Design, synthesis, and biological evaluation of anti-HIV double-drugsconjugates of HIV protease inhibitors with a reverse transcriptase inhibitor through spontaneously cleavable linkers

Article abstract of DOI:10.1016/S0968-0896(01)00045-1

Based o the prodrug concept as well as the combination of two different classes of anti-HIV agents, we designed and synthesized a series of anti-HIV double-drugs consisitng of HIV protease inhibitors conjugated with a nucleoside reverse transcriptase inhibitor in an effort to enhance the antiviral activity. For the conjugation, a series of linkers that conjoins the two different classes of inhibitors has been investigted. Double-drugs using a succinyl amino acid linker were shown to release the parent drugs via spontaneous imide formation at a faster rate compared ring. Among the double-drugs, KNI-1039 (3B) with a glutaryl-glycine linker exhibited extremely potent anti-HIV activity compared with that of the individual components. Double-drug 3b was relatively stable in culture medium, whereas it regenerated active species in cell homogenate. These results suggested that the synergistic enhancement of anti-HIV activities of 3b may be due to their ability to penetrate into the target cell and subsequent regeneration of two diferent classes of anti-HIV agents in the cytoplasm. Copyright

Full text of DOI:10.1016/S0968-0896(01)00045-1

Bioorganic & Medicinal Chemistry 9 (2001) 1589–1600
Design, Synthesis, and Biological Evaluation of Anti-HIV
Double-Drugs: Conjugates of HIV Protease Inhibitors with a
Reverse Transcriptase Inhibitor through Spontaneously
Cleavable Linkers
Hikaru Matsumoto,a Tooru Kimura,a Tomonori Hamawaki,a Akira Kumagai,a
Toshiyuki Goto,b Kouichi Sano,b Yoshio Hayashia and Yoshiaki Kisoa,*
aDepartment of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University,
Yamashina-ku, Kyoto 607-8412, Japan
bDepartment of Microbiology, Osaka Medical College, Takatsuki-shi, Osaka 569-8686, Japan
Received 9 January 2001; accepted 10 February 2001
Abstract—Based on the prodrug concept as well as the combination of two different classes of anti-HIV agents, we designed and
synthesized a series of anti-HIV double-drugs consisting of HIV protease inhibitors conjugated with a nucleoside reverse tran-
scriptase inhibitor in an effort to enhance the antiviral activity. For the conjugation, a series of linkers that conjoins the two dif-
ferent classes of inhibitors has been investigated. Double-drugs using a succinyl amino acid linker were shown to release the parent
drugs via spontaneous imide formation at a faster rate compared to compounds using a glutaryl amino acid linker, as expected
from the energetically favorable cyclization to the five-membered ring. Among the double-drugs, KNI-1039 (3b) with a glutaryl-
glycine linker exhibited extremely potent anti-HIV activity compared with that of the individual components. Double-drug 3b was
relatively stable in culture medium, whereas it regenerated active species in cell homogenate. These results suggested that the
synergistic enhancement of anti-HIV activities of 3b may be due to their ability to penetrate into the target cell and subsequent
regeneration of two different classes of anti-HIV agents in the cytoplasm. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
inhibitory activity, but also on their intracellular con-
centrations closely related to the membrane perme-
ability.12 The poor antiviral activities of PR inhibitors
containing a carboxyl group were probably due to their
insufficient cell membrane permeability caused by the
presence of the carboxyl group. To improve antiviral
activity, we have suggested ‘double-drug’ strategy that
combined an HIV PR inhibitor and a nucleoside RT
inhibitor (RTI) in a single molecule.13ꢀ15 The following
advantages were envisaged for this strategy: (1) the
undesirable physicochemical property such as low
membrane permeability would be improved. (2) The
conjugation of HIV PR inhibitors with the nucleoside
RT inhibitor may facilitate the penetration through the
biological membrane mediated by the nucleoside trans-
porter.16ꢀ18 In addition, the nucleosides have the affinity
to cell membranes.19 (3) Once the double-drug escapes
extracellular hydrolysis, it would enter the cell wherein
the intracellular hydrolysis regenerates the parent inhi-
bitors. They can act on two separate targets and exhibit
synergistic anti-HIV efficacy. Based on these premises,
HIV protease (HIV PR) and reverse transcriptase (RT)
are major targets for the prevention of HIV prolifera-
tion, and inhibitors of these enzymes are widely
employed for the chemotherapy of AIDS.1ꢀ3 In order to
maintain the efficient antiviral effect and to prevent
emergence of a drug-resistant virus, a combination of
HIV PR inhibitors and RT inhibitors has become the
clinical practice.4ꢀ7 We have studied the structure–
activity relationship of dipeptide-based HIV PR inhibi-
tors.8 Among them, we have obtained several HIV PR
inhibitors containing a carboxyl group, which exhibited
potent HIV PR inhibition but poor antiviral
activity.8ꢀ10
Bilello et al.11 have reported that the antiviral efficacy of
HIV PR inhibitors depends not only on the enzyme
*Corresponding author. Tel.:+81-75-595-4635; fax: +81-75-591-
9900; e-mail: kiso@mb.kyoto-phu.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00045-1

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H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
we had developed potent hybrid-type anti-HIV
agents,13ꢀ15 in which the carboxyl group of HIV
protease inhibitors was directly esterified with the
50-hydroxyl group of a nucleoside RT inhibitor, 30-
azido-30-deoxythimidine (AZT).20 The anti-HIV activ-
ities of these hybrid-type prodrugs were found to be
more potent than those of AZT and the parent HIV PR
inhibitors. These results suggested that the ‘double-
drug’ would be useful to enhance the anti-HIV efficacy
synergistically and improve the physicochemical char-
acteristics. However, this double-drug strategy using
direct-esterification was only applicable to the inhibitors
containing a carboxyl group.
HIV PR inhibitor 1 contains the hydroxyl group, which
is essential for its enzyme inhibitory activity,8 and
nucleoside RT inhibitor AZT also contains the hydroxyl
group. Therefore, we connected both the hydroxyl
groups of HIV PR inhibitor 1 and RT inhibitor (AZT)
through the spontaneously cleavable linker (Fig. 2).
The anti-HIV activities of the resulting double-drugs
were found to be more potent than those of AZT and
KNI-727 (1).21 These results suggested that the pro-
drugs would penetrate the cell membrane and exhibit
synergistic anti-HIV activity (Fig. 3). In order to con-
firm whether the double-drugs regenerate the parent
drugs inside the cell, the disintegration profile of the
prodrugs was determined in various media such as cell
homogenate and cell culture. Several disintegrated pro-
ducts were characterized and the in vitro anti-HIV effi-
cacy was evaluated. Furthermore, we synthesized the
conjugates of AZT and an HIV PR inhibitor KNI-840
(2), which showed more potent HIV PR inhibitory
activity compared with inhibitor 1. In this paper, we
describe the design, synthesis and biological evaluation
of these anti-HIV double-drugs.
We have applied the ‘double-drug’ concept to the
dipeptide HIV PR inhibitor KNI-727 (1),8ꢀ10 which is
devoid of a carboxyl group (Fig. 1). Inhibitor (1)
exhibited excellent HIV PR inhibitory activity but poor
anti-HIV activity. The poor water solubility and the
high lipophilic nature of 1 might restrict its penetration
across the cell membrane, thus resulting in poor anti-
HIV activity. To enhance the anti-HIV activity and
improve the physicochemical characteristics of inhibitor
1, we designed and synthesized the hybrid-type pro-
drugs of KNI-727 (1) conjugated with AZT (Fig. 1).
Conjugation Strategy
For the conjugation, a series of linkers connecting the
hydroxyl group of KNI-727 (1) and AZT was investi-
gated. At first, we incorporated succinic acid as a linker,
but the resulting compound 4 (KNI-935) (Fig. 4) was
ineffective (EC50=188.9 nM, HIV-1IIIB/CEM-SS) in the
antiviral cell culture assay. Since the succinyl ester link-
age was stable towards enzymatic cleavage,22 the parent
drugs were not regenerated inside the cell, and hence the
anti-HIV activity was poor.
Figure 1. Structure of HIV protease inhibitors (HIV PRI) 1, 2, and the
nucleoside reverse transcriptase inhibitor (RTI) AZT.
The essential criteria in the design of prodrugs are that
the prodrugs should be stable outside the target cell and
after the penetration across the cell membrane, it should
regenerate the parent inhibitors.23 Based on these
premises, we studied a series of linkers having the ability
to release the parent drugs spontaneously in the
physiological environment (Fig. 3).
The intramolecular displacement of benzyl alcohol in
b-benzyl-aspartyl peptide with the concomitant formation
Figure 2. Structure of double-drugs containing spontaneously clea-
vable linkers.
Figure 3. Concept of anti-HIV double-drugs containing sponta-
neously cleavable linkers.

H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
1591
of aspartylimide derivatives is one of the frequent reac-
tions in peptide synthesis.24,25 Therefore we tried to uti-
lize this reaction to the design of spontaneously
cleavable linker, and employed succinyl amino acid or
glutaryl amino acid as a linker. Under mild alkaline
conditions, these linkers were expected to release the
parent drug KNI-727 (1) via intramolecular nucleo-
philic attack by the amide nitrogen with an imide for-
mation (Fig. 5, path A).26 The resulting imide fragment
would be hydrolyzed and AZT be released. In order to
vary the rate of drug release, prodrugs with various lin-
kers such as 3a–3d were synthesized and the stability
was analyzed.
methylamino) phosphonium hexafluorophosphate/1-
hydroxybenzotriazole (BOP/HOBt) in the presence of
triethylamine, and the following deprotection by tri-
fluoroacetic acid (TFA) afforded 7b. Compound 4 was
prepared by condensation of 8a and AZT by use of 1,3-
dicyclohexylcarbodiimide/dimethylaminopyridine (DCC/
DMAP).
Prodrugs 3a–3d were synthesized as shown in Scheme 2.
AZT was coupled with Boc-Gly-OH or Boc-b-Ala-OH
using DCC in the presence DMAP, and the following
deprotection by 4 N HCl/dioxane afforded 9a,b. Con-
densation of 9a,b with 8a,b using 1-ethyl-3-(3-(dimethy-
lamino)propyl)carbodiimide hydrochloride (EDCꢁ HCl)
in the presence of HOBt resulted in the prodrugs 3a–3d,
which were purified by silica gel column chromato-
graphy and preparative HPLC using binary solvent
system (linear gradient of CH3CN in 0.1% aqueous
TFA). Disintegration products 6a,b were prepared by
coupling 9a with succinic anhydride or glutaric anhy-
dride in the presence of triethylamine.
Chemistry
Inhibitor 1 was prepared according to the method
described previously.10 Scheme 1 illustrates the synthetic
procedure of intermediates 8a,b, disintegrated product
7b and KNI-935 (4). Compound 1 was coupled with
succinic anhydride or glutaric anhydride in THF–ether
(1:2) in the presence of dicyclohexylamine (DCHA) to
yield the half-ester 8a,b. Condensation of 8b and tert-butyl
ester of glycine by use of benzotriazole-1-yloxy-tris (di-
Disintegration product 5a was prepared as shown in
Scheme 3. Condensation of compound 10, which was
obtained by the treatment of succinic anhydride with
benzyl alcohol, with 9a afforded compound 11. Com-
pound 11 was stirred in 50% DMSO in phosphate buf-
fer (pH 7.4) in the presence of triethylamine at 40 ꢂC.
The intramolecular cyclization reaction occurred to
afford imide 5a.
Scheme 4 illustrates the synthetic procedure of pro-
drugs 3e–3h consisting of another dipeptide HIV PR
inhibitor KNI-840 (2) and AZT. Dipeptide 2-methyl-
benzylamide
derivative
12
(Apns-Dmt-NH-(2-
methyl)Bzlꢁ HCl; Apns=AHPBA=(2S, 3S)-3-amino-2-
hydroxy-4-phenylbutyric acid; Dmt=l-dimethylthio-
proline=l-1,3-thiazolidine-4-carboxylic acid) and 2,6-
dimethylphenoxyacetic acid were prepared according to
the methods described previously.10 Condensation of 12
with 2,6-dimethylphenoxyacetic acid using the EDC-
HOBt method gave 2. The resulting inhibitor 2 was
converted to the prodrugs 3e–3h in a manner similar to
that described for 3a–3d. All the prodrugs 3e–3h were
finally purified by preparative HPLC.
Figure 4. Structure of the conjugate 4 (KNI-935) using succinic acid
linker.
Results and Discussion
Disintegration studies of prodrugs
The prodrugs were designed to release the parent drug
KNI-727 (1) spontaneously via intramolecular cycliza-
tion through an imide formation. However, there is a
possibility of another disintegration pathway such as
simple hydrolysis of the ester bond. Therefore, the dis-
integration profiles in various media were determined by
HPLC (Fig. 5).
In phosphate buffered saline (pH 7.4, 37 ꢂC), the spon-
taneous imide formation with the release of KNI-727 (1)
and several other intermediates was observed for all the
prodrugs. The typical HPLC chromatogram and a
Figure 5. Disintegration pathway of double-drugs to parent inhibitors.

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H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
Scheme 1. Reagents and conditions: (a) succinic anhydride or glutaric anhydride, DCHA, THF–ether (1:2); (b) HClꢁ H-Gly-OBut, Et3N, BOP,
HOBt, DMF; (c)TFA; (d) AZT, DCC, DMAP, DMF.
Scheme 2. Reagents and conditions: (a) Boc-Gly-OH or Boc- b-Ala-OH, DCC, DMAP, DMF; (b) 4 N HCl/dioxane; (c) 8a or 8b, EDCꢁ HCl, HOBt,
DMF; (d) succinic anhydride or glutaric anhydride, Et3N, DMF.
Scheme 3. Reagents and conditions: (a) 9a, Et3N, EDCꢁ HCl, HOBt, DMF; (b) Et3N, DMSO, phosphate buffer, 40 ꢂC.
Scheme 4. Reagents and conditions: (a) 2,6-dimethylphenoxyacetic acid, Et3N, HOBt, EDCꢁ HCl, DMF; (b) succinic anhydride or glutaric anhy-
dride, DCHA, THF–ether (1:2); (c) 9a or 9b, Et3N, HOBt, BOP, DMF.

H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
1593
kinetic profile for the disintegration of prodrug 3a are
shown in Figures 6 and 7, respectively. All of the peaks
in the chromatograms were unambiguously assigned by
mass spectrometry. At first, prodrug 3a released com-
pound 1 through the imide formation, and the resulting
5a disintegrated to AZT directly or indirectly via 6a
(path A, Fig. 5).
and the kinetic profile was approximately the same as in
PBS, pH 7.4 (Fig. 8A). In Molt-4 cell homogenate, the
disintegration pattern of 3b was different from that
observed in PBS, and compound 7b, KNI-727 (1) and
AZT were observed (Fig. 8B). In this medium, the
esterase-mediated hydrolysis (path B) as well as sponta-
neous cleavage (path A) as shown in Figure 5 would
occur. The resulting compound 7b was stable, and was
not converted to the parent drug 1.
The drug release rate varied depending on the linkers
(Table 1). The rate was indicated as the t1/2, which is the
time required for 50% release of KNI-727 (1) in PBS
(pH 7.4, 37 ꢂC). Prodrug 3a conjugated by the succi-
nylglycine linker had the faster releasing rate (t1/2=0.7 h)
compared with 3b (KNI-1039) containing glutar-
ylglycine linker (t1/2=12 h), as expected from energeti-
cally favorable cyclization to the five-membered ring.
The t1/2 of the prodrugs (3c, 3d) containing b-alanine in
the linker were longer than those of prodrugs (3a, 3b)
containing glycine.
In order to examine the sensitivity of the KNI-1039 (3b)
to the esterase-mediated hydrolysis, the stability in the
presence of carboxyesterase was determined. Porcine
liver esterase (PLE) was used in our study, since this
esterase has been widely used in similar studies and also
the results correlated well with those of plasma
studies.15,27ꢀ29 The prodrug 3b was completely hydro-
lyzed within 2 h resulting in compound 7b and AZT, but
the parent drug KNI-727 (1) was not regenerated. Con-
sequently, the ester linked to AZT would be readily
hydrolyzed, but that of KNI-727 (1) was resistant to
hydrolysis with the esterase. These results suggested that
prodrug KNI-1039 (3b) would regenerate the parent drug
KNI-727 (1) through the spontaneous intramolecular
In order to confirm whether the double-drugs regener-
ate the parent drugs inside the cell, the disintegration
behavior of the KNI-1039 (3b), which exhibited potent
anti-HIV activity, was examined in a culture medium
(RPMI-1640 containing 10% of heat-inactivated fetal
calf serum) and a Molt-4 cell homogenate. In the culture
medium, the prodrug 3b disintegrated through path A
Figure 6. Typical HPLC chromatograms of the disintegration of pro-
drug 3a in PBS (pH 7.4, 37 ꢂC), at 0, 1, 7 h and 72 h.
Figure 7. Kinetic profile of disintegration of prodrug 3a and the
appearance of parent drugs in PBS, pH 7.4 at 37 ꢂC.
Table 1. Biological activities and disintegration rate of ‘double-drugs’
b
Compound
m
n
R
HIV protease
inhibition (%)a
EC50HIV-1LAI
Molt-4 (nM)
Relative
potency
t1/2 (h)
3a (KNI-1038)
3b (KNI-1039)
3c (KNI-1046)
1
2
1
1
1
2
tert-Butyl
tert-Butyl
tert-Butyl
37
55
7
5.3
0.1
1.0
1.2
62
6.2
0.7
12
1.9
3d (KNI-1047)
3e
3f (KNI-1043)
3g
3h
2
1
2
1
2
2
1
1
2
2
tert-Butyl
12
38
15
7
3.4
11.4
0.8
23.1
30.0
1.8
0.5
7.8
0.3
0.2
20
–
–
–
–
2-Methylbenzyl
2-Methylbenzyl
2-Methylbenzyl
2-Methylbenzyl
28
AZT
1 (KNI-727)
2 (KNI-840)
—
—
—
—
—
—
—
—
—
—
100 (92)
100 (97)
6.2
92.0
168.9
1.0
0.07
0.04
–
–
–
a% of HIV-1 protease inhibition in the presence of 5 mM or 50 nM (in parentheses) of inhibitors.
bt1/2 is the time required for 50 release of parent drug (1) at 37 ꢂC in phosphate buffered saline (pH 7.4).

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H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
Figure 8. Disintegration profiles of prodrug 3b (KNI-1039) in cell culture medium (RPMI-1640 containing 10% of heat-inactivated fetal calf serum)
and Molt-4 cell homogenate at 37 ꢂC.
cyclization reaction, and AZT by the hydrolysis inside
the cell.
As mentioned above, in Molt-4 homogenate, prodrug
KNI-1039 (3b) disintegrated via spontaneous cleavage
(path A) as well as esterase-mediated hydrolysis (path
B). Therefore, the disintegrated compounds 5a, 6b and
7b were synthesized and evaluated for biological activ-
ity. It was difficult to synthesize 5b, which is the disin-
tegration material of prodrug 3b. Therefore, 5a was
used instead of 5b. All the compounds except for 7b did
not show the RT and HIV PR inhibitory activity.
Compound 7b exhibited moderate HIV PR inhibitory
activity (5 mM inhibition=99%, 50 nM inhibi-
tion=17%), but the antiviral activity was low
(EC50=3800 nM). The antiviral activity of 5a was
almost the same (EC50=13 nM) as that of AZT
(EC50=15 nM),33 and 6b was 2 times less potent
(EC50=34 nM) than AZT. These results support our
hypothesis, that is the prodrug KNI-1039 (3b) pene-
trates across the cell membrane and then the regener-
ated active species inhibit different targets with
synergistic effect. The antiviral efficacy of prodrugs
depends on many factors, such as enzyme inhibition,
cell membrane permeability, extracellular stability and
intracellular disintegration, and the correlation between
them is very complicated.
Biological activities
The enzyme inhibitory potencies of these prodrugs
against HIV PR and RT were evaluated as reported
previously.10,30 All of the prodrugs 3a–3h exhibited no
HIV-1 RT inhibitory activity and poor HIV PR inhibi-
tory activity, whereas the parent HIV PR inhibitor
KNI-727 (1) showed potent enzyme inhibition. Since
both of the hydroxyl groups of the HIV PR inhibitor
and the RT inhibitor are essential for their enzyme
inhibitory activity,8,31 masking the hydroxyl moieties of
these inhibitors results in poor enzyme inhibitory activity.
The anti-HIV activities of the prodrugs were evaluated
against HIV-1 strain LAI in Molt-4 cells.32 All of the
prodrugs 3a–3d showed more potent anti-HIV activity
than that of the parent inhibitor KNI-727 (1). Interest-
ingly, the anti-HIV activities of prodrugs seemed to be
affected by the structure of linkers. We suppose that the
differences of the activities were due to the stability of
the prodrugs. Prodrug 3a (KNI-1038) containing a suc-
cinylglycine linker showed a similar antiviral activity to
that of AZT, which might be due to the instability of
compound 3a (t1/2=0.7 h). Prodrug 3a might con-
siderably disintegrate outside the cell. On the other
hand, prodrug 3b (KNI-1039) containing glutaryl-
glycine linker exhibited remarkable anti-HIV activity
(EC50=0.1 nM, therapeutic index >2000) that was 920
and 62 times more potent than those of KNI-727 (1)
and AZT, respectively. These results suggested that the
conjugates would penetrate the cell membrane and then
the two classes of enzyme inhibitors were generated,
which could attack different targets in the infected cell,
thus exhibiting synergistic anti-HIV activity. The t1/2 of
3c was longer than that of 3a, and the anti-HIV activity
of 3c was 5 times more potent than that of 3a. Prodrug
3d exhibited longer half-life (t1/2=20 h), but its antiviral
activity was poor. This is probably due to the high sta-
bility of 3d, which prevents the disintegration to its
parent compounds.
We also applied this double-drug strategy to another
dipeptide HIV PR inhibitor KNI-840 (2), which exhib-
ited more potent HIV PR inhibitory activity (50 nM
inhibition=97%) compared with KNI-727 (1) but poor
antiviral activity (Table 1). Prodrugs 3e–3h consisting of
KNI-840 (2) and AZT showed better antiviral activity
than parent HIV PR inhibitor 2, but less potent than
AZT except for 3f. Prodrug 3f containing a glutaryl-
glycine linker exhibited the most potent anti-HIV activ-
ity in 3e–3h, and the activity was 7.8 times more potent
than that of AZT.
Conclusion
Based on the ‘double-drug’ strategy, we synthesized and
evaluated a series of prodrug-type anti-HIV agents
conjugating HIV PR inhibitors to a nucleoside-type RT

H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
1595
inhibitor using spontaneously cleavable linkers. Among
the prodrugs studied, KNI-1039 (3b), a ‘double-drug’ of
an HIV PR inhibitor KNI-727 (1) conjugated with a RT
inhibitor AZT by a glutarylglycine linker, exhibited
remarkable anti-HIV activity, which was 920 and 62
times more potent than those of KNI-727 (1) and AZT,
respectively. Prodrug 3b was relatively stable in culture
medium, whereas it regenerated active species in cell
homogenate. These results suggested that the double-
drug KNI-1039 (3b) penetrates across the cell mem-
brane and then the regenerated active species inhibit
different targets with synergistic effect. We believe that
this ‘double-drug’ approach in the field of medicinal
chemistry would pave the way for the development of
more potent drugs.
J=8.6 Hz, 1H), 4.52 (s, 1H), 4.50 (br m, 1H), 4.21 (d,
J=14.1 Hz, 1H), 3.99 (d, J=14.1 Hz, 1H), 2.98–2.89 (m,
2H), 2.75–2.55 (m, 4H, partially covered by DMSO
peaks), 2.14 (s, 6H), 1.47 (s, 3H), 1.40 (s, 3H), 1.25 (s,
9H); MS (FAB) m/z=656 [M+H]+. Anal. calcd for
C34H41N3O5S: C, 62.27; H, 6.92; N, 6.41. Found: C,
61.98; H, 6.94; N, 6.37.
(R)-N-tert-Butyl-3-[(2S,3S)-3-(2,6-dimethylphenoxyacetyl)-
amino-2-glutaryloxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (2,6-dimethylphenoxyacetyl-
Apns(glutaryl)-Dmt-NHBut, 8b). Compound 8b was
prepared from glutaric anhydride and compound 1 in a
manner similar to that described for compound 8a.
Yield 60%: mp 112–113 ꢂC; 1H NMR (300 MHz,
DMSO-d6) d 8.12 (d, J=9.0 Hz, 1H), 7.66 (s, 1H), 7.35
(d, J=6.6 Hz, 2H), 7.25–7.15 (m, 3H), 7.01–6.89 (m,
3H), 5.36–5.33 (m, 1H), 4.97 (d, J=8.7 Hz, 1H), 4.93 (d,
J=8.7 Hz, 1H), 4.52 (s, 1H), 4.48 (br m, 1H), 4.18 (d,
J=14.1 Hz, 1H), 3.97 (d, J=14.1 Hz, 1H), 2.91–2.88
(m, 2H), 2.77–2.74 (m, 2H), 2.30–2.20 (m, 4H), 2.13 (s,
6H), 1.48 (s, 3H), 1.46 (s, 3H), 1.26 (s, 9H); HRMS
(FAB): m/z 670.3170 for [M+H]+ (calcd 670.3162 for
C35H48N3O8S).
Experimental
Reagents and solvents were used as purchased from
Wako Pure Chemical Ind Ltd. (Osaka, Japa), nacalai
tesque (Kyoto, Japan), and Aldrich Chemical Co. Inc
(Milwaukee, WI) without further purification. Column
chromatography was performed on Merck 107734 silica
gel 60 (70–230 mesh). TLC was performed using Merck
Silica gel 60F254 precoated plates. Melting points were
measured on a Yanagimoto micro melting apparatus
without correction. Analytical HPLC was performed
using a C18 reverse phase column (4.6ꢃ150 mm; YMC
Pack ODS AM302) with binary solvent system: linear
gradient of CH3CN (0–100%, 40 min) in 0.1% aqueous
TFA at a flow rate of 0.9 mL/min, detected at UV
230 nm. Preparative HPLC was carried out on a C18
reverse phase column (20ꢃ250 mm; YMC Pack ODS
SH343-5) with binary solvent system: linear gradient of
CH3CN in 0.1% aqueous TFA at a flow rate of 5.0 mL/
min, detected at UV 230 nm. 1H NMR spectra were
obtained on a JEOL 300 MHz or Varian 400 MHz
spectrometer with TMS as an internal standard. FAB–
MS was performed on a JEOL JMS-SX102A spectro-
meter equipped with the JMA-DA7000 data system.
Optical rotation was determined with a Horiba SEPA-
300 polarimeter.
2,6-Dimethylphenoxyacetyl-Apns(glutaryl-Gly-OH)-Dmt-
NHBut (7b). To a solution of 8b (100 mg, 0.149 mmol)
in DMF (2 mL) was added HClꢁ H-Gly-OBut (25 mg,
0.149 mmol), triethylamine (50 mL, 0.358 mmol), HOBt
(46 mg, 0.298 mmol) and BOP (93 mg, 0.209 mmol) at
0 ꢂC, and the mixture was stirred for 18 h at rt. After
removal of the solvent in vacuo, the residue was dis-
solved in EtOAc (30 mL), washed with 10% citric acid
and saturated NaCl, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3–MeOH) and
reprecipitated from n-hexane. The product was redis-
solved in trifluoroacetic acid (2 mL) and stirred for 1 h.
The reaction mixture was concentrated in vacuo, and
the residue was dissolved in EtOAc (30 mL), washed
with saturated NaCl, dried over anhydrous Na2SO4,
and concentrated in vacuo. The product was obtained
as a colorless oil (51 mg). Yield 47%: TLC Rf 0.10
(CHCl3:MeOH=10:1); 1H NMR (300 MHz, DMSO-d6)
d 8.26 (d, J=8.7 Hz, 1H), 8.16 (t, J=6.0 Hz, 1H), 7.67
(s, 1H), 7.38 (d, J=6.6 Hz, 2H), 7.27–7.16 (m, 4H),
7.01–6.89 (m, 1H), 5.33 (d, J=3.9 Hz, 1H), 5.11 (d,
J=8.7 Hz, 1H), 4.92 (d, J=8.7 Hz, 1H), 4.55 (s, 1H),
4.49 (br m, 1H), 4.52 (d, J=14.1 Hz, 1H), 4.00 (d,
J=14.1 Hz, 1H), 3.72 (d, J=5.7 Hz, 2H), 2.98–2.84 (m,
2H), 2.45 (t, J=7.4 Hz, 2H), 2.21 (t, J=7.4 Hz, 1H),
2.13 (s, 6H), 1.85–1.78 (m, 2H), 1.47 (s, 3H), 1.40 (s,
3H), 1.25 (s, 9H); MS (FAB): m/z 727 for [M+H]+.
(R)-N-tert-Butyl-3-[(2S,3S)-3-(2,6-dimethylphenoxyacetyl)-
amino-2-succinyloxy-4-phenylbutanoyl]-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide (2,6-dimethylphenoxyacetyl-
Apns(succinyl)-Dmt-NHBut, 8a). To the solution of
protease inhibitor 1 (KNI-727) (500 mg, 0.90 mmol) in
THF–ether (1:2) were added succinic anhydride
(108 mg, 1.08 mmol) and DCHA (0.215 mL, 1.08 mmol),
and stirred for 18 h at room temperature. After removal
of the solvent in vacuo, the residue was dissolved in
EtOAc (30 mL), washed with 10% citric acid and satu-
rated NaCl, dried over anhydrous Na2SO4, and con-
centrated in vacuo. Purification of the product by silica
gel column chromatography (CHCl3–MeOH) and
reprecipitation from n-hexane gave 440 mg of the title
compound as a white solid. Yield 75%: mp 108–112 ꢂC;
1H NMR (300 MHz, DMSO-d6) d 8.23 (d, J=8.6 Hz,
1H), 7.64 (s, 1H), 7.38 (d, J=6.8 Hz, 2H), 7.28–7.18 (m,
3H), 7.00 (d, J=7.7 Hz, 2H), 6.94–6.89 (m, 1H), 5.34 (d,
J=3.9 Hz, 1H), 5.08 (d, J=8.6 Hz, 1H), 4.90 (d,
2,6-Dimethylphenoxyacetyl-Apns(succinyl-AZT)-Dmt-
NHBut (4, KNI-935). To a solution of 8a (100 mg,
0.152 mmol) in DMF (2 mL) was added AZT (45 mg,
0.167 mmol), DCC (35 mg, 0.167 mmol) and DMAP
(4 mg, 0.030 mmol) at 0 ꢂC, and the mixture was stirred
for 18 h at rt. After removal of the solvent in vacuo, the
residue was dissolved in EtOAc (30 mL), washed with
10% citric acid, 5% NaHCO3 and saturated NaCl,
dried over anhydrous Na2SO4, and concentrated in

1596
H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
vacuo. Purification of the product by silica gel column
chromatography (CHCl3–MeOH) and reprecipitation
from n-hexane gave 52 mg of the title compound as a
white solid. Yield 38%: mp 64–65 ꢂC; 1H NMR
2.28 (m, 6H), 1.85 (s, 3H); HRMS (FAB): m/z 425.1425
for [M+H]+ (calcd 425.1421 for C16H21N6O8).
Glutaryl-Gly-AZT (6b). Compound 6b was prepared
from glutaric anhydride and 9a in a manner similar to
that describe for compound 6a, and obtained as a TFA
salt. Yield 37%: TLC Rf 0.17 (CHCl3:MeOH=10:1);
1H NMR (300 MHz, DMSO-d6) d 12.1 (br s, 1H), 11.4
(s, 1H), 8.33 (t, J=5.8 Hz, 1H), 7.95 (s, 1H), 7.47 (s,
1H), 6.16–6.11 (m, 1H), 4.49–4.42 (m, 1H), 4.39–4.19
(m, 2H), 4.00–3.95 (m, 1H), 3.86 (t, J=5.4 Hz, 2H),
2.46–2.30 (m, 2H), 2.27–2.14 (m, 4H), 1.80 (s, 3H),
1.74–1.67 (m, 2H); MS (FAB): m/z 438 for [M+H]+.
(300 MHz, DMSO-d6)
d 11.30 (s, 1H), 8.21 (d,
J=8.4 Hz, 1H), 7.64 (s, 1H), 7.45 (d, J=1.2 Hz, 1H),
7.38 (d, J=7.2 Hz, 2H), 7.27–7.16 (m, 3H), 7.01–6.89
(m, 3H), 6.14–6.09 (m, 1H), 5.35 (d, J=3.9 Hz, 1H),
5.08 (d, J=8.7 Hz, 1H), 4.89 (d, J=8.7 Hz, 1H), 4.52 (s,
1H), 4.51–4.41 (m, 2H), 4.32–4.15 (m, 3H), 4.01–3.96
(m, 2H), 2.98–2.84 (m, 2H), 2.77–2.64 (m, 4H), 2.45–
2.28 (m, 2H), 2.13 (s, 6H), 1.79 (s, 3H), 1.46 (s, 3H), 1.40
(s, 3H), 1.25 (s, 9H); HRMS (FAB): m/z 905.3880 for
[M+H]+ (calcd 905.3868 for C44H57N8O11S).
2,6-Dimethylphenoxyacetyl-Apns(succinyl-Gly-AZT)-Dmt-
NHBut (3a, KNI-1038). To a solution of 8a (200 mg,
0.31 mmol) in DMF was added compound 9a (155 mg,
0.37 mmol), HOBt (98 mg, 0.77 mmol), triethylamine
(0.105 mL, 0.64 mmol) and BOP (202 mg, 0.46 mmol) at
0 ꢂC, and the solution was stirred for 18 h at rt. After
removal of the solvent in vacuo, the residue was dis-
solved in EtOAc (50 mL), washed with 5% NaHCO3,
10% citric acid and saturated NaCl, dried over anhy-
drous Na2SO4, and concentrated in vacuo. Purification
of the product by silica gel column chromatography
(CHCl3–MeOH) and preparative HPLC, and reprecipi-
tation from n-hexane gave 231 mg of the title compound
as a white solid. Yield 78%: mp 113–117 ꢂC; [a]D32
ꢀ4.69 (c 0.06, MeOH); H NMR (300 MHz, DMSO-
d6) d 11.34 (s, 1H), 8.45 (t, J=5.6 Hz, 1H), 8.21 (d,
J=8.4 Hz, 1H), 7.64 (s, 1H), 7.45 (s, 1H), 7.38 (d,
J=7.2 Hz, 2H), 7.27–7.16 (m, 3H), 7.00–6.89 (m, 3H),
6.15–6.11 (m, 1H), 5.34 (d, J=3.6 Hz, 1H), 5.08 (d,
J=8.7 Hz, 1H), 4.89 (d, J=8.7 Hz, 1H), 4.52 (s, 1H),
4.50–4.41 (m, 2H), 4.32–4.14 (m, 3H), 4.06–3.94 (m,
2H), 3.90–3.87 (m, 2H), 2.98–2.84 (m, 2H), 2.74–2.53
(m, 4H), 2.45–2.28 (m, 2H, partially covered by DMSO
peaks), 2.13 (s, 6H), 1.79 (s, 3H), 1.47 (s, 3H), 1.40 (s,
3H), 1.25 (s, 9H); HRMS (FAB): m/z 962.4090 for
[M+H]+ (calcd 962.4068 for C46H60N9O12S). Anal.
calcd for C46H59N9O12Sꢁ H2Oꢁ CF3COOH: C, 52.69; H,
5.71; N, 11.52. Found: C, 52.44; H, 5.74; N, 11.15.
Gly-AZT-HCl (9a). To a solution of Boc-Gly-OH
(175 mg, 1.00 mmol) in DMF (2 mL) were added AZT
(267 mg, 1.00 mmol), DCC (206 mg, 1.00 mmol) and
DMAP (12 mg, 0.1 mmol) at 0 ꢂC, and the mixture was
stirred at rt for 18 h. After removal of the solvent in
vacuo, the residue was dissolved in EtOAc (30 mL),
washed with 10% citric acid, 5% NaHCO3 and satu-
rated NaCl, dried over anhydrous Na2SO4, and con-
centrated in vacuo. Purification of the product by silica
gel column chromatography (CHCl3–MeOH) and
reprecipitation from n-hexane gave the Boc protected
title compound as a white solid. The resulting product
was redissolved in 4 N HCl–dioxane at 0 ꢂC, and was
added anisole (95.3 mL, 0.87 mmol), and the mixture was
stirred at rt for 1 h. After removal the solvent in vacuo,
the residue was precipitated from ether to give 260 mg
of the title compound as a white solid. Yield 79%: mp
ꢂ
1
80–83 ꢂC; H NMR (300 MHz, DMSO-d6) d 11.37 (s,
1
1H), 8.52 (br s, 3H), 7.54 (s, 1H), 6.17–6.09 (m, 1H),
4.63–4.56 (m, 1H), 4.46–4.36 (m, 2H), 4.00–3.95 (m,
1H), 3.83 (d, J=8.4 Hz, 2H), 2.57–2.48 (m, 1H), 2.40–
2.30 (m, 1H), 1.82 (s, 3H); HRMS (FAB): m/z 325.1255
for [M+H]+ (calcd 325.1260 for C12H17N6O5).
ꢀ-Ala-AZT.HCl (9b). Compound 9b was prepared from
Boc-b-Ala-OH and AZT in a manner similar to that
described for compound 9a. Yield 89%: mp 77–80 ꢂC;
1H NMR (300 MHz, DMSO-d6) d 11.37 (s, 1H), 8.04 (br
s, 3H), 7.48 (d, J=0.9 Hz, 1H), 6.15–6.07 (m, 1H), 4.55–
4.49 (m, 1H), 4.31–4.30 (m, 2H), 4.01–3.96 (m, 1H), 3.03
(t, J=7.0 Hz, 2H), 2.77–2.71 (m, 2H), 2.53–2.44 (m,
1H), 2.53–2.44 (m, 1H), 2.39–2.30 (m, 1H), 1.81 (s, 3H);
HRMS (FAB): m/z 339.1413 for [M+H]+ (calcd
339.1417 for C13H19N6O5).
2,6-Dimethylphenoxyacetyl-Apns(glutaryl-Gly-AZT)-Dmt-
NHBut (3b, KNI-1039). Compound 3b was prepared
from compound 8b and 9a in a manner similar to that
described for compound 3a. Yield 25%: mp 94–96 ꢂC;
[a]3D2 ꢀ4.77ꢂ (c 0.06, MeOH); 1H NMR (400 MHz,
DMSO- d6) d 11.43 (s, 1H, AZT-NH), 8.33 (t,
J=5.9 Hz, 1H, Gly-NH), 8.24 (d, J=8.6 Hz, 1H,
AHPBA-NH), 7.66 (S, 1H, tert-butyl-NH), 7.44 (d,
J=1.1 Hz, 1H, AZT-6H), 7.37 (d, J=7.0 Hz, 2H, aro-
matic), 7.25–7.15 (m, 3H, aromatic), 6.98 (d, J=7.1 Hz,
2H, aromatic), 6.92–6.88 (m, 1H, aromatic), 6.13–6.10
(m, 1H, AZT-10-H), 5.31 (d, J=3.7 Hz, 1H, AHPBA-2-
CH), 5.09 (d, J=8.9 Hz, 1H, phenoxyacetyl-2-CH2),
4.90 (d, J=8.9 Hz, 1H, phenoxyacetyl-2-CH2), 4.53 (s,
1H, Dmt-4-CH), 4.51–4.46 (m, 1H, AHPBA-3-CH),
4.45–4.41 (m, 1H, AZT-30-H), 4.31–4.24 (m, 2H, AZT-
50-H), 4.20 (d, J=14.2 Hz, 1H, Dmt-2-CH2), 3.98 (d,
J=14.2 Hz, 1H, Dmt-2-CH2), 3.96–3.90 (m, 1H, AZT-
40-H), 3.87–3.84 (m, 2H, Gly-CH2), 2.97–2.84 (m, 2H,
AHPBA-4-CH2), 2.46–2.40 (m, 1H, AZT-20-H), 2.45 (t,
Succinyl-Gly-AZT (6a). To a solution of 9a in DMF
was added triethylamine (0.232 mL, 1.668 mmol) and
succinic anhydride (61 mg, 0.62 mmol) at 0 ꢂC, and the
mixture was stirred for 18 h at rt. After removal of the
solvent in vacuo, the residue was dissolved in EtOAc
(50 mL), washed with saturated NaCl, dried over anhy-
drous Na2SO4, and concentrated in vacuo. Purification
of the product by preparative HPLC gave 108 mg of the
title compound as white foam. Yield 46%: TLC Rf 0.31
(CHCl3–MeOH–H2O=8:3:1); 1H NMR (300 MHz,
DMSO-d6) d 11.4 (s, 1H), 8.36 (t, J=5.7 Hz, 1H), 7.46
(s, 1H), 6.15–6.05 (m, 1H), 4.49–4.43 (m, 1H), 4.36–4.19
(m, 2H), 4.00–3.94 (m, 1H), 3.90–3.86 (m, 2H), 2.46–

H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
1597
J=7.4 Hz, 2H, glutaryl-a-CH2), 2.35–2.27 (m, 1H,
AZT-20-H), 2.22 (t, J=7.3 Hz, 2H glutaryl-g-CH2), 2.11
(s, 6H, 2,6-dimethylphenoxyacetyl-CH3), 1.84–1.80 (m,
2H, glutaryl-b-CH2), 1.78 (d, J=0.9 Hz, 3H, AZT-5-
CH3), 1.46 (s, 3H, Dmt-5-CH3) 1.39 (s, 3H, Dmt-5-
CH3), 1.23 (s, 9H, tert-butyl-CH3); HRMS (FAB): m/z
976.4229 for [M+H]+ (calcd 976.4224 for
C47H62N9O12S). Anal. calcd for C47H64N9O12Sꢁ H2Oꢁ
CF3COOH: C, 53.11; H, 5.82; N, 11.38. Found: C,
53.17; H, 5.91; N, 10.88.
5H), 5.10 (s, 2H), 2.60–2.52 (m, 4H, partially covered by
DMSO peaks); MS (FAB): m/z 209 [M+H]+.
Bzl-succinyl-Gly-AZT (11). To a solution of 10 (127 mg,
0.61 mmol) in DMF was added 9a (200 mg, 0.55 mmol),
triethylamine (77 mL, 0.55 mmol), HOBt (93 mg,
0.61 mmol) and EDCꢁ HCl (117 mg, 0.61 mmol) at 0 ꢂC,
and the mixture was stirred for 18 h at rt. After removal
of the solvent in vacuo, the residue was dissolved in
EtOAc (50 mL), washed with saturated NaCl, dried
over anhydrous Na2SO4, and concentrated in vacuo.
Purification of the product by silica gel column chro-
matography (CHCl3–MeOH) gave 108 mg of the title
compound as a colorless oil. Yield 46%: TLC Rf 0.34
(CHCl3–MeOH=10:1); 1H NMR (300 MHz, DMSO-
d6) d 11.35 (s, 1H), 8.42–8.38 (t, J=5.9 Hz, 1H), 7.45 (d,
J=1.2 Hz, 1H), 7.39–7.28 (m, 5H), 6.15–6.05 (m, 1H),
5.07 (s, 2H), 4.49–4.42 (m, 1H), 4.38–4.17 (m, 2H),
4.00–3.94 (m, 1H), 3.90–3.86 (m, 2H), 2.59–2.43 (m,
5H), 2.38–2.28 (m, 1H), 1.79 (d, J=0.6 Hz, 3H); MS
(FAB): m/z 515 [M+H]+.
2,6-Dimethylphenoxyacetyl-Apns(succinyl-ꢀ-Ala-AZT)-
Dmt-NHBut (3c, KNI-1046). Compound 3c was pre-
pared from compounds 8a and 9b in a manner similar to
thatꢂ described for compound 3a. Yield 54%: mp 98–
100 C; [a]3D3 ꢀ7.50ꢂ (c 0.09, MeOH); 1H NMR
(300 MHz, DMSO-d6) d 11.35 (s, 1H), 8.25–8.20 (m,
1H), 8.05 (t, J=5.4 Hz, 1H), 7.64 (s, 1H), 7.44 (s, 1H),
7.38 (d, J=7.2 Hz, 2H), 7.27–7.16 (m, 3H), 7.01–6.89
(m, 3H), 6.14–6.10 (m, 1H), 5.34 (d, J=3.9 Hz, 1H),
5.08 (d, J=8.7 Hz, 1H), 4.89 (d, J=8.7 Hz, 1H), 4.51 (s,
1H), 4.50–4.42 (m, 2H), 4.25–4.14 (m, 3H), 4.03–3.95
(m, 2H), 3.29–3.24 (m, 2H), 2.97–2.84 (m, 2H), 2.71–
2.51 (m, 4H), 2.42–2.28 (m, 4H, partially covered by
DMSO peaks), 2.13 (s, 6H), 1.79 (s, 3H), 1.46 (s, 3H),
1.40 (s, 3H), 1.24 (s, 9H); HRMS (FAB): m/z 976.4244
for [M+H]+ (calcd 976.4224 for C47H62N9O12S).
Succinimidylmethylcarbonyl-AZT (5a). To a solution of
11 (160 mg, 0.31 mmol) in DMSO (3 mL) was added
phosphate buffer (pH 7.4) (3 mL) and triethylamine
(43 mL, 0.31 mmol), and the mixture was stirred for 18 h
at 40 ꢂC. After removal of the solvent in vacuo, the
residue was the residue was dissolved in EtOAc (50 mL),
washed with saturated NaCl, dried over anhydrous
Na2SO4, and concentrated in vacuo. Purification of the
product by preparative HPLC gave 38 mg of the title
compound as white foam. Yield 30%: TLC Rf 0.56
(CHCl3–MeOH=10:1); 1H NMR (300 MHz, DMSO-
d6) d 11.36 (s, 1H), 7.44 (d, J=1.1 Hz, 1H), 6.15–6.05
(m, 1H), 4.51–4.45 (m, 1H), 4.39–4.17 (m, 4H), 4.02–
3.97 (m, 1H), 2.78–2.71 (m, 4H), 2.54–2.27 (m, 2H), 1.80
(s, 3H); HRMS (FAB): m/z 407.1320 for [M+H]+
(calcd 407.1315 for C16H19N6O7).
2,6-Dimethylphenoxyacetyl-Apns(glutaryl-ꢀ-Ala-AZT)-
Dmt-NHBut (3d, KNI-1047). Compound 3d was pre-
pared from compounds 8b and 9b in a manner similar to
that described for compound 3a, and obtained as a TFA
salt. Yield 83%: mp 93–96 ꢂC; [a]D31 ꢀ57.0ꢂ (c 0.02,
MeOH); 1H NMR (300 MHz, DMSO-d6) d 11.36 (s,
1H), 8.25 (d, J=8.7 Hz, 1H), 7.94 (t, J=5.3 Hz, 1H),
7.67 (s, 1H), 7.45 (s, 1H), 7.38 (d, J=7.2 Hz, 2H), 7.27–
7.16 (m, 3H), 7.01–6.89 (m, 3H), 6.14–6.10 (m, 1H), 5.33
(d, J=3.3 Hz, 1H), 5.09 (d, J=9.0 Hz, 1H), 4.90 (d,
J=9.0 Hz, 1H), 4.54 (s, 1H), 4.50–4.43 (m, 2H), 4.30–
4.13 (m, 3H), 4.06–3.94 (m, 2H), 3.29–3.16 (m, 2H),
2.98–2.84 (m, 2H), 2.64–2.54 (m, 2H), 2.44–2.28 (m, 4H,
partially covered by DMSO peaks), 2.18–2.10 (m, 2H),
2.13 (s, 6H), 1.79 (s, 3H), 1.80–1.76 (m, 2H), 1.47 (s, 3H),
1.40 (s, 3H), 1.25 (s, 9H); HRMS (FAB): m/z 990.4404
for [M+H]+ (calcd 990.4380 for C48H64N9O12S). Anal.
calcd for C48H63N9O12Sꢁ H2Oꢁ CF3COOH: C, 54.39; H,
5.84; N, 11.42. Found: C, 54.86; H, 6.39; N, 10.52.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-(2,6-dimethylphen-
oxyacetyl)amino-2-hydroxy-4-phenylbutanoyl]-5,5-dime-
thyl-1,3-thiazolidine-4-carboxamide (2,6-dimethylphen-
oxyacetyl-Apns-Dmt-NH-(2-Me)Bzl, 2, KNI-840). To a
solution of 2-methylbenzylamide derivative 12 (400 mg,
0.84 mmol) in DMF was added 2,6-dimethylphenox-
yacetic acid (166 mg, 0.92 mmol), HOBt (141 mg,
0.92 mmol) and EDCꢁ HCl (177 mg, 0.92 mmol) at 0 ꢂC,
and the mixture was stirred for 18 h at rt. After removal
of the solvent in vacuo, the residue was dissolved in
EtOAc (50 mL), washed with 5% NaHCO3, 10% citric
acid and saturated NaCl, dried over anhydrous Na2SO4,
and concentrated in vacuo. Purification of the product
by silica gel column chromatography (CHCl3–MeOH)
and reprecipitation from n-hexane gave 450 mg of the
title compound as a white solid. Yield 89%: mp 80–
81 ꢂC; 1H NMR (300 MHz, DMSO-d6) d 8.38 (t,
J=5.6 Hz, 1H), 8.15 (d, J=8.7 Hz, 1H), 7.32–6.90 (m,
12H), 5.52 (d, J=7.2 Hz, 1H), 4.98 (d, J=9.6 Hz, 1H),
4.95 (d, J=9.6 Hz, 1H), 4.51 (s, 1H), 4.48–4.46 (m, 1H),
4.41 (d, J=14.4 Hz, 1H), 4.39 (d, J=14.4 Hz, 1H), 4.18
(dd, J=14.7, 6.6 Hz, 1H), 4.00 (d, J=14.4 Hz, 1H),
2.84–2.73 (m, 2H), 2.26 (s, 3H), 2.15 (s, 6H), 1.51 (s,
3H), 1.36 (s, 3H) ; MS (FAB): m/z 604 [M+H]+. Anal.
3-(Benzyloxycarbonyl)propionic acid (10). To a solution
of succinic anhydride (1.0 g, 10 mmol) in DMF was
added benzyl alcohol (0.94 mL, 9.09 mol) and DCHA
(2.39 mL, 11 mmol) at 0 ꢂC, and the mixture was stirred
for 18 h at rt. After removal of the solvent in vacuo, the
residue was dissolved in EtOAc (30 mL) and washed
with saturated NaCl. The organic layer was extracted
with saturated NaHCO3. The aqueous phase was acid-
ified with citric acid to pH 3–4, and extracted with
EtOAc (30 mL). The organic layer was washed with
saturated NaCl, dried over anhydrous Na2SO4 and
evaporated in vacuo. The residue was obtained as a
white solid (1.77 g, 94%) and was used without further
ꢂ
1
purification in the next step: mp 52–53 C; H NMR
(300 MHz, DMSO-d6) d 12.22 (s, 1H), 7.35–7.32 (m,

1598
H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
calcd for C34H41N3O5S: C, 67.64; H, 6.84; N, 6.96.
Found: C, 67.36; H, 6.94; N, 7.05.
100 ꢂC; [a]D27 ꢀ2.50ꢂ (c 0.04, MeOH); 1H NMR (300
MHz, DMSO-d6) d 11.34 (s, 1H, AZT-NH), 8.41 (t,
J=5.6 Hz, 1H, benzylamine-NH), 8.35 (t, J=5.9 Hz,
1H, Gly-NH), 8.25 (d, J=8.4 Hz, 1H, AHPBA-NH),
7.45 (d, J=0.9 Hz, 1H, AZT-6H), 7.35–7.15 (m, 6H,
aromatic), 7.12–6.89 (m, 6H, aromatic), 6.13 (m, 1H,
AZT-10-H), 5.38 (d, J=3.9 Hz, 1H, AHPBA-2-CH),
5.09 (d, J=9.0 Hz, 1H, phenoxyacetyl-2-CH2), 4.95 (d,
J=9.0 Hz, 1H, phenoxyacetyl-2-CH2), 4.63–4.52 (m,
1H, AHPBA-3-CH), 4.55 (s, 1H, Dmt-4-CH), 4.48–4.15
(m, 6H, AZT-30-H, benzylamine-CH2, AZT-50-H, Dmt-
2-CH2), 4.10–3.92 (m, 2H, Dmt-2-CH2, AZT-40-H),
3.88–3.85 (m, 2H, Gly-CH2), 2.97–2.85 (m, 2H,
AHPBA-4-CH2), 2.46–2.41 (m, 3H, partally covered by
DMSO peaks, glutaryl-a-CH2, AZT-20-H), 2.37–2.32
(m, 1H, AZT-20-H), 2.28–2.21 (m, 2H, glutaryl-g-CH2),
2.25 (s, 3H, benzylamine-CH3), 2.13 (s, 6H, 2,6-dime-
thylphenoxyacetyl-CH3), 1.86–1.81 (m, 2H, glutaryl-b-
CH2), 1.79 (s, 3H, AZT-5-CH3), 1.49 (s, 3H, Dmt-5-
CH3), 1.36 (s, 3H, Dmt-5-CH3); HRMS (FAB): m/z
1024.4235 for [M+H]+ (calcd 1024.4224 for
C51H62N9O12S). Anal. calcd for C51H61N9O12Sꢁ H2Oꢁ
CF3COOH: C, 55.06; H, 5.58; N, 10.90. Found: C,
55.34; H, 5.67; N, 10.48.
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-(2,6-dimethylphen-
oxyacetyl)amino-2-succinyloxy-4-phenylbutanoyl]-5,5-di-
methyl-1,3-thiazolidine-4-carboxamide (2,6-dimethylphe-
noxyacetyl-Apns(succinyl)-Dmt-NH-(2-Me)Bzl, 13a).
Compound 13a was prepared from compound 2 and
succinic anhydride in a manner similar to that described
ꢂ
1
for compound 8a. Yield 82%: mp 69–70 C; H NMR
(300 MHz, DMSO-d6) d 8.40 (t, J=5.4 Hz, 1H), 8.26 (d,
J=8.8 Hz, 1H), 7.34–6.87 (m, 12 H), 5.40 (d, J=3.7 Hz,
1H), 5.06 (d, J=8.8 Hz, 1H), 4.93 (d, J=8.8 Hz, 1H),
4.61–4.49 (m, 1H), 4.53 (s, 1H), 4.41–4.34 (m, 1H), 4.38
(dd, J=15.2, 6.3 Hz, 1H), 4.22–4.12 (m, 1H), 4.17 (d,
J=14.4 Hz, 1H), 4.04 (d, J=14.4 Hz, 1H), 2.97–2.82 (m,
2H), 2.73–2.61 (m, 2H), 2.57–2.50 (m, 2H), 2.25 (s, 3H),
2.14 (s, 6H), 1.48 (s, 3H), 1.36 (s, 3H); HRMS (FAB):
m/z 704.3008 for [M+H]+ (calcd 704.3006 for
C38H46N3O8S).
(R)-N-(2-Methylbenzyl)-3-[(2S,3S)-3-(2,6-dimethylphen-
oxyacetyl)amino-2-glutaryloxy-4-phenylbutanoyl]-5,5-di-
methyl-1,3-thiazolidine-4-carboxamide (2,6-dimethylphe-
noxyacetyl-Apns(glutaryl)-Dmt-NH-(2-Me)Bzl,
13b).
Compound 13b was prepared from compound 2 and
2,6-Dimethylphenoxyacetyl-Apns(succinyl-ꢀ-Ala-AZT)-
Dmt-NH-(2-Me)Bzl (3g). Compound 3g was prepared
from compounds 13a and 9b in a manner similar to
that described for compound 3a. Yield 65%: mp
94–96 ꢂC; [a]3D1+6.80ꢂ (c 0.05, MeOH); 1H NMR
glutaric anhydride in a manner similar to that described
for compound 8a. Yield 60%: mp 74–76 ꢂC; H NMR
1
(300 MHz, DMSO-d6) d 12.13 (br s, 1H), 8.42 (t,
J=5.6 Hz, 1H), 8.27 (d, J=8.4 Hz, 1H), 7.35–6.90 (m,
12H), 5.37 (d, J=3.3 Hz, 1H), 5.08 (d, J=8.9 Hz, 1H),
4.96 (d, J=8.9 Hz, 1H), 4.60–4.52 (br m, 1H), 4.56 (s,
1H), 4.39 (dd, J=15.0, 6.0 Hz, 1H), 4.21–4.12 (m, 1H),
4.17 (d, J=14.4 Hz, 1H), 4.05 (d, J=14.4 Hz, 1H),
2.97–2.85 (m, 2H), 2.47–2.44 (m, 2H), 2.34–2.28 (m,
2H), 2.25 (s, 3H), 2.14 (s, 6H), 1.85–1.75 (m, 2H), 1.49
(s, 3H), 1.36 (s, 3H); MS (FAB): m/z 718 [M+H]+.
Anal. calcd for C39H47N3O8S: C, 65.25; H, 6.60; N,
5.85. Found: C, 64.98; H, 6.77; N, 5.80.
(300 MHz, DMSO-d6)
d 11.35 (s, 1H), 8.39 (t,
J=5.7 Hz, 1H), 8.23 (d, J=8.7 Hz, 1H), 8.05 (t,
J=5.7 Hz, 1H), 7.44 (d, J=1.2 Hz, 1H), 7.34–7.15 (m,
6H), 7.11–6.89 (m, 6H), 6.14–6.10 (m, 1H), 5.40 (d,
J=3.9 Hz, 1H), 5.06 (d, J=8.7 Hz, 1H), 4.93 (d,
J=8.7 Hz, 1H), 4.61–4.52 (m, 1H), 4.52 (s, 1H), 4.49–
4.43 (m, 1H), 4.37 (dd, J=15.0, 6.3 Hz, 1H), 4.30–4.15
(m, 3H), 4.18 (d, J=14.4 Hz, 1H), 4.03 (d, J=14.4 Hz,
1H), 4.00–3.95 (m, 1H), 3.28 (dd, J=12.3, 6.3 Hz, 2H),
2.98–2.85 (m, 2H), 2.73–2.58 (m, 2H), 2.54–2.27 (m,
6H, partially covered by DMSO peaks), 2.25 (s, 3H),
2.14 (s, 6H), 1.79 (s, 3H), 1.48 (s, 3H), 1.36 (s, 3H);
HRMS (FAB): m/z 1024.4227 for [M+H]+ (calcd
1024.4239 for C51H62N9O12S).
2,6-Dimethylphenoxyacetyl-Apns(succinyl-Gly-AZT)-Dmt-
NH-(2-Me)Bzl (3e). Compound 3e was prepared from
compounds 13a and 9a in a manner similar to that
described for compound 3a. Yield 50%: mp 107–109 ꢂC;
[a]2D7+8.08ꢂ (c 0.05, MeOH); 1H NMR (300 MHz,
DMSO-d6) d 11.35 (s, 1H), 8.45 (t, J=5.9 Hz, 1H), 8.39
(t, J=5.4 Hz, 1H), 8.24 (d, J=8.7 Hz, 1H), 7.45 (d,
J=0.9 Hz, 1H), 7.34–7.15 (m, 6H), 7.11–6.90 (m, 6H),
6.13 (t, J=6.6 Hz, 1H), 5.40 (d, J=3.6 Hz, 1H), 5.06 (d,
J=8.9 Hz, 1H), 4.93 (d, J=8.9 Hz, 1H), 4.62–4.53 (m,
1H), 4.53 (s, 1H), 4.48–4.17 (m, 5H), 4.18 (d,
J=14.4 Hz, 1H), 4.03 (d, J=14.4 Hz, 1H), 3.99–3.94 (m,
1H), 3.88 (dd, J=5.7, 2.7 Hz, 2H), 2.97–2.85 (m, 2H),
2.74–2.62 (m, 2H), 2.58–2.53 (m, 2H, partially covered
by DMSO peaks), 2.45–2.41 (m, 1H), 2.37–2.27 (m,
1H), 2.25 (s, 3H), 2.13 (s, 6H), 1.79 (s, 3H), 1.48 (s, 3H),
1.36 (s, 3H); HRMS (FAB): m/z 1010.4097 for
[M+H]+ (calcd 1010.4082 for C50H60N9O12S).
2,6-Dimethylphenoxyacetyl-Apns(glutaryl-ꢀ-Ala-AZT)-
Dmt-NH-(2-Me)Bzl (3h). Compound 3h was prepared
from compounds 13b and 9b in a manner similar to that
described for compound 3a. Yield 65%: mp 97–99 ꢂC;
[a]3D1 ꢀ3.92ꢂ (c 0.05, MeOH); 1H NMR (300 MHz,
DMSO-d6) d 11.32 (s, 1H), 8.39 (t, J=5.9 Hz, 1H), 8.24
(t, J=8.3 Hz, 1H), 7.92 (t, J=5.1 Hz, 1H), 7.44 (d,
J=0.9 Hz, 1H), 7.34–7.15 (m, 6H), 7.12–6.89 (m, 6H),
6.14–6.09 (m, 1H), 5.38 (d, J=3.5 Hz, 1H), 5.07 (d,
J=8.7 Hz, 1H), 4.94 (d, J=8.7 Hz, 1H), 4.62–4.54 (m,
1H), 4.55 (s, 1H), 4.49–4.34 (m, 2H), 4.29–4.17 (m,
3H), 4.17 (d, J=14.5 Hz, 1H), 4.04 (d, J=14.5 Hz, 1H),
3.99–3.95 (m, 1H), 3.35–3.26 (m, 2H), 2.97–2.84 (m,
2H), 2.53–2.15 (m, 8H, partially covered by DMSO
peaks), 2.25 (s, 3H), 2.13 (s, 6H), 1.82–1.76 (m, 2H),
1.79 (s, 3H), 1.49 (s, 3H), 1.36 (s, 3H); HRMS (FAB):
m/z 1038.4382 for [M+H]+ (calcd 1038.4395 for
C52H64N9O12S).
2,6-Dimethylphenoxyacetyl-Apns(glutaryl-Gly-AZT)-Dmt-
NH-(2-Me)Bzl (3f, KNI-1043). Compound 3f was pre-
pared from compound 13b and 9a in a manner similar
to that described for compound 3a. Yield 69%: mp 98–

H. Matsumoto et al. / Bioorg. Med. Chem. 9 (2001) 1589–1600
1599
Disintegration studies of prodrugs in PBS buffer. The
disintegration profile of the prodrugs 3a–3d were deter-
mined in phosphate buffered saline (PBS, pH 7.4). To
12 mL of PBS (pH 7.4) was added 120 mL of prodrug
solution (0.5 mM in DMSO) and the mixture was incu-
bated at 37 ꢂC in a water bath. At different points of
time, 1 mL of the samples was withdrawn and directly
analyzed by HPLC. HPLC was performed using C18
reverse phase column (4.6ꢃ150 mm; YMC Pack ODS
AM302) with binary solvent system: linear gradient of
CH3CN (0–100%, 40 min) in 0.1% aqueous TFA at a
flow rate of 0.9 mL/min, detected at UV 230 nm.
and 2 mM EDTA was mixed with 5 mL of prodrug
(50 mM or 500 nM) dissolved in DMSO and 10 mL of
HIV-1 protease (2 mg/mL) in 50 mM MES–NaOH, pH
6.0 containing 2.5 mM DTT, 1 mM EDTA-2Na, 0.2%
Nonidet P-40, and 15% glycerol. The enzyme reaction
was initiated by addition of 10 mL of a 1.0 mM substrate
solution in the above-described assay buffer. After
incubation for 15 min at 37 ꢂC, the reaction was termi-
nated by addition of 75 mL of 1 N HCl, and the N-
terminal cleavage fragment (H-Lys-Ala-Arg-Val-Tyr-
OH) was separated by reverse-phase HPLC on a C18
column (3.0ꢃ75 mm; YMC Pack ODS AS-3E7) with
linear gradient of CH3CN (6–16%, 5 min) in 0.1%
aqueous TFA at a flow rate of 1.0 mL/min, and deter-
mined the quantity by monitoring fluorescence intensity
(Ex, 275 nm; Em, 305 nm).
Disintegration studies of prodrugs in the presence of
esterase. Porcine liver esterase (PLE; carboxylic-ester
hydrolase; EC 3.1.1.1; E-2884) was obtained from
Sigma Chemical Co. (St Louis, MO) as a suspension in
a 3.2 M (NH4)2SO4 solution (pH 8). 3.2 mL of this sus-
pension (containing 3750 units of enzyme per mL) was
diluted with 12 mL of PBS (pH7.4). In this solution, the
disintegration profile of the prodrug 3b was determined
in a manner similar to that described above.
HIV RT inhibition. The prodrugs dissolved in DMSO
were mixed with standard RT solution containing
20 mU/mL recombinant HIV-RT, and assayed with
non-RI RT assay kit (Asahikasei, Japan).30
Antiviral activity. Antiviral activity of test compounds
was determined based on inhibition of HIV-1 IIIB-
induced cytopathic effect in Molt-4 cells in vitro.
HIV-1LAI (100 TCID50) and each 4-fold diluted pro-
drug were inoculated to 5000/well Molt-4 cells in a
microplate and cultured for 7 days. EC50 values were cal-
culated from the ratios of surviving cells, which were
quantitated by WST-8 assay kit32 (Dojin Lab., Kuma-
moto, Japan).
Disintegration studies of conjugates in cell culture medium.
To 50 mL of cell culture medium (RPMI-1640 contain-
ing 10% of heat-inactivated fetal calf serum) was added
0.5 mL of a solution of prodrug 3b (10 mM in DMSO)
and the mixture was incubated at 37 ꢂC. At different
points of time, 10 mL of the samples was withdrawn and
added to 100 mL of cold MeOH. The solution of the
samples were stirred vigorously and filtered through a
membrane filter (0.45 mm), and 75 mL of the filtrate was
analyzed by HPLC in a manner similar to that described
above.
Acknowledgements
Disintegration studies of conjugates in Molt-4 cell
homogenate. Molt-4 cells were suspended to 2 mL of
ice-cold Hank’s balanced salt solution (HBSS), and was
homogenized on ice using a 15 mL Wheaton glass
homogenizer (15 strokes, pestle/wall clearance 0.64–
0.76 mm). The cell debris and nuclei were removed at
4 ꢂC by centrifugation for 10 min at 10,000 rpm (9000g)
using a Marathon 21K/BR centrifuge (Herle AG,
Gosheim, FRG). To 50 mL of Molt-4 homogenate was
added 0.5 mL of a solution of prodrug (10 mM in
DMSO) and the mixture was incubated at 37 ꢂC. At
different points of time, 10 mL of the samples was with-
drawn and added to 100 mL of cold MeOH. The solu-
tion of the samples were stirred vigorously and filtered
through a membrane filter (0.45 mm), and 75 mL of the
filtrate was analyzed by HPLC in a manner similar to
that described above.
This research was supported in part by the Frontier
Research Program of the Ministry of Education,
Science and Culture of Japan, and grants from the
Ministry of Education, Science and Culture of Japan
and Japan Health Science Foundation. We thank Dr. S.
N. Rajesh for useful discussion and Ms. Y. Matsui, Mr.
D. Shuto, and Mr. Y. Sohma for technical assistance.
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