Design, synthesis and structure-activity study of shorter hexa peptide analogues as HIV-1 protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.10.052

Inhibition of HIV-1 protease enzyme can render the Human Immunodeficiency Virus (HIV-1) non-infectious in vitro. Previous studies have shown that several shorter peptides were discovered as HIV-1 protease inhibitors. In this context, a series of shorter synthetic hexapeptides, Leu-Leu-Glu-Tyr-Val-Xaa (Xaa = Phe, Met, Tyr and Trp), were designed. The synthesized hexa peptides were screened for their HIV-1 protease inhibition. These peptides showed moderately good HIV-1 protease inhibition when compared to acetyl pepstatin.

Full text of DOI:10.1016/j.bmc.2007.10.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 874–880
Design, synthesis and structure–activity study of shorter
hexa peptide analogues as HIV-1 protease inhibitors
S. N. Narendra Babu and K. S. Rangappa*
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India
Received 20 April 2007; revised 5 October 2007; accepted 10 October 2007
Available online 22 October 2007
Abstract—Inhibition of HIV-1 protease enzyme can render the Human Immunodeficiency Virus (HIV-1) non-infectious in vitro.
Previous studies have shown that several shorter peptides were discovered as HIV-1 protease inhibitors. In this context, a series
of shorter synthetic hexapeptides, Leu-Leu-Glu-Tyr-Val-Xaa (Xaa = Phe, Met, Tyr and Trp), were designed. The synthesized hexa
peptides were screened for their HIV-1 protease inhibition. These peptides showed moderately good HIV-1 protease inhibition when
compared to acetyl pepstatin.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(Matrix P17, Capsid P24, and Nucleocapsid P9) and re-
lease of viral enzymes (Reverse Transcriptase, RnaseH,
Integrase, and Protease). It has been shown that bud-
ding immature viral particle that contains catalytically
inactive protease cannot undergo maturation into an
infective form.5 The necessity of this enzyme in the virus
life cycle makes it a promising target for the design of
inhibitors against HIV-1 protease enzyme.
The pandemic spread of Human Immunodeficiency
Virus (HIV), the etiologic agent of Acquired Immunode-
ficiency Syndrome (AIDS),1 has promoted an unprece-
dented scientific and clinical effort to understand and
combat this lethal disease. According to the United Na-
tions AIDS organization,2 by the end of 2007, there
would be 75 million people living with HIV/AIDS glob-
ally and an estimated 30 million may die since the begin-
ning of the epidemic. The identification of the HIV
retrovirus and the accumulated knowledge about the
role of different elements in the viral life cycle made it
possible to identify numerous intervention points in
the HIV-1 viral life cycle that could be exploited in the
development of drugs for AIDS therapy.3
Several methodologies have been applied for drug dis-
covery of HIV-1 protease inhibitors. In these methodol-
ogies, the structure of enzyme substrates as a starting
point for drug discovery (substrate-based approach),
biological screening approach developed to test hun-
dreds/thousands of compounds, and X-ray crystallogra-
phy data of HIV-1 protease-inhibitor complexes
(biostructural approach or ‘‘rational drug design’’) were
the prominent ones. Mainly the peptidomimetics drug
discovery was achieved with the substrate-based ap-
proach. Systematic modification of the substrate-based
peptides using rational design techniques led to pepti-
domimetic compounds lacking much of the peptide
backbone while retaining the essential functionalities
for HIV-1 protease active site inhibition.6 Among them
the marketed drugs Invirase (Saquinavir mesylate, Hoff-
mann-LaRoche), Norvir (Ritonavir, Abott Laborato-
ries), Crixivan (Indinavir sulfate, Merck) and Viracept
(Nelfinavir mesylate, Agouron Pharmaceuticals), as well
as Amprenavir (VX-478, Glaxo Wellcome), are cur-
rently in clinical trials. These compounds present sub-
nanomolar activities versus the enzyme7 and demon-
strate high selectivity for HIV-1 protease over other
Very detailed crystallographic analysis combined with
extensive biochemical characterization and site-specific
mutagenesis studies made on the HIV-1 protease made
it perhaps the best characterized enzyme to date.4 The
virally encoded homodimeric aspartyl protease (HIV-1
protease) is responsible for the processing of the gag
(Pr 55gag) and gag/pol (Pr 160gag-pol) gene products that
allow for the organization of the core structural proteins
Keywords: Human Immunodeficiency Virus; HIV-1 protease; Hexa
peptides; Oyester peptides; Structural analogues; Design and synthesis.
*
Corresponding author. Tel.: +91 821 2419661; fax: +91 821
2412191; e-mail addresses: rangappaks@yahoo.com; rangap-
paks@chemistry.uni-mysore.ac.in
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.052

S. N. Narendra Babu, K. S. Rangappa / Bioorg. Med. Chem. 16 (2008) 874–880
875
aspartyl proteases. Non-peptide HIV-1 protease inhibi-
tors, irreversible inhibitors, dimerization inhibitors,
and metal based inhibitors were the other class of
HIV-1 protease inhibitors.
The studies suggest that aspartic proteases may be inhib-
ited by peptide aldehydes acting in their tetrahedral hy-
drated forms as transition state analogues. Valine and
Phenylalanine, the two C-terminus amino acid residues
of a-MAPI, were among the best suited to occupy S2
and S1 sub sites of the HIV-1 protease enzyme, respec-
tively. The studies based on substrate analogues show
that hydrophobic/aromatic moieties were preferable in
the S1 sub site of the enzyme, with Phe, Tyr, and Met
as best residues.
Another major class of HIV-1 protease inhibitors is the
peptide inhibitors. The rapid identification of HIV-1
protease inhibitors through the synthesis and screening
of defined tripeptide and acetylated tetrapeptide amides
was reported by Owens et al.8 The inhibition of HIV-1
protease by short peptides derived from the terminal
segment of the protease was reported.9 At the same time
the inhibition of HIV-1 protease by its own interface
peptides was also reported.10 An evaluation of the inhi-
bition of the HIV-1 protease by its C- and N-terminal
peptides at 100 lM concentration was also reported.11
Noever12 had reported the HIV-1 protease inhibitory
activity of the naturally occurring common protease
inhibitors. The fluorinated peptides incorporating a
4-fluoroproline residue as potential inhibitor of HIV-1
protease was reported by Tran et al.13 Louis et al.14 re-
ported the hydrophilic peptides derived from the trans-
frame region of Gag-Pol as potent HIV-1 protease
inhibitors. Recently it has been shown that the HIV-1
auxiliary protein Vif, and especially the N-terminal half
of Vif, inhibits the HIV-1 protease.15 Based on this fact
various Vif derived peptides were synthesized chemically
and screened for their HIV-1 protease inhibitory activ-
ity. The in vitro HIV-1 protease inhibition by the mod-
ified peptides, N-glyoxylyl peptides,16 and boronated
peptides17 has opened up a new vista in understanding
the HIV-1 protease enzyme inhibition.
In the above context we designed a hexapeptide series.
The hexapeptide series contains Leu-Leu-Glu-Tyr-Val
as the main pentapeptide sequence to which hydrophobic
amino acids such as Phe, Met, Tyr, and Trp were attached
at the C-terminal end to obtain a set of hexapeptides.
These peptides were structurally characterized and ana-
lyzed for HIV-1 protease inhibitory activity.
2. Results and discussion
2.1. Design of peptides
The various literature reports on the synthesis and
HIV-1 protease inhibitory activity of smaller peptides
made us to design and evaluate some small peptides as
HIV-1 protease inhibitors. The hexapeptides Leu-Leu-
Glu-Tyr-Ser-Ile and Leu-Leu-Glu-Tyr-Ser-leu, isolated
from the hydrolysate of Oyster (Crassostrea Gigas),
were taken as the platform for the design of the hexa-
peptide series. Valine and Phenylalanine, the two C-ter-
minus amino acid residues of a-MAPI, were among the
best suited to occupy S2 and S1 sub sites of the HIV-1
PR enzyme, respectively.19 The studies based on sub-
strate analogues show that hydrophobic/aromatic moie-
ties are preferable in the S1 sub site of the enzyme, with
Phe, Tyr, and Met as best residues. In this study, we
wanted to study the structure–activity of the above
hexapeptides. In this context we designed the hexapep-
tide Leu-Leu-Glu-Tyr-Val-Phe by replacing the Ser-Ile
C-terminal end with the more hydrophobic amino acids
Val-Phe. As the literature reports say that the hydropho-
bic amino acids play a prominent role in the HIV-1
protease inhibitors, we designed a hexapeptide series
Leu-Leu-Glu-Tyr-Val-(Xaa)-OH by replacing the Phe
with Met, Tyr, and Trp.
Several inhibitors of HIV-1 protease are currently ap-
proved and some are under clinical trials. However,
the therapeutic effect of protease inhibitors is limited
by the rapid development of inhibitor resistant variants
of the protease.
The peptides inhibiting HIV-1 protease were isolated
from the hydrolysate of Oyster (Crassostrea Gigas)
proteins prepared with thermolysin.18 The amino acid
sequences of peptides were determined as Leu-Leu-
Glu-Tyr-Ser-Ile and Leu-Leu-Glu-Tyr-Ser-leu. These
sequences exist in some proteins of various major viruses
or human cytomegalovirus. Chemically synthesized
Leu-Leu-Glu-Tyr-Ser-Ile and Leu-Leu-Glu-Tyr-Ser-
Leu showed IC50 values of 20 and 15 nM, respectively,
as competitive inhibitors of HIV-1 protease with Ki val-
ues of 13 and 10 nM, respectively. These peptides were
approximately 100-fold more potent as an HIV-1 prote-
ase inhibitor than acetyl pepstatin (IVal-Val-Val-Sta-
Ala-Sta), a characteristic inhibitor of aspartic protease.
The structure–activity studies on these peptides suggest
that the C- and N-terminal hydrophobic amino acids
or the length of the peptides is important for their inhib-
itory activity. Sarubbi et al.19 have reported the isolation
and structural characterization of an HIV-1 protease
inhibitor from the fermentation broth of a Streptomyces
strain, namely a-microbial alkaline protease inhibitor
(a-MAPI). a-MAPI is a tetrapeptide derivative with a
C-terminal Phenylalanine in the aldehyde form. This
function is essential for the inhibition of HIV-1 protease.
2.2. HIV-1 protease inhibition studies
The HIV-1 protease inhibitory activity of the synthesized
peptides was carried out at a concentration of 100 lg/ml
of each of the peptides (Table 3). As the initial screening,
we carried the assay at only one concentration of the pep-
tides. The hexapeptide series displayed a very good inhi-
bition of up to 64.92%. The overall order of HIV-1 PR
inhibition was found to be Leu-Leu-Glu-Tyr-Val-Trp-
OH > Leu-Leu-Glu-Tyr-Val-Tyr-OH > Leu-Leu-Glu-
Tyr-Val-Phe-OH > Leu-Leu-Glu-Tyr-Val-Met-OH.
The data shown above clearly show that some of the
above peptides may serve as promising lead compounds
for a novel type of HIV-1 protease inhibitors. Careful

876
S. N. Narendra Babu, K. S. Rangappa / Bioorg. Med. Chem. 16 (2008) 874–880
examination of the sequence of these peptides shows
that there is no similarity to any of the natural HIV-1
protease cleavage sites. Furthermore, the fact that the
above peptides were not cleaved by HIV-1 protease
strongly indicates that they do not serve as HIV-1 pro-
tease substrates, but they are the inhibitors. The mecha-
nism in which these peptides inhibit HIV-1 protease is
not clear. It is possible that, being non-substrate mole-
cules, they do not function in the active site of the en-
zyme, but inhibit its action by some allostearic
mechanism.
methyl morpholine (NMM), and EDCI were purchased
from Sigma Chemicals (St. Louis, USA). Recombi-
nantly produced HIV-1 protease enzyme and Acetyl
pepstatin were purchased from Sigma Aldrich (India)
Ltd. HIV-1 Substrate III was purchased from Severn
Biotech Ltd (UK). All solvents and reagents were of
analytical grade or were purified according to the stan-
dard procedure recommended for peptide synthesis. Sil-
ica gel (60–120 mesh) for column chromatography was
purchased from Merck India Ltd (India).
The thin layer chromatography (TLC) was carried out
on silica gel plates obtained from Whatman Inc., with
the following solvent systems:
2.3. Structure–activity relationship studies
The peptides related to the Oyster derived hexapeptides
were synthesized to study the structure–activity relation-
ship. The results showed that the hexapeptide series
exhibited very good HIV-1 protease inhibitory activity
compared to acetyl pepstatin. The results revealed that
the substitution of the Phe of the C-terminal, with more
hydrophobic amino acid residues like Trp and Tyr, re-
sulted in increase in the HIV-1 protease inhibiting
activity.
1:
2:
3:
CHCl3:CH3OH:CH3COOH
CHCl3:CH3OH:CH3COOH
CHCl3:CH3OH:CH3COOH
(95:05:3)
(90:10:3)
(85:15:3)
The compounds on TLC plates were detected by UV
light, by ninhydrin or chlorine/toluidine spray. The pur-
ity of the peptides was determined by HPLC analysis by
using Thermo Electron with RP C18 column (5CN Cos-
mogel 215–4.6 mm) and UV detector—UV-VISL7400.
Peptide sample (25 ll) in water was injected for analysis.
The analysis was carried out using appropriate 0–100%
water/acetonitrile linear gradients in the presence of
0.1% TFA (flow rate 1.0 ml/min). The melting points
of peptides were determined with Thomas Hoover
melting point apparatus and were uncorrected. The
optical rotation was measured using Perkin-Elmer 243
digital polarimeter. Elemental analysis was carried
out on VARIO EL III CHNOS Elementar. The com-
pounds were dried over P2O5 under reduced pressure
for 24 h prior to the preparation of samples for all the
analyses. 1H NMR was recorded on AMX-400 MHz
spectrometer.
3. Conclusions
Since the outbreak of the AIDS epidemic, tremendous
efforts have been directed toward the development of
antiretroviral therapies that target HIV-1 in particular
(HIV-1). Although there are six HIV-1 protease inhibi-
tors and commercially available reverse transcriptase
inhibitors, their effectiveness is hampered by the emer-
gence of drug resistance and cross-resistance mutants,
rendering AIDS with no definite cure. In general, the
HIV-1 protease inhibitors used today are all substrate-
based. Here we presented a novel type of HIV-1 prote-
ase inhibitors, non-substrate based, which can serve as
lead compounds for the development of a novel type
of HIV-1 protease inhibitors. This new approach may
be an answer to the acute problem of drug resistance
caused by active site mutations, a problem which pre-
vents the long-term usage of HIV-1 protease inhibitors
as anti HIV-1 drugs. Inhibitors which are not sub-
strate-based and will not function in the active site
should not be affected by the active site mutations.
These inhibitors may be active against protease-mutant
HIV-1 strains. The potential peptides described above
may become promising drug candidates and warrant
further development of peptides and peptidomimetics
as drug candidates against AIDS.
4.2. Peptide synthesis
Synthesis of peptides was achieved by classical solution
phase method20 using an efficient and well established
Boc-chemistry instead of more expensive Fmoc-chemis-
try. The hexapeptide series Leu-Leu-Glu-Tyr-Val-
(Xaa)-OH was synthesized by the stepwise solution phase
approach as shown in Scheme 1. The Boc group was cho-
sen for temporary Na-protection and its removal was
achieved with 4 N HCl in dioxan or trifluoroaceticacid
(TFA). The C-terminal carboxyl group was protected
by methyl ester, and its removal was effected by saponifi-
cation with 1 N NaOH. The side chain active hydrogen of
the Serine and Glutamic acid was protected by benzyl
group. The active hydrogen of OH group on the aromatic
side chain of Tyrosine was protected by 2, 6, dichloro ben-
zyl group. The side chain protecting group was deprotec-
ted by the catalytic transfer hydrogenolysis by using 10%
Pd/C and HCOONH4. All the coupling reactions were
achieved with the standard coupling agents isobutylchlo-
roformate (IBCF) and EDCI. The protected peptides
were purified by column chromatography over silica gel
and characterized by physical and analytical techniques.
4. Materials and methods
4.1. General
All the amino acids used were of L-configuration unless
otherwise specified. All tert-butyloxycarbonyl (Boc)
amino acids, amino acid derivatives, 1-hydroxybenzot-
rizole (HOBt), and trifluoroacetic acid (TFA) were pur-
chased from Advanced Chem. Tech. (Louisville,
Kentucky, USA). Isobutylchloroformate (IBCF), N-

S. N. Narendra Babu, K. S. Rangappa / Bioorg. Med. Chem. 16 (2008) 874–880
Glu
Tyr
Val
877
Leu
Leu
2,6-diClBzl
Boc
Boc
H OH
2,6-diClBzl
OMe
OMe
OMe
OMe
OMe
(i)
OBzl
2,6-diClBzl
2,6-diClBzl
(ii)
(i) IBCF/ NMM
(ii) HCl/ Dioxane
(iii) EDCI/ HoBt/ NMM
(iv) 1N NaOH
Boc
Boc
OH
H
OBzl
(iii)
OBzl
2,6-diClBzl
(ii)
Boc
Boc
OH H
(v) 10% Pd/C / HCOONH4
(vi) TFA
2,6-diClBzl
2,6-diClBzl
OBzl
OBzl
OBzl
OBzl
(iii)
(ii)
OMe
OMe
H
Boc
OH
2,6-diClBzl
(iii)
Xaa
OMe
OH
Boc
Boc
2,6-diClBzl
2,6-diClBzl
(iv)
OMe
OMe
OH
H
OBzl
OBzl
(iii)
Boc
Boc
2,6-diClBzl
(iv)
(v)
H
H
Boc
H
OH
(vi)
OH
Scheme 1. Scheme of synthesis for the hexapeptide analogues.
4.2.1. Boc-Tyr(2,6-diClBzl)-Val-OMe. To Boc-Tyr(2,6-
diClBzl)-OH (5.23 g, 12 mmol) dissolved in acetonitrile
(53 mL) and cooled to 0 ꢁC was added NMM
(1.21 mL, 12 mmol). The solution was cooled to
ꢀ15 1 ꢁC and IBCF (1.63 mL, 12 mmol) was added
under stirring while maintaining the temperature at
ꢀ15 ꢁC. The reaction mixture was stirred for an addi-
tional 10 min and a pre-cooled solution of HClÆH-Val-
OMe (2.01 g, 12 mmol) and NMM (1.21 mL, 12 mmol)
in DMF (21 mL) was added slowly. After 20 min, the
pH of the solution was adjusted to 8 by the addition
of NMM and the reaction mixture stirred overnight at
room temperature. Acetonitrile was removed under re-
duced pressure and the residual DMF solution was
poured into about 100 mL ice-cold 90% saturated
KHCO3 solution and stirred for 30 min. The peptide
precipitated was extracted into CHCl3. The organic
layer was washed with water, 10% citric acid, water,
5% NaHCO3 solution, water and dried over anhydrous
Na2SO4. The solvent was removed under reduced pres-
sure, the obtained peptide was triturated with dry ether,
petroleum ether and dried under vacuum. The yields and
physical constants are given in Table 1.
lBzl)-Val-OMe in DMF (37 mL) was neutralized with
NMM (0.64 mL, 6.42 mmol) and coupled to Boc-
Glu(OBzl)-OH (2.16 g, 6.42 mmol) in acetonitrile
(22 mL) and NMM (0.64 mL, 6.42 mmol) using IBCF
(0.87 mL, 6.42 mmol) and worked up the same as Boc-
Tyr(2,6-diClBzl)-Val-OMe to obtain Boc-Glu(OBzl)-
Tyr(2,6-diClBzl)-Val-OMe. The sample was recrystal-
lized from ether/petroleum ether. The yields and physi-
cal constants are given in Table 1.
4.2.3. Boc-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe.
Boc-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe
(2.94 g,
3.8 mmol) was deblocked with 4 N HCl/dioxane
(29.4 mL) for 1.5 h. Excess HCl and dioxane were
removed under reduced pressure, triturated with ether,
filtered, washed with ether, and dried (yield, 100%).
The HClÆH-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe in
DMF (30 mL) was neutralized with NMM (0.38 mL,
3.8 mmol) and coupled to Boc-Leu-OH. H2O (0.94 g,
3.8 mmol) in acetonitrile (10 mL) and NMM (0.38 mL,
3.8 mmol) using EDCI (0.72 g, 3.8 mmol), HOBt
(0.51 g, 3.8 mmol) and worked up the same as Boc-
Tyr(2,6-diClBzl)-Val-OMe
to
obtain
Boc-Leu-
Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe. The sample was
recrystallized from ether/petroleum ether. The yields
and physical constants are given in Table 1.
4.2.2. Boc-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe. Boc-
Tyr(2,6-diClBzl)-Val-OMe (3.64 g, 6.76 mmol) was de-
blocked with 4 N HCl/dioxane (36.4 mL) for 1.5 h. Ex-
cess HCl and dioxane were removed under reduced
pressure, triturated with ether, filtered, washed with
ether and dried (yield, 100%). The HClÆH-Tyr(2,6-diC-
4.2.4. Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe.
Boc-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe (2.54 g,
2.86 mmol) was deblocked with 4 N HCl/dioxane

878
S. N. Narendra Babu, K. S. Rangappa / Bioorg. Med. Chem. 16 (2008) 874–880
Table 1. Physical and analytical data of protected hexapeptides
Peptide Yield Mp
R1f R2f
(%)
(ꢁC)
25
½aꢁD (C 1) Molecular
Rf3
Elemental analysis found (calculated)
% C % H % N
MeOH
formula
Boc-Tyr(2,6-diClBzl)-Val-OMe 87
48–49 0.61 0.73 0.72 ꢀ82
C
27H34N2O6Cl2
37H47N3O9Cl2
57.51 (57.54) 6.00 (6.03) 4.94 (4.97)
59.20 (59.27) 6.24 (6.27) 5.56 (5.60)
Boc-Glu(OBzl)-Tyr
(2,6-diClBzl)-Val-OMe
90
86
79
97
80
92
89
60
0.93 0.94 0.96 ꢀ56
0.83 0.92 0.94 ꢀ64
C
Boc-Leu-Glu(OBzl)-Tyr
(2,6-diClBzl)-Val-OMe
49
C43H58N4O9Cl2
51H69N5O11Cl2
C60H78N6O12Cl2
58.94 (58.97) 6.60 (6.62) 6.38 (6.40)
61.30 (61.32) 6.89 (6.91) 7.00 (7.01)
62.85 (62.88) 6.81 (6891) 7.32 (7.33)
Boc-Leu-Leu-Glu(OBzl)-
Tyr(2,6-diClBzl)-Val-OMe
Boc-Leu- Leu-Glu(OBzl)-
Tyr(2,6-diClBzl)-Val-Phe-OMe
Boc-Leu-Leu-Glu(OBzl)-
Tyr(2,6-diClBzl)-Val-Met-OMe
Boc-Leu-Leu-Glu(OBzl)-
Tyr(2,6-diClBzl)-Val-Tyr-OMe
Boc-Leu-Leu-Glu(OBzl)-
Tyr(2,6-diClBzl)-Val-Trp-OMe
49–50 0.82 0.91 0.90 ꢀ63
58–59 0.90 0.95 0.98 ꢀ73
48–49 0.84 0.94 0.95 ꢀ44
C
C56H78N6O12SCl2 59.50 (59.52) 6.89 (6.90) 7.40 (7.44)
56
0.92 0.94 0.95 ꢀ67
C60H75N6O13Cl2
62H78N7O12Cl2
62.10 (62.17) 6.42 (6.47) 7.20 (7.25)
62.84 (62.89) 6.54 (6.59) 8.20 (8.24)
57–58 0.86 0.92 0.95 ꢀ63
C
R1f : Rf value in solvent 1.
R2f : Rf value in solvent 2.
R3f : Rf value in solvent 3.
(26 mL) for 1.5 h. Excess HCl and dioxane were re-
moved under reduced pressure, triturated with ether,
filtered, washed with ether, and dried (yield, 100%).
The HClÆH-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe
in DMF (30 mL) was neutralized with NMM
(0.28 mL, 2.86 mmol) and coupled to Boc-Leu-OH.
H2O (0.71 g, 2.86 mmol) in acetonitrile (10 mL) and
NMM (0.28 mL, 2.86 mmol) using EDCI (0.54 g,
2.86 mmol), HOBt (0.51 g, 2.86 mmol) and worked
up the same as Boc-Tyr(2,6-diClBzl)-Val-OMe to
obtain Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-
OMe. The sample was recrystallized from ether/petro-
leum ether. The yields and physical constants are gi-
ven in Table 1.
4.2.6. Leu-Leu-Glu-Tyr-Val-(Xaa)-OH (Xaa = Phe,
Met, Tyr, or Trp). The protected peptides were hydro-
genolyzed separately using ammonium formate (2 equiv-
alents) and 10% Pd/C (1 equivalent) in methanol
(10 mL/1 g) for 2 h at room temperature. The catalyst
was filtered and washed with methanol. The combined
washings and filtrate were evaporated in vacuo and
the residue taken into CHCl3, washed with water, and
dried over Na2SO4. The solvent was removed under re-
duced pressure and triturated with ether, filtered,
washed with ether, and dried. The resulting peptides
were saponified with 1 N NaOH as described in above
steps to remove the methyl protecting group. The result-
ing peptides were treated individually with TFA (10 mL/
1 g for 40 min) for final Boc deprotection. The solvent
was removed under reduced pressure, triturated with
ether to obtain TFA salt of Leu-Leu-Glu-Tyr-Val-
(Xaa)-OH. The free peptides were purified by gel filtra-
tion using Sephadex G-10 and checked the purity by
HPLC, using a linear gradient of 0–100% acetonitrile/
0.1% trifluoroacetic acid.
4.2.5. Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-(Xaa)-
OMe (Xaa = Phe, Met, Tyr, or Trp). Boc-Leu-Leu-
Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OMe (2.55 g, 2.5 mmol)
was taken in 26 mL of methanol and saponified with
1 N NaOH (5.0 mL, 5.00 mmol) to remove the methyl
ester group. The reaction was stirred up to the com-
pletion of the reaction as monitored by TLC, and
the MeOH was evaporated, dissolved the product in
25 mL water, washed with CHCl3, then neutralized
with cold 1 N HCl (5.0 mL). The precipitate was ta-
ken into CHCl3 layer, washed with saturated NaCl,
and dried over sodium sulfate anhydrous. The Boc-
Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-OH was neu-
tralized with NMM (0.05 mL, 0.49 mmol) and coupled
with individual amino acids HCl. 2HNÆXaa-OMe
(0.49 mmol) in DMF (10 mL/1 g of peptide) and
NMM (0.05 mL, 0.49 mmol) using EDCI (0.09 g,
0.49 mmol), HOBt (0.06 g, 0.49 mmol) and worked
up the same as Boc-Tyr(2,6-diClBzl)-Val-OMe to
obtain Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-
(Xaa)-OMe. The samples were purified over silica gel
using chloroform–methanol as elutant. The yields
and physical constants are given in Table 1. The 1H
NMR spectral data of protected peptides are provided
in Table 2.
4.3. HIV-1 protease inhibition studies
This assay followed the method previously described by
Min et al.21 Recombinant HIV-1 protease enzyme was
diluted with buffer composed of 50 mM sodium acetate
at pH 5.0, 1 mM ethylenediamine disodium (EDTAÆ2-
Na), and 2 mM 2-mercaptoethanol and glycerol in the
ratio of 75:25. The HIV-1 protease Substrate III, His-
Lys-Ala-Arg-Val-Leu-(pNO2-Phe)-Glu-Ala-Nle-Ser-NH2,
was diluted with a buffer solution of 50 mM sodium ace-
tate (pH 5.0). To a reaction mixture containing 2 ll
50 mM buffer solution (pH 5.0) and 2 ll of substrate
solution (1 mg/ml), 2 ll of the samples prepared in water
at 100 lg/ml concentration and 4 ll of HIV-1 protease
enzyme solution in buffer (0.025 mg/ml) were added.
The reaction mixture (10 ll) was incubated at 37 ꢁC
for 1 h. A control reaction was performed under the
same condition, without the addition of the sample solu-

S. N. Narendra Babu, K. S. Rangappa / Bioorg. Med. Chem. 16 (2008) 874–880
Table 2. 1H NMR data of protected hexapeptides in CDCl3
Peptide
879
Components
Chemical shift (d, ppm)
Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-Phe-OMea Boc1
Leu2
1.40 (s, 9H, (CH3)3)
4.50 (d, 1H, aCH), 2.25 (m, 1H, bCH), 1.0 (m, 3H, b1CH), 0.90
c
(m, 3H, CH), 7.65 (s, 1H, NH)
4.10 (d, 1H, aCH), 1.9 (m, 1H, bCH), 1.0 (m, 3H, b1CH),
Leu3
c
1.22(m, 3H, CH), 7.6 (s, 1H, NH)
3.0 (d, 1H, aCH), 1.6 (m, 2H, bCH), 1.58 (t, 2H, CH),
Glu(OBzl)4
c
5.1–5.3(m, 2H, CH), 7.2–7.3(m, 5H, Ar–H), 8.0 (s, 1H, NH)
Tyr(2,6-diClBzl)5 3.7 (d, 1H, aCH), 2.35 (m, 2H, bCH), 5.2 (t, 2H, CH),
7.0–7.2(m, 3H, Ar–H), 8.0 (s, 1H, NH)
4.60 (d, 1H, aCH), 3.0 (m, 2H, bCH), 3.0 (m, 3H, b1CH), 1.4
Val6
Phe
c
(m, 3H, CH), 8.0 (s, 1H, NH)
4.9 (d, 1H, aCH), 3.2 (m, 2H, bCH), 7.4(m, 5H, Ar–H), 8.0 (s,
1H, NH)
3.65 (s, 3H, CH3)
OMe7
Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-Met-OMeb Met
4.5 (m, 1H, aCH), 2.65(m, 2H, bCH), 2.2 (m, 2H, cCH), 1.6 (s,
6H, CH3), 8.45 (s, 1H, NH)
4.6 (d, 1H, aCH), 2.85 (m, 2H, bCH), 6.8–7.0(m, 5H, Ar–H),
8.35(s, 1H, NH)
5.2 (d, 1H, aCH), 3.0 (m, 2H, bCH), 8.25 (s, 1H, NH), 7.2–
7.35(m, 5H, Ar–H), 6.85(m, 1H, indole–H), 8.85 (s, 1H, ring
NH),
Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-Tyr-OMec Tyr
Boc-Leu-Leu-Glu(OBzl)-Tyr(2,6-diClBzl)-Val-Trp-OMed Trp
The chemical shift values for the residues Boc1, Leu2, Leu3, Glu(OBzl)4, Tyr(2,6-diClBzl)5, Val6, and OMe7of hexapeptides b, c, and d were almost
same as obtained for the hexapeptide a.
References and notes
Table 3. HIV-1 protease inhibition study of the synthesized peptides at
100 lg/ml
1. Gallo, R. C.; Montagnier, L. Sci. Am. 1988, 259, 40.
2. URL. www.unaids.org, Global estimates and projections
2005.
3. Mitsuya, H.; Yarchohan, R.; Brodar, S. Science 1990, 249,
1533.
Peptide
% of inhibition
Leu-Leu-Glu-Tyr-Val-Phe-OH
Leu-Leu-Glu-Tyr-Val-Met-OH
Leu-Leu-Glu-Tyr-Val-Tyr-OH
Leu-Leu-Glu-Tyr-Val-Trp-OH
Acetyl pepstatin (positive control)
52.3 4.3
47.2 7.7
58.6 2.5
64.92 4.7
98.47 0.27
4. Sathyanarayana, B. K.; Wlodawer, A. Curr. Sci. 1993, 65,
835.
5. Brik, A.; Wong, C. Org. Biomol. Chem. 2003, 1, 5.
6. Marshall, G. R. Tetrahedron 1993, 49, 3547.
7. Roberts, N. A.; Martin, J. A.; Kinchington, D.; Broad-
hurst, A. V.; Craig, J. C.; Duncan, I. B.; Galpin, S. A.;
Handa, B. K.; Kay, J.; Krohn, A.; Lambert, R. W.;
Merret, J. H.; Mills, J. S.; Parkes, K. E. B.; Redshaw, S.;
Ritchie, A. J.; Taylor, D. L.; Thomas, G. J.; Machin, P. J.
Science 1990, 248, 358.
8. Owens, R. A.; Gesellchen, P. D.; Houchins, B. J.;
DiMarchi, R. D. Biochem. Biophy. Res. Commun. 1991,
181, 402.
9. Schramm, H. J.; Breipohl, G.; Hansen, J.; Henke, S.;
Jaeger, E.; Meichsner, C.; Riess, G.; Ruppert, D.; Ruck-
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10. Schramm, H. J.; Billich, A.; Jaeger, E.; Rucknagel, K-P.;
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mun. 1993, 194, 595.
11. Franciskovich, J.; Houseman, K.; Mueller, R.; Chmielew-
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125.
The results are means SD (n = 3).
tion. The reaction was stopped by heating the reaction
mixture at 90 ꢁC for 1 min. Then 20 ll of double dis-
tilled water was added and an aliquot of 10 ll was ana-
lyzed by HPLC using RP-18 column. 10 ll of the
reaction mixture was injected to RP-18 column and gra-
diently eluted with acetonitrile (15–40%) and 0.1% tri-
fluoro acetic acid (TFA) in water, at a flow rate of
1 ml/min. The elution profile was monitored at
280 nm. The retention times of the substrate and
pNO2-Phe bearing hydrolysate were recorded at 10.32
and 8.97 min, respectively. The inhibitory activity of
HIV-1 protease was calculated as: % inhibition = (ACon-
trol ꢀ ASample) · 100/AControl, where A is the relative area
of the product hydrolysate. Acetyl pepstatin, which
showed 50% inhibitory activity at a concentration of
29 lg/ml, was used as a positive control of inhibition.
13. Tran, T. T.; Condom, R.; Froger, T.; Guedj, R. J. Fluorine
Chem. 1997, 82, 125.
Acknowledgments
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The authors are grateful to UGC-SAP Phase-I, DST-
FIST programmes, New Delhi, India, for financial assis-
tance. The spectral data obtained by the instrumenta-
tion facility provided by the above funding agencies
are greatly acknowledged. S.N.N.B. is grateful to CSIR,
New Delhi, India, for the award of S.R.F.

880
S. N. Narendra Babu, K. S. Rangappa / Bioorg. Med. Chem. 16 (2008) 874–880
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