Discovery of nitropyridine derivatives as potent HIV-1 non-nucleoside reverse transcriptase inhibitors via a structure-based core refining approach

Article abstract of DOI:10.1016/j.ejmech.2014.02.047

As a continuation of our efforts to discover and develop back-up analogs of DAPYs, novel substituted nitropyridine derivatives were designed via a structure-based core refining approach, synthesized and evaluated for their in vitro HIV-1 activity in MT-4 cells. Preliminary biological evaluation indicated that most of the compounds exhibited marked inhibitory activity against wild-type HIV-1 III_B. Most notably, the compound 7b was identified as the most promising candidate in inhibiting HIV-1 replication with an EC ₅₀ value of 0.056 μM and a selective index (SI) of 1251, which were much better than those of NVP (EC₅₀ = 0.23 μM) and DLV (EC₅₀ = 0.51 μM). Some other compounds, 7k, 7c, 7j and 7e, were also endowed with a favorable anti-HIV-1 potency (EC₅₀ = 0.034, 0.11, 0.11 and 0.16 μM, respectively). Some antivirally active compounds also showed moderate inhibitory activity against RT. Preliminary structure-activity relationships (SARs) and molecular modeling of these new analogs provide valuable avenues for future molecular optimization.

Full text of DOI:10.1016/j.ejmech.2014.02.047

European Journal of Medicinal Chemistry 76 (2014) 531e538
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of nitropyridine derivatives as potent HIV-1 non-nucleoside
reverse transcriptase inhibitors via a structure-based core refining
approach
,
a
c
b
Jun Wang a, Peng Zhan a , Zhenyu Li , Huiqing Liu , Erik De Clercq ,
*
Christophe Pannecouque b, 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 Jinan, Shandong, PR China
b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
c Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
As a continuation of our efforts to discover and develop back-up analogs of DAPYs, novel substituted
nitropyridine derivatives were designed via a structure-based core refining approach, synthesized and
evaluatedfor their invitro HIV-1 activity in MT-4 cells. Preliminary biological evaluation indicated that most
of the compounds exhibited marked inhibitory activity against wild-type HIV-1 IIIB. Most notably, the
compound 7b was identified as the most promising candidate in inhibiting HIV-1 replication with an EC50
Received 19 October 2013
Received in revised form
13 February 2014
Accepted 17 February 2014
Available online 19 February 2014
value of 0.056
m
M and a selective index (SI) of 1251, which were much better than those of NVP
M). Some other compounds, 7k, 7c, 7j and 7e, were also endowed
M, respectively). Some antivirally
(EC50 ¼ 0.23 M) and DLV (EC50 ¼ 0.51
m
m
Keywords:
HIV-1
with a favorable anti-HIV-1 potency (EC50 ¼ 0.034, 0.11, 0.11 and 0.16
m
active compounds also showed moderate inhibitory activity against RT. Preliminary structureeactivity
relationships (SARs) and molecular modeling of these new analogs provide valuable avenues for future
molecular optimization.
Reverse transcriptase
NNRTIs
Drug design
Organic synthesis
Pyridine
Ó 2014 Elsevier Masson SAS. All rights reserved.
Bioactivity
ꢀ
1. Introduction
about 10 A away from the RT catalytic site [3]. HIV-1 NNRTIs is a key
component of HAART regimens for the long-term management of
Human immunodeficiency virus (HIV) is the causative agent of
acquired immunodeficiency syndrome (AIDS). From the data of
AIDS Epidemic Update reported by World Health Organization, 1.7
million patients died from AIDS and related diseases, and 34.0
million peoples were living with AIDS in 2011 [1]. Currently, there is
no effective vaccine against HIV, but the generally adopted highly
active antiretroviral therapy (HAART) has significantly reduced the
morbidity and mortality of HIV-infected people. HIV type 1 reverse
transcriptase (HIV-1 RT) as a key enzyme in the HIV replicative
cycle represents one of the main targets for the treatment of HIV/
AIDS [2]. Among RT inhibitors, non-nucleoside RT inhibitors
(NNRTIs) can directly inhibit RT by binding to an allosteric site,
termed as the NNRTIs binding pocket (NNIBP), which is located
HIV infection because of their unique antiviral potency, high
specificity and low toxicity. However, during the clinical use of the
first generation NNRTIs (nevirapine, delavirdine and efavirenz),
significant resistance has been generated by the emergence of
mutant viral strains. Therefore, there is still need for discovery of
novel HIV-1 NNRTIs with an improved potency against the wild and
clinically observed mutant virus strains [4e9].
The diarylpyrimidine (DAPY) family, represented by the newly
approved anti-HIV-1 drugs etravirine (TMC125) and rilpivirine
(TMC278), has been regarded as promising HIV-1 NNRTIs with
robust anti-HIV-1 potency against both the wild type (wt) and
drug-resistant strains carrying multiple mutations [10]. Over the
past few years, considerable efforts have been devoted to the
structural modification of DAPYs to discover more potent back-up
congeners.
* Corresponding authors.
E-mail addresses: zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn
(X. Liu).
As determined by X-raycrystal structures of DAPYs-RTcomplexes
and molecular simulation, DAPYs resembled a horseshoe or “U”
http://dx.doi.org/10.1016/j.ejmech.2014.02.047
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

532
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
shape when bound in the NNIBP in contrast to the typical butterfly-
like binding shape of the first-generation NNRTIs (NVP, DLV and
EFV). The relative flexibility of the six-membered heterocycle core
might allow DAPYs to better adapt to the plasticity and changes of
the NNIBP, which appear to be critical for maintaining potency
against wt and a wide range of drug-resistant mutant HIV-1 RTs
[11,12]. As shown in Fig. 1, the DAPYs also shared a similar pharma-
cophore as mentioned for the first-generation NNRTIs, including a
compounds are also discussed to give further insight in their
probable binding modes in the allosteric binding site.
2. Chemistry
The synthesis of the newly designed nitropyridines compounds
was accomplished using an expeditious straight-forward route
described in Scheme 1. Chlorination of commercially available
picolinic acid (1) with SOCl2 followed by esterification with MeOH
resulted in the formation of methyl 4-chloropicolinate (2). The latter
was further converted to 4-chloropicolinamide (3) by amidation
using saturated ammonia. Then, 4-chloropyridin-2-amine (4) was
generated from intermediate 3 using standard procedures of Hof-
mann rearrangement. Further, nitration reaction of compound 4 in
the presence of HNO3/H2SO4 formed the key intermediate 4-chloro-
5-nitropyridin-2-amine (5) in 41% yield according to the methods
previously reported [19,20]. Compound 5 was subjected to treat
with substituted phenols or substituted anilines to obtain com-
pounds 6 [21], which was converted into the final nitropyridine
derivatives 7 with various 4-substituted phenyl bromides using
K2CO3 as a base and Pd(OAc)2 and Xantphos as catalysts, in good
yields [22,23]. The synthesized compounds were characterized by
physicochemical and spectral means. The MS and NMR spectral data
are in full agreement with the proposed structures.
hydrophobic center able to participate in pep stacking interactions
(C ring) as well as hydrogen bond donor and acceptor interactions.
The six-membered heterocyclic moiety not only stays in the center of
the NNIBP, anchoring the functional groups for optimal engagement
with the residues around NNIBP, but also serves as a critical
hydrogen bond acceptor (the pyrimidine nitrogen at 1-position) to
generate hydrogen bonding with the a-amino of LYS101. Besides, the
NH linker connecting the central B ring and the right A wing can
function as a hydrogen bond donor able to interact with the back-
bone carbonyl of LYS101 [13]. These conclusions provided critical
insights for further structural studies or scaffold refining towards
improving their activity and drug resistance profiles.
Scaffold refining via the replacement of the central core in
bioactive molecules combined with introducing privileged sub-
stituents is a common practice in contemporary medicinal chem-
istry to obtain intellectual property and novel hits, as well as to
improve synthetic accessibility. To discover highly potent DAPY
derivatives that are easier to synthesize or to avoid existing patent
property, further modifications were focused on the replacement of
the pyrimidine core with an array of aromatic scaffolds [14,15]. In
this regard, previous investigations in our laboratories using
structure-based drug design and isosterism resulted in the dis-
covery of a novel series of potent DAPYs back-up analogs, such as
pyridazine derivatives, with proved to be highly effective in
inhibiting HIV-1 replication at double-digit nanomolar concentra-
tions acting as RT inhibitors [16e18]. According to the molecular
modeling and structureeactivity relationship (SAR) results, the
central pyrimidine ring of DAPYs is relatively tolerant.
As an extension of these investigations and with the aim to
generate novel NNRTIs with desirable potency and properties, we
designed and synthesized novel nitropyridine derivatives in which
the pyrimidine ring of the original DAPY scaffold was replaced with
a synthetically accessible nitropyridine structural motif to adhere
to the optimal pharmacophore moieties, while the NH linker con-
necting the central ring and the right wing, and the constant sub-
stitutions of A and C ring were maintained in view of their
paramount importance in the previous series. The general structure
of such a new scaffold is illustrated in Fig. 1.
3. Results and discussion
3.1. Anti-HIV activity evaluation
All of the newly synthesized nitropyridine derivatives were
evaluated for their anti-HIV activity and cytotoxicity in MT-4 cells
infected by wt HIV-1 strain IIIB and the double mutant strain
RES056 (K103N þ Y181C) along with HIV-2 (ROD). The results,
expressed as CC50 (cytotoxicity), EC50 (anti-HIV activity) and SI
(selectivity index, given by the CC50/EC50 ratio), are summarized in
Table 1 together with those of nevirapine (NVP), delaviridine (DLV),
efavirenz (EFV) and zidovudine (azidothymidine, AZT) as the
reference drugs.
As listed in Table 1, the majority of these compounds showed
high activity against wt HIV-1(IIIB) with EC50 values in the range of
0.034e30.17
them are of low toxicity in MT-4 cells with CC50 values more than
125 M, SI ¼ 1251),
M. Encouragingly, compound 7b (EC50 ¼ 0.056
compound 7k (EC50 0.034
M, SI ¼ 691), compound 7c
(EC50 ¼ 0.11 M,
mM, except for the two compounds, 7m and 7q. Half of
m
m
¼
m
m
M, SI ¼ 339) and compound 7h (EC50 ¼ 0.17
m
SI ¼ 97) were more active than reference drugs NVP and DLV
against wt HIV-1(IIIB), but were still inferior to the clinically used
EFV and AZT. It was also observed that some compounds, 7a and 7r,
Herein, we will further report the synthesis, anti-HIV evaluation
against wt HIV-1 and HIV-2 ROD, as well as against a double-
mutated HIV-1 strain RES056 (K103N þ Y181C). In addition, pre-
liminary SAR data and molecular modeling results of these new
had high anti-HIV-1 potency (EC50 ¼ 0.58 and 0.72
m
M, SI ¼ 214 and
200, respectively). These promising results demonstrated that the
CN
CN
CN
R1
R
CN
NH
C
A
C
A
R2
R3
Scaffold refining
O
N
NH
X
NH
B
HN
N
N
B
Br
N
N
O2N
X = O, NH
NH2
Rilpivirine (TMC278)
Etravirine (TMC125, ETV)
Newly designed nitropyridine derivatives
Lead compounds
Fig. 1. Design of nitropyridine derivatives as back-up DAPYs via a scaffold refining approach.

J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
533
Cl
Cl
N
Cl
Cl
NH2
i
iii
ii
iv
OH
NH2
N
O
O2N
N
N
N
NH2
O
O
O
1
4
2
3
5
R1
R1
R
R2
R3
R2
R3
R1, R2, R3 = H, F, Cl, Br,
CH3, OCH3, CN, NO3
R = CN, CH3, NO2
X = O, NH
X
O2N
NH2
X
NH
v
vi
N
N
O2N
7
6
Scheme 1. Reagents and conditions. i: (a) SOCl2; (b) MeOH; ii: NH3 (aq); iii: Br2, NaOH(aq); iv: HNO3/H2SO4; v: Substituted phenols, K2CO3, DMF; or substituted anilines, K2CO3,
DMF; vi: Pd(OAc)2, Xantphos, Cs2CO3, dioxane.
analogs, indicating that perhaps the R1 substituents may
have some unfavorable aspects regarding cytotoxicity.
isosteric replacement of pyridine for pyrimidine in the central
B-ring of DAPY compounds was gratifying and afforded a series of
potent nitropyridine NNRTIs.
Based on the results of antiviral assay (Table 1), several impor-
tant structural features for conferring optimum HIV inhibition and
the cytotoxicity were investigated and summarized as follows:
(d) Thenatureof thesubstituentsattheR2/R3 positionoftheC-ring
essentiallyinfluences the anti-HIV-1 activity. The R2/R3-methyl
derivative 7a showed activity in the low micromolecular range
with EC50 value (0.58
chlorine derivative 7g (EC50 ¼ 0.62
of the methoxy counterpart 7f (EC50 ¼ 4.28
compound 7f was a compelling moleculewith the highest CC50
value (318.78 M) of all the tested compounds.
m
M) comparable to that of the R2/R3-
M), much lower than that
M). Even so,
m
m
(a) The biological assay results clearly indicated that the activ-
ities of the compounds where X is O are generally better
than the compounds where X is NH. For instance, the active
sequence of the compounds with the same substitutions in A
and C ring was as follows: 7a > 7m, 7b > 7n, 7j > 7o.
m
(e) In the case of 2,4,6-trihalogen-phenoxy derivatives (7h, 7j,
7l), we found that their antiviral potency was in the order
2,4,6-tribromo > 2,4,6-trichloro > 2,4,6-trifluoro. Compared
with their congeners, significantly increased cytotoxicities
were observed. Compound 7l characterized by the absence of
the 2,4,6-trifluoro substitution at the phenoxy showed the
highest toxicity among all the nitropyridine derivatives.
(b) Next we turned our attention to SAR of the R substituent at
the para position of the A-ring. Like the DAPY analogs
TMC125 and TMC278, the compound 7c (EC50 ¼ 0.11
CC50 ¼ 36.06 M) containing a cyano group as the R sub-
stituent was found to be more active than the analogs con-
taining NO2 (7r, EC50 ¼ 0.72 M, CC50 ¼ 141.44 M) and CH3
(7q, EC50 > 206.87 M). These results in
M, CC50 ¼ 206.87
mM,
m
m
m
With the aim to define the resistance profiles of these com-
pounds, they were also assayed against the double mutant strain
RES056 (K103N þ Y181C), the often encountered clinical strain in
the treatment of AIDS with HIV-1 NNRTIs (results shown in
Table 1). Unfortunately, all the compounds lost their activity against
the double mutant strain RES056. Furthermore, none of the de-
rivatives tested showed inhibition of HIV-2 (ROD) replication in
MT-4 cells, which might suggest that this new series of nitro-
pyridine derivatives was specific for HIV-1.
m
m
conjunction with previous studies suggested that the para-
cyano moiety would be a favorable structural moiety for anti-
HIV activity. Besides, it is noteworthy that the cytotoxicity of
these congeners increased in the same order (7c > 7r > 7q).
After determining that the O atom was the favorable linker
connecting the central ring and the left wing and a nitrile was the
optimal substituent at the R-position on the right wing, we then
focused our attention on the SAR of the left C wing.
3.2. Inhibition of HIV-1 RT
(c) As highlighted in Table 1, the anti-HIV activity is strongly
dependent on the nature of the substituents at the R1 posi-
tion of the C-ring. In the case of 2,6-dimethylphenoxy ana-
logs (7ae7e), a clear order of R1-substituent for anti-HIV
activity was observed by direct comparison: CH3 (7b) > CN
(7c) > Cl (7e) > Br (7d) > H (7a). This conclusion is in
agreement with 2,6-dichlorophenoxy analogs (Cl > H > NO2)
and 2,6-dibromophenoxy analogs (CH3 > Br). This set of re-
sults seemed to indicate that R1 substituents with appro-
priate bulk are beneficial for potency. The negative impact
on the potency was greatly exacerbated with the replace-
ment of the H in 7g by NO2 (7i). It was also observed that,
compound 7a with the lowest potency in the 2,6-
dimethylphenoxy series demonstrated the lowest cytotox-
With aim to verify their binding target, three selected title
compounds (7b, 7c, and 7k) were tested in enzyme assays against
highly purified recombinant HIV-1 RT using poly (rC)-oligo (dG) as
template primer. IC50 values corresponded to the concentration of
the substituted nitropyridine derivatives required to inhibit biotin-
dUTP incorporation into the HIV-1 RT by 50%. The three compounds
exhibited moderate inhibition of enzymatic activity with IC50
values of 6.9
mM, 8.5
mM and 10.4
m
M, respectively, which was
slightly higher than that of TMC125 (6.5
that the newly synthesized nitropyridine derivatives bind to HIV-1
RT and belong to the genuine NNRTIs.
What needs to be stressed is that the anti-RT potency of com-
pound 7k was slightly weaker than that of 7b and 7c even though
compound 7k was more potent against HIV-1 replication (Table 1).
The discrepancy in potency between anti-HIV-1 replication and
mM) (Table 2). It suggests
icity (CC50 ¼ 125.85
mM), and likewise, this will also be the
case for compound 7h among three 2,6-dichlorophenoxy

534
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
Table 1
HIV-1 wt (IIIB) inhibition effect and cytotoxicity of the title compounds.
Compds
X
R
R1
R2
R3
HIV-1 IIIB
EC50
M)a
RES056
EC50
M)a
ROD
EC50
CC50(m
M)b
(
m
SIc
(
m
SIc
(
m
M)a
SIc
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
NVP
DLV
EFV
AZT
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
O
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CH3
NO2
H
CH3
CH3
CH3
CH3
CH3
OCH3
Cl
Cl
Cl
Br
Br
CH3
CH3
CH3
CH3
CH3
OCH3
Cl
Cl
Cl
Br
Br
0.58
0.056
0.11
0.27
0.16
4.28
0.62
0.17
214
1251
339
45
70
74
44
97
2
65
691
5
<1
9
43
>125.84
>69.86
>36.06
>12.26
>11.06
>318.78
>27.69
>16.24
>26.51
>7.49
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
>4
<1
>21
>125.84
>69.86
>36.06
>12.26
>11.06
>318.78
>27.69
>16.24
>26.51
>7.49
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
125.84
69.86
36.06
12.26
11.06
318.78
27.69
16.24
26.51
7.49
CH3
CN
Br
Cl
H
H
Cl
NO2
Br
CH3
F
16.8
0.11
0.034
0.72
>12.78
30.17
3.85
3.05
>206.87
0.72
>23.55
>3.54
>23.55
>3.54
23.55
3.54
12.78
F
F
H
CH3
CH3
Br
Br
CH3
CH3
CH3
CH3
Br
F
CH3
CH3
>12.78
>262.36
>166.52
>149.25
>206.87
>141.44
4.09
>36.19
0.32
0.016
>12.78
>262.36
>166.52
>149.25
>206.87
>141.44
CH3
CH3
Br
CN
CN
262.36
166.52
149.25
206.87
141.44
>15.02
>36.19
>6.34
>93.55
49
<1
O
200
>67
>71
>1112
>12,473
0.23
0.51
0.0057
0.0075
>5959
0.012
>7716
a
EC50: concentration required to protect cells against the cytopathogenicity of HIV by 50%.
CC50: concentration required to inhibit uninfected cells proliferation by 50%.
SI: selective index (CC50/EC50); NVP: nevirapine; DLV: Delavirdine; EFV: efavirenz; AZT: zidovudine.
b
c
anti-RT activity could be due to the fact that the physicochemical
properties of different compounds probably showed some impact
on the celluar assay.
residues. These interactions would lead to an improved binding
affinity and increased anti-HIV-1 activity of these inhibitors; (2) The
new compounds formed two hydrogen bonds: the NH moiety linker
at the 2-position and the N atom of the nitropyridine ring interacted
with the backbone carbonyl and backbone
a-amino of residue
3.3. Molecular simulation analysis
LYS101, similar to those observed in the DAPY compounds; (3) The
right A ring of compounds 7b and 7k located between amino acid
VAL106 and PRO236, almost completely overlapped with the right
side of TMC125. The antiviral potency of 7b and 7k were retained
owing to nearly the same binding mode as TMC125.
To investigate the HIV inhibitory potencies of the newly syn-
thesized compounds at a molecular level and to understand the
structural basis of their binding mode, molecular docking was per-
formed by docking the representative compounds 7b and 7k into
the NNRTIs binding pocket (NNIBP) of wt HIV-1 RT (PDB code:
3M8Q) and the double mutant (K103N þ Y181C) RT (PDB code:
3BGR) using the software Surflex-Dock SYBYL-X 1.1 and the docking
results were shown by PyMOL 0.99 (www.pymol.org). These mo-
lecular modeling studies (Fig. 2) suggested that the presence of the
central nitropyridine motif could assure a similar molecular topol-
ogy and flexibility, and hence the new compounds were able to bind
into NNIBP of the RT in a very similar manner as TMC125 (Fig. 2B).
Detailed analysis of the docking results revealed the following
notable features that may clarify the mechanism of action of
nitropyridine derivatives. (1) The left C ring of compounds 7b and 7k
fitted into the aromatic-rich binding pocket, which is surrounded by
the aromatic side chains of aromatic amino acid residues TYR181,
As shown in Fig. 3, the most active compound 7k was docked
into the double mutant (K103N þ Y181C) NNIBP. Docking studies
revealed the predominant binding mode of compound 7k that was
distinct from the binding mode of TMC278, and suggest a deviation
in the binding site. Consequently, compound 7k lost the key
hydrogen bonds with residue LYS101 which were necessary for
anti-HIV activity. Obviously, the right A ring of compound 7k was
parallel to VAL106, ASN103 and PRO236, which was completely
different from TMC278. Therefore, compound 7k does not interact
significantly with some of the key amino acids implicated in anti-
viral activity. The results clearly indicated that the small modifi-
cations in the heterocyclic core could affect the hydrogen bonds
strength with the LYS101 residue of mutant NNIBP which are
indispensable for anti-HIV-1 RT activity.
TYR188 and TRP229, exhibiting p-p stacking interactions with these
It should be specially mentioned that, our studies also suggested
that the nitro group in the pyridine ring, situated in the “entrance
channel” extending into the solvent-exposed region where struc-
tural alternations could be tolerated [24], was considered to be
highly amenable for chemical modifications optimizing anti-HIV
activity. A report of the ongoing studies will be communicated in
the near future.
Table 2
Activity of selected nitropyridine derivatives against wild-type HIV-1 RT.
Compound
7b
6.9
7c
8.5
7k
10.4
TMC125
6.5
IC50
(m
M)a
a
IC50: Inhibitory concentration required to inhibit biotin deoxyuridine triphos-
phate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.

J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
535
Fig. 2. Predicted binding modes of compound 7b, 7k in the NNIBP of wild-type HIV-1 RT (PDB code: 3M8Q) showed by PyMOL. (A) Predicted binding mode and molecular docking
of compound 7b; (B) Superimposition of the docked conformations of 7b (pink) and TMC125 (yellow); (C) Predicted binding mode and molecular docking of compound 7k; (D)
Superimposition of the docked conformations of 7k (green) and TMC125 (yellow). Hydrogen bonds are indicated by dashed lines.
Fig. 3. (A) Binding mode of 7k (green) with K103N/Y181C mutant RT (PDB: 3BGR). (B) TMC278 (white) was superposed into the same pocket for comparison. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
In summary, the computational modeling study has helped in
better understanding the interactions between these inhibitors and
the HIV-1 RT and rationalized the SAR conclusions. The molecular
binding model of compounds 7b and 7k provided valuable infor-
mation for design of novel effective anti-HIV drugs. Further opti-
mization of DAPY analogs aimed at improving drug resistance
profiles will take into account these molecular modeling results.
at low micromolar concentrations. Among them, compound 7b was
identified as the most promising candidate with favorable inhibi-
tory activity against the wt HIV-1 (EC50 ¼ 0.056 M, SI ¼ 1251).
m
Selected compounds were also found to inhibit RT. These results
suggested that the nitropyridine is an adequate motif to replace the
pyrimidine ring of DAPY compounds. Taking full use of the valuable
information from SAR analysis and molecular simulation, further
optimization of this series of compounds aimed at improving drug
resistance profiles are ongoing in our lab and will be the subject of
future communications.
4. Conclusion
In the present paper, a series of novel nitropyridine derivatives
were designed as HIV-1 NNRTIs based on the structure-based
scaffold refining approach, and synthesized via a convenient and
efficient method. Furthermore, those compounds were evaluated
for their antiviral activity against of HIV-1 IIIB strain in MT-4 cells,
as well as HIV-1 RT inhibitory activity (some selected compounds).
The bioactivity results showed that most derivatives proved to be
highly effective in inhibiting wt HIV-1 replication with EC50 values
5. Experimental section
5.1. Chemistry
The melting points of the compounds were determined in a
micromelting point apparatus. Infrared spectra were recorded with
a Nexus 470 FT-IR Spectrometer. 1H NMR spectra were determined

536
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
by a Bruker Avance (600 MHz) NMR spectrometer in the indicated
solvents. Chemical shifts were reported in ppm (parts per million)
ether ¼ 1:1) to give compound 4-chloro-5-nitropyridin-2-amine
(5) as a yellow solid [28,29]. Yield: 2.88 g, 41%, mp: 257e259 ꢁC.
d
relative to Tetramethyl Silane (TMS) and signals are expressed as s
(singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). Mass
spectra were recorded with an LC Autosampler Device: Standard
G1313A instrument. All reactions were monitored by thin-layer
chromatography (TLC) and viewed with UV light (254 nm). Flash
column chromatography was performed on a column packed with
silica gel 60 (230e400 mesh). Solvents were reagent grade and,
when necessary, were purified and dried by standard methods.
1H NMR (DMSO-d6, ppm)
d: 8.81 (s, 1H, CH), 7.76 (s, 1H, CH), 6.59 (s,
2H, NH2). ESI-MS: m/z 174.3 (M þ 1), C5H4ClN3O2 [173.00].
5.3. General procedure for the synthesis of target compounds 7ae7r
Compound 5 was dissolved in anhydrous DMF (10 mL) in the
presence of anhydrous K2CO3 (2 eq), followed by the addition of
appropriate substituted phenol (1.1 eq) or substituted phenylamine
(1.1 eq). The reaction mixture was stirred at room temperature
overnight. The solvent was removed under reduced pressure, and
water (20 mL) was added. Extracted with ethyl acetate (2 ꢀ 10 mL),
and the organic phase was washed with saturated sodium chloride
(10 mL), and dried over anhydrous Na2SO4 to give the corre-
sponding crude product, which was purified by flash column
chromatography (EtOAc : petroleum ether ¼ 1:1) to afford com-
pounds 6 [21].
4-(2,4,6-Trisubstituted-phenoxy)-5-nitropyridin-2-amines
(compounds 6), Pd(OAc)2 (5%), Xantphos (10%), cesium carbonate
(2 eq), and para-substituted bromobenzene (1.1 eq) were pre-
mixed in dioxane (10 mL) under a nitrogen atmosphere and the
reaction was heated at reflux under nitrogen overnight. The reac-
tion was poured directly onto a column of silica gel (EtOAc : pe-
troleum ether ¼ 1:1) to give the target compounds [22,23].
5.2. General procedure for the synthesis of key intermidate 4-
chloro-5-nitropyridin-2-amine (5)
Picolinic acid (1) (30.84 g, 250 mmol) was added dropwise to the
stirring thionyl chloride (50 mL). The resulting mixture was heated
at reflux for 3 days giving a clear orange solution. After cooling to
ambient temperature, the solution was concentrated to small vol-
ume and the residue coevaporated with toluene (2 ꢀ 20 mL). The
residue was dissolved in toluene (40 mL) and cooled to 0e5 ꢁC.
Methanol (16 mL) was then added dropwise to the cold reaction
mixture and stirred for 1 h giving a white precipitate. This pre-
cipitate was collected by filtration, washed with toluene
(3 ꢀ 10 mL), and dried at 40 ꢁC for 24 h to give compound methyl 4-
chloropicolinate (2) as a white crystal [25]. Yield: 32.49 g, 76%.
mp:131e132 ꢁC. 1H NMR (DMSO-d6, ppm)
d
: 8.72 (d, 1H, J ¼ 5.4 Hz,
CH), 8.09 (s,1H, CH), 7.84(d,1H, J ¼ 2.0 Hz, CH), 3.85(s, 3H, CH3). ESI-
5.3.1. 4-(4-(2,6-Dimethylphenoxy)-5-nitropyridin-2-ylamino)
benzonitrile (7a)
MS: m/z 172.1 (M þ 1), C7H6ClNO2 [171.01].
Methyl 4-chloropicolinate (2) (32.49 g, 190 mmol) was stirred in
100 mL water for 15 min, then concentrated ammonium hydroxide
(50 mL, 25% w 28%) was added slowly. After the addition of
concentrated ammonium hydroxide was complete, the reaction
mixture was stirred for 1 h at room temperature. The mixture was
filtered and washed with water, then dried at 55e60 ꢁC to give
compound 4-chloropicolinamide (3) as a white solid [26]. Yield:
Yellow solid. Yield: 74%, mp: 240e242 ꢁC. 1H NMR (DMSO-d6,
ppm) d: 9.01 (s, 1H, CH), 7.84 (s, 2H, CH), 7.74 (s, 2H, CH), 7.28 (s, 2H,
CH), 7.24 (s, 1H, CH), 6.01 (s, H, CH), 2.14 (s, 6H, 2CH3). ESI-MS: m/z
361.4 (M þ 1). C20H16N4O3 [360.12].
5.3.2. 4-(4-(Mesityloxy)-5-nitropyridin-2-ylamino)benzonitrile
(7b)
24.61 g, 83%. mp: 160e162 ꢁC. 1H NMR (DMSO-d6, ppm)
d: 8.72 (d,
Yellow solid. Yield: 78%, mp: 251e252 ꢁC. 1H NMR (DMSO-d6,
1H, J ¼ 5.3 Hz, CH), 8.47 (s, H, CH), 8.35 (s, 2H, NH2), 7.79e7.84 (m,
ppm)
d
: 8.99 (s, 1H, CH), 7.85 (d, 2H, J ¼ 9.0 Hz, PhH), 7.74 (d, 2H,
H, CH). ESI-MS: m/z 157.5 (M þ 1), C6H5ClN2O [156.01].
J ¼ 9.0 Hz, PhH), 7.08 (s, 2H, PhH), 6.02 (s, H, CH), 2.31 (s, 3H, CH3),
2.09 (s, 6H, 2CH3). ESI-MS: m/z 375.2 (M þ 1). C21H18N4O3 [374.14].
Sodium hydroxide (39.6 g, 990 mmol) was dissolved in water
(200 mL) and cooled to 0e5 ꢁC. Bromine (10 mL) was slowly
dropped at
0
ꢁC and stirred for half hour. Then 4-
5.3.3. 4-(2-(4-Cyanophenylamino)-5-nitropyridin-4-yloxy)-3,5-
dimethylbenzonitrile (7c)
chloropicolinamide (3) (13.47 g, 86.3 mmol) was added at 0 ꢁC
and stirred for 5 min. The reaction mixture was heated to 100 ꢁC
and maintained at reflux to get a clear solution. The progress of the
reaction was monitored by TLC. After completion of the reaction,
the reaction mixture was cooled to 0e5 ꢁC in 1h, filtered the
precipitated solid, washed with chilled water, and dried at 50e
55 ꢁC to give the corresponding crude product [27], which was
purified by flash column chromatography (EtOAc : petroleum
ether ¼ 1:1) to afford compound 4-chloropyridin-2-amine (4) as a
white solid. Yield: 6.18 g, 56%. mp: 129e131 ꢁC. 1H NMR (DMSO-d6,
Yellow solid. Yield: 69%, mp: 236e238 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 9.04 (s, 1H, CH), 7.87 (s, 2H, PhH), 7.84 (d, 2H, J ¼ 9.0 Hz,
PhH), 7.76 (d, 2H, J ¼ 8.4 Hz, PhH), 5.96 (s, H, CH), 2.18 (s, 6H, 2CH3).
ESI-MS: m/z 386.3 (M þ 1). C21H15N5O3 [385.12].
5.3.4. 4-(4-(4-Bromo-2,6-dimethylphenoxy)-5-nitropyridin-2-
ylamino)benzonitrile (7d)
Light yellow solid. Yield: 63%, mp: 258e259 ꢁC. 1H NMR (DMSO-
d6, ppm)
d
: 9.02 (s, 1H, CH), 7.85 (d, 2H, J ¼ 9.0 Hz, PhH), 7.75 (d, 2H,
ppm)
d
: 7.93 (d,1H, J ¼ 5.5 Hz, CH), 7.74 (s, 2H, NH2), 6.72 (s,1H, CH),
J ¼ 9.0 Hz, PhH), 7.55 (s, 2H, PhH), 6.01 (s, H, CH), 2.13 (s, 6H, 2CH3).
ESI-MS: m/z 439.4 (M þ 1), 441.4 (M þ 3). C20H15BrN4O3 [438.03].
6.65 (d, 1H, J ¼ 1.9 Hz, CH). ESI-MS: m/z 129.4 (M þ 1), C5H5ClN2
[128.01].
4-Chloropyridin-2-amine (4) (5.2 g, 40.6 mmol) was carefully
dissolved in conc. sulfuric acid (25 mL). The reaction mixture was
cooled to 0 ꢁC and then fuming nitric acid (1.5 mL) was added
dropwise with stirring. The reaction mixture was allowed to stand
for overnight at room temperature, or until a drop mixed with
water, gave no precipitate (the nitro-amine is insoluble in dilute
acid). The red reaction mixture was then poured into crushed ice
and bought to pH 5e6 by adding ammonia solution (28%). The
separated yellow precipitate was removed by filtration, washed
well with ice water and dried. The solids were impregnated onto
silica gel and purified by flash chromatography (EtOAc : petroleum
5.3.5. 4-(4-(4-Chloro-2,6-dimethylphenoxy)-5-nitropyridin-2-
ylamino)benzonitrile (7e)
Light yellow solid. Yield: 67%, mp: 260e262 ꢁC. 1H NMR (DMSO-
d6, ppm)
d
: 9.02 (s, 1H, CH), 7.84 (d, 2H, J ¼ 9.0 Hz, PhH), 7.75 (d, 2H,
J ¼ 9.0 Hz, PhH), 7.41 (s, 2H, PhH), 6.01 (s, H, CH), 2.13 (s, 6H, 2CH3).
ESI-MS: m/z 395.2 (M þ 1), 397.3 (M þ 3). C20H15ClN4O3 [394.08].
5.3.6. 4-(4-(2,6-Dimethoxyphenoxy)-5-nitropyridin-2-ylamino)
benzonitrile (7f)
Yellow solid. Yield: 56%, mp: 247e248 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 8.97 (s, 1H, CH), 7.83 (d, 2H, J ¼ 9.0 Hz, PhH), 7.74 (d, 2H,

J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
537
J ¼ 8.4 Hz, PhH), 7.34 (t, H, J ¼ 8.4 Hz, PhH), 6.91 (d, 2H, J ¼ 9.0 Hz,
5.3.15. 4-(4-(2,6-Dibromo-4-methylphenylamino)-5-nitropyridin-
2-ylamino)benzonitrile (7o)
Yellow solid. Yield: 50%, mp: 263e265 ꢁC. 1H NMR (DMSO-d6,
ppm)
J ¼ 9.0 Hz, PhH), 7.55 (s, 2H, PhH), 7.08 (s, H, CH), 2.36 (s, 3H, CH3).
ESI-MS: m/z 501.9 (M þ 1), 503.9 (M þ 3). C19H13Br2N5O2 [500.94].
PhH), 6.12 (s, H, CH), 3.78 (s, 6H, 2OCH3). ESI-MS: m/z 393.2 (M þ 1),
415.3 (M þ 23). C20H16N4O5 [392.11].
d
: 9.03 (s, 1H, CH), 7.89 (d, 2H, J ¼ 8.4 Hz, PhH), 7.81 (d, 2H,
5.3.7. 4-(4-(2,6-Dichlorophenoxy)-5-nitropyridin-2-ylamino)
benzonitrile (7g)
Yellow solid. Yield: 54%, mp: 255e257 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 9.08 (s, 1H, CH), 7.84 (d, 2H, J ¼ 9.0 Hz, PhH), 7.80 (d, 2H,
5.3.16. 4-(4-(2,4-Dibromo-6-fluorophenylamino)-5-nitropyridin-2-
ylamino)benzonitrile (7p)
J ¼ 7.8 Hz, PhH), 7.77 (d, 2H, J ¼ 9.0 Hz, PhH), 7.54 (t, H, J ¼ 8.4 Hz,
PhH), 6.09 (s, H, CH). ESI-MS: m/z 401.2 (M þ 1), 403.3 (M þ 3),
405.4 (M þ 5). C18H10Cl2N4O3 [400.01].
Yellow solid. Yield: 43%, mp: 268e270 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 9.03 (s, 1H, CH), 7.89 (d, 2H, J ¼ 8.4 Hz, PhH), 7.80 (d, 2H,
J ¼ 9.0 Hz, PhH), 7.72 (s, H, CH), 7.66 (d, H, J ¼ 9.0 Hz, PhH), 7.08 (s, H,
CH). ESI-MS: m/z 505.9 (M þ 1), 507.9 (M þ 3), 509.9 (M þ 5).
5.3.8. 4-(5-Nitro-4-(2,4,6-trichlorophenoxy)pyridin-2-ylamino)
benzonitrile (7h)
C18H10Br2FN5O2 [504.92].
Yellow solid. Yield: 51%, mp: 260e261 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 9.08 (s, 1H, CH), 8.08 (s, 2H, PhH), 7.84 (d, 2H, J ¼ 8.4 Hz,
5.3.17. 3,5-Dimethyl-4-(5-nitro-2-(p-tolylamino)pyridin-4-yloxy)
benzonitrile (7q)
PhH), 7.78 (d, 2H, J ¼ 8.4 Hz, PhH), 6.12 (s, H, CH). ESI-MS: m/z 435.2
(M þ 1), 437.2 (M þ 3), 439.2 (M þ 5). C18H9Cl3N4O3 [433.97].
Yellow solid. Yield: 63%, mp: 237e240 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 8.80 (s, 1H, CH), 7.82 (s, 2H, PhH), 7.57 (d, 2H, J ¼ 8.4 Hz,
PhH), 7.48 (d, 2H, J ¼ 9.0 Hz, PhH), 5.44 (s, H, CH), 2.36 (s, 3H, CH3),
2.17 (s, 6H, 2CH3). ESI-MS: m/z 375.2 (M þ 1). C21H18N4O3 [374.14].
5.3.9. 4-(4-(2,6-Dichloro-4-nitrophenoxy)-5-nitropyridin-2-
ylamino)benzonitrile (7i)
Brown yellow solid. Yield: 45%, mp: 215e217 ꢁC. 1H NMR
(DMSO-d6, ppm)
d
: 9.04 (s, 1H, CH), 8,21 (s, 2H, PhH), 7,92 (d, 2H,
5.3.18. 3,5-Dimethyl-4-(5-nitro-2-(4-nitrophenylamino)pyridin-4-
yloxy)benzonitrile (7r)
J ¼ 9.0 Hz, PhH), 7.81 (d, 2H, J ¼ 9.0 Hz, PhH), 7.09 (s, H, CH), 4.24 (s,
H, NH). ESI-MS: m/z 446.1 (M þ 1), 448.2 (M þ 3), 450.1 (M þ 5).
Yellow solid. Yield: 51%, mp: 223e225 ꢁC. 1H NMR (DMSO-d6,
C
18H9Cl2N5O5 [445.00].
ppm)
d
: 8.80 (s, 1H, CH), 8.25 (d, 2H, J ¼ 8.4 Hz, PhH), 7.82 (s, 2H,
PhH), 7.13 (d, 2H, J ¼ 8.4 Hz, PhH), 5.44 (s, H, CH), 2.18 (s, 6H, 2CH3).
ESI-MS: m/z 406.3 (M þ 1). C20H15N5O5 [405.11].
5.3.10. 4-(5-Nitro-4-(2,4,6-tribromophenoxy)pyridin-2-ylamino)
benzonitrile (7j)
Yellow solid. Yield: 47%, mp: 245e247 ꢁC. 1H NMR (DMSO-d6,
5.4. Biological activity
ppm)
d
: 9.09 (s, 1H, CH), 8.27 (s, 1H, PhH), 7.85 (d, 2H, J ¼ 8.4 Hz,
PhH), 7.78 (d, 2H, J ¼ 8.4 Hz, PhH), 6.09 (s, H, CH). ESI-MS: m/z 568.9
5.4.1. In vitro anti-HIV assay
(M þ 3), 571.0 (M þ 5), 572.9 (M þ 7). C18H9Br3N4O3 [565.82].
The anti-HIV assay has been previously described [30,31]. Stock
solutions (10 ꢀ final concentration) of test compounds were added
in 25-mL volumes to two series of triplicate wells in 96-well mi-
5.3.11. 4-(4-(2,6-Dibromo-4-methylphenoxy)-5-nitropyridin-2-
ylamino)benzonitrile (7k)
crotiter plates so as to allow simultaneous evaluation of their ef-
fects on mock- and HIV-infected cells at the beginning of each
experiment. Serial fivefold dilutions of test compounds were made
directly in flat-bottomed 96-well microtiter trays using a Biomek
3000 robot (Beckman instruments, Fullerton, CA). Untreated con-
trol HIV- and mock-infected cell samples were included for each
Light yellow solid. Yield: 59%, mp: 251e253 ꢁC. 1H NMR (DMSO-
d6, ppm)
d: 9.07 (s, 1H, CH), 7.84 (d, 2H, J ¼ 9.0 Hz, PhH), 7.80 (s, 2H,
PhH), 7.77 (d, 2H, J ¼ 9.0 Hz, PhH), 6.07 (s, H, CH), 2.39 (s, 3H, CH3).
ESI-MS: m/z 505.1 (M þ 1), 507.1 (M þ 3). C19H12Br2N4O3[504.13].
sample. HIV-1(IIIB) stock (50 mL) at 100e300 CCID50 (50% cell cul-
5.3.12. 4-(5-Nitro-4-(2,4,6-trifluorophenoxy)pyridin-2-ylamino)
benzonitrile (7l)
ture infectious dose) or culture medium was added to either the
infected or mock-infected wells of the microtiter tray [32]. Mock-
infected cells were used to evaluate the effect of the test com-
pounds on uninfected cells in order to assess the cytotoxicity of the
test compounds. Exponentially growing MT-4 cells were centri-
fuged for 5 min at 1000 rpm and the supernatant was discarded.
Yellow solid. Yield: 69%, mp: 230e231 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 9.04 (s, 1H, CH), 7.85 (d, 2H, J ¼ 8.4 Hz, PhH), 7.77 (d, 2H,
J ¼ 8.4 Hz, PhH), 7.68 (t, 2H, J ¼ 9.0 Hz, PhH), 6.34 (s, H, CH). ESI-MS:
m/z 387.1 (M þ 1). C18H9F3N4O3 [386.06].
The MT-4 cells were resuspended at 6 ꢀ 105 cells/mL, and 50-
mL
5.3.13. 4-(4-(2,6-Dimethylphenylamino)-5-nitropyridin-2-
ylamino)benzonitrile (7m)
volumes were transferred to the microtiter tray wells [33]. Five days
after infection, the viability of mock- and HIV-infected cells was
examined spectrophotometrically by the MTT assay. The MTT assay
is based on the reduction of yellow colored 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros Organics,
Geel, Belgium) by mitochondrial dehydrogenase of metabolically
active cells to a blue-purple formazan that can be measured spec-
trophotometrically. The absorbances were read in an eight-channel
computer-controlled photometer (Multiscan Ascent Reader, Lab-
systems, Helsinki, Finland), at two wavelengths (540 and 690 nm).
All data were calculated using the median OD (optical density)
value of three wells. The 50% cytotoxic concentration (CC50) was
defined as the concentration of the test compound that reduced the
absorbance (OD540) of the mock-infected control sample by 50%.
Yellow solid. Yield: 53%, mp: 223e225 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 8.63 (s, 1H, CH), 7.89 (d, 2H, J ¼ 8.4 Hz, PhH), 7.84 (d, 2H,
J ¼ 8.4 Hz, PhH), 7.76 (d, 2H, J ¼ 8.4 Hz, PhH), 7.71 (d, H, J ¼ 7.8 Hz,
PhH), 6.34 (s, H, CH), 4.24 (s, 2H, 2NH), 2.88 (s, 6H, 2CH3). ESI-MS:
m/z 360.2 (M þ 1). C20H17N5O2 [359.14].
5.3.14. 4-(4-(Mesitylamino)-5-nitropyridin-2-ylamino)benzonitrile
(7n)
Yellow solid. Yield: 50%, mp: 214e216 ꢁC. 1H NMR (DMSO-d6,
ppm)
d
: 8.22 (s, 1H, CH), 7.87 (d, 2H, J ¼ 8.4 Hz, PhH), 7.72 (d, 2H,
J ¼ 9.0 Hz, PhH), 7.00e6.98 (m, 2H, PhH), 6.10 (s, H, CH), 2.34 (s, 3H,
CH3), 2.12 (s, 6H, 2CH3). ESI-MS: m/z 374.2 (M þ 1). C21H19N5O2
[373.15].
The 50% effective concentration (EC50) was defined as the

538
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
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tase (RT) enzyme in the lysis buffer with or without inhibitors. After
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peroxide substrate (ABST) was added to the MTP. A colored reaction
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Acknowledgment
The financial support from the National Natural Science Foun-
dation of China (No. 81273354, 81102320), Key Project of NSFC for
International Cooperation (No. 30910103908), Research Fund for
the Doctoral Program of Higher Education of China (No.
20110131130005), and Natural Science Foundation of Shandong
Province (ZR2009CM016) is gratefully acknowledged.
The authors declare no conflict of interest.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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