Structure-activity and structure-metabolism relationships of HIV protease inhibitors containing the 3-hydroxy-2-methylbenzoyl-allophenylnorstatine structure

Article abstract of DOI:10.1016/j.bmc.2003.10.037

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-metabolism relationship of this type of HIV protease inhibitors, the compounds having para substitution of the phenyl ring of Apns and/or 2,6-disubstitution of the P2′ benzylamine were found to be able to avoid the P2 phenol glucuronidation that occurs with SM-319777 (formerly named JE-2147, KNI-764); one of the main metabolic pathways of SM-319777. These new analogues, such as SM-322377, had more desirable pharmacokinetic profiles and more potent antiviral activity against not only wild type HIV-1 but also the multi-drug-resistant HIV-1 than SM-319777.

Full text of DOI:10.1016/j.bmc.2003.10.037

Bioorganic & Medicinal Chemistry 12 (2004) 281–293
Structure–activity and structure–metabolism relationships of HIV
protease inhibitors containing the 3-hydroxy-2-methylbenzoyl-
allophenylnorstatine structure
Tsutomu Mimoto,* Keisuke Terashima, Satoshi Nojima, Haruo Takaku,
Mitsunobu Nakayama, Makoto Shintani, Takashi Yamaoka and Hideya Hayashi
Research Division, Sumitomo Pharmaceuticals Co., Ltd, 1-98, Kasugade Naka 3-Chome, Konohanaku, Osaka 554-0022, Japan
Received 11 August 2003; accepted 12 September 2003
Abstract—A series of peptidomimetic human immunodeficiency virus (HIV) protease inhibitors containing substituted allo-
phenylnorstatine [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were designed and synthesized. From the structure–
metabolism relationship of this type of HIV protease inhibitors, the compounds having para substitution of the phenyl ring of Apns
and/or 2,6-disubstitution of the P20 benzylamine were found to be able to avoid the P2phenol glucuronidation that occurs with
SM-319777 (formerly named JE-2147, KNI-764); one of the main metabolic pathways of SM-319777. These new analogues, such as
SM-322377, had more desirable pharmacokinetic profiles and more potent antiviral activity against not only wild type HIV-1 but
also the multi-drug-resistant HIV-1 than SM-319777.
# 2003 Elsevier Ltd. All rights reserved.
nelfinavir, amprenavir, and lopinavir.5 As SM-319777
showed a good pharmacokinetic profile in dogs,4 we
1. Introduction
In the last decade, a number of agents have become avail-
able for the treatment of individuals infected with HIV-1,
and combination chemotherapy with reverse transcriptase
inhibitors and protease inhibitors has been shown to sup-
press HIV-1 replication to an undetectable level in
patients.1 However, accumulated data indicate that the
development of HIV-1 variants with reduced suscept-
ibility to reverse transcriptase inhibitors and protease
inhibitors is directly related to clinical deterioration in
those individuals receiving such therapy.2 Thus, the
development of novel compounds that are active against
multidrug-resistant HIV-1 variants is urgently needed.3
decided to choose this compound as a clinical candi-
date, which should be useful for salvage therapy; and at
present SM-319777 has been examined in an early P-II
clinical trial stage. Coadministration of each HIV-1
protease inhibitor with ritonavir is well-known to ele-
vate and sustain the plasma drug levels because of the
inhibitiory activity of the latter toward cytochrome
P450 (CYP) 3A4-mediated metabolism.6 The coadmin-
istration regimen, for example Kaletra (lopinavir plus
ritonavir),7 shows the favorable pharmacokinetic profile
and a strong anti-HIV efficacy. The results of in vitro
metabolism studies indicated that ritonavir did not give
a remarkable improvement of the metabolism of
SM-319777 because SM-319777 was found to be meta-
bolized by not only CYP3A4 but also UDP-glucur-
onosyl transeferase (UGT) 2B7 in human hepatocytes;8
and the activity of UGT could not be inhibited by rito-
navir. These data suggested that the coadministration of
SM-319777 with ritonavir could not be expected to give
effective improvement of the pharmacokinetic profile of
SM-319777 in humans. In the present study, we disclose
the results of our continuing efforts to understand the
structure–activity and structure–metabolism relation-
ships of the HMC-based HIV protease inhibitors con-
taining the substituted Apns structure. Ultimately we
Previously, we have reported that SM-3197774 (Fig. 1)
represents a class of dipeptide HIV protease inhibitors
containing allophenylnorstatine with
a
hydroxy-
methylcarbonyl (HMC) isostere as the transition-state
mimetic. SM-319777 completely suppressed all HIV-1
and HIV-2strains as well as clinical HIV-1 variants that
were highly resistant against all currently available pro-
tease inhibitors including saquinavir, ritonavir, indinavir,
* Corresponding author. Tel.: +81-6-6466-5265; fax: +81-6-6466-
5287; e-mail: tmimoto@sumitomopharm.co.jp
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.037

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T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
Figure 1. Structures of SM-319777 (left) and nelfinavir (right).
found SM-322377 (12n) to display more desirable phar-
macokinetic profile and antiviral activity against the
multi-drug-resistant HIV-1 than SM-319777.
3. Chemistry
All commercially available optically active amino acids
were purchased. The 4-alkoxyphenylalanines were
derived from l-tyrosine by alkylation. The other l-phe-
nylalanines were prepared by the acetamidomalonate
method or by the Erlenmeyer method, followed by
resolution with aminoacylase. Boc-protected (2S,3S)-3-
amino-2-hydroxy-4-arylbutanoic acids, without the
3-methoxy and 3,4-methylenedioxy derivatives, were
prepared by the cyanohydrin procedure12 using amino-
aldehydes derived from l-phenylalanines. The Boc-pro-
tected (2RS,3S) mixture thus obtained was converted to
the benzyl ester, and the (2S,3S) enantiomer was easily
separated by recrystallization from n-hexane/ethyl ace-
tate, and then debenzylation by hydrogenation (H2–Pd/
C) gave the desired Boc-Apns derivatives, (2S,3S)
enantiomers. In the case of the compounds having
2. Concept of the drug design
The main metabolites of SM-319777 produced by
human hepatocytes in vitro were the phenol glucur-
onide, M1, and the thiazolidine sulfoxide, M2 (Fig. 2).8
In the in vitro metabolism study using human liver
microsomes, SM-319777 was highly metabolized in the
presence of uridine 50-diphosphoglucuronic acid
(UDPGA), a co-factor of UGT (Table 1). On the other
hand, nelfinavir9 (Fig. 1), having the same building
block at the P2site and a phenylthio-structure at the P1
site, was not glucuronized under the same in vitro con-
ditions (Table 1). We speculated that the steric hin-
drance of the extended aromatic ring system in
nelfinavir might be effective to prevent the glucuronida-
tion of the P2phenol. The X-ray crystallography of
HIV protease inhibitors of our Apns series complexed
with HIV-1 protease10,11 showed that the meta or para
position of phenyl moiety of Apns faced the outside of
the HIV protease-binding pocket. These data suggested
to us that the meta or para substitution on phenyl moi-
ety of Apns might lead to improvement of the pharma-
cokinetic property of SM-319777 without changing the
binding mode to HIV-1 protease and its excellent viral
resistance profile.
Table 1. In vitro metabolism by human liver microsomes
Compd
Structure
R2R3
H
Metabolism: remaining (%)
R1
H
OMe
H
OEt
OnPr
OiPr
UDPGA
NADPH
SM-319777
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
55
>95
63
20
27
47
18
25
13
62
43
55
41
43
42
53
34
51
45
52
H
OMe
H
H
H
91
>95
>95
>95
>95
68
>95
53
38
71
>95
>95
>95
>95
–O–CH2–O–
Me
H
Cl
H
F
H
H
Me
H
Cl
H
F
OMe
H
–O–CH2–O– Me
H
12n (SM-322377)
12o
Nelfinavir
OMe Me
Data show the mean residual percent of duplicates. A given test drug
(5 mM) 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.
Residual drugs were measured by RP-HPLC.
Figure 2. Main metabolites of SM-319777 generated by human
hepatocytes.

T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
283
3-methoxy or 3,4-methylenedioxy group, the cyanohy-
drin method could not give the desired products in
satisfactory yield due to their instability under acid
hydrolysis condition of cyanohydrin intermediates, so
another method reported by Ajinomoto’s group was
applied to the synthesis of these derivatives (Scheme
1).13 The reaction of Boc-protected l-3-methox-
yphenylalanine methyl ester (2) derived from the corre-
sponding amino acid 1 with the carbanion derived from
dimethylsulfoxide gave the diastereomixture of b-keto-
obtained were deprotected under acidic conditions gave
the dipetide amine derivatives 10a–o. The coupling of
3-acetoxy-2-methylbenzoic acid to the dipeptide amine
derivatives 10a–o by the mixed anhydride method using
diphenylphosphoryl chloridate gave 11a–o, which after
saponification, gave compounds 12a–o in good yield
(Scheme 2).
4. Results and discussion
sulfoxide 3. a-Keto-hemimercaptal acetate
4 was
obtained by reacting 3 with acetic anhydride in CH2Cl2
in the presence of pyridine (Pummerer rearrangement),
and then 4 was rearranged into a b-amino-a-acylox-
ythioester 5 possibly via 1,4-acyl transfer in enolate
form in the presence of 1,8-diazabicyclo[5.4.0]undec-7-
ene in N,N-dimethylformamide (DMF) at À20 ꢀC. The
diastereomeric ratio (2S/2R) of 5 was about 3:2, and the
desired (2S,3S) enantiomer 6 was easily separated by
recrystallization from n-hexane/ethyl acetate. Saponifi-
cation of 6 gave the desired Boc-Apns derivative 7.
4.1. Structure–metabolism relationship by human liver
microsomes in vitro
Table 1 shows the results of our in vitro metabolism
study on these compounds. The compounds containing
the para substituent (R1) on the phenyl ring, except 12k,
were stable against the glucuronidation; whereas the
compounds having the meta substitution (R2), 12b, h, j,
and l, or a small para substitution such as fluorine 12k,
on the phenyl ring remained susceptible to the glucur-
onidation of the P2phenol. These data suggest that the
steric bulk of the para substituent of the Apns ring could
prevent the glucuronidation of the P2phenol. Moreover,
the introduction of a lower alkoxy substituent to the
phenyl ring provided an increase in solubility (data not
shown). However, the para alkoxy-substituted analogues
did not show improved stability against CYP oxidation
when their in vitro metabolism was examined with
human liver microsomes in the presence of b-nicotin-
amide adenine dinucleotide phosphate (NADPH). In
contrast, the meta alkoxy analogues 12b,f were more
stable than SM-319777 against the CYP oxidation. In
the case of the compounds containing the 2,6-dimethyl-
benzyl group at the P20 site (R3=Me), the meta alkoxy
analogues 12n,o also had such stability against the CYP
Boc-Apns derivatives thus obtained were coupled
with previously reported3 5,5-dimethyl-1,3-thiazoli-
dine-4-carboxamides 9a,b. Hayashi et al. reported14
that low yields were often observed in the coupling
of N-protected Apns derivatives and 5,5-dimethyl-1,3-
thiazolidine-4-carboxamides, caused by the formation
of Boc-Apns-homobislactone. In their report, 1-ethyl-
3 - (dimethylaminopropyl)carbodiimide - 1 - hydroxy - 7 -
azabenzotriazole (HOAt) method in DMF was recom-
mended. In the course of our scale-up synthesis of
SM-319777, N,N0-dicyclohexylcarbodiimide (DCC)-1-
hydroxybenzotriazole (HOBt) method in ethyl acetate
was found out to be effective for the formation of this
amide bond.15 The Boc-protected dipeptide amide thus
Scheme 1. (a) di-tert-Butyl dicarbonate, triethylamine, THF–H2O; (b) methyl iodide, K2CO3, DMF; (c) DMSO, sodium amide, THF; (d) acetic
anhydride, pyridine, 4-(dimethylamino)pyridine, CH2Cl2; (e) 1,8-diazabicyclo[5.4.0]undec-7-ene, DMF; (f) recrystalization from n-hexane/ethyl
acetate; (g) NaOH aq, methanol.

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T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
Scheme 2. (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 acid, N,N0-dicyclohexylcarbodiimide, 1-hydroxybenztriazole, ethyl acetate; (d) 4 N
HCl in ethyl acetate (or dioxane); (e) 3-acetoxy-2-methylbenzoic acid, diphenylphosphoryl chloridate, triethylamine, ethyl acetate; (f) NaOH aq,
methanol.
oxidation. Surprisingly, the compound of this class
Table 2. HIV-1 protease (HIV-1 PR) inhibition and anti-HIV activity
having no para substitution, 12n, was not affected under
the in vitro glucuronizing condition. These data suggest
that the para substitution of Apns or the additional
ortho methyl group on the P20 benzyl group of
SM-319777 could reduce the affinity of SM-319777 for
UGT. Compound 12n had stability comparable to that
of nelfinavir against both CYP oxidation and UDP
glucuronidation.
Compd
Structure
HIV-1 PR
inhibition
HIV-1 IIIB; EC50
(nM)
4.2. Structure–activity relationship of HIV-1 protease
inhibition and antiviral activity
R1
R2R3
Ki (pM)
15% fetal 50% human
bovine serum
Table 2 represents the results of the study on HIV-1
protease inhibitory activity16 and anti-HIV activity of
the selected compounds that were stable against the
UDP glucuronidation. As the inhibitory activity of
SM-319777 toward HIV protease was not affected by
the introduction of the meta- or para subsituent on the
phenyl moiety of Apns, the P1 phenyl-substituted com-
pounds, 12a,c–f, had activity comparable to that of
SM-319777 in terms of HIV-1 protease inhibition
(Ki=31–59 pM). Moreover, the series of compounds
containing the additional ortho methyl group at the P20
site (R3=Me), 12m–o, showed slightly more potent
activity than SM-319777 (Ki=22–29 pM).
serum
SM-319777
12a
12c
12d
12e
12f
12m
12n (SM-
322377)
12o
H
H
H
H
H
H
H
H
H
H
H
35
31
38
59
40
37
29
29
26
6.6
6.5
52
28
9.7
OMe
OEt
OnPr
OiPr
5
3.22
7.9
4.8
7.6
2.4
17
52
14
9.0
–O–CH2–O–
H
H
Me
OMe
H
OMe Me
–O–CH2–O– Me
22
931
3.0
19
14
293
Nelfinavir
Assay of HIV-1 protease was performed by measuring the fluorescence
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, 2mM dithiothreitol, and 2mM
EDTA-2Na, with incubation at 37 ꢀC for 15 min. Evaluation of in
vitro antiviral activities of test compounds against wild-type HIV-1
(strain IIIB) was conducted as described previously16 (details are
written in the Experimental).
Although these compounds had nearly the same HIV
protease inhibitory activity as SM-319777, cell culture
assays under the condition of 15% fetal bovine serum
demonstrated that almost all compounds of this study

T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
285
were more effective than SM-319777 in suppressing the
Table 3. Sensitivity of mutant HIV-1s to HIV protease inhibitors
spread of acute HIV-1 infection, probably due to their
superior permeation of the cell membrane. A previous
paper reported4 that the in vitro antiviral efficacy of
SM-319777 was not affected by the addition of the
human relevant protein, which was different from the
case for other HIV protease inhibitors such as nelfinavir
and ritonavir. Some compounds in this study showed
the anti-HIV activity superior to that of SM-319777
even in the presence of 50% human serum. We con-
firmed that the substitution of a lower alkoxy group on
the Apns ring reduced the protein binding affinity,
especially for a-acid glycoprotein.17 Above all, the
compounds containing the 2,6-dimethylbenzyl group at
the P20 site, 12m–o, which had the lower Ki values,
showed extremely potent anti-HIV activity even in the
presence of 50% human serum.
Compd
HIV-1/PBMC; EC50 (nM)
1002-60 1026-40 052-52
Wild type
SM-319777
12a
12n (SM-322377) 1.0 (1)
17
(1)
37
(2) 103
(3) 99
8.9 (9) <1 (1)
(38) 169 (14)
1.2 (1) 1235 (1029) 335 (279) 176 (147)
26 (1) 194 (7) 752 (29) 535 (21)
1.3 (1) 573 (441) 398 (306) 685 (527)
(6) 17 (1)
6.6 (1)
17
(15) 33 (5)
4.5 (5)
Indinavir
12(1) 985 ) (84250
Nelfinavir
Amprenavir
Lopinavir
Evaluation of in vitro antiviral activities of test compounds against
wild-type HIV-1 (strain IIIB) and drug-resistant HIV containing
multi-mutations isolated from patients conferring to indinavir were
conducted as described previously18 (details are written in the Experi-
mental). The mutation sites of each strain were as follow: 1002-60:
L10I/M46I/I54V/L63P/V82F/L90M; 1026-40: L10I/M46I/I54V/L63P/
A71V/V82A/L90M; 052-52: L10R/M46I/L63P/A71V/V82T/I84V.
Numbers in parentheses represent fold changes of EC50 values against
each isolate compared with EC50 values against the wild type.
4.3. Antiviral activity against multi-drug resistant HIV-1
As shown in Table 3, the indinavir-resistant clinical
isolates used in this experiment were also resistant to
nelfinavir, amprenavir, and lopinavir. All isolates had a
high level of resistance to indinavir (14–82-fold), nelfi-
navir (147–1029-fold), amprenavir (7–29-fold), and
lopinavir (306–527-fold), compared with that against a
wild-type clinical isolate. In contrast, SM-319777 sup-
pressed the replication of all isolates, with EC50 values
of less than 100 nM; less than single figure-fold change
in EC50 was found compared with the EC50 against
wild-type HIV-1. Compound 12a, having a para meth-
oxy group at the P1 site of SM-319777, showed nearly the
same antiviral property as SM-319777. This result sug-
gests that the introduction of para methoxy group did not
affect the mode of binding of SM-319777 to HIV-1 pro-
tease, and 12a retained the unique resistance profile of
SM-319777. Moreover, SM-322377 (12n) had more
potent anti-HIV activity than SM-319777 and maintained
the same resistance profile as SM-319777; and SM-322377
showed the greatest antiviral activity with EC50 values of
less than 10nM against all three mutants tested.
Table 4. In vitro metabolism by liver microsomes in the presence of
ritonavir
Compd
Human
Rat
Dog
(À)
(+)
RTV
(À)
(+)
RTV
(À)
(+)
RTV
RTV
RTV
RTV
SM-319777
12a
12n (SM-322377)
Lopinavir
13
59
2 69
87
>95
1231
38
9
11
46
>95
>95
>95
>95
32893
46
10
34
>95
53
16
Data show the mean residual percent of duplicates. Drug (5 mM) with
or without 2.5 mM ritonavir (RTV) were incubated with liver micro-
somes (0.5 mg/mL) and cofactors (1 mM NADPH, 5 mM UDPGA) at
37 ꢀC for 30 min. Residual drug was measured by RP-HPLC.
4.4. In vitro human liver microsomal metabolism in the
presence of ritonavir and pharmacokinetics of coadminis-
tration with ritonavir
According to the in vitro metabolism study on
SM-319777 incubated with human liver microsomes,
ritonavir (CYP3A4 inhibitor6) did not show the inhib-
itory activity against the metabolism of SM-319777,
because the phenol-glucuronidation was one of the
main metabolic pathway of SM-319777 and ritonavir
could not inhibit UGT enzyme. When compound 12a or
n, which had resistance against the glucuronidation, was
incubated with human or canine liver microsomes in the
absence or presence of ritonavir (Table 4), the metabo-
lism of these compounds was effectively inhibited by the
addition of 2.5 mM ritonavir, however the metabolism
of 12a,n, and SM-319777 was not effectively inhibited in
rat microsomes. These data indicate that the informa-
tion from canine microsomes is a good predictor of the
pharmacokinetic profiles of 12a, and 12n coadminis-
tered with ritonavir in humans.
Figure 3. Plasma concentrations of SM-322377 (12n) after intravenous
or oral administration to beagle dogs. SM-322377 was dissolved in 50%
PEG. The plasma concentration was measured by HPLC method as
described in the Experimental. Each point represents the meanÆSE of
three dogs. *, 5 mg/kg intravenous injection; *, 15 mg/kg oral admin-
istration; ~, 15 mg/kg oral administration with 7.5 mg of ritonavir.

286
T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
Table 5. Pharmacokinetic parameters of SM-322377 (12n) in beagle dogs
Route
Dose (mg/kg)
AUC
(mM*h)
Cmax
(mM)
Tmax
(h)
T1=2
(h)
Vdss
(l/kg)
CL
(l/h/kg)
F
(%)
12n
(SM-322377)
Ritonavir
iv
po
po
5
15
15
0
0
7.5
5.0
3.0
23.7
0.5
1.4
1.6
0.6
2.7
1.0
1.0
20
Data are given as mean values of triplicates. F(%) was determined by comparing the mean areas under the curves (AUC) after intravenous and oral
doses. CL, plasma clearance rate; Vdss, volume of distribution; T1=2, half-life in plasma; Cmax, maximum plasma concentration; Tmax, time of max-
imum plasma concentration; F(%), percent orally bioavailable.
Compound 12n was orally administered to dogs at
15 mg/kg with or without 7.5 mg ritonavir/kg. Plasma
concentrations are shown in Figure 3, and pharmacoki-
netic parameters are given in Table 5. The concentration
of 12n in the plasma was enhanced by coadministration
with ritonavir, because CYP3A4, the major metabolic
enzyme of 12n, was inhibited by ritonavir. The value for
the area under the plasma concentration curve (AUC)
was enhanced by about 8-fold, and the coadministration
of 12n with ritonavir provided a plasma concentration
of 12n that exceeded the antiviral EC50 (measured in
vitro in the presence of 50% human serum) by 30-fold
for at least 12 h. These data suggest that SM-322377
(12n) coadministered with ritonavir can be expected to
show a favorable pharmacokinetic profile and the
strong anti-HIV efficacy in human use.
were recorded on a JEOL GSX270 FT NMR spectro-
meter, and chemical shifts were expressed in d ppm from
the internal standard tetramethylsilane.
6.1.1.
(2S,3S)-2-Acetoxy-3-(N-tert-butoxycarbonyl)
amino-4-(3-methoxyphenyl)butyric acid methylthioester
(6). Sodium amide (3.43 g, 88.0 mmol) was suspended
in tetrahydrofuran (THF, 60 mL) and DMSO (10.4 mL,
147 mmol), and heated at 50 ꢀC for 3 h. After cooling, a
THF (30 mL) solution of N-tert-butoxycarbonyl-(l)-3-
methoxyphenylalanine methyl ester (9.05 g, 29.3 mmol)
was added and stirred at 0 ꢀC for 2h. The reaction
mixture was extracted with 1 N HCl (100 mL) and ethyl
acetate (100 mL) and the organic layer was washed with
50 mL of brine, dried over MgSO4, and concentrated.
The resulting residue was purified by silica-gel chroma-
tography to obtain 6.15 g of the crude product 3 as an
oil (yield 59%). Compound 3 (6.15 g, 17.3 mmol) was
dissolved in CH2Cl2 (160 mL), pyridine (16.3 mL), and
acetic anhydride (16.3 mL, 173 mmol), and then
4-(dimethylamino)pyridine (104 mg, 0.87 mmol) was
added, and the mixture was stirred at 25 ꢀC for 16 h. To
the reaction mixture was added 200 mL of water, with
stirring for 1 h. The organic layer was washed with 1 N
HCl, 5% NaHCO3, and brine, and then dried over
Na2SO4 and concentrated. The resulting residue was pur-
ified by silica-gel chromatography to obtain 5.19g of the
crude compound 4. Compound 4 (5.19 g, 13.1 mmol) was
dissolved in DMF (25 mL) and cooled to À30ꢀC, and
1,8-diazabicyclo[5.4.0]undec-7-ene (1.99 mL, 13.1mmol)
was then added thereto. Having been stirred at À10ꢀC for
3 h, this mixture was partitioned between ethyl acetate and
1 N HCl. The organic layer was washed with 1 N HCl, 5%
NaHCO3, and 5% NaCl, dried over MgSO4, and then
concentrated. The residue thus obtained was recrystallized
from n-hexane (three times), giving 1.70g of the title
compound in 25% yield (total, 15%): mp 114–116 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.29 (s, 9H), 2.21 (s, 3H), 2.26
(s, 3H), 2.6–2.7 (m, 2H), 3.72 (s, 3H), 4.09–4.19 (m, 1H),
5.31 (d, 1H, J=4.3 Hz), 6.73–6.78 (m, 3H), 7.13–7.20 (m,
2H). Anal. calcd for C19H27NO6S: C, 57.41, H, 6.85, N,
3.52; Found C, 57.49; H, 6.84; N, 3.36.
5. Conclusions
In summary, we designed and synthesized a series of the
substituted-Apns-containing HIV protease inhibitors.
From the structure-metabolism relationships studies on
these types of HIV protease inhibitior, we found out
that the para substitution of Apns phenyl ring and/or
the 2, 6-disubstitution of the P20 benzylamine could pre-
vent the P2phenol glucuronidation by human liver
microsomes, which gives rise to one of the main meta-
bolites of SM-319777 by human hepatocytes. In the in
vitro metabolism by human liver microsomes, the meta-
bolism of 12a,n was effectively inhibited by the addition
of ritonavir. The plasma levels of SM-322377 (12n)
coadministered with ritonavir exceeded the antiviral
EC50 (measured in vitro in the presence of 50% human
serum) by 30-fold for at least 12h. As these compounds
maintained a potent antiviral activity against the multi-
drug resistant HIV-1, the coadministration regimen with
ritonavir should be useful for salvage therapy.
6. Experimental
6.1. Chemistry general
6.1.2. (2S,3S) - 3 - (N - tert - butoxycarbonyl)amino - 2-
hydroxy-4-(3-methoxyphenyl)butyric acid (7). Com-
pound 6 (1.65 g, 4.16 mmol) was dissolved in methanol
(30 mL), and 1.5 N NaOH (11 mL) was added, and the
mixture was stirred at room temperature overnight.
The reaction mixture was partitioned between ethyl
In general, reagents and solvents were used as purchased
without further purification. Column chromatography
was performed on FL60D (Fuji Silysia Chemical Ltd).
Melting points were measured with a Buchi 535 melting
point apparatus and uncorrected. 1H NMR spectra

T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
287
acetate and 1 N HCl, and the organic layer was washed
with brine, dried over MgSO4, and evaporated. Recrys-
tallization from n-hexane/ethyl acetate gave 1.19 g of the
title compound, in 88% yield: mp 121–123 ꢀC, 1H NMR
(DMSO-d6): d (ppm) 1.27 (s, 9H), 2.63–2.69 (m, 2H),
3.72(s, 3H), 3.85–3.99 (m, 2H), 6.69–6.75 (m, 4), 7.15 (t,
1H, J=6.8 Hz). Anal. calcd for C16H23NO6: C, 59.06;
H, 7.13; N, 4.31; Found C, 59.01; H, 7.14; N, 4.19.
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.
6.1.5. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-methoxyphenyl)-
butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(12a). To a solution of 3-acetoxy-2-methylbenzoic acid
(79 mg, 0.41 mmol) and triethylamine (57 mL,
0.41 mmol) in ethyl acetate (2mL), diphenylphosphoryl
chloridate (84 mL, 0.41 mmol) was added in an ice-bath,
and the reaction mixture was stirred for 1 h. Then to the
reaction mixture, compound 10a (174 mg, 0.37 mmol),
and triethylamine (62 mL, 0.45 mmol) were added in an
ice-bath followed by stirring overnight. The reaction
mixture was washed sequentially with 3% K2CO3, 1 N
HCl, and brine, and then concentrated. To the solution
of the resulting residue in methanol (1.5 mL), 1 N
NaOH (0.75 mL, 0.75 mmol) was added; and the mix-
ture was stirred for 1 h. The mixture was acidified with
1 N HCl, and the aqueous phase was extracted with
ethyl acetate. The organic extract was washed sequen-
tially with 1 N HCl, 3% K2CO3, and brine; dried over
MgSO4; filtered; and concentrated. The crude product,
recrystallized from n-hexane/ethyl acetate, gave 176 mg
of the title compound, with a yield of 79%: mp
6.1.3. (R)-N-(2-Methylbenzyl)-5,5-dimethyl-1,3-thiazoli-
dine-4-carboxamide (9a). To a solution of (R)-N-tert-
butoxycarbonyl- 5,5 -dimethyl-1,3 -thiazolidine- 4- car-
boxylic acid (5.22 g, 20.0 mmol) and triethylamine
(3.06 mL, 22.0 mmol) in ethyl acetate (50 mL), diphe-
nylphosphoryl chloridate (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 the mixture
was 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 stirred for 2h
with 4 N HCl in dioxane (30 mL). To the reaction mix-
ture, water was added, and aqueous phase was washed
with toluene, neutralized with 2N NaOH, and extracted
with ethyl acetate. The organic phase was washed with
brine, dried over MgSO4, filtered, and concentrated to
give a crude product. Recrystallization from n-hexane/
ethyl acetate gave 3.75 g of the title compound, with a
147–150 ꢀC, H NMR (DMSO-d6): d (ppm) 1.35 (s, 3H,
1
Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.83 (s, 3H,
benzoyl-CH3), 2.26 (s, 3H, benzylamine-CH3), 2.6–2.8
(m, 2H, Apns-4-CH2), 3.70 (s, 3H, Apns-OCH3), 4.09
(dd, 1H, J=5.1 Hz, 15.1 Hz, benzylamine-CH2), 4.3–4.5
(m, 4H, benzylamine-CH2, Dmt-4-CH, Apns-2-CH, and
Apns-3-CH), 5.01 (d, 1H, J=9.5 Hz, Dmt-2-CH2), 5.13
(d, 1H, J=8.6 Hz, Dmt-2-CH2), 5.45 (d, 1H, J=6.8 Hz,
Apns-2-OH), 6.58 (d, 1H, J=7.0 Hz, aromatic), 6.7–6.9
(m, 3H, aromatic), 6.96 (t, 1H, J=7.8 Hz, aromatic),
7.0–7.4 (m, 6H, aromatic), 8.11 (d, 1H, J=8.1 Hz, Apns-
NH), 8.32(t, 1H, J=5.4 Hz, benzylamine-NH), 9.40 (s,
1H, benzoyl-OH). Anal. calcd for C33H39N3O6: C, 65.43;
H, 6.49; N, 6.94; Found C, 65.28; H, 6.52; N, 6.88.
yield of 71%: mp 77–79 ꢀC, H NMR (DMSO-d6): d
1
(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.
6.1.4. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-methoxyphenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10a). To a solution of com-
pound 9a (0.97 g, 9.08 mmol), (2S,3S)-3-(N-tert-butoxy-
carbonyl)amino-2-hydroxy-4-(4-methoxyphenyl)butyric
acid (2.95 g, 9.08 mmol), and 1-hydroxybenztriazole
(1.23 g, 9.08 mmol) in ethyl acetate (50 mL), N,N0-dicy-
clohexylcarbodiimide (2.15 g, 10.44 mmol) was added,
and the mixture was stirred overnight. The reaction
mixture was 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 stirred for 1 h
with 4 N HCl in dioxane (10 mL). The reaction mixture
was concentrated under reduced pressure and then
redissolved in water. The aqueous phase was filtered,
and the filtrate was neutralized with 2N NaOH to give a
precipitate. The crude product was recrystallized from
ethyl acetate–n-hexane to provide 4.28 g of the title
compound, in 88% yield: 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=13.2Hz), 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–
6.1.6. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(3-methoxyphenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10b). 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), 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.
6.1.7. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(3-methoxyphenyl)-
butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(12b). Mp 132–135 ꢀC, H NMR (DMSO-d6): d (ppm);
1
1.35 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.86
(s, 3H, benzoyl-CH3), 2.26 (s, 3H, benzylamine-CH3),
2.6–2.8 (m, 2H, Apns-4-CH2), 3.68 (s, 3H, Apns-
OCH3), 4.12(dd, 1H, J=4.3 Hz, 15.1 Hz, benzylamine-
CH2), 4.3–4.5 (m, 4H, benzylamine-CH2, Dmt-4-CH,
Apns-2-CH, and Apns-3-CH), 5.01 (d, 1H, J=9.2Hz,

288
T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
Dmt-2-CH2), 5.15 (d, 1H, J=8.6 Hz, Dmt-2-CH2), 5.41
(d, 1H, J=6.8 Hz, Apns-2-OH), 6.57 (d, 1H, J=7.3 Hz,
aromatic), 6.7–6.8 (m, 2H, aromatic), 6.9–7.0 (m, 3H,
aromatic), 7.1–7.2(m, 4H, aromatic), 7.3–7.4 (m, 1H,
aromatic), 8.18 (d, 1H, J=8.1 Hz, Apns-NH), 8.36 (t,
1H, J=5.4 Hz, benzylamine-NH), 9.40 (s, 1H, benzoyl-
OH). Anal. calcd for C33H39N3O6S 0.5EtOAc: C,
64.69; H, 6.67; N, 6.47; Found C, 64.71; H, 6.62; N,
6.81.
2H, OCH2–CH2–CH3), 1.86 (s, 3H, benzoyl-CH3), 2.26
(s, 3H, benzylamine-CH3), 2.6–2.8 (m, 2H, Apns-4-
CH2), 3.87 (t, 2H, J=6.3 Hz, Apns-OCH2), 4.14 (dd,
1H, J=4.3 Hz, 15.1 Hz, benzylamine-CH2), 4.3–4.5 (m,
4H, benzylamine-CH2, Dmt-4-CH, Apns-2-CH, and
Apns-3-CH), 5.00 (d, 1H, J=8.9 Hz, Dmt-2-CH2), 5.13
(d, 1H, J=9.2Hz, Dmt-2-CH ), 5.44 (d, 1H, J=7.0 Hz,
2
Apns-2-OH), 6.58 (d, 1H, J=7.6 Hz, aromatic), 6.7–6.8
(m, 3H, aromatic), 6.96 (t, 1H, J=7.8 Hz, aromatic),
7.1–7.2 (br, 3H, aromatic), 7.26 (d, 2H, J=8.9 Hz, aro-
matic), 7.25–7.35 (m, 1H, aromatic), 8.09 (d, 1H,
J=8.4 Hz, Apns-NH), 8.31 (br, 1H, benzylamine-NH),
9.39 (s, 1H, benzoyl-OH). Anal. calcd for C35H43N3O6S
0.5 EtOAc: C, 65.56; H, 6.99; N, 6.20; Found C, 65.89;
H, 7.05; N, 6.49.
6.1.8. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-ethoxyphenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10c). 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=13.2Hz), 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.
6.1.12. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-isopropoxyphenyl)butanoyl]-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (10e). Mp 207–209 ꢀC,
1H NMR (DMSO-d6): d (ppm) 1.1–1.2 (br, 2H), 1.26 (d,
6H, J=5.9 Hz), 1.33 (s, 3H), 1.52(s, 3H), 2.17 (s, 3H),
2.2–2.3 (m, 1H), 2.6–2.7 (m, 1H), 3.00 (d, 1H,
J=13.2Hz), 4.0–4.1 (m, 1H), 4.1–4.3 (m, 2H), 4.35 (s,
1H), 4.5–4.6 (m, 1H), 4.90 (s, 2H), 5.28 (d, 1H,
J=7.8 Hz), 6.83 (d, 2H, J=8.6 Hz), 6.9–7.1 (m, 5H),
7.1–7.2(m, 1H), 8.53 (t, 1H, J=5.0 Hz). Anal. calcd for
C27H37N3O4S: C, 64.90; H, 7.46; N, 8.41; Found C,
64.70; H, 7.58; N, 8.30.
6.1.9. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-ethoxyphenyl)-
butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
ꢀ
1
(12c). Mp 137–139 C, H NMR (DMSO-d6): d (ppm)
1.29 (t, 3H, J=7.0 Hz, OEt–CH3), 1.35 (s, 3H, Dmt-5-
CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.86 (s, 3H, benzoyl-
CH3), 2.26 (s, 3H, benzylamine-CH3), 2.6–2.8 (m, 2H,
Apns-4-CH2), 3.96 (q, 2H, J=6.9 Hz, Apns-OCH2),
4.14 (dd, 1H, J=4.3 Hz, 15.1 Hz, benzylamine-CH2),
4.2–4.5 (m, 4H, benzylamine-CH2, Dmt-4-CH, Apns-2-
CH, and Apns-3-CH), 5.00 (d, 1H, J=9.5 Hz, Dmt-2-
6.1.13. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-i-propoxyphe-
nyl)butanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 -carbox-
amide (12e). Mp 133–137 ꢀC, H NMR (DMSO-d6): d
1
CH2), 5.13 (d, 1H, J=9.2Hz, Dmt-2-CH ), 5.43 (d, 1H,
2
(ppm) 1.22 (s, 3H, iPr-CH3), 1.24 (s, 3H, iPr-CH3), 1.35
(s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.84 (s,
3H, benzoyl-CH3), 2.26 (s, 3H, benzylamine-CH3),
2.6–2.8 (m, 2H, Apns-4-CH2), 4.14 (dd, 1H, J=4.3 Hz,
15.1Hz, benzylamine-CH2), 4.3–4.6 (m, 5H, benzylamine-
CH2, Dmt-4-CH, Apns-2-CH, Apns-OCH, and Apns-3-
CH), 5.01 (d, 1H, J=8.9Hz, Dmt-2-CH2), 5.14 (d, 1H,
J=9.2Hz, Dmt-2-CH 2), 5.44 (d, 1H, J=6.8 Hz, Apns-2-
OH), 6.57 (d, 1H, J=7.6 Hz, aromatic), 6.75–6.79 (m,
3H, aromatic), 6.95 (t, 1H, J=7.8 Hz), 7.10–7.14 (m,
3H, aromatic), 7.20 (d, 2H, J=8.1 Hz, aromatic), 7.28–
7.31 (m, 1H, aromatic), 8.09 (d, 1H, J=8.4 Hz, Apns-
NH), 8.31 (bt, 1H, benzylamine-NH), 9.34 (s, 1H, ben-
zoyl-OH). Anal. calcd for C35H43N3O6S 0.5 EtOAc: C,
65.56; H, 6.99; N, 6.20; Found C, 65.41; H, 6.90; N,
6.47.
J=7.0 Hz, Apns-2-OH), 6.57 (d, 1H, J=6.8 Hz, aro-
matic), 6.78 (d, 2H, J=8.6 Hz, aromatic), 6.96 (t, 1H,
J=7.6 Hz, aromatic), 7.1–7.2(m, 3H, aromatic), 7.2
(d, 2H, J=8.4 Hz, aromatic), 7.3–7.4 (m, 1H, aromatic),
8.09 (d, 1H, J=8.4 Hz, Apns-NH), 8.3 (br, 1H, benzyl-
amine-NH), 9.39 (s, 1H, benzoyl-OH). Anal. calcd for
C34H41N3O6S: C, 65.89; H, 6.67; N, 6.78; Found C,
65.62; H, 7.00; N, 6.74.
6.1.10. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-n-propoxyphenyl)butanoyl]-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (10d). Mp 203–205 ꢀC,
1H NMR (DMSO-d6): d (ppm) 0.98 (t, 3H, J=7.3 Hz),
1.2–1.3 (br, 2H), 1.33 (s, 3H), 1.52 (s, 3H), 1.73 (q, 2H,
J=6.9 Hz), 2.17 (s, 3H), 2.1–2.3 (m, 1H), 2.6–2.7 (m,
1H), 3.01 (d, 1H, J=13.2Hz), 3.91 (t, 2H, J=6.5 Hz),
4.02–4.08 (m, 1H), 4.1–4.3 (m, 2H), 4.35 (s, 1H), 4.90
(s, 2H), 5.29 (d, 1H, J=7.8 Hz), 6.84 (d, 3H,
J=8.6 Hz), 6.94–7.07 (m, 4H), 7.11–7.16 (m, 2H), 8.55
(t, 1H, J=7.8 Hz). Anal. calcd for C27H37N3O4S: C,
64.90; H, 7.46; N, 8.41; Found C, 64.67; H, 7.64; N,
8.47.
6.1.14. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(3,4-methylenedioxyphenyl)butanoyl]-5,5-di-
methyl-1,3-thiazolidine-4-carboxamide (10f). 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.
6.1.11. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-n-propoxyphe-
nyl)butanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 - carbox-
amide (12d). Mp 132–135 ꢀC, H NMR (DMSO-d6): d
1
(ppm) 0.96 (t, 3H, J=7.3 Hz, OnPr-CH3), 1.35 (s, 3H,
Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.66–1.73 (m,

T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
289
6.1.15. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(3,4-methylenedioxy-
phenyl)butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxa-
mide (12f). Mp 133–137 ꢀC, 1H NMR (DMSO-d6): d
(ppm) 1.35 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-
CH3), 1.87 (s, 3H, benzoyl-CH3), 2.27 (s, 3H, benzyl-
amine-CH3), 2.6–2.8 (m, 2H, Apns-4-CH2), 4.12(dd,
1H, J=4.3 Hz, 15.1 Hz, benzylamine-CH2), 4.3–4.6 (m,
4H, benzylamine-CH2, Dmt-4-CH, Apns-2-CH, and
tanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 - carboxamide
(12h). Mp 179–181 ꢀC, H NMR (DMSO-d6): d (ppm)
1
1.35 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.87
(s, 3H, benzoyl- CH3), 2.24 (s, 3H, Apns-CH3), 2.26 (s,
3H, benzylamine-CH3), 2.65–2.85 (m, 2H, Apns-4-
CH2), 4.11 (dd, 1H, J=4.3 Hz, 15.1 Hz, benzylamine-
CH2), 4.3–4.5 (m, 4H, benzylamine-CH2, Dmt-4-CH,
Apns-2-CH, and Apns-3-CH), 5.01 (d, 1H, J=9.2Hz,
Dmt-2-CH2), 5.13 (d, 1H, J=9.2Hz, Dmt-2-CH ), 5.49
2
Apns-3-CH), 5.00 (d, 1H, J=9.2Hz, Dmt-2-CH ), 5.15
2
(d, 1H, J=9.2Hz, Dmt-2-CH ), 5.37 (d, 1H, J=6.5 Hz,
2
(d, 1H, J=6.8 Hz, Apns-2-OH), 6.57 (d, 1H, J=6.8 Hz,
aromatic), 6.79 (d, 1H, J=7.3 Hz, aromatic), 6.96 (t,
2H, J=7.6 Hz, aromatic), 7.0–7.2(m, 6H, aromatic),
7.29–7.30 (m, 1H, aromatic), 8.13 (d, 1H, J=8.4 Hz,
Apns-NH), 8.32(bt, 1H, benzylamine-NH), 9.40 (s, 1H,
benzoyl-OH). Anal. calcd for C33H39N3O5S 0.5 EtOAc:
C, 66.33; H, 6.84; N, 6.63; Found C, 66.52; H, 6.73; N,
6.82.
Apns-2-OH), 5.93 (s, 2H, Apns-O-C H2–), 6.59 (d, 1H,
J=7.6 Hz, aromatic), 6.7–6.9 (m, 3H, aromatic), 6.9–7.0
(m, 2H, aromatic), 7.1–7.2 (m, 3H, aromatic), 7.3–7.4
(m, 1H, aromatic), 8.14 (d, 1H, J=8.9 Hz, Apns-NH),
8.35 (t, 1H, J=5.4 Hz), 9.40 (s, 1H, benzoyl-OH). Anal.
calcd for C33H37N3O7S 0.5 EtOAc: C, 63.33; H, 6.23; N,
6.33; Found C, 63.06; H, 5.99; N, 6.68.
6.1.20. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-chlorophenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10i). Mp 201–203 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.2–1.4 (br, 2H), 1.33 (s,
3H), 1.52 (s, 3H), 2.17 (s, 3H), 2.28 (t, 1H, J=11.6 Hz),
2.65 (t, 1H, J=8.2Hz), 3.0–3.1 (br, 1H), 4.0–4.1 (m,
1H), 4.1–4.3 (m, 2H), 4.36 (s, 1H), 4.90 (s, 2H), 5.33 (d,
1H, J=7.8 Hz), 6.98 (s, 2H), 7.16 (d, 2H, J=5.9 Hz),
7.1–7.3 (br, 1H), 7.35 (d, 2H, J=8.1 Hz), 8.51 (t, 1H,
J=5.1 Hz). Anal. calcd for C24H30ClN3O3S: C, 60.55;
H, 6.35; N, 8.83; Found C, 60.20; H, 6.46; N, 8.75.
6.1.16. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-methylphenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10g). Mp 207–209 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.1–1.3 (br, 2H), 1.33 (s,
3H), 1.52 (s, 3H), 2.16 (s, 3H), 2.2–2.3 (m, 1H), 2.29 (s,
3H), 2.6–2.7 (br, 1H), 3.04 (d, 1H, J=12.7 Hz), 4.0–4.1
(m, 1H), 4.1–4.3 (m, 2H), 4.35 (s, 1H), 4.90 (s, 2H),
5.92–5.32(br, 1H), 6.93–7.12(m, 8H), 8.54 (t, 1H,
J=5.1 Hz). Anal. calcd for C25H33N3O3S: C, 65.90; H,
7.30; N, 9.22; Found C, 65.77; H, 7.48; N, 9.41.
6.1.17. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-methylphenyl)-
butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
6.1.21. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-chlorophenyl)bu-
tanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 - carboxamide
(12g). Mp 142–146 ꢀC, H NMR (DMSO-d6): d (ppm)
(12i). Mp 143–146 ꢀC, H NMR (DMSO-d6): d (ppm)
1
1
1.35 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.86
(s, 3H, benzoyl-CH3), 2.25 (s, 3H, Apns-CH3), 2.26 (s,
3H, benzylamine-CH3), 2.6–2.9 (m, 2H, Apns-4-CH2),
4.14 (dd, 1H, J=4.3 Hz, 15.1 Hz, benzylamine-CH2),
4.3–4.5 (m, 4H, benzylamine-CH2, Dmt-4-CH, Apns-2-
CH, and Apns-3-CH), 5.00 (d, 1H, J=9.5 Hz, Dmt-2-
1.35 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.82
(s, 3H, benzoyl- CH3), 2.26 (s, 3H, benzylamine-CH3),
2.7–2.9 (m, 2H, Apns-4-CH2), 4.14 (dd, 1H, J=4.3 Hz,
15.1 Hz, benzylamine-CH2), 4.3–4.5 (m, 4H, benzyl-
amine-CH2, Dmt-4-CH, Apns-2-CH, and Apns-3-CH),
5.00 (d, 1H, J=9.5 Hz, Dmt-2-CH2), 5.15 (d, 1H,
CH2), 5.12(d, 1H, J=9.2Hz, Dmt-2-CH ), 5.47 (d, 1H,
2
J=9.2Hz, Dmt-2-CH ), 5.47 (d, 1H, J=6.5 Hz, Apns-
2
J=6.2Hz, Apns-2-OH), 6.57 (d, 1H, J=7.3 Hz, aro-
matic), 6.78 (d, 1H, J=7.8 Hz, aromatic), 6.96 (t, 1H,
J=7.8 Hz), 7.0–7.2 (m, 5H, aromatic), 7.20 (d, 2H,
J=7.6 Hz, aromatic), 7.28–7.31 (m, 1H, aromatic), 8.08
(d, 1H, J=7.8 Hz, Apns-NH), 8.30 (br, 1H, benzyl-
amine-NH), 9.39 (s, 1H, benzoyl-OH). Anal. calcd for
C33H39N3O5S 0.5 EtOAc: C, 66.33; H, 6.84; N, 6.63;
Found C, 66.67; H, 6.93; N, 6.96.
2-OH), 6.57 (d, 1H, J=7.3 Hz, aromatic), 6.78 (d, 1H,
J=7.8 Hz, aromatic), 6.96 (t, 1H, J=7.8 Hz, aromatic),
7.0–7.2(m, 3H, aromatic), 7.2–7.4 (m, 5H, aromatic),
8.17 (d, 1H, J=8.6 Hz, Apns-NH), 8.34 (bt, 1H, benzyl-
amine-NH), 9.40 (s, 1H, benzoyl-OH). Anal. calcd for
C32H36ClN3O5S: C, 62.99; H, 5.95; N, 6.89; Found C,
62.74; H, 6.17; N, 6.65.
6.1.22. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(3-chlorophenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10j). Mp 161–163 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.2–1.4 (br, 2H), 1.34 (s,
3H), 1.52 (s, 3H), 2.17 (s, 3H), 2.23–2.32 (m, 1H), 2.66 (t,
1H, J=8.2Hz), 3.06 (d, 1H, J=11.1 Hz), 4.0–4.1 (m, 1H),
4.1–4.3 (m, 2H), 4.36 (s, 1H), 4.90 (s, 2H), 5.32 (d, 1H,
J=8.1Hz), 6.96 (s, 2H), 7.09–7.36 (m, 6H), 8.48 (t, 1H,
J=5.1Hz). Anal. calcd for C24H30ClN3O3S: C, 60.55; H,
6.35; N, 8.83;; Found C, 59.72; H, 6.48; N, 8.72.
6.1.18. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(3-methylphenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10h). Mp 173–175 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.1–1.3 (br, 2H), 1.33 (s,
3H), 1.52 (s, 3H), 2.16 (s, 3H), 2.2–2.3 (m, 1H), 2.29 (s,
3H), 2.6–2.7 (br, 1H), 3.06 (d, 1H, J=12.9 Hz), 4.0–4.1
(m, 1H), 4.1–4.3 (m, 2H), 4.36 (s, 1H), 4.91 (s, 2H), 5.3–
5.4 (br, 1H), 6.91–7.04 (m, 5H), 7.13–7.20 (m, 3H), 8.54
(t, 1H, J=5.1 Hz). Anal. calcd for C25H33N3O3S: C,
65.90; H, 7.30; N, 9.22; Found C, 65.67; H, 7.42; N, 9.39.
6.1.23. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(3-chlorophenyl)bu-
tanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 - carboxamide
6.1.19. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(3-methyphenyl)bu-

290
T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
(12j). Mp 201–202 ꢀC, H NMR (DMSO-d6): d (ppm)
1.35 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.83
(s, 3H, benzoyl-CH3), 2.27 (s, 3H, benzylamine-CH3),
2.67–2.85 (m, 2H, Apns-4-CH2), 4.16 (dd, 1H,
J=4.6 Hz, 15.1 Hz, benzylamine-CH2), 4.3–4.6 (m, 4H,
benzylamine-CH2, Dmt-4-CH, Apns-2-CH, and Apns-3-
CH), 5.00 (d, 1H, J=8.9 Hz, Dmt-2-CH2), 5.17 (d, 1H,
J=8.9 Hz, Dmt-2-CH2), 5.44 (d, 1H, J=6.8 Hz, Apns-2-
OH), 6.58 (d, 1H, J=7.0 Hz, aromatic), 6.79 (d, 1H,
J=7.6 Hz, aromatic), 6.97 (t, 2H, J=7.7 Hz, aromatic),
7.0–7.2(m, 3H, aromatic), 7.20–7.33 (m, 4H, aromatic),
7.40 (s, 1H, aromatic), 8.24 (d, 1H, J=8.4 Hz, Apns-
NH), 8.38 (bt, 1H, benzylamine-NH), 9.41 (s, 1H, ben-
zoyl-OH). Anal. calcd for C32H36ClN3O5S: C, 62.99; H,
5.95; N, 6.89; Found C, 62.86; H, 5.99; N, 6.80.
(s, 3H, benzoyl-CH3), 2.26 (s, 3H, benzylamine-CH3),
2.66–2.84 (m, 2H, Apns-4-CH2), 4.12(dd, 1H, J=4.3 Hz,
15.1 Hz, benzylamine-CH2), 4.3–4.6 (m, 4H, Apns-3-
CH, benzylamine-CH2, Dmt-4-CH, and Apns-2-CH),
5.01 (d, 1H, J=8.9 Hz, Dmt-2-CH2), 5.16 (d, 1H,
J=8.9 Hz, Dmt-2-CH2), 5.44 (d, 1H, J=6.8 Hz, Apns-2-
OH), 6.56 (d, 1H, J=7.0 Hz, aromatic), 6.78 (d, 1H,
J=7.6 Hz, aromatic), 6.96 (t, 1H, J=7.7 Hz, aromatic),
7.0–7.2(m, 6H, aromatic), 7.2–7.4 (m, 3H, aromatic),
8.18 (d, 1H, J=8.4 Hz, Apns-NH), 8.35 (t, 1H,
J=5.4 Hz, benzylamine-NH), 9.40 (s, 1H, benzoyl-OH).
Anal. calcd for C32H36FN3O5S 0.5 EtOAc: C, 64.03; H,
6.32; N, 6.59; Found C, 64.30; H, 6.13; N, 6.99.
1
6.1.28. (R)-N-(2,6-Dimethylbenzyl)-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (9b). To a solution of (R)-N-
tert-butoxycarbonyl-5,5-dimethyl-1,3-thiazolidine-4-
carboxylic acid (15.7 g, 60 mmol) and triethylamine
(8.76 mL, 63 mmol) in ethyl acetate (230 mL), diphenyl-
phosphoryl chloridate (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-methylbenzylamine hydro-
chloride (12.1 g, 63 mmol), and triethylamine (18.3 mL,
132mmol) were added in an ice-bath, followed by over-
night stirring. The reaction mixture was then washed
sequentially with 1 N HCl, 3% K2CO3 and brine; dried
over MgSO4: filtered: and concentrated. The residue
was redissolved in CH2Cl2 (100 mL), to which was
added 4 N HCl in dioxane (100 mL); and this mixture
was stirred for 2h. After the reaction mixture had been
concentrated, the residue was redissolved in CH2Cl2–
H2O, and neutralized with 2N NaOH. The organic
phase was washed with brine, dried over MgSO4, fil-
tered, and concentrated to give a crude product.
Recrystallization from n-hexane gave 15.7ꢀg of the title
compound, with a yield of 94%: mp 97–99 C, 1H 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.
6.1.24. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(4-fluorophenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10k). Mp 197–199 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.1–1.3 (br, 2H), 1.33 (s, 3H),
1.52 (s, 3H), 2.17 (s, 3H), 2.2–2.3 (m, 1H), 2.6–2.7 (m,
1H), 3.05 (d, 1H, J=11.1 Hz), 4.0–4.1 (m, 1H), 4.1–4.3
(m, 2H), 4.36 (s, 1H), 4.90 (s, 2H), 5.31 (d, 1H,
J=8.1 Hz), 6.95–6.99 (m, 3H), 7.07–7.20 (m, 5H), 8.52 (t,
1H, J=5.0 Hz). Anal. calcd for C24H30FN3O3S: C,
62.72; H, 6.58; N, 9.14; Found C, 62.47; H, 6.80; N, 9.18.
6.1.25. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(4-fluorophenyl)bu-
tanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 - carboxamide
(12k). Mp 190–195 ꢀC, H NMR (DMSO-d6): d (ppm)
1
1.36 (s, 3H, Dmt-5-CH3), 1.51 (s, 3H, Dmt-5-CH3), 1.82
(s, 3H, benzoyl-CH3), 2.27 (s, 3H, benzylamine-CH3),
2.68–2.86 (m, 2H, Apns-4-CH2), 4.14 (dd, 1H,
J=4.9 Hz, 14.9 Hz, benzylamine-CH2), 4.3–4.5 (m, 4H,
Apns-3-CH, benzylamine-CH2, Dmt-4-CH, and Apns-
2-CH), 5.01 (d, 1H, J=8.9 Hz, Dmt-2-CH2), 5.17 (d,
1H, J=8.9 Hz, Dmt-2-CH2), 5.43 (d, 1H, J=7.0 Hz,
Apns-2-OH), 6.56 (d, 1H, J=7.3 Hz, aromatic), 6.79 (d,
1H, J=8.1 Hz, aromatic), 6.94–7.01 (m, 2H, aromatic),
7.09–7.18 (m, 5H, aromatic), 7.23–7.32 (m, 3H, aro-
matic), 8.23 (d, 1H, J=8.4 Hz, Apns-NH), 8.38 (t, 1H,
J=5.5 Hz, benzylamine-NH), 9.40 (s, 1H, benzoyl-OH).
Anal. calcd for C32H36FN3O5S: C, 64.74; H, 6.11; N,
7.08; Found C, 64.58; H, 6.12;N, 7.21.
6.1.29. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-amino-
2-hydroxy-4-(4-methoxyphenyl)butanoyl]-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (10m). To a solution of
compound 9b (153 mg, 0.55 mmol), (2S,3S)-3-(N-tert-
butoxycarbonyl)amino - 2- hydroxy - 4 - (4 - methoxy-
phenyl)butyric acid (163 mg, 0.50 mmol), and 1-hydroxy-
benztriazole (68 mg, 0.50 mmol) in ethyl acetate (5 mL),
N,N0-dicyclohexylcarbodiimide (113 mg, 0.55 mmol)
was added, followed by stirring overnight. The reaction
mixture was filtered, and the filtrate was washed
sequentially with 3% K2CO3, 1 N HCl, and brine. Then
it was dried over MgSO4, filtered, and concentrated.
The residue was redissolved in ethyl acetate (5 mL), and
subsequently stirred for 1 h with 4 N HCl in ethyl ace-
tate (5 mL). The reaction mixture was concentrated
under reduced pressure and then redissolved in water.
The aqueous phase was filtered, and the filtrate was
neutralized with 2N NaOH to give a precipitate. The
crude product was recrystallized from ethyl acetate,
giving 129 mg of the title compound, in 53% yield: mp
6.1.26. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-amino-2-
hydroxy-4-(3-fluorophenyl)butanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (10l). Mp 187–189 ꢀC, 1H
NMR (DMSO-d6): d (ppm) 1.2–1.3 (br, 2H), 1.34 (s,
3H), 1.52 (s, 3H), 2.17 (s, 3H), 2.2–2.3 (m, 1H), 2.6–2.7
(m, 1H), 3.07 (d, 1H, J=10.5 Hz), 4.0–4.1 (m, 1H),
4.1–4.3 (m, 2H), 4.37 (s, 1H), 4.90 (s, 2H), 5.32 (d, 1H,
J=8.4 Hz), 6.94–7.06 (m, 6H), 7.1–7.2(m, 1H),
7.29–7.37 (m, 1H), 8.50 (t, 1H, J=5.1 Hz). Anal. calcd
for C24H30FN3O3S: C, 62.72; H, 6.58; N, 9.14;; Found
C, 62.47; H, 6.67; N, 9.04.
6.1.27. (R)-N-(2-Methylbenzyl)-3-[(2S,3S)-2-hydroxy-3-
(3-hydroxy-2-methylbenzoyl)amino-4-(3-fluorophenyl)bu-
tanoyl] - 5,5 - dimethyl - 1,3 - thiazolidine - 4 - carboxamide
ꢀ
1
(12l). Mp 171–173 C, H NMR (DMSO-d6): d (ppm)
1.36 (s, 3H, Dmt-5-CH3), 1.50 (s, 3H, Dmt-5-CH3), 1.82
199–201 ꢀC, H NMR (DMSO-d6): d (ppm) 0.6–0.8 (br,
1

T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
291
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; Found C, 62.00; H, 7.40; N, 8.34.
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=3.2Hz), 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.
6.1.30. (R) - N - (2,6 - Dimethylbenzyl) - 3 - [(2S,3S) - 2 -
hydroxy-3-(3-hydroxy-2-methylbenzoyl)amino-4-(4-meth-
oxyphenyl)butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-car-
boxamide (12m). Mp 218–220 ꢀC, 1H NMR (DMSO-
d6): d (ppm) 1.35 (s, 3H, Dmt-5-CH3), 1.45 (s, 3H, Dmt-
5-CH3), 1.84 (s, 3H, benzoyl-CH3), 2.31 (s, 6H, benzyl-
amine-CH3), 2.6–2.8 (m, 2H, Apns-4-CH2), 3.72(s, 3H,
Apns-OCH3), 4.17 (dd, 1H, J=2.7 Hz, 13.2 Hz, benzyl-
amine-CH2), 4.2–4.4 (m, 1H, Apns-3-CH), 4.43–4.54
(m, 3H, benzylamine-CH2, Dmt-4-CH, and Apns-2-
CH), 4.98 (d, 1H, J=8.9 Hz, Dmt-2-CH2), 5.17 (d, 1H,
6.1.34. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-2-hydroxy
-3-(3-hydroxy-2-methylbenzoyl)amino-4-(3,4-methylene-
dioxyphenyl)butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (12o). Mp 201–203 ꢀC, H NMR (DMSO-
1
d6): d (ppm) 1.36 (s, 3H, Dmt-5-CH3), 1.46 (s, 3H, Dmt-
5-CH3), 1.86 (s, 3H, benzoyl-CH3), 2.31 (s, 6H, benzyl-
amine-CH3), 2.56–2.73 (m, 2H, Apns-4-CH2), 4.18 (dd,
1H, J=2.7 Hz, 13.8 Hz, benzylamine-CH2), 4.2–4.3 (m,
1H, Apns-2-CH), 4.45–4.54 (m, 3H, benzylamine-CH2,
Dmt-4-CH, and Apns-3-CH), 4.98 (d, 1H, J=9.2Hz,
Dmt-2-CH2), 5.17–5.24 (m, 2H, Dmt-2-CH2, Apns-2-
OH), 5.94 (s, 2H, O-CH2-O), 6.59 (d, 1H, J=6.8 Hz,
aromatic), 6.81 (t, 3H, J=8.1 Hz, aromatic), 6.86–7.11
(m, 6H, aromatic), 8.09–8.13 (br, 1H, benzylamine-
NH), 8.23 (d, 1H, J=8.6 Hz, Apns-NH), 9.41 (s, 1H,
benzoyl-OH). Anal. calcd for C34H39N3O7S 0.5 EtOAc:
C, 63.79; H, 6.39; N, 6.20; Found C, 63.70; H, 6.23; N,
6.53.
J=9.2Hz, Dmt-2-CH ), 5.28 (d, 1H, J=7.0 Hz, Apns-2-
2
OH), 6.58 (d, 1H, J=6.8 Hz, aromatic), 6.81 (t, 3H,
J=9.3 Hz, aromatic), 6.93–7.13 (m, 4H, aromatic), 7.30
(d, 1H, J=8.6 Hz, aromatic), 8.05–8.11 (br, 1H, benzyl-
amine-NH), 8.21 (d, 1H, J=8.4 Hz, Apns-NH), 9.39 (s,
1H, benzoyl-OH). Anal. calcd for C34H41N3O6S: C,
65.89; H, 6.67; N, 6.78; Found C, 65.49; H, 6.74; N, 6.75.
6.1.31. (R)-N-(2,6-Dimethylbenzyl)-3-[(2S,3S)-3-amino-
2-hydroxy-4-(3-methoxyphenyl)butanoyl]-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (10n). Mp 185–187 ꢀC,
1H 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=3.2Hz), 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.
6.2. In vitro metabolism experiment
Metabolic stability was determined by using liver
microsomes isolated from humans, rats or dogs. The
reaction mixture contained 0.1 M phosphate buffer (pH
7.4), 5 mM test compound, 5 mM MgCl2, 1 mM
NADPH and/or 5 mM UDPGA, and 0.5 mg/mL
microsomes in the presence or absence of ritonavir (0.5
or 2.5 mM) in a total volume of 0.2mL. The incubation
was initiated by the addition of microsomes and was
performed for 30 min at 37 ꢀC. The reaction was termi-
nated by adding 0.4 mL of ice-cold methanol, and the
reaction mixture was then centrifuged at 12,000 rpm for
10 min at 4 ꢀC. The supernatant was filtered through a
PTFE filter, and the residual amount of the test com-
pound in the supernatant was determined by LC/UV
analysis. The mean percentage (%) of the test com-
pound was remaining calculated from the data of
duplicate experiments.
6.1.32. (R) - N - (2,6 - Dimethylbenzyl) - 3 - [(2S,3S) - 2 -
hydroxy-3-(3-hydroxy-2-methylbenzoyl)amino-4-(3-meth-
oxyphenyl)butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-car-
boxamide (12n). Mp 200–202 ꢀC, H NMR (DMSO-d6):
1
d (ppm) 1.35 (s, 3H, Dmt-5-CH3), 1.46 (s, 3H, Dmt-5-
CH3), 1.85 (s, 3H, benzoyl-CH3), 2.31 (s, 6H, benzyla-
mine-CH3), 2.6–2.8 (m, 2H, Apns-4-CH2), 3.75 (s, 3H,
Apns-OCH3), 4.18 (dd, 1H, J=3.5 Hz, 14.0 Hz, benzyl-
amine-CH2), 4.2–4.4 (m, 1H, Apns-3-CH), 4.42–4.55 (m,
3H, benzylamine-CH2, Dmt-4-CH, and Apns-2-CH),
4.99 (d, 1H, J=8.6 Hz, Dmt-2-CH2), 5.18 (d, 1H,
J=8.6 Hz, Dmt-2-CH2), 5.27 (d, 1H, J=7.0 Hz, Apns-2-
OH), 6.59 (d, 1H, J=6.8 Hz, aromatic), 6.72–6.80 (m,
2H, aromatic), 6.93–7.11 (m, 6H, aromatic), 7.16 (t, 1H,
J=7.8 Hz, aromatic), 8.08–8.12(br, 1H, benzylamine-
NH), 8.25 (d, 1H, J=8.4 Hz, Apns-NH), 9.39 (s, 1H,
benzoyl-OH). Anal. calcd for C34H41N3O6S H2O: C,
64.03; H, 6.80; N, 6.59; Found C, 64.19; H, 6.50; N, 6.38.
6.3. Pharmacokinetics
Pharmacokinetics parameters of the drugs were studied
in dogs. The pharmacokinetic behavior of 12n was
evaluated following a single oral and intravenous dose
to three male beagle dogs (7–10 kg, HRP, Inc., Cum-
berland, USA). Compound 12n dissolved in 50% PEG
was administered intravenously via the cepatic vein as a
bolus injection at the dose of 25 mg/kg, and the same
dose was administered orally by gavage. Heparinized
blood samples were collected from a bolus via the
cepatic vein at designated times after the dosing. Two
hundred microliters of plasma was obtained by
immediate centrifugation and kept frozen at À80 ꢀC
until analyzed. The plasma sample was vortexed with
4 mL of tert-buthylmethylether, and centrifuged at
6.1.33. (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 (10o). 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,

292
T. Mimoto et al. / Bioorg. Med. Chem. 12 (2004) 281–293
3500 rpm for 20 min. A portion of the organic layer
obtained (3.6 mL) was evaporated to dryness at 40 ꢀC,
and then reconstituted in 0.3 mL of 50% methanol. Two
hundred microliters of the reconstituted sample was
injected into an HPLC system equipped with a Zorbax
bonus RP column (4.6 Â 150 mm), and the drug was
eluted with a linear gradient from 50 to 74% of aceto-
nitrile in 0.1% trifluoroacetic acid (TFA) at a flow rate
of 1.0 mL/min and monitored with UV detection at
210 nm. The drug concentration was calculated from the
peak area using the calibration curve obtained from
determination of standard plasma samples (The lower
quantification limit was 10 ng/mL, and a maximum
for the range was 10,000 ng/mL). Pharmacokinetic
parameters for inhibitors were estimated by using a
non-compartmental method. Maximum plasma con-
centration (Cmax), and time of maximum plasma
concentration (Tmax) were determined by inspection
of individual subject concentration–time curves, and
the mean area under the plasma concentration–time
curve (AUC) was determined by the linear trapezoi-
pounds were fitted by nonlinear regression analysis to
Eq. 1 with KaleidaGraph software for Macintosh,
where V is the initial velocity with different concentra-
tions of test compounds; V0 is the initial velocity in the
absence of the inhibitor; Km is the Michaelis constant;
and S, Et, and It are the concentrations of substrate,
active enzyme, and inhibitor, respectively:
0
(
)
1=2
ꢂ
ꢀ
ꢁ
ꢃ
ꢀ
ꢁ
2
VO
2Et
ꢀ
S
S
@
V¼
Ki 1þ
þ It À Et þ 4Ki 1þ
Et
Km
Km
ꢂ
ꢁ
ꢃꢁ
S
À Ki 1 þ
þ It À Et
Km
ð1Þ
6.5. Antiviral activity
Evaluation of the in vitro antiviral activities of the test
compounds against a wild-type HIV-1 (strain IIIB) and
drug-resistant HIV containing multi-mutations isolated
from patients were conducted as described previously.18
The virus-infected CEM-SS cells were incubated with
different concentrations of test compounds in the
absence or presence of 50% human serum at 37 ꢀC for 6
days in a 5% CO2 incubator. Then virus-induced cyto-
pathic effects were analyzed by staining with the tetra-
zolium dye XTT. The concentration of test compounds
affording 50% protection from the cytopathic effect of
the virus in infected cells was defined as the 50% effec-
tive concentration (EC50). In the case of the experiments
with drug-resistant HIV, fresh human peripheral blood
lymphocytes (PBMC) were used for the target cells. The
virus infected PBMC were incubated at 37 ꢀC for 7 days
in a 5% CO2 incubator with different concentrations of
test compounds in RPMI 1640 medium containing 15%
fetal bovine serum. Then transcriptase activity derived
from the virus in the cell-free supernatant was measured
by polymerase chain reaction assay. The concentration
of test compounds achieving 50% inhibition of the
transcriptase activity in the cell-free supernatant was
defined as the EC50.
À
Á
dal rule. The apparent plasma half-life t1=2ꢀ was
estimated from the slope of the terminal phase fitted
to the log plasma concentration–time data by the
method of least squares. The apparent distribution
volume (Vdss) of the inhibitor was determined by the
following equation.
Vdss ¼ Dose iv  AUMCð0À1Þ=AUC2 ivð0À1Þ
where AUMC (0–1) is the total area under the first
moment of the drug concentration curve from zero to
infinity. The plasma clearance (CL) was calculated as
the dose divided by the AUC from zero to infinity
[AUC]10 .
6.4. HIV protease inhibition
Recombinant wild-type HIV protease (NY5-type
sequence) was expressed in Escherichia coli and purified
by column chromatography to a single band on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis.
Synthetic peptide substrate [H-Lys-Ala-Arg-Val-Tyr-
Phe(4-NO2)-Glu-Ala-Nle-NH2] was purchased from
Bachem Inc., Switzerland. In the inhibition assay, dif-
ferent concentrations of test compounds dissolved in
dimethylsulfoxide were pre-incubated at 37 ꢀC for 5 min
with HIV-1 proteases in 200 mM 2-[N-morpholi-
no]ethanesulfonic acid (MES-NaOH) buffer, pH 5.5,
containing 1 M NaCl, 2mM dithiothreitol and 2mM
EDTA-2Na. Then the enzymic reactions were initiated
by the addition of 0.1 mM synthetic peptide substrate,
and incubated at 37 ꢀC for 15 min. The reactions were
stopped by the addition of 1 N HCl. The C-terminal
cleavage fragment was separated by reverse-phase
HPLC on a C18 column operated under isocratic con-
ditions (2mL/min, 10% acetonitrile aq containing 0.1%
TFA), detected by fluorometry (excitation at 275 nm;
emission at 305 nm), and quantified by using a synthetic
product standard. The inhibition constant, Ki, was ana-
lyzed by a mathematical model for tight-binding inhibi-
tors.19 Briefly, the initial velocity data on HIV protease
in the presence of different concentrations of test com-
Acknowledgements
We are sincerely grateful to members of Southern
Research Institute for anti-HIV assays. We appreciate
assistance from Mr. Shinji Matsumoto, Mr. Yasuki
Hiyama, and Ms. Rena Sekine for the chemical synth-
esis of HIV protease inhibitors, the evaluation of meta-
bolism, and the determination of HIV-1 protease
activity, respectively.
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