the thiazolidine sulfoxide
M2 (Fig. 2) and that JE-2147 is highly metabolized by
human liver microsomes in the presence of uridine 50-
diphosphoglucuronic acid (UDPGA), a co-factor of
UDP-glucuronosyltransferase (UGT). These findings
suggest that co-administration of JE-2147 with ritonavir
cannot improve the pharmacokinetic profile of JE-2147
in human, because ritonavir cannot inhibit UGT activ-
ity. In a previous paper, we have shown that compounds
having a para substitution at the phenyl ring of Apns
and/or 2, 6-disubstitution at the P20 benzylamine are
Keywords: HIV; Protease; Inhibitor; SM-309515.
*
Corresponding author. Tel.: +81 6 6337 5934; fax: +81 6 6337
6010; e-mail: tsutomu-mimoto@ds-pharma.co.jp
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.062

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T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
R1
R2
O
O
O
Cl
O
O
O
R3
HO
H2N
N
N
N
N
N
N
H
H
H
H
OH
OH
S
S
JE-2147 (1)
JE-4181 (2) : R1 = R2 = R3 = H
SM-309515 (6f) : R1 = H, R2 = OMe, R3 = Me
Figure 1. Structures of JE-2147 (1), JE-4181(2), and SM-309515 (6f).
in rats (F = 51%, data not shown). Although inhibitory
activity of JE-4181 against HIV-1 protease is moderate
(Ki = 353 pM: approximately 10 times lower than that
of JE-2147, Table 2), its pharmacokinetic profile makes
it a suitable lead compound. In a previous study, we
found that substitution at the meta or para position of
the P1 phenyl ring of JE-2147 does not affect this com-
pound’s inhibitory activity against HIV-1 protease and
that lower-alkoxy substituted compounds show en-
hanced antiviral activity due to their superior perme-
ation across cell membrane.6 These findings suggest
that introduction of a lower alkoxy group into the P1
phenyl ring of the lead JE-4181 might enhance this com-
pound’s antiviral activity, while maintaining its pharma-
cokinetic advantage.
O
O
O
HO
N
H
N
N
H
OH
S
JE-2147
P2-site
O
P2'-site
O
-OOC
HO
HO
O
O
N
N
H
HO
S
O
M1
M2
Figure 2. Main metabolites of JE-2147 generated by human
hepatocytes.
3. Chemistry
Boc-protected Apns derivatives, the key intermediates of
HMC type compounds, were prepared from the corre-
sponding L-phenylalanines as described previously.8
Hayashi et al. reported that coupling of N-protected
Apns derivatives to 5,5-dimethyl-1,3-thiazolidine-4-car-
boxamides often produces low yields due to the forma-
tion of Boc-Apns-homobislactone.9 In our synthesis of
JE-2147, the formation of this type of amide bond was
promoted by the use of N,N0-dicyclohexylcarbodiimide
(DCC)-1-hydroxybenzotriazole (HOBt) in ethyl acetate
as a solvent.6 The thiazolidine dipeptide amine deriva-
tives 5a–h were prepared by coupling Boc-Apns
derivatives to the 5,5-dimethyl-1,3-thiazolidine-4-car-
boxamides 4a, b, followed by removal of the Boc group
under acidic conditions (Scheme 1). The (RS)-N-tert-
butoxycarbonyl-3,3-dimethylpyrrolidine-2-carboxylic acid
7, an intermediate of the pyrrolidine type compounds, was
prepared using a procedure described in the literature.10
The carboxyl group was coupled to 2,6-dimethylbenzyl-
amine by mixed anhydration with diphenylphosphoryl
chloridate (DPP-Cl). After removal of the Boc group,
Boc-Apns derivatives were coupled by use of 1-ethyl-3-
(dimethylaminopropyl)carbodiimide (EDC)-HOBt in
N,N-dimethylformamide (DMF). Pyrrolidine dipeptides
in the diastereomixture were separated by silica gel chro-
matography after removal of the Boc group, and the less
polar diastereoisomers of the pyrrolidine dipeptide
amine derivatives 9a–c were forwarded to the final
step.11 Coupling of 3-amino-2-chlorobenzoic acid to
the dipeptide amine derivatives 5a–h and 9a–c by use
not subjected to the P2 phenol glucuronidation that
occurs with JE-2147.6 In this communication, we report
another strategy to improve the pharmacokinetic profile
of our HMC-based HIV protease inhibitors. Ultimately,
we found SM-309515 (6f) to display a desirable pharma-
cokinetic profile and a strong antiviral activity.
2. Concept of drug design
Information on the interaction between JE-2147 and
HIV-1 protease based on X-ray crystallography of their
complex4 provided the basis for the design of novel HIV
protease inhibitors that are effective against PI-resistant
HIV-1. X-ray crystallography revealed that the hydrox-
ymethylcarbonyl group of Apns (P1) interacts with the
aspartic acid carbonyl group of HIV-1 protease active
site in essentially the same hydrogen-bonding mode as
in the transition state.4,7 This favorable interaction with
the active center of HIV protease contributes to the high
activity of JE-2147 against mutant proteases, because
the sequence Asp-Thr-Gly in the active center of HIV
protease is conserved in any HIV-1 mutant. Another
structural feature of JE-2147 is its flexible P20 benzyl
moiety, which can adapt to structural changes in mutant
enzymes.4 Putting this information together, we were
able to find JE-4181 (2, Fig. 1) as a new HIV protease
inhibitor that resists not only glucuronidation but also
CYP oxidation by human liver microsomes in vitro (Ta-
ble 1). JE-4181 also shows good pharmacokinetic profile

T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
Table 1. In vitro metabolism by human liver microsomes
1301
R1
R2
Cl
O
O
O
R3
H2N
N
H
N
N
H
OH
X
Compound
Structure
Metabolism remaining (%)
NADPH UDPGA
X
R1
R2
R3
2 (JE-4181)
6a
S
H
H
H
H
46
60
66
65
74
58
64
54
53
63
83
53
20
>95
>95
93
S
OMe
H
OEt
H
6b
6c
S
OMe
H
H
S
H
89
6d
6e
S
OCH2
OMe
H
O
H
>95
>95
>95
>95
>95
>95
>95
>95
55
S
H
Me
Me
Me
Me
Me
Me
Me
6f (SM-309515)
6g
S
OMe
H
S
OEt
OCH2
OMe
H
6h
10a
S
O
CH2
CH2
CH2
H
10b
10c
OMe
H
OEt
1 (JE-2147)
Data show the mean residual percent of duplicates. A given test drug (5 lM) was incubated with human liver microsomes (0.5 mg/ml) and cofactor
(1 mM NADPH or 5 mM UDPGA) at 37 °C for 30 min. Concentration of residual drug was measured by RP-HPLC.
Table 2. Inhibitory activity against HIV-1 protease and antiviral activity
R1
R2
Cl
O
O
O
R3
H2N
N
H
N
N
H
OH
X
Compound
Structure
HIV-1 PR Ki (pM)
HIV-1 IIIB EC50 (nM)
+50% Human serum
X
R1
R2
R3
2 (JE-4181)
6a
6b
S
H
H
H
353
311
255
310
163
138
134
152
70
77
18
21
6
165
88
138
35
77
14
6
S
OMe
H
OEt
H
H
S
OMe
H
H
6c
6d
S
H
S
OCH2
OMe
H
O
H
20
24
4
6e
S
H
Me
Me
Me
Me
Me
Me
Me
6f (SM-309515)
6g
6h
S
OMe
H
S
OEt
OCH2
OMe
H
20
17
40
36
63
26
7
66
20
80
87
270
52
50
41
87
293
95
8
S
O
10a
10b
CH2
CH2
CH2
H
804
750
861
35
OMe
H
10c
OEt
1 (JE-2147)
Saquinavir
Ritonavir
Indinavir
Nelfinavir
Amprenavir
Lopinavir
Atazanavir
138
98
2
739
931
359
16
23
19
16
8
50
<4
9
Assay for inhibition of HIV-1 protease was performed by measuring fluoresence intensity of the peptide fragment produced from H-Lys-Ala-Arg-
Val-Tyr-Phe(4-NO2)-Glu-Ala-Nle- NH2 as a substrate in 200 mM MES buffer, pH 5.5, containing 1 M NaCl, 2 mM dithiothreitol, and 2 mM
EDTA-2Na, after incubation at 37 °C for 15 min. Evaluation of in vitro antiviral activity of each test compound against wild-type HIV-1 (strain
IIIB) was conducted as described previously.6

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T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
of EDC-HOBt in DMF gave the target compounds 6a–h
and 10a–c, respectively (Schemes 1 and 2).
which the thiazolidine was not oxidized, only compound
10b having 3-OMe substituent on the P1 phenyl group
was highly stable against CYP oxidation. This might
be due to the unstableness of 4-alkoxy substituent on
the P1 phenyl group.
4. Results and discussion
4.1. In vitro metabolism of alkoxy-substituted Apns
analogues by human liver microsomes
4.2. Inhibitory activity of alkoxy-substituted Apns ana-
logues and other marketed drugs against HIV-1 protease
and wild type HIV-1
Results of in vitro metabolism of the alkoxy-substituted
Apns analogues by human liver microsomes in the pres-
ence of UDPGA, a co-factor of UGT, or b-nicotinamide
adenine dinucleotide phosphate (NADPH), a co-factor
of CYP, are shown in Table 1. All compounds, including
the lead JE-4181 (2), showed high metabolic resistance
to glucuronidation. On the other hand, JE-2147 was
strongly glucuronized under our assay conditions. The
prepared compounds also showed higher metabolic sta-
bility against CYP oxidation than JE-4181. Against our
expectation, among the pyrrolidine derivatives 10a–c, in
The inhibitory activity of the prepared HMC-based HIV
protease inhibitors as well as that of some marketed drugs
against HIV-1 protease is shown in Table 2. The inhibi-
tory activity of JE-4181 against HIV-1 protease was
approximately 10 times lower than that of JE-2147
(Ki = 353 and 35 pM, respectively). Substitution by the
4-methoxy group (6a), 3-methoxy group (6b), 4-ethoxy
group (6c), or 3,4-methylenedioxy group (6d) at the P1
phenyl did not enhance the inhibitory activity of com-
pound 2 against HIV-1 protease (Ki = 163–311 pM). As
O
O
O
a), b)
c), d )
O
N
OH
HN
N
H
S
S
R3
4a : R3 = H
b : R3 = Me
3
R1
R1
R2
S
R2
S
O
O
Cl
O
O
O
e )
H2N
H2N
N
N
N
H
N
N
H
H
OH
OH
6a-h
R3
R3
5a-h
R1, R2 = H or Alkoxy
R1, R2 = H or Alkoxy
R3 = H or Me
R3 = H or Me
Scheme 1. Reagents: (a) Diphenylphosphoryl chloridate, triethylamine, ethyl acetate, and then benzylamines; (b) 4 N HCl in ethyl acetate or dioxane;
(c) (2S,3S)-3-(N-tert-butoxycarbonyl)amino-2-hydroxy-4-arylbutyric acids, DCC, HOBt, ethyl acetate; d) 4 N HCl in ethyl acetate or dioxane; (e) 3-
amino-2-chlorobenzoic acid, EDC Æ HCl, HOBt, DMF.
O
O
O
(a) (b)
(c)(d)(e)
O
N
OH
HN
N
H
7
8
R1
R1
R2
R2
O
O
Cl
O
O
O
(f)
H2N
H2N
N
N
H
N
H
N
N
H
OH
OH
9a: R1 = OMe, R2 = H
b: R1 = H, R2 = OMe
c: R1 = OEt, R2 = H
10a: R1 = OMe, R2 = H
b: R1 = H, R2 = OMe
c: R1 = OEt, R2 = H
Scheme 2. Reagents: (a) diphenylphosphoryl chloridate, triethylamine, ethyl acetate, and then 2,6-dimethybenzylamine hydrochloride, triethylamine;
b) 4 N HCl in dioxane, CH2Cl2; (c) (2S,3S)-3-(N-tert-butoxycarbonyl)amino-2-hydroxy-4-arylbutyric acids, EDC Æ HCl, HOBt, DMF; (d) 4 N HCl in
ethyl acetate; (e) separation of diastereomer by silica gel chromatography; (f) 3-amino-2-chlorobenzoic acid, EDC Æ HCl, HOBt, DMF.

T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
1303
we have shown in previous studies that an additional
ortho methyl on the P20 benzyl group of this type of
HIV protease inhibitors enhances the inhibitory activity
against HIV-1 protease,3,6 compounds having 2,6-
dimethylbenzyl at the P20 site (6e–h) were synthesized
and their inhibitory activity against HIV-1 protease was
evaluated. Compounds 6e–h all showed enhanced HIV-
1 protease inhibitory activity. Transformation to the pyr-
rolidine ring decreased the inhibitory activity against
HIV-1 protease with compounds 10a–c showing approx-
imately 5 times lower activity than the corresponding ana-
logues (Ki = 750–861 pM).12 Although the thiazolidine
based compounds 6a–h had less potent HIV protease
inhibitory activity than JE-2147, they showed an anti-
HIV activity (EC50 = 4–24 nM) superior or comparable
to that of JE-2147 due to their better cell permeability.
We have previously reported that the in vitro antiviral effi-
cacy of JE-2147 is not affected by addition of human ser-
um. This finding is contrary to what has been reported
with other HIV protease inhibitors, such as nelfinavir.
Unlike most other protease inhibitors, in vitro IC50 values
of 6e–h did not increase in the presence of 50% human ser-
um and their antiviral activity was comparable to that of
lopinavir or atazanavir. Although the pyrrolidine com-
pounds had relatively weak HIV protease inhibitory
activity, compounds 10a, b showed antiviral activity in
the presence of 50% human serum comparable to that
of JE-2147 or other marketed protease inhibitors due to
their superior cell permeability.
6f and 10b would have a better pharmacokinetic profile
than JE-2147 or atazanavir in human.
4.4. Co-administration of the synthesized HIV-1 protease
inhibitors with ritonavir as pharmacokinetics enhancer
Co-administration of JE-2147 with ritonavir could not
be expected to improve the pharmacokinetic profile of
JE-2147 in human, because one of the main metabolites
of JE-2147 is the phenol glucuronide. Compound 6f,
which showed resistance against glucuronidation, was
incubated with human liver microsomes in the absence
or presence of ritonavir. Although in the presence of
ritonavir the metabolism of 6f was not effectively inhib-
ited in rat microsomes, it was completely suppressed in
canine or human microsomes (Table 5). These findings
indicate that pharmacokinetics data collected from the
dog are useful to predict the pharmacokinetic profile
of 6f co-administered with ritonavir in humans. In our
experiments, concomitant treatment with ritonavir at
7.5 mg/kg considerably enhanced the pharmacokinetics
of 6f (15 mg/kg, po) as indicated by a sixfold increase
in AUC (Fig. 3 and Table 3). Plasma drug (6f) concen-
tration at 12 h after dosing (C12h) was also enhanced by
co-treatment with ritonavir to 2.95 lM, which is over
490 times higher than EC50 value in the presence of
50% human serum. As shown in Table 6, 6f showed anti-
viral activity against multi-drug-resistant HIV-1s with
10
4.3. Animal pharmacokinetic profile
The pharmacokinetic profiles of compounds 6f, 10b, JE-
2147, and atazanavir in dogs are summarized in Table 3.
Compounds 6f and 10b, having 3-methoxy substituent
on the P1 phenyl ring and 2, 6-dimethylbenzyl group,
had favorable pharmacokinetic profiles exceeding that
of JE-2147 or atazanavir (Fig. 3 and Table 3). In addi-
tion, plasma concentrations of 6f and 10b at 12 h
(C12h) after po administration (6f: 0.13 lM and 10b:
0.71 lM) exceeded their antiviral EC50s (measured in
the presence of 50% human serum in vitro) by 20- and
8-fold, respectively. These compounds were fully resis-
tant to glucuronidation by human liver microsomes
and improved the JE-2147 susceptibility to oxidation
by human liver microsomes (Table 4). In addition, 6f
and 10b showed better stability in human microsomes
than in canine microsomes. These findings indicate that
1
0.1
0.01
0
2
4
6
8
10
12
Time after administration (hr)
Figure 3. Plasma concentrations of HIV protease inhibitors in Beagle
dogs after oral dosing with test compound (15 mg/kg) dissolved in 50%
PEG. Each point represents the mean SE (n = 3), 6f (s), 10b (d), 6f
with ritonavir (7.5 mg/kg po) (n), JE-2147 (m), and atazanavir (h).
Table 3. Pharmacokinetic parameters of 6f, 10b, JE-2147, and atazanavir in beagle dogsa
Compound
Route Dose (mg/kg) AUC (lM Æ min) Cmax (lM) Tmax (min) Vdss (l/kg) CLb (l/h/kg) F (%) C12h (lM)
b
6f (SM-309515) po
poc
15
15
15
15
15
936
3167
2203
390
2.8
5.3
5.0
3.1
4.2
120
180
60
1.3
1.1
71
0.13
2.95
0.71
0.03
0.05
10b
JE-2147
atazanavir
po
po
po
1.9
1.6
2.0
1.0
0.9
1.6
146
25
30
30
900
102
F (%) was determined by comparing the mean areas under the curve (AUC) after intravenous or oral dosing. Cmax, maximum plasma concentration;
Tmax, time of maximum plasma concentration; Vdss, volume of distribution; CL, plasma clearance rate; F (%), percent oral bioavailability; C12h
,
plasma concentration at 12 h after po administration.
a Data are given as mean values.
b Calculated from i.v. data.
c Co-administered with 7.5 mg/kg of ritonavir (po).

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T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
Table 4. Comparison of in vitro metabolism profiles of 6f, 10b, JE-
2147, and atazanavir
JE-2147. From the results of in vitro metabolism of the
synthesized compounds using human and canine liver
microsomes, SM-309515 is expected to have a good phar-
macokinetic profile and anti-HIV activity in humans.
Moreover, plasma levels of SM-309515 co-administered
with ritonavir exceeded its antiviral EC50 against multi-
drug-resistant HIV-1s. These findings indicate that co-
administration of SM-309515 and ritonavir could be use-
ful as salvage therapy for HIV infection.
Compound
In vitro metabolism (remaining [%])
Dog Human
NADPH UDPGA NADPH UDPGA
6f (SM-309515) 47
>95
>95
91
64
83
27
83
>95
>95
42
10b
JE-2147
Atazanavir
69
49
85
>95
>95
Data show the mean residual of duplicates. Concentration of residual
drug was measured by RP-HPLC.
6. Experimental
6.1. Chemistry general
Table 5. In vitro metabolism profile in the presence of ritonavir
RTV conc (lM)
In vitro metabolism (remaining [%])
In general, reagents and solvents were used as purchased
without further purification. Column chromatography
was performed on an FL60D (Fuji Silysia Chemical
Rat
Dog
Human
0
0.5 2.5
0
0.5 2.5
0
0.5 2.5
LTD). Melting points were measured with a Buchi 535
¨
and are uncorrected. H NMR spectra were recorded
on a JEOL GSX270 FT NMR spectrometer, and chem-
ical shifts were expressed in d ppm from the internal
standard, tetramethylsilane.
6f (SM-309515) 21 41 55
12 16 31
36 90 >95 49 93
46 92 93 13 56
>95
59
1
JE-2147
Lopinavir
11 83 >95 16 83 >95 10 >95 >95
Data show the mean residual of duplicates. Drug (5 lM) with or
without ritonavir (RTV) was incubated with liver microsomes (0.5 mg/
ml) and cofactors (1 mMNADPH, 5 mM UDPGA) at 37 °C for
30 min. Concentration of residual drug was measured by RP-HPLC.
6.1.1. (R)-N-(2-Methylbenzyl)-5,5-dimethyl-1,3-thiazoli-
dine-4-carboxamide (4a). To a solution of (R)-N-tert-
butoxycarbonyl-5,5-dimethyl-1,3-thiazolidine-4-carbox-
ylic acid (3, 5.22 g, 20.0 mmol) and triethylamine
(3.06 mL, 22.0 mmol) in ethyl acetate (50 mL), DPP-Cl
(4.55 mL, 22.0 mmol) was added in an ice-bath, and
the mixture was stirred for 1 h. Then to the reaction
mixture, 2-methylbenzylamine (2.73 mL, 22.0 mmol)
and triethylamine (3.06 mL, 22.0 mmol) were added in
an ice-bath and stirred overnight. The reaction mixture
was washed sequentially with 1 N HCl, 3% K2CO3,
and brine, dried over MgSO4, filtered, and concentrated.
The residue was redissolved in CH2Cl2 (30 mL) and
added to 4 N HCl in dioxane (30 mL). After the reaction
mixture had been stirred for 2 h, water was added to it;
and the aqueous phase was washed with toluene, neu-
tralized with 2 N NaOH, and extracted with ethyl ace-
tate. The organic phase was washed with brine, dried
over MgSO4, filtered, and concentrated to give a crude
product. Recrystallization from n-hexane/ethyl acetate
IC50 values ranging from 0.022 to 0.214 lM, and the
fold shift of IC50 (mutant/wild type) was smaller than
that of each marketed protease inhibitor. These findings
indicate that 6f, given in combination with ritonavir,
would be useful as salvage therapy for HIV infection.
5. Conclusions
In summary, a series of 3-amino-2-chlorobenzoyl-all-
ophenylnorstatine derivatives, asHIV protease inhibitors,
were designed and synthesized. From the structure–activ-
ity relationship and metabolism of these compounds, we
found that compounds 6f, 10b, having 3-methoxy substi-
tuent on the P1 phenyl ring and 2,6-dimethylbenzyl group,
have a favorable pharmacokinetic profile exceeding that
of JE-2147 or atazanavir. Compound 6f (SM-309515)
had more potent inhibitory activity against HIV-1 than
Table 6. Sensitivity of mutant HIV-1s to HIV protease inhibitors in Phenosence assay13
EC50 (lM)
6f
ATV
LPV
AMP
NFV
IDV
RTV
SQV
Wild type
Strain 1
Strain 2
Strain 3
Strain 4
Strain 5
Strain 6
Strain 7
0.0056 (1)
0.15 (27)
0.106 (19)
0.122 (22)
0.022 (4)
0.214 (38)
0.096(17)
0.042 (7)
0.0015 (1)
0.092 (62)
0.048 (32)
0.023 (16)
0.095 (64)
0.266 (180)
0.134(90)
0.074 (50)
0.0037 (1)
0.214 (57)
0.251 (68)
0.657 (176)
0.066 (18)
0.807 (216)
0.411 (110)
0.212 (57)
0.014 (1)
0.098 (7)
0.350 (25)
0.143 (10)
0.051 (4)
0.615 (43)
0.432 (30)
0.165 (12)
0.0054 (1)
0.452 (82)
0.346 (64)
0.413 (75)
>1.5 (>273)
0.776 (141)
>1.5 (>273)
0.515 (94)
0.0076 (1)
0.401 (52)
0.464 (61)
0.753 (97)
0.113 (15)
>1.5 (>194)
0.819 (106)
0.145 (19)
0.016 (1)
0.0019 (1)
>0.5 (>265)
0.097 (51)
>0.5 (>265)
>0.5 (>265)
>0.5 (>265)
0.158 (83)
0.141 (75)
1.733 (106)
>3 (>184)
>3 (>184)
1.003 (61)
>3 (>184)
>3 (>184)
>3 (>184)
Numbers in parentheses represent fold change of EC50 values against each isolate compared with EC50 values against the wild type. Mutation sites of
each strain were as follows: strain l: L10L N37S, M46L, G48V, I54V, Q58E, Q61Q/E, I62I/V, L63P, A71V, V77I, V82A, L90M, I93L; strain 2: L1 0I,
G16A, K45V, F53L, I54V, K55N, D60E, L63P, A71V, I72K, V77I, V82F, I84V, L90M, I93L; strain 3: L10I, T12I, E34Q, R41K, M46I, G48V, I50V,
F53Y, I54S, I62V, L63P, H69Q, A71V, I72V, V77I, V82A, I93M; strain 4: L10F, K20R, D30N,E35D, M36V, N37T, G51G/E, F53L, I54V, R57K,
Q58E, I62V, L63P, A71V, V77I, N88D, L90M; strain 5: L10I, I13V, L33F, R41K, M46L, G48V, F53Y, I54N, Q61E, I62V, L63P, A71V, V77I,
V82A, L89V; strain 6: L10I, I13V, K20R, L33F, E35D, M36I, K43T, M46I, I54S, I62V, L63T, I64V, I72V, T74S, V82A, N83D; strain 7: T4A, L10I,
K20R, L23I, L33F, E35D, M36I, K43T, M46L.

T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
1305
gave 3.75 g of the title compound with a yield of 71%:
mp 77–79 °C; H NMR (DMSO-d6) d (ppm): 1.15 (s,
3H), 1.52 (s, 3H), 2.28 (s, 3H), 3.27 (s, 3H), 3.66 (s,
1H), 4.03 (d, 1H, J = 9.6 Hz), 4.22–4.33 (m, 3H), 7.12–
7.22 (m, 4H), 8.32–8.33 (br, 1H); Anal. Calcd for
C14H20N2OS: C, 63.60; H, 7.62; N, 10.60; found: C,
63.53; H, 7.65; N, 10.46.
3.06 (d, 1H, J = 10.5 Hz), 3.75 (s, 3H), 4.03–4.09 (m, 1H),
4.1–4.3 (m, 2H), 4.36 (s, 1H), 4.90 (s, 2H), 5.29 (d, 1H,
J = 8.4 Hz), 6.70–6.80 (m, 3H), 6.96 (s, 3H), 7.13–7.23 (m,
2H), 8.53 (t, 1H, J = 5.13 Hz); Anal. Calcd for
C25H33N3O4S: C, 63.67; H, 7.05; N, 8.91; found: C, 63.39;
H, 7.13; N, 8.87;
1
6.1.5. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-(3-
amino-2-chlorobenzoyl)amino-4-(3-methoxyphenyl)butan-
6.1.2. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-hydro-
xy-4-(4-methoxyphenyl)butanoyl]-5,5-dimethyl-1,3-thiazol-
idine-4-carboxamide (5a). To a solution of compound 4a
(0.97 g, 9.08 mmol), (2S,3S)-3-(N-tert-butoxycarbonyl)-
amino-2-hydroxy-4-(4-methoxyphenyl)butyric acid (2.95 g,
9.08 mmol), HOBt (1.23 g, 9.08 mmol) in ethyl acetate
(50 mL), and DCC (2.15 g, 10.44 mmol) were added and
the mixture was stirred overnight. The reaction mixture
was then filtered and the filtrate was washed sequentially
with 3% K2CO3, 1 N HCl, and brine, dried over MgSO4,
filtered, and concentrated. The residue was redissolved in
CH2Cl2 (10 mL) and added to 4 N HCl in dioxane
(10 mL), and the mixture was stirred for 1 h. It was then
concentrated under reduced pressure and redissolved in
water. The aqueous phase was filtered, and the filtrate
was neutralized with 2 N NaOH to give the precipitate.
The crude product was recrystallized from ethyl acetate-
n-hexane to give 4.28 g of the title compound with a yield
of 88%: mp 202–204 °C; 1H NMR (DMSO-d6) d (ppm):
1.15–1.25 (br, 2H), 1.33 (s, 3H), 1.52 (s, 3H), 2.17 (s,
3H), 2.2–2.3 (m, 1H), 2.64 (t, 1H, J = 8.0 Hz), 3.02 (d,
1H, J = 13.2 Hz), 3.74 (s, 3H), 4.02–4.09 (br, 1H), 4.14
(d, 1H, J = 4.9 Hz), 4.20 (d, 1H, J = 4.9 Hz), 4.36 (s,
1H), 4.90 (s, 2H), 5.30 (d, 1H, J = 7.8 Hz), 6.95 (s, 3H),
6.86 (d, 2H, J = 8.6 Hz), 6.94–7.15 (m, 6H), 8.55 (t, 1H,
J = 5.1 Hz); Anal. Calcd for C25H33N3O4S 0.25EtOAc:
C,63.26;H,7.15;N,8.51;found:C,63.17;H,7.18;N,8.79.
oyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(6b).
1
Mp 138–140 °C; H NMR (DMSO-d6) d (ppm): 1.36
(s, 3H), 1.51 (s, 3H), 2.27 (s, 3H), 2.7–2.8 (m, 2H),
3.67 (s, 3H), 4.14 (dd, 1H, J = 4.3 Hz, 15.1 Hz), 4.2–
4.5 (m, 4H), 4.99 (d, 1H, J = 9.2 Hz), 5.14 (d, 1H,
J = 8.9 Hz), 5.35 (d, 1H, J = 7.0 Hz), 5.41 (s, 2H),
6.35–6.37 (m, 1H), 6.6–6.8 (m, 2H), 6.9–7.0 (m, 3H),
7.1–7.2 (m, 4H), 7.3–7.4 (m, 1H), 8.3–8.4 (m, 2H); Anal.
Calcd for C32H37ClN4O5S 0.5EtOAc: C, 61.02; H, 6.18;
N, 8.37; found: C, 61.09; H, 6.01; N, 8.45.
6.1.6. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-hydro-
xy-4-(4-ethoxyphenyl)butanoyl]-5,5-dimethyl-1,3-thiazoli-
dine-4-carboxamide (5c). Mp 208–210 °C; 1H NMR
(DMSO-d6) d (ppm): 1.1–1.3 (br, 2H), 1.30–1.35 (m,
6H), 1.52 (s, 3H), 2.17 (s, 3H), 2.1–2.3 (m, 1H), 2.63 (t,
1H, J = 8.1 Hz), 3.00 (d, 1H, J = 13.2 Hz), 4.00 (q, 3H,
J = 6.8 Hz), 4.1–4.3 (m, 2H), 4.35 (s, 1H), 4.90 (s, 2H),
5.28 (d, 1H, J = 7.8 Hz), 6.84 (d, 2H, J = 8.4 Hz), 6.9–
7.1 (m, 5H), 7.1–7.2 (m, 1H), 8.55 (t, 1H, J = 5.1 Hz);
Anal. Calcd for C26H35N3O4S: C, 64.30; H, 7.26; N,
8.65; found: C, 63.98; H, 7.29; N, 8.59.
6.1.7. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-(3-
amino-2-chlorobenzoyl)amino-4-(4-ethoxyphenyl)butanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (6c). Mp 137–
139 °C; 1H NMR (DMSO-d6) d (ppm): 1.29 (t, 3H,
J = 7.0 Hz), 1.35 (s, 3H), 1.50 (s, 3H), 2.27 (s, 3H), 2.6–2.8
(m, 2H), 3.96 (q, 2H, J = 6.8 Hz), 4.14 (dd, 1H, J = 4.3 Hz,
15.1 Hz), 4.2–4.5 (m, 4H), 4.98 (d, 1H, J = 9.2 Hz), 5.12 (d,
1H, J = 9.5 Hz), 5.3–5.5 (m, 3H), 6.36 (d, 1H, J = 6.2 Hz),
6.7–6.9 (m, 3H), 6.98 (t, 1H, J = 7.8 Hz), 7.1–7.2 (br, 3H),
7.23 (d, 2H, J = 8.9 Hz), 7.3–7.4 (m, 1H), 8.28–8.34 (m,
2H); Anal. Calcd for C33H39ClN4O5S: C, 62.01; H, 6.15;
N, 8.77; found: C, 61.57; H, 6.16; N, 8.63.
6.1.3. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-(3-
amino-2-chlorobenzoyl)amino-4-(4-methoxyphenyl)butan-
oyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (6a). To
a solution of 3-amino-2-chlorobenzoic acid (70 mg,
0.41 mmol) and 5a (174 mg, 0.37 mmol) in DMF
(4 mL), HOBt (57 mg, 0.42 mmol) and EDCHCl (82 mg,
0.43 mmol) were added in an ice-bath and the mixture
was stirred overnight. The reaction mixture was then
washed sequentially with 3% K2CO3, 1 N HCl, and brine,
and dried over MgSO4, filtered, and concentrated. The
crude product was recrystallized from n-hexane-ethyl ace-
tate to give 180 mg of the title compound with a yield of
6.1.8. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-hydro-
xy-4-(3,4-methylenedioxyphenyl)butanoyl]-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (5d). Mp 202–204 °C; 1H
NMR (DMSO-d6) d (ppm): 1.1–1.2 (br, 2H), 1.34 (s,
3H), 1.52 (s, 3H), 2.17 (s, 3H), 2.2–2.3 (m, 1H), 2.6–
2.8 (m, 1H), 2.98 (d, 1H, J = 11.1 Hz), 4.0–4.1 (m,
1H), 4.1–4.3 (m, 2H), 4.35 (s, 1H), 4.90 (s, 2H), 5.26
(d, 1H, J = 8.1 Hz), 5.97 (s, 2H), 6.59 (d, 1H,
J = 7.6 Hz), 6.70 (s, 1H), 6.82 (d, 1H, J = 7.8 Hz),
6.90–7.00 (m, 3H), 7.15–7.17 (m, 1H), 6.70–6.80 (m,
3H), 6.96 (s, 3H), 7.13–7.23 (m, 2H), 8.56 (t, 1H,
J = 5.0 Hz); Anal. Calcd for C25H31N3O5S: C, 61.83;
H, 6.43; N, 8.65; found: C, 61.67; H, 6.52; N, 8.63.
1
78%: mp 120–122 °C; H NMR (DMSO-d6) d (ppm):
1.35 (s, 3H), 1.50 (s, 3H), 2.27 (s, 3H), 2.6–2.8 (m, 2H),
3.70 (s, 3H), 4.14 (dd, 1H, J = 4.3 Hz, 15.1 Hz), 4.2–4.5
(m, 4H), 4.98 (d, 1H, J = 9.2 Hz), 5.12 (d, 1H,
J = 8.9 Hz), 5.3–5.5 (m, 3H), 6.37 (d, 1H, J = 6.8 Hz),
6.7–6.9 (m, 3H), 6.98 (t, 1H, J = 7.8 Hz), 7.1–7.2 (br,
1H), 7.2–7.4 (m, 3H), 8.2–8.4 (m, 2H); Anal. Calcd for
C32H37ClN4O5S: C, 61.48; H, 5.97; N, 8.96; found: C,
61.48; H, 6.12; N, 8.72.
6.1.4. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-hydro-
xy-4-(3-methoxyphenyl)butanoyl]-5,5-dimethyl-1,3-thiazoli-
dine-4-carboxamide (5b). Mp 173–175 °C; 1H NMR
(DMSO-d6) d (ppm): 1.1–1.2 (br, 2H), 1.34 (s, 3H), 1.53
(s, 3H), 2.17 (s, 3H), 2.1–2.3 (m, 1H), 2.6–2.8 (m, 1H),
6.1.9. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-(3-
amino-2-chlorobenzoyl)amino-4-(3,4-methylenedioxy-
phenyl)butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-car-
boxamide (6d). Mp 170–172 °C; 1H NMR (DMSO-d6) d

1306
T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
(ppm): 1.36 (s, 3H), 1.51 (s, 3H), 2.27 (s, 3H), 2.6–2.8 (m,
2H), 4.14 (dd, 1H, J = 4.3 Hz, 15.1 Hz), 4.2–4.5 (m, 4H),
4.98 (d, 1H, J = 8.6 Hz), 5.14 (d, 1H, J = 8.9 Hz), 5.32 (d,
1H, J = 6.8 Hz), 5.42 (s, 2H), 5.93 (s, 2H), 6.38 (d, 1H,
J = 7.6 Hz), 6.7–6.8 (m, 3H), 6.9–7.0 (m, 2H), 7.1–7.2
(m, 4H), 7.3–7.4 (m, 2H), 8.3–8.4 (m, 2H); Anal. Calcd
for C32H35ClN4O6S: C, 60.13; H, 5.52; N, 8.77; found:
C, 59.99; H, 5.42; N, 8.67.
6.1.12. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-amino-2-chlorobenzoyl)amino-4-(4-methoxyphenyl)butan-
oyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (6e). Mp
175–177 °C; H NMR (DMSO-d6) d (ppm): 1.36 (s, 3H),
1.46 (s, 3H), 2.32 (s, 6H), 2.6–2.7 (m, 2H), 3.72 (s, 3H),
4.1–4.3 (m, 2H), 4.44–4.54 (m, 3H), 4.96 (d, 1H,
J = 9.5 Hz), 5.15 (d, 1H, J = 8.9 Hz), 5.21 (d, 1H,
J = 6.8 Hz), 5.40 (s, 2H), 6.38 (d, 1H, J = 7.3 Hz), 6.7–
6.9 (m, 3H), 6.9–7.1 (m, 4H), 7.31 (d, 2H, J = 8.4 Hz),
8.10 (m, 1H), 8.39 (d, 1H, J = 8.4 Hz); Anal. Calcd for
C33H39ClN4O5S: C, 62.01; H, 6.15; N, 8.77; found: C,
61.88; H, 6.04; N, 8.59.
1
6.1.10. (R)-N-(2,6-Dimethylbenzyl)-5,5-dimethyl-1,3-thia-
zolidine-4-carboxamide (4b). To a solution of (R)-N-tert-
butoxycarbonyl-5,5-dimethyl-1,3-thiazolidine-4-carboxylic
acid (3, 15.7 g, 60 mmol) and triethylamine (8.76 mL,
63 mmol) in ethyl acetate (230 mL), DPP-Cl (13.0 mL,
63 mmol) was added in an ice-bath; and the mixture
was stirred for 4 h. Then to the reaction mixture, 2,6-dim-
ethylbenzylamine hydrochloride (12.1 g, 63 mmol) and
triethylamine (18.3 mL, 132 mmol) were added in an
ice-bath, followed by stirring overnight. The reaction
mixture was washed sequentially with 1 N HCl, 3%
K2CO3, and brine, dried over MgSO4, filtered, and con-
centrated. The residue was redissolved in CH2Cl2
(100 mL) and added to 4 N HCl in dioxane (100 mL),
and this mixture was stirred for 2 h. After the reaction
mixture had been concentrated, the residue was redis-
solved in CH2Cl2-H2O and neutralized with 2 N NaOH.
The organic phase was washed with brine, dried over
MgSO4, filtered, and concentrated to give a crude prod-
uct. Recrystallization from n-hexane gave 15.7 g of the ti-
6.1.13. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(3-methoxyphenyl)butanoyl]-5,5-dimethyl-1,3-thi-
1
azolidine-4-carboxamide (5f). Mp 185–187 °C; H NMR
(DMSO-d6) d (ppm): 0.65–0.75 (br, 2H), 1.34 (s, 3H),
1.52 (s, 3H), 2.0–2.1 (m, 1H), 2.11 (s, 6H), 2.4–2.5 (m,
1H), 3.07 (d, 1H, J = 11.1 Hz), 3.79 (s, 3H), 3.99 (t, 1H,
J = 8.6 Hz), 4.10 (d, 2H, J = 3.2 Hz), 4.27 (s, 1H), 4.84
(s, 2H), 5.24 (d, 1H, J = 8.1 Hz), 6.64–6.75 (m, 5H),
6.83–6.87 (m, 1H), 7.25 (t, 1H, J = 8.1 Hz), 8.15 (br,
1H); Anal. Calcd for C26H35N3O4S: C, 64.30; H, 7.26;
N, 8.65; found: C, 64.04; H, 7.37; N, 8.61.
6.1.14. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-amino-2-chlorobenzoyl)amino-4-(3-methoxyphenyl)butan-
oyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (SM-
1
309515, 6f). Mp 183–185 °C; H NMR (DMSO-d6) d
1
tle compound with a yield of 94%: mp 97–99 °C; H
(ppm): 1.36 (s, 3H, Dmt-5-CH3), 1.46 (s, 3H, Dmt-5-
CH3), 2.32 (s, 6H, benzylamine-CH3), 2.6–2.7 (m,
2H, Apns-4-CH2), 3.76 (s, 3H, Apns-OCH3), 4.19
(dd, 1H, J = 3.0 Hz, 13.5 Hz, benzylamine-CH2), 4.2–
4.4 (m, 1H, Apns-3-CH), 4.44–4.55 (m, 3H, benzyl-
amine-CH2, Dmt-4-CH, and Apns-2-CH), 4.97 (d,
1H, J = 8.4 Hz, Dmt-2-CH2), 5.16 (d, 1H, J = 9.5 Hz,
Dmt-2-CH2), 5.22 (d, 1H, J = 6.8 Hz, Apns-2-OH),
5.41 (s, 2H, benzoyl-NH2), 6.36 (d, 1H, J = 7.3 Hz,
aromatic), 6.71–6.80 (m, 2H, aromatic), 6.96–7.18
(m, 7H, aromatic), 8.09–8.11 (br, 1H, benzylamine-
NH), 8.45 (d, 1H, J = 8.9 Hz, Apns-NH); Anal. Calcd
for C33H39ClN4O5S: C, 62.01; H, 6.15; N, 8.77; found:
C, 61.93; H, 6.04; N, 8.65.
NMR (DMSO-d6) d (ppm): 1.15 (s, 3H), 1.45 (s, 3H),
2.32 (s, 6H), 3.23 (s, 1H), 3.69 (br, 1H), 3.99 (d, 1H,
J = 8.9 Hz), 4.20–4.39 (m, 3H), 7.01–7.13 (m, 3H), 7.99
(br, 1H); Anal. Calcd for C15H22N2OS: C, 64.71; H,
7.96; N, 10.06; found: C, 64.4; H, 8.05; N, 10.04.
6.1.11. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-methoxyphenyl)butanoyl]-5,5-dimethyl-1,3-thi-
azolidine-4-carboxamide (5e). To a solution of compound
4b (153 mg, 0.55 mmol), (2S,3S)-3-(N-tert-butoxycar-
bonyl)amino-2-hydroxy-4-(4-methoxyphenyl)butyric acid
(163 mg, 0.50 mmol), HOBt (68 mg, 0.50 mmol) in ethyl
acetate (5 mL), and DCC (113 mg, 0.55 mmol) were
added; and the mixture was stirred overnight. The reaction
mixture was then filtered, and the filtrate was washed
sequentially with 3% K2CO3, 1 N HCl, and brine, dried
over MgSO4, filtered, and concentrated. The residue was
redissolved in ethyl acetate (5 mL) and added to 4 N
HCl in ethyl acetate (5 mL), and the mixture stirred for
1 h. The reaction mixture was then concentrated under re-
duced pressure and then redissolved in water. The aqueous
phase was filtered, and the filtrate was neutralized with 2 N
NaOH to give the precipitate. The crude product was
recrystallized from ethyl acetate to give 129 mg of the title
compound with a yield of 53%: mp 199–201 °C; 1H NMR
(DMSO-d6) d (ppm): 0.6–0.8 (br, 2H), 1.34 (s, 3H), 1.52
(s, 3H), 2.09 (s, 6H), 2.0–2.1 (m, 1H), 2.3–2.5 (m, 1H),
3.02 (d, 1H, J = 11.3 Hz), 3.79 (s, 3H), 3.95–4.01 (br,
1H), 4.10 (br, 2H), 4.27 (s, 1H), 4.84 (s, 2H), 5.22 (d,
1H, J = 8.1 Hz), 6.66–6.77 (m, 3H), 6.90 (d, 2H,
J = 8.6 Hz), 6.99 (d, 2H, J = 8.4 Hz), 8.17 (br, 1H); Anal.
Calcd for C26H35N3O4S H2O: C, 61.60; H, 7.08; N, 8.20;
C, 62.00; H, 7.40; N, 8.34.
6.1.15. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-amino-
2-hydroxy-4-(4-ethoxyphenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (5g). Mp 214–216 °C; 1H
NMR (DMSO-d6) d (ppm): 0.70 (br, 2H), 1.34 (s, 3H),
1.36 (t, 3H, J = 8.1 Hz), 1.52 (s, 3H), 2.12 (s, 6H), 2.0–
2.1 (m, 1H), 2.4–2.5 (m, 1H), 3.02 (d, 1H, J = 11.1 Hz),
3.95–4.01 (br, 1H), 4.02–4.08 (br, 2H), 4.10–4.11 (br,
2H), 4.27 (s, 1H), 4.84 (s, 2H), 5.21 (d, 1H, J = 8.1 Hz),
6.67–6.75 (m, 3H), 6.89 (d, 2H, J = 8.6 Hz), 6.97 (d, 2H,
J = 8.4 Hz), 8.16 (br, 1H); Anal. Calcd for C27H37N3O4S:
C, 64.90; H, 7.46; N, 8.41; found: C, 64.70; H, 7.50; N, 8.30.
6.1.16. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-amino-2-chlorobenzoyl)amino-4-(4-ethoxyphenyl)butan-
oyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (6g). Mp
1
120–122 °C; H NMR (DMSO-d6) d (ppm): 1.29 (t, 3H,
J = 7.0 Hz), 1.35 (s, 3H), 1.50 (s, 3H), 2.27 (s, 3H), 2.6–
2.8 (m, 2H), 3.96 (q, 2H, J = 6.8 Hz), 4.14 (dd, 1H,
J = 4.3 Hz, 15.1 Hz), 4.2–4.5 (m, 4H), 4.98 (d, 1H,

T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
1307
J = 9.2 Hz), 5.12 (d, 1H, J = 9.5 Hz), 5.3–5.5 (m, 3H),
6.36 (d, 1H, J = 6.2 Hz), 6.7–6.9 (m, 3H), 6.98 (t, 1H,
J = 7.8 Hz), 7.1–7.2 (br, 3H), 7.23 (d, 2H, J = 8.9 Hz),
7.3–7.4 (m, 1H), 8.28–8.34 (m, 2H); Anal. Calcd for
C34H41ClN4O5S: C, 62.51; H, 6.33; N, 8.58; C, 62.24;
H, 6.38; N, 8.26.
5.3 mmol), and EDC Æ HCl (1.11 g, 5.8 mmol) were
added in an ice-bath. After stirring for 16 h, the reaction
mixture was poured into ethyl acetate, washed sequen-
tially with 3% K2CO3, 5% citric acid, and brine, dried
over MgSO4, and then concentrated. The residue was
redissolved in ethyl acetate (10 mL) and added to 4 N
HCl in ethyl acetate (10 mL). After stirring for 2 h, the
reaction mixture was neutralized with 2 N NaOH. The
organic phase was washed with brine, dried over
MgSO4, filtered, and concentrated to give a crude prod-
uct. The crude product was purified by silica gel chroma-
tography, the less polar product having higher Rf value
in CH2Cl2-MeOH TLC system was collected and recrys-
tallized from n-hexane-ethyl acetate to give 1.06 g of the
6.1.17. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-amino-
2-hydroxy-4-(3,4-methylenedioxyphenyl)butanoyl]-5,5-di-
methyl-1,3-thiazolidine-4-carboxamide (5h). Mp 183–
1
185 °C; H NMR (DMSO-d6) d (ppm): 0.65–0.75 (br,
2H), 1.34 (s, 3H), 1.52 (s, 3H), 2.0–2.1 (m, 1H), 2.14
(s, 6H), 2.3–2.4 (m, 1H), 3.00 (d, 1H, J = 11.1 Hz),
3.97 (t, 1H, J = 8.2 Hz), 4.11 (br, 2H), 4.27 (s, 1H),
4.83 (s, 2H), 5.20 (d, 1H, J = 8.4 Hz), 6.05 (d, 1H,
J = 3.2 Hz), 6.52 (d, 1H, J = 7.8 Hz), 6.62 (s, 1H),
6.70–6.82 (m, 3H), 6.87 (d, 1H, J = 7.8 Hz), 8.20 (br,
1H); Anal. Calcd for C26H33N3O5S 0.5H2O: C, 61.40;
H, 6.74; N, 8.26; found: C, 61.26; H, 6.64; N, 8.13.
1
title compound with a yield of 86%: mp 153–155 °C, H
NMR (DMSO-d6) d (ppm): 0.95 (s, 3H), 1.09 (s, 3H),
1.6–1.8 (m, 2H), 2.1–2.2 (m, 1H), 2.15 (s, 6H), 2.5–2.6
(m, 3H), 3.03 (d, 1H, J = 11.1 Hz), 3.5–3.6 (m, 1H),
3.7–3.8 (m, 1H), 3.78 (s, 3H), 3.86–3.93 (m, 2H), 4.0–
4.3 (m, 2H), 5.03 (d, 1H, J = 8.4 Hz), 6.71–6.83 (m,
6H), 7.23 (t, 1H, J = 8.0 Hz), 8.07 (br, 1H). Anal. Calcd
for C27H37N3O4: C, 69.35; H, 7.98; N, 8.99; found: C,
69.18; H, 8.01; N, 8.88.
6.1.18. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-(3-amino-2-chlorobenzoyl)amino-4-(3,4-methylenedioxy-
phenyl)butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carbox-
1
amide (6h). Mp 158–160 °C; H NMR (DMSO-d6) d
(ppm): 1.36 (s, 3H), 1.46 (s, 3H), 2.32 (s, 6H), 2.6–2.7
(m, 2H), 4.1–4.3 (m, 2H), 4.44–4.54 (m, 3H), 4.95 (d,
1H, J = 8.6 Hz), 5.15–5.19 (m, 2H), 5.41 (s, 2H), 5.94
(s, 2H), 6.37–6.40 (m, 1H), 6.76–6.87 (m, 3H), 6.96–
7.09 (m, 5H), 8.12 (br, 1H), 8.42 (d, 1H, J = 8.4 Hz);
Anal. Calcd for C33H37ClN4O6S: C, 60.68; H, 5.71;
N, 8.58; found: C, 60.69; H, 5.78; N, 8.61.
6.1.21. (S)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-(3-amino-2-chlorobenzoyl)amino-4-(3-methoxyphenyl)-
butanoyl]-3,3-dimethylpyrrolidine-2-carboxamide (10b).
To a solution of 3-amino-2-chlorobenzoic acid (390 mg,
2.3 mmol) and 9b (1.06 g, 2.3 mmol) in DMF (5 mL),
HOBt (310 mg, 2.3 mmol) and EDC Æ HCl (480 mg,
2.5 mmol) were added in an ice-bath; and the mixture
was stirred overnight. The reaction mixture was poured
into ethyl acetate, washed sequentially with 3% K2CO3,
5% citric acid, and brine, and then dried over MgSO4.
After filtered, and concentrated, the crude product was
purified by silica gel chromatography and then recrystal-
lized from n-hexane-ethyl acetate to give 1.06 g of the title
compound with a yield of 75% mp 175–177 °C, 1H NMR
(DMSO-d6) d (ppm): 1.00 (s, 3H), 1.04 (s, 3H), 1.6–1.7 (br,
1H), 1.9–2.1 (br, 1H), 2.31 (s, 6H), 2.6–2.8 (m, 2H), 3.6–
3.7 (br, 1H), 3.76 (s, 3H), 4.0–4.2 (m, 3H), 4.2–4.3 (br,
1H), 4.4–4.6 (m, 2H), 4.90 (d, 1H, J = 6.8 Hz), 5.41 (s,
2H), 6.36 (d, 1H, J = 6.2 Hz), 6.71–6.79 (m, 2H), 6.96–
7.11 (m, 6H), 7.15 (t, 1H, J = 7.8 Hz), 8.09 (br, 1H), 8.43
(d, 1H, J = 8.1 Hz). Anal. Calcd for C34H41ClN4O5: C,
65.74; H, 6.65; N, 9.02; found: C, 65.46; H, 6.62; N, 8.84.
6.1.19. (RS)-N-(2,6-Dimethylbenzyl)-3,3-dimethylpyrrol-
idine-2-carboxamide (8). To a solution of (RS)-N-tert-
butoxycarbonyl-3,3-dimethylpyrrolidine-2-carboxylic
acid (7, 1.05 g, 4.3 mmol) and triethylamine (0.72 mL,
5.2 mmol) in ethyl acetate (30 mL), DPP-Cl (0.98 mL,
4.8 mmol) was added in an ice-bath; and the mixture
was stirred for 4 h. Then to the reaction mixture, 2,6-dim-
ethylbenzylamine hydrochloride (0.81 g, 4.8 mmol) and
triethylamine (1.50 mL, 10.8 mmol) were added in an
ice-bath, followed by stirring overnight. The reaction
mixture was washed sequentially with 1 N HCl, 3%
K2CO3, and brine, dried over MgSO4, filtered, and con-
centrated. The residue was redissolved in ethyl acetate
(20 mL) and added to 4 N HCl in ethyl acetate (20 mL),
and this mixture was stirred for 2 h. The reaction mixture
was neutralized with 2 N NaOH. The organic phase was
washed with brine, dried over MgSO4, filtered, and con-
centrated to give a crude product. Recrystallization from
n-heptane gave 0.92 g of the title compound with a yield of
82%: mp 62–64 °C, 1H NMR (DMSO-d6) d (ppm): 0.79 (s,
3H), 1.11 (s, 3H), 2.31 (s, 6H), 2.78–2.91 (m, 3H), 3.11 (s,
1H), 4.27 (d, 2H, J = 3.8 Hz), 7.01–7.11 (m, 3H), 7.62 (t,
1H, J = 4.5 Hz). Anal. Calcd for C16H24N2O: C, 73.81;
H, 9.29; N, 10.76; found: C, 73.47; H, 9.30; N, 10.64.
6.1.22. (S)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-amino-4-(4-methoxyphenyl)butanoyl]-3,3-dimethylpyr-
rolidine-2-carboxamide (9a). Mp 186–188 °C, (DMSO-
d6) d (ppm): 0.95 (s, 3H), 1.08 (s, 3H), 1.6–1.7 (m, 2H),
2.1–2.2 (m, 1H), 2.16 (s, 6H), 2.50–2.53 (m, 3H), 2.98
(d, 1H, J = 11.2 Hz), 3.5–3.6 (m, 1H), 3.7–3.8 (m, 1H),
3.75 (s, 3H), 3.85–3.93 (m, 2H), 4.0–4.3 (m, 2H), 5.00
(d, 1H, J = 8.2 Hz), 6.75–6.85 (m, 3H), 6.89 (d, 2H,
J = 8.4 Hz), 7.04 (d, 2H, J = 8.4 Hz), 8.07 (br, 1H). Anal.
Calcd for C27H37N3O4: C, 69.35; H, 7.98; N, 8.99; found:
C, 69.14; H, 8.00; N, 8.94.
6.1.20. (S)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-amino-4-(3-methoxyphenyl)butanoyl]-3,3-dimethylpyrrol-
idine-2-carboxamide (9b). To a solution of compound 8
(1.37 g, 5.3 mmol) in DMF (10 mL), (2S,3S)-3-(N-tert-
butoxycarbonyl)amino-2-hydroxy-4-(3-methoxyphenyl)-
butyric acid (1.72 g, 5.3 mmol), HOBt (0.71 g,
6.1.23. (S)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-(3-amino-2-chlorobenzoyl)amino-4-(4-methoxyphenyl)-
butanoyl]-3,3-dimethylpyrrolidine-2-carboxamide (10a).
Mp 195–196 °C, 1H NMR (DMSO-d6) d (ppm): 1.00 (s,

1308
T. Mimoto et al. / Bioorg. Med. Chem. 16 (2008) 1299–1308
3H), 1.03 (s, 3H), 1.6–1.7 (br, 1H), 1.9-2.1 (br, 1H), 2.31
(s, 6H), 2.6–2.8 (m, 2H), 3.6–3.7 (br, 1H), 3.72 (s, 3H),
4.0–4.3 (m, 4H), 4.3–4.4 (br, 1H), 4.50 (dd, 1H,
J = 6.5 Hz, 13.8 Hz), 4.89 (d, 1H, J = 7.0 Hz), 5.41 (s,
2H), 6.37 (d, 1H, J = 6.2 Hz), 6.80 (t, 3H, J = 8.6 Hz),
6.96–7.11 (m, 4H), 7.32 (d, 2H, J = 8.4 Hz), 8.09 (br,
1H), 8.38 (d, 1H, J = 8.4 Hz). Anal. Calcd for
C34H41ClN4O5: C, 65.74; H, 6.65; N, 9.02; found: C,
65.46; H, 6.70; N, 8.77.
Hiyama, and Ms. Rena Sekine for the chemical synthe-
sis of HIV protease inhibitors, evaluation of compounds
metabolism, and determination of HIV-1 protease activ-
ity, respectively. The main part of this work was per-
formed when the authors worked for Pharmaceutical
&
Corporation.
Biotechnology
Laboratory,
Japan
Energy
References and notes
6.1.24. (S)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-amino-4-(4-ethoxyphenyl)butanoyl]-3,3-dimethylpyrrol-
idine-2-carboxamide (9c). Mp 181–183 °C, 1H NMR
(DMSO-d6) d (ppm): 0.95 (s, 3H), 1.08 (s, 3H), 1.32 (t,
3H, J = 7.6 Hz), 1.6–1.8 (m, 2H), 2.2–2.2 (m, 1H), 2.16
(s, 6H), 2.5–2.6 (m, 3H), 2.9–3.0 (m, 1H), 3.5–3.6 (m,
1H), 3.7–3.8 (m, 1H), 3.85–3.96 (m, 2H), 4.0–4.3 (m,
4H), 4.99 (d, 1H, J = 8.4 Hz), 6.75–6.83 (m, 3H), 6.87
(d, 2H, J = 8.6 Hz), 7.02 (d, 2H, J = 8.6 Hz), 8.07 (br,
1H). Anal. Calcd for C28H39N3O4: C, 69.83; H, 8.16;
N, 8.72; found: C, 69.57; H, 8.19; N, 8.63.
1. Gulick, R. M. Quality Life Res. 1997, 6, 471.
2. Swainston, H. T.; Scott, L. J. Drugs 2005, 65, 2309.
3. Mimoto, T.; Kato, R.; Takaku, H.; Nojima, S.; Terashi-
ma, K.; Misawa, S.; Fukazawa, T.; Ueno, T.; Sato, H.;
Shintani, M.; Kiso, Y.; Hayashi, H. J. Med. Chem. 1999,
42, 1789.
4. Yoshimura, K.; Kato, R.; Yusa, K.; Kavlick, M. F.;
Maroun, V.; Nguyen, A.; Mimoto, T.; Ueno, T.; Shintani,
M.; Falloon, J.; Masur, H.; Hayashi, H.; Erickson, J.;
Mitsuya, H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8675.
5. Unpublished data.
6. Mimoto, T.; Terashima, K.; Nojima, S.; Takaku, H.;
Nakayama, M.; Shintani, M.; Yamaoka, T.; Hayashi, H.
Bioorg. Med. Chem. 2004, 12, 281.
7. Balden, E. T.; Bhar, T. N.; Gulnik, S.; Liu, B.; Topol, I.
A.; Kiso, Y.; Mimoto, T.; Mitsya, H.; Erickson, J. W.
Structure 1995, 3, 581.
6.1.25. (S)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy-
3-(3-amino-2-chlorobenzoyl)amino-4-(4-ethoxyphenyl)-
butanoyl]-3,3-dimethylpyrrolidine-2-carboxamide (10c).
1
Mp 172–174 °C, H NMR (DMSO-d6) d (ppm): 1.01 (s,
8. (a) Nishizawa, R.; Saino, T.; Takita, T.; Suda, H.; Aoyagi,
T. J. Med. Chem. 1977, 20, 510; (b) Yuan, W.; Munoz, B.;
Wong, C. H.; Haeggstrom, J. Z.; Wetterholm, A.; Sam-
uelsson, B. J. Med. Chem. 1993, 36, 211; (c) Suzuki, T.;
Honda, Y.; Izawa, K.; EP 0767168A2., 1997; (d) Mat-
sumoto, S.; Matsuo, K.; Sugawa, T.; Moroshima, T.;
Inoue, K.; WO98/07687, 1998; (e) Nishiyama, A.; Inoue,
K.; WO2000/53575, 2000.
3H), 1.03 (s, 3H), 1.31 (t, 3H, J = 6.9 Hz), 1.6–1.7 (br,
1H), 1.9–2.1 (br, 1H), 2.31 (s, 6H), 2.6–2.8 (m, 2H), 3.5–
3.7 (br, 1H), 3.98 (q, 2H, J = 6.9 Hz), 4.0–4.3 (m, 4H),
4.3–4.4 (br, 1H), 4.49 (dd, 1H, J = 6.5 Hz, 13.5 Hz), 4.89
(d, 1H, J = 7.3 Hz), 5.41 (s, 2H), 6.37 (d, 1H,
J = 5.7 Hz), 6.76–6.81 (m, 3H), 6.96–7.09 (m, 4H), 7.30
(d, 2H, J = 8.4 Hz), 8.09 (br, 1H), 8.38 (d, 1H,
J = 8.4 Hz). Anal. Calcd for C35H43ClN4O5: C, 66.18;
H, 6.82;N, 8.82; found: C, 66.00; H, 6.86;N, 8.65.
9. Hayashi, Y.; Kinoshita, Y.; Hidaka, K.; Kiso, A.;
Uchibori, H.; Kimura, T.; Kiso, Y. J. Org. Chem. 2001,
66, 5537.
10. Morgan, B. A.; Schafer, D. J., US4060603, 1977.
11. The desired S-pyrrolidine diastereoisomer was preserved
based on the chemical behavior of corresponding L-
proline and R-thiazolidine derivatives. In addition, HIV-1
protease inhibitory activity of the target compounds 10a-c
derived from the less polar diastereoisomers was far
superior to that of the polar diastereoisomers.
6.2. Biological evaluations
HIV protease inhibitory activities, metabolic stabilities,
plasma concentrations, and pharmacokinetic parame-
ters of inhibitors were determined by the methods re-
ported previously.6
12. Similar structure–activity relationships were observed in
tripeptide type HMC compounds Mimoto, T.; Hattori,
N.; Takaku, H.; Kisanuki, S.; Fukazawa, T.; Terashima,
K.; Kato, R.; Nojima, S.; Misawa, S.; Ueno, T.; Imai, J.;
Enomoto, H.; Tanaka, S.; Sakikawa, H.; Shintani, M.;
Hayashi, H.; Kiso, Y. Chem. Pharm. Bull. 2000, 48, 1310.
13. Petropoulos, C. J.; Parkin, N. T.; Limoli, K. L.; Lie, Y. S.;
Wrin, T.; Huang, W.; Tian, H.; Smith, D.; Winslow, G.
A.; Capon, D. J.; Whitcomb, J. M. Antimicrob. Agents
Chemother. 2000, 44, 920.
Acknowledgments
The authors thank Southern Research Institute and
ViroLogic Incorporation for conducting the wild-type
HIV inhibition assay and the mutant HIV inhibition as-
say (Phenosence assay13), respectively. We appreciate
the assistance of Mr. Shinji Matsumoto, Mr. Yasuki