Design and synthesis of highly potent HIV-1 protease inhibitors with novel isosorbide-derived P2 ligands

Article abstract of DOI:10.1016/j.bmcl.2014.04.008

The design, synthesis, and biological evaluation of a series of six HIV-1 protease inhibitors incorporating isosorbide moiety as novel P2 ligands are described. All the compounds are very potent HIV-1 protease inhibitors with IC₅₀ values in the nanomolar or picomolar ranges (0.05-0.43 nM). Molecular docking studies revealed the formation of an extensive hydrogen-bonding network between the inhibitor and the active site. Particularly, the isosorbide-derived P2 ligand is involved in strong hydrogen bonding interactions with the backbone atoms.

Full text of DOI:10.1016/j.bmcl.2014.04.008

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of highly potent HIV-1 protease inhibitors with
novel isosorbide-derived P2 ligands
⇑
Xin Qiu , Guo-Dong Zhao , Long-Qiang Tang, Zhao-Peng Liu
Institute of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012,
Shandong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and biological evaluation of a series of six HIV-1 protease inhibitors incorporating
isosorbide moiety as novel P2 ligands are described. All the compounds are very potent HIV-1 protease
inhibitors with IC50 values in the nanomolar or picomolar ranges (0.05–0.43 nM). Molecular docking
studies revealed the formation of an extensive hydrogen-bonding network between the inhibitor and
the active site. Particularly, the isosorbide-derived P2 ligand is involved in strong hydrogen bonding
interactions with the backbone atoms.
Received 11 February 2014
Revised 3 April 2014
Accepted 5 April 2014
Available online xxxx
Keywords:
HIV-1 protease inhibitors
Isosorbides
Ó 2014 Elsevier Ltd. All rights reserved.
P2 ligand
Drug-resistant
Design
Synthesis
HIV/AIDS a chronic, potentially life-threatening disease that
interferes with the immune system, and approximately 1.8 million
people died of AIDS-related illnesses in 2010.1 Although there is no
effective vaccine available,2 therapies involving more than two
drugs from reverse transcriptase inhibitors, protease inhibitors,
and a fusion inhibitor, called HAART (highly active antiretroviral
therapy), form an effective options to reduce plasma HIV mRNA
to undetectable level and thus improve the lives of AIDS patients.2,3
HIV-1 protease (PR) is critical for viral particle maturation because
it cleaves the viral precursor polypeptides Gag and Gag-Pol into the
mature structural and enzymatic proteins.4,5 Therefore, PR is an
effective target for antiviral drugs, and in fact, the FDA has
approved 10 different protease inhibitors (PIs) since 1995.6
However, clinically used HIV-1 PIs have been associated with the
emergence of drug resistant viruses, unfavorable side effects, poor
ADMET properties, and long-term high dose requirements.7–9 Con-
sequently, the development of structurally new and highly potent
HIV protease inhibitors with different resistance profiles is still of
interest.10
Analysis of the structural and biochemical properties of PR
mutants suggests that resistant mutations act by multiple mecha-
nisms, including mutations in the binding site that directly lower
inhibitor affinity, mutations at the dimer interface that destabilize
the catalytically active dimer, and flap mutations that alter the
conformational flexibility.12 Drug resistant PR mutants exhibit
decreased binding affinity for inhibitors while maintaining the crit-
ical PR function in viral replication.13–15 Two HIV-1 PIs drugs,
Tipranavir16 and Darunavir,17,18 have recently been approved by
the FDA for use in salvage therapies against the emergence of
HIV mutants that are resistant to available drugs. X-ray structural
studies of Darunavir-bound HIV-1 protease revealed the formation
of an extensive hydrogen-bonding network between the inhibitor
and the active site.19–21 Particularly, the bis-THF P2 ligand is
involved in strong hydrogen bonding interactions with the back-
bone amides of conserved residues Asp 29 and Asp 30 in the S2
subsite. Such tight interactions are consistently observed with
mutant proteases and responsible for the unusually high resistance
profile of Darunavir. Thus, the concept of targeting the protein
backbone in current structure-based drug design offers a reliable
strategy for combating drug resistance.22 In the design of novel
PIs with nonpeptidal bis-tetrahydrofuran P2 ligands based upon
sorbitol, Ghosh’s group concluded that the position of ring oxy-
gens, ring size, and stereochemistry are all crucial to potency.23
In our efforts to utilize naturally derived P2 ligands, we replaced
the P2 bis-THF moiety in Darunavir with a stereochemically
HIV-1 PR is a C2 symmetrical homodimer with 99 residues per
monomer. Structural regions critical for PR activity and stability
are the dimer interface including the catalytic Asp25 from each
subunit and the flexible flaps comprising residues 45–55.11
⇑
Corresponding author. Tel.: +86 531 88382006; fax: +86 531 88382548.
E-mail address: liuzhaop@sdu.edu.cn (Z.-P. Liu).
defined trans-4-hydroxy-L-prolinamides and discovered two
These authors contributed equally to this project.
http://dx.doi.org/10.1016/j.bmcl.2014.04.008
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Qiu, X.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.008

2
X. Qiu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
R1
R
OH
OH
Ph
H
N
H
N
H
O
H
O
N
O
H
N
S
S
O
H
R2O
O
O2
O2
O
O
Ph
3a−f
H
O
1 R = NH2 (Darunavir)
2 R = OMe (TMC-126)
Figure 1. Darunavir 1, TMC-126 2 and novel HIV-1 PIs 3a–f.
Table 1
O
HO
H
H
Inhibitory activities of compounds 3a–f against wild-type HIV-1 protease
O2N
O
O
6
H
H
O
R1
R2
IC50
Compds
R1
R2
IC50
a
a
3
Compds
a
O
O
3a
3b
3c
Indinavir
OMe
OMe
OMe
NO2
H
Me
0.11 nM
0.10 nM
0.05 nM
<10.0 nMb
3d
3e
3f
NH2
NH2
NH2
NO2
Me
H
0.12 nM
0.43 nM
0.36 nM
O
ONO2
ONO2
ISMN
4
a
ref. 24
A mean value of three parallel experiments with a deviation within 10%.
96.9% inhibition at 10 nM.
b
O
O2N
TBSO
O
H
H
H
O
b
O
O
through TBS ether formation followed by the removal of the nitrate
under reductive conditions (H2, Pd/C), according to the reported
procedures.25 Treatment of alcohol 5 with NaH followed by reac-
tion with MeI provided a methyl ether, which was converted to
the corresponding p-nitrophenyl carbonate 6 in a total of 56% yield
(3 steps) after the removal of the TBS protective group and subse-
quent carbonate formation.
O
O
H
OH
OMe
6
5
Scheme 1. Reagents and conditions: (a) p-NO2-PhOCOCl, Py, DMAP, CH2Cl2, (b) (i)
NaH, MeI, CH2Cl2, (ii) TBAF, THF, (iii) p-NO2-PhOCOCl, Py, DMAP.
The syntheses of designed inhibitors were carried out by
coupling the activated carbonates 4 and 6 with various hydroxy-
ethylsulfonamide isosteres containing functionalized P20-phenyl-
sulfonamide ligands. As shown in Scheme 2, amines 7, 8, and 9
were readily prepared according to the literature method.26–29
Reaction of amine 7 with carbonate 4 or 6 in the presence of trieth-
ylamine provided inhibitor 3a or 3c in 88% and 66% yield, respec-
tively. Inhibitors 3d and 3e were prepared similarly by reaction
of carbonates 4 and 6 with amine 8 in good yields (79% and
85%). Coupling reaction of amine 9 with carbonate 4 afforded com-
pound 10 in 89% yield. Catalytic hydrogenation of 3a or 10 over
10% Pd/C in MeOH gave inhibitors 3b and 3f in 86% and 94% yield,
respectively. PIs 3a–f were obtained in high purity (>98.5, by
HPLC).
potent PIs with IC50 values in the nanomolar range.24 Since isosor-
bide is a biodegradable, thermally stable and non-toxic diol, it is
used as medicines or isosorbide-derived medications (isosorbide
dinitrate, isosorbide mononitrate, etc). Besides its drug-like prop-
erties, the isosorbide moiety can act as both H-bond donor and
acceptor, and if properly positioned as a P2 ligand, it will be
expected to make maximum hydrogen bonding interactions with
the backbone of the wild-type enzyme. Herein we report the syn-
thesis and preliminary enzyme inhibitory activities of six novel PIs
applying the isosorbide-derived P2 ligands to mimic the bis-THF
moiety in Darunavir and TMC-126 (Fig. 1).
For the efficient synthesis of the target PIs, the isosorbide ligand
alcohols were converted to the respective mixed activated carbon-
ates. As shown in Scheme 1, the commercially available isosorbide
mononitrate (ISMN) was reacted with 4-nitrophenyl chloroformate
in the presence of pyridine and a catalytic amount of 4-dimethyl-
aminopyridine (DMAP) in CH2Cl2 to give the activated carbonate
4 in 66% isolated yield. The TBS-protected alcohol 5 was prepared
The enzyme inhibitory potencies of the synthetic compounds
3aꢀf were measured against wild-type HIV-1 protease using
fluorometric assays with the commercial drug Indinavir as a con-
trol,24,29,30 and the results are given in Table 1. All the six com-
pounds with the isosorbide-derived P2 ligands are very potent
R1
R1
OH
H
OH
O
H
O
N
N
a
H2N
Ph
N
S
O
H
R2O
S
O2
O
O2
Ph
7 R1 = OMe
8 R1 = NH2
9 R1 = NO2
3a R1 = OMe, R2 = NO2
3b R1 = OMe, R2 = H
3c R1 = OMe, R2 = Me
3d R1 = NH2, R2 = NO2
3e R1 = NH2, R2 = Me
10 R1 = NO2, R2 = NO2
3f R1 = NH2, R2 = H
b
b
Scheme 2. Reagents and conditions: (a) 4 or 6, Et3N, CH2Cl2, (b) H2, Pd/C, MeOH.
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X. Qiu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Figure 2. The binding mode of 3c (red) and Darunavir (green) with the wild HIV-1 protease (PDB code: 4HLA).
Figure 3. The binding mode of 3e (orange) and 3f (green) with the wild HIV-1 protease (PDB code: 4HLA).
HIV-1 PIs, displaying nanomolar or subnanomolar inhibitory
potency against the enzyme. The most potent compound 3c had
an IC50 of 50 pM. In general, the protease inhibitors 3b and 3c with
It is bound in the active-site cavity through a series of hydrogen-
bond interactions with the main-chain atoms of the HIV-1
protease. The inhibitor hydroxyl group interacts with the two car-
boxylate oxygen atoms of the catalytic Asp25 and Asp250. A tetra-
coordinated water mediates hydrogen bonds with both NH atoms
of the flap residues Ile50/500 as well as the inhibitor’s urethane car-
bonyl and one of the sulfonamide (SO2) oxygens. The oxygen atom
(OMe) of sulfonamide isostere in the P20 position forms a hydrogen
bond with the amide NH of Asp300. A hydrogen bond between the
inhibitor urethane amide and the carbonyl oxygen atom of Gly27 is
also observed. Moreover, one of the isosorbide ring oxygen in
inhibitor 3c is involved in hydrogen bondings with both the
4-methoxybenzenesulfonamide at P20 exhibited
a significant
increase in potency compared to the corresponding 4-amino ana-
logues 3f and 3e.
To gain molecular insight into the ligand-binding site interac-
tions responsible for their potent inhibitory activity against the
HIV-1 protease, we made the docking study of compound 3c with
the wild HIV-1 enzyme (PDB code: 4HLA) by utilizing software
Sybyl-x1.3. As shown in Figure 2, compound 3c can interact with
the HIV-1 protease in a similar way as the drug Darunavir does.22
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X. Qiu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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Acknowledgments
Thanks for the National Center for Drug Screening of China for
the enzyme inhibitory assay.
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Supplementary data
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30. The HIV-1 protease was cloned and heterologously expressed in Escherichia coli
and purified. All inhibitors were dissolved in DMSO and diluted to appropriate
concentrations, and the incubations were performed at 30 °C in 0.1 M sodium
acetate-1 M NaCl-1 mM dithiothreitol (DTT)-1 mM EDTA-3% DMSO at pH 5.0
Supplementary data (experimental details and spectroscopic
data for compounds 3, 4, 6, 7, 8a–i, 2a–i, procedures for enzyme
inhibitory assays) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.008.
References and notes
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Please cite this article in press as: Qiu, X.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.008