Design and synthesis of potent HIV-1 protease inhibitors incorporating hydroxyprolinamides as novel P2 ligands

Article abstract of DOI:10.1016/j.bmcl.2011.04.070

A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyprolinamide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent enzyme inhibitory activity with IC₅₀ values in the nanomolar range. An active site binding model for inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the hydroxyprolinamide-derived novel P2-ligand.

Full text of DOI:10.1016/j.bmcl.2011.04.070

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3730–3733
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of potent HIV-1 protease inhibitors incorporating
hydroxyprolinamides as novel P2 ligands
⇑
Bing-Lei Gao, Cheng-Mei Zhang, Yi-Zhen Yin, Long-Qian Tang, Zhao-Peng Liu
School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhua Xilu, 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:
A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different hydroxyproli-
namide P2 ligands were designed and synthesized. Variation of substitutions at the P2 significantly
affected the enzyme inhibitory potency of the inhibitors. Compounds 2a and 2d showed excellent
enzyme inhibitory activity with IC50 values in the nanomolar range. An active site binding model for
inhibitors 2a and 2d was suggested based upon the computational-docking results of the ligand with
HIV-1 protease. This model offers molecular insights regarding ligand-binding site interactions of the
hydroxyprolinamide-derived novel P2-ligand.
Received 9 March 2011
Revised 14 April 2011
Accepted 18 April 2011
Available online 24 April 2011
Keywords:
HIV-1 protease inhibitors
Hydroxyprolinamide
P2 ligand
Ó 2011 Elsevier Ltd. All rights reserved.
Drug-resistant
Design
Synthesis
Acquired immunodeficiency syndrome (AIDS), a degenerative
disease of the immune system, is caused by the human immunode-
ficiency virus (HIV). The current statistics for global HIV/AIDS are
staggering, as an estimated 40 million people worldwide are ailing
with HIV/AIDS, and an estimated two million people die from this
disease each year.1 In the absence of curative or preventative treat-
ments, suppressing HIV-1 replication in infected patients has be-
come a critical goal in managing the disease. HIV-1 protease
plays a critical role in the virus life cycle by processing the viral
Gag and Gag-Pol polyproteins into structural and functional pro-
teins essential for viral maturation. Inhibition of HIV-1 protease
leads to the production of noninfectious virus particles and hence
is a promising therapeutic target for antiviral therapy in AIDS pa-
tients. The HIV-1 protease inhibitors (PIs), introduced in 1995,
are certainly a blessing for HIV/AIDS infected patients.2,3 Among
many strategies to combat this disease, highly active antiretroviral
therapy (HAART) with HIV protease inhibitors in combination with
reverse transcriptase inhibitors continues to be the first line treat-
ment for control of HIV infection.4 This treatment regimen pro-
vides prolonged lifetime and better quality of life by efficiently
suppressing the formation of new infectious virus particles in
patients.
saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir,
fosamprenavir, atazanavir, tipranavir, and darunavir, are all com-
petitive inhibitors that bind in the active site of the enzyme. Except
the newly approved drug tipranavir, all approved inhibitors have
been developed on the basis of the transition state mimetic con-
cept and contain various noncleavable dipeptide isosteres as core
scaffolds to mimic the transition state of HIV-1 protease substrates.
Nowadays, HIV-1 protease inhibitors are integral parts of HAART.
Despite this undeniable success, PI regimens suffer from a number
of other drawbacks including high pill burden, treatment cost, poor
ADMET properties, debilitating side effects, and toxicity issues.6–9
In addition, the rapid emergence of drug-resistant HIV-1 strains
and the appearance of cross-resistance are severely limiting long-
term treatment options.5,10,11 An estimated 10–25% of newly in-
fected patients harbor at least one viral strain that is resistant to
current medications.10–13 Therefore, the development of novel PIs
with broad-spectrum activity against multidrug-resistant HIV-1
variants remains a major therapeutic objective.
Darunavir ( Fig. 1), which combines the bis-tetrahydrofuran
(bis-THF) P2 ligand with a sulfonamide isostere, displayed excellent
antiviral activity against multidrug-resistant HIV-1 variants. It was
approved by the United States Food and Drug Administration for
treatment of drug-resistant HIV in 2006. Extensive studies of dar-
unavir-bound HIV-1 protease crystal structures have consistently
revealed tight hydrogen bonding between the inhibitor and the pro-
tease backbone.14–16 The bis-THF P2 ligand in darunavir forms a
strong hydrogen bonding network between its two cyclic ether
oxygens and the backbone amide NH bonds of the protease residues,
Asp29 and Asp30. Such tight interactions were consistently
In the past 26 years, structure-based drug design has led to the
discovery of ten FDA-approved PIs and several others in advanced
clinical trials.5 Currently marketed HIV-1 protease inhibitors,
⇑
Corresponding author. Tel.: +86 531 88382006; fax: +86 531 88382548.
E-mail address: liuzhaop@sdu.edu.cn (Z.-P. Liu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.070

B.-L. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3730–3733
3731
NH2
NH2
OH
OH
H
N
H
H
N
O
O
H
N
O
N
S
S
O
O2
O2
O
O
RHN
N
H
H
Ph
1 Darunavir
Ph
O
2a−i
Figure 1. Darunavir 1 and novel HIV-1 protease inhibitors (PIs) 2a–i.
observed with mutant proteases and might therefore account for the
unusually high resistance profile of darunavir. Optimization at-
tempts of the backbone binding in other subsites of the enzyme,
through rational modifications of the isostere or tailor made P2 li-
gands, led to equally impressive inhibitors with excellent resistance
profiles.17 Thus, the concept of targeting the protein backbone in
current structure-based drug design may offer a reliable strategy
for combating drug resistance. Upon this presumption, a series of
protease inhibitors were designed based on the (R)-(hydroxyethyl-
amino)sulfonamide isostere, the core scaffold of darunavir, where
the P2 bis-THF moiety was replaced with a stereochemically defined
trans-4-hydroxy-L-prolinamide (Fig. 1). The trans-4-hydroxyproli-
namide moiety can act as both H-bond donor and acceptor, and is
expected to exhibit maximum hydrogen bonding interactions with
the backbone of the wild-type enzyme. Herein, we report the syn-
thesis and preliminary enzyme inhibitory activities of these novel
PIs.
shown in Scheme 2. The hydroxyethylamine sulfonamide inhibitor
scaffold 5 was prepared according to the literature method.19 The
removal of Boc-group was effected by exposure of the nitrosulf-
onamide 5 to trifluoroacetic acid in CH2Cl2 to furnish the corre-
sponding amine. Alkoxycarbonylation of the aliphatic amine with
succinimidyl carbonate 4 provided the methyl ester 6 in 80.8%
yield in the two-step sequence. Hydrolysis of ester 6 with aqueous
LiOH in THF yielded the acid 7 in a modest yield of 59%. Treatment
the acid 7 with EDC, HOBt and aqueous ammonia generated the
amide 8a in 55.8% yield. Coupling the acid 8 with other amines
led to the synthesis of 8b–i in 58.6%, 53.9%, 70.2%, 55.2%, 65.4%,
72.3%, 65.6%, and 59.7% yield, respectively. Catalytic hydrogenation
of 8aꢀi over 10% Pd/C in MeOH afforded the corresponding amino-
sulfonamide derivatives. Following the removal of Boc-group, PIs
2a–i were obtained in high purity (>98.5, HPLC) and in 52.7%,
65.2%, 60.2%, 67.5%, 64.3%, 61.8%, 57.7%, 53.8% and 63.1% yield,
respectively.
For the efficient synthesis of the target PIs, the trans-4-hydroxy-
L-proline moiety was prepared as its active carbonate. As shown in
Scheme 1, the commercially available methyl ester of trans-
The enzyme inhibitory potencies of the synthetic compounds
2a–i were measured against wild-type HIV-1 protease using fluo-
rometric assays with the commercial drug indinavir as the con-
trol,20 and the results are given in Table 1.
4-hydroxy-L-proline hydrochloride was reacted with (Boc)2O in a
solution of 1,4-dioxane/H2O (2:1) to give the alcohol 3 in 90.1%
isolated yield.18 Treatment of 3 with N,N0-disuccinimidyl carbonate
in the presence of Et3N afforded the active carbonate 4 as a white
solid in 61.5% yield.
In general, the substituents on the trans-4-hydroxy-L-prolina-
mide P2 ligands have great influences on their activities against
the HIV-1 protease. Compound 2a, incorporating the unsubstituted
prolinamide moiety at the P2 position, displayed nanomolar inhib-
itory potency against the enzyme, while the corresponding N-tert-
butyl-prolinamide analog 2b was much less potent. In the N-aryl
The synthesis of inhibitors 2a–i with trans-4-hydroxy-L-prolina-
mide as the P2 ligands and 4-aminosulfonamide as the P20 ligand is
O
OH
a
OH
O
O
b
N
MeO2C
Boc
MeO2C
MeO2C
O
N
H
N
N
O
•HCl
Boc
3
4
Scheme 1. Reagents and conditions: (a) (Boc)2O, Et3N, dioxane/H2O; (b) DSC, Et3N, MeCN.
NO2
OH
Ph
NO2
H
N
OH
O
O
N
H
N
a
S
N
Boc
O2
S
R1
O
O2
N
Boc
Ph
5
6 R1 = CH3O
7 R1 = OH
NO2
OH
Ph
H
N
O
b
O
N
c
S
2a−i
O2
O
RHN
N
Boc
8a−i
Scheme 2. Reagents and conditions: (a) (i) 30% CF3CO2H/CH2Cl2, (ii) 4, Et3N, CH2Cl2, (iii) LiOH, THF; (b) EDC, HOBt, NH3, MeCN or RNH2, EDC, HOBt, NMM, CH2Cl2; (c) (i) H2,
Pd/C, MeOH, (ii) 30% CF3CO2H/CH2Cl2.

3732
B.-L. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3730–3733
Table 1
Enzymatic inhibitory activity of compounds 2a–i
Compounds
R
IC50
Compounds
R
IC50
2a
2b
2c
2d
2e
H
t-Bu
Ph
4-MeO–Ph
4-Me–Ph
16.6 nM
2f
2g
2h
2i
2-MeO–Ph
3-MeO–Ph
2,5-(MeO)2–Ph
3,4,5-(MeO)3–Ph
13.0
15.2
11.8
10.1
l
l
l
l
M
M
M
M
10.1
14.8
l
l
M
M
15.4 nM
139.0 nM
Indinavir
<10.0 nMa
a
98.8% inhibition at 10 nM.
Figure 2. The binding mode of 2d with HIV-protease.
Figure 3. The binding mode of 2a (green) and darunavir (gray) with HIV-protease.
substituted prolinamide series, when the phenyl is unsubstituted
(2c), or substituted by a 2-methoxy (2f), 3-methoxy (2g), 2,5-dime-
thoxy (2h), and 3,4,5-trimethoxy (2i) group, these compounds
exhibited low enzyme inhibitory activities. However, the 4-meth-
oxy- and 4-methyl-phenyl derivatives 2d and 2e were high active,
with the IC50 values of 15.4 and 139.0 nM, respectively. It is appar-
ent that the substituents on the prolinamide P2 ligands may
change the conformations and binding potency and modes of these
PIs.
To gain molecular insight into the ligand-binding site interac-
tions responsible for their potent inhibitory activity against the
HIV-1 protease, compounds 2d and 2e were selected for molecular
docking study by utilizing Sybyl 7.0. The binding mode of 2d in the
active site of the enzyme (PDB code: 3EBZ) is proposed based on

B.-L. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3730–3733
3733
Figure 4. The binding mode of 2e with HIV-protease.
the computational-docking results (Fig. 2). Compound 2d can inter-
Acknowledgment
act with the HIV-1 protease in a similar way as the drug darunavir
does [17]. It forms strong interaction with the two catalytic aspar-
tates Asp25/Asp250 through the hydrogen bonding of the hydroxyl
group in the chiral center. The catalytic water is also incorporated
into the inhibitor through the H-bonding with the two Ile50/Ile500
in the flap part of the protease together with the sulfonyl and car-
bonyl group in 2d. In addition, the prolinamide P2 ligand can form
H-bondings with the backbone Arg80 through the 4-methoxy oxy-
gen, with Gly1480 through the proline NH, with Asp300 through the
amide NH. Compound 2a exhibited a similar interaction pattern
with the enzyme (Fig. 3). In addition, the interactions of 2d and
2a both in the protease active site and the backbone part were in
a similar way like that of drug darunavir, as represented by the
comparison of compound 2a with darunavir (Fig. 3). The additional
interactions of the P2 ligand with the backbone part of the protease
not only enhance its inhibitory potency, but also suggest that
inhibitors 2d and 2a be active towards the mutant enzyme.
As shown in Figure 4, the 4-methylpthenyl substituted analog
2e can not form the additional H-bonds with the backbone parts
of the protease, thus it is much less potent than 2d and 2a.
In summary, a series of nine novel HIV-1 protease inhibitors
with the hydroxyethylamine core and different hydroxyprolina-
mide P2 ligands were designed and synthesized. Compounds 2a
and 2d showed excellent enzyme inhibitory activity with IC50 val-
ues in the nanomolar range. The substituents on the prolinamide
P2 ligands may affect the conformations of these molecules, and
these conformational differences at P2 moieties alter the com-
pounds’ interactions with the protease in the S2 binding pocket,
which likely account for their significantly different binding affin-
ities. Based on our preliminary finding, more PIs incorporating
hydroxyprolinamides as P2 ligands will be synthesized and evalu-
ated for their enzyme inhibitory potencies a against wild-type HIV-
1 protease and drug-resistant variants, and their antiviral activities
against wild-type and MDR HIV-1 strains. We hope that further
optimization of these compounds using structure-based design
may lead to the development of novel protease inhibitors with
improved activity against drug-resistant strains of HIV-1.
This work was supported partially by the National Natural Sci-
ence Foundation of China (NSFC, Grant No. 81072517).
Supplementary data
Supplementary data (Experimental details and spectroscopic
data for compounds 3, 4, 6, 7, 8a–i, 2a–i, procedures for enzyme
inhibitory assays, are provided.) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2011.04.070.
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