New water-soluble prodrugs of HIV protease inhibitors based on O→N intramolecular acyl migration

Article abstract of DOI:10.1016/S0968-0896(02)00322-X

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns-Thz core structure (Apns; allophenylnorstatine, Thz; thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs based on the O→N intramolecular acyl migration reaction at the α-hydroxy-β-amino acid residue, that is allophenylnorstatine. The synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300 mg/mL) more than 4000-fold in comparison with the parent compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the aqueous condition from slightly acidic to basic pH at 37°C, with the suitable migration rate, via a five-membered ring intermediate. Using a similar method, we synthesized a prodrug (12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-membered ring intermediate, with the t_1/2 value of 32 h that may not be suitable for practical use.

Full text of DOI:10.1016/S0968-0896(02)00322-X

Bioorganic & Medicinal Chemistry 10 (2002) 4155–4167
New Water-Soluble Prodrugs of HIV Protease Inhibitors Based
on O!N Intramolecular Acyl Migration
Yoshio Hamada, Jun Ohtake, Youhei Sohma, Tooru Kimura, Yoshio Hayashi
and Yoshiaki Kiso*
DepartmentofMedicinalChemistry,CenterforFrontierResearchinMedicinalScience,KyotoPharmaceuticalUniversity,Yamashina-Ku,
Kyoto 607-8412, Japan
Received 13 June 2002; accepted 20 July 2002
Abstract—To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and
KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns–Thz core structure (Apns; allophenylnorstatine, Thz;
thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with
an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs
based on the O!N intramolecular acyl migration reaction at the a-hydroxy-b-amino acid residue, that is allophenylnorstatine. The
synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300 mg/mL) more than 4000-fold in comparison with the parent
compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a
strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the
aqueous condition from slightly acidic to basic pH at 37 ꢀC, with the suitable migration rate, via a five-membered ring intermediate.
Using a similar method, we synthesized a prodrug (12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS
drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-
membered ring intermediate, with the t1/2 value of 32 h that may not be suitable for practical use.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
KNI-27213À15 (1a) and KNI-27913 (2a), which contain
an allophenylnorstatine (Apns) residue as a hydro-
xymethylcarbonyl (HMC) isostere derived from a nat-
ural scissile amino acid sequence ‘Phe-Pro’ based on the
substrate–transition strategy13À17 (Fig. 1). However,
these peptidomimetics as well as other HIV-1 PR inhibi-
tors such as ritonavir18,19 and amprenavir20 exhibited sig-
nificantly low water-solubility in the physiological media.
Human immunodeficiency virus type 1 (HIV-1) encodes
an aspartic protease that is responsible for the proces-
sing of viral precursor proteins such as gag and gag-pol
polyproteins to form mature structural proteins and
functional enzymes required in the production of infec-
tive viral particles.1À3 Therefore, HIV-1 protease (HIV-1
PR) has been an attractive target for the design of inhi-
bitors for effective antiviral therapy. Recently, many
potent protease inhibitors that bind to the active site of
HIV-1 PR have been developed based on the strategy of
a substrate transition-state mimic, and several com-
pounds are clinically used in combination therapies for
AIDS.4À8 However, the low water-solubility of these
anti-AIDS drugs is a serious problem9À12 causing unde-
sirable pharmaceutical properties such as erratic oral
absorption and poor oral bioavailability. Recently,
Mimoto et al. reported potent HIV-1 PR inhibitors,
To overcome this low water-solubility, one effective
strategy is to convert the water-insoluble parent drugs
into hydrophilic prodrugs by covalently attaching the
appropriate solubilizing moieties such as phos-
phates,21,22 sugars23,24 and amines,25,26 which can elim-
inate the parent drugs enzymatically or chemically
under physiological conditions. The phosphate-type
water-soluble prodrugs of HIV-1 PR inhibitors were
reported by Thaisrivongs et al.11 We recently reported
distinct water-soluble prodrugs27 derived from HIV-1
PR inhibitors which contain an Apns core. These pro-
drugs were designed to enable a chemical regeneration
of the parent compound adopting a unique pH sensitive
self-cleavable linker.
*Corresponding author. Tel.: +81-75-595-4636; fax: +81-75-595-
4787; e-mail: kiso@mb.kyoto-phu.ac.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00322-X

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Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
On the other hand, as shown in Figure 2a, it is well
known that an acyl migration reaction is observed
between adjacent amino and hydroxyl groups, and the
formation of O-acylpeptides occurs when the peptides
containing b-hydroxyamino acids such as serine and
threonine residues are exposed to strong acids.28 The
solubility of these O-acylpeptides in aqueous media
generally increases by the newly produced amino group,
and the reverse reaction to the peptides can be achieved
by the pH shift to weak basic condition in aqueous
media.29
By introducing this pH-dependent reversible group into
prodrugs, Hurley et al.30 reported prodrugs of peptido-
mimetic inhibitors of renin, which is an aspartic pro-
tease responsible for the regulation of blood pressure in
the cardiovascular system. This strategy seemed to be
applicable to HIV-1 PR inhibitors such as KNI-272 (1a)
and KNI-279 (2a), since the Apns residue in these com-
pounds has both adjacent a-hydroxyl and b-amino
groups, and an acyl group connected to hydroxyl group
of the Apns residue would be interconvertible through
the formation of an energetically favorable five-mem-
bered ring intermediate (Fig. 2a). Thus, we
designed31À33 prodrugs 3 and 4 of KNI-272 and -279
based on O!N intramolecular acyl migration reaction.
In this paper, the design and synthesis of a series of
prodrugs 3–11 (Table 1), which are O-acyl derivatives
derived from the structures of KNI-272 (1a) and KNI-
279 (2a), are described. We evaluated the solubility and
Figure 1. Structure of KNI-272 (1a), KNI-279 (2a) and these analo-
gues.
Figure 2. Design of prodrug based in the O!N acyl migration. (a) N!O acyl migration and O!N acyl migration reaction; (b) prodrug of KNI-
272; (c) Prodrug of ritonavir.

Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
Table 1. Water-solubility and O!N migration rate of prodrugs
4157
Prodrug
Solubility (mg/mL)
Ratio of solubility
t1/2a(min)
Prodrug
Parent drug
(prodrug/parent drug)
pH 4.9
pH 5.5
pH 7.4
pH 8.0
3
4
5
6
7
8
9
10
11
>300
>300
—
>300
>300
0.075
0.079
—
0.055
0.041
0.0008
0.0025
0.0015
0.002
>4000
>3797
—
>5455
>7317
4625
1600
2000
1300
28
820
—
34
822
35
800
47
816
7
290
28
<1
7.7
<1
<1
8.6
<1
10
<1
5.9
<1
<1
6.6
<1
8.7
<1
13
8
305
7.8
291
8.6
310
3.7
3
4
2.6
<1
22
at1/2 is the time required for 50% release of parent drugs at 37 ꢀC in PBS (pH 7.4).
Chemistry
the ability of the O!N intramolecular acyl migration
reaction of these prodrugs to regenerate the parent
compounds. For comparison, a prodrug 12 of ritonavir,
a clinically approved inhibitor, was also synthesized,
since the O!N intramolecular acyl migration reaction
in the prodrug of ritonavir was expected to proceed
through the formation of an energetically less favorable
six-membered ring intermediate (Fig. 2c).
The synthesis of prodrugs 3–11 is shown in Scheme 1.
The Boc groupof the starting materials 13a and 13b13,34
was deprotected with 4 N-HCl/dioxane, followed by
coupling with Boc-Apns-OH35,36 using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide HCl (EDC HCl) in
the presence of HOBt,37À40 resulting in protected
.
.
.
Scheme 1. Reagents: (a) 4 N-HCl/dioxane; (b) Boc-Apns-OH, EDC HCl, HOBt, Et3N, DMF; (c) Z-OSu, Et3N, DMF; (d) N-Boc-amino acids (16a;
.
Boc-Mta-OH, 16b; Boc-Val-OH, 16c; Boc-Nva-OH), DCC, DMAP, CH2Cl2; (e) iQoa or Noa, EDC HCl, HOBt, Et3N, DMF; (f) TFA, dimethyl-
sulfide, anisole; (g) 4 N-HCl/EtOAc.

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Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
dipeptides 14a and 14b. To convert the Boc groupto Z
groupin 14a and 14b, the deprotection of the Boc group
and subsequent introduction of a Z groupusing N-
(benzyloxycarbonyloxy)succinimide (Z-OSu) were per-
formed to give 15a and 15b. In the next step, three kinds
of Boc-amino acids were introduced to the a-hydroxyl
groupof Apns in 15a and 15b using DCC in the pre-
sence of a catalytic amount of DMAP to afford O-acyl
dipeptides 17–19. Although partial racemization (less
than 1–3%) at the a-carbon of the introduced Boc-
amino acids was observed during this condensation, the
racemized compounds could be removed by preparative
HPLC purification at the last step of the synthesis. After
the Boc groupdeprotection of 17–19, condensation with
5-isoquinoyloxyacetic acid (iQoa) or 1-naphtoxyacetic
acid (Noa) was carried out to afford the protected pro-
drugs 20–24. Final deprotection of the Z group in 20–24
using TFA-anisole-dimethylsulfide by ‘push–pull’
mechanism,41 and subsequent purification by pre-
parative HPLC. Then ion-exchange treatment using
4 N-HCl/EtOAc gave a hydrochloride salt of prodrugs
3–11.
ester 30. The deprotection of 30 using 4N-HCl/dioxane
gave the desired ritonavir prodrug 12.
Results and Discussion
Water-solubility of prodrugs
Water-solubility of synthetic prodrugs was determined
and compared to that of their parent compounds (Table
1). In all cases, prodrugs exhibited more than 1000-fold
higher water-solubility than the corresponding parent
drugs, due to the existence of a hydrophilic amine
structure in these O-acyl structures. Especially, the
solubility of compounds 3, 4, 6, and 7 was >300 mg/
mL, which is a practicable value as water-soluble pro-
The parent compounds 1a–d, 2a, and 2c were synthe-
sized according to Mimoto et al.13 The parent com-
pounds 2b and 2d were synthesized from 25 by a
conventional protocol13 using EDC-HOBt for the
amide bond formation (Scheme 2).
The synthesis of ritonavir prodrug 12 is shown in
Scheme 3. (2S,3S,5S)-2-Amino-5-Boc-amino-1,6-diphe-
nyl-3-hydroxyhexane (26)42 was coupled with 5-hydroxy-
methylthiazole-4-nitrophenyl carbonate hydrochloride
(27)43 to give the compound 28, which was condensed
N-[N-[(2-isopropyl-4-thiazole)methyl]-N-methyl]car-
bonyl]-l-valine44À46 (29) using DCC-DMAP to give the
Scheme 2. Reagents: (a) 4 N-HCl/dioxane; (b) iQoa or Noa,
.
EDC HCl, HOBt, Et3N, DMF.
Scheme 3. Reagents: (a) THF–DMF; (b) DCC, DMAP, CH2Cl2; (c) 4 N-HCl/dioxane.

Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
4159
drugs. However, the compounds 8–11 exhibited lower
solubility with the values about 2–4 mg/mL, probably
resulting from the naphthalene structure at the P3 moi-
ety of the parent compounds. This result demonstrated
that the P3 moiety is also important to obtain water-
soluble prodrugs, since an isoquinoline structure of the
P3 position in 3, 4, 6, and 7 is able to form an HCl salt.
Under acidic conditions, a slower migration was
observed than in neutral or basic condition, probably
due to the decrease of nucleophilicity of the amino
group. Compound 3 showed rapid migration under
neutral conditions, that is, 74% of 3 was converted to
KNI-272 (1a) during the first 30 s after incubation at pH
7.4 with the t1/2 value of less than 1 min (Table 1 and
Fig. 4). However, the t1/2 value was 28 min at pH 4.9.
On the other hand, compound 4 exhibited relatively
slower migration with t1/2 values of 7.7 and 820 min,
which were more than 8- and 30-times longer than those
of 3 at pH 7.4 and 4.9, respectively. The structural dif-
ference of compounds 3 and 4 is in a side chain at the P2
position, and the compound 3, having a less bulky sul-
fur atom at this position, showed faster migration. To
investigate which factors are more significant for the
migration rate, compound 5, possessing a norvaline
(Nva) residue at the P2 position, was synthesized and
compared with compounds 3 and 4. The sulfur atom of
methylthioalanine (Mta) is substituted to a methylene
groupin Nva. A similar rapid migration of 5 as that of 3
was observed at pH 7.4, although compound 5 exhibited
Effects of pH and structures on the O!N acyl migration
of prodrugs
To study the O!N acyl migration, water-soluble pro-
drugs 3–11 were dissolved in phosphate-buffered saline
(PBS) with different pH values ranging from 4.9 to 8.0
and incubated at 37 ꢀC. The migration was monitored
by HPLC. Typical HPLC charts for compound 3 (a
prodrug of KNI-272 1a) in PBS (pH 7.4) are shown in
Figure 3 and the time course of the migration in pro-
drugs 3 and 4 (a prodrug of 2a) is shown in Figure 4. A
time-dependent O!N acyl migration reaction to parent
drugs was observed in both compounds 3 and 4. The
migration rate was dependent on the pH values used.
Figure 3. HPLC profiles of the O!N migration of compound 3 in
PBS (pH 7.4) at 37 ꢀC. A peak with a retention time of about 8 min
corresponds to compound 3 and a peak with a retention time of about
9 min corresponds to parent compound 1a.
Figure 4. Formation of parent compound in various pH conditions.
(a) prodrug 3; (b) prodrug 4.

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Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
a small increase of t1/2 value at pH 5.5. This result indi-
cated that the steric effect is more significant to accel-
erate the migration, although the existence of an
electrostatic sulfur atom at the P2 position may have
some effect to prompt migration. Thus, a sterically less
hindered side chain is effective for increasing the migra-
tion rate, and it is possible to control the migration rate
by modifying the side chain of the P2 site.
a six-membered ring intermediate in the migration step,
while the KNI-series prodrugs form an energetically
favorable five-membered ring intermediate,47 suggesting
that this strategy may not be useful for the water-solu-
ble prodrugs via the six-membered ring intermediate in
the formation of parent drugs.
Inhibitory activity of synthetic prodrugs
To investigate other structural factors which influence
the migration speed, the P3 and P10 sites were modified.
When the iQoa groupat the P3 site was substituted to a
more hydrophobic and less electrostatic Noa group in
compounds 8 and 9, almost the same t1/2 values were
obtained as those of 3 and 4, respectively, indicating
that this modification at the P3 position had no influ-
ence on the migration rate. In the P10 site, it is known
that the modification of Thz (R1=H) to Dmt
(R1=CH3, 5,5-dimethyl-1,3-thiazolidine-4-carboxylic
acid) improves HIV-1 PR inhibitory and antiviral
activity. Hence, compounds 6 and 7 with the Dmt resi-
due were synthesized and the effect of Dmt at the P10
position on the migration speed was evaluated. As
shown in Table 1, the t1/2 values of 6 and 7 did not show
any significant change compared to 3 and 4, suggesting
that this modification is also insignificant and applicable
to develop more potent compounds possessing the
Apns–Dmt core structure. Compounds 10 and 11 con-
taining both modifications at the P3 and P10 sites, also
did not show any significant change in migration time
with 3 and 4.
HIV-1 PR inhibitory activity of prodrugs 3, 4, 8, 9 and
12 was determined and shown in Table 2. About 60% of
the enzymatic activity was inhibited by 50 nM of
prodrugs 3 and 8. This observed HIV-1 PR inhibi-
tory activity of 3 and 8 is probably due to the
activity of their parent compounds formed during
incubation in the assay, since the incubation period
(5 min) is similar to the t1/2 values of these prodrugs
under the same incubation conditions (pH 5.5). Fur-
thermore, no inhibitory activity was observed in pro-
drugs 4 and 9, which exhibited longer t1/2 values than
3 and 8. These results indicate that the prodrugs have
no activity themselves.
On the other hand, when the prodrugs were pre-incu-
bated for 1 h under the same condition (pH 5.5 at
37 ꢀC), most of 3 and 8 was converted to the corre-
sponding parent drugs and exhibited higher inhibitory
From these results, it is suggested that the derivatization
of the compounds at a position away from the migra-
tion point such as the P3 and P10 sites will be tolerated,
and the strategy for the water-soluble prodrugs using
the N!O acyl migration reaction can be applicable as a
general method to increase water solubility of HIV-1
PR inhibitors based on the Apns–Thz and Apns–Dmt
core structures.
A prodrug (12) of ritonavir18,19 based on the O!N acyl
migration was also synthesized and the migration was
examined in the same manner as that of KNI-series
prodrugs. Although ritonavir has a Val residue at the P2
site as compound 4, prodrug 12 exhibited a t1/2 value of
32 h at pH 7.4 (Fig. 5) that was 250-fold longer than
that of 4. This significantly slow migration can be
attributed to the fact that the prodrug of ritonavir forms
Figure 5. Formation of ritonavir from prodrug 12 in various pH con-
ditions.
Table 2. HIV-1 protease inhibition of prodrugs
Prodrug
Inhibitiona (%)
Inhibitionb (%) (pre-incubation)
Conversionc (%) (pre-incubation)
3
4
8
9
12
66.6
3.9
55.9
À0.3
1.2
95.4
23.0
93.8
33.3
2.7
98
16
97
13
Not detectedd
a% of HIV-1 protease inhibition in the presence of 50 nM of prodrugs (enzyme and prodrugs were added at the same time).
b% of HIV-1 protease inhibition in the presence of 50 nM of prodrugs (prodrugs were pre-incubated for 1 h at 37 ꢀC before enzyme addition).
cFormation of parent compounds (%) from prodrugs after 1 h incubation at pH 5.5 at 37 ꢀC.
dRitonavir prodrug (12) which was indicated a t1/2 value of >1800 min in PBS (pH5.5) at 37 ꢀC, was rarely converted to ritonavir after 5 min
(enzyme assay) and 1 h (pre-incubation time) incubation.

Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
4161
activity of more than 90%. In more stable 4 and 9, less
than 20% was converted and exhibited relatively low
(20–30%) inhibitory activity. Prodrug 12, which has an
excessively longer t1/2 value of >30 h, was hardly con-
verted to ritonavir at pH 5.5 and did not show any
inhibitory activity in this enzyme assay. These data
suggested that the anti-HIV-1 PR activity was well cor-
related with the formation of the biologically active
parent compounds.
acid, 5% NaHCO3 and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The residue was
applied to a silica gel column (3.5ꢁ18 cm) and eluted
with hexane–EtOAc (1:1) to give 11.7 g (94%) of the
title compound 14a as a white solid: mp79–82 ꢀC; H
1
NMR (300 MHz, CDCl3) d 1.29 (s, 9H), 1.34 (s, 9H),
2.53–2.87 (m, 2H), 3.19 (dd, J=7.5, 11.7 Hz, 1H), 3.32
(br s, 1H), 3.55 (dd, J=6.9, 11.7 Hz, 1H), 3.72 (d,
J=6.6 Hz, 1H), 4.04 (m, 1H), 4.50–4.77 (m, 2H), 4.81
(dd, J=6.9, 7.5 Hz, 1H), 4.90–5.15 (m, 2H), 6.30 (br s,
1H), 7.09–7.37 (m, 5H); MS (FAB): m/z 466 [M+H]+.
Anal. calcd for C23H35N3O5S: H, 7.58; C, 59.33; N,
9.02. Found: H, 7.70; C, 59.01; N, 8.86.
Conclusion
New water-soluble prodrugs of HIV-1 PR inhibitors
KNI-272 and KNI-279 based on the N!O acyl migra-
tion reaction were developed, and the ability of these
prodrugs was evaluated. The prodrugs 3, 4, 6, and 7
exhibited high solubility in aqueous media and a rapid
conversion to the parent drugs under physiological
conditions. This suggested that these prodrugs are use-
ful as practical water-soluble prodrugs. This strategy is
applicable to other HIV-1 PR inhibitors13À17,34 with an
Apns–Dmt core structure and might be able to over-
come the problem of the low water-solubility of HIV-1
PR inhibitors.
Boc-Apns-Dmt-NH-But (14b). Compound 14b was pre-
pared from Boc-Dmt-NH-But (13b)13,34 (6.33 g,
20 mmol) according to the similar procedure described
ꢀ
for compound 14a. Yield 7.97 g (82%); mp84–87 C; 1H
NMR (300 MHz, CDCl3) d 1.27–1.43 (m, 18H), 1.48–
1.60 (m, 6H), 2.60–2.84 (m, 2H), 3.65–3.76 (m, 1H), 3.91
(m, 1H), 4.11 (m, 1H), 4.28–5.08 (m, 5H) 7.07–7.36 (m,
5H); MS (FAB): m/z 494 [M+H]+. Anal. calcd for
C25H39N3O5S: H, 7.96; C, 60.82; N, 8.51. Found: H,
8.05; C, 60.57; N, 8.54.
Z-Apns-Thz-NH-But (15a). To compound 14a (3.6 g,
7.75 mmol) was added 4 N-HCl/dioxane (20 mL) and
stirred for 2 h at room temperature. After the reaction
mixture was concentrated in vacuo, ether was added to
give precipitate, which was dissolved in DMF (20 mL)
and neutralized with Et3N (1.09 mL). To this reaction
mixture were added Z-OSu (1.93 g, 7.75 mmol) and
Et3N (1.09 mL, 7.75 mmol) stepwise at 0 ꢀC and stirred
at room temperature for 4 h. After removal of the sol-
vent in vacuo, the residue was dissolved in EtOAc,
washed sequentially with 5% citric acid, 5% NaHCO3
and saturated NaCl, dried over MgSO4, and con-
centrated in vacuo. The residue was applied to a silica
gel column (3ꢁ18 cm) and eluted with hexane–EtOAc
(1:1) to give 3.45 g (89%) of the title compound 14a as a
Experimental
Reagents and solvents used were purchased from Wako
Pure Chemical Ind., Ltd. (Osaka, Japan), Nacalai Tes-
que (Kyoto, Japan) and Watanabe Chem. Ind., Ltd.
(Hiroshima, Japan) without further purification. TLC
was performed using Merck Silica gel 60 F254 precoated
plate. Column chromatography was performed on
Merck 107734 silica gel 60 (70–230 mesh). Melting
points were measured on Yanagimoto micro melting
apparatus. Analytical HPLC was performed using a
C18 reverse phase column (4.6ꢁ150 mm; YMC Pack
ODS AM302) with binary solvent system: linear gra-
dient of CH3CN in 0.1% aqueous TFA at a flow rate of
white solid: mp69–70 ꢀC; H NMR (300 MHz, CDCl3)
1
1
1.0 mL/min, detected 230 nm. H NMR spectra were
d 1.29 (s, 9H), 2.55–2.86 (m, 2H), 3.19 (dd, J=7.6,
11.7 Hz, 1H), 3.314 (m, 1H), 3.52 (dd, J=6.6, 11.7 Hz,
1H), 3.75 (d, 1H), 4.10 (m, 1H), 4.65 (d, J=9.6 Hz, 1H),
4.70–5.10 (m, 5H), 5.26 (d, J=7.8 Hz, 1H), 6.26 (br s,
1H), 7.09–7.42 (m, 10H); MS (FAB): m/z 500 [M+H]+.
Anal. calcd for C26H33N3O5S: H, 6.66; C, 62.50; N,
8.41. Found: H, 6.81; C, 62.31; N, 8.30.
obtained on a JEOL AL300 spectrometer with TMS as
an internal standard. FAB-MS was performed on a
JEOL JMS-SX102A spectrometer equipped with the
JMA-DA7000 data system. TOF-MS was performed on
a Voyager-DE RP spectrometer (PerSeptive Biosystems,
inc.).
Z-Apns-Dmt-NH-But (15b). Compound 15b was pre-
pared from Boc-Apns-Dmt-NH-But (14b) (1.95 g,
4.53 mmol) according to the similar procedure described
Boc-Apns-Thz-NH-But (14a). To compound 13a13,34
(10 g, 28.4 mmol) were added anisole (7.5 mL) and 4 N-
HCl/dioxane (110 mL), and stirred for 2 h at room tem-
perature. After the reaction mixture was concentrated in
vacuo, ether was added to give precipitate (7.33 g). To
the solution of the obtained precipitate (6 g) and Boc-
Apns-OH (8.7 g, 29.5 mmol) in DMF (100 mL) were
ꢀ
for compound 15a. Yield 1.91 g (92%): mp79–83 C; 1H
NMR (300 MHz, DMSO-d6) d 1.27 (s, 9H), 1.40 (s, 3H),
1.49 (s, 3H), 2.53–2.78 (m, 2H), 3.91 (m, 1H), 4.47 (m,
1H), 4.51 (s, 1H), 4.87–5.06 (m, 3H), 5.14 (d, J=7.2 Hz,
1H), 7.09–7.46 (m, 10H); TOF-Ms: m/z 528 [M+H]+.
Anal. calcd for C28H37N3O5S: H, 7.07; C, 63.73; N,
7.96. Found: H, 7.24; C, 63.81; N, 7.91.
.
added HOBt (4.5 g, 29.5 mmol), EDC HCl (5.63 g,
29.5 mmol) and Et3N (2.7 g, 26.7 mmol) stepwise at 0 ꢀC
and stirred overnight at room temperature.13,34 After
removal of the solvent in vacuo, the residue was dis-
solved in EtOAc, washed sequentially with 5% citric
Z-Apns(Boc-Mta)-Thz-NH-But (17a). To a solution of
15a (700 mg, 1.44 mmol) and Boc-Mta-OH (16a)
(509 mg, 2.16 mmol) in dichloromethane (9.3 mL) were

4162
Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
added DMAP (25 mg, 0.216 mmol) and
a
DMF
according to the similar procedure described for com-
pound 17a. Yield 920 mg (93%); mp160–161 ꢀC; 1H
NMR (300 MHz, CDCl3) d 0.89–1.11 (m, 6H), 1.26–
1.62 (m, 24H), 2.20 (m, 1H), 2.80–3.21 (m, 2H), 3.98–
4.38 (m, 3H), 4.73–5.11 (m, 5H), 5.39–5.68 (m, 3H),
7.10–7.40 (m, 10H), 6.53 (br s, 1H); TOF-Ms: m/z 749
[M+Na]+. Anal. calcd for C38H54N4O8S: H, 7.49; C,
62.79; N, 7.71. Found: H, 7.54; C, 62.70; N, 7.95.
(1.1 mL) solution of DCC (433 mg, 2.16 mmol) at 0 ꢀC
and stirred overnight at room temperature. After
removal of the solvent in vacuo, the residue was dis-
solved in EtOAc, washed sequentially with 5% citric
acid, 5% NaHCO3 and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The residue was
applied to a silica gel column (2.5ꢁ18 cm) and eluted
with hexane–EtOAc (1:1) to give 883 mg (89%) of the
title compound 17a as a white solid: mp69–72 ꢀC;
[a]2D1=À66.07 (c 0.56, MeOH); 1H NMR (300 MHz,
CDCl3) d 1.31 (s, 9H), 1.43 (s, 9H), 2.13 (s, 3H), 2.86–
3.26 (m, 5H), 3.45 (dd, J=5.4, 11.2 Hz, 1H), 4.11 (br s,
1H), 4.32 (m, 1H), 4.50 (m, 1H), 4.57 (m, 1H), 4.76 (t,
J=6.4 Hz, 1H), 4.89 (d, J=8.7 Hz, 1H), 5.03 (s, 2H),
5.19–5.62 (m, 3H), 6.21 (br s, 1H), 7.11–7.47 (m, 10H);
MS (FAB): m/z 717 [M+H]+. Anal. calcd for
Z-Apns(iQoa-Mta)-Thz-NH-But (20a). To compound
19a (747 mg, 1.06 mmol) were added anisole (1.5 mL)
and 4N-HCl/dioxane (10 mL) and stirred for 2 h at
room temperature. After removal of the solvent in
vacuo, the residue was triturated with ether at 0 ꢀC to
give precipitate (695 mg). After the reaction mixture was
concentrated in vacuo, ether was added to give pre-
cipitate (7.33 g). To a solution of the obtained pre-
cipitate (400 mg, 0.61 mmol) and iQoa-OH (137 mg,
0.67 mmol) in DMF (5 mL) were added HOBt (103 mg,
0.67 mmol), DCC (139 mg, 0.67 mmol) and Et3N
(85 mg, 0.61 mmol) stepwise at 0 ꢀC and stirred over-
night at room temperature. After removal of the solvent
in vacuo, the residue was dissolved in EtOAc, washed
sequentially with 5% citric acid, 5% NaHCO3 and
saturated NaCl, dried over MgSO4, and concentrated in
vacuo. The residue was applied to a silica gel column
(2.5ꢁ20 cm) and eluted with hexane–EtOAc (1:2) to give
414 mg (84%) of title compound 20a: mp92–95 ꢀC;
.
C35H48N4O8S2 1.2H2O: H, 6.88; C, 56.92; N, 7.59.
Found: H, 6.54; C, 56.88; N, 7.70.
Z-Apns(Boc-Val)-Thz-NH-But (17b). Compound 17b
was prepared from Z-Apns-Dmt-NH-But (15b) (600 mg,
1.24 mmol) and Boc-Val-OH (16b) (391 mg, 1.85 mmol)
according to the similar procedure described for com-
pound 17a. Yield 840 mg (97%); mp77–80 ꢀC;
22
1
[a]D =À118.26 (c 0.58, MeOH); H NMR (300 MHz,
CDCl3) d 0.92–1.10 (m, 6H), 1.30 (s, 9H), 1.42 (s, 9H),
2.22 (m, 1H), 2.78–3.23 (m, 3H), 3.46 (dd, J=6.2,
12 Hz, 1H), 4.02–5.01 (m, 6H), 5.03 (s, 2H), 5.10–5.58
22
1
[a]D =À105.36 (c 0.56, MeOH); H NMR (300 MHz,
CDCl3) d 1.31 (s, 9H), 2.11 (s, 3H), 2.72–3.59 (m, 7H),
4.28–5.17 (m, 8H), 5.41–5.70 (m, 2H), 6.19 (s, 1H), 7.01
(d, J=6.9 Hz, 1H), 7.09–7.39 (m, 10H), 7.09–7.39 (m,
10H), 7.52 (dd, J=8.4, 8.4 Hz, 1H), 7.64 (d, J=8.4 Hz,
1H), 8.06 (d, J=5.6 Hz, 1H), 8.57 (d, J=5.6 Hz, 1H),
9.25 (s,1H); MS (FAB): m/z 802 [M+H]+. Anal. calcd
(m, 2H), 6.21 (s, 1H), 7.09–7.42 (m, 10H); MS (FAB):
+
.
m/z 699 [M+H] . Anal. calcd for C36H50N4O8S 9/
4H2O: H, 7.43; C, 58.48; N, 7.58. Found: H, 6.70; C,
58.42; N, 7.67.
Z-Apns(Boc-Nva)-Thz-NH-But (18). Compound 18 was
prepared form Z-Apns-Dmt-NH-But (15b) (350 mg,
0.67 mmol) and Boc-Nva-OH (16c) (235 mg, 1.00 mmol)
according to the similar procedure described for com-
pound 17a. Yield 443 mg (91%); mp73–75 ꢀC; 1H
NMR (300 MHz, CDCl3) d 0.96 (t, 3H), 1.22–1.38 (m,
9H), 1.41 (br.s, 11H), 1.55–1.75 (m, 1H), 1.76–2.00 (m,
1H), 2.73–3.22 (m, 3H), 3.46 (dd, 1H), 4.09 (s, 1H),
4.20–4.43 (m, 2H), 4.61 (d, 1H), 4.79 (t, 1H), 4.84–4.95
(m, 2H), 5.03 (s, 2H), 5.23–5.62 (m, 2H), 6.22 (s, 1H),
7.12–7.42 (m, 10H); TOF-Ms: m/z 721 [M+Na]+.
Anal. calcd for C36H50N4O8S: H, 7.21; C, 61.87; N,
8.02. Found: H, 7.35; C, 61.86; N, 8.01.
.
for C41H47N5O8S2 5/4H2O: H, 6.05; C, 59.73; N, 8.49.
Found: H, 5.98; C, 59.85; N, 8.10.
Z-Apns(iQoa-Val)-Thz-NH-But (20b). Compound 20b
was prepared from Z-Apns(Boc-Val)-Thz-NH-But (19b)
(440 mg, 0.63 mmol) and iQoa-OH (141 mg, 0.69 mmol)
according to the similar procedure described for com-
pound 20a. Yield 372 g (75%); mp98–102 ꢀC;
22
1
[a]D =À137.63 (c 0.47, MeOH); H NMR (300 MHz,
CDCl3) d 0.73–1.08 (m, 6H), 1.30 (s, 9H), 2.22–2.46 (m,
1H), 2.70–3.28 (m, 3H), 3.33–3.63 (m, 1H), 4.10 (br s,
1H), 4.31 (br s, 1H), 4.53–5.20 (m, 6H), 5.29–5.68 (m,
2H), 6.18 (br s, 1H), 6.92–7.41 (m, 11H), 7.51 (t,
J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.89–8.02 (m,
Z-Apns(Boc-Mta)-Dmt-NH-But (19a). Compound 19a
was prepared from Z-Apns-Dmt-NH-But (15b) (700 mg,
1.33 mmol) and Boc-Mta-OH (16a) (468 mg, 1.99 mmol)
according to the similar procedure described for com-
pound 17a. Yield 950 g (94%); mp121–123 ꢀC; 1H
NMR (300 MHz, CDCl3) d 1.30–160 (m, 24H), 2.12 (s,
3H), 2.78–3.19 (m, 4H), 4.05 (br s, 1H), 4.21 (s, 1H),
4.27–4.58 (m, 2H), 4.76–4.94 (m, 2H), 5.04 (s, 2H),
5.26–5.81 (m, 3H), 7.11–7.39 (m, 10H); TOF-Ms: m/z
745 [M+H]+. Anal. calcd for C37H52N4O8S2: H, 7.04;
C, 59.65; N, 7.52. Found: H, 7.26; C, 59.81; N, 7.47.
1H), 8.60 (d, J=6.0 Hz, 1H), 9.28 (s, 1H); MS (FAB):
+
.
m/z 784 [M+H] . Anal. calcd for C42H49N5O8S 1/
2H2O: H, 6.36; C, 63.61; N, 8.83. Found: H, 6.19; C,
63.66; N, 8.84.
Z-Apns(iQoa-Nva)-Thz-NH-But (21). Compound 21
was prepared from Z-Apns(Boc-Nva)-Thz-NH-But (20)
(220 mg, 0.31 mmol) and iQoa-OH (70.4 mg, 0.35 mmol)
according to the similar procedure described for com-
pound 20a. Yield 238 mg (97%); mp72–76 ꢀC; 1H
NMR (300 MHz, CDCl3) d 0.89–1.06 (m, 3H), 1.31 (s,
9H), 1.36–1.61 (m, 2H), 1.65–2.12 (m, 2H), 2.72–3.28
(m, 3H), 3.45 (dd, J=5.3, 12.0 Hz, 1H), 4.02–4.48 (m,
1H), 4.53–4.88 (m, 4H), 4.93 (d, J=9.6 Hz, 2H), 5.02 (s,
Z-Apns(Boc-Val)-Dmt-NH-But (19b). Compound 19b
was prepared from Z-Apns-Dmt-NH-But (15b) (700 mg,
1.33 mmol) and Boc-Val-OH (16b) (432 mg, 1.99 mmol)

Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
4163
+
.
2H), 5.38–5.66 (m, 2H), 6.16 (s, 1H), 6.90–7.40 (m,
12H), 7.50 (t, J=8.1 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H),
8.00 (d, J=5.8 Hz, 1H), 8.60 (d, J=5.8 Hz, 1H), 9.25 (s,
1H); TOF-Ms: m/z 784 [M+H]+. Anal. calcd for
C42H49N5O8S2 1/4H2O: H, 6.33; C, 63.98; N, 8.88.
Found: H, 6.58; C, 63.98; N, 8.64.
[M+H] . Anal. calcd for C43H50N4O8S 3/4H2O: H,
6.52; C, 64.85; N, 7.03. Found: H, 6.42; C, 64.99; N,
7.05.
Z-Apns(Noa-Mta)-Dmt-NH-But (24a). Compound 24a
was prepared form Z-Apns(Boc-Mta)-Dmt-NH-But
(19a) (437 mg, 0.59 mmol) and Noa-OH (131 mg,
0.65 mmol) according to the similar procedure described
.
Z-Apns(iQoa-Mta)-Dmt-NH-But (22a). Compound 22a
was prepared from Z-Apns(Boc-Mta)-Dmt-NH-But
(19a) (410 mg, 0.59 mmol) and iQoa-OH (131 mg,
0.64 mmol) according to the similar procedure described
ꢀ
for compound 20a. Yield 434.6 mg (89%); mp79–83 C;
1H NMR (300 MHz, CDCl3) d 1.32 (s, 9H), 1.40–1.62
(m, 6H), 2.05–2.16 (m, 3H), 2.78–3.29 (m, 4H), 4.06 (br
s, 1H), 4.22 (s, 1H), 4.34 (br s, 1H), 4.44–5.18 (m, 7H),
5.49–5.89 (m, 2H), 6.51–6.87 (m, 1H), 7.09–7.42 (m,
11H), 7.48–7.59 (m, 3H), 7.69–7.90 (m, 1H), 8.23–8.39
(m, 1H); TOF-Ms: m/z 830 [M+H]+. Anal. calcd for
C44H52N4O8S2: H, 6.32; C, 63.75; N, 6.76. Found: H,
6.51; C, 63.49; N, 6.66.
ꢀ
for compound 20a. Yield 414 g (85%); mp88–92 C; 1H
NMR (300 MHz, CDCl3) d 1.33 (s, 9H), 1.42–1.61 (m,
6H), 2.01–2.18 (m, 3H), 2.73–3.30 (m, 4H), 4.07 (br s,
1H), 4.22 (s, 1H), 4.35 (br s, 1H), 4.45–5.17 (m, 8H),
5.48–5.68 (m, 2H), 5.78–5.81 (m, 1H), 6.56 (s, 1H),
6.85–7.04 (m, 1H), 7.09–7.37 (m, 10H), 7.41–7.57 (m,
1H), 7.58–7.71 (m, 1H), 7.98–8.10 (m, 1H), 8.51–8.62
(m, 1H), 9.23 (s, 1H); TOF-Ms: m/z 831 [M+H]+.
Z-Apns(Noa-Val)-Dmt-NH-But (24b). Compound 24b
was prepared from Z-Apns(Boc-Val)-Dmt-NH-But
(19b) (425 mg, 0.59 mmol) and Noa-OH (134 mg,
0.66 mmol) according to the similar procedure described
.
Anal. calcd for C43H51N5O8S2 1/4H2O: H, 6.19; C,
62.22; N, 8.44. Found: H, 6.37; C, 61.99; N, 8.21.
ꢀ
Z-Apns(iQoa-Val)-Dmt-NH-But (22b). Compound 22b
was prepared from Z-Apns(Boc-Val)-Dmt-NH-But
(19b) (440 mg, 0.65 mmol) and iQoa-OH (141 mg,
0.69 mmol) according to the similar procedure described
for compound 20a. Yield 422 mg (86%); mp91–95 ꢀC;
1H NMR (300 MHz, CDCl3) d 0.88–1.09 (m, 6H), 1.33
(s, 9H), 1.41–1.60 (m, 6H), 2.22–2.42 (m, 1H), 2.76–2.97
(m, 1H), 3.02–3.21 (m, 1H), 4.08 (br s, 1H), 4.19–4.39
(m, 1H), 4.52–5.11 (m, 7H), 5.41–5.75 (m, 2H), 6.51 (br
s, 1H), 6.81–7.02 (m, 2H), 7.04–7.36 (m, 11H), 7.50 (t,
J=8.0 Hz, 1H), 7.63 (d, J=8.0, 1H), 7.89–8.00 (m, 1H),
8.53–8.65 (m, 1H), 9.25 (s, 1H); TOF-Ms: m/z 813
for compound 20a. Yield 459 g (94%); mp77–82 C; 1H
NMR (300 MHz, CDCl3) d 1.88 (m, 6H), 1.32 (s, 9H),
1.39–1.59 (m, 6H), 2.23–2.39 (m, 1H), 2.84–2.97 (m,
1H), 3.00–3.22 (m, 1H), 3.99–4.12 (m, 1H), 4.19–4.52
(m, 2H), 4.58–5.12 (m, 6H), 5.39–5.80 (m, 2H), 6.47–
6.82 (m, 1H), 7.04–7.40 (m, 10H), 7.48–7.61 (m, 3H),
7.78–7.89 (m, 1H), 8.13–8.27 (m, 1H); TOF-Ms: m/z 811
[M+H]+. Anal. calcd for C45H54N4O8S: H, 6.71; C,
66.64; N, 6.91. Found: H, 6.93; C, 66.62; N, 6.68.
t
.
Apns(iQoa-Mta)-Thz-NH-Bu 2HCl (3). To compound
20a (100 mg, 0.125 mmol) was added dimethylsulfide
(0.366 mL, 5 mmol), anisole (136 mL, 1.25 mmol) and
TFA (4.8 mL, 62.3 mmol) at À5 ꢀC and stirred for 1 h at
0–10 ꢀC and overnight at room temperature. After the
reaction mixture was concentrated in vacuo at room
temperature, ether was added to give precipitate
(85 mg). This crude product was purified using pre-
parative HPLC (YMC 120A ODS; 20ꢁ250 mm) eluted
in a linear gradient with 30–80% MeCN in 0.1% aqu-
eous TFA over 30 min at a flow rate of 1 mL/min and
the desired fraction was lyophilized to give a white
powder as a TFA salt. To a solution of this powder in
EtOAc was added 4 N-HCl/EtOAc at 0 ꢀC to form a
crystal and separation by centrifuge gave the 25 mg
(29%) of desired compound 24 as a white fluffy powder:
mp166–170 ꢀC; [a]D23=À131.87 (c 0.46, MeOH); 1H
NMR (300 MHz, DMSO-d6) d 1.26 (s, 9H), 2.13 (s, 3H),
2.78–3.19 (m, 5H), 3.40 (dd, J=7.6, 11.6 Hz, 1H), 3.99
(br s, 1H), 4.69 (d, J=8.8 Hz, 1H), 4.76 (t, J=6.9 Hz,
1H), 4.81–5.05 (m 4H), 5.64 (br s, 1H), 7.19–7.46 (m,
6H), 7.68 (t, J=8.0 Hz, 1H), 7.82 (d, J=8.0 Hz, 1H),
7.89 (s, 1H), 8.24 (br s, 1H), 8.32 (d, J=8.0 Hz, 1H),
8.58 (d, J=6.0 Hz, 1H), 8.78 (d, J=8.1 Hz, 1H) 9.46 (s,
1H); TOF-MS: m/z 668 [M+H]+. Anal. calcd for
C33H43Cl2N5O6S2: H, 5.82; C, 53.50; N, 9.46. Found:
H, 5.95; C, 53.41; N, 9.67.
+
.
[M+H] . Anal. calcd for C44H53N5O8S 1/4H2O: H,
6.60; C, 64.70; N, 8.58. Found: H, 6.71; C, 64.83; N,
8.32.
Z-Apns(Noa-Mta)-Thz-NH-But (23a). Compound 23a
was prepared from Z-Apns(Boc-Mta)-Thz-NH-But
(17a) (430 mg, 0.61 mmol) and Noa-OH (131 mg,
0.65 mmol) according to the similar procedure described
for compound 20a. Yield 434 mg (89%); mp80–82 ꢀC;
1H NMR (300 MHz, CDCl3) d 1.31 (s, 9H), 2.10 (s, 3H),
2.87–3.28 (m, 5H), 3.40–3.58 (m, 2H), 4.12 (m, 1H), 4.32
(m, 1H), 4.54–5.18 (m, 7H), 5.40–5.72 (m, 2H), 6.16 (br
s, 1H), 6.79 (d, J=7.5 Hz, 1H), 7.08–7.48 (m, 11H),
7.49–7.66 (m, 3H), 7.81–7.88 (m, 1H), 8.27–8.36 (m,
1H); MS(FAB): m/z 801 [M+H]+. Anal. calcd for
C42H48N4O8S2: H, 6.04; C, 62.98; N, 6.99. Found: H,
6.21; C, 62.83; N, 7.03.
Z-Apns(Noa-Val)-Thz-NH-But (23b). Compound 23b
was prepared from Z-Apns(Boc-Val)-Thz-NH-But (17b)
(425 mg, 0.59 mmol) and Noa-OH (134 mg, 0.66 mmol)
according to the similar procedure described for com-
pound 20a. Yield 476 g (97%); mp81–83 ꢀC; H NMR
1
(300 MHz, CDCl3) d 0.92–1.11 (m, 6H), 1.30 (s, 9H),
2.20–2.46 (m, 1H), 2.70–3.27 (m, 3H), 3.36–3.70 (m,
2H), 4.08 (br s, 1H), 4.30 (br s, 1H), 4.53–5.12 (m, 7H),
5.23–5.67 (m, 2H), 6.16 (br s, 1H), 6.78 (d, J=7.5 Hz,
1H), 7.03–7.41 (m, 11H), 7.44–7.62 (m, 3H), 7.78–7.91
(m, 1H), 8.12–8.28 (m,1H); MS(FAB): m/z 783
Compounds 4–11 were prepared according to the simi-
lar procedure described for compound 3 starting from
the corresponding intermediates 20–24.

4164
Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
t
.
Apns(iQoa-Val)-Thz-NH-Bu 2HCl (4). 32% yield from
20b; mp164–170 ꢀC; [a]2D2=À124.77 (c 0.55, MeOH);
1H NMR (300 MHz, DMSO-d6) d 0.89–1.03 (m, 6H),
1.27 (s, 9H), 2.22–2.38 (m, 1H), 2.70–3.11 (m, 3H), 3.41
(dd, J=7.4, 11.9 Hz, 1H), 4.62–4.75 (m, 2H), 4.80 (t,
J=7.0 Hz, 1H), 4.86 (d, J=9.2 Hz, 1H), 4.94 (br s, 2H),
5.57 (br s, 1H), 7.17–7.46 (m, 6H), 7.65 (t, J=8.0 Hz,
1H), 7.77 (d, J=8.0 Hz, 1H), 7.90 (s, 1H), 8.08–8.29 (m,
3H), 8.48 (d, J=8.7 Hz, 1H), 8.56 (d, J=5.7 Hz, 1H),
9.41 (s, 1H); TOF-MS: m/z 650 [M+H]+. Anal. calcd
for C34H45Cl2N5O6S: H, 6.28; C, 56.50; N, 9.69. Found:
H, 6.24; C, 56.44; N, 9.84.
5.04 (m, 5H), 5.64 (d, J=2.7 Hz, 1H), 6.96 (d,
J=7.5 Hz, 1H), 7.18–7.60 (m, 6H), 7.78–7.92 (m, 2H),
8.27–8.48 (m, 2H), 8.89 (d, J=8.7 Hz, 1H); TOF-MS:
m/z 696 [M+H] . Anal. calcd for C36H47ClN4O6S2 3/
2H2O: H, 6.64; C, 57.01; N, 7.38. Found: H, 6.38; C,
57.19; N, 7.00.
+
.
t
.
Apns(Noa-Val)-Dmt-NH-Bu HCl (10). 37% yield from
24b; mp171–174 ꢀC; H NMR (300 MHz, DMSO-d6) d
1
0.88–1.01 (m, 6H), 1.28 (s, 9H), 1.41 (s, 3H), 1.50 (s,
3H), 2.22–2.38 (m, 1H), 2.85 (dd, J=9.8, 14.3 Hz, 1H)
3.06 (dd, J=3.0, 14.3 Hz, 1H), 3.89 (br s, 1H), 4.55 (s,
1H), 4.71 (dd, J=4.8, 9.0 Hz, 1H), 4.78–5.01 (m, 4H),
5.51 (br s, 1H), 6.89 (d, J=7.8 Hz, 1H), 7.20–7.42 (m,
6H), 7.44–7.57 (m, 3H), 7.81 (s, 1H), 7.83–7.90 (m, 1H),
8.08–8.28 (m, 3H), 8.43 (d, J=9.0 Hz, 1H); TOF-MS:
t
.
Apns(iQoa-Mta)-Dmt-NH-Bu 2HCl (5). 26% yield
ꢀ
from 22a; mp164–170 C; 1H NMR (300 MHz, DMSO-
d6) d 1.27 (s, 9H), 1.41 (s, 3H), 1.49 (s, 3H), 2.12 (s, 3H),
2.80–3.22 (m, 4H), 3.82 (br s, 1H), 4.49 (s, 2H), 4.83–
5.09 (m, 5H), 5.72 (s, 1H), 7.13–7.42 (m, 6H), 7.53 (d,
J=7.8 Hz, 1H), 7.77–7.92 (m, 2H), 7.99 (t, J=7.8 Hz,
1H), 8.40–8.81 (m, 5H), 9.06 (d, J=8.7 Hz, 1H), 9.79 (s,
1H); TOF-MS: m/z 696 [M+H]+. Anal. calcd for
m/z
677
[M+H]+.
Anal.
calcd
for
.
C37H49ClN4O6S 2H2O: H, 7.12; C, 59.30; N, 7.48.
Found: H, 6.98; C, 59.57; N, 7.24.
t
.
Apns(iQoa-Nva)-Thz-NH-Bu 2HCl (11). 35% yield
from 21; mp154–159 ꢀC; H NMR (300 MHz, DMSO-
1
.
C35H47Cl2N5O6S2 3/2H2O: H, 6.33; C, 52.82; N, 8.79.
Found: H, 6.25; C, 53.19; N, 8.23.
d6) d 0.90 (t, J=7.2 Hz, 3H), 1.27 (s, 9H), 1.32–1.52 (m,
2H), 1.69–1.96 (m, 2H), 2.82–3.10 (m, 4H), 3.42 (dd,
J=7.2, 11.4 Hz, 1H) 3.79 (br s, 1H), 4.61–4.82 (m, 3H),
4.95 (d, J=9.0 Hz, 1H), 5.01 (s, 2H), 5.76 (s, 1H), 7.18–
7.56 (m, 7H), 7.80 (t, J=8.3 Hz, 1H), 7.91–8.06 (m, 2H),
8.43–8.60 (m, 4H), 8.64 (d, J=6.6 Hz, 1H), 9.00 (d,
t
.
Apns(iQoa-Val)-Dmt-NH-Bu 2HCl (6). 48% yield from
ꢀ
1
22b; mp158–162 C; H NMR (300 MHz, DMSO-d6) d
0.99–1.01 (m, 6H), 1.28 (s, 9H), 1.41 (s, 3H), 1.50 (s,
3H), 2.23–2.38 (m, 1H), 2.83 (dd, J=9.8, 14.4 Hz, 1H),
3.05 (dd, J=3.0, 14.4 Hz, 1H), 3.89 (br s, 1H), 4.55 (s,
2H), 4.69 (dd, J=5.0, 8.8 Hz, 1H), 4.82–5.05 (m, 4H),
5.51 (d, J=2.7 Hz, 1H), 7.22–7.43 (m, 6H), 7.69 (t,
J=8.0 Hz, 1H), 7.78–7.88 (m, 2H), 8.12–8.28 (m, 3H),
8.52–8.64 (m, 2H), 9.49 (s, 1H); TOF-MS: m/z 678
J=8.7 Hz, 1H), 9.72 (s, 1H); TOF-Ms: m/z 650
+
.
[M+H] . Anal. calcd for C34H45Cl2N5O6S 2H2O: H,
6.51; C, 53.82; N, 9.23. Found: H, 6.36; C, 53.56; N,
9.03.
+
iQoa-Val-Apns-Dmt-NH-But (2b). To compound 25
(300 mg, 0.53 mmol) were added anisole (0.15 mL)
and 4 N-HCl/dioxane (5 mL), and stirred for 2 h at
room temperature. After the reaction mixture was
concentrated in vacuo, n-hexane was added to give
precipitate at 0 ꢀC. To a solution of the obtained
precipitate (140 mg, 0.28 mmol) and iQoa-OH
(62.4 mg, 0.31 mmol) in DMF (2 mL) were added
HOBt (47 mg, 0.31 mmol), EDC-HCl (59 mg,
0.31 mmol) and Et3N (39 mL, 0.28 mmol) stepwise at
0 ꢀC and stirred overnight at room temperature. After
removal of the solvent in vacuo, the residue was dis-
solved in EtOAc, washed sequentially with 5% citric
acid, 5% NaHCO3 and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The residue was
applied to a silica gel column (2.5ꢁ18 cm) and eluted
with CHCl3–MeOH (20:1) to give 140 mg (78%) of
title compound 2b; mp122–125 ꢀC; 1H NMR
(300 MHz, CDCl3) d 0.68–0.96 (m, 6H), 1.30 (s, 9H),
1.42–1.63 (m, 6H), 1.99–2.20 (m, 1H), 2.63–2.92 (m,
2H), 3.99 (br s, 1H), 4.18–4.40 (m, 2H), 4.40–4.80
(m, 4H), 4.85 (m, 1H), 6.85–7.28 (m, 7H), 7.46–7.58
(m, 1H), 7.60–7.69 (m, 1H), 7.94 (d, J=5.4 Hz, 1H),
8.61 (t, J=6.2 Hz, 1H), 9.26 (d, J=5.0 Hz, 1H);
TOF-MS: m/z 678 [M+H]+. Anal. calcd for
.
[M+H] . Anal. calcd for C36H49Cl2N5O6S 2H2O: H,
6.79; C, 54.96; N, 8.90. Found: H, 6.84; C, 54.86; N,
8.58.
t
.
Apns(Noa-Mta)-Thz-NH-Bu HCl (7). 54% yield from
23a; mp174–177 ꢀC; [a]3D0=À103.1 (c 0.485, MeOH);
1H NMR (300 MHz, DMSO-d6) d 1.27 (s, 9H), 2.14 (s,
3H), 2.80–3.19 (m, 5H), 3.57 (s, 1H), 3.84 (br s, 1H),
4.62–5.02 (m, 6H), 5.72 (br s, 1H), 6.96 (d, J=7.5 Hz,
1H), 7.19–7.58 (m, 9H), 7.78–7.95 (m, 2H), 8.20–8.51
(m, 3H), 8.77 (d, J=8.7 Hz, 1H); TOF-MS: m/z 667
+
.
[M+H] . Anal. calcd for C34H43ClN4O6S2 7/4H2O: H,
6.38; C, 55.57; N, 7.62. Found: H, 6.23; C, 55.99; N,
7.14.
t
.
Apns(Noa-Val)-Thz-NH-Bu HCl (8). 30% yield from
23b; mp121–126 ꢀC; [a]3D0=À83.76 (c 0.485, MeOH);
1H NMR (300 MHz, DMSO-d6) d 0.88–1.03 (m, 6H),
1.24 (s, 9H), 2.20–2.36 (m, 1H), 2.83–3.15 (m, 3H), 3.57
(s, 1H), 3.83 (br s, 1H), 4.67–4.98 (m, 6H), 5.70 (br s,
1H), 6.91 (d, J=7.5 Hz, 1H), 7.20–7.58 (m, 9H), 7.80–
7.95 (m, 2H), 8.17–8.24 (m, 1H), 8.36 (br s, 3H), 8.49–
8.58 (m, 1H); TOF-MS: m/z 649 [M+H]+. Anal. calcd
.
for C35H45ClN4O6S H2O: H, 6.85; C, 58.28; N, 7.77.
Found: H, 6.48; C, 58.57; N, 7.27.
.
C36H47N5O6S 1/2H2O: H, 7.04; C, 62.95; N, 10.20.
Found: H, 7.04; C, 62.91; N, 10.09.
t
.
Apns(Noa-Mta)-Dmt-NH-Bu HCl (9). 33% yield from
24a; mp132–136 ꢀC; H NMR (300 MHz, DMSO-d6) d
1.27 (s, 9H), 1.41 (s, 3H), 1.49 (s, 3H), 2.14 (s, 3H),
2.84–3.17 (m, 4H), 3.87 (br s, 1H), 4.50 (s, 1H), 4.88–
1
Noa-Val-Apns-Dmt-NH-But (2d). 70% yield from 25;
mp105–108 ꢀC; H NMR (300 MHz, CDCl3) d 0.62–
1

Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
4165
0.73 (s, 3H), 0.80–0.95 (s, 3H), 1.30 (s, 9H), 1.46–1.62
(m, 6H), 2.11 (m, 1H), 2.59–2.82 (m, 2H), 3.73–3.98 (m,
1H), 4.10–4.38 (m, 2H), 4.40–5.18 (m, 5H), 6.39 (br s,
1H), 6.80 (d, J=7.5 Hz, 1H), 6.82–7.20 (m, 6H), 7.35–
7.46 (m, 1H), 7.48–7.63 (m, 3H), 7.79–7.92 (m, 1H),
8.15–8.28 (m, 1H); TOF-MS: m/z 677 [M+H]+. Anal.
calcd for C37H48N4O6S: H, 7.15; C, 65.65; N, 8.28.
Found: H, 7.17; C, 65.42; N, 8.40.
compound 30 (200 mg, 0.24 mmol) was added anisole
(0.15 mL) and 4 N-HCl/dioxane (2 mL) and the reaction
mixture was stirred for 3 h at 0 ꢀC. After the reaction
mixture was concentrated in vacuo, ether was added to
form precipitate at 0 ꢀC. The resulting precipitate was
applied to a silica gel column (2ꢁ18 cm) and eluted with
EtOAc–MeOH (10:1) to give 148.5 mg (81%) of the title
compound 12 as a powder: mp 121–125 ꢀC; H NMR
1
(300 MHz, CDCl3) d 0.92–1.05 (m, 6H), 1.30 (d,
J=6.9Hz, 6H), 1.68–1.83 (m, 1H), 1.89–2.03 (m, 1H),
2.18–2.32 (m, 1H), 2.67–3.07 (m, 5H), 3.23 (heptalet,
J=6.9Hz, 1H), 3.32–3.52 (m, 1H), 3.90–4.07 (m, 2H),
4.40–4.58 (m, 2H), 4.92–5.37 (m, 11H), 6.68 (d, J=7.0Hz,
1H), 7.06–7.30 (m, 11H), 7.42 (d, J=9.3Hz, 1H), 7.85 (s,
1H), 8.16 (br s, 3H), 9.10 (s, 1H); TOF-MS: m/z 722
(2S,3S,5S)-5-[N-[(tert-butyloxy)carbonyl]amino]-2-[N-
[(5-thiazolyl)methoxy]carbonyl]amino]-1,6-diphenyl-3-hy-
droxyhexane] 28. To a solution of [(2S,3S,5S)-2-amino-
5-[N-[(tert-butyloxy)carbonyl]amino]-1,6-diphenyl-3-
hydroxyhexane] 2642 (1.0 g, 2.6 mmol) in THF–DMF
(1:1, 10 mL) was added 4-nitrophenyl (5-thiazo-
lyl)methyl carbonate hydrochloride 2743 (0.91 g,
2.8 mmol) and stirred overnight at room temperature.
After the reaction mixture was concentrated under
reduced pressure, the resulting residue was dissolved in
EtOAc, washed sequentially with 5% citrate, 5%
NaHCO3 and saturated NaCl, dried over MgSO4, and
concentrated in vacuo. The residue was applied to a
silica gel column (2.5ꢁ20 cm) and eluted with n-hexane–
EtOAc (1:1) to give 1.1 mg (81%) of title compound 28:
+
.
[M+H] . Anal. calcd for C37H49ClN6O5S2 14/3H2O: H,
6.99; C, 52.81; N, 9.98. Found: H, 6.56; C, 52.86; N, 9.61.
Water-solubility of prodrugs and parent compounds
Water-solubility of the prodrugs and parent compounds
was determined by HPLC analysis. HPLC was per-
formed using C18 reverse phase column (YMC AM-302
ODS; 4ꢁ150 mm) with a binary solvent system at a flow
rate of 1 mL/min, detected at UV 230 nm. An excessive
amount of the prodrugs or the parent drugs was sus-
pended in pure water under supersonification for 5 min
at rt, and filtered by centrifugal membrane filtration of
0.45 mm pore size. Water-solubility was calculated from
each peak area of the above solution by HPLC compared
with the sample, the concentration of which is already
known. The conversion of prodrugs to the parent drugs
was not observed during the experiment, since the com-
pound solution was slightly acidic due to an HCl salt.
mp72–81 ꢀC; H NMR (300 MHz, CDCl3) d 1.38 (s,
1
9H), 1.60 (m, 2H), 2.72 (d, J=6.6 Hz, 2H), 2.85 (d,
J=7.5 Hz, 2H), 3.49 (br s, 1H), 3.64 (br s, 1H), 3.71–
3.98 (m, 2H), 4.53 (br s, 1H), 5.16 (d, J=9.0 Hz, 1H),
5.23 (s, 2H), 7.00–7.37 (m, 10H), 7.82 (s, 1H), 8.78 (s,
1H); TOF-MS: m/z 526 [M+H]+. Anal. calcd for
C28H35N3O5S: H, 6.71; C, 63.98; N, 7.99. Found: H,
6.74; C, 63.78; N, 7.80.
(2S,3S,5S)-3-[O-[N-[N-methyl-N-[(2-isopropyl-4-thiazo-
lyl)methyl]amino]carbonyl]-
oxy)carbonyl]amino]-2-[N-[(5-thiazolyl)methoxy]carbony-
l]amino]-1,6-diphenyl-3-hydroxyhexane] 30. To
L-valyl]oxy]-5-[N-[(tert-butyl-
Determination of the reaction rate of O!N acyl
a
migration of synthetic prodrugs
solution of compound 28 (600 mg, 1.14 mmol) and N-
[N-methyl-N-[(2-isopropyl-4-thiazolyl)methyl] amino]-
carbonyl]-l-valine 2944À46 (537 mg, 1.71 mmol) in
CHCl3 (2 mL) were added DMAP (27 mg, 0.22 mmol)
and DCC (400 mg, 1.94 mmol) at 0 ꢀC and the reaction
mixture was stirred overnight at room temperature.
After the reaction mixture was concentrated under
reduced pressure, the resulting residue was dissolved in
EtOAc, washed sequentially with 5% citrate, 5%
NaHCO3 and saturated NaCl, dried over MgSO4, and
concentrated in vacuo. The residue was recrystallized
from hexane to give 250 mg (27%) of the title com-
The migration rate of prodrugs of KNI-compounds and
ritonavir determined by RP-HPLC. 5 mL of methanol
solutions (1 mg/mL) of prodrugs was poured into
500 mL PBS buffer (pH 4.9, 5.5, 7.4, 8.0) and incubated
at 37 ꢀC. At different points of time, 200 mL of the mix-
ture was analyzed directly by HPLC.
HPLC was performed using C18 reverse-phase col-
umn (YMC AM-302 ODS; 4ꢁ150 mm) with a binary
solvent system at a flow rate of 1 mL/min, detected at
UV 230 nm. Prodrugs 3–7 were flowed out by linear
gradient of MeCN 25–80% (15 min) and prodrugs 8–
11 were flowed out by linear gradient of MeCN 30–
100%(15 min) in 0.1% aqueous TFA. Because rito-
navir prodrug and it’s parent compound showed
similar retention times under the above eluent condition,
ritonavir prodrug was flowed out by linear gradient of
MeCN 40–80% (15 min) in 0.1 M ammonium acetate
buffer (pH 7.5). Half-life of prodrugs was calculated from
peak areas at each incubation time.
pound as a solid: mp 72–76 ꢀC; H NMR (300 MHz,
1
CDCl3) d 1.01 (d, J=6.9 Hz, 3H), 1.07 (d, J=6.9 Hz,
3H), 1.36 (s, 15H), 1.68 (m, 2H), 2.28 (dq, J=6.9,
11.2 Hz, 1H), 2.72 (br s, 4H), 2.95 (s, 3H), 3.25 (hepta-
let, J=6.9 Hz, 1H), 3.70–4.60 (m, 6H), 4.90–5.28 (m,
4H), 6.41 (br s, 1H), 6.97 (s, 1H), 7.00–7.40 (m, 10H),
7.78 (s, 1H), 8.77 (s, 1H); TOF-MS: m/z 844 [M+Na]+.
Anal. calcd for C42H56N6O7S2: H, 6.87; C, 61.44; N,
10.24. Found: H, 6.92; C, 61.21; N, 10.06.
(2S,3S,5S)-5-amino-3-[O-[N-[N-methyl-N-[(2-isopropyl-
4-thiazolyl)methyl]amino]carbonyl]-L-valyl]oxy]-2-[N-[(5-
thiazolyl)methoxy]carbonyl]amino]-1,6-diphenyl-3-hydro-
xyhexane] hydrochloride (Ritonavir prodrug) 12. To
HIV-1 PR inhibition
HIV-1 PR inhibitory activity of prodrugs 3, 4, 8, 9 and
12 was determined based on the previously reported

4166
Y. Hamada et al. / Bioorg. Med. Chem. 10 (2002) 4155–4167
method34,48,49 using a substrate S1050À52 [H-Lys-Ala-
S.; Saldivar, A.; Wideburg, N. E.; Kati, W. M.; Ruiz, L.;
Zhao, C.; Fino, L.; Patterson, J.; Molla, A.; Plattner, J. J.;
Norbeck, D. W. J. Med. Chem. 1998, 41, 602.
Arg-Val-Tyr-Phe(p-NO2)-Glu-Ala-Nle-NH2]
recombinant HIV-1 PR (NY5-type sequence).
and
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Acknowledgements
This research was supported, in part, by the Frontier
Research Program of the Ministry of Education,
Science and Culture of Japan and the Japan Health
Science Foundation. We thank Mr. T. Hamada for
measurement of mass spectroscopy. We thank Dr. S. N.
Rajesh and Dr. M. Skwarczynski for their helpin pre-
paring the manuscript.
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