The discovery of novel diarylpyri(mi)dine derivatives with high level activity against a wide variety of HIV-1 strains as well as against HIV-2

Article abstract of DOI:10.1016/j.bmc.2018.03.003

By means of structure-based molecular hybridization strategy, a series of novel diarylpyri(mi)dine derivatives targeting the entrance channel of HIV-1 reverse transcriptase (RT) were designed, synthesized and evaluated as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs). Encouragingly, all the tested compounds showed good activities against wild-type (WT) HIV-1 (IIIB) with EC₅₀ in the range of 1.36 nM–29 nM, which is much better than those of nevirapine (NVP, EC₅₀ = 125.42 nM) and azidothymidine (AZT, EC₅₀ = 11.36 nM). Remarkably, these compounds also displayed effective activity against the most of the single and double-mutated HIV-1 strains with low EC₅₀ values, which is comparable to the control drugs. Besides, these compounds were also exhibited favorable enzymatic inhibitory activity. Moreover, preliminary structure-activity relationships (SARs) and molecular modeling study were investigated and discussed in detail. Unexpectedly, four diarylpyrimidines yielded moderate anti-HIV-2 activities. To our knowledge, this is rarely reported that diarylpyrimidine-based NNRTIs have potent activity against both HIV-1 and HIV-2 in cell culture.

Full text of DOI:10.1016/j.bmc.2018.03.003

Accepted Manuscript
The discovery of novel diarylpyri(mi)dine derivatives with high level activity
against a wide variety of HIV-1 strains as well as against HIV-2
Xueyi Lu, Jiapei Yang, Dongwei Kang, Ping Gao, Dirk Daelemans, Erik De
Clercq, Christophe Pannecouque, Peng Zhan, Xinyong Liu
PII:
S0968-0896(17)32352-0
https://doi.org/10.1016/j.bmc.2018.03.003
BMC 14241
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 December 2017
27 February 2018
2 March 2018
Please cite this article as: Lu, X., Yang, J., Kang, D., Gao, P., Daelemans, D., De Clercq, E., Pannecouque, C., Zhan,
P., Liu, X., The discovery of novel diarylpyri(mi)dine derivatives with high level activity against a wide variety of
HIV-1 strains as well as against HIV-2, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.bmc.2018.03.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The discovery of novel diarylpyri(mi)dine derivatives with high level
activity against a wide variety of HIV-1 strains as well as against
HIV-2
a
a
a
a
b
b
Xueyi Lu , Jiapei Yang , Dongwei Kang , Ping Gao , Dirk Daelemans , Erik De Clercq ,
b
a,*
a,*
Christophe Pannecouque , Peng Zhan , Xinyong Liu
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
a
250012 Ji'nan, Shandong, PR China
b
Rega Institute for Medical Research, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
Abstract
By means of structure-based molecular hybridization strategy, a series of novel
diarylpyri(mi)dine derivatives targeting the entrance channel of HIV-1 reverse
transcriptase (RT) were designed, synthesized and evaluated as potent non-nucleoside
reverse transcriptase inhibitors (NNRTIs). Encouragingly, all the tested compounds
showed good activities against wild-type (WT) HIV-1 (IIIB) with EC50 in the range of
1
1
.36 nM to 29 nM, which is much better than those of nevirapine (NVP, EC50
=
25.42 nM) and azidothymidine (AZT, EC50 = 11.36 nM). Remarkably, these
compounds also displayed effective activity against the most of the single and
double-mutated HIV-1 strains with low EC50 values, which is comparable to the
control drugs. Besides, these compounds were also exhibited favorable enzymatic
inhibitory activity. Moreover, preliminary structure-activity relationships (SARs) and
molecular modeling study were investigated and discussed in detail. Unexpectedly,
four diarylpyrimidines yielded moderate anti-HIV-2 activities. To our knowledge, this
is rarely reported that diarylpyrimidine-based NNRTIs have potent activity against
both HIV-1 and HIV-2 in cell culture.
Keywords: HIV-1, HIV-2, NNRTIs, DAPY, Drug design, Entrance channel
1
. Introduction
Nowadays, highly active antiretroviral therapy (HAART), which uses a
combination of three to four drugs, can distinctly defer people dying from
AIDS-related causes[1, 2]. As an indispensable ingredient of HAART, HIV-1 NNRTIs
show impressive potency, modest toxicity and high selectivity. Meanwhile they
interact with the allosteric site (NNRTIs binding pocket, NNIBP) in a noncompetitive
manner and thereby distort the enzyme’s active conformation, thus perturb the

function of the enzyme [3-5]. Especially, diarylpyrimidine (DAPY) derivatives, such
as dapivirine (TMC120, 1), etravirine (TMC125, ETV, 2) and rilpivirine (TMC278, 3),
have attracted attentions in structural modification research over the past decade
(
Figure 1) [6, 7].
Figure 1.The structures of the representative diarylpyrimidine (DAPY)-based HIV-1 NNRTIs.
Figure 2. The structures of representative DAPYs-based NNRTIs discovered in our lab
targeting the entrance channel of HIV-1 reverse transcriptase (RT).
Our prior studies discovered novel series of diarylpyri(mi)dine derivatives
targeting the entrance channel (one solvent-accessible region) of the NNIBP of RT,

exemplified by piperidinylaminodiarylpyrimidines (pDAPYs) 4 and 5, and
diarylpyridine derivatives 6-11 [8-10]. Notably, compounds 5 (EC50 = 38 nM), 6
(
EC50 = 35 nM), 7 (EC50 = 43 nM) and 8 (EC50 = 41 nM) showed high potency
against WT HIV-1 (Figure 2). Besides, compounds 5 (K103N/Y181C, EC50 = 0.95
μM) displayed potent activity against the most common clinically observed HIV-1
double mutations while 9 (K103N, EC50 = 0.14 μM), 10 (K103N, EC50 = 49 nM) and
11 (K103N, EC50 = 0.18 μM; E138K, EC50 = 0.94 μM) exhibited valid activity against
the most common clinically observed HIV-1 single mutations in micromolar
concentration [8-11]. Notably, there were large differences in activity between
compounds having minor structural changes. These encouraging results provided
rational support for further modification at C-6 position of the pyri(mi)dine ring.
Previous SAR studies also indicated that 6-substituted diarylpyridines adopted
binding orientations and horseshoe conformations in the NNIBP. The left substituted
phenoxy ring moiety (A-ring) possessed the hydrophobic sub-pocket comprised of
Tyr181, Tyr188, Phe227 and Trp229, forming π-π interaction with these amino acid
residues. The para-cyanoaniline moiety (B-ring) was oriented towards the solvent
exposed region formed by Leu234, Pro236 and Tyr318 [12]. Moreover, the existence
of the O and NH group could permit the adaptation to changes caused by the mutation
of amino acid via reorienting itself in the NNIBP. Besides, NH linker connecting C
ring and B ring and pyridine/pyrimidine nitrogen developed dual H-bonds with the
main chain backbone of Lys101 [13]. The amino functionality at C-6 position (R )
2
established additional H-bonds with Glu138 and Lys101 at the entrance of the binding
pocket and led to the better accommodation in the NNIBP [10, 14, 15].
Figure 3. Design of new diarylpyri(mi)dine derivatives.
On the other hand, the linear cyanovinyl in rilpivirine was embedded deeper into
the cylindrical hydrophobic tunnel than the cyano in etravirine, which was favored to

form interaction with the conservable amino acid W229. Thus, the para-cyanovinyl
group on the A-ring was considered to be crucial for enhancing activity [16].
In continuation of our lab’s efforts, we reported herein the design, synthesis and
biological evaluation of a novel series of 6-amino-substituted diarylpyri(mi)dines.
Notably, based on the structure-based molecular hybridization strategy, the favorable
para-cyanovinyl group on the A-ring was adopted to deeply utilize RT hydrophobic
region (Figure 3). Additionally, pyri(mi)dine rings were still employed to probe SARs
in this region. Meanwhile, with aim to capture the entrance channel of the NNIBP,
multitudinous substituents endowed of diverse electronic and steric properties were
introduced in C-6 position of the core ring. Moreover, preliminary SARs and
molecular modeling of these new diarylpyri(mi)dines will be discussed in detail.
2
2
. Results and discussion
.1.Chemistry
Scheme 1. Reagents and conditions: (i) 30% H
2
O
2
, CF
DMF,
, XantPhos, Cs
, t-BuOK, THF, 0 °C; (vi) NHR, Pd (dba)
3
COOH, reflux; (ii) POCl
3
, reflux; (iii)
°C; (iv)
, 1,4-dioxane,
CO
4
4
9
-hydroxy-3,5-dimethylbenzaldehyde,
-aminobenzonitrile/4-chlorobenzonitrile, Pd(OAc)
0 °C; (v) CNCH P(O)(OCH
K
2
CO
3
,
N
2
,
60
2
2
CO
3
, N
2
2
3
)
2
2
3
, XantPhos, Cs
2
3
,
2
N , 1,4-dioxane, reflux.
The synthetic pathways employed to prepare the title compounds 6-substituted
diarylpyri(mi)dines were straightforwardly depicted in Scheme 1. The compounds
F-a ~ F-i were readily prepared from the commercially available starting material
4
,6-dichloropyrimidin-2-amine (F-1). Firstly intermediate F-2 was achieved from F-1
by cross-coupling reaction with cyanoaniline in the presence of Pd(OAc) [17]. Then
in the presence of CO F-2 was selectively substituted by
-hydroxy-3,5-dimethylbenzaldehyde to produce F-3 [18]. Afterwards, F-3 was
2
K
2
3
,
4

converted to F-4 followed by CNCH
2
P(O)(OCH
3
)
2
with t-BuOK. Finally,
intermediate F-4 underwent Pd (dba) catalyzed C-N cross-coupling processes with a
2
3
set of aliphatic amine, respectively, to obtain the desired compounds F-a ~ F-i [19].
As for E-a ~ E-j, commercially available material 2,6-dichloro pyridine (E-1) was
treated with 30% aqueous H
E-2, which was transformed into 2,4,6-trichloro pyridine (E-3) by chlorination with
POCl [20]. Afterwards, E-3 followed the same routes as F-1 to afford the target
compounds E-a ~ E-j.
2
O
2
solution in CF
3
COOH to provide the intermediate
3
2
.2.Biological activity
The newly synthesized DAPY analogues including intermediates E-6 and F-4
were tested in MT-4 cells for their cytotoxicities and activities against WT HIV-1
strain (IIIB), HIV-2 strain (ROD), a panel of RT single-mutated strains (L100I,
K103N, E138K, Y181C, Y188L) and two double-mutated strains (K103N/Y181C
(
RES056) and F227L/V106A), respectively. Nevirapine (NVP), Efavirenz (EFV),
ETV and AZT were chosen as reference drugs. Table 1 and Table 2 depicted the
comparisons of the antiviral 50% inhibitory concentration (EC50), 50% cytotoxic
concentration (CC50), and SI values (selectivity index, given by the CC50/EC50 ratio)
for different agents.
Table 1. Activity and cytotoxicity of diarylpyri(mi)dine derivatives against HIV-1 (IIIB) and
HIV-2 (ROD) strains in MT-4 cells.
a
c
Compd
s
EC50
SI
b
NHR
CC50(μM)
IIIB(nM)
9.99±2.39
ROD(μM)
IIIB
d
E-a
E-b
>82.11
82.11±64.75
8221
d
6.24±4.28
>58.16
-
9326

E-d
1.91±0.50
>4.27
4.27±0.48
2235
d
d
E-f
E-g
E-h
3.35±1.34
28.79±16.91
24.82±1.49
>24.8
>73.63
>7.96
24.80±20.39
7395
2558
321
73.63±57.18
d
7.96±6.28
d
E-i
E-j
1.68±0.67
4.76±1.11
>42
-
25638
≥4.72
≥4.72
≥992
F-a
F-b
F-c
1.52±0.49
1.56±0.080
1.36±0.75
>3.05
3.05±1.37
3.19±1.04
1±0.060
2004
2043
736
1.56±0.93
0.32±0.087
F-d
F-e
1.48±0. 59
2.78±1.33
>3.04
3.04±1.82
3.47±1.24
2051
1250
0.32±0.047
F-f
2.68±1.51
>3.00
>268
3.00±0.86
>268
1117
F-g
29.01±5.27
>9236
F-h
F-i
24.70±1.70
1.54±0.28
>3.65
3.65±0.57
3.54±0.95
148
1.42±0.73
2306
Cl
Cl
E-6
F-4
7.81±1.35
1.69±0.26
>311.83
>4.31
>311.83
4.31±0.97
>15.02
>39936
2556
1
25.42±12.48
NVP
EFV
ETV
AZT
>120
>4012
765
1
2
.58±0.48
.81±0.28
>6.34
2.15±0.11
>7.48
11.36±5.72
0.0081±0.002
>658

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
-
: Because these compounds have multiple hydrogen bond donor and acceptor sites, there is a
high probability of formation of cocrystals, salts or aggregators. Therefore, the
physicochemical properties of these compounds may have changed significantly with the
increase of the solution concentration, which would lead to the deviations of the data.
Table 2 Activity of diarylpyri(mi)dines against HIV-1 (single and double mutant) strains in
MT-4 cells
a
EC50(μM)
Compds
CC50(μM)
L100I
K103N
Y181C
Y188L
>82.11
41.52±3.70
>4.27
E138K
F227L+V106A
RES056
30.45±10.62
4.79±0.48
0.23±0.034
2.82±1.61
31.58±4.84
1.32±0.20
0.43±0.21
0.7±0.14
E-a
E-b
E-d
E-f
0.63±0.37
0.74±0.34
0.19±0.007
0.17±0.043
2.6±0.24
0.027±0.004
0.028±0.001
0.0027±0.001
0.008±0.002
0.048±0.004
0.028±0.001
0.0017±0.000
0.0056±0.001
1.09±0.072
1.37±0.12
0.18±0.034
0.21±0.004
4.54±0.076
1.43±0.14
0.22±0.013
0.76±0.16
0.043±0.001
0.057±0.022
0.97±0.13
≥57.42
82.11±64.75
d
d
22.34±19.89
58.16±58.08
>4.27
5.35±0.32
>73.63
4.27±0.48
d
>24.8
0.027±0.019
0.17±0.041
0.77±0.098
0.077±0.026
0.11±0.016
24.80±20.39
E-g
E-h
E-i
>73.63
>7.96
73.63±57.18
2.02±0.91
0.24±0.21
0.53±0.10
5.7±0.77
7.96±6.28
d
>43.19
≥4.72
4.43±0.51
5.01±0.67
0.53±0.062
0.22±0.003
0.19±0.003
0.31±0.014
0.23±0.030
0.35±0.094
172.12±111.25
>3.65
43.19±57.24
≥4.72
E-j
F-a
F-b
F-c
0.021±0.013 0.0048±0.004 0.013±0.004 0.056±0.006 0.0044±0.000
0.0093±0.001 0.0017±0.000 0.0094±0.002 0.051±0.008 0.0064±0.003
0.011±0.000 0.0012±0.001 0.0099±0.000 0.031±0.000 0.0065±0.001
0.044±0.003
0.042±0.004
0.014±0.006
0.027±0.014
0.059±0.013
0.058±0.012
44.37
3.05±1.34
3.19±1.04
1±0.060
3.04±1.82
3.47±1.24
3.00±0.86
>268
F-d
F-e
0.01±0.001
0.0024±0.000 0.009±0.001
0.9±0.061
0.041±0.002
0.021±0.007 0.0046±0.002 0.028±0.022 0.083±0.032 0.0093±0.000
F-f
0.023±0.003 0.0026±0.001 0.035±0.001
0.11±0.058
>268
0.01±0.001
0.15±0.051
0.94±0.082
F-g
F-h
F-i
2.89±0.045
0.79±0.036
0.01±0.002
0.71±0.001
0.091±0.023
0.031±0.001
6.97±1.88
0.61±0.082
>3.65
0.77±0.038
0.021±0.006
>311.83
3.65±0.57
3.54±0.95
>311.83
4.31±0.97
>15.02
0.0017±0.000 0.0072±0.000 0.098±0.036 0.0075±0.000
0.033±0.007 1.17±0.014 >311.83 0.043±0.001
0.18±0.036
>311.83
E-6
F-4
NVP
EFV
ETV
AZT
0.0088±0.000 0.0024±0.000 0.011±0.001 0.048±0.001 0.0093±0.000
0.16±0.030
0.044±0.004
>15.02
3.25±2.46
0.047±0.015
0.010±0.003
10.26±3.70
0.11±0.012
0.003±0.000
0.008±0.001
0.28±0.06
0.006±0.001
0.13±0.016
0.049±0.001
0.12±0.16
0.024±0.012
0.005±0.001
0.11±0.012
0.026±0.004
0.012±0.003
>6.34
0.020±0.007 0.021±0.003 0.014±0.003
0.006±0.001 0.008±0.002 0.018±0.006
2.15±0.11
>7.48
0.005±0.000
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
Generally, all the compounds were active against the IIIB strain with EC50 values
in low nanomolar to sub-micromolar concentration range. All compounds were more
effective than the control drug NVP (EC50 = 125.42 nM); eight of them were more

valid than ETV (EC50 = 2.81 nM); five compounds (F-a, F-b, F-c, F-d and F-i) were
more potent than EFV (EC50 = 1.58 nM). In particular, F-c exhibited most prominent
inhibitory activity with an EC50 value of 1.36 nM and a SI value of 736. Additionally,
F-a (EC50 = 1.58 nM, SI = 2004), F-b (EC50 = 1.56 nM, SI = 2043), F-d (EC50 = 1.48
nM, SI = 2051) and F-i (EC50 = 1.54 nM, SI = 2306), compared with F-c, showed
similar activities but higher SI than F-c. Furthermore, nearly all the tested compounds
inhibited the frequently encountered mutations (L100I, K103N, E138K, Y181C,
F227L+V106A, RES056) with nanomolar to micromolar concentration range (EC50
.2 nM-31.58 μM). Besides, most of the F series inhibited Y181L mutation in
sub-micrimolar concentration range (EC50 = 0.031 μM – 0.9 μM).
=
1
Compared with the previous reported DAPYs, the newly synthesized compounds
demonstrated remarkable potency. Thus, modification at C-6 position, which extended
into the entrance channel, provided new alternatives for further optimization.
A concise investigation of SARs and structure-cytotoxicity relationships (SCRs)
were summarized as follows:
1
) By comparison with the activities between E series and F series, it was found
that the pyrimidine motif was more favorable to the anti-HIV-1 activities than the
pyridine motif as the core ring. However, the pyrimidine motif increased the
cytotoxicity since F series showed higher cytotoxicity than those of E series.
2
) Further SAR investigation validated that the C-6 position (R) of the core ring
is highly tolerated by diverse substituents. The size and length of the substituents are
responsible for the anti-HIV-1 activity, which can be supported in Tables 1 and 2. i)
When the linear amino group was introduced into R group, it looks like that the
anti-HIV activities was slightly increased. ii) Compared with olefin and methoxy, the
N,N-dimethylamino group at the end of the side chain could gently increase both
activity and cytotoxicity against WT HIV-1, as well as the activitiy against HIV-1
mutations (E-d compared with E-a, E-b; F-d compared with F-a, F-b). iii) Contrast
with N,N-dimethylamino group, cyano-containing compounds demonstrated slightly
increased activity but fairly enhanced cytotoxicity (F-c with F-d). iv) Reduction of
the length of the side chain was capable to significantly increase potency against both
WT and mutated HIV-1 strains (E-i with E-j). v) The order of potency of the cyclo
group was as follows: morpholinyl (E-i, F-i) > cyclopropyl (E-f, F-f) > piperazinyl
(
E-h, F-h) > furanyl (E-g, F-g), indicating that hydrophilic heterocycles like
morpholine ring in the side chain of the C-6 position could enhance the activities

against WT as well as mutations.
) According to CC50 values, all the tested compounds showed lower cytotoxicity
3
than ETV (CC50 = 2.15 μM) with the exception of F-c (CC50 = 1 μM), therefore,
modification at the C-6 position may apprise superiority in cytotoxicity but cyano
group.
4
) As shown in Table 2, generally speaking, the regulations of SARs of antiviral
activities against mutated strains were consistent with those of IIIB strain. i) For
L100I strain, all compounds showed better inhibition than NVP (EC50 = 3.25 μM)
while F series exhibited higher potencies than EFV (EC50 = 0.047 μM) except F-g and
F-h. F-b (EC50 = 0.0093 μM), F-c, F-d and F-i were equipotent with ETV (EC50
=
0
.010 μM), or more potent. ii) With respect to K103N strain, all compounds showed
better inhibition than NVP (EC50 = 10.26 μM) and EFV (EC50 = 0.11 μM). Among
them, E-d and E-f showed similarly potent with ETV (EC50 = 0.003 μM), and E-i,
F-b, F-c (EC50 = 0.0012 μM), F-d and F-i were more potent. iii) In regard to Y181C
strain, F-a, F-b F-c, F-d and F-i displayed better inhibition than ETV (EC50 = 0.020
μM). Particularly, F-i (EC50 = 0.0072 μM) was the most potent, which was
comparable to EFV (EC50 = 0.006 μM). iv) In terms of Y181L strain, E series were
inactive while most compounds of F series were more potent than EFV (EC50 = 0.13
μM) but less potent than ETV (EC50 = 0.021 μM). v) Regarding E138K strain, F-a,
F-b F-c, F-e, F-i and F-f exhibited higher inhibitory activities than ETV (EC50
=
0
.014 μM). vi) As far as double mutated F227L+V106A strain was concerned, none of
the tested compounds was better than the control drugs. Among them, F-i displayed
moderate inhibition with an EC50 value of 0.18 μM. vii) Finally, for RES056 strain, E
series were less potent than the control drugs while F series exhibited higher
potencies than EFV (EC50 = 0.11 μM) except F-g and F-h. Encouragingly, F-c (EC50
=
0.014 μM) and F-i (EC50 = 0.021 μM) were more potent than ETV (EC50 = 0.026
μM).
Overall, the introduction of the linear amino groups at C-6 position of the core
ring was preferred to enhance the activities against mutations, which may resulted
from their flexibility to adapt themselves into the entrance channel. When it came to
morpholine ring in the side chain of the C-6 position, its good water solubility may
lead to the achievement in activities.
5
) To our surprise, intermediates E-6 and F-4 were endowed with high potency
against both WT and mutated HIV-1 strains.

To sum up, some constructive information were provided by the biological
evaluation results and the SAR analysis above to be beneficial in the future
modification of DAPYs targeting at the entrance channel.
Unexpectedly, F-b (EC50 = 1.56 μM), F-c (EC50 = 0.32 μM), F-e (EC50 = 0.32 μM)
and F-i (EC50 = 1.42 μM) yielded moderate antiviral activities against HIV-2 ROD
strain. To our knowledge, this is rarely reported that diarylpyrimidine-based NNRTIs
have potent activity against both HIV-1 and HIV-2 in cell culture [21].
At present, treatment of HIV-2 infection has progressed at a tardy pace. HIV-2
infection and HIV-1/2 dual infection, accounting for 3 to 5% of the global burden of
HIV, are largely confined to West Africa as well as a few regions tied with
socioeconomic [22]. The antiretroviral therapy (ART) for HIV-2 draws exclusively on
compounds with anti-HIV-1 activities. HIV-2 is sensitive to integrase strand transfer
inhibitors (INSTIs) [23-25] and nucleoside reverse transcriptase inhibitors (NRTIs)
used for HIV-1 ART but is per se resistant to NNRTIs, the fusion inhibitor and the
majority of the protease inhibitors (PIs) [26-28]. Besides, the emergence of drug
resistance hampers treatment of HIV-2 infection. Therefore, novel HIV-2-active
antiretrovirals (ARVs) are urgently needed.
Collectively, our findings suggest that F-b, F-c, F-e and F-i, unlike INSTIs
(
raltegravir, etc.), NRTIs (zidovudine, emdtricitabine, tenofovirdisoproxil, etc.) and
PIs, should be considered as potential components, with divergent anti-HIV-2
mechanism, for further optimization, thus help fill the need for newer HIV-1/2-active
ARVs.
2
.3.HIV-1 RT inhibition assay
Table 3. Inhibitory activity of diarylpyri(mi)dine derivatives against HIV-1 RT
a
Compd
s
IC50 (µM)
Compd
s
IC50 (µM)
WT
K103N+Y181C
WT
K103N+Y181C
0.19±0.003
0.23±0.04
E-a
E-b
E-d
E-f
0.27±0.01
0.92±0.07
0.83±0.11
0.35±0.002
0.63±0.01
2.19±0.62
0.74±0.03
31.31±2.43
18.15±3.40
2.47±0.49
5.39±0.56
41.20±9.50
6.24±1.51
6.27±0.40
F-a
F-b
F-c
F-d
F-e
F-f
0.03±0.000
0.04±0.01
0.02±0.003
0.01±0.001
0.17±0.003
0.05±0.01
0.44±0.04
0.10±0.06
0.02±0.01
E-g
E-h
E-i
0.50±0.14
0.24±0.04
F-g
86.93±10.10

E-j
E-6
0.97±0.09
0.18±0.04
0.02±0.01
0.90±0.33
7.80±0.12
0.08±0.02
F-h
F-i
0.44±0.04
0.14±0.02
0.02±0.01
1.12±0.05
0.34±0.06
0.36±0.17
0.02±0.005
F-4
EFV
ETV
b
NVP
ND
a
IC50: 50% of the inhibitory concentration of tested compound required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.
b
ND: not determined.
For the sake of confirming the binding target of these diarylpyri(mi)dine
derivatives, an HIV-1 RT inhibitory assay was performed via an ELISA assay using
hybrid poly (A)-oligo (dT)15 (9A260 nm/mL), lyophilizate as the template/primer [29,
3
0]. The result indicated that all the desired compounds showed moderate affinity to
HIV-1 WT and double-mutated (K103N+Y181C) RT enzymes. In WT enzymatic
assay, most of them exhibited higher inhibition than the reference drugs NVP (IC50
=
0
0
.90 µM), while merely F-d was superior to EFV (IC50 = 0.02 µM) with IC50 value of
.01 µM. In terms of K103N+Y181C strain enzymatic assay, half of the tested
compounds showed better affinity than the reference drugs EFV (IC50 = 0.36 µM),
and only F-d inhibited the mutated enzyme with the same IC50 value to ETV (IC50
.02 µM). Generally, the SAR for inhibitory potency of RT was consistent with that of
=
0
HIV-1 replication activity. These results manifested that the active compounds
inhibited the replication of HIV-1 as NNRTIs. Meanwhile, the results suggested that
F-d with 6-dimethylaminoethylamino-diarylpyrimidine deserves further optimization.
2
.4 Molecular modeling analysis
To gain a deeper understanding of structure-affinity relationships for
diarylpyri(mi)dine derivatives, molecular docking studies was performed for the
representative compounds F-d and F-i with NNRTIs binding pocket (PDB code:
3
MEC) as well as K103N mutant NNRTIs binding pocket (PDB code: 3MED) by
virtue of the software Surflex-Dock SYBYL-X 2.0. Docking results were shown by
PyMOL. The theoretical binding behavior is illustrated in Figure 4. On the other hand,
we got the predicted dihedral angle (Table 4) and torsion angles (Table 5) of F-i and
ETV in the site of HIV-1 K103N RT (PDB code: 3MED) by virtue of the software
Diamond 3.2.

a
b
c
Figure 4. a, b. Predicted binding mode and molecular docking of compound F-d (pink, a)
and F-i (pink, b) with 8 (yellow) into the allosteric site of HIV-1 RT (PDB code: 3MEC),
respectively. 8 (yellow) was for comparison. c. Predicted binding mode and molecular
docking of compound F- i (pink) with ETV (yellow) into the allosteric site of HIV-1 K103N
RT (PDB code: 3MED). The dashed lines (yellow) was on behalf of the H-bonds.
Table 4. Predicted dihedral angle of F- i and ETV (PDB code: 3MED)
ETV
F-i
Dihedral angle
40.8°
80.9°
Table 5. Predicted torsion angles of F- i and ETV
PDB code: 3MED)
(
τ
1
τ
2
τ
3
τ
4
τ
5
τ
6
ETV 179.1 179.8 178.2 -179.1 178.9 -179.6
F-i 172.9 178.6 175.0 179.8 179.9 -176.1
Compounds F-d and F-i shared resembling binding mode with 8 in the NNIBP.
Details are as follows: 1) The left 4-cyanovinyl-2,6-dimethylphenyl group of F-d and
F-i located in the hydrophobic sub-pocket, exhibiting π-π interaction and hydrophobic
interactions with the aromatic amino acid residues Tyr181, Tyr188, Phe227 and

Trp229. The 4-cyanovinyl group extended deeper into this region and formed
characteristic π-π interaction with the indol ring of conserved amino acid Trp229,
which might be the main reason of giving rise to the high-affinity binding to the
NNBIP thereby improved the anti-HIV-1 activity and prevented the loss of activity
specifically caused by Y181C, Y181L and F227L RT mutations; 2) The right
cyanoaniline ring pointed towards a hydrophobic/π-electron pocket formed by the side
chain of amino acids Leu234, Pro236 and Tyr318; 3) The NH linker, connecting the
pyri(mi)dine (8(F-d and F-i)) ring and the right ring, and pyri(mi)dine nitrogen
formed dual hydrogen bonds with the main chain backbone of Lys101; 4) The
6
-(2-dimethylaminoethyl)amino and 6-(2-morpholinoethyl)amino functionality at
pyri(mi)dine efficiently anchored into the tolerant region II of NNIBP (NNRTI
entrance channel), which exploited Van der Waals forces and electrostatic interactions
directly or via water-bridge with Glu138 and Lys101, so as to further give the
advantage of the affinity between inhibitor and RT.
Notably, most of the compounds inhibited the replication of K103N strain, one of
the most prevalent mutant strains in clinical, in nanomolar concentration, which was
comparable to ETV. According to the predicted dihedral angles and torsion angles of
compound F-i and ETV in HIV-1 K103N RT, the predicted dihedral angle of F-i was
twice than that of ETV, on the other hand, the predicted torsion angles of F-i (τ
1
6
-τ )
were different from that of ETV. The NH linker of the compounds could form a
hydrogen bond with the backbone C=O of Asn103, while the resilience of
6
-substituted-amino also improved the structurally intrinsic torsional flexibility of the
compounds, thus improved the affinity. And at the same time, these inhibitor agents
might not rely heavily on interactions with Lys101 for their binding to the enzyme, it
is therefore not surprising that substitutions by 6 position of some groups, like
6
6
-(2-methoxyethyl)amino (F-b, 1.7 nM), 6-(2-cyanoethyl)amino (F-c, 1.2 nM) and
-(2-(dimethylamino)ethyl)amino (F-d, 2.4 nM), might improve the binding affinity
of the mutant RT.
In addition, the 6-(2-morpholinoethyl)amino counterpart showed high potency
against E138K, which probably resulted from its flexibility and good water solubility
as well as the flexibility of the residue of Lys138.
On the whole, the molecular modeling studies elucidated that the
6
-substituted-amino modification of diarylpyri(mi)dine exhibited high potency and
favorable resistance profiles, which provided guidance for further optimization.

3
. Conclusion
In summary, based on the structure-based molecular hybridization strategy, a
novel series of diarylpyri(m)idine derivatives targeting the entrance channel of the
NNIBP of RT were designed and synthesized. Biological evaluation revealed that
most of the desired compounds displayed good activities against WT HIV-1 (IIIB
strain), single/double-mutated strains in MT-4 cells. All compounds were proved to
have efficacy in inhibiting HIV-1 replication, among which E-d (EC50 = 1.91 nM),
E-i (EC50 = 1.64 nM), F-a (EC50 = 1.52 nM), F-b (EC50 = 1.56 nM), F-c (EC50 = 1.36
nM), F-d (EC50 = 1.48 nM) and F-i (EC50 = 1.54 nM) showed promising inhibitory
potency against IIIB. In addition, F-b, F-c, F-d and F-i were beneficial in
anti-single/double-mutations activity. Besides, F-d displayed high affinity to WT RT
(
IC50 = 0.01 µM).
Moreover, the preliminary SARs, SCRs and docking studies provide vital
information for further functionalization at C-6 position to discover new series of
compounds aiming at improving drug resistance profiles.
Finally, in this study, we also reported the identification and characterization of
four new HIV inhibitors that possessed high level activity against a wide variety of
HIV-1 strains as well as against HIV-2. Furthermore, these four new agents were
structurally distinguished with the INSTIs, NRTIs and PIs, and hence they may take
advantage of new mechanism in anti-HIV-2 activitiy.
4
4
. Experimental section
.1.Chemistry
Melting points (mp) were achieved by a micromelting point apparatus (Tian Jin
Analytical Instrument Factory, Nankai, Tianjin, China). Mass spectra were carried out
1
13
on a G1313A Standard LC Autosampler (Agilent). H NMR and C NMR spectra
were performed on a Bruker AV-400 spectrometer (Bruker BioSpin, Switzerland),
making use of solvents as indicated (d
6
-DMSO or CDCl ). Chemical shifts were
3
recorded in δ values (ppm) with tetramethylsilane as the internal reference, while J
values were recorded in hertz (Hz). Thin layer chromatography was performed on
R○
pre-coated HUANGHAI HSGF254, 0.15-0.2 mm TLC-plates (Yantai Jiangyou
Silica Gel Development Co., Ltd., Yantai, Shandong, China). Flash column
chromatography took advantage of columns packed with silica gel 60 (200-300 mesh)

(
Qingdao waves silica gel desiccant Co., Ltd, Qingdao, China). Solvents were of
reagent grade and were purified and dried by standard methods when necessary.
Concentration of the reaction solutions involved the use of rotary evaporator at
reduced pressure. Chemical reagents were obtained commercially and were used
without further purification.
4
.1.1 General procedure for the synthesis of 2,6-dichloropyridine 1-oxide (E-2)
,6-dichloropyridine (E-1) (10.0 g, 0.067 mol) was dissolved in a mixture of
COOH (80 mL) and 30% aq H (25 mL), which was heated at reflux for 3 h.
solution was added to the above solution until hydrogen
2
CF
3
2
O
2
Then, saturated Na
2
S
2
O
4
peroxide was depleted completely via an inspection with potassium iodide-starch test
paper. Afterwards, the reaction mixture was concentrated in vacuo to about 10 mL and
then added 50 mL water, filtered, basified the filtrate to pH = 8-9 with solid Na
2
CO .
3
The obtained precipitation was filtered and dried, affording the crude
2
,6-dichloropyridine-1-oxide (E-2) as a white solid, yield 54%, mp: 137-138 °C.
.1.2 General procedure for the synthesis of 2,4,6-trichloropyridine (E-3)
4
A mixture of 2,6-dichloropyridine-1-oxide (E-2) (8.20 g, 0.050 mol) in POCl (25
3
mL) was refluxed for 4 h. Then, the solvent was removed by evaporation. The residue
was poured into crushed ice and extracted with petroleum ether (3×50 mL). The
organic layers were combined and dried over K
2
CO , filtered, and evaporated in
3
vacuum. The residue was further purified by flash column chromatography to give
2
4
4
,4,6-trichloropyridine (E-3)as colorless needles, yield 85%, mp: 32-33 °C.
.1.3 General procedure for the synthesis
-((2,6-dichloropyridin-4-yl)oxy)-3,5-dimethylbenzaldehyde (E-4)
A mixture of 4-hydroxy-3,5-dimethylbenzaldehyde (1.82 g, 12.1 mmol) and
CO (1.83 g, 13.2 mmol) in 15 mL DMF was stirred at room temperature for 5 min,
of
K
2
3
to which E-3 (11 mmol) was added with stirring under 40 °C overnight. Then, the
solvent was removed and the residue was redissolved in water and extracted with
ethyl acetate (3×15 mL). Afterwards, the organic phase was dried over MgSO and
4
concentrated in vacuo. The residue was purified by column chromatography using
ethyl acetate and petroleum ether and recrystallized from ethyl acetate and petroleum
1
to give E-4. White solid, yield 37%, mp: 118-120 °C. H NMR (400 MHz, CDCl
3
) δ:
);
); δ: 191.1, 166.0, 153.9, 152.0, 134.6, 132.0, 131.0,
(295.02)
9
.99 (s, 1H, CHO), 7.70 (s, 2H, PhH), 6.67 (s, 2H, pyridine-H), 2.21 (s, 6H, 2CH
3
1
3
C NMR (100 MHz, CDCl
3
1
09.7, 16.2. C14
H
11
Cl2NO
2

4
.1.4
-((6-chloro-4-(4-formyl-2,6-dimethylphenoxy)pyridin-2-yl)amino)benzonitrile (E-5)
In a schlenk-type flask, a mixture of Pd(OAc) (0.08 g, 0.34 mmol) and XantPhos
0.20 g, 0.34 mmol) in dry dioxane (25 mL) was stirred at room temperature for 5 min.
Then, 4-aminobenzonitrile (1.0 g, 8.2 mmol) and Cs CO (3.32 g, 10.1 mmol) were
General
procedure
for
the
synthesis
of
4
2
(
2
3
added and stirred for another 5 min at ambient temperature. Afterwards, E-4 (6.8
mmol) was added, and the flask was evacuated and backfilled with nitrogen for three
times. The reaction mixture was stirred at 90 °C for 9 h and followed by TLC until
completion. After cooling, the solvent was removed under reduced pressure. The
residue was purified by flash column chromatography with ethyl acetate/petroleum
1
ether to afford compounds E-5. White solid, yield 68%, mp: 155-157 °C. H NMR
(
400 MHz, d
6
-DMSO) δ: 10.00 (s, 1H, COH), 9.77 (s, 1H, NH), 7.82 (s, 2H, PhH),
7
1
1
1
4
4
.72 (t, J = 8.1 Hz, 4H, PhH), 6.71 (d, J = 1.8 Hz, 1H, pyridine-H), 6.03 (d, J = 1.8 Hz,
1
3
H, pyridine-H), 2.20 (s, 6H, 2CH
3
); C NMR (100 MHz, d -DMSO) δ: 192.7, 165.6,
6
56.4, 154.4, 149.9, 145.3, 134.6, 133.9, 133.7, 132.3, 131.2, 120.0, 118.2, 113.9,
04.2, 102.8, 94.8, 16.2. C21
.1.5 General
H
16ClN
3
O
2
(377.09)
procedure
for
the
synthesis
of
-((4,6-dichloropyrimidin-2-yl)amino)benzonitrile (F-2)
In a schlenk-type flask, a mixture of Pd(OAc) (0.08 g, 0.34 mmol) and XantPhos
0.20 g, 0.34 mmol) in dry dioxane (25 mL) was stirred at room temperature for 5 min.
Then, 4-chlorobenzonitrile (8.2 mmol) and Cs CO (3.32 g, 10.1 mmol) were added
and stirred for another min at ambient temperature. Afterwards,
,6-dichloropyrimidin-2-amine (F-1) (6.8 mmol) was added, and the flask was
2
(
2
3
5
4
evacuated and backfilled with nitrogen for three times. The reaction mixture was
stirred at 90 °C for 9 h and followed by TLC until completion. After cooling, the
solvent was removed under reduced pressure. The residue was purified by flash
column chromatography with ethyl acetate/petroleum ether to afford compounds F-2.
F-2, white solid, yield 59%, mp: 114-116 °C.
4
4
.1.6
General
procedure
for
the
synthesis
of
-((4-chloro-6-(4-formyl-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzonitrile
(
F-3)
A mixture of 4-hydroxy-3,5-dimethylbenzaldehyde (1.82 g, 12.1 mmol) and
CO (1.83 g, 13.2 mmol) in 15 mL DMF was stirred at room temperature for 5 min,
to which F-2 (11 mmol) was added with stirring under 40 °C overnight. Then, the
K
2
3

solvent was removed and the residue was redissolved in water and extracted with
ethyl acetate (3×15 mL). Afterwards, the organic phase was dried over MgSO and
4
concentrated in vacuo. The residue was purified by column chromatography using
ethyl acetate and petroleum ether and recrystallized from ethyl acetate and petroleum
to give F-3. White solid, yield 42%, mp: 147-148 °C. white solid, yield 54%, mp:
1
1
7
37-138 °C; H NMR (400 MHz, CDCl
3
) δ: 10.01 (s, 1H, NH), 7.70 (s, 2H, PhH),
1
3
.37 (m, 5H, 4PhH, =CH), 6.57 (s, 1H, pyrimidine-H), 2.22 (s, 6H, 2CH
3
); C NMR
(
100 MHz, CDCl
3
) δ: 191.4, 169.5, 162.4, 158.2, 154.5, 142.4, 134.2, 133.08, 132.2,
+
1
30.3, 119.0, 118.4, 105.6, 98.6, 16.5; ESI – MS: 340.1 [M + H ]. C20
H15ClN
4
O
2
(
378.81)
.1.7
4
General
procedure
for
the
synthesis
of
(
E)-4-((6-chloro-4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyridin-2-yl)amino)benzon
itrile (E-6) and
E)-4-((4-chloro-6-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benz
onitrile (F-4)
To a solution of CNCH
(
2
P(O)(OCH
3
) (1.4 g, 7.5 mmol) in THF was added
2
t-BuOK (1.12 g, 10 mmol) in ice bath and stirred for 30 min. Then E-5 (F-3) (5 mmol)
was added dropwise to the above solution. The reaction mixture was stirred for
another 2 h at room temperature. Afterwards, the solvent was removed and the residue
was redissolved in water and extracted with ethyl acetate (3×15 mL). The organic
phase was dried over MgSO and concentrated in vacuo. The residue was purified by
4
recrystallization from ethyl acetate and petroleum to give E-6 (F-4). E-6: white solid,
1
yield 90%, mp: 278-280 °C. H NMR (400 MHz, CDCl
3
) δ: 7.59 (d, J = 8.8 Hz, 2H,
PhH), 7.50 (d, J = 8.8 Hz, 2H, PhH), 7.34 (d, J = 16.7 Hz, 1H, =CH), 7.23 (s, 2H,
PhH), 6.76 (s, 1H, NH), 6.30 (d, J = 1.6 Hz, 1H, pyridine-H), 6.14 (d, J = 1.5 Hz, 1H,
1
3
pyridine-H), 5.85 (d, J = 16.6 Hz, 1H, =CH), 2.17 (s, 6H, 2CH
3
); C NMR (100 MHz,
CDCl ) δ:166.4, 151.9, 149.6, 143.9, 133.6, 132.0, 128.4, 118.4, 103.8, 96.4, 94.2,
3
+
6
8
3.8, 16.3; ESI – MS: 401.5 [M + H ]. C23
H
17ClN O (400.11). F-4: white solid, yield
4
+
7%, mp: 283-285 °C; ESI – MS: 402.6 [M + H ]. C22
H
16ClN O (401.85)
5
4
.1.8
General
procedure
for
the
synthesis
of
(
E)-4-((6-substituted-amino-4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyridin-2-yl)a
mino)benzonitrile (E-a E-j) and
E)-4-((4-substituted-amino-6-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)
~
(

amino)benzonitrile (F-a ~ F-i)
In a schlenk-type flask, a mixture of Pd
2
(dba) (0.023 g, 0.025 mmol) and
3
XantPhos (0.015 g, 0.025 mmol) in dry dioxane was stirred at room temperature for 5
min. Then, NHR (2 mmol) and Cs CO (0.24 g, 0.75 mmol) were added and stirred
2
3
for another 5 min at ambient temperature. Afterwards, E-6 (F-4) (0.5 mmol) was
added, and the flask was evacuated and backfilled with nitrogen for three times. The
reaction mixture was stirred at 100 °C for 9 h and followed by TLC until completion.
After cooling, the solvent was removed under reduced pressure. The residue was
purified by flash column chromatography with ethyl acetate/petroleum ether to afford
compounds E-a ~ E-k (F-a ~ F-j).
(
E)-4-((6-(Allylamino)-4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyridin-2-yl)amino)
benzonitrile (E-a)
White solid, yield 53%, mp: 136-138 °C; H NMR (400 MHz, CDCl
J = 7.2 Hz, 2H, PhH), 7.50 (m, 3H, PhH, =CH), 7.32 (d, J = 16.8 Hz, 1H, =CH), 7.20
m, 3H, PhH, NH), 6.75 (s, 1H, NH), 6.30 (s, 1H, pyridine-H), 6.14 (s, 1H,
pyridine-H), 5.83 (m, 1H, =CH), 2.17 (s, 8H, 2CH , CH ), 1.68 (s, 1H, =CH), 1.26 (s,
1
3
) δ: 7.57 (d,
(
3
2
1
3
1
1
H, =CH); C NMR (100 MHz, CDCl
3
) δ: 166.4, 155.1, 149.5, 143.9, 133.5, 132.0,
+
28.4, 118.4, 103.8, 96.6, 94.2, 16.3; ESI – MS: 422.8 [M + H ]. C26
H
23
5
N O (421.49)
(
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2-methoxyethyl)amino)pyridin-
-yl)amino)benzonitrile (E-b)
White solid, yield 43%, mp: 116-118 °C; H NMR (400 MHz, CDCl
2
1
3
) δ: 7.52 (d,
J = 10.0 Hz, 4H, PhH), 7.34 (d, J = 16.6 Hz, 1H, =CH), 7.20 (s, 2H, PhH), 6.63 (s, 1H,
NH), 5.83 (d, J = 16.6 Hz, 1H, =CH), 5.61 (d, J = 1.6 Hz, 1H, pyridine-H), 5.36 (d, J
=
=
1.6 Hz, 1H, pyridine-H), 4.75 (s, 1H, NH), 3.57 (t, J = 5.2 Hz, 2H, CH
2
), 3.43 (d, J
1
3
5.2 Hz, 2H, CH
2
), 3.38 (s, 3H, CH
3
), 2.18 (s, 6H, 2CH
3
); C NMR (100 MHz,
CDCl
3
) δ: 159.5, 152.7, 149.9, 145.1, 133.3, 132.4, 130.9, 128.1, 118.2, 117.8, 95.9,
+
8
6.4, 85.5, 71.1, 58.8, 41.8, 29.7, 16.3; ESI – MS: 439.6 [M + H ]. C26
H
25
N
5
O
2
(
439.20)
(E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2-(dimethylamino)ethyl)amino)
pyridin-2-yl)amino)benzonitrile (E-d)
White solid, yield 63%, mp: 156-158 °C; H NMR (400 MHz, CDCl
1
3
) δ: 7.51 (d,

J = 4.7 Hz, 4H, PhH), 7.33 (d, J = 16.6 Hz, 1H, =CH), 7.19 (s, 2H, PhH), 6.66 (s, 1H,
NH), 5.83 (d, J = 16.6 Hz, 1H, =CH), 5.61 (d, J = 1.4 Hz, 1H, pyridine-H), 5.34 (d, J
=
1.4 Hz, 1H, pyridine-H), 4.98 (s, 1H, NH), 3.34 – 3.27 (m, 2H, CH
2
), 2.52 (t, J =
); C NMR (100 MHz, CDCl
δ: 166.3, 159.6, 158.9, 154.6, 152.8, 149.9, 145.3, 133.2, 132.4, 130.8, 128.1, 119.8,
18.2, 117.7, 102.9, 95.9, 86.2, 85.4, 59.3, 58.0, 45.2, 39.4, 16.3; ESI – MS: 453.5 [M
1
3
5
.9 Hz, 2H, CH
2
), 2.25 (s, 6H, CH
3
), 2.18 (s, 6H, CH
3
3
)
1
+
+
H ]. C27
H
28
6
N O (452.23)
(E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-(cyclopropylamino)pyridin-2-yl)
amino)benzonitrile (E-f)
1
White solid, yield 29%, mp: 244-246 °C; H NMR (400 MHz, CDCl ) δ: 7.50 (q,
3
J = 9.0 Hz, 4H, PhH), 7.34 (d, J = 16.6 Hz, 1H, =CH), 7.21 (s, 2H, PhH), 6.50 (s, 1H,
NH), 5.83 (d, J = 16.6 Hz, 1H, =CH), 5.79 (s, 1H, pyridine-H), 5.50 (s, 1H,
pyridine-H), 4.96 (s, 1H, NH), 2.45 (s, 1H, CH), 2.19 (s, 6H, CH
3
), 0.76 – 0.67 (m,
1
3
2
1
9
H, CH
2
), 0.54 (s, 2H, CH
2
); C NMR (100 MHz, CDCl
3
) δ: 166.6, 160.6, 154.6,
52.8, 149.9, 145.1, 133.3, 132.4, 130.8, 128.3, 128.1, 119.7, 118.2, 117.7, 103.1,
+
5.9, 86.7, 85.4, 24.0, 16.3, 7.5; ESI – MS: 422.4 [M + H ]. C26
H
23
5
N O (421.19)
(
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-(((tetrahydrofuran-2-yl)methyl)a
mino)pyridin-2-yl)amino)benzonitrile (E-g)
White solid, yield 42%, mp: 108-110 °C; H NMR (400 MHz, CDCl
H, PhH), 7.26 (d, J = 16.6 Hz, 1H, =CH), 7.12 (s, 2H, PhH), 6.53 (s, 1H, NH), 5.75
d, J = 16.6 Hz, 1H, =CH), 5.52 (s, 1H, pyridine-H), 5.29 (d, J = 1.5 Hz, 1H,
pyridine-H), 4.69 (s, 1H, NH), 4.01 (dd, J = 7.1, 3.5 Hz, 1H, CH), 3.79 (dd, J = 10.8,
.0 Hz, 1H, CH), 3.70 (t, J = 7.5 Hz, 1H, CH), 3.41 (ddd, J = 13.0, 6.4, 3.7 Hz, 1H,
CH), 3.23 – 3.04 (m, 1H, CH), 2.09 (s, 6H, 2CH ), 1.88 – 1.82 (m, 2H, CH ), 0.79 (dd,
); C NMR (100 MHz, CDCl ) δ: 166.4, 159.5, 154.4,
52.7, 149.9, 145.1, 133.5, 133.2, 132.4, 132.0, 130.9, 128.4, 128.1, 119.7, 118.3,
1
3
) δ: 7.43 (s,
4
(
4
3
2
1
3
J = 10.7, 7.8 Hz, 2H, CH
2
3
1
+
1
17.8, 103.1, 95.9, 86.3, 85.6, 68.1, 46.2, 29.0, 25.8, 16.3; ESI – MS: 466.7 [M + H ].
(465.22)
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2-(4-methylpiperazin-1-yl)ethy
l)amino)pyridin-2-yl)amino)benzonitrile (E-h)
C
28
H
27
N
5
O
2
(

1
White solid, yield 31%, mp: 144-146 °C; H NMR (400 MHz, CDCl ) δ: 7.51 (d,
3
J = 1.6 Hz, 4H, PhH), 7.33 (d, J = 16 Hz, 1H, =CH), 7.20 (s, 2H, PhH), 6.96 (s, 1H,
NH), 5.83 (d, J = 16.6 Hz, 1H, =CH), 5.62 (s, 1H, pyridine-H), 5.35 (s, 1H,
pyridine-H), 3.34 (s, 2H, CH
2
), 2.80 (s, 4H, CH
2
), 2.68 (s, 2H, CH
2
), 2.45 (s, 3H,
1
3
CH
3
), 2.14 (s, 6H, CH
3
), 1.89 (s, 2H, CH
2
), 1.25 (s, 2H, CH
2
); C NMR (100 MHz,
CDCl
3
) δ: 152.7, 149.8, 133.2, 132.3, 128.2, 117.9, 96.0, 86.2, 51.7, 44.9, 16.3; ESI –
+
MS: 508.2 [M + H ]. C30
H
33
7
N O (507.27)
(
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2-morpholinoethyl)amino)pyri
din-2-yl)amino)benzonitrile (E-i)
White solid, yield 75%, mp: 201-202 °C; H NMR (400 MHz, CDCl
1
3
) δ: 7.55 –
7
6
.48 (dd, J = 24, 8 Hz, 4H, PhH), 7.34 (d, J = 16.6 Hz, 1H, =CH), 7.21 (s, 2H, PhH),
.54 (s, 1H, NH), 5.83 (d, J = 16.6 Hz, 1H, =CH), 5.59 (d, J = 1.6 Hz, 1H,
pyridine-H), 5.38 (d, J = 1.6 Hz, 1H, pyridine-H), 4.98 (s, 1H, NH), 3.78 – 3.66 (m,
4
2
1
6
H, 2CH
2
), 3.31 (d, J = 5.5 Hz, 2H, CH
2
), 2.60 (t, J = 5.9 Hz, 2H, CH
2
), 2.48 (s, 4H,
1
3
CH ), 2.19 (s, 6H, 2CH
2
3
); C NMR (100 MHz, CDCl ) δ: 166.4, 159.6, 154.6, 152.7,
3
49.8, 145.2, 133.3, 132.4, 130.9, 128.1, 119.7, 118.2, 117.7, 103.2, 96.0, 86.3, 85.3,
+
6.9, 57.2, 53.4, 38.3, 16.3; ESI – MS: 495.5 [M + H ]. C29
H
30
N
6
2
O (494.24)
(E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((3-morpholinopropyl)amino)pyr
idin-2-yl)amino)benzonitrile (E-j)
1
White solid, yield 73%, mp: 182-184 °C; H NMR (400 MHz, CDCl
dd, J = 23.6, 8.8 Hz, 4H, PhH), 7.33 (d, J = 16.6 Hz, 1H, =CH), 7.20 (s, 2H, PhH),
.50 (s, 1H, NH), 5.83 (d, J = 16.6 Hz, 1H, =CH), 5.53 (s, 1H, NH), 5.47 (d, J = 1.3
Hz, 1H, pyridine-H), 5.40 (d, J = 1.3 Hz, 1H, pyridine-H), 3.73 (t, J = 4.4 Hz, 4H,
3
) δ: 7.53
(
6
2
1
1
6
CH
2
), 3.30 (d, J = 5.7 Hz, 2H, CH
2
), 2.59 – 2.37 (m, 6H, 3CH
2
), 2.18 (s, 6H, 2CH ),
3
1
3
.85 – 1.74 (m, 2H, CH
2
); C NMR (100 MHz, CDCl ) δ: 166.4, 159.8, 154.7, 152.8,
3
49.9, 145.2, 133.2, 132.4, 130.8, 128.1, 119.7, 118.2, 117.6, 103.0, 95.9, 85.9, 84.8,
+
7.0, 57.5, 53.8, 41.7, 25.4, 16.3; ESI – MS: 509.2 [M + H ]. C30
H
32
N
6
2
O (508.26)
(
E)-4-((4-(Allylamino)-6-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amin
o)benzonitrile (F-a)
White solid, yield 57%, mp: 142-144 °C; H NMR (400 MHz, CDCl
1
3
) δ: 7.46 (d, J

=
8.8 Hz, 2H, PhH), 7.36 (m, 3H, PhH, =CH), 7.21(s, 2H, PhH), 7.04 (s, 1H, NH),
5
.84 (d, J = 16.8 Hz, 1H, =CH), 5.90(m, 1H, =CH) 5.40 (s, 1H, pyrimidine-H), 5.20
1
3
(
m, 2H, =CH
2
), 5.02 (s, 1H, NH), 3.96(s, 2H, CH
2
), 2.17 (s, 6H, CH ); C NMR (100
3
MHz, CDCl
3
) δ: 165.3, 158.5, 152.6, 149.9, 143.9, 133.0, 132.4, 130.8, 127.7, 119.5,
+
1
18.3, 118.0, 117.0, 104.0, 95.9, 44.2, 16.6; ESI – MS: 423.2 [M + H ]. C25
H
22
6
N O
(
422.48)
(E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2-methoxyethyl)amino)pyrimid
in-2-yl)amino)benzonitrile (F-b)
White solid, yield 38%, mp: 112-114 °C; H NMR (400 MHz, CDCl
8.8 Hz, 2H, PhH), 7.38 (m, 3H, 2PhH, =CH), 7.21(s, 2H, PhH), 7.05 (s, 1H, NH),
.84 (d, J = 16.4 Hz, 1H, =CH), 5.38 (s, 1H, NH), 5.24(s, 1H, pyrimidine-H), 3.58 (m,
1
3
) δ: 7.46 (d, J
=
5
3
1
3
H, CH
3
), 3.53 (s, 2H, CH
2
), 2.18 (s, 2H, CH
2
), 2.17 (s, 6H, CH
3
); C NMR (100
MHz, CDCl
19.5, 118.2, 118.0, 103.9, 95.9, 70.8, 58.9, 41.3, 16.5; ESI – MS: 441.2 [M + H ].
(440.50)
E)-4-((4-((2-Cyanoethyl)amino)-6-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)pyrimidin-
3
) δ: 165.3, 158.6, 149.9, 144.0, 132.9, 132.4, 132.2, 130.8, 129.4, 127.7,
+
1
C
25
H
24
N
6
O
2
(
2
-yl)amino)benzonitrile (F-c)
White solid, yield 35%, mp: 140-142 °C; H NMR (400 MHz, CDCl
H, PhH, =CH), 7.23 (s, 2H, PhH), 6.96 (s, 1H, NH), 5.85 (d, J = 16.4 Hz, 1H, =CH),
.46 (s, 1H, NH), 5.06 (s, 1H, pyrimidine-H), 2.75 (t, J = 6.4 Hz, 2H, CH ), 2.17 (s,
1
3
) δ: 7.41 (m,
5
5
6
1
2
1
3
H, CH
3
), 0.88 (t, 2H, CH
2
); C NMR (100 MHz, CDCl
3
) δ: 133.0, 132.4, 127.7,
+
21.8, 118.0, 96.0, 58.5, 35.9, 18.5, 16.4; ESI – MS: 436.2 [M + H ].
C
25
H
21
N
7
O(435.48)
E)-4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)-6-((2-(dimethylamino)ethyl)amino)
pyrimidin-2-yl)amino)benzonitrile (F-d)
White solid, yield 58%, mp: 160-162 °C; H NMR (400 MHz, CDCl
(
1
3
) δ: 7.49 (m,
2
1
2
H, PhH), 7.36 (m, 3H, PhH, =CH), 7.21 (s, 2H, PhH), 7.09 (s, 1H, NH), 5.84 (d, J =
6.4 Hz, 1H, =CH), 5.53 (s, 1H, NH), 5.35 (s, 1H, pyrimidine-H), 2.54 (t, J = 5.6 Hz,
1
3
H, CH
2
), 2.28 (s, 6H, CH
3
), 2.17 (s, 6H, CH
3
), 0.88 (t, J = 6.4 Hz, 2H, CH
2
); C
NMR (100 MHz, CDCl
3
) δ: 165.2, 158.6, 152.6, 149.9, 144.1, 133.0, 132.4, 130.8,

119.5, 118.3, 118.0, 103.8, 95.8, 65.3, 57.5, 45.1, 42.0, 38.7, 30.2, 29.1, 23.4, 23.1,
+
1
6.6, 14.1, 11.1; ESI – MS: 454.3 [M + H ]. C26
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2,2-dimethoxyethyl)amino)pyri
midin-2-yl)amino)benzonitrile (F-e)
White solid, yield 42%, mp: 120-122 °C; H NMR (400 MHz, CDCl
8.8 Hz, 2H, PhH), 7.36 (m, 3H, 2PhH, =CH), 7.21(s, 2H, PhH), 7.06 (s, 1H, NH),
.84 (d, J = 16.4 Hz, 1H, =CH), 5.40 (s, 1H, NH), 5.01(s, 1H, pyrimidine-H), 4.52(t, J
H
27
N
7
O(453.54)
(
1
3
) δ: 7.46 (d, J
=
5
1
3
=
4.8 Hz, 1H, CH), 3.50(s, 2H, CH
2
), 3.43 (s, 6H, CH
3
), 2.17 (s, 6H, CH ); C NMR
3
(
100 MHz, CDCl
3
) δ: 165.2, 158.6, 152.6, 149.9, 143.9, 133.0, 132.4, 130.8, 127.7,
+
1
19.5, 118.2, 118.0, 104.0, 102.3, 95.9, 54.2, 16.5; ESI – MS: 471.2 [M + H ].
C
26
H
26
N
6
3
O (470.52)
(
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-(cyclopropylamino)pyrimidin-2-
yl)amino)benzonitrile (F-f)
White solid, yield 27%, mp: 235-237 °C; H NMR (400 MHz, CDCl
H, PhH, =CH), 7.35 (m, 2H, PhH), 7.22 (s, 2H, PhH), 7.03 (s, 1H, NH), 5.84 (d, J =
6.4 Hz, 1H, =CH), 5.78 (s, 1H, NH), 5.31 (s, 1H, pyrimidine-H), 2.18 (s, 6H, CH ),
) δ: 169.6,
1
3
) δ: 7.40 (m,
3
1
0
1
1
3
1
3
.83 (m, 3H, CH, CH
2
), 0.62 (m, 2H, CH
2
); C NMR (100 MHz, CDCl
3
66.7, 158.3, 152.7, 149.9, 143.9, 132.9, 132.4, 130.8, 127.6, 119.5, 118.3, 117.9,
+
03.9, 95.8, 23.5, 16.6, 7.6; ESI – MS: 423.2 [M + H ]. C25
H
22
6
N O(422.48)
(E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-(((tetrahydrofuran-2-yl)methyl)a
mino)pyrimidin-2-yl)amino)benzonitrile (F-g)
1
White solid, yield 35%, mp: 140-142 °C; H NMR (400 MHz, DMSO) δ: 9.56 (s,
1
7
3
2
1
4
H, NH), 7.66 (m, 3H, 2PhH, NH), 7.62 (d, J = 8 Hz, 1H, =CH), 7.49 (s, 2H, 2PhH),
.45 (m, 3H, 2PhH, =CH), 6.42 (d, J = 8.8 Hz, 1H, =CH), 5.51 (s, 1H, pyrimidine-H),
.96 (m, 1H, CH), 3.76 (m, 2H, CH
2
), 3.61 (m, 2H, CH
2
), 2.10 (s, 6H, CH
3
), 1.81 (m,
1
3
H, CH
2
); C NMR (100 MHz, DMSO) δ:152.7, 150.6, 145.8, 133.0, 132.0, 131.4,
28.6, 120.1, 119.4, 118.5, 102.1, 96.5, 77.6, 67.6, 29.1, 25.6, 16.6, 16.5; ESI – MS:
+
67.2 [M + H ]. C27
H
26
N
6
2
O (466.53)
(E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-(piperidin-4-ylamino)pyrimidin-
2
-yl)amino)benzonitrile (F-h)

1
White solid, yield 29%, mp: 152-154 °C; H NMR (400 MHz, CDCl
8.4 Hz, 2H, PhH), 7.36 (m, 3H, 2PhH, =CH), 7.21 (d, J = 8.8 Hz, 2H, PhH), 5.84 (d,
J = 16.8 Hz, 1H, =CH), 4.86 (s, 1H, pyrimidine-H), 3.68 (m, 2H, 2NH), 3.16 (m, 2H,
3
) δ: 7.46 (d, J
=
1
3
CH
CDCl
03.8, 95.8, 58.4, 51.6, 45.2, 33.0, 28.9, 18.4, 16.6; ESI – MS: 466.3 [M + H ].
O(465.55)
E)-4-((4-(4-(2-Cyanovinyl)-2,6-dimethylphenoxy)-6-((2-morpholinoethyl)amino)pyri
midin-2-yl)amino)benzonitrile (F-i)
White solid, yield 62%, mp: 168-170 °C; H NMR (400 MHz, CDCl
8.4 Hz, 2H, PhH), 7.36 (m, 3H, PhH, =CH), 7.22 (s, 2H, PhH), 7.07 (s, 1H, NH),
.84 (d, J = 16.8 Hz, 1H, =CH), 5.48 (s, 1H, NH), 5.41 (s, 1H, pyrimidine-H), 3.72 (m,
H, CH ), 2.61 (t, J = 16.6 Hz, 2H, CH ), 2.50 (s, 4H, CH ), 2.17 (s, 6H, CH ), 1.81 (s,
H, CH ); C NMR (100 MHz, CDCl ) δ: 165.2, 158.6, 152.6, 149.9, 144.0, 133.0,
2
), 2.74 (m, 2H, CH
2
), 2.14 (m, 4H, CH
2
), 1.45 (s, 6H, CH
3
); C NMR (100 MHz,
3
) δ: 165.2, 164.3, 149.9, 139.6, 132.9, 132.4, 131.7, 128.0, 127.7, 121.7, 118.0,
+
1
C
27
H
27
N
7
(
1
3
) δ: 7.45 (d, J
=
5
4
2
1
–
2
2
2
3
1
3
2
3
32.4, 130.8, 127.7, 119.5, 118.2, 117.9, 103.9, 95.9, 66.9, 56.7, 53.3, 37.6, 16.6; ESI
+
MS: 496.7 [M + H ]. C28
H
29
N
7
O (495.58)
2
4
.2. In vitro anti-HIV assay
By taking advantage of MTT method described previously [31, 32], the
compounds were evaluated for their activity against WT HIV-1 [33] (strain HIV-IIIB),
double RT mutant strain HIV-1 IIIB (RES056 and F227L/V106A), five single RT
mutant strain HIV-1 IIIB (L100I, K103N, E138K, Y181C, Y188L), and HIV-2 [34]
(
strain ROD) in MT-4 cells.
Stock solutions (10× final concentration) of test compounds were added in 25-μL
volumes to two series of triplicate wells so as 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
wells 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(IIIB), HIV-1 (RES056, F227L/V106A, L100I, K103N, E138K, Y181C
and Y188L) or HIV-2 (ROD) 24 stock (50 μL) at 100-300 CCID50 (50% 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 test
compound. Exponentially growing MT-4 cells were centrifuged for 5 min at 1000 rpm
5
and the supernatant was discarded. The MT-4 cells were resuspended at 6×10
cells/mL, and 50-μL 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 an eight-channel computer-controlled photometer
(
Multi-scan Ascent Reader, Labsystems, Helsinki, Finland), at two wave-lengths (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%. The concentration achieving 50% protection from the cytopathic
effect of the virus in infected cells was defined as the 50% effective concentration
(
EC50). The symbol ‘>’ indicated the highest concentration at which the compound
was tested and still found to be non-cytotoxic.
4
.3. HIV-1 RT inhibition assay
A RT assay kit produced by Roche was selected for the RT inhibition assay. All
the reagents for performing the RT reaction came with the kit, and the ELISA
procedures for RT inhibition assay was performed following the description in the kit
protocol [35]. Briefly, the reaction mixture containing template/primer complex, viral
nucleotides (dNTPs) and RT in the incubation buffer with or without inhibitors was
incubated for 1 h at 37 °C. After that, the reaction mixture was transferred to a

streptavidin-coated microtiter plate and incubated for another 1 h at 37 °C to make
sure retranscriptional cDNA chain that consisted biotin-labeled dNTPs bound to
streptavidin. Then removed un-bound dNTPs using washing buffer and added
anti-DIG-POD working solution. After incubation for 1 h at 37 °C, the DIG-labeled
dNTPs incorporated in cDNA were bound to the anti-DIG-POD antibody. The
unbound anti-DIG-PODs were removed and the peroxidesubstrate (ABST) solution
was added to the MTPs. A colored reaction proceed during cleavage of the substrate
catalyzed by POD. The absorbance of the sample was determined at OD 405 nm
using a microtiter plate ELISA reader. The percentage inhibitory activity of RT
inhibitors was calculated by formula as given below:
%
Inhibition = [OD value with RT but without inhibitors - OD value with RT and
inhibitors]/[OD value with RT and inhibitors - OD value without RT and inhibitors].
The IC50 values corresponded to the concentrations of the inhibitors required to
inhibit biotin-dUTP incorporation by 50%.
4
.4.Molecular simulation
Molecular modeling was performed with the Tripos molecular modeling packages
Sybyl-X 2.0. All the molecules for docking were built using standard bond lengths
and angles from Sybyl-X 2.0/base Builder. Then the molecules were optimized using
the Tripos force field for 1000 generations two times or more, until the minimized
conformers of the ligand were the same. The flexible docking method (Surflex-Dock)
[
36] docks the ligand automatically into the ligand binding site of the receptor by
using a protocol-based approach and an empirically-derived scoring function [37].
The protocol is a computational representation of a putative ligand that binds to the
intended binding site and is a unique and essential element of the docking algorithm
[
38]. The scoring function in Surflex-Dock, containing hydrophobic, polar, repulsive,
entropic, and solvation terms, was trained to estimate the dissociation constant (Kd)
2
expressed in -log(Kd) . Before docking, the protein was prepared by removing water
molecules, the ligand and other unnecessary small molecules from the crystal
structure of the ligand HIV-1 RT complex (PDB code: 3MEC and 3MED); meantime,
polar hydrogen atoms were added to the protein. Surflex-Dock default settings were
used for other parameters, such as the maximum number of rotatable bonds per
molecule (set to 100), and the maximum number of poses per ligand (set to 20).
During the docking procedure, all of the single bonds in residue side-chains inside the
defined RT binding pocket were regarded as rotatable or flexible, and the ligand was

allowed to rotate at all single bonds and move flexibly within the tentative binding
pocket. The atomic charges were recalculated using the Kollman all-atom approach
for the protein and the Gasteiger-Hückel approach for the ligand. The binding
interaction energy was calculated, including van der Waals, electrostatic, and torsional
energy terms defined in the Tripos force field. The structure optimization was
performed for 10,000 generations using a genetic algorithm, and the 20-best-scoring
2
ligand-protein complexes were kept for further analysis. The -log(Kd) values of the
2
0-best-scoring complexes, representing the binding affinities of ligand with RT,
-2
-9
encompassed a wide scope of functional classes (10 - 10 ). Thus, only the
highest-scoring 3D structural model of the ligand-bound RT was chosen to define the
binding interaction [39].
Acknowledgements
Financial support from the Key Project of NSFC for International Cooperation (No.
8
2
1420108027), Young Scholars Program of Shandong University (YSPSDU No.
016WLJH32), The Fundamental Research Funds of Shandong University
(
No.2017JC006), Key research and development project of Shandong Province (No.
017CXGC1401) and Major Project of Science and Technology of Shandong
2
Province (No. 2015ZDJS04001) is gratefully acknowledged.
Author Contributions
The manuscript was written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest. No writing assistance was
utilized in the production of this manuscript.
References
[
5
[
1] T. Mathes, D. Pieper, S.L. Antoine, M. Eikermann, Hiv Medicine, 14 (2013), p.
83.
2] L. Slama, L.I. Xiuhong, T. Brown, L.P. Jacobson, G. Pialoux, B. Macatangay, R.
Bolan, J. Phair, F.J. Palella, J Acquir Immune Defic Syndr (1999), 65.1 (2014), p. 57.
[
(
[
3] P. Zhan, X. Chen, D. Li, Z. Fang, E. De Clercq, X. Liu, Med Res Rev, 33 Suppl 1
2013) E1.
4] Y. Song, Z. Fang, P. Zhan, X. Liu, Curr Med Chem, 21 (2014), p. 329.

[
1
[
5] X. Li, L. Zhang, Y. Tian, P. Zhan, X. Liu, Expert Opin Ther Pat, 24 (2014), p.
199.
6] W. Chen, P. Zhan, D. Rai, E. De Clercq, C. Pannecouque, J. Balzarini, Z. Zhou, H.
Liu, X. Liu, Bioorg Med Chem, 22 (2014), p.1863.
[
[
7] D. Jochmans, Virus Research, 134 (2008), p. 171.
8] X. Chen, X. Liu, Q. Meng, D. Wang, H. Liu, E. De Clercq, C. Pannecouque, J.
Balzarini, X. Liu, Bioorg Med Chem Lett, 23 (2013), p. 6593.
9] Z. Liu, W. Chen, Z. Peng, E. De Clercq, C. Pannecouque, X. Liu, Eur J Med Chem,
7 (2014), p. 52.
10] J. Yang, W. Chen, D. Kang, X. Lu, X. Li, Z. Liu, B. Huang, D. Daelemans, C.
Pannecouque, E. De Clercq, Eur J Med Chem, 109 (2016), p.294.
11] W. Chen, P. Zhan, D. Daelemans, J. Yang, B. Huang, E. De Clercq, C.
Pannecouque, X. Liu, Eur J Med Chem, 121 (2016), p.352.
[
8
[
[
[
[
12] H. Gunaydin, M.D. Bartberger, ACS Med Chem Lett, 7 (2016), p. 341.
13] D.J. Kertesz, C. Brotherton-Pleiss, M. Yang, Z. Wang, X. Lin, Z. Qiu, D.R.
Hirschfeld, S. Gleason, T. Mirzadegan, P.W. Dunten, Bioorg Med Chem Lett, 20
(
[
[
2010), p.4215.
14] E.B. Lansdon, J Med Chem, 53 (2010), p.4295.
15] S. Yang, C. Pannecouque, D. Daelemans, X.D. Ma, Y. Liu, F.E. Chen, E.D.
Clercq, Eur J Med Chem, 65 (2013), p. 134.
16] X. Tian, B. Qin, Z. Wu, X. Wang, H. Lu, S.L. Morrisnatschke, C.H. Chen, S.
Jiang, K.H. Lee, L. Xie, J Med Chem, 53 (2010), p.8287.
17] V. Kumar, J.A. Dority, E.R. Bacon, B. Singh, G.Y. Lesher, J.org.chem, 57 (1992),
p.6995.
18] S.G. Ruggeri, B.C. Vanderplas, B.G. Anderson, R. Breitenbach, F.J. Urban,
A.M.S. Iii, G.R. Young, Org.process Res.dev, 12 (2008), p.411.
19] K.W. Anderson, R.E. Tundel, I. Takashi, R.A. Altman, S.L. Buchwald,
Cheminform, 45 (2006), p. 6523.
20] C. Kaneko, K. Uchiyama, M. Sato, N. Katagiri, Chem Pharm Bull, 18 (2008), p.
658.
21] Y. Liu, G. Meng, A. Zheng, F. Chen, W. Chen, E. De Clercq, C. Pannecouque, J.
Balzarini, Eur J Med Chem, 62 (2014), p. 334.
22] T.I. de Silva, M. Cotten, S.L. Rowland-Jones, Trends Microbiol, 16 (2008),
p.588.
23] C. Charpentier, L. Larrouy, G. Collin, F. Damond, S. Matheron, G. Chêne, T. Nie,
R. Schinazi, F. Brun-Vézinet, D. Descamps, Aids, 24 (2010), p. 2753.
24] R.A. Smith, D.N. Raugi, C. Pan, P.S. Sow, M. Seydi, J.I. Mullins, G.S. Gottlieb,
Retrovirology, 12 (2015), p.10.
25] R.A. Smith, D.N. Raugi, N.B. Kiviat, S.E. Hawes, J.I. Mullins, P.S. Sow, G.S.
Gottlieb, Aids, 25 (2011), p. 2235.
26] D. Desbois, B. Roquebert, G. Peytavin, F. Damond, G. Collin, A. Bénard, P.
Campa, S. Matheron, G. Chêne, F. Brunvézinet, Antimicrob Agents Ch, 52 (2008), p.
545.
27] D.N. Raugi, R.A. Smith, S. Ba, M. Toure, F. Traore, F. Sall, C. Pan, L.
[
[
[
[
[
3
[
[
[
[
[
[
1
[

Blankenship, A. Montano, J. Olson, Antimicrob Agents Ch, 57 (2013), p.2751.
[
[
28] L. Menéndez-Arias, J. Tözsér, Trends Pharmacol Sci, 29 (2008), p. 42.
29] K. Suzuki, B.P. Craddock, N. Okamoto, T. Kano, R.T. Steigbigel, J Virol
Methods, 44 (1993), p. 189.
30] P. Zhan, X. Chen, X. Li, D. Li, Y. Tian, W. Chen, C. Pannecouque, E. De Clercq,
X. Liu, Eur J Med Chem, 46 (2011), p. 5039.
[
[
[
[
[
31] C. Pannecouque, D. Daelemans, E. De Clercq, Nature protocols, 3 (2008), p.427.
32] T. Mosmann, J Immunol Methods, 65 (1983), p. 55.
33] M. Popovic, M.G. Sarngadharan, E. Read, R.C. Gallo, in, 1984, p. 497.
34] F. Clavel, D. Guetard, F. Brun-Vezinet, S. Chamaret, M.A. Rey, M.O.
Santos-Ferreira, A.G. Laurent, C. Dauguet, C. Katlama, C. Rouzioux, et al., Science,
33 (1986), p. 343.
35] X. Li, W. Chen, Y. Tian, H. Liu, P. Zhan, E. De Clercq, C. Pannecouque, J.
Balzarini, X. Liu, Eur J Med Chem, 80 (2014), p. 112.
2
[
[
[
[
[
36] R. Spitzer, A.N. Jain, J Comput Aid Mol Des, 26 (2012), p.687.
37] A.N. Jain, J Comput Aid Mol Des, 21 (2007), p. 281.
38] A.N. Jain, J Med Chem, 46 (2003), p.499.
39] J. Ruppert, W. Welch, A.N. Jain, Protein Science, 6 (1997), p. 524.

Highlights
All diarylpyri(mi)dines exhibited robust activities against wild-type (WT) HIV-1 with
EC50 ranging from 1.36 nM to 29 nM.
Most compounds displayed significant activity against a wide variety of HIV-1
mutated strains.
·
·
·
·
Unexpectedly, four diarylpyrimidines demonstrate moderate anti-HIV-2 activities.
Preliminary SARs and molecular modeling analysis were detailed.