Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC) isostere-based dipeptidomimetics targeting malarial aspartic protease plasmepsin

Article abstract of DOI:10.1016/j.bmc.2008.10.011

Plasmepsin (Plm) is a potential target for new antimalarial drugs, but most reported Plm inhibitors have relatively low antimalarial activities. We synthesized a series of dipeptide-type HIV protease inhibitors, which contain an allophenylnorstatine-dimethylthioproline scaffold to exhibit potent inhibitory activities against Plm II. Their activities against Plasmodium falciparum in the infected erythrocyte assay were largely different from those against the target enzyme. To improve the antimalarial activity of peptidomimetic Plm inhibitors, we attached substituents on a structure of the highly potent Plm inhibitor KNI-10006. Among the derivatives, we identified alkylamino compounds such as 44 (KNI-10283) and 47 (KNI-10538) with more than 15-fold enhanced antimalarial activity, to the sub-micromolar level, maintaining their potent Plm II inhibitory activity and low cytotoxicity. These results suggest that auxiliary substituents on a specific basic group contribute to deliver the inhibitors to the target Plm.

Full text of DOI:10.1016/j.bmc.2008.10.011

Bioorganic & Medicinal Chemistry 16 (2008) 10049–10060
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC)
isostere-based dipeptidomimetics targeting malarial aspartic
protease plasmepsin
Koushi Hidaka a, Tooru Kimura a, Adam J. Ruben b, Tsuyoshi Uemura a, Mami Kamiya a, Aiko Kiso a,
Tetsuya Okamoto a, Yumi Tsuchiya a, Yoshio Hayashi a,c, Ernesto Freire b, Yoshiaki Kiso a,
*
a Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto
607-8412, Japan
b Department of Biology and Johns Hopkins Malaria Research Institute, Johns Hopkins University, Baltimore, MD 21218, USA
c Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Plasmepsin (Plm) is a potential target for new antimalarial drugs, but most reported Plm inhibitors have
relatively low antimalarial activities. We synthesized a series of dipeptide-type HIV protease inhibitors,
which contain an allophenylnorstatine-dimethylthioproline scaffold to exhibit potent inhibitory activi-
ties against Plm II. Their activities against Plasmodium falciparum in the infected erythrocyte assay were
largely different from those against the target enzyme. To improve the antimalarial activity of peptidom-
imetic Plm inhibitors, we attached substituents on a structure of the highly potent Plm inhibitor KNI-
10006. Among the derivatives, we identified alkylamino compounds such as 44 (KNI-10283) and 47
(KNI-10538) with more than 15-fold enhanced antimalarial activity, to the sub-micromolar level, main-
taining their potent Plm II inhibitory activity and low cytotoxicity. These results suggest that auxiliary
substituents on a specific basic group contribute to deliver the inhibitors to the target Plm.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 9 September 2008
Revised 3 October 2008
Accepted 4 October 2008
Available online 10 October 2008
Keywords:
Antimalarial drug
Aspartic protease
Plasmepsin inhibitor
Hydoxymethylcarbonyl
Allophenylnorstatine
Peptidomimetics
1. Introduction
of P. falciparum, ten aspartic proteases called plasmepsins (Plms)
were identified in its genome,5 and four of them participate in
The World Health Organization (WHO) estimated the malaria-
infected population to have reached around 350–500 million peo-
ple in 2004.1 Over one million people lose their lives to the disease
annually, especially children in sub-Saharan Africa. The growth of
malaria’s endemic region by the development of transportation
and global warming make the problem even more severe. The most
lethal malarial parasite, Plasmodium falciparum, is increasingly
building resistance to available drugs such as chloroquine or sulfa-
doxine-pyrimethamine.2 Therefore, new anti-malarial drugs are
needed with different mechanisms of action from those of existing
drugs.3
Malaria parasites invade the human body via the bite of the
Anopheles mosquito; through hematocytic multiplication, the par-
asites proliferate by infecting and multiplying inside erythrocytes.
During their trophozoite stage, parasites obtain amino acids by
hemoglobin digestion, a process essential for their propagation.4
Hemoglobin is transported to the parasite’s acidic food vacuole,
where it is cleaved into small peptides by proteases. In the case
hemoglobin degradation.6 Plm I7,8 and II8,9 are known to start the
cleavage; recent knockout studies suggested a requisite inhibition
of all the four Plms, that is, Plm I, II, III10 (known as histo-aspartic
protease, HAP) and IV11, to prevent proliferation.12–15 Treatment of
mouse malaria using pepstatin A suggested the potential of Plm
inhibitors as antimalarial drugs.16 Several groups reported Plm
inhibitors derived from statine, cathepsin D inhibitor libraries, or
HIV protease inhibitors.17–20 Most of these compounds, however,
possessed low antimalarial activities except aldehyde21 and non-
peptidic22 and primaquine-conjugated compounds,23 highlighting
a difficulty of the development of antimalarial agents. Analysis of
cleaved fragments of hemoglobin has revealed that Plm I and II rec-
ognize Phe33 at the P1 position and Leu34 at the P1 position.24 The
0
Phe-Leu cleavage site is seen also in the HIV protease substrates.25
We previously identified HIV protease inhibitors containing all-
ophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phen-
ylbutyric acid] with a hydroxymethylcarbonyl (HMC) as an ideal
transition state mimic,26,27 which exhibited potent Plm II inhibi-
tory activities.28 Among them, we found some HIV protease inhib-
itors such as KNI-76429–31 (1, Fig. 1) possessing antimalarial
activity in an infected erythrocyte assay. These findings led us to
* Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591 9900.
E-mail address: kiso@mb.kyoto-phu.ac.jp (Y. Kiso).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.011

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K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
S2
S1'
S2
S1'
Apns
OH
Dmt
S
OH
H
N
NH
O
N
O
NH
O
N
O
O
NH
O
OH
S
Dmt
OH
Apns
S1
S2'
S1
S2'
KNI-764 (1) in HIV protease
In Plm IV
Figure 1. Binding directions of 1 in HIV protease and Plm IV.
P1'
S2
S1'
P
2
O
O
O
O
R4
NH
NH
N
H
N
N
H
N
O
O
HO
HO
OH
OH
S
O
S
O
R3
R1
R2
P2'
P1
S1
S2'
KNI-10006 (2) in Plm
Figure 2. Lead compound 2 and its phenoxyacetyl derivatives.
design KNI-10006 (2, Fig. 2) using (1S,2R)-aminoindanol, which
resulted in a remarkable improvement in Plm inhibitory activity.32
Compound 2 exhibited potent inhibitory activity against other
Plms related to hemoglobin degradation. Despite its prominent
molecular potency, however, KNI-10006 exhibited relatively low
prepared without protection from the corresponding nitro deriva-
tives 4n and 4o. Some of the p-substituted 2,6-dimethylphenoxy-
acetic acids were synthesized from 2,6-dimethyl-4-nitrophenol 5,
followed by alkylation and hydrogenation to obtain 6 (Scheme
2). The intermediate was protected with Boc, then hydrolyzed to
activity (EC50 = 6.8 l
M) against live parasites.33 We hypothesized
that this attenuation comes from its unbalanced hydrophobic-
hydrophilic nature because the compound must pass through four
membranes to bind to its targets in the parasite food vacuole.6
Further extension of the inhibitor size using tripeptidomimetics
revealed superior potencies against Plm II but negligible improve-
ment in antimalarial activity.34 In order to overcome the disadvan-
tage of peptidomimetic character, we modified a specific moiety of
2, that is, the 2,6-dimethylphenoxyacetyl group that caused a
similar problem in our previous HIV protease inhibitor study.35
An X-ray crystallographic study disclosed the unique binding of 1
to Plm IV from P. malariae, oriented opposite to that of pepstatin
A and also to that of 1 in HIV protease (Fig. 1).36 The HMC inter-
acted with two catalytic Asp residues, but the Apns fit in the S1´
pocket, and (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
(Dmt) was in the S1 space. The 2-methylbenzyl group did not fit
R1
R1
O
R2
R3
OH
R4
R2
R3
O
a, b
OH
R4
3
4a—w
4a: R1 = CH3,
4b: R1 = H,
4c: R1 = H,
4d: R1 = OCH3,
4e: R1 = H,
4f: R1 = H,
R2 = H,
R3 = H,
R3 = H,
R4 = H
R4 = H
R4 = H
R4 = H
R4 = H
R2 = CH3,
R
R
2 = H,
2 = H,
R
R
3 = CH3,
3 = H,
R2 = OCH3, R3 = H,
R2 = H,
2 = H,
R2 = OH,
2 = H,
R2 = H,
R3 = OCH3, R4 = H
4g: R1 = OH,
4h: R1 = H,
4i: R1 = H,
R
R
3 = H,
R3 = H,
3 = OH,
R3 = H,
3 = H,
R4 = H
R4 = H
R4 = H
R4 = H
R4 = H
R
R
4j: R1 = CH3OH,
4k: R1 = H,
4l: R1 = H,
0
in the S1 pocket. These observations suggested that compound 2
R
2 = CH3OH,
R
would bind to other Plms in a similar manner to that of 1 in Plm
R2 = H,
R2 = H,
R3 = CH3OH, R4 = H
IV.37 Therefore, the dimethylphenoxyacetyl group of 2 is expected
c,d
4m: R1 = NO2,
4n: R1 = H,
4o: R1 = H,
R3 = H,
R4 = H
0
to bind to the S1 pocket of any Plm (Fig. 2). We herein report the
R
2 = NO2,
R
3 = H,
R4 = H
R2 = H,
R3 = NO2,
R3 = H,
R4 = H
structure-activity relationships of dipeptide-type Plm inhibitors
and the enhancement of antimalarial activity in these analogues.
c
c
4p: R1 = NH(Boc), R2 = H,
R4 = H
4q: R1 = H,
4r: R1 = H,
R2 = NH2,
2 = H,
R2 = H,
2 = H,
R2 = H,
R3 = H,
R4 = H
R
R
3 = NH2,
R4 = H
4s: R1 = F,
4t: R1 = Cl,
4u: R1 = OCH3,
R3 = H,
3 = H,
R3 = H,
3 = H,
R3 = F,
R4 = F
2. Synthesis
R
R
R4 = Cl
R4 = OCH3
R4 = CH(CH3)2
R4 = CH3
Phenoxyacetic acid derivatives 4aꢀw were synthesized from
commercially available phenols 3, following the general procedure
in Scheme 1. o-Nitro derivative 4m was hydrogenated to form the
amino group, which was further protected by tert-butyloxycar-
bonyl (Boc) to obtain 4p. Other amino derivatives 4q and 4r were
4v: R1 = CH(CH3)2, R2 = H,
R
4w: R1 = CH3,
R2 = H,
Scheme 1. Reagents: (a) BrCH2COOEt, K2CO3, DMF; (b) 1M-NaOH, MeOH; (c) 10%
Pd–C, H2, MeOH; (d) (Boc)2O, Et3N, THF–H2O.

K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
10051
O
O
O
O
O
O
OH
OEt
OEt
OH
c
h
a, b
g, d
Boc
Boc
H2N
N
H
N
R
O2N
6
5
7
11: R = methyl
12: R = ethyl
d
13: R = pyridin-2-ylmethyl
14: R = pyridin-3-ylmethyl
15: R = pyridin-4-ylmethyl
O
O
O
O
O
O
OH
OH
e, f
OH
Boc
N
HO
N
H
10
9
8
Scheme 2. Reagents and condition: (a) BrCH2COOEt, K2CO3, DMF; (b) 10% Pd–C, H2, MeOH; (c) (Boc)2O, Et3N, THF–H2O; (d) 1 M NaOH, MeOH; (e) 4 M HCl/dioxane; (f) NaNO2,
H2SO4, then H2O, reflux 3 h; (g) HCHO aq, 10% Pd–C, H2, MeOH; (h) NaH, CH3I or R–Br, THF.
HO
HO
O
O
O
O
a
b, c, b
Boc
Boc
N
OH
N
N
H
H2N
HCl•
N
N
H
OH
S
S
S
16
17
18
HO
R1
O
O
O
d
(or d, b)
R2
R3
O
N
H
N
N
H
OH
R4
S
19—49
Scheme 3. Reagents: (a) (1S,2R)-1-amino-2-indanol, BOP, Et3N, DMF; (b) 4 M HCl/dioxane, anisole; (c) Boc-Apns-OH, EDCꢁHCl, HOBtꢁH2O, Et3N, DMF; (d) 4a–4w or 8–15, BOP,
Et3N, DMF.
HO
HO
O
O
O
O
O
O
a
O
O
N
H
N
N
H
N
H
N
N
H
OH
R
OH
H2N
HCl•
N
H
S
S
43
50: R = n-butyl
51: R = n-pentyl
Scheme 4. Reagents: (a) Et3N, MeOH, butanal or pentanal, NaBH3CN.
result in Boc-amino derivative 8. Intermediate 8 was transformed
to the hydroxyl derivative 9 via diazonium salt formation. Dimeth-
ylamino analogue 10 was prepared by hydrogenation with formal-
dehyde and saponification. Mono-alkylamino derivatives 11–15
were synthesized using iodomethane or alkylbromides with NaH,
hydrolyzed simultaneously. Intermediate 18 was prepared as de-
scribed in previous reports33,34 from Boc-Dmt-OH 16 with subse-
quent couplings of aminoindanol and Boc-Apns-OH using
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) or N-ethyl-N0-[3-(dimethylamino)pro-
pyl]carbodiimide hydrochloride (EDCꢁHCl) plus 1-hydroxy-
benzotriazole (HOBt) methods and deprotections of the Boc groups
using HCl-dioxane, shown in Scheme 3. Phenoxyacetic acid deriva-
tives were then condensed with 18 using BOP. Additional depro-
tection of the Boc group was performed in the case of
compounds 31, 43, 44, and 46–49. p-Butylamino and p-pentylami-
no derivatives 50 and 51 were synthesized from compound 43 by
reductive alkylation with aldehydes and NaBH3CN (Scheme 4). All
of the final products were identified by MALDI-TOF MS and FAB-
MS after purifications by preparative HPLC.
3. Results and discussion
Various substituents were attached at each position of the
phenoxyacetyl group of KNI-10006 without the inherent dimethyl
groups. The inhibitory activity results are summarized in Table 1.
Methyl substitution showed Plm II’s preference of the m- and
p-positions (20 and 21). Particularly noteworthy is the large differ-
ence in Ki value between o-methyl and o-dimethyl groups, 145 (19)
and 0.5 nM (2), respectively. The result indicates the importance of

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K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
Table 1
Inhibitory activity of phenoxyacetyl derivatives
O
O
NH
N
H
N
O
HO
OH
S
O
R3
R1
R2
Compound
R1
R2
R3
Plm II Ki (nM)
HIV-1 PR % inhibition (at 50 nM)
Anti-P. falciparum EC50
(
lM)
TD50 (lM)
19 (KNI-10079)
20 (KNI-10080)
21 (KNI-10081)
22 (KNI-10092)
23 (KNI-10093)
24 (KNI-10094)
25 (KNI-10369)
26 (KNI-10216)
27 (KNI-10217)
28 (KNI-10113)
29 (KNI-10124)
30 (KNI-10125)
31 (KNI-10106)
32 (KNI-10087)
33 (KNI-10088)
34 (KNI-10095)
35 (KNI-10255)
36 (KNI-10256)
CH3
H
H
OCH3
H
H
OH
H
H
CH2OH
H
H
NH2
H
H
NO2
H
H
H
CH3
H
H
OCH3
H
H
OH
H
H
H
CH3
H
H
OCH3
H
H
OH
H
H
CH2OH
H
H
NH2
H
H
145
42
55
153
4
479
112
10
10
132
9
10
126
29
72
13
27
27
10
38
34
13
31
61
24
37
62
29
27
9
>8
>8
>8
>8
>8
H
CH2OH
H
H
NH2
H
H
>8
1.1
>120
>8
>8
28
55
38
25
NO2
H
12
53
2.8
4.2
58
63
NO2
the o-dimethyl structure to fit in the pocket of Plm II with some
hydrophobic interactions. A methoxy group was preferred only at
the m-position (compound 23). Less adaptation of o-methoxy
(22) was similar to that of the methyl group. Compound 24 with
a p-methoxy group was the worst among these analogues. The
extension of the methyl group may cause a hydrophobic repulsion
that affects the binding of the whole ligand. Modification of the
methyl with a hydroxyl, hydroxymethyl, or amino group resulted
in similar potency to methyl substitution with a slight improve-
ment of Plm II inhibitory activity (compounds 25–33). In the case
of nitro substitutions (34–36), similar preference at the m- and
p-positions was observed, but the difference between these and
the o-position was not considerable. We also tested the HIV-1 pro-
tease inhibition of these analogues. Preferences of o-substitutions
such as methyl (19), hydroxymethyl (28), or amino group (31)
were specific for HIV protease compared to Plm II, in which only
the nitro group (34) was accepted for Plm II, highlighting the dif-
ferences of the Plm and HIV protease binding pockets. Compounds
with relatively potent Plm II inhibitory activity, with Ki values less
than 100 nM, were tested against P. falciparum. Among them, com-
pound 30 with a p-hydroxymethyl group exhibited relatively po-
except diisopropyl, which had no activity. Besides the lower
potency against Plm II compared to 2, the antimalarial activity of
difluoro, dichloro, and diisopropyl derivatives were improved. Be-
cause the dimethyl groups of KNI-10006 appeared indispensable
for Plm II inhibition, we decided to attach an additional substituent
at the 4-position of the phenyl group. Although fluoro derivative 41
exhibited a moderate decrease in Plm II inhibition, the antimalarial
activity was improved to 1.4 lM. This small modification with
fluorine yielded fourfold improvement of activity against the para-
site. Attachment of a hydroxyl group at the 4-position resulted in
slight improvement in Ki compared to fluorine but less antimalarial
activity (compound 42). The HIV protease inhibitory activity
showed no difference without the hydroxyl group (42 and 2),
suggesting no interference to its binding.
p-Amino substitution on 2,6-dimethylphenoxyacetyl moiety
was previously studied with HIV protease inhibitors to improve
cellular activity.38,39 Plm II inhibition of free amino and its methyl-
ated analogues, compounds 43–45, was moderate around 20 nM
with about a 40-fold decrease compared to 2. Besides its moderate
activity, an attachment of a monomethylamino group, compound
44, dramatically enhanced antimalarial activity, 0.45 lM, with
tent inhibition, EC50 = 1.1
Compound 29, with a slightly different substituent position from
30, did not show antimalarial activity. The TD50 value of 30 was
>120 lM with more than 100-fold selectivity. Compounds 35 and
l
M, more potent than
1
and 2.
15-fold improvement compared to that of 2. The dimethylamino
derivative also exhibited relatively potent antimalarial activity.
These results suggested that addition of some basicity would con-
tribute to inhibitory activity in the parasite-infected erythrocyte
assay. The low cytotoxicity of 44 led us to vary the monomethyla-
mino moiety (Table 3).
We elongated the carbon chain length with ethyl, butyl, and
pentyl (compounds 46, 50 and 51). Among them, compound 46
with the ethyl moiety exhibited the best Plm II inhibition. Antima-
larial activity increased along with the chain length; however,
cytotoxicity increased as well. Therefore, the first methylamino
analogue was the best among the compounds modified in this
manner.
36, with nitro substituents, also exhibited moderate antimalarial
activity, but they showed some cytotoxicity. These results suggest
that small changes in chemical properties affect a compound’s abil-
ity to reach the Plms in the food vacuole. The mono-substitution
studies resulted in an attenuation of Plm inhibition, suggesting
an importance of 2,6-disubstitution for inhibitory activity. There-
fore, we shifted our focus to 2,6-substituted derivatives of KNI-
10006.
We changed the dimethyl moiety to difluoro, dichloro, dime-
thoxy, or diisopropyl groups (37–40). All of the modifications failed
to improve Plm II inhibitory activity; in particular, dimethoxy sub-
stitutions resulted in 1500-fold decrease in potency (Table 2). The
o-disubstituents moderately affected HIV protease inhibition,
Addition of a pyridine group is an option to reduce hydropho-
bicity, and its basicity would contribute enhancement of antima-
larial activity. Therefore, we decided to incorporate a pyridine
group on the methyl of 44. Resultant three pyridinylmethyl

K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
10053
Table 2
Inhibitory activity of 2,6- and 2,4,6-substituted phenoxyacetyl derivatives
O
O
R3
NH
N
H
N
O
HO
OH
S
O
R2
R1
Compound
R1
R2
R3
Plm II Ki (nM)
HIV-1 PR % inhibition (at 50 nM)
Anti-P. falciparum EC50
(
lM)
TD50
55
(lM)
2
CH3
F
Cl
OCH3
CH(CH3)2
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
F
OH
NH2
NHCH3
N(CH3)2
CH3
F
Cl
OCH3
CH(CH3)2
CH3
CH3
CH3
CH3
CH3
0.5
14
39
782
23
19
5
23
25
21
98
58
95
54
0
87
98
96
91
86
6.8
3.3
5.2
37 (KNI-10265)
38 (KNI-10074)
39 (KNI-10260)
40 (KNI-10152)
41 (KNI-10266)
42 (KNI-10155)
43 (KNI-1293)
44 (KNI-10283)
45 (KNI-10282)
2.9
1.4
3.3
6.3
29
0.45
1.2
>120
45
Table 3
Inhibitory activity of monosubstituted-amino KNI-10006 derivatives
O
O
NH
O
N
H
N
HO
OH
R
S
O
N
H
Compound
R
Plm II Ki (nM)
HIV-1 PR % inhibition (at 50 nM)
91
Anti-P. falciparum EC50
(lM)
TD50
>120
(lM)
CH3
44
25
0.45
46 (KNI-10529)
50 (KNI-10539)
51 (KNI-10541)
7
30
26
84
85
78
0.72
0.54
0.43
64
22
12
N
47 (KNI-10538)
10
91
0.30
>120
N
N
48 (KNI-10526)
49 (KNI-10527)
21
6
91
91
0.50
0.49
>120
>120
analogues 47–49 exhibited potent Plm II inhibition, especially 49,
with a Ki value of 6 nM.
is, the hydroxyl group of HMC interacted with the carboxylate an-
ion of one of the Asps, and the carbonyl interacted with the carbox-
ylic acid proton of the other Asp.40,41 However, these interactions
were collapsed in the MD simulation, and the both Asp residues
tended to interact only with the hydroxyl group of HMC. Hydrogen
bond interactions except HMC were similar to our previous study
of tripeptidic inhibitors34 and those suggested by Åqvist’s group.37
The difference was found at the phenoxyacetyl moiety, that is, the
p-position of 2,6-dimethylphenoxyacetyl group, was oriented to
water outside; therefore, further extension of the pyridine-4-yl-
methyl moiety from the p-amino group was favored to be sur-
rounded mostly by water molecules (Fig. 3B and C). This is
consistent with Plm II inhibitory activity of 49, suggesting that it
is easier for the nitrogen of the 4-pyridine-ring to contact the water
than 2- and 3-pyridine. A significant movement was observed in
the phenoxyacetyl moiety during the MD simulation with RMSD
1.341 Å 0.366, suggesting a loss of contact with the enzyme,
To explore the moderate attenuation in Plm II inhibitory activity
of 49 compared to 2, we constructed a binding model of 49 with
Plm II (PDB ID, 1SME) using the molecular modeling package
MOE 2007.09 (Chemical Computing Group, Inc., Montreal, Canada).
Pepstatin A was replaced with compound 49. The X-ray crystallo-
graphic data of inhibitor 1 in Plm IV (PDB ID, 2ANL) was used to
generate the initial docking conformation of 49 in Plm II. According
to the binding mode of 1, Asp214 was intentionally protonated.
After several energy minimization processes with an MMFF94x
force field including a 20 Å of water soak around the inhibitor, a
molecular dynamics (MD) simulation was performed (Fig. 3). The
binding direction of 49 was same with that of 1 but opposite to
that of pepstatin A (Fig. 3A). The hydrogen bond interactions of
HMC with the two catalytic Asps were deliberately kept in a simi-
lar configuration to those observed in HIV protease complexes, that

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K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
tional to Plm inhibitory activities. This indicates the strong depen-
dence on other factors for the compounds to reach the target Plm.
Similar attempts using basic nitrogen were reported by Hallberg’s
group.43,44 However, the effect of this basic group could be caused
by a different inhibition mechanism without Plm inhibition.45 Fur-
ther investigations to clarify the effect of compound basicity for
delivery of the drug to the acidic food vacuole are required.
4. Conclusions
We synthesized a series of derivatives of 2 with specific substi-
tutions on the phenoxyacetyl moiety, which contain the Apns-Dmt
scaffold to exhibit potent inhibitory activity against the Plms. We
observed the enhancement of antimalarial activity in cases of com-
pounds modified by attaching both lipophilic and hydrophilic moi-
eties, suggesting an importance of the balance of inhibitor
characteristics. In particular, attachment of basic moieties such
as a monoalkylamino group contributed to enhance antimalarial
activity up to the submicromolar level. In this study, we identified
some alkylamino compounds with promising antimalarial activity,
maintaining their potent Plm II inhibitory activity and low cytotox-
icity. Evaluations of these analogues using malaria parasite-in-
fected mouse models are underway to develop potential
antimalarial drugs.
5. Experimental
5.1. Materials
Reagents and solvents were purchased from Wako Pure Chem-
ical Ind., Ltd (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), Aldrich
Chemical Co. Inc. (Milwaukee, WI, USA), and Tokyo Kasei Kogyo
Co., Ltd (Tokyo, Japan) and used without further purification. TLC
was performed using Merck Silica gel 60 F254 precoated plates. Col-
umn chromatography was performed on Merck 107734 silica gel
60 (70–230 mesh). Melting points were measured on a Yanagimoto
micro melting apparatus. Preparative HPLC was carried out on a
C18 reverse phase column (20 ꢂ 250 mm; YMC Pack ODS SH343-
5) with a binary solvent system: a linear gradient of CH3CN in
C
S2
S1'
0.1% aqueous trifluoroacetic acid (TFA) with
a flow rate of
O
O
5.0 mL/min and detection at 230 nm. 1H NMR spectra were ob-
tained on a JEOL AL300 (300 MHz) spectrometer with TMS as an
internal standard. Mass spectra (electrospray ionization, methanol
as the mobile phase) were analysed with SHIMADZU LCMS-2010
spectrometer. MALDI-TOF MS was performed on a Voyager-DE
RP spectrometer (PerSeptive Biosystems, Inc.). High resolution
FAB-MS was performed on a JEOL JMS-SX102A spectrometer
equipped with the JMA-DA7000 data system. Analytical HPLC
was performed using a C18 reverse phase column (4.6 ꢂ 150
mm; YMC Pack ODS AM-302) with a binary solvent system: linear
gradient of CH3CN 20–80% in 0.1% aqueous TFA in 30 min at a flow
rate of 0.9 mL/min, detected at 230 nm. The purity of the desired
compounds was greater than 95%.
H2O
H2O
NH
N
H
N
H2O
H2O
H2O
O
HO
OH
S
O
N
NH
H2
S1
S2'
H2O
Figure 3. MD simulated pose of compound 49 bound to Plm II. Asp214 was
protonated. (A) Superimposition of pepstatin A (cyan), 1 (yellow) and 49 (red). (B)
Pyridinylmethyl group of 49 (green stick) was surrounded by water molecules
(lines, shown only 5 Å from the ligand), not in the Plm II binding sites (red surface).
(C) Schematic representation of 49. Figures
A and B were generated using
MacPyMOL (DeLano Scientific LLC, CA, USA).42
resulting in moderate reduction in the activity compared to that of
2. The moderate reduction of 42 with only a hydroxyl group differ-
ent from 2 may be explained by the same reasoning.
Antimalarial activities of pyridinylmethyl derivatives 47–49
were maintained at the sub-micromolar level. The EC50 value of
5.2. Chemistry
5.2.1. Ethyl 4-(tert-butyloxycarbonylamino)-2,6-
dimethylphenoxyacetate (7)
To a mixture of 2,6-dimethyl-4-nitrophenol (10.0 g, 60 mmol),
K2CO3 (10.0 g, 72 mmol) in DMF (100 mL) was added ethyl bromo-
acetate (9.27 mL, 84 mmol) in an ice-water bath, and the mixture
was stirred overnight at room temperature. After removal of the
solvent in vacuo, the residue was dissolved in EtOAc, filtered insol-
uble solid, washed sequentially with 10% citric acid, 5% NaHCO3,
and saturated NaCl, dried over MgSO4, and concentrated in vacuo.
0.3 lM of 47 is the best among tested derivatives, about a 20-fold
improvement over 2. The cytotoxicity of these pyridinemethyl ana-
logues was in the acceptable range for clinical inhibitors. These re-
sults suggest that the pyridinemethyl analogues are promising for
the development of new antimalarial agents. Interestingly, all of
the tested monoalkylamino derivatives exhibited sub-micromolar
potency against parasites even though the activities are not propor-

K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
10055
A mixture of the residue and 10% Pd–C in MeOH (100 mL) was vig-
orously stirred with a H2 balloon. After 4 h stirring, the reaction
mixture was filtered, concentrated in vacuo to give amino interme-
diate 6; yield 96%. MS (ES+) m/z: 224 for [M+H]+.
5.2.7. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-amino-
2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide hydrochloride salt (18)
To a solution of Boc-Dmt-OH (16, 8.47 g, 32.4 mmol) in DMF
(80 mL) were added triethylamine (6.79 mL, 48.6 mmol), (1 S,2R)-
1-amino-2-indanol (5.33 g, 35.7 mmol) and BOP (15.74 g,
35.6 mmol) in an ice-water bath, and the mixture was stirred over-
night at room temperature. After removal of the solvent in vacuo,
the residue was mixed with EtOAc, washed sequentially with
10% citric acid, 5% NaHCO3, and saturated NaCl, dried over MgSO4,
and concentrated in vacuo to give 13.6 g of 17 as a white solid (MS
(ES+) m/z: 393 for [M+H]+).
To a solution of the residue and triethylamine (12.1 mL) in THF–
H2O (1:1, 100 mL) was added (Boc)2O (14.0 g, 64 mmol) and stirred
overnight at room temperature. The reaction mixture was concen-
trated in vacuo, diluted with EtOAc, washed with acid and base
mentioned above, dried over MgSO4, and concentrated in vacuo.
Precipitation with n-hexane gave 18.0 g of the titled compound
as white solid; yield 92%; mp 106–108 °C. 1H NMR (300 MHz,
DMSO-d6) d (ppm): 9.10 (s, 1H), 7.09 (s, 2H), 4.38 (s, 2H), 4.18 (q,
J = 7.1 Hz, 2 H), 2.16 (s, 6 H), 1.46 (s, 9 H) 4.18 (t, J = 7.1 Hz, 3H);
MS (ES+) m/z: 346 for [M+Na]+.
A mixture of 19, anisole (7.04 mL, 64.8 mmol), and 4 M HCl in
dioxane (100 mL) was stirred at room temperature for 1.5 h. After
removal of the solvent in vacuo, the residue was precipitated from
ether and filtered to give the hydrochloride salt. To a solution of
the salt in DMF (80 mL) were added HOBtꢁH2O (5.45 g, 35.6 mmol),
Boc-Apns-OH (10.51 g, 35.6 mmol), and EDCꢁHCl (6.82 g,
35.6 mmol). Under stirring in an ice-water bath, triethylamine
(4.53 mL, 32.4 mmol) was slowly added for 20 min. The mixture
was stirred overnight at room temperature. After removal of the
solvent in vacuo, the residue was mixed with EtOAc, washed
sequentially with 10% citric acid, 5% NaHCO3, and saturated NaCl,
dried over MgSO4, and concentrated in vacuo. Purification of the
product by silica gel column chromatography (n-hexane–EtOAc)
gave 14.85 g of white solid; yield 80% (MS (ES+) m/z: 593 for
5.2.2. 4-(N-tert-Butyloxycarbonyl-N’-methyl)amino-2,6-
dimethylphenoxyacetic acid (11)
Compound 11 was prepared from intermediate 7 in a manner
similar to that described for compound 15; yield 74%; mp 97–
99 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 12.8 (br, 1H), 6.91
(s, 2H), 4.35 (s, 2H), 3.11 (s, 3H), 2.21 (s, 6H), 1.38 (s, 9H); MS
(ES+) m/z: 308 for [MꢀH]ꢀ.
5.2.3. 4-(N-tert-Butyloxycarbonyl-N’-ethyl)amino-2,6-
dimethylphenoxyacetic acid (12)
Compound 12 was prepared from intermediate 7 in a manner
similar to that described for compound 15; yield 97%; mp 121–
123 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 6.80 (s, 2H), 3.90
(s, 2H), 3.51 (q, J = 6.9 Hz, 2H), 2.18 (s, 6H), 1.36 (s, 9H), 1.03
(t, J = 6.9 Hz, 3H); MS (ES+) m/z: 322 for [MꢀH]ꢀ.
[M+Na]+).
A mixture of the intermediate, anisole (5.67 mL,
52.2 mmol), and 4 M HCl in dioxane (85 mL) was stirred at room
temperature for 1.5 h. After removal of the solvent in vacuo, the
residue was precipitated from ether to give 12.34 g of the titled
compound; yield 75% from 18; mp 141–143 °C. 1H NMR
(300 MHz, DMSO-d6) d (ppm): 8.19 (d, J = 9.0 Hz, 1H), 8.02 (br,
3H), 7.41–7.12 (m, 9H), 6.22 (br, 1H), 5.25 (dd, J = 8.6 Hz, 5.0 Hz,
1H), 5.09 (br, 1H), 4.91 (d, J = 8.7 Hz, 1H), 4.71–4.68 (m, 2H),
4.56–4.51 (m, 1H), 4.44–4.38 (m, 1H), 3.58–3.49 (m, 1H, over-
lapped with H2O), 3.04 (dd, J = 16.5 Hz, 4.5 Hz, 1H), 2.96–2.78
(m, 3H), 1.55 (s, 3H), 1.46 (s, 3H); MS (ES+) m/z: 470 for [M+H]+.
5.2.4. 4-(N-tert-Butyloxycarbonyl-N’-pyridin-2-yl)amino-2,6-
dimethylphenoxyacetic acid (13)
Compound 13 was prepared from intermediate 7 in a manner
similar to that described for compound 15; yield 90%; mp 156–
158 °C. 1H NMR (300 MHz, DMSO-d6) d (ppm): 8.50–8.46 (m,
1H), 7.80–7.73 (m, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.26–7.22 (m,
1H), 6.97 (s, 2H), 4.80 (s, 2H), 4.32 (s, 2H), 2.16 (s, 6H), 1.29 (s,
9H); MS (ES+) m/z: 385 for [MꢀH]ꢀ.
5.2.8. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2-
methylphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (19)
5.2.5. 4-(N-tert-Butyloxycarbonyl-N’-pyridin-3-yl)amino-2,6-
dimethylphenoxyacetic acid (14)
To a solution of 18 (100 mg, 0.20 mmol) in DMF (3 mL) were
Compound 14 was prepared from intermediate 7 in a manner
similar to that described for compound 15; yield 95% as oil. 1H
NMR (300 MHz, DMSO-d6) d (ppm): 8.44 (dd, J = 4.5 Hz, 1.5 Hz,
1H), 8.40 (d, J = 2.1 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.34 (dd,
J = 7.8 Hz, 4.8 Hz, 1H), 6.87 (s, 2H), 4.78 (s, 2H), 4.33 (s, 2H), 2.17
(s, 6H), 1.35 (s, 9H); MS (ES+) m/z: 385 for [MꢀH]ꢀ.
added triethylamine (55.2 lL, 0.40 mmol), 2-methylphenoxyacetic
acid (36.1 mg, 0.22 mmol) and BOP (96.1 mg, 0.22 mmol) in an ice-
water bath and the mixture was stirred overnight at room temper-
ature. After removal of the solvent in vacuo, the residue was dis-
solved in EtOAc, washed sequentially with 10% citric acid, 5%
NaHCO3, and saturated NaCl, dried over MgSO4, and concentrated
in vacuo. The residue was precipitated from hexane to give the
crude product. Purification of the product by preparative HPLC
gave the titled compound as a white foam; yield 72%; 1H NMR
(DMSO-d6) d (ppm): 8.23 (d, J = 9.0 Hz, 1H), 8.11 (d, J = 8.7 Hz,
1H), 7.36–6.99 (m, 11H), 6.85–6.81 (m, 1H), 6.59 (d, J = 7.8 Hz,
1H), 5.43 (d, J = 7.2 Hz, 1H), 5.29 (dd, J = 8.6 Hz, 5.0 Hz, 1H), 5.03
(d, J = 4.5 Hz, 1H), 4.94 (s, 2H), 4.74 (s, 1H), 4.49–4.25 (m, 4H),
3.09–3.01 (m, 1H), 2.84–2.68 (m, 3H), 2.20 (m, 3H), 1.56 (s, 3H),
1.47 (s, 3H); MS (TOF) m/z: 619 [M+H]+; HRMS (FAB+) calcd for
C34H39N3O6SNa [M+Na]+ 640.2457, found m/z 640.2452.
5.2.6. 4-(N-tert-Butyloxycarbonyl-N’-pyridin-4-yl)amino-2,6-
dimethylphenoxyacetic acid (15)
To a solution of compound 7 (100 mg, 0.31 mmol) in dry THF
(5 mL) was added sodium hydride (74 mg, 3.1 mmol) and stirred
for 1 h under ice-water cooling and Ar atmosphere, then 4-(bromo-
methyl)pyridine hydrobromide (235 mg, 0.93 mmol) was added
and stirred overnight at room temperature. Additional sodium hy-
dride (74 mg, 3.1 mmol) was added and stirred overnight at room
temperature. The reaction mixture was diluted with ether, ex-
tracted with 5% NaHCO3. The aqueous layer was neutralized with
citric acid, saturated with NaCl, extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
in vacuo. Precipitation with n-hexane gave 102 mg of the titled
compound as a brownish solid; yield 86%; mp 188–190 °C. 1H
NMR (300 MHz, DMSO-d6) d (ppm): 8.50 (d, J = 4.5 Hz, 2H), 7.22
(d, J = 4.5 Hz, 2H), 6.92 (s, 2H), 4.78 (s, 2H), 4.33 (s, 2H), 2.17 (s,
6H), 1.33 (s, 9H); MS (ES+) m/z: 385 for [MꢀH]ꢀ.
5.2.9. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(3-
methylphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (20)
Compound 20 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.26 (d, J = 9.0 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.34–7.06
(m, 10H), 6.75 (d, J = 7.8 Hz, 1H), 6.67 (s, 1H), 6.57 (d, J = 8.7 Hz,

10056
K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
1H), 5.40 (d, J = 6.9 Hz, 1H), 5.31–5.24 (m, 1H), 5.04 (d, J = 3.9 Hz,
1H), 4.95 (s, 2H), 4.75 (s, 1H), 4.49–4.21 (m, 5H), 3.07–3.02 (m,
1H), 2.85–2.68 (m, 3H), 2.24 (m, 3H), 1.55 (s, 3H), 1.46 (d,
J = 5.7 Hz, 3H); MS (TOF) m/z: 619 [M+H]+; HRMS (FAB+) calcd
for C34H39N3O6SNa [M+Na]+ 640.2457, found m/z 640.2454.
J = 9.0 Hz, 1H), 4.97 (d, J = 9.3 Hz, 1H), 4.76 (s, 1H), 4.55 (d,
J = 3.0 Hz, 1H), 4.43–4.26 (m, 4H), 3.06 (dd, J = 6.6 Hz, 4.7 Hz, 1H),
2.90–2.71 (m, 3 H), 1.56 (d, J = 15.3 Hz, 3H), 1.47 (s, 3H); MS
(TOF) m/z: 621 [M+H]+; HRMS (FAB+) calcd for C33H37N3O7SNa
[M+Na]+ 642.2250, found m/z 642.2256.
5.2.10. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
methylphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (21)
5.2.15. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(3-
hydroxyphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (26)
Compound 21 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.26 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.34–6.99
(m, 11H), 6.68 (d, J = 8.7 Hz, 2H), 5.40 (d, J = 6.9 Hz, 1H), 5.31–
5.26 (m, 1H), 5.04 (d, J = 4.5 Hz, 1H), 4.95 (s, 2H), 4.75 (s, 1H),
4.49–4.19 (m, 5H), 3.08–3.02 (m, 1H), 2.85–2.68 (m, 3H), 2.20 (s,
3H), 1.55 (s, 3H), 1.47 (s, 3H); MS (TOF) m/z: 619 [M+H]+; HRMS
(FAB+) calcd for C34H39N3O6SNa [M+Na]+ 640.2457, found m/z
640.2451.
Compound 26 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 9.39 (s, 1H), 8.17 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 9.0 Hz,
1H), 7.31 (t, J = 6.2 Hz, 1H), 7.25–7.06 (m, 6H), 6.98 (t, J = 8.1 Hz,
1H), 6.38–6.35 (m, 1H), 6.30 (d, J = 2.3 Hz, 1H), 6.22 (dd,
J = 8.0 Hz, 2.0 Hz, 1H), 5.31–5.26 (m, 1H), 4.95 (s, 1H), 4.74 (s,
1H), 4.48 (d, J = 3.0 Hz, 1H), 4.44–4.40 (m, 1H), 4.35–4.22 (m,
3H), 3.05 (dd, J = 16.2 Hz, 4.5 Hz, 1H), 2.89–2.71 (m, 3H), 1.55 (s,
3H), 1.47 (s, 3H); MS (TOF) m/z: 621 [M+H]+; HRMS (FAB+) calcd
for C33H37N3O7SNa [M+Na]+ 642.2250, found m/z 642.2245.
5.2.11. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2-
methoxyphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (22)
5.2.16. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
hydroxyphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (27)
Compound 22 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.18 (d, J = 8.7 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.29–7.05
(m, 9H), 7.00–6.90 (m, 2H), 6.79–6.69 (m, 2H), 5.39 (br, 1H), 5.28
(dd, J = 8.4 Hz, 5.1 Hz, 1H), 5.04 (br, 1H), 4.96 (s, 2H), 4.75 (s, 1H),
4.50 (s, 1H), 4.39–4.22 (m, 4H), 3.78 (s, 3H), 3.08–3.02 (m, 1H),
2.87–2.79 (m, 2H), 2.71–2.62 (m, 1H), 1.56 (s, 3H), 1.47 (s, 3H);
MS (TOF) m/z: 635 [M+H]+; HRMS (FAB+) calcd for C34H39N3O7SNa
[M+Na]+ 656.2406, found m/z 656.2399.
Compound 27 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.92 (br, 1H), 8.13 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 9.0 Hz,
1H), 7.22–7.11 (m, 5H), 7.06 (t, J = 7.2 Hz, 1H), 6.64–6.57 (m, 4H),
5.26 (dd, J = 8.6 Hz, 4.8 Hz, 1H), 4.93 (s, 2H), 4.72 (s, 1H), 4.45 (d,
J = 3.3 Hz, 1H), 4.42–4.38 (m, 1H), 4.33–4.22 (m, 3H), 3.03 (dd,
J = 15.9 Hz, 4.5 Hz, 1H), 2.87–2.66 (m, 3H), 1.54 (s, 3H), 1.45 (s,
3H); MS (TOF) m/z: 621 [M+H]+; HRMS (FAB+) calcd for
C33H37N3O7SNa [M+Na]+ 642.2250, found m/z 642.2257.
5.2.12. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(3-
methoxyphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (23)
5.2.17. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2-
hydroxymethylphenoxyacetyl)amino-2-hydroxy-4-phenyl-
butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (28)
Compound 28 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.28 (d, J = 8.7 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.41–7.28
(m, 4H), 7.22–7.08 (m, 7H), 6.98–6.91 (m, 1H), 6.64 (d, J = 7.8 Hz,
1H), 5.35–5.23 (m, 2H), 5.17–5.09 (m, 1H), 5.04–4.93 (m, 3H),
4.75 (s, 1H), 4.56–4.41 (m, 7H), 4.33–4.21 (m, 1H), 3.07–3.02 (m,
1H), 2.84–2.65 (m, 3H), 1.55 (s, 3H), 1.47 (s, 3H); MS (TOF) m/z:
657 [M+Na]+; HRMS (FAB+) calcd for C34H39N3O7SNa [M+Na]+
656.2406, found m/z 656.2411.
Compound 23 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.26 (d, J = 9.0 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.34–7.29
(m, 2H), 7.23–7.08 (m, 8H), 6.54–6.50 (m, 1H), 6.45 (s, 1H), 6.37–
6.34 (m, 1H), 5.40 (br, 1H), 5.28 (dd, J = 8.7 Hz, 5.1 Hz, 1H), 5.03
(br, 1 H), 4.95 (s, 2H), 4.75 (s, 1H), 4.48–4.24 (m, 5H), 3.69 (s,
3H), 3.08–3.02 (m, 1H), 2.85–2.73 (m, 3H), 1.55 (s, 3H), 1.47 (s,
3H); MS (TOF) m/z: 635 [M+H]+; HRMS (FAB+) calcd for
C34H39N3O7SNa [M+Na]+ 656.2406, found m/z 656.2415.
5.2.13. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
methoxyphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (24)
5.2.18. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(3-
hydroxymethylphenoxyacetyl)amino-2-hydroxy-4-phenylbuta-
noyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (29)
Compound 29 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.27 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 8.7 Hz, 1H), 7.35–7.08
(m, 10H), 6.90–6.83 (m, 2H), 6.63 (d, J = 8.1 Hz, 1H), 5.40 (d,
J = 6.9 Hz, 1H), 5.31–5.15 (m, 2H), 5.04 (d, J = 4.5 Hz, 1H), 4.96 (s,
2H), 4.75 (s, 1H), 4.78–4.19 (m, 7H), 3.07–3.03 (m, 1H), 2.84–2.68
(m, 3H), 1.55 (s, 3H), 1.47 (s, 3H); MS (TOF) m/z: 635 [M+H]+;
HRMS (FAB+) calcd for C34H39N3O7SNa [M+Na]+ 656.2406, found
m/z 656.2402.
Compound 24 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.24 (d, J = 8.7 Hz, 1H), 8.11 (d, J = 9.3 Hz, 1H), 7.35–7.29
(m, 3H), 7.22–7.08 (m, 9H), 6.86–6.82 (m, 4H), 5.41 (d, J = 7.2 Hz,
1H), 5.28 (dd, J = 8.9 Hz, 5.0 Hz, 1H), 5.04 (d, J = 4.5 Hz, 1H), 4.95
(s, 2H), 4.75 (s, 1H), 4.49–4.41 (m, 2H), 4.33–4.24 (m, 3H), 3.68
(s, 3H), 3.09–3.02 (m, 1H), 2.85–2.73 (m, 3H), 1.56 (s, 3H), 1.47
(s, 3H); MS (TOF) m/z: 635 [M+H]+; HRMS (FAB+) calcd for
C34H39N3O7SNa [M+Na]+ 656.2406, found m/z 656.2404.
5.2.14. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2-
hydroxyphenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (25)
Compound 25 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 9.19 (s, 1H), 8.45 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 8.4 Hz,
1H), 7.43–7.28 (m, 3H), 7.22–7.05 (m, 6H), 6.82–6.77 (m, 3H),
6.70–6.44 (m, 1H), 5.28 (dd, J = 8.4 Hz, 5.1 Hz, 1H), 5.05 (d,
5.2.19. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
hydroxymethylphenoxyacetyl)amino-2-hydroxy-4-phenylbuta-
noyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (30)
1H NMR (DMSO-d6) d (ppm): 8.28 (d, J = 8.7 Hz, 1H), 8.11 (d,
J = 9.0 Hz, 1H), 7.35–7.06 (m, 11H), 6.73 (d, J = 8.7 Hz, 2H), 5.40
(d, J = 6.9 Hz, 1H), 5.31–5.26 (m, 1H), 5.07–5.03 (m, 2H), 4.95 (s,
2H), 4.75 (s, 1H), 4.49–4.22 (m, 7H), 3.07–3.03 (m, 1H), 2.84–2.68

K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
10057
(m, 3H), 1.55 (s, 3H), 1.46 (s, 3H); MS (TOF) m/z: 635 [M+H]+;
HRMS (FAB+) calcd for C34H39N3O7SNa [M+Na]+ 656.2406, found
m/z 656.2400.
(s, 3H); MS (TOF) m/z: 650 [M+H]+; HRMS (FAB+) calcd for
C33H36N4O8SNa [M+Na]+ 671.2152, found m/z 671.2159.
5.2.25. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
nitrophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (36)
5.2.20. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2-
aminophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (31)
Compound 36 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.40 (d, J = 9.0 Hz, 1H), 8.14–8.06 (m, 3H), 7.35–7.04 (m,
9H), 6.92 (d, J = 9.0 Hz, 2H), 5.27 (dd, J = 8.7 Hz, 5.1 Hz, 1H), 4.96
(d, J = 2.4 Hz, 2H), 4.75 (s, 1H), 4.63 (s, 2H), 4.50 (d, J = 3.3 Hz,
1H), 4.43–4.39 (m, 1H), 4.32–4.22 (m, 1H), 3.04 (dd, J = 16.1 Hz,
4.7 Hz, 2H), 2.89–2.68 (m, 3H), 1.55 (s, 3H), 1.46 (s, 3H); MS
(TOF) m/z: 650 [M+H]+; HRMS (FAB+) calcd for C33H36N4O8SNa
[M+Na]+ 671.2152, found m/z 671.2148.
Compound 31 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.16 (d, J = 9.0 Hz, 1H), 7.30–7.12 (m, 10H), 6.95–6.87 (m,
4H), 5.42 (br, 1H), 5.23–5.18 (m, 1H), 5.05 (br, 1H), 4.93 (s, 2H),
4.74 (d, J = 6.6 Hz, 1H), 4.58 (s, 1H), 4.55 (s, 2H), 4.42–4.35 (m,
1H), 4.14 (d, J = 6.9 Hz, 1H), 3.07–2.98 (m, 3H), 2.85–2.72 (m,
1H), 1.56 (s, 3H), 1.45 (s, 3H); MS (TOF) m/z: 620 [M+H]+; HRMS
(FAB+) calcd for C33H38N4O6SNa [M+Na]+ 641.2410, found m/z
641.2415.
5.2.26. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
difluorophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (37)
5.2.21. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(3-
aminophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (32)
Compound 37 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.17 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.33–7.27
(m, 3H), 7.22–7.04 (m, 9H), 5.28 (dd, J = 9.0 Hz, 4.8 Hz, 1H), 4.94 (s,
2H), 4.74 (s, 1H), 4.51–4.47 (m, 3H), 4.43–4.39 (m, 1H), 4.33–4.24
(m, 1H), 3.08–3.01 (m, 1H), 2.87–2.78 (m, 2H), 2.75–2.66 (m, 1H),
1.56 (s, 3H), 1.46 (s, 3H); MS (TOF) m/z: 641 [M+H]+; HRMS (FAB+)
calcd for C33H35F2N3O6SNa [M+Na]+ 662.2112, found m/z 662.2108.
Compound 32 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.37 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.35–7.08
(m, 10H), 6.95–6.81 (m, 2H), 6.64 (d, J = 8.1 Hz, 1H), 5.29–5.25
(m, 1H), 4.97 (d, J = 3.6 Hz, 2H), 4.75 (s 1H), 4.51 (d, J = 3.0 Hz,
3H), 4.46–4.37 (m, 3H), 4.33–4.25 (m, 2H), 3.05–3.01 (m, 1H),
2.85–2.73 (m, 3H), 1.55 (s, 3H), 1.47 (s, 3H); MS (TOF) m/z: 620
[M+H]+; HRMS (FAB+) calcd for C33H38N4O6SNa [M+Na]+
641.2410, found m/z 641.2415.
5.2.27. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dichlorophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide (38)
5.2.22. (R)-N-[(1S,2R)-2-Hhydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
aminophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (33)
Compound 38 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.17 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.48 (d,
J = 4.1 Hz, 2H), 7.36 (d, J = 7.2 Hz, 2H), 7.29 (d, J = 7.5 Hz, 1H),
7.24–7.11 (m, 6H), 7.04 (t, J = 7.1 Hz, 1H), 5.42 (br, 1H), 5.31–5.26
(m, 1H), 5.17 (br, 1H), 4.96 (d, J = 5.1 Hz, 2H), 4.75 (s, 1H), 4.54–
4.50 (m, 1H), 4.44–4.36 (m, 3H), 4.25 (d, J = 13.8 Hz, 1H), 3.05
(dd, J = 16.1 Hz, 5.0 Hz, 1H), 2.91–2.71 (m, 3H), 1.56 (s, 3H), 1.48
(s, 3H); MS (TOF) m/z: 673 [M+H]+; HRMS (FAB+) calcd for
C33H35Cl2N3O6SNa [M+Na]+ 694.1521, found m/z 694.1526.
Compound 33 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.58 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.35–7.08
(m, 11H), 6.86–6.80 (m, 2H), 5.30–5.25 (m, 1H), 4.96 (d,
J = 3.0 Hz, 2H), 4.76 (s, 1H), 4.49–4.41 (m, 4H), 4.34–4.24 (m, 1H),
3.05 (d, J = 11.4 Hz, 1H), 2.85–2.72 (m, 3H), 1.55 (s, 3H), 1.47 (s,
3H); MS (TOF) m/z: 620 [M+H]+; HRMS (FAB+) calcd for
C33H38N4O6SNa [M+Na]+ 641.2410, found m/z 641.2403.
5.2.23. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2-
nitrophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (34)
5.2.28. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethoxyphenoxyacetyl)amino-2-hydroxy-4-phenylbuta-
noyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (39)
Compound 34 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.18 (d, J = 8.7 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.88 (dd,
J = 8.1 Hz, 1.8 Hz, 1H), 7.33–7.27 (m, 3H), 7.27–7.06 (m, 7H), 6.86
(d, J = 8.4 Hz, 1H), 5.35 (br, 1H), 5.27 8.18 (dd, J = 8.9 Hz, 5.0 Hz,
1H), 5.15 (br, 1H), 4.96 (s, 2H), 4.75 (s, 1H), 4.64 (s, 2H), 4.54–4.49
(m, 1H), 4.43–4.38 (m, 1H), 4.32–4.23 (m, 1H), 3.04 (dd,
J = 15.9 Hz, 4.8 Hz, 1H), 2.90–2.78 (m, 2H), 2.75–2.63 (m, 1H), 1.55
(s, 3H), 1.47 (s, 3H); MS (TOF) m/z: 650 [M+H]+; HRMS (FAB+) calcd
for C33H36N4O8SNa [M+Na]+ 671.2152, found m/z 671.2155.
Compound 39 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.27 (d, J = 9.3 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.28 (d,
J = 7.2 Hz, 2H), 7.23–7.03 (m, 10H), 6.73 (d, J = 8.4 Hz, 2H), 5.28
(dd, J = 8.4 Hz, 5.1 Hz, 1H), 5.00 (d, J = 9.0 Hz, 1H), 4.91 (d,
J = 9.0 Hz, 1H), 4.77 (s, 1H), 4.52 (d, J = 2.7 Hz, 1H), 4.44–4.37 (m,
2H), 4.30 (d, J = 15.9 Hz, 1H), 4.17 (d, J = 15.6 Hz, 1H), 3.84 (s,
6H), 3.05 (dd, J = 16.2 Hz, 4.5 Hz, 1H), 2.92–2.60 (m, 3H), 1.56 (s,
3H), 1.48 (s, 3H); MS (TOF) m/z: 665 [M+H]+; HRMS (FAB+) calcd
for C33H41N3O8SNa [M+Na]+ 686.2512, found m/z 686.2505.
5.2.24. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(3-
nitrophenoxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (35)
5.2.29. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
diisopropylphenoxyacetyl)amino-2-hydroxy-4-phenylbuta-
noyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (40)
Compound 35 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.34 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.83–7.78
(m, 1H), 7.65 (t, J = 2.3 Hz, 1H), 7.50 (t, J = 8.3 Hz, 1H), 7.31–7.04
(m, 10H), 5.30–5.24 (m, 1H), 4.96 (d, J = 3.3 Hz, 2H), 4.75 (s, 1H),
4.57 (s, 2H), 4.50 (d, J = 3.3 Hz, 1H), 4.43–4.39 (m, 1H), 4.33–4.22
(m, 1H), 3.08–3.00 (m, 1H), 2.88–2.68 (m, 3H), 1.55 (s, 3H), 1.46
Compound 40 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.14 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.36 (d,
J = 6.9 Hz, 2H), 7.30–7.09 (m, 9H), 7.02 (t, J = 7.5 Hz, 1H), 5.27 (dd,
J = 8.4 Hz, 5.1 Hz, 1H), 4.99 (s, 2H), 4.76 (s, 1H), 4.54 (d, J = 3.3 Hz,
1H), 4.46–4.37 (m, 2H), 4.13 (d, J = 14.1 Hz, 1H), 3.96 (d, J = 14.1 Hz,
1H), 3.23–3.01 (m, 3H), 2.94–2.74 (m, 3H), 1.56 (s, 3H), 1.49 (s, 3H),

10058
K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
1.14–1.10(m, 12H);MS(TOF)m/z:689[M+H]+;HRMS(FAB+)calcdfor
C39H49N3O6SNa [M+Na]+ 710.3240, found m/z 710.3237.
J = 6.9 Hz, 2H), 7.30–7.11 (m, 6H), 7.03 (t, J = 6.9 Hz, 1H), 6.66 (s,
2H), 5.28 (dd, J = 8.6 Hz, 5.0 Hz, 1H), 4.96 (d, J = 2.7 Hz, 2H), 4.75
(s, 1H), 4.50 (d, J = 3.3 Hz, 1H), 4.44–4.34 (m, 2H), 4.15 (d,
J = 14.4 Hz, 1H), 3.94 (d, J = 14.1 Hz, 1H), 3.04 (dd, J = 16.1 Hz,
5.3 Hz, 1H), 2.91 (s, 6H), 2.88–2.75 (m, 3H), 2.14 (s, 6H), 1.56 (s,
3H), 1.48 (s, 3H); MS (TOF) m/z: 676 [M+H]+; HRMS (FAB+) calcd
for C37H46N4O6SNa [M+Na]+ 697.3036, found m/z 697.3042.
5.2.30. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-fluorophenoxyacetyl)amino-2-hydroxy-4-phenyl-
butanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (41)
Compound 41 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.20 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.37 (d,
J = 6.9 Hz, 2H), 7.29–7.11 (m, 6H), 7.02 (d, J = 7.1 Hz, 1H), 6.86 (s,
1H), 6.83 (s, 1H), 5.27 (dd, J = 8.4 Hz, 5.1 Hz, 1H), 4.96 (s, 2H),
4.75 (s, 1H), 4.50 (d, J = 3.3 Hz, 1H), 4.44–4.29 (m, 2H), 4.18 (d,
J = 14.1 Hz, 1H), 3.99 (d, J = 14.1 Hz, 1H), 3.04 (dd, J = 15.9 Hz,
5.1 Hz, 1H), 2.92–2.71 (m, 3H), 2.15 (s, 6H), 1.56 (s, 3H), 1.48 (s,
3H); MS (TOF) m/z: 650 [M+H]+; HRMS (FAB+) calcd for
C35H40FN3O6SNa [M+Na]+ 672.2520, found m/z 672.2516.
5.2.35. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-ethylamino-phenoxyacetyl)amino-2-hydroxy-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(46)
Compound 46 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.17 (d, J = 8.7 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.37 (d,
J = 6.9 Hz, 2H), 7.31–7.10 (m, 6H), 7.03 (t, J = 6.9 Hz, 1H), 6.59 (s,
2H), 5.28 (dd, J = 8.1 Hz, 5.1 Hz, 1H), 4.96 (d, J = 2.4 Hz, 2H), 4.75
(s, 1H), 4.50 (d, J = 3.3 Hz, 1H), 4.44–4.34 (m, 2H), 4.16 (d,
J = 13.8 Hz, 1H), 3.95 (d, J = 13.8 Hz, 1H), 3.12–3.00 (m, 3H), 2.90–
2.72 (m, 3H), 2.11 (s, 6H), 1.56 (s, 3H), 1.48 (s, 3H), 1.15 (t,
J = 7.1 Hz, 3H); MS (TOF) m/z: 676 [M+H]+; HRMS (FAB+) calcd
for C37H46N4O6SNa [M+Na]+ 697.3036, found m/z 697.3030.
5.2.31. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-hydroxyphenoxyacetyl)amino-2-hydroxy-4-phen-
ylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (42)
Compound 42 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.97 (s, 1H), 8.15 (d, J = 9.0 Hz, 1H), 8.10 (d, J = 9.0 Hz, 1H),
7.37 (d, J = 7.2 Hz, 2H), 7.30–7.11 (m, 6H), 7.06–7.00 (m, 1H), 6.37
(s, 2H), 5.44 (d, J = 6.6 Hz, 1H), 5.30–5.26 (m, 1H), 5.04 (d,
J = 4.2 Hz, 1H), 4.96 (s, 2H), 4.76 (s, 1H), 4.52–4.46 (m, 1H), 4.42–
4.26 (m, 1H), 4.11 (d, J = 14.4 Hz, 1H), 3.89 (d, J = 14.4 Hz, 1H),
3.08–3.01 (m, 1H), 2.89–2.70 (m, 3H), 2.09–2.07 (m, 6H), 1.56 (s,
3H), 1.48 (s, 3H); MS (TOF) m/z: 649 [M+H]+; HRMS (FAB+) calcd
for C35H41N3O7SNa [M+Na]+ 670.2563, found m/z 670.2558.
5.2.36. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-(pyridine-2-ylmethyl)amino-phenoxyacetyl)amino-
2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (47)
Compound 47 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.61 (d, J = 5.4 Hz, 1H), 8.12–8.08 (m, 2H), 7.98 (t,
J = 7.2 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.46 (t, J = 6.6 Hz, 1H), 7.36
(d, J = 7.2 Hz, 2H), 7.30–7.09 (m, 6H), 7.02 (t, J = 7.8 Hz, 1H), 6.26
(s, 2H), 5.30–5.26 (m, 1H), 4.95 (d, J = 3.0 Hz, 2H), 4.75 (s, 1H),
4.49 (d, J = 3.6 Hz, 1H), 4.44–4.37 (m, 4H), 4.09 (d, J = 15.0 Hz,
1H), 3.87 (d, J = 14.4 Hz, 1H), 3.04 (dd, J = 16.8 Hz, 5.4 Hz, 1H),
2.86–2.77 (m, 3H), 2.03 (s, 6H), 1.56 (s, 3H), 1.48 (s, 3H); MS
(TOF) m/z: 739 [M+H]+; HRMS (FAB+) calcd for C41H47N5O6SNa
[M+Na]+ 760.3145, found m/z 760.3148.
5.2.32. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(4-
amino-2,6-dimethylphenoxyacetyl)amino-2-hydroxy-4-phen-
ylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (43)
Compound 43 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.21 (d, J = 8.8 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.37 (d,
J = 7.3 Hz, 2H), 7.31–7.10 (m, 8H), 7.03 (t, J = 7.2 Hz, 1H), 6.78 (s,
2H), 5.28 (dd, J = 8.1 Hz, 4.7 Hz, 1H), 4.96 (s, 2H), 4.76 (s, 1H),
4.50 (d, J = 2.9 Hz, 1H), 4.45–4.27 (m, 2H), 4.19 (d, J = 14.3 Hz,
1H), 4.07–3.96 (m, 1H), 3.05 (dd, J = 16.0 Hz, 5.1 Hz, 1H), 2.92–
2.71 (m, 3H), 2.14 (s, 6H), 1.56 (s, 3H), 1.48 (s, 3H); HRMS (FAB+)
calcd for C35H42N4O6SNa [M+Na]+ 669.2723, found m/z 669.2728.
5.2.37. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-(pyridine-3-ylmethyl)amino-phenoxyacetyl)amino-
2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (48)
Compound 48 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.66 (s, 1H), 8.57 (d, J = 3.6 Hz, 1H), 8.10 (d, J = 8.4 Hz,
2H), 8.01 (t, J = 6.9 Hz, 1H), 7.62–7.53 (m, 2H), 7.36 (d, J = 6.6 Hz,
2H), 7.30–7.12 (m, 8H), 7.01 (t, J = 3.6 Hz, 1H), 6.25 (s, 2H), 5.28
(dd, J = 8.1 Hz, 4.2 Hz, 1H), 4.96 (s, 2H), 4.76 (s, 1H), 4.49 (d,
J = 3.6 Hz, 1H), 4.44–4.28 (m, 4H), 4.08 (d, J = 14.4 Hz, 1H), 3.86
(d, J = 14.4 Hz, 1H, overlapped with water), 3.04 (dd, J = 15.9 Hz,
3.9 Hz, 1H), 2.86–2.77 (m, 3H), 2.22–2.03 (m, 6H), 1.55 (s, 3H),
1.48 (s, 3H); MS (TOF) m/z: 739 [M+H]+; HRMS (FAB+) calcd for
C41H47N5O6SNa [M+Na]+ 760.3145, found m/z 760.3148.
5.2.33. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-methylamino-phenoxyacetyl)amino-2-hydroxy-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(44)
Compound 44 was prepared from intermediate 18 in a manner
similar to that described for compound 49. 1H NMR (DMSO-d6) d
(ppm): 8.14 (d, J = 9.0 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.37 (d,
J = 6.9 Hz, 2H), 7.30–7.11 (m, 6H), 7.03 (t, J = 7.2 Hz, 1H), 6.54 (s,
2H), 5.28 (dd, J = 8.6 Hz, 5.0 Hz, 1H), 4.96 (d, J = 2.7 Hz, 2H), 4.75
(s, 1H), 4.50 (d, J = 3.3 Hz, 1H), 4.44–4.34 (m, 2H), 4.15 (d,
J = 14.4 Hz, 1H), 3.94 (d, J = 14.1 Hz, 1H), 3.05 (dd, J = 16.1v,
5.3 Hz, 1H), 2.91–2.77 (m, 3H), 2.73 (s, 3H), 2.11 (s, 6H), 1.56 (s,
3H), 1.48 (s, 3H); MS (TOF) m/z: 662 [M+H]+; HRMS (FAB+) calcd
for C36H44N4O6SNa [M+Na]+ 683.2879, found m/z 683.2876.
5.2.38. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-(pyridine-4-ylmethyl)amino-phenoxyacetyl)amino-
2-hydroxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (49)
5.2.34. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-dimethylamino-phenoxyacetyl)amino-2-hydroxy-
4-phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (45)
Compound 45 was prepared from intermediate 18 in a manner
similar to that described for compound 19. 1H NMR (DMSO-d6) d
(ppm): 8.15 (d, J = 8.7 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.37 (d,
To a solution of 18 (50.6 mg, 0.10 mmol) in DMF (2 mL) were
added triethylamine (34.9 lL, 0.25 mmol), compound 15 (42.5
mg, 0.11 mmol) and BOP (48.7 mg, 0.11 mmol) in an ice-water
bath and the mixture was stirred overnight at room temperature.
After removal of the solvent in vacuo, the residue was diluted with
EtOAc, washed sequentially with 5% NaHCO3, and saturated NaCl,
dried over MgSO4, and concentrated in vacuo. A mixture of the

K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
10059
intermediate, thioanisole (35.2
l
L, 0.3 mmol), m-cresol (31.4
l
L,
NO2)-Glu-Ala-Nle-NH2] was synthesized by conventional solid-
phase method. In the inhibition assay, 25 L of 200 mM 2-(N-mor-
pholino)ethanesulfonic acid (MES)–NaOH buffer (pH 5.5), contain-
0.3 mmol) and 4M HCl (1 mL) in dioxane was stirred at room tem-
perature for 1 h. After removal of the solvent in vacuo, the residue
was precipitated from ether to give the crude product. Purification
of the product by preparative HPLC gave the titled compound as a
yellowish white foam; yield 38%; 1H NMR (DMSO-d6) d (ppm):
8.80–8.68 (m, 2H), 8.08 (d, J = 8.7 Hz, 1H), 7.90–7.75 (m, 2H),
7.36 (d, J = 8.7 Hz, 2H), 7.31–7.11 (m, 8H), 7.07–7.00 (m, 1H),
6.19 (s, 2H), 5.30–5.24 (m, 1H), 4.97 (s, 2H), 4.74 (s, 1H), 4.52–
4.33 (m, 5H), 4.08 (d, J = 13.5 Hz, 1H, overlapped with water),
3.85 (d, J = 13.5 Hz, 1H, overlapped with water), 3.09–3.00 (m,
1H), 2.86–2.75 (m, 3H), 2.22–2.01 (m, 6H), 1.56 (s, 3H), 1.47 (s,
3H); MS (TOF) m/z: 739 [M+H]+; HRMS (FAB+) calcd for
C41H47N5O6SNa [M+Na]+ 760.3145, found m/z 760.3140.
l
ing 2 mM dithiothreitol, 2 mM EDTA–2Na and 1 M NaCl was mixed
with 5
lL of the inhibitor (500 nM) dissolved in DMSO and 10
lL of
HIV-1 protease (2
lg/mL) in 50 mM AcOH (pH 5.0) containing
1 mM EDTA-2Na, 25 mM NaCl, 0.2% 2-mercaptoethanol, 0.2% Non-
idet P-40, and 10% glycerol. The reaction was initiated by adding of
10 lL of a 1.0 mM substrate solution. After incubation for 15 min at
37 °C, the reaction was terminated by addition of 1M HCl, and the
amount of cleaved N-terminal fragment (H-Lys-Ala-Arg-Val-Tyr-
OH) was measured by reversed-phase HPLC on a C18 column
(3.0 ꢂ 75 mm; YMC Pack ODS AS-3E7) with a linear gradient of
CH3CN in 0.8% aqueous TFA at a flow rate of 1.0 mL/min and the
quantity was determined by monitoring fluorescence intensity
(Ex, 275 nm; Em, 305 nm). Percent inhibition was obtained com-
pared to intensity without inhibitor.
5.2.39. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-butylamino-phenoxyacetyl)amino-2-hydroxy-4-phen-
ylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide (50)
To a solution of crude 43 (50 mg, 0.07 mmol) in MeOH (0.2 mL)
5.5. Anti-malarial activity
were added triethylamine (12.3
lL, 0.09 mmol), butyraldehyde
(6.6 L, 0.07 mmol) and NaBH3CN (27.6 mg, 0.44 mmol) in an
l
Activity against P. falciparum was determined as described pre-
viously.28 Briefly, chloroquine-sensitive infected cells (NF54) were
maintained in a 2.4% suspension of type O+ human erythrocytes in
RPMI 1640, supplemented with 25 mM HEPES, 27 mM NaHCO3,
and 10% heat inactivated human type O+ serum, under 3% O2, 4%
CO2, and 93% N2. DMSO solution of test compounds was serially di-
luted in 0.2% DMSO in medium. Parasite culture was added, incu-
ice-water bath and the mixture was stirred for 6 h at room temper-
ature. After removal of the solvent in vacuo, the residue was di-
luted with EtOAc, washed 5% NaHCO3, and saturated NaCl, dried
over MgSO4, and concentrated in vacuo. Purification of the product
by preparative HPLC gave the titled compound as a white foam;
yield 20%; 1H NMR (DMSO-d6) d (ppm): 8.18 (d, J = 9.0 Hz, 1H),
8.10 (d, J = 8.7 Hz, 1H), 7.37 (d, J = 7.2 Hz, 2H), 7.31–7.10 (m, 6H),
7.03 (t, J = 7.8 Hz, 1H), 6.64 (s, 2H), 5.28 (dd, J = 8.6 Hz, 4.7 Hz,
1H), 4.96 (s, 2H), 4.76 (s, 1H), 4.50 (d, J = 3.3 Hz, 1H), 4.44–4.33
(m, 2H), 4.16 (d, J = 14.1 Hz, 1H), 3.96 (d, J = 14.7 Hz, 1H, over-
lapped with water), 3.10–3.00 (m, 3H), 2.92–2.72 (m, 3H), 2.12
(s, 6H), 1.58–1.46 (m, 8H), 1.35 (apparent q, J = 7.8 Hz, 1H), 0.89
(t, J = 7.4 Hz, 3H); MS (TOF) m/z: 704 [M+H]+; HRMS (FAB+) calcd
for C39H50N4O6SNa [M+Na]+ 725.3349, found m/z 725.3355.
bated for 48 h prior. After addition of 0.6 lCi of
[3H]hypoxanthine, the mixture was incubated for 20 h. Cells were
harvested onto GF-C glass filter, washed, dried, and counted in
scintillation cocktail. Drug concentration of 50% reduced the level
of incorporation of [3H]hypoxanthine was determined from dose-
response curves. The cytotoxicity of the inhibitors was evaluated
using L6 cells (rat skeletal myoblasts) using standard procedures.46
5.6. Molecular dynamics simulation of plasmepsin II binding
models
5.2.40. (R)-N-[(1S,2R)-2-Hydroxyindan-1-yl]-3-[(2S,3S)-3-(2,6-
dimethyl-4-pentylamino-phenoxyacetyl)amino-2-hydroxy-4-
phenylbutanoyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(51)
An X-ray crystal structure of Plm IV complexed with compound
1 (PDB, 2ANL) was aligned with Plm II (PDB, 1SME), and 1 was used
to build initial conformation of inhibitor 49 in Plm II using the
Molecular Operating Environment modeling package (MOE
2007.09, Chemical Computing Group, Inc., Montreal, Canada) with
MMFF94x force field. A carboxyl group of an Asp214 close to the
HMC carbonyl group was protonated. Anilinyl and pyridinyl nitro-
gens of 49 were protonated to simulate acidic assay condition. The
binding area was immersed in a 20 Å sphere of TIP3P water from
the ligand. The inhibitor, contacted residues and flap region of
the enzyme, and surrounded water were energy-minimized while
the other atoms were fixed. MD simulations were performed with-
out heating at 298 K for 300 ps equilibration with 1.5 fs time step.
An additional 50 ps of MD simulation was performed for data col-
lection. Alignment with crystal data was performed using MOE
Align. Pictures were generated using MacPyMOL (DeLano Scientific
LLC, CA, USA).
Compound 51 was prepared from intermediate 43 in a manner
similar to that described for compound 50. 1H NMR (DMSO-d6) d
(ppm): 8.18 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 7.37 (d,
J = 6.9 Hz, 2H), 7.31–7.10 (m, 6H), 7.03 (t, J = 7.2 Hz, 1H), 6.60 (s,
2H), 5.31–5.25 (m, 1H), 4.96 (s, 2H), 4.76 (s, 1H), 4.50 (d,
J = 3.3 Hz, 1H), 4.44–4.34 (m, 2H), 4.16 (d, J = 14.1 Hz, 1H), 3.95
(d, J = 14.1 Hz, 1H), 3.09–3.00 (m, 3H), 2.87–2.77 (m, 3H), 2.11 (s,
6H), 1.58–1.46 (m, 8H), 1.34–1.27 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H);
MS (TOF) m/z: 718 [M+H]+; HRMS (FAB+) calcd for C40H52N4O6SNa
[M+Na]+ 739.3505, found m/z 739.3511.
5.3. Plasmepsin II inhibitory activity
Inhibition constants (Ki) against Plm II were obtained as de-
scribed previously.28 Briefly, the rate of substrate hydrolysis at
25 °C was measured using 400 nM protease in 10 mM sodium for-
mate (pH 4.0), 163
l
M chromogenic substrate [H-Ala-Leu-Glu-
Acknowledgments
Arg-Thr-Phe-Phe(p-NO2)-Ser-Phe-Pro-Thr-OH] which is purchased
from California Peptide Research Inc., Napa, CA and 2% DMSO with
increasing amounts of inhibitor. Ki were estimated by fitting the
data to standard equations for tight binding competitive inhibitors.
This research was supported in part by the Frontier Research
Program, the 21st Century COE Program of Ministry of Educa-
tion, Culture, Sports, Science, and Technology of Japan (MEXT),
and grants from MEXT. E.F. and A.J.R. also thank the Johns Hop-
kins Malaria Institute for a Pilot Research Grant and a Graduate
Student Fellowship as well as grants from the National Institutes
of Health (GM57144 and GM56550) and the National Science
Foundation (MCB-0641252). We gratefully acknowledge Mr. T.
5.4. HIV protease inhibitory activity
Recombinant HIV-1 PR was purchased from Bachem AG, Buben-
dorf-Switzerland. HIV PR substrate [H-Lys-Ala-Arg-Val-Tyr-Phe(p-

10060
K. Hidaka et al. / Bioorg. Med. Chem. 16 (2008) 10049–10060
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