Design, synthesis and anti-HIV evaluation of novel diarylpyridine derivatives as potent HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.ejmech.2017.07.012

As a continuation of our efforts to discover and develop “me-better” drugs of DAPYs, novel diarylpyridine derivatives were designed, synthesized and evaluated for their anti-HIV activities in MT-4 cells. The majority of these compounds showed high activity against wild-type HIV-1 strain (IIIB) with EC₅₀ values in the range of 0.04–4.41 μM. Among them, compound 5b2 (EC₅₀ = 0.04 μM, SI = 3963) was the most potent. This compound showed anti-HIV-1_IIIB activity superior than of Nevirapine but still inferior than of Etravirine. Selected compounds were also evaluated for the activity against reverse transcriptase (RT), and most of the compounds exhibited submicromolar IC₅₀ values indicating they are specific RT inhibitors. Preliminary structure-activity relationships and modeling studies of these new analogues provide valuable avenues for future molecular optimization.

Full text of DOI:10.1016/j.ejmech.2017.07.012

Accepted Manuscript
Design, synthesis and anti-HIV evaluation of novel diarylpyridine derivatives as potent
HIV-1 NNRTIs
Zhaoqiang Liu, Ye Tian, Jinghan Liu, Boshi Huang, Dongwei Kang, Erik De Clercq,
Dirk Daelemans, Christophe Pannecouque, Peng Zhan, Xinyong Liu
PII:
S0223-5234(17)30534-2
10.1016/j.ejmech.2017.07.012
EJMECH 9575
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 January 2017
Revised Date: 6 July 2017
Accepted Date: 9 July 2017
Please cite this article as: Z. Liu, Y. Tian, J. Liu, B. Huang, D. Kang, E. De Clercq, D. Daelemans,
C. Pannecouque, P. Zhan, X. Liu, Design, synthesis and anti-HIV evaluation of novel diarylpyridine
derivatives as potent HIV-1 NNRTIs, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.07.012.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and anti-HIV evaluation of
novel diarylpyridine derivatives as potent HIV-1
NNRTIs
1
,a
1,a
b
a
a
c
Zhaoqiang Liu , Ye Tian , Jinghan Liu , Boshi Huang , Dongwei Kang , Erik De Clercq , Dirk
c
c
∗ ,a
∗ ,a
Daelemans , Christophe Pannecouque , Peng Zhan , Xinyong Liu
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, P.R. China;
b
Shool of life science and technology, China Pharmaceutical University, 639 Longmian Avenue, 210009, Nanjing,
P.R. China;
c
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Abstract
As a continuation of our efforts to discover and develop “me-better” drugs of DAPYs, novel
diarylpyridine derivatives were designed, synthesized and evaluated for their anti-HIV activities in
MT-4 cells. The majority of these compounds showed high activity against wild-type HIV-1 strain
(
IIIB) with EC values in the range of 0.04-4.41 ꢀM. Among them, compound 5b2 (EC = 0.04
5
0
5
0
ꢀM, SI = 3963) was the most potent. This compound showed anti-HIV-1IIIB activity superior than
of Nevirapine but still inferior than of Etravirine. Selected compounds were also evaluated for the
activity against reverse transcriptase (RT), and most of the compounds exhibited submicromolar
IC50 values indicating they are specific RT inhibitors. Preliminary structure-activity relationships
and modeling studies of these new analogues provide valuable avenues for future molecular
optimization.
Keywords: HIV-1; NNRTIs; pyridine; bioisosteric principle; drug design
∗
Corresponding authors. Tel: +86-531-88380270; Fax: +86-531-88382731; E-mail: zhanpeng1982@sdu.edu.cn (P.
Zhan), xinyongl@sdu.edu.cn (X. Liu).
1
Zhaoqiang Liu and Ye Tian contributed equally to this article.

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1
. Introduction
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of
highly active antiretroviral therapy (HARRT) due to their excellent potency, remarkable selectivity
and relatively low toxicity. Nevertheless, the unavoidable emergence of drug-resistant viruses and
severe side effects associated with the long-term administration of the clinical approved NNRTIs
demand unremitting efforts to discover novel NNRTIs candidates with different resistance profiles
and improved drug-like properties [1-3].
Among more than 50 different series of NNRTIs that have been reported [3-5],
diarylpyrimidine (DAPY) derivatives, as second generation NNRTIs, have attracted considerable
attention over the past few years due to their potent activity against wild and various mutant
HIV-1 strains. Two DAPY derivatives, Etravirine (TMC125, ETR) and Rilpivirine (TMC278,
RPV), have been approved by FDA for the treatment of HIV infection in 2008 and 2011
respectively (Fig. 1). However, newly reported adverse effects of ETR and RPV [6] along with the
imperfect pharmacokinetics profiles of the most DAPY derivatives [7, 8] encouraged researches
on exploring novel NNRTI agents with better drug-like properties for the treatment of HIV
infections [4]. According to the results obtained from crystallography [9] and structure-activity
relationship (SAR) [10, 11] studies, this central pyrimidine ring of DAPY derivatives is tolerant
for isosteric replacement. In our previous studies [12-18], replacement of the central pyrimidine
ring using structure-based drug design and bioisosteric principle had led to the discovery of novel
series of potent NNRTIs, such as pyridazine derivatives [17] and 2-pyridone derivatives [18],
which are proved to be highly effective in inhibiting HIV-1 replication.
As an extension of these investigations and with the aim to generate novel NNRTIs with
desirable potency and drug-like properties, novel diarylpyridine derivatives were designed via
replacing the pyrimidine core present in DAPY derivatives by the bioisosteric pyridine which is
one of the heterocycles with significant and privileged bioactivity and pharmacokinetic properties
[19]. In the target structures, the active para-cyanoaniline moiety was retained, along with various
substituents at the 5-position of the central pyridine as the left wing of the structure. Besides, we
incorporated a nitro or amino group at the 2-position on the pyridine ring trying to provide a
stronger H-bonding with the lysine 101 (K101) of the viral enzyme RT, as either an H-bond

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acceptor or donor, than the nitrogen on the original pyrimidine ring of TMC125 (Fig. 1). Herein,
we report the synthesis, anti-HIV evaluation and preliminary SAR of these diarylpyridine
derivatives.
Figure 1. FDA approved DAPYs as NNRTIs and the design of novel diarylpyridine derivatives
2
. Results and discussion
2
.1. Chemistry
The newly designed diarylpyridine derivatives were synthesized straightforwardly [20] as
depicted in Scheme 1. Reaction of commercially available 3-amino-5-bromopyridine with propyl
chlorocarbonate afforded propyl (5-bromopyridin-3-yl)carbamate (1). The regioselective nitration
of 1 [21] in the presence of HNO /H SO , resulted in the formation of propyl
3
2
4
(5-bromo-2-nitropyridin-3-yl)carbamate 2 whose hydrolysis, using KOH-EtOH-H2O, afforded
5
-bromo-2-nitropyridin-3-amine (3). Buchwald-Hartwig reaction of 3 with 4-aminobenzonitrile
gave 4-((5-bromo-2-nitropyridin-3-yl)amino)benzonitrile (4). Then derivatives 5a1-10 were
obtained from 4 by Ullman condensation with different substituted phenols, while 5b1-2 were

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obtained by Buchwald-Hartwig reaction with appropriate anilines. Further treatment of 5a1-9 with
SnCl ·2H O gave compounds 6a1-9.
2
2
Scheme 1. Reagents and conditions: i: Propyl chlorocarbonate, NaHCO ; ii: H SO -HNO ; iii:
3
2
4
3
KOH, H O-EtOH; iv: 4-aminobenzonitrile, palladium acetate, Xantphos, Cs CO 90 °C; v: ArOH,
2
2
3,
CuI, picolinic acid, K PO 90 °C or ArNH , palladium acetate, Xantphos, Cs CO , 90 °C; vi:
3
4,
2
2
3
SnCl ·2H O, EtOH, reflux.
2
2
2
.2. Anti-HIV activity evaluation
The newly synthesized diarylpyridine derivatives were evaluated for anti-HIV activity
against wild-type (wt) HIV-1 strain (IIIB), K103N+Y181C double mutant HIV-1 strain (RES056)
and HIV-2 strain (ROD) in MT-4 cells using the MTT method [22-24]. Nevirapine (NVP),
Zidovudine (azidothymidine, AZT), Efavirenz (EFV) and Etravirine were used as reference drugs.
The cytotoxicity of these compounds was determined in parallel. The results, expressed as EC ,
5
0
CC and SI, are illustrated in Table 1.
5
0
Table 1. Activity and cytotoxicity of the title compounds in MT-4 cells.
a
b
Compds
R
1
R
2
EC50 (ꢀM)
CC50 (ꢀM)
SI

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c
IIIB
ROD
>324.4
>333.9
>84.34
>346.9
>318.6
>316.6
>284.6
>286.9
>219.7
>323.6
>6.27
RES056
>324.4
>333.9
>84.34
>346.9
-
(IIIB)
5
5
5
5
5
5
5
5
5
a1
a2
a3
a4
a5
a6
a7
a8
a9
CN
CH
CH
Br
3
4.12 ± 0.55
0.29 ± 0.024
0.0 5± 0.003
2.03 ± 0.36
>318.6
>324.4
>333.9
>79
CH
CH
H
3
3
>1161
1678
3
84.34 ± 19.36
>346.9
CH
3
>171
d
H
OCH
3
>318.6
X1
Cl
Br
Cl
Br
F
CH
CH
Cl
3
1.41 ± 0.42
4.41 ± 1.14
0.31 ± 0.049
0.79 ± 0.19
1.01 ± 0.16
1.06 ± 0.20
0.04 ± 0.006
0.07 ± 0.015
0.14 ± 0.028
0.07 ± 0.02
0.60 ± 0.28
35.73 ± 6.83
0.37 ± 0.14
0.84 ± 0.32
0.26 ± 0.02
0.50 ± 0.14
>316.6
>284.6
>286.9
>219.7
>323.6
>6.27
>316.6
>171
>64
>924
>277
>320
6
3
>284.6
>286.9
Br
>219.7
5
a10
F
>323.6
5
b1
b2
a1
a2
a3
a4
a5
a6
a7
a8
a9
H
CH
CH
CH
CH
Br
3
3
3
3
6.27 ± 0.34
167.7 ± 56.32
39.53 ± 6.97
32.09 ± 4.05
>263.6
5
CH
3
>167.7
>39.53
>32.09
>263.6
>378.3
>344.9
>35.06
>26.68
≥57.68
>27.68
>167.7
≥11.45
>32.09
>263.6
>378.3
>344.9
>35.06
>26.68
>178.6
>27.68
3963
577
227
>3803
>628
>10
95
6
6
6
6
6
6
6
6
6
CN
CH
CH
H
3
3
CH
3
>378.3
H
OCH
3
>344.9
Cl
Br
Cl
Br
CH
CH
Cl
3
35.06 ± 13.26
26.68 ± 0.53
178.6 ± 85.58
27.68 ± 9.05
3
32
680
55
Br

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NVP
EFV
ETR
AZT
0.25 ± 0.066
0.005 ± 0.002
0.004 ± 0.0002
0.006 ± 0.002
-
-
-
-
>15.02
>15.02
>6.34
>4.59
>7.48
>60
0.31 ± 0.16
0.017 ± 0.002
0.011 ± 0.005
>1258
>1128
>1288
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity,
as determined by the MTT method.
b
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
c
SI: selectivity index, the ratio of CC50/EC50
.
d
X1: stands for ≥ 1 or <1.
The majority of the synthesized compounds showed high activities against wt HIV-1 strain
(IIIB) with EC50 values in the range of 0.04-4.41 ꢀM. Totally, 4 compounds were found more
potent than the reference drug NVP (EC50 = 0.25 ꢀM) against wt HIV-1. They are 5a3 (EC50
.05 ꢀM), 6a1 (EC = 0.07 ꢀM), 6a3 (EC = 0.07 ꢀM) and the most effective 5b2 with an EC
=
0
5
0
50
50
value of 0.04 ꢀM, which was about 6 times more potent than NVP. Unfortunately, these
compounds were still less active than other tested control drugs, i.e. EFV (EC = 0.005 ꢀM), ETR
5
0
(
EC = 0.004 ꢀM) and AZT (EC = 0.006 ꢀM). For example, 5b2 was about 10 times less active
50 50
than ETR.
Most compounds showed low cytotoxicity, and 12 out of them had CC50 > 200 ꢀM. Taken
the safety profile into consideration, compounds 5b2 and 6a3 with the largest SI (= 3963 or >
3
807, respectively) were the best two compounds in this series.
The contribution of the 2-pyridine substituent (NH or NO ) to the biological activity was
2
2
firstly determined. Except for 5a3 and 6a3, the compounds of the 6a sub-series (NH at the 2
2
position of the pyridine ring) showed better activity than those of 5a sub-series (NO group at the
2
2
position of the pyridine ring). This result suggests that the hydrogen bond donor/acceptor
properties of the NH group were better than the hydrogen bond acceptor properties of the NO .
2
2
However, the NH moiety contributes more than the NO to the cytotoxicity because most of the
2
2
compounds in 6a sub-series are more toxic than their counterparts in 5a sub-series.

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Next, we turned the attention to the SAR on the left ring, the compounds substituted with
anilines (X = NH) at the 5 position of the pyridine ring (5b1, EC = 1.06 ꢀM; 5b2, EC = 0.04
5
0
50
ꢀM) were more potent than their counterparts with phenols (X= O) (5a4, EC50 = 2.03 ꢀM; 5a2,
EC = 0.29 ꢀM). However, compounds 5b1, 5b2 were also more cytotoxic than 5a4, 5a2.
5
0
Within 5a sub-series (X = O), compound 5a3 (R = CH , R = Br) is the most active. When
1
3
2
R = CH , a clear order of R substituents for anti-HIV-1 activity was observed by comparison:
2
3
1
CH (5a2, EC = 0.29 ꢀM) > Cl (5a6, EC = 1.41 ꢀM) > H (5a4, EC = 2.03 ꢀM) > CN (5a1,
3
50
50
50
EC = 4.12 ꢀM) > Br (5a7, EC = 4.41 ꢀM).
5
0
50
In 6a sub-series, the order of R substituents when R = CH is slightly different: CN (6a1,
1
2
3
EC = 0.07 ꢀM) > CH (6a2, EC = 0.14 ꢀM) > Cl (6a6, EC = 0.37 ꢀM) > H (6a4, EC = 0.60
5
0
3
50
50
50
ꢀ
M) > Br (6a7, EC = 0.84 ꢀM).
50
In both 5a and 6a series, the anti-HIV-1 activity of the compounds was influenced by the
substituents at R position as well. When the R group was fixed, there were some interesting
2
1
aspects related with the influence of R on the activity which might reveal more SAR information.
2
Thus, when R = CH , compounds in which R = Br (5a3, EC = 0.05 ꢀM; 6a3, EC = 0.07 ꢀM)
1
3
2
50
50
showed better activity than those having R = CH (5a2, EC = 0.29 ꢀM; 6a2, EC = 0.14 ꢀM);
2
3
50
50
when R = H, compounds in which R = OCH (5a3, EC > 318.6 ꢀM; 6a3, EC50 = 35.73 ꢀM)
1
2
3
50
were significantly less active than those with R = CH (5a4, EC = 2.03 ꢀM; 6a4, EC50 = 0.60
2
3
50
ꢀ
M); when R = Cl, compounds in which R = Cl (5a8, EC = 0.31 ꢀM; 6a8, EC = 0.26 ꢀM)
1 2 50 50
showed better activity than those with R = CH (5a6, EC = 1.41 ꢀM; 6a6, EC50 = 0.37 ꢀM);
2
3
50
when R = Br, compounds in which R = Br (5a9, EC = 0.79 ꢀM; 6a9, EC = 0.50 ꢀM) showed
1
2
50
50
better activity than those with R = CH (5a7, EC = 4.41 ꢀM; 6a7, EC = 0.84 ꢀM). In general,
2
3
50
50
comparing to CH , Br and Cl appeared to be advantageous substituents for the activity (e.g., 5a3
3
and 6a8), while the bulky methoxy group at this position led to a significant loss of antiviral
potency (e.g., 5a5).
Finally, all the compounds lack potency against the double mutant strain RES056. In addition,
none of the compounds showed inhibition of HIV-2 (ROD), which suggested these derivatives
was specific for HIV-1.

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2
.3. HIV-1 RT inhibition assay
Eleven selected compounds were tested in enzymatic assays against wt HIV-1 RT using
poly(rA)-oligo(dT) as template/primer in order to confirm the binding target [25-27]. Nevirapine
1
6
NVP and EFV were used as reference drugs (Table 2). Except for 6a5, all the compounds
exhibited good inhibitory activity against wt HIV-1 RT with IC values in the range of 0.05-0.91
5
0
ꢀM, which was greater than that of NVP (IC = 2.32 ꢀM), comparable to that of EFV (IC = 0.04
50 50
ꢀM), but still 4 times lower than ETR (IC50 = 0.01 ꢀM). Among them, compound 6a3 exhibited
the highest enzymatic inhibition activity, which was about 45 times higher than that of NVP. The
results suggest that these diarylpyridine derivatives bind to HIV-1 RT and belong to the HIV-1
NNRTIs class. Basically, it reflected some SAR found in cell-based assay, like the NH derivatives
2
are better than the corresponding NO counterpart. The difference between SAR in the two assays
2
may due to the membrane permeability of the compounds.
Table 2. Activity of the title compounds against wt HIV-1 RT polymerization.
a
No.
IC50 (ꢀM)
0.28 ± 0.01
0.65 ± 0.19
0.17 ± 0.05
0.09 ± 0.03
0.05 ± 0.02
0.91 ± 0.54
145.90 ± 93.15
0.31 ± 0
5a3
5b2
6a1
6a2
6a3
6a4
6a5
6a6
6a7
6a8
0.26 ± 0.05
0.16 ± 0.004

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6
a9
0.17 ± 0.01
2.32 ± 0.59
0.04 ± 0.01
NVP
EFV
ETR
b
0.01 ± 0.00
a
IC50: Inhibitory concentration of tested compounds required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP)
incorporation into the HIV-1 RT by 50%. The data were obtained from Rega institute for medical research, KU Leuven, Belgium.
b
The data were obtained from the same laboratory of Prof. Erik De Clercq [28].
2
.4. Molecular modeling analysis
(a)

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(b)
Figure 2. (a) Predicted binding mode of compound 5b2 (orange) in the allosteric site of HIV-1 wt RT (PDB code: 3MEC) in
comparison with the original ligand of this crystal structure ETR (yellow); (b) Predicted binding mode of compound 6a3 (pink)
in the allosteric site of HIV-1 wt RT (PDB code: 3MEC) with the comparison with the original ligand of this crystal structure
ETR (yellow). The docking results are shown by PyMOL.H-bonding interactions are indicated by dashed lines.
To better understand the structure-affinity relationship for this series of derivatives, the best
compound in cell-based HIV-1 infection assay 5b2 and the most potent one in HIV-1 RT inhibition
assay 6a3 was docked into the NNRTIs binding pocket (NNIBP, PDB code: 3MEC) using the
Surflex-Dock of SYBYL-X 2.0. Default parameters were used as described in the Sybyl-2.0
manual unless otherwise specified, and the result was displayed by PyMOL. The proposed binding
modes of these compounds to the NNIBP are shown in Fig. 2.
The results indicated a similar binding mode between 5b2, 6a3 and the original ligand ETR
with NNIBP in reverse transcriptase: 1) The left substituted aniline of 5b2 or phenoxy ring of 6a3
all fitted in the hydrophobic cavity surrounded by P95, Y181, Y188, F227 and W229, especially

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having a π-π stacking with residue Y188 of RT. Furthermore, the imperfect adaption of the relative
bulky OCH of 5a5 and 6a5 as R into the space limited pocket displaced by the modeling may be
3
2
the reason why OCH led to a decrease in the potency. 2) The right 4-cyanoaniline was located in
3
another pocket and interacted with residues V106, L234, P236 and Y318. 3) The NH connecting
the right aniline and the central ring has a strong H-bond with the key residue K101 of RT. Further
analysis showed the differences of the polar interactions between the central rings of 5b2 and 6a3
and K101 of RT. The O atom of the nitro group of 5b2 formed a H-bond with the NH on the main
2
chain of K101 and it also had a polar interaction with the C=O of K101. With respect to 6a3, the
NH can also form a H-bond with K101. Since these two groups are out of the pyridine ring, they
2
are closer to K101 than the N atom of pyrimidine of ETR, which may lead to enhanced polar
interactions with the binding pocket. However, a H-bond between E138 and ETR was missing
from 5b2 or 6a3 causing the observed decreased in potency.
4
. Conclusion
In summary, a series of novel diarylpyridine derivatives as novel NNRTIs have been
synthesized and evaluated for their bioactivities against HIV-1 (IIIB strain and K103N+Y181C
double-mutated strains) and HIV-2 (ROD) in MT-4 cells, as well as against HIV-1 RT activity.
These compounds have been designed by a structure-based bioisosterism strategy. Screening
results indicated that some compounds showed excellent activity against wt HIV-1 at
submicromolar concentrations ranging from 40 nM to 4.41 µM. Taking full advantage of the
valuable information from SARs analysis, further optimization of this series of compounds are
ongoing in our lab and will be reported in due course.
5
. Experimental section
5
.1. Chemistry
All melting points were determined on a micromelting point apparatus and are uncorrected.
1
13
H-NMR and C-NMR spectra were recorded on a Bruker AV-400 spectrometer using DMSO-d6
as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts are reported in parts
per million (δ), and signals are expressed as s (singlet), d (doublet), t (triplet), q (quartet) or m
(multiplet). Mass spectra were taken on a LC Autosampler Device: Standard G1313A instrument.

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TLC was performed on Silica Gel GF254 for TLC (Merck) and spots were visualized by iodine
vapors or by irradiation with UV light (254 nm). Flash column chromatography was performed on
column packed with Silica Gel 60 (200-300 mesh). Solvents were reagent grade and, when
necessary, were purified and dried by standard methods. Concentration of the reaction solutions
involved the use of rotary evaporator at reduced pressure.
5
.1.1. Propyl (5-bromopyridin-3-yl)carbamate (1)
To a mixture of 3-amino-5-bromopyridine (5.0 g, 28.9 mmol) and NaHCO (7.3 g, 86.9
3
mmol) in THF (20 mL), propyl chlorocarbonate (9.3 mL, 86.9 mmol) was added dropwise at 0 °C.
After stirring for 30 min, THF was removed under reduced pressure. Water (30 mL) was added
and extracted with ethyl acetate (3 × 10 mL). The combined organic phase was dried over
anhydrous Na SO and filtered. The solvent was removed under reduced pressure and the crude
2
4
propyl (5-bromopyridin-3-yl)carbamate (1) was obtained and used in the next step without further
purification.
5
.1.2. Propyl (5-bromo-2-nitropyridin-3-yl)carbamate (2)
To a mixture of concentrated H SO (15 mL) and propyl (5-bromopyridin-3-yl)carbamate (1),
2
4
fuming HNO (5 mL) was added dropwise at 0 °C. After stirring at 0 °C for 15 min, the mixture
3
was stirred at rt overnight. The mixture was poured into iced water, basified with KOH to pH > 8
and extracted with ethyl acetate (3 × 10 mL). The combined organic phase was dried over
anhydrous Na SO , filtered and concentrated in vacuum. The residue was purified by silica gel
2
4
column chromatography using ethyl acetate-petroleum ether. Pure fractions were collected and
concentrated, giving the desired compound 2 as a yellow solid. Total yield of the above two steps:
1
8
0.8%, mp: 74-76°C. H NMR (400 MHz, DMSO-d6, ppm) δ: 10.11 (s, 1H), 8.48-8.46 (m, 2H),
.08 (t, 2H, J = 6.7 Hz, OCH ), 1.64 (sext, 2H, J = 7.3 Hz, CH ), 0.93 (t, 3H, J = 7.4 Hz, CH ).
4
2
2
3
1
3
C-NMR (100 MHz, DMSO-d6, ppm) δ: 153.76, 148.01, 143.82, 136.15, 129.79, 124.92, 67.64,
2
2.15, 10.56.
5
.1.3. 5-Bromo-2-nitropyridin-3-amine (3)
To the reaction mixture of intermediate 2 (0.55g, 9.8 mmol) in 1.5 mL EtOH, KOH (0.55 g,
9
.8 mmol) in 8 mL H O was added. After stirring at 90 °C for 1 h, the mixture was stirred at rt for
2

ACCEPTED MANUSCRIPT
another 1 h. Water (20 mL) was added, and the precipitate was collected by filtration and dried
under reduced pressure to give 5-bromo-2-nitropyridin-3-amine (3) as a yellow solid. Yield:
1
9
4.1%, mp: 183-185 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 7.84 (d, J = 2.0 Hz, 1H,
1
3
pyridine-H), 7.77 (d, 1H, J = 2.0 Hz, pyridine-H), 7.45 (s, 2H, NH2). C-NMR (100 MHz,
DMSO-d6, ppm) δ: 142.53, 138.93, 136.06, 130.27, 126.43.
5
.1.4. 4-((5-Bromo-2-nitropyridin-3-yl)amino)benzonitrile (4)
Palladium acetate (0.1541 g, 0.69 mmol) and Xantphos (0.3977 g, 0.69mmol) were dissolved
in 15 mL dioxane and stirred at ambient temperature for 15 min. 4-Aminobenzonitrile (3.3 g, 14.4
mmol), intermediate 3 (3.0 g, 13.8 mmol) and Cs CO (6.7 g, 20.6 mmol) were added. Then the
2
3
reaction flask was evacuated and backfilled with nitrogen for three times, the resulting mixture
was stirred at 100 °C for 12 h. The mixture was filtrated, and the filtrate was collected and
concentrated. The crude residue was purified by silica gel column chromatography to provide the
1
title product 4 as a yellow solid. Yield: 78.6%, mp: 241-244 °C. H NMR (400 MHz, DMSO-d6,
ppm) δ: 9.43 (s, 1H, NH), 8.27 (d, J = 1.9 Hz, pyridine-H), 8.19 (d, J = 2.0 Hz, pyridine-H), 7.80
1
3
(
d, J = 8.7 Hz, Ph-H), 7.41 (d, J = 8.7 Hz, Ph-H). C-NMR (100 MHz, DMSO-d6, ppm) δ:
1
45.33, 144.95, 140.96, 135.15, 134.27 (2 × C), 132.30, 126.21, 120.77 (2 × C), 119.54, 105.37.
-
ESI -MS: m/z 317.2 (M-1), 319.2 (M+1). C H BrN O (317.98).
1
2
7
4
2
5
.1.5. General procedure for the synthesis of 5a1-10.
To a solution of different substituted phenols (1.03 mmol), 2-picolinic acid (0.0231 g, 0.19
mmol), CuI (0.0179 g, 0.094mmol) and K PO (0.4 g, 1.9 mmol) in DMSO (2 mL), intermediate 4
3
4
(0.3 g, 0.94 mmol) were added. The reaction flask was evacuated and backfilled with nitrogen for
three times, and the resulting mixture was stirred at 90 °C for 24 h. Water (20 mL) was added, the
precipitate was collected by filtration and dried under reduced pressure. The precipitate was
purified by silica gel column chromatography to provide the title products 5a1-10.
5
.1.5.1. 4-((5-((4-cyanophenyl)amino)-6-nitropyridin-3-yl)oxy)-3,5-dimethylbenzonitrile (5a1)
1
Yellow solid, yield: 40.1%, mp: 238-240 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.45 (s,
1
H, NH), 7.84 (d, 1H, J = 2.5 Hz, pyridine-H), 7.75 (s, 2H, Ph-H), 7.73 (d, 2H, J = 8.8 Hz, Ph-H),

ACCEPTED MANUSCRIPT
7
.30 (d, 2H, J = 8.7 Hz, Ph-H), 7.01 (d, 1H, J = 2.5 Hz, pyridine-H ), 2.15 (s, 6H, 2 × CH3).
C-NMR (100 MHz, DMSO-d6, ppm) δ: 157.28, 153.43, 144.90, 141.11, 136.67, 134.05 (2 × C),
33.87 (2 × C), 132.95 (2 × C), 129.01, 120.83 (2 × C), 119.43, 118.74, 112.85, 109.61, 105.32,
1
3
1
1
+
5.96 (2 × C). ESI -MS: m/z 386.5 (M+1), 403.6 (M+18), 408.5 (M+23). C H N O (385.12).
2
1
15
5
3
5
.1.5.2. 4-((5-(mesityloxy)-2-nitropyridin-3-yl)amino)benzonitrile (5a2)
1
Yellow solid, yield: 51.2%, mp: 191-193 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.45 (s,
1
H, NH), 7.85 (d, 1H, J = 2.4 Hz, pyridine-H), 7.72 (d, 2H, J = 8.7 Hz, Ph-H), 7.29 (d, 2H, J = 8.7
Hz, Ph-H), 6.98 (s, 2H, Ph-H), 6.89 (d, 1H, J = 2.5 Hz, pyridine-H), 2.24 (s, 3H, CH ), 2.06 (s, 6H,
3
1
3
2
1
1
× CH3). C-NMR (100 MHz, DMSO-d6, ppm) δ: 158.51, 147.57, 144.87, 140.58, 136.78,
35.90, 134.01 (2 × C), 130.33 (2 × C), 130.10 (2 × C), 129.37, 120.89 (2 × C), 119.40, 111.95,
+
05.27, 20.75, 16.03 (2 × C). ESI -MS: m/z 375.4 (M+1), 397.5 (M+23). C H N O (374.14).
2
1
18
4
3
5
.1.5.3. 4-((5-(2,6-dibromo-4-methylphenoxy)-2-nitropyridin-3-yl)amino)benzonitrile (5a3)
1
Yellow solid, yield: 44.3%, mp: 200-203 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.47 (s,
1
H, NH), 7.93 (d, 1H, J = 2.5 Hz, pyridine-H), 7.74 (d, 2H, J = 8.7 Hz, Ph-H), 7.68 (s, 2H, Ph-H),
1
3
7
.28 (d, 2H, J = 8.8 Hz, Ph-H), 7.08 (d, 1H, J = 2.5 Hz, pyridine-H), 2.33 (s, 3H, CH ). C-NMR
3
(100 MHz, DMSO-d6, ppm) δ: 156.87, 144.90, 144.87, 141.49, 140.59, 136.21, 134.31 (2 × C),
-
1
5
34.12 (2 × C), 129.32, 120.59 (2 × C), 119.42, 116.94, 113.46, 105.31, 20.22. ESI -MS: m/z
03.2 (M+1), 505.2 (M+3). C H Br N O (501.93).
1
9
12
2
4
3
5
.1.5.4. 4-((5-(2,6-dimethylphenoxy)-2-nitropyridin-3-yl)amino)benzonitrile (5a4)
1
Yellow solid, yield: 47.3%, mp: 190-192 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.45 (s,
1
H, NH), 7.88 (d, 1H, J = 2.4 Hz, pyridine-H), 7.71 (d, 2H, J = 8.7 Hz, Ph-H), 7.29 (d, 2H, J = 8.8
Hz, Ph-H), 7.20-7.12 (m, 3H, Ph-H), 6.87 (d, 1H, J = 2.5 Hz, pyridine-H), 2.10 (s, 6H, 2 × CH3).
1
3
C-NMR (100 MHz, DMSO-d6, ppm) δ: 158.29, 149.69, 144.78, 140.54, 136.84, 133.99 (2 × C),
1
30.55 (2 × C), 129.91 (2 × C), 129.37, 126.88, 120.95 (2 × C), 119.40, 111.73, 105.33, 16.10 (2 ×
+
C). ESI -MS: m/z 361.4 (M+1), 383.4 (M+23). C H N O (360.12).
2
0
16
4
3
5
.1.5.5. 4-((5-(2,6-dimethoxyphenoxy)-2-nitropyridin-3-yl)amino)benzonitrile (5a5)
1
Yellow solid, yield: 43.4%, mp: 218-222 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.42 (s,

ACCEPTED MANUSCRIPT
1
H, NH), 7.92 (d, 1H, J = 2.5 Hz, pyridine-H), 7.75 (d, 2H, J = 8.7 Hz, Ph-H), 7.26 (d, 2H, J = 8.8
Hz, Ph-H), 7.25 (t, 1H, J = 8.5 Hz, Ph-H), 6.93 (d, 1H, J = 2.5 Hz, pyridine-H), 6.82 (d, 2H, J =
1
3
8
.5 Hz, Ph-H), 3.78 (s, 6H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 158.65, 152.58 (2
3
×
C), 144.92, 140.57, 136.34, 134.07 (2 × C), 129.72, 127.61, 120.52 (2 × C), 119.43, 111.95,
-
1
05.94 (2 × C), 105.16, 56.59 (2 × C). ESI -MS: m/z 391.5 (M-1). C H N O (392.11).
2
0
16
4
5
5
.1.5.6. 4-((5-(4-chloro-2,6-dimethylphenoxy)-2-nitropyridin-3-yl)amino)benzonitrile (5a6)
1
Yellow solid, yield: 44.4%, mp: 230-233 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.45 (s,
1
H, NH), 7.86 (d, 1H, J = 2.4 Hz, pyridine-H), 7.73 (d, 2H, J = 8.7 Hz, Ph-H), 7.30 (d, 2H, J = 8.6
Hz, Ph-H), 7.30 (s, 2H, Ph-H), 6.95 (d, 1H, J = 2.5 Hz, pyridine-H), 2.10 (s, 6H, 2 × CH3).
1
3
C-NMR (100 MHz, DMSO-d6, ppm) δ: 157.88, 148.62, 140.90, 140.86, 136.72, 134.03 (2 × C),
1
33.10 (2 × C), 130.54, 129.37 (2 × C), 129.18, 120.88 (2 × C), 119.42, 112.39, 105.30, 16.00 (2 ×
+
C). ESI -MS: m/z 395.3 (M+1), 397.4 (M+3), 417.5 (M+23). C H ClN O (394.08).
2
0
15
4
3
5
.1.5.7. 4-((5-(4-bromo-2,6-dimethylphenoxy)-2-nitropyridin-3-yl)amino)benzonitrile (5a7)
1
Yellow solid, yield: 47.2%, mp: 237-240 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.45 (s,
1
H, NH), 7.86 (d, 1H, J = 2.4 Hz, pyridine-H), 7.73 (d, 2H, J = 8.7 Hz, Ph-H), 7.43 (s, 2H, Ph-H),
.30 (d, 2H, J = 8.7 Hz, Ph-H), 6.95 (d, 1H, J = 2.4 Hz, pyridine-H), 2.10 (s, 6H, 2 × CH3).
C-NMR (100 MHz, DMSO-d6, ppm) δ:157.80, 149.13, 144.90, 140.85, 136.71, 134.02 (2 × C),
7
1
3
1
33.47 (2 × C), 132.20 (2 × C), 129.18, 120.88 (2 × C), 119.42, 118.92, 112.42, 105.31, 15.90 (2 ×
-
C). ESI -MS: m/z 437.4 (M-1). C H BrN O (438.03).
2
0
15
4
3
5
.1.5.8. 4-((2-nitro-5-(2,4,6-trichlorophenoxy)pyridin-3-yl)amino)benzonitrile (5a8)
1
Yellow solid, yield: 54.9%, mp: 213-216 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.47 (s,
1
7
1
1
H, NH), 7.97 (d, 1H, J = 2.5 Hz, pyridine-H), 7.93 (s, 2H, Ph-H), 7.76 (d, 2H, J = 8.8 Hz, Ph-H),
1
3
.33-7.30 (m, 3H). C-NMR (100 MHz, DMSO-d6, ppm) δ: 156.52, 145.03, 144.50, 141.92,
36.17, 134.11 (2 × C), 132.35, 130.23 (2 × C), 129.40, 128.96, 120.48 (2 × C), 119.46, 114.15,
-
05.23. ESI -MS: m/z 433.4 (M-1), 435.3 (M+1), 437.3 (M+3). C H C N O (433.97).
1
8
9
l3
4
3
5
.1.5.9. 4-((2-nitro-5-(2,4,6-tribromophenoxy)pyridin-3-yl)amino)benzonitrile (5a9)
1
Yellow solid, yield: 44.9%, mp: 235-240 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.47 (s,

ACCEPTED MANUSCRIPT
1
H, NH), 8.14 (s, 2H, Ph-H), 7.96 (d, 1H, J = 2.5 Hz, pyridine-H), 7.75 (d, 2H, J = 8.7 Hz, Ph-H),
1
3
7
.29 (d, 2H, J = 8.8 Hz, Ph-H), 7.23 (d, 1H, J = 2.5 Hz, pyridine-H). C-NMR (100 MHz,
DMSO-d6, ppm) δ: 156.37, 147.01, 145.02, 141.81, 136.15, 136.09, 134.13 (2 × C), 129.31,
-
1
5
20.88, 120.43 (2 × C), 119.45, 118.68, 114.16, 105.22. ESI -MS: m/z 565.2 (M-1), 567.2 (M+1),
71.2 (M+5). C H Br N O (565.82).
1
8
9
3
4
3
5
.1.5.10. 4-((2-nitro-5-(2,4,6-trifluorophenoxy)pyridin-3-yl)amino)benzonitrile (5a10)
1
Yellow solid, yield: 44.0%, mp: 218-221 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.48 (s,
1
H, NH), 8.04 (d, 1H, J = 2.5 Hz, pyridine-H), 7.77 (d, 2H, J = 8.7 Hz, Ph-H), 7.56-7.51 (m, 2H,
1
3
Ph-H), 7.45 (d, 1H, J = 2.5 Hz, pyridine-H), 7.34 (d, 2H, J = 8.7 Hz, Ph-H). C-NMR (100 MHz,
DMSO-d6, ppm) δ: 159.40 (dt, J = 238.5 Hz, J = 14.6 Hz), 157.32, 155.33 (ddd, J = 248.8 Hz,
1
2
1
J = 15.9 Hz, J = 6.3 Hz), 144.98, 141.86, 136.25, 134.11 (2 × C), 128.87, 126.60 (td, J = 15.0
2
3
1
-
Hz, J = 5.5 Hz), 120.62 (2 × C), 119.47, 114.15, 105.31, 102.93 (t, J = 27.3 Hz,). ESI -MS: m/z
2
3
85.3 (M-1). C H F N O (386.06).
18 9 3 4 3
5
.1.6. General procedure for the synthesis of 5b1 and 5b2.
Palladium acetate (0.0176 g, 0.079 mmol) and Xantphos (0.0453 g, 0.079mmol) were
dissolved in 20 mL dioxane and stirred at ambient temperature for 15 min. Intermediate 4 (0.50 g,
.57 mmol), substituted aniline (1.73 mmol) and Cs CO (0.77 g, 236 mmol) were added. Then
1
2
3
the reaction flask was evacuated and backfilled with nitrogen for three times, the resulting mixture
was stirred at 100 °C for 12 h. The reaction mixture was filtrated, and the filtrate was collected
and concentrated. The crude residue was purified by silica gel column chromatography to provide
the title product 5b1-2.
5
.1.6.1. 4-((5-((2,6-dimethylphenyl)amino)-2-nitropyridin-3-yl)amino)benzonitrile (5b1)
1
Yellow solid, yield: 76.4%, mp: 251-254 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.44 (s,
1
H, NH), 8.87 (s, br, 1H), 7.72 (d, 2H, J = 8.1 Hz, Ph-H), 7.53 (s, br, 1H), 7.32 (d, 2H, J = 6.8 Hz,
1
3
Ph-H), 7.18-7.11 (m, 3H, Ph-H), 6.31 (s, br, 1H), 2.15 (s, 6H, 2 × CH3). C-NMR (100 MHz,
DMSO-d6, ppm) δ: 149.11, 144.84, 137.89, 136.68, 136.14 (2 × C), 135.71, 133.94 (2 × C),
+
1
3
29.07 (2 × C), 127.66, 121.40 (2 × C), 119.46, 105.05, 18.19 (2 × C). ESI -MS: m/z 360.5 (M+1)
82.8 (M+23). C H N O (359.14).
20
17
5
2

ACCEPTED MANUSCRIPT
5
.1.6.2. 4-((5-(mesitylamino)-2-nitropyridin-3-yl)amino)benzonitrile (5b2)
1
Yellow solid, yield: 67.2%, mp: 217-222 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.42 (s,
1
H, NH), 8.78 (s, br, 1H), 7.73 (d, 2H, J = 5.9 Hz), 7.31-7.23 (m, 3H), 6.96 (s, 2H, Ph-H), 6.07 (s,
1
3
br, 1H), 2.23 (s, 3H, CH ), 2.10 (s, 6H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ:149.37,
3
3
1
44.95, 137.84, 136.77, 136.68, 135.85 (2 × C), 133.96 (2 × C), 133.07, 129.65 (2 × C), 121.30,
+
1
19.47, 104.96, 20.96, 18.10 (2 × C).ESI -MS: m/z 374.5 (M+1), 396.4 (M+23). C H N O
2
1
19
5
2
(373.15).
5
.1.7. General procedure for the synthesis of 6a1-9.
To a solution of derivatives 5a1-9 (0.45 mmol) in EtOH (5 mL), SnCl .2H O (2.25 mmol)
2
2
was added. The reaction flask was evacuated and backfilled with nitrogen for three times, the
reaction mixture was stirred at reflux overnight. After removal of the solvent under reduced
pressure, water (15 mL) was added. The mixture was alkalized with K CO to pH >8 and
2
3
extracted with ethyl acetate (3×10 mL). The combined organic phase was dried over anhydrous
Na SO , filtered and concentrated in vacuum. Recrystallization from ethyl acetate afforded the
2
4
title products 6a1-9.
5
.1.7.1. 4-((6-amino-5-((4-cyanophenyl)amino)pyridin-3-yl)oxy)-3,5-dimethylbenzonitrile (6a1)
1
White solid, yield: 70.0%, mp: 238-240 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 8.16 (s,
1
6
6
H, NH), 7.67 (s, 2H, Ph-H), 7.56 (d, 2H, J = 8.7 Hz, Ph-H), 7.40 (d, 1H, J = 2.7 Hz, pyridine-H),
.91 (d, 1H, J = 2.7 Hz, pyridine-H), 6.76 (d, 2H, J = 8.8 Hz, Ph-H), 5.56 (s, 2H, NH ), 2.15 (s,
2
1
3
H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 155.47, 150.96, 149.33, 145.72, 133.97
3
(2 × C), 133.62 (2 × C), 133.19 (2 × C), 130.46, 121.61, 120.42, 119.07, 119.03, 114.82 (2 × C),
+
1
08.27, 99.52, 16.29 (2 × C). ESI -MS: m/z 356.5 (M+1). C H N O (355.14).
2
1
17
5
5
.1.7.2. 4-((2-amino-5-(mesityloxy)pyridin-3-yl)amino)benzonitrile (6a2)
1
Light yellow solid, yield: 64.2%, mp: 210-212 °C. H NMR (400 MHz, DMSO-d6, ppm) δ:
8
.15 (s, 1H, NH), 7.55 (d, 2H, J = 8.8 Hz, Ph-H), 7.42 (d, 1H, J = 2.7 Hz, pyridine-H), 6.92 (s, 2H,
Ph-H), 6.78 (d, 1H, J = 2.7 Hz, pyridine-H), 6.74 (d, 2H, J = 8.8 Hz, Ph-H), 5.44 (s, 2H, NH2),
1
3
2
.23 (s, 3H, CH ), 2.06 (s, 6H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 150.40, 149.49,
3 3

ACCEPTED MANUSCRIPT
1
49.15, 146.65, 134.49, 133.96 (2 × C), 130.61, 130.55, 130.08 (2 × C), 121.38, 120.44, 118.68,
+
1
14.68 (2 × C), 99.34, 20.79, 16.37 (2 × C). ESI -MS: m/z 345.4 (M+1). C H N O (344.16).
2
1
20
4
5
.1.7.3. 4-((2-amino-5-(2,6-dibromo-4-methylphenoxy)pyridin-3-yl)amino)benzonitrile (6a3)
1
White solid, yield: 66.5%, mp: 229-230 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 8.17 (s,
1
6
6
1
2
H, NH), 7.61 (s, 2H, Ph-H), 7.56 (d, 2H, J = 8.7 Hz, Ph-H), 7.46 (d, 1H, J = 2.7 Hz, pyridine-H),
.86 (d, 1H, J = 2.7 Hz, pyridine-H), 6.77 (d, 2H, J = 8.8 Hz, Ph-H), 5.58 (s, 2H, NH ), 2.31 (s,
2
1
3
H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 151.08, 149.37, 146.65, 145.43, 139.20,
3
34.10 (2 × C), 133.99 (2 × C), 130.92, 121.25, 120.41, 119.09, 117.77, 114.75 (2 × C), 99.51,
+
0.16. ESI -MS: m/z 475.1 (M+1). C H Br N O (471.95).
1
9
14
2
4
5
.1.7.4. 4-((2-amino-5-(2,6-dimethylphenoxy)pyridin-3-yl)amino)benzonitrile (6a4)
1
White solid, yield: 65.5%, mp: 224-226 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 8.15 (s,
1
6
6
H, NH), 7.55 (d, 2H, J = 8.6 Hz, Ph-H), 7.42 (d, 1H, J = 2.6 Hz, pyridine-H), 7.14-7.04 (m, 3H),
.80 (d, 1H, J = 2.5 Hz, pyridine-H), 6.74 (d, 2H, J = 8.7 Hz, Ph-H), 5.46 (s, 2H, NH ), 2.11 (s,
2
1
3
H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 151.38, 150.42, 149.41, 146.48, 1333.96
3
(2 × C), 131.05, 130.46, 129.61 (2 × C), 125.64, 121.44, 120.43, 118.58, 114.72 (2 × C), 99.39,
+
1
6.45 (2 × C). ESI -MS: m/z 331.5 (M+1). C H N O (330.15).
2
0
18
4
5
.1.7.5. 4-((2-amino-5-(2,6-dimethoxyphenoxy)pyridin-3-yl)amino)benzonitrile (6a5)
1
White solid, yield: 68.9%, mp: 205-207 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 8.13 (s,
1
H, NH), 7.56 (d, 2H, J = 8.7 Hz, Ph-H), 7.51 (d, 1H, J = 2.7 Hz, pyridine-H), 7.15 (t, 1H, J = 8.4
Hz, Ph-H), 6.79 (d, 1H, J = 2.7 Hz, pyridine-H), 6.80-6.73 (m, 4H), 5.42 (s, br, 2H, NH ), 3.74 (s,
2
1
3
6
H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 153.46, 150.24, 149.54, 147.29, 133.96
3
(2 × C), 132.27, 131.12, 126.12, 120.87, 120.46, 119.00, 114.52 (2 × C), 106.02 (2 × C), 99.26,
+
5
6.43 (2 × C). ESI -MS: m/z 363.4 (M+1). C H N O (362.14).
2
0
18
4
3
5
.1.7.6. 4-((2-amino-5-(4-chloro-2,6-dimethylphenoxy)pyridin-3-yl)amino)benzonitrile (6a6)
1
Light yellow solid, yield: 76.4%, mp: 227-231 °C. H NMR (400 MHz, DMSO-d6, ppm) δ:
8
.16 (s, 1H, NH), 7.56 (d, 2H, J = 8.6 Hz, Ph-H), 7.42 (d, 1H, J = 2.4 Hz, pyridine-H), 7.23 (s, 2H,
Ph-H), 6.86 (d, 1H, J = 2.4 Hz, pyridine-H), 6.76 (d, 2H, J = 8.6 Hz, Ph-H), 5.50 (s, 2H, NH2),

ACCEPTED MANUSCRIPT
1
3
2
.11 (s, 6H, 2 × CH3). C-NMR (100 MHz, DMSO-d6, ppm) δ:150.66, 150.33, 149.38, 146.14,
1
33.96 (2 × C), 133.49 (2 × C), 130.36, 129.22, 129.06 (2 × C), 121.52, 120.43, 118.74, 114.77 (2
+
×
C), 99.45, 16.31 (2 × C). ESI -MS: m/z 365.4 (M+1), 367.3 (M+3). C H ClN O (364.11).
2
0
17
4
5
.1.7.7. 4-((2-amino-5-(4-bromo-2,6-dimethylphenoxy)pyridin-3-yl)amino)benzonitrile (6a7)
1
Light yellow solid, yield: 64.4%, mp: 222-225 °C. H NMR (400 MHz, DMSO-d6, ppm) δ:
8
.16 (s, 1H, NH), 7.56 (d, 2H, J = 8.7 Hz, Ph-H), 7.41 (d, 1H, J = 2.7 Hz, pyridine-H), 7.36 (s, 2H,
Ph-H), 6.85 (d, 1H, J = 2.7 Hz, pyridine-H), 6.75 (d, 2H, J = 8.8 Hz, Ph-H), 5.50 (s, 2H, NH2),
1
3
2
1
.10 (s, 6H, 2 × CH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 150.85, 150.68, 149.39, 146.07,
3
33.96 (2 × C), 133.92 (2 × C), 131.99 (2 × C), 130.40, 121.52, 120.43, 118.78, 117.55, 114.78 (2
+
×
C), 99.45, 16.23 (2 × C). ESI -MS: m/z 409.5 (M+1). C H BrN O (408.06).
2
0
17
4
5
.1.7.8. 4-((2-amino-5-(2,4,6-trichlorophenoxy)pyridin-3-yl)amino)benzonitrile(6a8)
1
White solid, yield: 65.4%, mp: 245-250 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 8.19 (s,
1
7
H, NH), 7.83 (s, 2H, Ph-H), 7.57 (d, 2H, J = 8.7 Hz, Ph-H), 7.51 (d, 1H, J = 2.8 Hz, pyridine-H),
1
3
.02 (d, 1H, J = 2.7 Hz, pyridine-H), .6.78 (d, 2H, J = 8.8 Hz, Ph-H), 5.65 (s, 2H, NH ). C-NMR
2
(100 MHz, DMSO-d6, ppm) δ: 151.52, 149.36, 146.51, 145.35, 133.98 (2 × C), 130.94, 130.80,
+
1
29.96, 129.95, 121.37, 120.42, 119.41, 114.78 (2 × C), 99.54. ESI -MS: m/z 405.4 (M+1) 407.4
(
M+3) 409.4 (M+5). C H Cl N O (404.00).
18 11 3 4
5
.1.7.9. 4-((2-amino-5-(2,4,6-tribromophenoxy)pyridin-3-yl)amino)benzonitrile (6a9)
1
Light yellow solid, yield: 71.3%, mp: 255-258 °C. H NMR (400 MHz, DMSO-d6, ppm) δ:
8
.18 (s, 1H, NH), 8.06 (s, 2H, Ph-H), 7.56 (d, 2H, J = 8.7 Hz, Ph-H), 7.47 (d, 1H, J = 2.6 Hz,
pyridine-H), 6.94 (d, 1H, J = 2.6 Hz, pyridine-H), .6.78 (d, 2H, J = 8.7 Hz, Ph-H), 5.62 (s, 2H,
13
NH ). C-NMR (100 MHz, DMSO-d6, ppm) δ: 151.30, 149.36, 148.82, 145.06, 135.92 (2 × C),
2
+
1
33.99 (2 × C), 130.87, 121.34, 120.42, 119.40 (2 × C), 119.30, 114.79 (2 × C), 99.52. ESI -MS:
m/z 539.2 (M+1) 541.2 (M+3) 543.1 (M+5). C H Br N O (535.85).
1
8
11
3
4
5
5
.2. Biological activity
.2.1. In vitro anti-HIV assay

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The methodology of the anti-HIV assay has been previously described [12, 22, 23]. Stock
solutions (10 × final concentration) of test compounds were added in 25 ꢀL volumes to two series
of triplicate wells to allow simultaneous evaluation of their effects 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 control HIV- and mock-infected cell samples were
included for each sample.
HIV-1 [29] (IIIB, RES056) or HIV-2 [30] (ROD) stock (50 ꢀL) at 100-300 CCID50 (cell
culture infectious dose) or culture medium was added to either the infected or mock-infected wells
of the microtiter tray. Mock-infected cells were used to evaluate the effect of test compound on
uninfected cells in order to assess the cytotoxicity of the tested compound. Exponentially growing
MT-4 cells were centrifuged for 5 min at 220 g and the supernatant was discarded. The MT-4 cells
5
were resuspended at 6×10 cells/mL, and 50 ꢀL volumes were transferred to the microtiter tray
wells. 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 spectrophotometrically. The absorbances were read in a computer-controlled
photometer (Infinite M1000, Tecan, Mechelen, Belgium), 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 (CC ) was defined as the concentration of the test compound that reduced
50
the absorbance (OD540) of the mock-infected control samples by 50%. The 50% effective
concentration (EC ) was defined as the compound concentration required for inhibiting
5
0
virus-induced syncytium formation by 50%.
5
.2.2. HIV-1 RT inhibition assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as described. The RT
assay is performed with the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes,

ACCEPTED MANUSCRIPT
Invitrogen), as described by the manufacturer. The assay is based on the dsDNA quantitation
reagent PicoGreen. This reagent shows a pronounced increase in fluorescence signal upon binding
to dsDNA or RNA-DNA heteroduplexes. Single-stranded nucleic acids generate only minor
fluorescence signal enhancement when a sufficiently high dye:base pair ratio is applied. This
condition is met in the assay.
A poly(rA) template of approximately 350 bases long, and an oligo(dT)16 primer, are
annealed in a molar ratio of 1:1.2 (60 min. at room temperature). 52 ng of the RNA/DNA is
brought into each well of a 96-well plate in a volume of 20 µL polymerization buffer (60 mM
Tris-HCl, 60 mM KCl, 8 mM MgCl , 13 mM DTT, 100 µM dTTP, pH 8.1). 5 µl of RT enzyme
2
solution, diluted to a suitable concentration in enzyme dilution buffer (50 mM Tris-HCl, 20%
glycerol, 2 mM DTT, pH = 7.6), is added. The reactions are incubated at 25°C for 40 minutes and
then stopped by the addition of EDTA (15 mM). Heteroduplexes are then detected by addition of
PicoGreen. Signals are read using an excitation wavelength of 490 nm and emission detection at
5
23 nm using a spectrofluorometer (Safire2, Tecan). To test the activity of compounds against RT,
µl of compound in DMSO is added to each well before the addition of RT enzyme solution.
1
Control wells without compound contain the same amount of DMSO. Results are expressed as
relative fluorescence i.e. the fluorescence signal of the reaction mix with compound divided by the
signal of the same reaction mix without compound.
5
.3. Molecular modeling
Structures of 5b2 and 6a3 was drawn, optimized and docked into the published
three-dimensional crystal structures of wt RT (complexes with ETR, PDB code: 3MEC, retrieved
from the Protein Data Bank) by means of surflex-docking module of Sybyl-2.0. Top scoring poses
were shown by PyMOL version 1.7.0 (http://www.pymol.org/), in overlap with the bound ligand
(ETR) in the binding site of RT. The secondary structure of RT is shown in cartoons, and only the
key residues for interactions with the inhibitors were shown in sticks and labeled. The potential
hydrogenbonds were presented by dashed lines. More detailed method is described previously
[13].
Note

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The authors declare no conflict of interest.
Acknowledgment
Financial support from the National Natural Science Foundation of China (NSFC Nos.
1273354), Key Project of NSFC for International Cooperation (Nos. 81420108027), Young
8
Scholars Program of Shandong University (YSPSDU, for P.Z.), and Major Project of Science and
Technology of Shandong Province (No. 2015ZDJS04001) and KU Leuven (GOA 10/014) is
gratefully acknowledged. We thank K. Erven, K. Uyttersprot and C. Heens for technical assistance
with the HIV assays.
Supplementary Material
1
13
MS, H NMR and C NMR of title compounds can be found in the Supplementary Material.
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Highlights
1
. Twenty-one 3,5-diaryl-pyridine derivatives were designed, synthesized and
evaluated for their anti-HIV activities.
2
3
. 5b2 (EC = 0.04 µM, SI = 3963) was the most potent inhibitor.
50
. The 2-NH pyridine derivatives showed better anti-HIV-1 activity than the
2
corresponding 2-NO counterparts.
2