Novel piperidinylamino-diarylpyrimidine derivatives with dual structural conformations as potent HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.10.059

A series of novel piperidinylamino-diarylpyrimidine (pDAPY) derivatives with dual structural conformations was designed through a molecular hybridization strategy and expected to bind into the non-nucleoside inhibitor binding pocket (NNIBP) of HIV-1 RT in

Full text of DOI:10.1016/j.bmcl.2013.10.059

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6593–6597
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel piperidinylamino-diarylpyrimidine derivatives with dual
structural conformations as potent HIV-1 non-nucleoside reverse
transcriptase inhibitors
Xuwang Chen a, Xin Liu a, Qing Meng a, Ding Wang a, Huiqing Liu b, Erik De Clercq c,
Christophe Pannecouque c, Jan Balzarini c, Xinyong Liu a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture
Road, 250012 Ji’nan, Shandong, PR China
b Institute of Pharmacology, School of Medicine, Shandong University, 44, West Culture Road, 250012 Ji’nan, Shandong, PR China
c Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel piperidinylamino-diarylpyrimidine (pDAPY) derivatives with dual structural conforma-
tions was designed through a molecular hybridization strategy and expected to bind into the non-nucle-
oside inhibitor binding pocket (NNIBP) of HIV-1 RT in a flexible manner. A cell-based antiviral screening
assay showed that some compounds were active against both wild-type and drug-resistant mutant virus
Received 29 August 2013
Revised 10 October 2013
Accepted 28 October 2013
Available online 1 November 2013
strains (K103N+Y181C RT) of HIV-1 (compound 10b3 with EC50 = 0.047 and 4.6 lM, selectivity
index = 2145 and 22, respectively). Molecular simulation studies indicated that compound 10b3 could
maintain the key hydrophobic interaction and hydrogen bonds with the NNIBP of two RT/ligand com-
plexes. In particular, it could simultaneously occupy the protein/solvent interface and the entrance chan-
nel. Exploring these hybrid molecules with dual binding conformations might provide optional chemical
scaffolds as novel HIV-1 reverse transcriptase inhibitors (HIV-1 NNRTIs).
Keywords:
HIV-1 NNRTIs
DAPY
Piperidin-4-yl-aminopyrimidines
Dual conformations
Molecular hybridization
Ó 2013 Elsevier Ltd. All rights reserved.
Acquired immunodeficiency syndrome (AIDS) that is caused by
human immunodeficiency virus type-1 (HIV-1), threatens human
health and life and spreads rapidly worldwide because of no effec-
tive vaccine.1 HIV-1 non-nucleoside reverse transcriptase inhibi-
tors (NNRTIs), as an ingredient of highly active antiretroviral
therapy (HAART), have played an indispensible role in first-line
drug regimens2 with five drugs approved by the US FDA. Especially,
diarylpyrimidine (DAPY) derivatives, such as dapivirine (TMC120,
1), etravirine (TMC125, 2) and rilpivirine (TMC278, 3), have been
the hotspot in structural modification research over the past
decade.3 Among these structurally diverse analogues, piperidin-
4-yl-aminopyrimidines (4) with piperidine replacing the 4-cyano-
phenyl group of DAPYs (Fig. 1), showed excellent activity against
HIV-1 replication,4,5 which have recently attracted our atten-
tion.6–8
towards the Pro236 and Val106 of the opening window of RT. Thus,
series of novel piperidinylamino-diarylpyrimidine (pDAPY)
a
derivatives with dual structural conformations was designed
through a molecular hybridization strategy10 by introducing both
a piperidine-linked benzyl group and a 4-cyanophenyl group into
the central pyrimidine ring (Fig. 2). The newly designed com-
pounds were expected to bind into the non-nucleoside inhibitor
binding pocket (NNIBP) of RT according to the binding modes of
the lead compounds 2 and 4. As depicted in Figure 2, the hydro-
phobic interaction and the key hydrogen bonds were retained in
both two modes. The piperidine-linked benzyl group and the 4-
cyanophenyl group were expected to fit into the protein/solvent
interface respectively close to Pro236/Val106/Leu234 and an open
region in front of Lys101/Glu138/Val179 which is considered as the
entrance channel11 for the NNRTI. The present study addresses
newly designed compounds with dual binding conformations, their
inhibitory activity against HIV-1 replication in cell culture and the
molecular simulation studies.
According to the reported crystal structures of the HIV-1 RT/
NNRTI complexes,4,9 both DAPY derivatives and piperidin-4-yl-
aminopyrimidines formed hydrogen bonds with Lys101, and
hydrophobic interaction (
rounded by Tyr181, Tyr188 and Trp229. The 4-cyanophenyl group
of DAPYs and the piperidine-linked benzyl group are oriented
p
–
p
stacking) with a sub-pocket sur-
The newly designed compounds were synthesized by an
expeditious method as depicted in Scheme 1. The intermediates
6a–b were readily prepared from 4,6-dichloropyrimidin-2-amine
(5) and 2,4,6-trisubstituted phenol under DMF/Cs2CO3 condition,12
and intermediates 7a–b were obtained via a palladium-catalyzed
coupling reaction.13 Treatment of 7a–b with 4-amino-1-Boc-
⇑
Corresponding author.
E-mail addresses: xinyongl@sdu.edu.cn, xinyongllab@163.com (X. Liu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.059

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X. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6593–6597
SO2CH3
CN
CN
CN
NH
CN
NH
CN
NH
CN
O
N
O
N
HN
N
HN
N
N
NH
N
Br
N
N
N
NH2
1
2
3
4
Dapivirine (TMC120)
Etravirine (TMC125)
Rilpivirine (TMC278)
Piperidine-4-yl-aminopyrimidines
Figure 1. The structures of the HIV-1 NNRTIs.
SO2CH3
CN
O
N
Ar
CN
CN
O
CN
NH
N
N
NH
N
O
NH
Molecular
N
hybridization
4
N
N
CN
CN
N
Hydrogen bond
interaction domain
NH
NH
Ar
N
NC
O
N
NH
Compounds with dual structural conformations
N
Br
NH2
Proposed binding
conformations
2
Lead compounds
Solvent/protein interface
Leu234
Leu234
Val106
Phe227
Val106
Pro236
Trp229
Phe227
CN
Trp229
Tyr188
Ar
CN
Tyr188
Tyr181
CN
N
Pro236
Tyr181
Hydrophobic
interaction
Hydrophobic
interaction
O
NH
O
N
NH
Hydrogen bond
O
Hydrogen bond
O
N
N
N
Glu138
Glu138
NH
NH
HN
HN
Lys101
Ar
N
Lys101
NC
Val179
Val179
Entrance channel
Bind into the pocket like lead compound 4
Bind into the pocket like lead compound 2
Figure 2. The design of new compounds via molecular hybridization and proposed binding conformations.
piperidine in refluxing DMSO gave the intermediate 8a–b, of which
the Boc group was removed in the presence of trifluoroacetic acid
with satisfactory yields.14 The exposed NH group of piperidine was
linked with substituted benzyl to obtain the target compounds

X. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6593–6597
6595
Ar
R
R
Boc
N
R
O
R
O
H
N
R
O
N
CN
O
Cl
O
NH
NH
NH
Cl
N
N
d
e
Br
c
N
N
N
N
N
N
b
a
N
N
NH
NH
NH
NH
NH2
Cl
Cl
NC
NC
NC
NC
N
N
6a, R = CH3
6b, R = CN
7a, R = CH3
7b, R = CN
8a, R = CH3
8b, R = CN
9a, R = CH3
9b, R = CN
10a1~a3, R = CH3
10b1~b3, R = CN
NH2
5
Ar
Boc
f
H
N
N
N
CN
HN
NH
HN
NH
HN
NH
HN
N
Cl
HN
Cl
e
d
N
N
Br
N
N
N
N
N
b
b
N
N
NH
NH
NH
NH
NH2
11
NC
NC
NC
NC
12
13
14
15
Scheme 1. Reagents and conditions: (a) 2,4,6-trisubstituted phenol, Cs2CO3, DMF, 100 °C, overnight, 66–71%; (b) PdAc2 (cat.), Xantphos (cat.), sodium tert-butoxide, dioxane,
80–100 °C, 6 h-overnight, 41–76%; (c) 4-amino-1-Boc-piperidine, K2CO3, DMSO, reflux, 11 h, 60–64%; (d) trifluoroacetic acid, CH2Cl2, room temperature, overnight, 84–87%;
(e) substituted benzyl chloride (or 4-picolyl chloride hydrochloride), K2CO3, DMF, room temperature, overnight, 54–67%; (f) 2,4,6-trimethylaniline, concentrated HCl (cat.),
dioxane, reflux, 20 h, 53%.
Table 1
Activity against HIV-1 (wild-type, IIIB) and resistant mutant strain RES056 (K103N+Y181C RT) in MT-4 cell cultures using the MTT method
Ar
R
X
R
X
H
N
N
NH
NH
N
N
N
N
NH
NH
NC
NC
9a, 9b
10a1~a3, 10b1~b3
SIc
a
b
Compd
R
X
Ar
EC50
(l
M)
CC50
(
lM)
IIIB
RES056
IIIB
RES056
9a
9b
CH3
CN
CH3
CH3
CH3
CN
CN
CN
CH3
O
O
O
O
O
O
O
O
0.14 0.05
0.038 0.0028
0.19 0.04
0.13 0.08
0.24 0.12
P1.8
0.95 0.42
>28
P16
>11
3.9 0.2
>206
4.6 2.3
>6.4
2.5 1.0
>36
0.52 0.02
9.6 10
4.2 1.7
11 5.8
124 49
11 9.2
12 6.3
>206
100 27
6.4 6.1
>15
69
112
61
934
44
93
>3551
2145
83
>89
>227
>855
65
4
<1
68
<1
3
X1
22
<1
>6
X1
>12
10a1
10a2
10a3
10b1
10b2
10b3
15
NVP
DLV
EFV
4-SO2NH2-Ph
4-SO2CH3-Ph
4-Pyridyl
4-SO2NH2-Ph
4-SO2CH3-Ph
4-Pyridyl
0.13 0.05
0.058 0.028
0.047 0.011
0.077 0.065
0.17 0.057
0.13 0.043
0.007 0.003
NH
4-SO2NH2-Ph
>36
>6.3
a
b
c
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathicity.
CC50: cytotoxic concentration of compound that reduces the normal uninfected cell viability by 50%.
SI: selectivity index, the ratio of CC50/EC50. X1 stands for P1 or <1.

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X. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6593–6597
Figure 3. Predicted binding modes of compound 10b3 in the NNIBP of HIV-1 RT showed by PyMOL:17 (A) and (B) RTwild-type (PDB code: 3M8Q);4 (C) RTwild-type (PDB code:
3MEC);9 (D) Superimposed conformation of the two binding modes (green, PDB code: 3M8Q; yellow, PDB code: 3MEC); (E) Superimposed conformations of RTwild-type (white,
PDB code: 3MEC)9 and RTK103N (magenta, PDB code: 3MED).9 Hydrogen bonds are indicated with dashed lines in yellow and hydrogen atoms are omitted for the sake of
clarity.
10a1–a3 and 10b1–b3. Compound 15 was prepared via a substitu-
tion reaction of starting material 5 and 2,4,6-trimethylaniline cat-
alyzed by concentrated hydrochloric acid, and then followed a
similar procedure as in the preparation of compounds 10a1–a3.
The title compounds including intermediates 9a and 9b were
tested in a cell-based antiviral assay against HIV-1 (wild-type, IIIB
and a resistant mutant HIV-1 strain, containing K103N+Y181C in
the RT) with non-nucleoside drugs nevirapine (NVP), delavirdine
mesylate (DLV) and efavirenz (EFV) as reference drugs.6–8,15 All
the new compounds were active against wild-type HIV-1 with
EC50 values in the low sub-micromolar concentration range (Ta-
ble 1). Compounds 10a1–a3 and 9a with a 2,4,6-trimethylphenyl
group (R = CH3) showed no activity against the frequently encoun-
tered drug-resistant mutant strain (containing the K103N+Y181C
RT).16 After para-methyl was replaced by a cyano group (R = CN),
compounds (9b and 10b1–b3) not only showed clearly improved
activity against wild-type HIV-1 but also had the ability to inhibit
the mutant virus strain except for compound 10b2. Introduction of
polar hydrophilic substituents (4-SO2NH2-Ph, 4-SO2CH3-Ph and
pyridin-4-yl) at the N atom of the piperidine ring decreased the
activity by comparing compound 9b with 10b1–b3, but lowered
the cytotoxicity (CC50 values) in MT-4 cell cultures. Compared to
10a1, compound 15 with a –NH– linker (X = NH) was more active
against wild-type HIV-1, but still not effective towards the drug-
resistant mutant virus strain. Overall, 10b3 was confirmed as a
promising compound with considerable antiviral potency
(EC50 = 0.047 (wild-type) and 4.6
lM (mutant)), low cytotoxicity
(CC50 = 100 M) and high selectivity (SI = 2145 and 22), respec-
l
tively, for the wild-type and mutant HIV-1 strains.
An HIV-1 RT inhibitory assay was performed via an ELISA assay
using a commercial kit (using poly rA:dT as the template/primer).
The result indicated that the representative compound 10b3
showed moderate affinity to HIV-1 RT, and inhibited the activity
of RT in vitro (IC50 = 33
ence drugs NVP (IC50 = 0.51
l
M), which was less potent than the refer-
M) and etravirine (IC50 = 0.12 M).
l
l
To further check the mode of interaction, compound 10b3 was
docked into two RT/ligand complexes PDB 3M8Q (the initial ligand
is lead compound 4)4 and PDB 3MEC (the initial ligand is lead

X. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6593–6597
6597
compound 2)9 using the software Surflex–Dock SYBYL-X. Appar-
ently, compound 10b3 displayed some well-known interactions
simulation studies supported our initial design hypothesis that the
hybrid compounds could occupy the protein/solvent interface and
the entrance channel simultaneously. Further studies on optimiza-
tion of this series of compounds are currently underway, such as
decreasing the molecular size and introducing other potential func-
tional groups. It is expected that the exploration of the unique mol-
ecules with dual binding conformations will provide optional
chemical scaffolds in the future design of HIV-1 NNRTIs.
with NNIBP in both two docking studies (Fig. 3A and D): the
p–p
interaction to the side chain phenyl of Tyr181 and the edge-to-face
interaction to the tail ring of the highly conserved Trp229. The fol-
lowing detailed analysis of the docking results revealed that the hy-
brid molecule 10b3 had achieved the main interactions as designed.
(1) Similar to lead compound 4,4 the 1-(pyridin-4-
ylmethyl)piperidine group occupied the groove lined by
Pro236, Val106 and Leu234 and extended the polar 4-pyri-
dyl head near the solvent/protein interface (Fig. 3A) and
the NH moiety at the 4-position of piperidine formed hydro-
gen-bonding interaction with the Lys101 backbone carbonyl
(Fig. 3B). The 4-cyanophenyl group was oriented into the
entrance channel11 in front of Lys101, Glu138 and Val179,
and the NH group between the pyrimidine ring and the 4-
cyanophenyl also formed a H-bond with Glu138 (Fig. 3B).
(2) Similar to lead compound 2,9 the 2-position NH linker and
the N atom of the pyrimidine ring interacted with the
Acknowledgments
The financial support from the National Natural Science Foun-
dation of China (NSFC Nos. 81273354, 81102320, 30873133,
30772629, 30371686), Key Project of NSFC for International Coop-
eration (No. 30910103908), Research Fund for the Doctoral Pro-
gram of Higher Education of China (Nos. 20110131130005,
20110131120037), Natural Science Foundation of Shandong prov-
ince (ZR2009CM016), Independent Innovation Foundation of Shan-
dong University (IIFSDU, No. 2010GN044), Shandong Postdoctoral
Innovation Science Research Special Program (No. 201002023),
China Postdoctoral Science Foundation funded project (No.
20100481282, 2012T50584) and KU Leuven (GOA 10/014) is grate-
fully acknowledged.
Lys101 backbone carbonyl and the a-amino, forming double
H-bonds. The newly introduced 1-(pyridin-4-ylmethyl)
piperidine group stretched out in the NNIBP through the
entrance channel (near Lys101, Glu138 and Val179) and
formed a hydrogen-bonding interaction between Glu138
and the NH moiety at 4-position of the piperidine (Fig. 3C).
(3) The superimposed conformation of the above two binding
modes was showed in Figure 3D (green: the mode similar
to lead compound 4; yellow: the mode similar to lead com-
pound 2). The Tyr181, Tyr188 and Trp229 were kept nearly
the same conformation, whereas, the Lys103, Leu234 and
Pro236 showed different poses between the two modes.
The 1-(pyridin-4-ylmethyl)piperidine group and 4-cyano-
phenyl group fitted into the protein/solvent interface and
an open region of the entrance channel respectively in both
two modes.
We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the anti-HIV assays.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.10.
059.
References and notes
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In addition, compound 10b3 was also docked into the NNIBP of
resistant mutant RTK103N (PDB code: 3MED).9 Through the super-
imposed conformations of mutant and wide-type RT in Figure 3E,
it was observed that the ligand had similar binding interactions
with NNIBP except for some minor differences. The Tyr181 of both
mutant (magenta) and the wild-type (white) RT kept nearly the
same orientation and was parallel to the 4-cyano-2,6-dimethyl-
phenyl group of compound 10b3. When the resistant mutation at
the Tyr181 side-chain emerged (usually into Cys181), the
p–p
interaction got lost. Moreover, the ligand formed double H-bonds
with Lys101 even when the Lys103 (white) got mutated to
Asn103 (magenta), meaning that the ligand did not interact with
Lys103 or Asn103. Based on the above two aspects, in order to im-
prove the activity against the frequently encountered drug-resis-
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compounds should strengthen
p–p interactions with Tyr188 and
Trp229 to weaken the dependence on Tyr181. Also, some func-
tional groups should be introduced as potential hydrogen bond
acceptor or donor to form an additional H-bond with Lys103.
In summary, based on a molecular hybridization strategy, we
have designed and synthesized a novel series of HIV-1 NNRTIs that
assume dual binding conformations by integration of the pharma-
cophoric groups respectively from piperidin-4-yl-aminopyrimi-
dines and DAPY derivatives. The representative hybrid molecule
10b3 showed good antiviral potency against wild-type and mutant
strains of HIV-1 in cell culture (EC50 = 0.047 and 4.6 lM). Molecular