A novel dipeptide-based HIV protease inhibitor containing allophenylnorstatine

Article abstract of DOI:10.1002/ardp.200400882

Dipeptide analogues incorporating allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] as a transition state mimic at the scissile bond were designed and synthesized in the hope of obtaining a novel KNI series of HIV protease inhibitors. The precursors, N-P₂′- 3-(2S,3S)-3-(tert-butyloxycarbonyl)amino-2-hydroxy-4-phenylbutanoyl)-5, 5-dimethylthiazolidine-4-carboxamide (N-Boc-Apns-Dmt-P₂′) 4a-p were prepared by deprotection of the synthones N-P₂′-(tert- butyloxycarbonyl)-5,5-dimethylthiazolidine-4-carboxamide (Boc-Dmt-P ₂′) 2a-p, then coupling with (2S,3S)3-(tert-butyloxycarbonyl) amino-2-hydroxy-4-phenylbutanoic acid (N-Boc-Apns-OH) 3. The deprotected intermediates 4 were coupled with the activated carboxyl groups of the P ₂ ligands to afford the target dipeptides. In this work, we fixed at the P₂ site either a 2,6-dimethylphenoxyacetyl or a 3-hydroxy-2-methylbenzoyl group. Substitutes at the P₂′ site were varied to afford the members of the series 7 and 8. Improved activity of most of the members of series 8 relative to their analogues of series 7 can be partially attributed to the differences in the structures of the P₂ moieties. Positional isomerism in the P₂′ moieties significantly affected the activity and polarity of the target.

Full text of DOI:10.1002/ardp.200400882

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
A Novel Dipeptide-based HIV Protease Inhibitor 587
Hamdy M. Abdel-Rahmana,
A Novel Dipeptide-based HIV Protease
Inhibitor Containing Allophenylnorstatine
Nawal A. El-Koussia,
Gamal S. Alkaramanya,
Adel F. Youssefa,
Yoshiaki Kisob
Dipeptide analogues incorporating allophenylnorstatine [Apns; (2S,3S)-3-
amino-2-hydroxy-4-phenylbutyric acid] as a transition state mimic at the scissile
bond were designed and synthesized in the hope of obtaining a novel KNI series
of HIV protease inhibitors. The precursors, N-P2Ј-3-(2S,3S)-3-(tert-butyloxy-
carbonyl)amino-2-hydroxy-4-phenylbutanoyl)-5,5-dimethylthiazolidine-4-carbox-
amide (N-Boc-Apns-Dmt-P2Ј) 4aϪp were prepared by deprotection of the syn-
a Pharmaceutical Medicinal
Chemistry Department,
Faculty of Pharmacy,
Assiut University,
Assiut 71526, Egypt
thones
N-P2Ј-(tert-butyloxycarbonyl)-5,5-dimethylthiazolidine-4-carboxamide
b Department of Medicinal
Chemistry, Kyoto
(Boc-Dmt-P2Ј) 2aϪp, then coupling with (2S,3S)3-(tert-butyloxycarbonyl)amino-
2-hydroxy-4-phenylbutanoic acid (N-Boc-Apns-OH) 3. The deprotected inter-
mediates 4 were coupled with the activated carboxyl groups of the P2 ligands to
afford the target dipeptides. In this work, we fixed at the P2 site either a 2,6-
dimethylphenoxyacetyl or a 3-hydroxy-2-methylbenzoyl group. Substitutes at the
P2Ј site were varied to afford the members of the series 7 and 8. Improved
activity of most of the members of series 8 relative to their analogues of series
7 can be partially attributed to the differences in the structures of the P2 moieties.
Positional isomerism in the P2Ј moieties significantly affected the activity and
polarity of the target.
Pharmaceutical University,
Kyoto 607-8412, Japan
Keywords: Dipeptide; Antiviral Drugs; HIV Protease Inhibitory Activity; SAR
Received: February 25, 2004; accepted: September 10, 2004 [FP882]
DOI 10.1002/ardp.200400882
Introduction
such as the clinically used drug ABT-378 [1, 2] (Fig-
ure 2).
Inhibition of human immunodeficiency virus (HIV)
protease is one of the most important and promising
approaches for the treatment of an HIV infection.
On the other hand, incorporation of the 2,6-dimeth-
ylphenoxyacetyl moiety into the KNI series of inhibitors
led to the development of highly potent compounds,
like the dipeptide KNI-901 [3].
The design and development of potent HIV protease
inhibitors have generated considerable interest among
the AIDS research field and HIV-positive patients. A
promising class of HIV protease inhibitors containing
allophenylnorstatine (Apns) as a transition state mimic
yielded a series of KNI derivatives of highly potent in-
hibitory properties. In this work, we reported the syn-
thesis of dipeptides with different substituents at the
P2 and P2Ј positions with respect to the leads KNI-577
and KNI-901 (see Figure 1) and their protease inhi-
bition activities.
Further challenging of the protease inhibitory activity
of the KNI series was practiced by introduction of the
The use of the 2,6-dimethylphenoxyacetyl moiety as
P2 ligand was already reported and provided com-
pounds with potent HIV protease inhibitory activity
Correspondence: Nawal A. El-Koussi, Pharmaceutical Medici-
nal Chemistry Department, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt; Phone: +20 88 411297, Fax:
+20 88 332776, e-mail: nawal-a@acc.aun.edu.eg
Figure 1. Lead protease inhibition of the KNI series.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

588 El-Koussi et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
the synthones 4aϪp (Table 4). These intermediates
were used as scaffolds for the synthesis of the targets
7 and 8. 2,6-Dimethylphenoxyacetic acid 5 was acti-
vated by EDC, HOBt in DMF or THF and coupled with
the deprotected 4aϪn to afford the series 7aϪn (Table
1). In series 8, 3-acetyl-2-methylbenzoic acid 6 was
activated by DPPCl in AcOEt and then coupled with
the deprotected synthones 4aϪh, o and p. Hydrolytic
cleavage of the 3-acetyl group by LiOH yielded 8aϪh,
o and p dipeptides in Table 2. The final compounds
obtained after crystallization were checked by analyti-
cal HPLC; from the resulting data, preparative HPLC
was established and carried out. The purity was
checked again by analytical HPLC. The fractions were
mixed and lyophilized to afford the analytically pure
final compounds; yields of the products were deter-
mined by reversed-phase HPLC (RP-HPLC). Within
each series, the parallelism between Rt of the com-
pounds and the polarity of the P2Ј moieties could be
easily observed. The effect of the location of the polar
groups in the P2Ј moieties on the Rt values of the posi-
tional isomers 7g, h, i, k, l, m, n and in 8g, h, o, p
was perceptible. Homogenity of targets 7 and 8 was
checked by TLC using two systems of different polarit-
ies: chloroform/methanol (10:1) and chloroform/meth-
anol/water (8:3:1).
Figure 2. ABT-378 (Lopinavir).
3-hydroxy-2-methylbenzoyl moiety at P2 sites, which
simultaneously provides effective capabilities of hydro-
phobic and hydrogen bonding interactions with the en-
zyme relative sites [4].
This approach yielded the highly active KNI-577 [4Ϫ7].
The improved activity may be correlated with the com-
bined capabilities of hydrophobic and hydrogen bond-
ing potentialities of their intact moiety.
Guided by the leads KNI-577 and KNI-901, we pre-
pared two series of dipeptide analogues. In one of
these series, the P2 ligand was the 2,6-dimethyl-
phenoxyacetyl moiety as shown by compounds 7 in
Table 1. Introduction of the 3-hydroxy-2-methylbenzoyl
moiety at the P2 site provided the second series of
dipeptides represented by the compounds 8 in Table
2. In series 7 and 8, P2Ј was changed in the hope
to improve protease inhibition potential and enhance
polarity. It is clear that the partition coefficient, rep-
resented by the Rt value, and solubility are closely re-
lated phenomena; therefore, the choice of moieties at
P2Ј with predominant polar functions was targeted in
both series [8, 9].
Structure activity relationship against HIV-1 pro-
tease
The effect of variation of the P2Ј moieties on HIV-1
protease inhibition of the potent leads KNI-901 and
KNI-577 was regarded as the main objective of the
present study. The benzylamino group at the P2Ј site
in KNI-901 was the target modified to afford the dipep-
tides 7, whereas the t-butyl group at the P2Ј site in
KNI-577 was replaced by the same modified benzyl-
amino derivatives to afford almost all members of
series 8.
Results and discussion
Chemistry
Three approaches were considered for modification of
the benzylamine moiety. First, the assumption that the
phenyl ring must be distanced by one atom from the
amidic NH represents an essential requisite. Groups
of different bulkiness and polarity, with one atom bridg-
ing the phenyl ring and the amidic NH, were inserted
to replace the α-methylene group in benzylamine. This
approach was challenged by the compounds 7a, b, e
and 8a, b, e. As shown in Table 5, compounds 7b and
8b with inserted α-N (CH3) were the least active ones.
On the other hand, the 2-isopropylene bridge signifi-
cantly improved the activity of 7a and 8a, which are
still less active than the corresponding leads. Com-
pound 8e revealed 91.2% inhibition potential, which is
higher than the potential of the lead KNI-577. Matching
the activities of 7a, e with their analogues 8a, e dem-
The target dipeptide inhibitors were prepared as illus-
trated in Scheme 1, starting from 5,5-dimethylthiazol-
idine-3-carboxylic acid (Dmt-OH), which was prepared
by cyclization of L-penicillamine with formaldehyde,
followed by N-protection by Boc to yield Boc-Dmt-OH
1 according to reported procedures [10]. Coupling of
the amino ligands P2ЈNH2 with 1 in the presence of 1-
hydroxybenzotriazole (HOBt) in DMF [11, 12] or di-
phenylphosphochloridate (DPPCl), Et3N and AcOEt
[13] afforded the intermediates 2aϪp (Table 3). N-
elimination of the Boc moiety and coupling with Boc-
Apns-OH 3 by either 1-ethyl-3-(3Ј-dimethylaminopro-
pyl) carbodiimide (EDC) or benzotriazole-1-yloxytris-
(dimethylamino) phosphonium hexafluorophosphate
(BOP) in the presence of HOBt in DMF [14] yielded
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
A Novel Dipeptide-based HIV Protease Inhibitor 589
onstrated a boosting effect of the P2 moiety in series
8. Secondly, introduction of a polar substitute at the
o-, m-, or p-position on the benzene ring was tried by
preparing 7c, d, f, kϪn and 8c, d, f dipeptides. All
these isomers were less active than the leads; how-
ever, the derivatives of the 8 series still revealed a
Table 1. Physical data of dipeptide-based HIV protease inhibitors 7.
No.
P2Ј
mp (°C)
Yield
(%)
HPLC§
Rt (min)
TLC
†
¥
Rf1
Rf2
7a
7b
89Ϫ90
85
27.44
24.81
0.98
0.81
0.96
0.90
115Ϫ117
54.5
7c
89Ϫ91
85Ϫ88
65
71
61
26.37
27.12
27.28
0.68
0.72
0.90
0.89
0.90
0.96
7d
7e#
103Ϫ105
7f
110Ϫ113
107Ϫ108
141Ϫ143
98Ϫ100
78
71
70
73
25.08
16.92
17.34
18.10
0.90
0.80
0.62
0.68
0.93
0.86
0.77
0.84
7g
7h
7i
7j
149Ϫ151
55
21.10
0.62
0.77
7k
7l
124Ϫ126
98Ϫ100
140Ϫ142
99Ϫ100
79
53
57
63
20.38
24.70
19.81
24.38
0.54
0.84
0.52
0.89
0.83
0.91
0.90
0.94
7m
7n
#
§
†
Mixture of diastereomers; 20Ϫ80% CH3CN in 0.1% aqueous TFA over 30 min; CHCl3/CH3OH (10:1);
¥
CHCl3/CH3OH/H2O (8:3:1).
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

590 El-Koussi et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
Table 2. Physical data of dipeptide-based HIV protease inhibitors 8.
No.
P2Ј
mp (°C)
Yield
(%)
HPLC§
Rt (min)
TLC
†
¥
Rf1
Rf2
8a
8b
128Ϫ130
150Ϫ152
65.5
78
20.86
18.08
0.69
0.37
0.88
0.76
8c
127Ϫ129
121Ϫ123
139Ϫ141
68
72
58
19.45
21.10
0.57
0.67
0.56
0.83
0.86
0.76
8d
8e#
20.42
20.52
8f
139Ϫ140
141Ϫ143
154Ϫ156
71
20.24
11.47
9.94
0.59
0.58
0.35
0.67
0.80
0.65
8g
8h
35.5
24
8o
153Ϫ155
159Ϫ161
76
66
27.15
26.64
0.56
0.54
0.66
0.70
8p
§
†
¥
#, , and as in Table 1.
higher pattern of activity that can only be attributed to
the different P2 moieties. Finally, the benzyl moiety
was replaced by the bioisosteric α-, β-, and γ-pico-
lines. This approach yielded the most active derivative
7g, which is equally active as the lead KNI-901, with
an apparently enhanced polarity. Improvement of po-
larity presumably may advantageously affect its ab-
sorption and distribution properties. Furthermore, the
presence of a basic pyridine center allows for forma-
tion of salts.
The significantly reduced activity of the α-picolinyl de-
rivative 7i may be attributed to the possible compe-
tition for intramolecular hydrogen bond formation be-
tween the pyridine N and the amidic NH, which partici-
pates in a crucial hydrogen bonding with the water
molecule bridging Ala28 and Asp29 in the S2Ј site of
the enzyme [15]. In the absence of such interaction,
higher activity of the β- and γ-picoline 7g and 7h was
observed. On the other hand, the lowered activity of
the alkoxy 7c, d, 8c, d and 4,6-dimethyl-1,2-dihydro-3-
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
A Novel Dipeptide-based HIV Protease Inhibitor 591
Scheme 1. Synthesis of dipeptides 7 and 8.
picolinyl-2- one 7j can be equally attributed to possible
intramolecular hydrogen bonding between the un-
shared pair of electrons on the o-oxygen and the
amidic NH.
Positional isomers of the substituted benzyl amines
and of the picolines revealed a significant impact on
activity. The β-picolinyl 7g, and 8g and the m-substi-
tuted benzyl derivatives 7k, l on the one hand exerted
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

592 El-Koussi et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
Table 3. Physical data of intermediate compounds N-Boc-Dmt-P2Ј 2.
No.
P2Ј
mp (°C)
HPLC§
Rt (min)
No.
P2Ј
mp (°C)
HPLC§
Rt (min)
2a
2b
102Ϫ104
185Ϫ188
23.24
19.71
2i
2j
116Ϫ119
128Ϫ130
10.29
13.30
2c
2d
112Ϫ113
113Ϫ114
23.34
22.92
2k
2l
88Ϫ89
13.48
20.14
‡
23.92
24.16
2e#
2f
60Ϫ62
2m
2n
2o
2p
107Ϫ109
115Ϫ116
155Ϫ156
178Ϫ179
13.92
20.96
30.56
30.10
124Ϫ126
21.68
7.68
9.86
‡
2g
2h
162Ϫ164
#
§
‡
Mixture of diastereomers; 20Ϫ80% CH3CN in 0.1% aqueous TFA over 30 min; not determined, sticky
semisolid material.
a relatively higher activity when matched with the γ-
picolines 7h, and 8h and the p-substituted benzyls
7m, n on the other hand. A common feature of these
isomers is the availability of an atom or group on the
aromatic ring that carries an unshared pair of elec-
trons. It seems feasible to correlate the observed dif-
ferences in activity with the unshared pair of electrons
that should be suitably oriented at a critical distance
from the amidic NH. Deviation from this critical dis-
tance might lead to decreased activity. Thus, a location
of the unshared pair of electrons separated by three
carbons from the amidic NH seems to give the opti-
mum activity, as in 7g. Shifting of the unshared pair of
electrons on N towards α- or γ-positions either gets it
involved in intramolecular hydrogen bonding, as in 7i,
or diminishes its supporting role of interaction with the
enzyme, as in 7h. By analogy, the differences in activi-
ties of the m- and p-isomers of benzylcarboxamides
7k and 7m and the methyl benzylcarboxylates 7l and
7n were found to parallel the distance separating the
carbonyl oxygen from the amidic NH.
The tolerance of the S2Ј site to accommodate a bulky
substituent was challenged by the attachment of 1- or
2- adamantyl groups, which can be regarded as the
constrained analogues of the tert-butyl moiety at the
P2Ј position in the lead KNI-577. The yielded dipep-
tides 8o and 8p were found to be the least active de-
rivatives.
Conclusions
KNI-577 and KNI-901 leads are substrate-based HIV
protease inhibitors. The two leads are members of the
KNI series containing allophenylnorstatine (Apns) with
hydroxymethylcarbonyl isostere as a transition state
mimic at the scissile peptide bond.
The activity of the prepared series of P2-Apns-Dmt-P2Ј
strongly depends on the simultaneous balance be-
tween electronic and steric properties of both the P2
and P2Ј moieties. As shown from Table 5, the ability of
interaction of P2 and P2Ј with the relevant enzyme
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
A Novel Dipeptide-based HIV Protease Inhibitor 593
Table 4. Physical data of intermediate compounds N-Boc-Dmt-P2Ј 4.
No.
P2Ј
mp (°C)
HPLC§
Rt (min)
No.
P2Ј
mp (°C)
HPLC§
Rt (min)
4a
4b
102Ϫ106
106Ϫ109
25.56
22.72
4i
4j
94Ϫ98
15.52
18.87
147Ϫ150
4c
99Ϫ102
23.70
25.57
4k
132Ϫ135
18.80
4d
4e#
4f
100Ϫ103
95Ϫ98
4l
117Ϫ120
96Ϫ99
25.44
17.78
22.66
32.75
25.06
25.23
4m
4n
4o
75Ϫ78
25.66
16.19
110Ϫ112
103Ϫ106
4g
96Ϫ100
4h
105Ϫ109
14.92
4p
107Ϫ109
31.94
#
§
and as in Table 3.
1H NMR spectra were recorded on a JEOL JNM-EX 270 (270
MHz) spectrometer. Chemical shifts are given in (δ ppm) rela-
tive to tetramethylsilane (TMS) as an internal standard. 13C
NMR spectra were recorded on a JEOL JNM-EX 270 (67.5
MHz) using solvents as internal standard. FAB mass spectra
(FAB-MS) and high-resolution FAB-MS (HRFAB-MS) were re-
corded on a JEOL JMS-SX102 AQQ/MS-HYB10 mass spec-
trometer using glycerol, thioglycerol or Magic Bullet as in-
ternal references. MALDI TOF mass spectra were measured
at Voyager-DETMRP BiospectrometryTM Workstation (Per-
Septive Biosystems). Commercially available chemicals were
purchased from Nacalai tesque, Waku Chemicals or Tokyo
Chemical Industries, Japan, and were used without further
purification. Commercially non-available chemicals were pre-
pared according to standard methods described in [16] and
showed 1H NMR and 13C NMR spectra in accordance with
the assigned structures.
sites S2 and S2Ј would significantly affect the fitting of
Apns to the enzyme catalytic site, which reflects the
HIV protease inhibitory activity.
Two derivatives were found to be equally or more ac-
tive than the leads KNI-577 and KNI-901 and present
promising candidates for further investigations.
Experimental
Melting points were determined on a micro hot plate of a Yan-
aco micro melting point apparatus and were uncorrected. The
optical rotations were measured on a Horiba model SEPA-
300 digital polarimeter. TLC was performed on precoated
Merck silica gel 60 F254 sheets. Column chromatography was
carried out on Merck silica gel 60 (particle size
0.063Ϫ0.200 mm).
(tert-Butyloxycarbonyl)-5,5-dimethylthiazolidine-4-carboxylic
acid Boc-Dmt-OH (1) [10]
Analytical RP-HPLC was performed with a Hitachi L-7100
pump and an L-7400 UV detector utilizing YMC Pack ODS-
AM AM 302.
Yield 81%, mp 124Ϫ127°C.
Preparative RP-HPLC was performed with a Shimadzu LC-
4A liquid chromatograph utilizing a YMC Pack ODS-AM type
SH-343-5AM column (250 ϫ 20 mm i.d., S-5 µm, 120 A).
N-P2Ј-(tert-Butyloxycarbonyl)-5,5-dimethylthiazolidine-4-
carboxamide Boc-Dmt-P2Ј (2aϪp)
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

594 El-Koussi et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
Table 5. HIV protease inhibitory activity of targeted dipeptides P2-Apns-Dmt-P2Ј 7 and 8.
No.
% HIV protease
inhibition
No.
% HIV protease
inhibition
(50 nM)
(50 nM)
7a
7b
72.5
15.3
8a
8b
82.2
12.9
7c
7d
24.0
41.7
8c
8d
65.8
49.7
7e#
64.5
8e#
91.2
7f
27.6
86.8
70.7
8f
67.6
62.6
34.5
7g
7h
8g
8h
7i
57.8
20.9
67.7
8o
7.1
7j
8p
5.5
87.6
7k
KNI-577
7l
45.0
3.3
7m
7n
28.6
86
KNI-901
#
mixture of diasteromers.
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
A Novel Dipeptide-based HIV Protease Inhibitor 595
Boc-Dmt-OH (1.5 g, 5.74 mmol) was dissolved and stirred in
AcOEt (20 mL); Et3N (0.88 mL, 6.33 mmol) and DPPCl (1.31
mL, 6.33 mmol) were added at 0°C. The mixture was stirred
at room temperature for 1 h, then appropriate amine (6.33
mmol) and Et3N (0.88 mL, 6.33 mmol) were added. Stirring
was continued at room temperature for 6 h. The mixture was
washed twice with 10% citric acid, 5% NaHCO3 and brine,
dried over anhydrous Na2SO4 and filtered. The filtrate was
evaporated under reduced pressure, crystallized from n-hex-
ane and dried in a dessicator [16, 17].
overnight at room temperature, and the solvent was removed
under reduced pressure. The residue was extracted with Ac-
OEt. The organic layer was washed with 10% citric acid, 5%
NaHCO3 and brine, dried over anhydrous Na2SO4, filtered
and evaporated. The residue was crystallized from n-hexane.
Physical data are listed in Table 1.
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-cumyl (7a)
1
[a]D28 Ϫ1.60 (c = 0.218, CH3OH), H NMR (DMSO-d6) δ: 1.43
(s, 3H), 1.49 (s, 1H), 1.54 (s, 3H), 1.65 (s, 3H), 2.16 (s, 6H),
2.70Ϫ2.73 (m, 2H), 3.99Ϫ4.05 (d, J = 14.51 Hz, 1H),
4.14Ϫ4.20 (d, J = 13.85 Hz, 1H), 4.28 (bs, 1H), 4.42 (s, 1H),
4.66 (s, 1H), 4.84Ϫ4.93 (m, 2H), 6.92Ϫ7.02 (m, 3H),
7.15Ϫ7.24 (m, 10H), 7.41Ϫ7.44 (d, J = 7.69 Hz, 1H),
8.24Ϫ8.27 (d, J = 8.58 Hz, 1H), HRFAB-MS: m/z 618.3004
for [M+H]+ (calcd. 618.3002 for C35H44N3O5S).
(2S,3S)3-(tert-Butyloxycarbonyl)amino-2-hydroxy-4-phenyl-
butanoic acid N-Boc-Apns-OH (3) [18]
Yield 8%, mp147Ϫ148°C.
N-P2Ј-3-(2S,3S)-3-(tert-butyloxycarbonyl)amino-2-hydroxy-
4-phenylbutanoyl)-5,5-dimethylthiazolidine-4-carboxamide
Boc-Apns-Dmt-P2Ј (4aϪp)
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-MPH (7b)
To the appropriate Boc-Dmt-P2Ј 2aϪp (0.5 mmol) in 4 N HCl/
dioxane solution (2 mL), anisol (108 µL, 1 mmol) was added
at 0°C. This solution was stirred for 2 h at room temperature.
The solvent was evaporated in vacuo, ether was added, the
mixture was centrifuged, and the residue was dissolved in
DMF (5 mL). Boc-Apns-OH 3 (134 mg, 0.45 mmol), HOBt.
H2O (76.6 mg, 0.5 mmol), BOP (211 mg, 0.5 mmol) and Et3N
(139 mL, 1 mmol) were added at 0°C. The mixture was
stirred overnight at room temperature. The solvent was then
removed under vacuum and the residue was extracted with
AcOEt. The organic layer was washed with 10% citric acid,
5% NaHCO3 and brine, dried over anhydrous Na2SO4 and
filtered. The filtrate was evaporated under reduced pressure.
The residue was purified by column chromatography, and the
appropriate fractions were pooled and evaporated to yield the
coupled peptide (Boc-Apns-Dmt-P2Ј), which was dried in a
desiccator [16, 17].
[a]D24.9 Ϫ11.42 (c = 0.07, CH3OH), 1H NMR (DMSO-d6) δ: 1.49
(s, 3H), 1.58 (s, 3H), 2.16 (s, 6H), 2.76Ϫ2.82 (m, 2H),
3.97Ϫ4.02 (d, J = 13.53 Hz, 1H), 4.15Ϫ4.20 (d, J = 13.52
Hz, 1H), 4.26Ϫ4.48 (m, 3H), 4.97Ϫ5.00 (m, 2H), 6.68Ϫ6.71
(t, J = 7.26 Hz, 1H), 6.81Ϫ7.03 (m, 4H), 7.12Ϫ7.25 (m, 8H),
8.17Ϫ8.20 (d, J = 8.91 Hz, 1H), 10.17 (s, 1H), HRFAB-MS:
m/z 605.2804 for [M+H]+ (calcd. 605.2798 for C33H41N4O5S).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-(2-OCH3)Bz (7c)
[a]D24 Ϫ14.85 (c = 0.202, CH3OH), 1H NMR (DMSO-d6) δ: 1.34
(s, 3H), 1.50 (s, 3H), 2.14 (s, 6H), 2.77Ϫ2.80 (m, 2H), 3.77
(s, 3H), 3.97Ϫ4.02 (d, J = 14.19 Hz, 1H), 4.13Ϫ4.18 (d, J =
14.18 Hz, 1H), 4.25Ϫ4.27 (d, J = 5.28 Hz, 2H), 4.37Ϫ4.40
(m, 1H), 4.46Ϫ4.49 (m, 2H), 4.91Ϫ5.00 (m, 2H), 6.79Ϫ6.85
(t, J = 7.59 Hz, 1H), 6.89Ϫ7.01 (m, 2H), 7.15Ϫ7.30 (m, 9H),
8.12Ϫ8.15 (d, J = 8.57 Hz, 1H), 8.27Ϫ8.35 (t, J = 5.6 Hz,
1H), HRFAB-MS: m/z 620.2854 for [M+H]+ (calcd. 620.2794
for C34H42N3O6S).
2,6-Dimethylphenoxyacetic acid (5)
Compound 5 was synthesized starting from 2,6-dimethyl phe-
nol by alkylation with ethyl-2-bromoacetate, then hydrolysis.
Yield 37%, mp138Ϫ139°C.
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-(2-OC2H5)Bz (7d)
[a]D27.3 Ϫ9.96 (c = 0.158, CH3OH), 1H NMR (DMSO-d6) δ:
1.31Ϫ1.35 (m, 6H), 1.51 (s, 3H), 2.14 (s, 6H), 2.77Ϫ2.80 (m,
2H), 3.93Ϫ4.05 (m, 3H), 4.12Ϫ4.18 (m, 1H), 4.26Ϫ4.28 (d,
J = 4.29 Hz, 2H), 4.39 (s, 1H), 4.47Ϫ4.50 (m, 2H), 4.92Ϫ5.00
(m, 2H), 6.78Ϫ6.83 (t, J = 6.27 Hz, 1H), 6.90Ϫ7.01 (m, 3H),
7.12Ϫ7.30 (m, 7H), 8.13Ϫ8.15 (d, J = 7.91 Hz, 1H), 8.30 (t,
J = 5.6 Hz, 1H), HRFAB-MS: m/z 634.2969 for [M+H]+ (calcd.
634.2951 for C35H44N3O6S).
3-Hydroxy-2-methylbenzoic acid [16]
Yield 86.5%, mp142Ϫ144°C.
3-Acetyloxy-2-methylbenzoic acid (6)
Acetylation of 3-hydroxy-2-methylbenzoic acid yielded 3-ace-
tyloxy-2-methylbenzoic acid 6 (quantitative, mp 147Ϫ148°C).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-Ind (7e)
[a]D25 +19.86 (c = 0.448, CH3OH), 1H NMR (DMSO-d6) δ: 1.44
(s, 3H), 1.51 (s, 3H), 1.79Ϫ1.84 (m, 1H), 2.15 (s, 6H),
2.34Ϫ2.40 (m, 1H), 2.81Ϫ2.91 (m, 4H), 3.95Ϫ4.02 (m, 1H),
4.16Ϫ4.22 (m, 1H), 4.39Ϫ4.50 (m, 3H), 4.98Ϫ4.99 (m, 2H),
5.32Ϫ5.35 (m, 1H), 6.90Ϫ7.08 (m, 4H), 7.17Ϫ7.38 (m, 8H),
8.14Ϫ8.17 (d, J = 8.24 Hz, 1H), 8.35Ϫ8.45 (dd, J = 8.24 Hz,
1H), HRFAB-MS: m/z 616.2878 for [M+H]+ (calcd. 616.2845
for C35H42N3O5S), TOF-MS 616.727 for [M+H]+.
Synthesis of dipeptides containing 2,6-dimethyl phenoxy-
acetic acid as P2 ligand (2,6-dimethylphenoxyacetyl-Apns-
Dmt-P2Ј) (7aϪn)
The titled compounds were prepared, starting from N-Boc-
Apns-Dmt-P2Ј 4a؊n (20.8 mmol) in 4 N HCl/dioxane (40 mL);
anisole (4.5 mL, 41.67 mmol) was added at 0°C. The reaction
mixture was stirred for 1 h at room temperature, and the sol-
vent was then removed in vacuo at room temperature; ether
was added, and the mixture was centrifuged. The formed pre-
cipitate was dissolved in DMF (40 mL), and then 2,6-dimeth-
ylphenoxyacetic acid 5 (22.85 mmol), HOBt.H2O (3.5 g, 22.85
mmol), EDC.HCl (4.3 g, 22.43 mmol) and Et3N (5.78 mL, 41.6
mmol) were added at 0°C. The reaction mixture was stirred
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-Pip (7f)
[a]D27 Ϫ2.00 (c = 0.15, CH3OH), 1H NMR (DMSO-d6) δ: 1.34
(s, 3H), 1.51 (s, 3H), 2.15 (s, 6H), 2.76Ϫ2.80 (m, 2H),
3.99Ϫ4.02 (m, 1H), 4.14Ϫ4.29 (m, 3H), 4.34Ϫ4.48 (m, 3H),
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

596 El-Koussi et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
1
4.97 (s, 2H), 5.88Ϫ5.92 (m, 2H), 6.77 (s, 2H), 6.85 (s, 1H),
6.95Ϫ7.02 (m, 3H), 7.19Ϫ7.30 (m, 4H), 8.09Ϫ8.13 (d, J =
9.17 Hz, 1H), 8.43Ϫ8.48 (t, J = 6.11 Hz, 1H), HRFAB-MS:
m/z 634.2583 for [M+H]+ (calcd. 634.2587 for C34H40N3O7S).
[a]D24 Ϫ13.33 (c = 0.03, CH3OH), H NMR (DMSO-d6) δ: 1.33
(s, 3H), 1.51 (s, 3H), 2.13 (s, 6H), 2.75Ϫ2.79 (m, 2H), 3.81
(s, 3H), 3.96Ϫ4.02 (d, J = 14.19 Hz, 1H), 4.13Ϫ4.17 (d, J =
14.19 Hz, 1H), 4.30Ϫ4.52 (m, 5H), 4.96Ϫ4.98 (m, 2H),
5.50Ϫ5.53 (d, J = 8.7 Hz, 1H), 6.92Ϫ7.01 (m, 3H), 7.19Ϫ7.26
(m, 5H), 7.36Ϫ7.42 (t, J = 7.2 Hz, 1H), 7.57Ϫ7.61 (d, J = 6.6
Hz, 1H), 7.75Ϫ7.78 (d, J = 7.58 Hz, 1H), 7.91 (s, 1H),
8.09Ϫ8.12 (d, J = 8.25 Hz, 1H), 8.58Ϫ8.62 (t, J = 5.8 Hz,
1H), HRFAB-MS: m/z 648.2753 for [M+H]+ (calcd. 648.2743
for C35H42N3O7S).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-3-picolyl (7g)
[a]D23.8 +47.14 (c = 0.28, CH3OH), 1H NMR (DMSO-d6) δ: 1.34
(s, 3H), 1.53 (s, 3H), 2.12 (s, 6H), 2.76Ϫ2.84 (m, 2H),
3.93Ϫ3.98 (d, J = 14.19 Hz, 1H), 4.11Ϫ4.17 (d, J = 14.19 Hz,
1H), 4.19Ϫ4.72 (m, 5H), 5.00 (m, 2H), 6.89Ϫ7.01 (m, 3H),
7.16Ϫ7.24 (m, 5H), 7.63Ϫ7.68 (m, 1H), 8.07Ϫ8.11 (d, J =
8.91 Hz, 1H), 8.14Ϫ8.17 (d, J = 7.59 Hz, 1H), 8.57Ϫ8.58 (d,
J = 3.96 Hz, 1H), 8.67 (s, 1H), 8.72Ϫ8.80 (t, J = 5.6 Hz, 1H),
HRFAB-MS: m/z 591.2639 for [M+H]+ (calcd. 591.2641 for
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-Bz(4-CONH2)
(7m)
1
[a]D25 +22.00 (c = 0.05, CH3OH), H NMR (DMSO-d6) δ: 1.35
C
32H39N4O5S). TOF-MS 591.543 for [M+H]+.
(s, 3H), 1.52 (s, 3H), 2.14 (s, 6H), 2.77Ϫ2.89 (m, 2H),
3.96Ϫ4.03 (m, 1H), 4.15Ϫ4.20 (m, 1H), 4.26Ϫ4.52 (m, 5H),
5.00 (s, 2H), 6.90Ϫ7.03 (m, 3H), 7.15Ϫ7.33 (m, 5H),
7.35Ϫ7.40 (d, J = 7.2 Hz, 2H), 7.76Ϫ7.81 (d, J = 7.2 Hz, 2H),
7.89 (s, 2H), 8.10Ϫ8.15 (d, J = 8.1 Hz, 1H), 8.55Ϫ8.60 (t, J =
5.4 Hz, 1H), HRFAB-MS: m/z 633.2763 for [M+H]+ (calcd.
633.2747 for C34H41N4O6S).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-4-picolyl (7h)
1
[a]D26 +36.36 (c = 0.55, CH3OH), H NMR (DMSO-d6) δ: 1.37
(s, 3H), 1.55 (s, 3H), 2.12 (s, 6H), 2.71Ϫ2.89 (m, 2H),
3.93Ϫ3.98 (d, J = 14.19 Hz, 1H), 4.09Ϫ4.15 (d, J = 14.19,
1H), 4.32Ϫ4.61 (m, 5H), 5.00 (s, 2H), 6.89Ϫ7.01 (m, 3H),
7.12Ϫ7.26 (m, 5H), 7.63Ϫ7.66 (d, J = 5.74 Hz, 2H),
8.09Ϫ8.13 (d, J = 9.24 Hz, 1H), 8.58Ϫ8.61 (d, J = 6.26 Hz,
2H), 8.77Ϫ8.81 (t, J = 5.94 Hz, 1H), HRFAB-MS: m/z
591.2628 for [M+H]+ (calcd. 591.2641 for C32H39N4O5S).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-Bz(4-COOCH3)
(7n)
[a]D26 +16.66 (c = 0.202, CH3OH), 1H NMR (DMSO-d6) δ: 1.35
(s, 3H), 1.52 (s, 3H), 2.14 (s, 6H), 2.76Ϫ2.79 (m, 2H), 3.81
(s, 3H), 3.96Ϫ4.01 (d, J = 14.19 Hz, 1H), 4.13Ϫ4.19 (d, J =
14.51 Hz, 1H), 4.29Ϫ4.48 (m, 5H), 4.98 (s, 2H), 6.90Ϫ7.01
(m, 3H), 7.18Ϫ7.24 (m, 5H), 7.43Ϫ7.46 (d, J = 7.26 Hz, 2H),
7.82Ϫ7.85 (d, J = 8.24 Hz, 2H), 8.11Ϫ8.15 (d, J = 8.75 Hz,
1H), 8.63 (t, J = 5.4 Hz, 1H), HRFAB-MS: m/z 648.2753 for
[M+H]+ (calcd. 648.2743 for C35H42N3O7S).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-2-picolyl (7i)
1
[a]D25 +21.80 (c = 0.61, CH3OH), H NMR (DMSO-d6) δ: 1.37
(s, 3H), 1.54 (s, 3H), 2.13 (s, 6H), 2.76Ϫ2.85 (m, 2H),
3.95Ϫ4.00 (d, J = 14.19 Hz, 1H), 4.12Ϫ4.17 (d, J = 14.19
Hz, 1H), 4.26Ϫ4.56 (m, 5H), 4.94Ϫ5.02 (q, J = 9.24 Hz, 2H),
6.89Ϫ7.01 (m, 3H), 7.12Ϫ7.26 (m, 5H), 7.32Ϫ7.37 (t, J = 6.6
Hz, 1H), 7.53Ϫ7.56 (d, J = 7.59 Hz, 1H), 7.81Ϫ7.87 (t, J =
7.58 Hz, 1H), 8.11Ϫ8.14 (d, J = 8.57 Hz, 1H), 8.53Ϫ8.54 (d,
J = 4.62 Hz, 1H), 8.71Ϫ8.75 (t, J = 5.94 Hz, 1H), HRFAB-MS:
m/z 591.2631 for [M+H]+ (calcd. 591.2641 for C32H39N4O5S).
Dipeptides containing the 3-hydroxy-2-methylbenzoyl moiety
(3-hydroxy-2-methyl benzoyl-Apns-Dmt-P2Ј) (8aϪh, o, p)
To a solution of the appropriate Boc-Apns-Dmt-P2Ј 4a؊h, o,
p (0.1 mmol) in 4 N HCl/dioxane solution (1 mL), anisole (22
µL, 0.2 mmol) was added at 0°C. The reaction mixture was
stirred for 1 h at room temperature. The solvent was removed
in vacuum at room temperature; ether was added. After cen-
trifugation, the residue was suspended in AcOEt (5 mL), then
Et3N (17 µL, 0.12 mmol) was added (solution A). 3-Acetyloxy-
2-methylbenzoic acid 6 (21.3 mg, 0.11 mmol) was dissolved
in AcOEt (5 mL), then Et3N (17 µL, 0.12 mmol) and DPPCl
(25 µL, 0.12 mmol) were added at 0°C. The mixture was
stirred at room temperature for 1 h, then mixed with solution
A. The mixture was further stirred at room temperature for 6
h, then washed with 10% citric acid, 5% NaHCO3 and brine.
The organic layer was dried and evaporated under reduced
pressure. The residue was dissolved in methanol (2 mL), then
1 M LiOH (3 mL) was added. Stirring was continued at room
temperature for 5 h, followed by addition of 10% citric acid
till pH 3. The mixture was extracted with AcOEt and washed
with 10% citric acid and brine. The organic extract was dried,
evaporated and crystallized from n-hexane. The crude prod-
uct was purified using analytical, preparative and analytical
HPLC. The pure product was lyophilized. Physical data are
listed in Table 2.
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-(2-hydroxy-4,6-
dimethyl)-3-picolyl (7j)
1
[a]2D6.3 Ϫ4.28 (c = 0.07, CH3OH), H NMR (DMSO-d6) δ: 1.31
(s, 3H), 1.45 (s, 3H), 2.05Ϫ2.24 (m, 12H), 2.73Ϫ2.80 (m, 2H),
3.98Ϫ4.20 (m, 4H), 4.29Ϫ4.40 (bs, 1H), 4.44Ϫ4.50 (m, 2H),
4.93 (m, 2H), 5.74 (s, 1H), 6.91Ϫ7.06 (m, 3H), 7.13Ϫ7.37
(m, 5H), 7.98Ϫ8.02 (t, J = 5.6 Hz, 1H), 8.13Ϫ8.16 (d, J =
7.71 Hz, 1H), HRFAB-MS: m/z 635.2897 for [M+H]+ (calcd.
635.2903 for C34H43N4O6S).
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-Bz(3-CONH2) (7k)
[a]D26 +42.66 (c = 0.075, CH3OH), 1H NMR (DMSO-d6) δ: 1.34
(s, 3H), 1.51 (s, 3H), 2.14 (s, 6H), 2.71Ϫ2.80 (m, 2H),
3.96Ϫ4.02 (d, J = 14.19 Hz, 1H), 4.14Ϫ4.18 (d, J = 14.19
Hz, 1H), 4.24Ϫ4.49 (m, 5H), 4.94Ϫ5.03 (m, 2H), 6.90Ϫ7.05
(m, 3H), 7.15Ϫ7.34 (m, 6H), 7.44Ϫ7.48 (d, J = 6.6 Hz, 1H),
7.68Ϫ7.70 (d, J = 7.58 Hz, 1H), 7.81 (s, 1H), 7.93 (s, 2H),
8.09Ϫ8.13 (d, J = 9.24 Hz, 1H), 8.55Ϫ8.62 (t, J = 5.8 Hz,
1H), HRFAB-MS: m/z 633.2756 for [M+H]+ (calcd. 633.2747
for C34H41N4O6S).
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-cumyl (8a)
[a]D23.5 +18.69 (c = 0.23, CH3OH), 1H NMR (DMSO-d6) δ: 1.43
(s, 3H), 1.50 (s, 3H), 1.53 (s, 3H), 1.65 (s, 3H), 1.82 (s, 3H),
2.71 (m, 2H), 4.20Ϫ4.38 (m, 2H), 4.50Ϫ5.51 (d, J = 2.97 Hz,
2,6-Dimethylphenoxyacetyl-Apns-Dmt-NH-Bz(3-COOCH3)
(7l)
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
A Novel Dipeptide-based HIV Protease Inhibitor 597
1H), 4.65 (s,1H), 4.93Ϫ4.97 (d, J = 8.9 Hz, 1H), 5.08Ϫ5.12
(d, J = 8.9 Hz, 1H), 6.55Ϫ6.58 (d, J = 7.26 Hz, 1H),
6.76Ϫ6.79 (d, J = 7.91 Hz, 1H), 6.92Ϫ7.04 (m, 2H),
7.11Ϫ7.19 (m, 7H), 7.40Ϫ7.43 (d, J = 8.25 Hz, 2H),
8.19Ϫ8.21 (d, J = 8.25 Hz, 1H), 9.22Ϫ9.32 (bs, 1H), HRFAB-
MS: m/z 590.2704 for [M+H]+ (calcd. 590.2689 for
C33H40N3O5S).
[a]D25.8 +15.62 (c = 0.224, CH3OH), 1H NMR (DMSO-d6) δ:
1.32 (s, 3H), 1.50 (s, 3H), 1.82 (s, 3H), 2.68Ϫ2.86 (m, 2H),
4.15Ϫ4.23 (m, 1H), 4.28Ϫ4.47 (m, 4H), 5.00Ϫ5.03 (d, J =
8.91, 1H), 5.13Ϫ5.17 (d, J = 9.23 Hz, 1H), 5.49Ϫ5.52 (d, J =
6.6 Hz, 1H), 6.53Ϫ6.56 (d, J = 7.26 Hz, 1H), 6.76Ϫ6.79 (d,
J = 7.91 Hz, 1H), 6.90Ϫ6.98 (m, 1H), 7.13Ϫ7.31 (m, 6H),
7.67Ϫ7.70 (d, J = 7.58 Hz, 1H), 8.12Ϫ8.15 (d, J = 7.91 Hz,
1H), 8.39Ϫ8.53 (m, 3H), 9.38 (bs, 1H), HRFAB-MS: m/z
563.2341 for [M+H]+ (calcd. 563.2328 for C30H35N4O5S).
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-MPH (8b)
[a]D26 +28.82 (c = 0.17, CH3OH), H NMR (DMSO-d6) δ: 1.47
1
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-4-picolyl (8h)
(s, 3H), 1.56 (s, 3H), 1.82 (s, 3H), 2.67Ϫ2.87 (m, 2H), 3.06
(s, 3H), 4.41 (s, 2H), 4.49 (s, 1H), 5.02Ϫ5.06 (d, J = 8.91 Hz,
1H), 5.14Ϫ5.18 (d, J = 9.24 Hz, 1H), 6.54Ϫ6.57 (d, J = 7.26
Hz, 1H), 6.66Ϫ6.72 (t, J = 7.26 Hz, 1H), 6.76Ϫ6.78 (d, J =
7.58 Hz, 1H), 6.87Ϫ6.96 (m, 3H), 7.11Ϫ7.27 (m, 7H),
8.15Ϫ8.19 (d, J = 8.25 Hz, 1H), 9.35 (s, 1H), 10.12 (s,1H),
HRFAB-MS: m/z 577.2492 for [M+H]+ (calcd. 577.2485 for
C31H37N4O5S).
[a]D27.5 +25.92 (c = 0.135, CH3OH), 1H NMR (DMSO-d6) δ:
1.37 (s, 3H), 1.55 (s, 3H), 1.80 (s, 3H), 2.70Ϫ2.90 (m, 2H),
4.20Ϫ4.66 (m, 5H), 5.01Ϫ5.05 (d, J = 8.7 Hz, 1H), 5.15Ϫ5.19
(d, J = 8.9 Hz, 1H), 6.53Ϫ6.55 (d, J = 7.26 Hz, 1H),
6.73Ϫ6.76 (d, J = 7.9 Hz,1H), 6.86Ϫ6.92 (t, J = 7.83 Hz 1H),
7.05Ϫ7.32 (m, 4H), 7.66Ϫ7.68 (d, J
= 5.74 Hz, 2H),
8.08Ϫ8.12 (d, J = 7.9 Hz, 1H), 8.26 (d, J = 1.7 Hz 1H),
8.56Ϫ8.60 (d,
J = 6.26 Hz, 2H), 8.70Ϫ8.76 (m, 1H),
9.35Ϫ9.45 (bs, 1H), HRFAB-MS: m/z 563.2337 for [M+H]+
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-(2-OCH3)Bz (8c)
(calcd. 563.2328 for C30H35N4O5S).
[a]D27.5 0.00 (c = 0.07, CH3OH), 1H NMR (DMSO-d6) δ: 1.35
(s, 3H), 1.51 (s, 3H), 1.84 (s, 3H), 2.69Ϫ2.88 (m, 2H), 3.79
(s, 3H), 4.20Ϫ4.38 (m, 2H), 4.40Ϫ4.48 (m, 3H), 4.99Ϫ5.03
(d, J = 9.24, 1H), 5.12Ϫ5.16 (d, J = 9.24, 1H), 6.54Ϫ6.57 (d,
J = 7.26 Hz, 1H), 6.76Ϫ6.94 (m, 4H), 7.14Ϫ7.33 (m, 7H),
8.12Ϫ8.15 (d, J = 8.24 Hz, 1H), 8.25Ϫ8.28 (m, 1H),
9.20Ϫ9.50 (bs, 1H), HRFAB-MS: m/z 592.2472 for [M+H]+
(calcd. 592.2481 for C32H38N3O6S).
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-1-Adam (8o)
[a]D18.5 +7.45 (c = 0.295, CH3OH), 1H NMR (DMSO-d6) δ: 1.41
(s, 3H), 1.48 (s, 3H), 1.61 (s, 6H), 1.82Ϫ1.96 (m, 12H),
2.69Ϫ2.82 (m, 2H), 4.24Ϫ4.42 (m, 1H), 4.53 (s, 2H),
4.94Ϫ4.97 (d, J = 9.23 Hz, 1H), 5.15Ϫ5.18 (d, J = 8.25 Hz,
1H), 6.55Ϫ6.57 (d, J = 6.93 Hz, 1H), 6.76Ϫ6.79 (d, J = 7.58
Hz, 1H), 6.91Ϫ6.94 (m, 1H), 7.07Ϫ7.23 (m, 3H), 7.37Ϫ7.39
(d, J = 6.92 Hz, 2H), 7.49 (s, 1H), 8.22Ϫ8.25 (d, J = 8.25
Hz, 1H), 9.20Ϫ9.60 (bs, 1H), HRFAB-MS: m/z 606.3011 for
[M+H]+ (calcd. 606.3002 for C34H44N3O5S).
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-(2-OC2H5)Bz (8d)
[a]D25.6 +20.32 (c = 0.123, CH3OH), 1H NMR (DMSO-d6) δ:
1.33Ϫ1.38 (m, 6H), 1.51 (s ,3H), 1.83 (s, 3H), 2.76Ϫ2.88 (m,
2H), 4.00Ϫ4.05 (m, 2H), 4.14Ϫ4.49 (m, 5H), 5.00Ϫ5.03 (d,
J = 9.24 Hz, 1H), 5.13Ϫ5.16 (d, J = 9.24 Hz, 1H), 6.53Ϫ6.56
(d, J = 7.26 Hz, 1H), 6.75Ϫ6.84 (m, 2H), 6.91Ϫ7.04 (m, 2H),
7.14Ϫ7.35 (m, 7H), 8.12Ϫ8.15 (d, J = 7.58 Hz, 1H),
8.20Ϫ8.30 (m, 1H), 9.35 (s, 1H), HRFAB-MS: m/z 606.2652
for [M+H]+ (calcd. 606.2638 for C33H40N3O6S).
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-2-Adam (8p)
[a]D20 +11.81 (c = 0.127, CH3OH), 1H NMR (DMSO-d6) δ: 1.38
(s, 3H), 1.48Ϫ1.52 (m, 6H), 1.69Ϫ2.09 (m, 15H), 2.65Ϫ2.81
(m, 2H), 3.89 (s, 1H), 4.31Ϫ4.37 (m, 1H), 4.51 (s, 1H), 4.70
(s, 1H), 4.99Ϫ5.02 (d, J = 8.91 Hz, 1H), 5.13Ϫ5.16 (d, J =
8.58 Hz, 1H), 6.53Ϫ6.56 (d, J = 7.59 Hz, 1H), 6.76Ϫ6.78 (d,
J = 7.25 Hz, 4H), 6.91Ϫ6.93 (t, J = 7.58 Hz, 1H), 7.08Ϫ7.32
(m, 5H), 7.81Ϫ7.84 (d, J = 6.26 Hz, 1H), 8.19Ϫ8.22 (d, J =
7.92 Hz, 1H), HRFAB-MS: m/z 606.3011 for [M+H]+ (calcd.
606.3002 for C34H44N3O5S).
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-Ind (8e)
[a]D24.2 +35.00 (c = 0.22, CH3OH), 1H NMR (DMSO-d6) δ: 1.44
(s, 3H), 1.51 (s, 3H), 1.82 (s, 3H), 2.27Ϫ2.49 (m, 2H),
2.75Ϫ2.91 (m, 4H), 4.46Ϫ4.51 (m, 3H), 5.00Ϫ5.04 (m, 1H),
5.06Ϫ5.18 (m, 1H), 5.24Ϫ5.37 (m, 1H), 6.54Ϫ6.57 (d, J =
7.26 Hz, 1H), 6.76Ϫ6.79 (d, J = 7.91 Hz, 1H), 6.84Ϫ6.95
(m, 1H), 7.10Ϫ7.39 (m, 9H), 8.16Ϫ8.19 (t, J = 7.59 Hz, 1H),
8.33Ϫ8.39 (t, J = 8.91 Hz, 1H), 9.37 (bs, 1H), HRFAB-MS:
m/z 588.2548 for [M+H]+ (calcd. 588.2532 for C33H38N3O5S).
HIV protease inhibition
HIV protease inhibition was determined by an HPLC method
using S10 peptide (H-Lys-Ala-Arg-Val-Tyr*Phe(p-NO2)-Glu-
Ala-Nle-NH2) as the enzyme substrate. The inhibitory poten-
tials were tested at 50 nM concentration of the inhibitor. The
assay protocol was followed as described by Mimoto et al.
[15].
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-Pip (8f)
[a]D24.5 +16.36 (c = 0.11, CH3OH), 1H NMR (DMSO-d6) δ: 1.34
(s, 3H), 1.50 (s, 3H), 1.83 (s, 3H), 2.73Ϫ2.88 (m, 2H),
4.03Ϫ4.10 (m, 1H), 4.28Ϫ4.36 (m, 1H), 4.42Ϫ4.47 (d, J =
12.7 Hz, 3H), 4.99Ϫ5.03 (d, J = 9.24 Hz, 1H), 5.12Ϫ5.16 (d,
J = 8.9 Hz, 1H), 5.90Ϫ5.96 (m, 2H), 6.54Ϫ6.57 (d, J = 7.26
Hz, 1H), 6.73Ϫ6.88 (m, 4H), 6.92Ϫ6.98 (t, J = 7.92 Hz, 1H),
References
[1] A.Carrillo, K. D. Stewart, H. L. Sham, D. W. Norbeck, W.
E. Kohlbrenner, J. M. Leonard, D. J. Kempf, A. Monna,
J. Virol. 1998, 72, 7532Ϫ7541.
7.16Ϫ7.33 (m, 5H), 8.11Ϫ8.14 (d, J
= 7.92 Hz, 1H),
8.37Ϫ8.39 (t, J = 5.61 Hz, 1H), 9.20Ϫ9.60 (bs, 1H), HRFAB-
MS: m/z 606.2269 for [M+H]+ (calcd. 606.2274 for
[2] L. Tong, S. Pav, S. Mui, D. Lamarre, C. Yoakim, P.
Beaulieu, P. C. Anderson, Structure 1995, 3, 33Ϫ40.
C32H36N3O7S).
[3] A. A. Bekhit, H. Matsumoto, H. M. M. Abdel-Rahman,
T. Mimoto, S. Nojima, H. Takaku, T. Kimura, K. Akaji,
3-Hydroxy-2-methylbenzoyl-Apns-Dmt-NH-3-picolyl (8g)
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

598 El-Koussi et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 587−598
Y. Kiso; “Peptide Science Ϫ Present and Future”, Pro-
[10] M.Schutkowski, C. Mrestani-Klaus, K. Neubert, Int. J.
Peptide Protein Res. 1995, 45, 257Ϫ265.
ceedings of the 1st International Peptide Symposium,
Ed. Y. Shimonishi, Kluwer Academic Publishers, Dord-
recht, 1999, pp. 660Ϫ661.
[11] D.Hudson, J. Org. Chem. 1988, 53, 617Ϫ624.
[12] T. Miyazawa, T. Yamada, S. Kuwata, Bull. Chem. Soc.
Jpn. 1988, 61, 606Ϫ608.
[4] R. S. Randad, L. Lubkowska, T. N. Bhat, S. Munshi, S.
V. Gulnik, B. Yu, J. W. Erickson, Bioorg. Med. Chem.
Lett. 1995, 5, 1707Ϫ1712.
[13] A. S. Kende, D. Scholz, J. Schneider, Synth. Commun.
1978, 8, 59Ϫ63.
[5] R. S. Randad, L. Lubkowska, A. M. Silva, D. M. A.
Guerin, S. V. Gulnik, B. Yu, J. W. Erickson, Bioorg. Med.
Chem. 1996, 4, 1471Ϫ1480.
[14] J. Dudash, Jr., J. Jiang, S. C. Mayer, M. M Joullie, Synth.
Commun. 1993, 23, 349Ϫ356.
[15] T. Mimoto, R. Kato, H. Takaku, S. Nojima, K. Terashima,
S. Misawa, T. Fukazawa, T. Ueno, H.Sato, M. Shintani,
Y. Kiso, H. Hayashi, J. Med. Chem. 1999, 42,
1789Ϫ1802.
[6] D. J. Kempf, C. A. Flentge, N. E. Wideburg, A. Saldivar,
S. Vasavanonda, D. W. Norbeck, Bioorg. Med. Chem.
Lett. 1995, 5, 2725Ϫ2728.
[16] H. M. M. Abdel-Rahman, Ph.D. Thesis, Assiut Univer-
[7] Y. Kiso, S.Yamaguchi, H. Matsumoto, T. Mimoto, R.
Kato, S. Nojime, H. Takaku, T. Fukkazawa, T. Kimura, K.
Akaji, Arch. Pharm. Pharm. Med. Chem. 1998, 331, 87.
sity, Egypt, 1999.
[17] Y. Kiso, Biopolymers (Peptide Science) 1996, 40,
235Ϫ244.
[8] M. S. Roger, H. B. Christina, J. Chromatogr. 1989,
475, 57.
[18] M. M. Sheha, N. M. Mahfouz, H. Y. Hassan, A. F. Yous-
sef, T. Mimoto, Y. Kiso, Eur. J. Med. Chem. 2000, 35,
887Ϫ894.
[9] P. Ruelle, W. K. Ulrich, J. Pharm. Sci. 1998, 87, 987.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim