Piperidine scaffold as the novel P2-ligands in cyclopropyl-containing HIV-1 protease inhibitors: Structure-based design, synthesis, biological evaluation and docking study

Article abstract of DOI:10.1371/journal.pone.0235483

A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the P2-ligands, 4-substituted phenylsulfonamides as the P2'-ligands and a hydrophobic cyclopropyl group as the P1'-ligand, were designed, synthesized and evaluated in this work. Among these twenty-four target compounds, many of them exhibited excellent activity against HIV-1 protease with half maximal inhibitory concentration (IC₅₀) values below 20 nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2'-ligand exhibited the most effective inhibitory activity with an IC₅₀ value of 3.61 nM. More importantly, 22a exhibited activity with inhibition of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respectively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into the ligand-binding properties, which was of great value for further study.

Full text of DOI:10.1371/journal.pone.0235483

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RESEARCH ARTICLE
Piperidine scaffold as the novel P2-ligands in
cyclopropyl-containing HIV-1 protease
inhibitors: Structure-based design, synthesis,
biological evaluation and docking study
1
Huiyu Zhou1, Mei Zhu1, Ling Ma1, Jinming Zhou2, Biao Dong1, Guoning Zhang
,
ID
1
Shan Cen1, Yucheng Wang1*, Juxian Wang
*
ID
1 Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical
College, Beijing, China, 2 Key Laboratory of the Ministry of Education for Advanced Catalysis Materials,
Department of Chemistry, Zhejiang Normal University, Jinhua, China
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
* wyc9999@126.com (YW); imbjxwang@163.com (JW)
Abstract
A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the
P2-ligands, 4-substituted phenylsulfonamides as the P2’-ligands and a hydrophobic cyclo-
propyl group as the P1’-ligand, were designed, synthesized and evaluated in this work.
Among these twenty-four target compounds, many of them exhibited excellent activity
against HIV-1 protease with half maximal inhibitory concentration (IC50) values below 20
nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand
and a 4-methoxylphenylsulfonamide as the P2’-ligand exhibited the most effective inhibitory
activity with an IC50 value of 3.61 nM. More importantly, 22a exhibited activity with inhibition
of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respec-
tively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into
the ligand-binding properties, which was of great value for further study.
OPEN ACCESS
Citation: Zhou H, Zhu M, Ma L, Zhou J, Dong B,
Zhang G, et al. (2020) Piperidine scaffold as the
novel P2-ligands in cyclopropyl-containing HIV-1
protease inhibitors: Structure-based design,
synthesis, biological evaluation and docking study.
PLoS ONE 15(7): e0235483. https://doi.org/
10.1371/journal.pone.0235483
Editor: A Ganesan, University of East Anglia,
UNITED KINGDOM
Received: April 10, 2020
Accepted: June 17, 2020
Published: July 22, 2020
Introduction
Copyright: © 2020 Zhou et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
HIV-1 infection has become a serious threat to human beings around the world since the first
case was reported in 1981 in the USA [1]. An estimated 37.9 million people lived with HIV-1
and 770 thousand people died from AIDS-related diseases in 2018, according to the Joint
United Nations Programme on HIV/AIDS (UNAIDS)’s 2019 fact sheet on global HIV &
AIDS statistics [2]. Fortunately, the emergence of a large variety of antiviral drugs, especially
the application of HIV-1 protease inhibitors (PIs) in highly active antiretroviral therapy
(HAART), made significant contributions to transforming HIV-1 infection from an inevitably
fatal disease into a manageable chronic ailment [3, 4]. HIV-1 PIs serve as a critical therapeutic
approach for the treatment of HIV-1 infection due to their ability to block the production of
viral proteins for mature virions [4–6]. So the design of potent PIs continues to be essential for
long-term control of HIV-1 infection and AIDS [7–10].
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This work is supported by National
Science & Technology Major Project “Key New
Drug Creation and Manufacturing Program”, China
(2019ZX09201001-003) and CAMS Innovation
Fund for Medical Sciences (CIFMS 2016-I2M-1-
011) to HZ. The funders had no role in study
In an effort to develop structurally novel PIs that exhibit potent inhibitory activity, one of
the major design strategies is to optimize ligand-binding site interactions with the active site
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design, data collection and analysis, decision to
publish, or preparation of the manuscript.
of HIV-1 protease (PR) [6, 11–15]. Recently, we reported a series of PIs incorporating a
cyclopropyl as the P1’-ligand and morpholine derivatives as the P2-ligands [16]. Among
which, compounds A and B in Fig 1 showed IC50 values of 53 nM and 47 nM, respectively.
The molecular docking of compound A revealed that the small hydrophobic cyclopropyl
group filled in the pocket of the S1 Appendix-subsite subtly [17–19]. However, the oxygen
atom of morpholine in the P2-ligand formed weak van der Waals interaction with the back-
bone atoms, while the wrapped nitrogen atom failed to make contact with the active site,
which might be amenable for the suboptimal activity. In view of the above phenomena,
piperidine—a flexible heterocycle containing exposed nitrogen atom—was introduced as the
P2-ligand in the newly designed HIV-1 PIs, with the aim of promoting extensive hydrogen
bonding interactions or favorable van der Waals interactions with the backbone atoms in the
corresponding S2 Appendix-subsite of PR. In addition, the effect of P2’-ligands incorporat-
ing functionalized 4-substituted phenylsulfonamides on HIV-1 protease inhibitory activity
was investigated (Fig 2).
Competing interests: The authors have declared
that no competing interests exist.
Fig 1. Structures of HIV-1 PIs A and B.
https://doi.org/10.1371/journal.pone.0235483.g001
Fig 2. Chemical structures of target molecules.
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Materials and methods
Chemistry
The syntheses of substituted piperidine carboxylic acids 9–14 are shown in Scheme 1. All
starting materials are commercially available. Chiral piperidine-3-carboxylic acids 1, 2 and
piperidine-4-carboxylic acid 3 were reacted with (Boc)2O in the presence of NaHCO3 to
obtain Boc-protected amine derivatives 9–11 in excellent yields (92.5–97.3%) [20]. Reaction
of the optically active ethyl piperidine-3-carboxylates 4, 5 with formaldehyde and formic
acid in methanol at reflux for 6 h afforded the corresponding derivatives 7, 8 in yields of
95.6% and 93.4%, respectively [21]. Saponification of 7 or 8 with aqueous sodium hydroxide
provided corresponding carboxylic acids 12 or 13 in nearly quantitative yield. Treatment of
piperidine 6 with bromoacetic acid and potassium carbonate in anhydrous DMF furnished
14 in 83.0% yield [11].
Scheme 1. Syntheses of substituted piperidine carboxylic acids 9–14. Reagents and
conditions: (a) (Boc)2O, NaHCO3, THF/H2O (1:1), Argon, 25 ˚C, overnight; (b) 40% form-
aldehyde, formic acid, MeOH, 0 ˚C to reflux, 6 h; (c) (ⅰ) NaOH, H2O, 25 ˚C, 1 h; (ⅱ) 1 N
HCl, 0 ˚C, 0.5 h; (d) bromoacetic acid, K2CO3, anhydrous DMF, Argon, 25 ˚C, overnight.
As depicted in Scheme 2, hydroxyethylamine sulfonamide isosteres 18a-d were synthesized
similarly according to the literature procedure [11, 16, 22]. Exposure of the commercially
available epoxide 15 to cyclopropanamine in acetonitrile afforded amino alcohol 16 in 87.5%
yield. Treatment of the resulting amino alcohol with 4-substituded-penzenesulfonly chlorides
provided compounds 17a-c in yields of 88.7–92.1%. They were subsequently converted to
sulfonamide derivatives 18a-c by deprotection of the Boc-group with trifluoroacetic acid in
moderate yields (74.2–80.4%). Catalytic hydrogenation of 18c over 10% Pd/C in a mixtre of
ethyl acetate and methanol (1:2) for 4 h furnished aminosulfonamide derivate 18d in 96.9%
yield.
Scheme 2. Syntheses of hydroxyethylamine sulfonamide isosteres 18a-d. Reagents and
conditions: (a) cyclopropanamine, acetonitrile, reflux, 7 h; (b) aryl sulfonyl chloride, DIEA,
DMAP (Cat.), THF, 0–25 ˚C, overnight; (c) CF3COOH/CH2Cl2 (1:3), 25 ˚C, 5 h; (d) H2 (gas),
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50 psi, 10% Pd/C, ethyl acetate/methanol (1:2), 25 ˚C, 4 h.
The syntheses of the target molecules 22a-27d were illustrated in Scheme 3. Coupling of
amines 18a-d with acids 9–14 in the presence of EDCI, HOBt and catalytic amounts of DMAP
obtained compounds 19a-21d, 24a-25d and 27a-d in yields of 34.7–92.4%. Removal of the
Boc-group by exposure of 19a-21d to hydrogen chloride gas in CH2Cl2 provided correspond-
ing piperidine derivatives 22a-23d and 26a-d in yields of 60.7–92.0%.
Scheme 3. Syntheses of inhibitors 22a-27d. Reagents and conditions: (a) EDCI, HOBt,
DMAP, anhydrous DMF, Argon, 0–25 ˚C, 8 h; (b) HCl (gas), CH2Cl2, 25 ˚C, 0.5 h.
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In vitro HIV-1 PR activity assay
The inhibitory effect of all new designed inhibitors were measured using fluorescence reso-
nance energy transfer (FRET) method. Peptide (Arg-Glu (EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-
Val-Gln- Lys(DABCYL)-Arg) purchased from AnaSpec was selected as the substrate. The
energy transfer donor (EDANS) and acceptor (DABCYL) dyes are labeled at two ends of the
peptide to perform FRET. Excitation and emission wavelengths were set at 340 nm and 490
nm. Inhibitors were dissolved in dimethylsulfoxide (DMSO) and diluted to appropriate con-
centrations. HIV-1 protease was cloned and heterologously expressed in Escherichia coli and
purified. The experiment was carried out in 96-well plates. The FRET assay reaction buffer
contained 0.1 M sodium acetate, 1 M sodium chloride, 1 mM ethylenediaminetetraacetic acid
(EDTA), 1 mM dithiothreitol (DTT), 2% DMSO and 1 mg/mL bovine serum albumin (BSA)
with an adjusted pH 4.7. Protease and inhibitor were mixed and incubated for 20–30 mins at
room temperature and then the substrate was added. Each reaction was recorded for about 10
mins. From plots of concentration versus the calculated percent inhibition, IC50 values were
determined.
HIV-1 infectivity assay
The inhibitory effect of compounds on HIV-1 infectivity were determined using a single-
round HIV-1 infectivity assay. 293T cells were co-transfected with either plasmid pNL4-3-E-R-
(pHIV-1NL4-3) or DRV-resistant pNL4-3-E-R- variants (pHIV-1DRV S) and pHCMV-G
R
(VSV-G) to produce VSV-G pseudotyped HIV-1. Inhibitors dissolved in dimethylsulfoxide
(DMSO) and diluted to appropriate concentrations, were added into culture medium at 5
hours of post-transfection. After incubating for 48 hours at temperature 37 ˚C, pseudotyped
viruses in 10 μL of supernatant were used to infect SupT1 cells for 48 hours, followed by mea-
suring luciferase activity of newly infected cells using Centro LB960 (Berthold).
For the assay using wild type HIV-1, 1×106 SupT1 cells were infected with 100 μL HIV-1
NL4-3 in the presence of 100 nM chemicals and 10 μg/mL polybrene, keeping a total volume
of 500 μL (Spin infection at 1800rpm, 45min). Cells were washed once in the next morning
and medium were replaced with fresh medium containing 100 nM chemicals. At 48 hpi,
viruses were harvested and 50 μL of viruses were used to infect TZM-bl cells, followed by mea-
suring luciferase activity in the infected cells.
Cytotoxicity assay
Selected inhibitors were further evaluated in cytotoxicity assay using a cell counting kit-8
assay. Plates were prepared with 20 000 293T cells per well. After 24h of culture, 1μL of drugs
were added to each well. After another 24h of culture, 10 μL of CCK-8 was added to each well.
Absorbance was quantified at wavelength 450 nm using an EnVision multilabel reader (Perki-
nElmer) after 2h at room temperature. The 50% cytotoxic concentrations (CC50) were deter-
mined as the concentration required to reduce the number of the cells by 50% compared to
that of drug-unexposed control cultures.
Molecular docking
In general, the docking was performed through “DOCK” module in the Molecular Operating
Environment (MOE) using the alpha triangle placement method. Refinement of the docked
poses was carried out using the Forcefield refinement scheme and scored using both the
affinity dG and london dG scoring system. The pose with the higher docking negative score
implied better binding.
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Results and discussion
Structure activity relationships
All target compounds were evaluated the inhibitory potency against wild-type HIV-1 PR using
the fluorescence resonance energy transfer (FRET) method, including DRV as the positive
control [23, 24]. The results are presented in Figs 3–5. As can be seen, the piperidine-derived
inhibitors exhibited potent enzymatic activity with IC50 values of submicromolar to nanomolar
Fig 3. Enzymatic inhibitory activity and cytotoxicity of inhibitors 22a-25d.
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Fig 4. Enzymatic inhibitory activity of inhibitors. (a) Enzymatic inhibitory activity of inhibitors with (R)-piperidine derivatives as the P2-ligands; (b) Enzymatic
inhibitory activity of inhibitors with (S)-piperidine derivatives as the P2-ligands.
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Fig 5. Enzymatic inhibitory activity of inhibitors 26a-27d.
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in general. Especially, inhibitor 22a, containing a (R)-piperidine-3-carboxamide as the
P2-ligand and a 4-methoxylphenylsulfonamide as the P2’-ligand, displayed the most impres-
sive activity with an IC50 value of 3.61 nM.
As it turned out in Figs 3 and 4, inhibitors with (R)-piperidine derivatives as the P2-ligands
exhibited superior activity than those with (S)-configuration as a whole. For instance, com-
pound 22a showed a 120-fold improvement of potency over 23a, which revealed the signifi-
cance of configurations. In addition, the activity decreased when the nitrogen atom of (R)-
piperidine scaffold was methylated, such as 22a vs 24a, 22b vs 24b, 22c vs 24c, and 22d vs 24d.
The results might be attributed to both the smaller volume of the P2-ligand which was more
suitable for the cavity of S2 Appendix-subsite and the capacity of the exposed nitrogen atom
which could form hydrogen bonding interactions with the carbonyl oxygen or amide NHs of
Asp30 and Asp29 [25–28].
The functional P2’-ligands also exerted great impact on the potency of inhibitors. Com-
pounds containing 4-methoxyl (22-25a), 4-trifluoromethyl (22-25b) and 4-amino (22-25d)
substituents exhibited more potent enzyme inhibitory than the corresponding 4-nitro substit-
uent compounds (22-25c). The oxygen atom of methoxyl group is capable of forming hydro-
gen bonds with the backbone NH and side-chain carboxylate of Asp30’ directly, which could
enhance the antiviral potency [29]. The 4-trifluoromethyl group in the P2’-ligand may cause
favorable halogen interactions and van der Waals interactions with the P2’-pocket [30, 31], in
spite of its weak electron-withdrawing inductive effect that impaired the binding affinity
slightly [15]. Although the amino of 4-aminobenzene sulfonamide could participate in direct
or water-mediated hydrogen bonds with Asp30’, these interactions were weaker than those
between 4-methoxybenzene sulfonamide and the cavity of S2 Appendix-subsite [10]. On the
contrary, the strong electron-withdrawing property of nitro in the P2’-ligand, including both
electron-withdrawing inductive effect and conjugation, was likely to weaken not only the
hydrogen bonds between the nitro oxygen and Asp30’, but also the water-mediated interac-
tions between the sulfonyl oxygen and Ile50’ [19, 15].
Furthermore, the cytotoxicity of selected inhibitors was assayed [32]. Surprisingly, all of
them exhibited low cytotoxicity. Therefore, this kind of inhibitors with potent activity and low
toxicity deserved in-depth study.
However, compounds 26a and 27a bearing 4- or 1-subsituted piperdine derivatives in Fig 5
showed more than 50-fold loss of potency over the 3-subsituted piperidine derivative 22a. The
same trend can also be observed in the other three groups (22 vs 26, 27b; 22 vs 26, 27c; 22 vs
26, 27d), which suggested that the shift of substituent position and the length of linker can
impact the activity remarkably. Moreover, the effect of functional P2’-ligands on potency is
similar to that shown in Fig 3.
HIV-1 infectivity assay
In the assays against HIV-1 wild-type and DRV-resistant variants [33, 34], selected com-
pounds 22a, 24a and 24b exhibited inhibition activity to some extent (Fig 6). Notably, 22a
exhibited the most remarkable activity with inhibition of 42% and 26% against wild-type and
DRV-resistant HIV-1 variants, respectively, which agreed with the activity tested in vitro.
Although compounds 24a and 24b showed inconspicuous inhibition (with 16% and 12%
against wild-type variants, and 8% and 7% against DRV-resistant variants, respectively), there
still revealed regularity. Generally, compounds with 4-methoxylphenylsulfonamide as the P2’-
ligand exhibited superior activity than those with 4-trifluoromethylphenylsulfonamide as the
P2’-ligand in vivo, which pointed the way for further study.
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Fig 6. Inhibition of inhibitors against wild-type and DRV-resistant HIV-1 variants. Inhibition of inhibitors 22a, 24a and 24b was
conducted at the concentration of 10 μM, and DRV at 20 nM.
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Molecular docking
In order to gain insight into the ligand-binding site interactions, the molecular modeling for
22a was done in the molecular modeling software MOE with a HIV-1 PR crystal structure
(PDB-ID: 4mc9) [35]. Remarkably, 22a fitted into the active site of PR perfectly. As illustrated
in Fig 7, several hydrogen bonding interactions were formed between the scaffold of the inhib-
itor and the residues Asp25, Ile50 (A chain) and Ile50 (B chain). Furthermore, plentiful van
der Waals interactions between atoms in the P2-ligand, P1-ligand, P1’-ligand or P2’-ligand
and external enzyme atoms were also observed. Especially, the newly introduced piperidine
could produce favorable interactions with the active site of PR and be inserted into the cavity
of S2 Appendix-subsite properly. All of the above mentioned might be responsible for the
promising inhibitory activity of 22a.
Conclusions
In summary, we have reported the structure-based design and synthesis of a series of novel
HIV-1 PIs incorporating flexible piperidine moieties as the P2-ligands, 4-substituted phenyl-
sulfonamides as the P2’-ligands and a cyclopropyl group as the P1’-ligand. Introduction of
poperidine in the P2-ligand was for the sake of promoting hydrogen bonding or van der Waals
interactions with the active site of HIV-1 PR backbone. A number of inhibitors exhibited
excellent potency and low cytotoxicity. In particular, inhibitor 22a containing a (R)-piperi-
dine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2’-ligand
showed the most remarkable enzyme inhibitory activity, with an IC50 value of 3.61 nM, as well
as activity with inhibition of 42% and 26% against wild-type and DRV-resistant HIV-1 vari-
ants, respectively. We demonstrated that the stereochemistry and substitution position of
piperidine derivatives in the P2-ligands, as well as functional phenylsulfonamides in the P2’-
ligands, are decisive for the potency. Moreover, the molecular docking of 22a showed that the
piperidine could fill in the pocket of S2 Appendix-subsite perfectly and make strong interac-
tions with residues of HIV-1 PR, which was consistent with its potent antiviral activity. Further
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Fig 7. Docking of 22a in HIV-1 PR. Ligand exposures are described as purple spheres. Hydrogen bonding interactions between
ligands and residues are represented as blue or green arrows.
https://doi.org/10.1371/journal.pone.0235483.g007
studies on the evaluation of other flexible N-containing heterocycle derivatives are currently in
progress.
Supporting information
S1 Appendix. Description of synthetic experiments.
(DOCX)
S2 Appendix. 1H NMR, 13C NMR and HR MS spectrums of compounds.
(DOCX)
S3 Appendix. Description of biological evaluation and docking study.
(DOCX)
Acknowledgments
We would like to thank the Department of Virology for in vitro HIV-1 PR activity assay and
cytotoxicity assay, Department of Immunology for HIV-1 infectivity assay and providing the
Molecular Operating Environment for molecular docking.
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Author Contributions
Data curation: Guoning Zhang.
Formal analysis: Ling Ma.
Investigation: Huiyu Zhou, Ling Ma, Jinming Zhou, Biao Dong, Shan Cen.
Methodology: Mei Zhu.
Resources: Shan Cen, Yucheng Wang.
Supervision: Juxian Wang.
Writing – original draft: Huiyu Zhou.
Writing – review & editing: Mei Zhu, Yucheng Wang, Juxian Wang.
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