Discovery of novel DAPY-IAS hybrid derivatives as potential HIV-1 inhibitors using molecular hybridization based on crystallographic overlays

Article abstract of DOI:10.1016/j.bmc.2017.06.022

Crystallographic overlap studies and pharmacophoric analysis indicated that diarylpyrimidine (DAPY)-based HIV-1 NNRTIs showed a similar binding mode and pharmacophoric features as indolylarylsulfones (IASs), another class of potent NNRTIs. Thus, a novel series of DAPY-IAS hybrid derivatives were identified as newer NNRTIs using structure-based molecular hybridization. Some target compounds exhibited moderate activities against HIV-1 IIIB strain, among which the two most potent inhibitors possessed EC₅₀ values of 1.48?μM and 1.61?μM, respectively. They were much potent than the reference drug ddI (EC₅₀?=?76.0?μM) and comparable to 3TC (EC₅₀?=?2.54?μM). Compound 7a also exhibited the favorable selectivity index (SI?=?80). Preliminary structure-activity relationships (SARs), structure-cytotoxicity relationships, molecular modeling studies, and in silico calculation of physicochemical properties of these new inhibitors were also discussed.

Full text of DOI:10.1016/j.bmc.2017.06.022

Accepted Manuscript
Discovery of novel DAPY-IAS hybrid derivatives as potential HIV-1 inhibitors
using molecular hybridization based on crystallographic overlays
Boshi Huang, Xueshun Wang, Xinhao Liu, Zihui Chen, Wanzhuo Li, Songkai
Sun, Huiqing Liu, Dirk Daelemans, Erik De Clercq, Christophe Pannecouque,
Peng Zhan, Xinyong Liu
PII:
S0968-0896(17)30837-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.022
BMC 13805
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
20 April 2017
31 May 2017
13 June 2017
Please cite this article as: Huang, B., Wang, X., Liu, X., Chen, Z., Li, W., Sun, S., Liu, H., Daelemans, D., De Clercq,
E., Pannecouque, C., Zhan, P., Liu, X., Discovery of novel DAPY-IAS hybrid derivatives as potential HIV-1
inhibitors using molecular hybridization based on crystallographic overlays, Bioorganic & Medicinal Chemistry
(
2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.06.022
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Discovery of novel DAPY-IAS hybrid derivatives as potential HIV-1
inhibitors using molecular hybridization based on crystallographic
overlays
a
a
a
a
a
Boshi Huang , Xueshun Wang , Xinhao Liu , Zihui Chen , Wanzhuo Li , Songkai
a
b
c
c
c
Sun , Huiqing Liu , Dirk Daelemans , Erik De Clercq , Christophe Pannecouque ,
a,*
a,*
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 Ji’nan, Shandong, PR China
b
Department of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road,
50012 Ji’nan, Shandong, PR China
2
c
Rega Institute for Medical Research, KU Leuven Minderbroedersstraat 10, B-3000 Leuven,
Belgium
Abstract
Crystallographic overlap studies and pharmacophoric analysis indicated that
diarylpyrimidine (DAPY)-based HIV-1 NNRTIs showed a similar binding mode and
pharmacophoric features as indolylarylsulfones (IASs), another class of potent
NNRTIs. Thus, a novel series of DAPY-IAS hybrid derivatives were identified as
newer NNRTIs using structure-based molecular hybridization. Some target
compounds exhibited moderate activities against HIV-1 IIIB strain, among which the
two most potent inhibitors possessed EC50 values of 1.48 μM and 1.61 μM,
respectively. They were much potent than the reference drug ddI (EC50 = 76.0 μM)
and comparable to 3TC (EC50 = 2.54 μM). Compound 7a also exhibited the favorable
selectivity index (SI = 80). Preliminary structure-activity relationships (SARs),
structure-cytotoxicity relationships, molecular modeling studies, and in silico
Corresponding authors. Tel: +86 531 88380270; fax: +86 531 88382731; E-mail address:
zhanpeng1982@sdu.edu.cn (Zhan P.); xinyongl@sdu.edu.cn (Liu X.Y.)
1

calculation of physicochemical properties of these new inhibitors were also discussed.
Keywords
HIV-1 inhibitors; DAPY-IAS hybrids; crystallographic overlays; drug design; SARs;
molecular modeling.
1
. Introduction
Currently, non-nucleoside reverse transcriptase inhibitors (NNRTIs) are major
components of highly active antiretroviral therapy (HAART) to treat HIV-1 infections
[
1-3]. However, the usage of the first-generation NNRTIs has been significantly
compromised by the emergence of drug-resistant HIV-1 strains (especially K103N
and Y181C) [4-6]. Considerable efforts have been made to address the low resistance
barrier issue over the past two decades, leading to the discovery of the
second-generation NNRTIs [7]. Diarylpyrimidine (DAPY) derivatives, as the most
dazzling pearl, exhibit remarkable potency against both the wild-type (wt) and
drug-resistant HIV-1 strains. Two DAPY-type drugs, etravirine (ETR, 2008) and
rilpivirine (RPV, 2011) have been approved for clinical use [8] (Fig. 1). Nevertheless,
DAPYs suffer from the issues of poor solubility, hyper sensitivity reactions, emerging
drug resistance and virologic failure [9-11]. Therefore, there is still a compelling need
to develop novel NNRTIs to address the current issues [12].
Moreover, indolylarylsulfone (IAS) derivatives (lead compound RS-80 and its
derivative ZZ-7e) represent another class of next generation NNRTIs with broad
spectrum of activities against wt and mutant HIV-1 strains or HIV-1 RTs in the
nanomolar range (Fig. 1). Besides, the reported crystallographic structure of
ZZ-7e/RT complexes (PDB code: 2RF2) and the extensive molecular modeling
studies have provided clear information on the binding modes of IASs [13-17].
2

Figure 1. Chemical structures of ETR, RPV, ZZ-7e and RS-80
Although IASs possess a structurally distinct scaffold from that of DAPYs, the two
types of NNRTIs share similar pharmacophoric features and binding conformations.
The co-crystal structure of ZZ-7e bound in the NNRTI binding site (NNBS) was
overlaid with the reported structure of RPV/RT complex (PDB code: 3MEE), which
demonstrated that most fragments in their structures can overlap well with each other
(
Fig. 2). The pyrrolidine ring of ZZ-7e and the 2,6-dimethyl-4-cyanovinylphenoxyl
moiety of RPV both participate in hydrophobic interactions with residues Y181, Y188
and W229 (region A). The 5-chloro phenyl group of ZZ-7e can highly overlap with
the right 4-cyano phenyl group of RPV, pointing toward the protein/solvent interface
(
region C). The 2-carbamoyl-pyrrole moiety functions as hydrogen bond acceptor and
donor, like the right NH linker and 1’-N of the pyrimidine ring of RPV, developed
dual key hydrogen bonds with the backbone of K101 (region B). These hydrogen
bonding interactions are critical for binding affinity of the two classes of NNRTIs,
especially in the cases of the appearance of K101 mutants. What’s more, as mentioned
above, it’s indicated that RS-80 bearing a 3,5-dimethylphenyl moiety presents almost
the same binding mode as that of ZZ-7e in NNBS by molecular modeling studies, as
the 3,5-dimethylphenyl moiety fits into the same sub-pocket surrounded by residues
Y181, Y188 and W229 as pyrrolidine ring of ZZ-7e.
3

Figure 2. Crystallographic overlays of the RT co-crystal structures of RPV (green) (PDB code:
MEE, resolution: 2.4 Å) and ZZ-7e (yellow) (PDB code: 2RF2, resolution: 2.4 Å) with RT
performed by SYBYL-X 1.1, shown by PyMOL version 1.5, http://www.pymol.org/).
3
(
Prompted by the analyses of the crystallographic overlay study and pharmacophoric
features of RPV and ZZ-7e, combined with the molecular simulation studies, a novel
series of DAPY-IAS hybrid derivatives was designed using molecular hybridization
based on crystallographic overlays[18-20]. Concretely, as shown in Fig. 3, the core
ring was pyrimidine or pyridine, which was expected to anchor the functional groups
and to serve as key hydrogen bond acceptor as well. Meanwhile, the preferred
p-aminobenzonitrile moiety of DAPYs was installed at the right-wing portion of the
central ring. In addition, substituted aryl group anticipated to fit into the aromatic-rich
sub-pocket as 3,5-dimethylphenyl group of IASs was connected to the core ring
through a sulfamide (SO NH) linker. Different substituents, varying in size, electronic
2
nature and position were linked to the left aryl ring to establish preliminary
structure-activity relationships (SARs) (Fig. 3). The yielded hybrid molecules are
expected to hold pre-selected characteristics of the parent ones. Herein, we report the
synthesis, biological evaluation and molecular modeling studies of these novel
DAPY-IAS hybrid derivatives as potential HIV-1 NNRTIs.
4

Figure 3. Newly designed DAPY-IAS hybrid derivatives
2
. Results and discussion
.1. Chemistry
2
Newly designed DAPY-IAS derivatives were expeditiously synthesized via two
different routes. As shown in Scheme 1, commercially available
2
-chloropyridin-4-amine 1 was chosen as the starting material, which first reacted
with 4-aminobenzonitrile to afford the key intermediate 2 by Buchwald–Hartwig
reaction[21]. Subsequently, construction of the sulfonamide functionality to yield
3
a-k could be implemented by stirring 2 with the corresponding arylsulfonyl chloride
in pyridine at room temperature[22]. However, the attempts to prepare the desired
compounds 7a-k through a similar direct two-step approach was not successful
(
Scheme 2). It was probably because the resulting product at the first step was quite
electron deficient for an attack of the electrophile. An alternative synthetic approach
has been established for 7a-k (Scheme 3), in which arylsulfonyl chlorides were
converted into corresponding arylsulfamides. Specifically, 2-methylthio-4-pyrimidone
4
5
was first transformed into 4-((4-oxo-4,5-dihydropyrimidin-2-yl)amino)benzonitrile
through reacting with excess amount of 4-aminobenzonitrile at a high temperature
of 150 °C under N
oxychloride (POC1
-((4-chloropyrimidin-2-yl)amino)benzonitrile 6. Finally, 6 underwent electrophilic
2
atmosphere[23]. And then, chlorination of 5 with phosphorous
3
)
at 100 °C produced the important intermediate
4
reactions with well prepared arylsulfamides under high temperature to give the target
compounds 7a-k. These new synthesized compounds were featured by
physicochemical and spectral means, and both analytical and spectral data of these
5

target compounds were totally consistent with their molecular structures.
Scheme 1. Reagents and conditions: (a) 4-aminobenzonitrile, Pd(OAc)
dioxane, N , 100℃; (b) ArSO Cl, pyridine, 0℃, r.t.
2 2 3
, Xantphos, Cs CO ,
2
2
Scheme 2. The initial designed synthetic route for 7a-k.
Scheme 3. Reagents and conditions: (a) 4-aminobenzonitrile, N
2
, 150℃; (b) POCl , 100℃; (c)
3
2 2
ArSO NH , NaH, DMSO, 130℃.
2
.2. Anti-HIV activity in MT-4 cells
All the target compounds were screened for their in vitro anti-HIV activity and
cytotoxicity in human T-lymphocyte (MT-4) cell cultures infected with the wt HIV-1
(
IIIB). Some compounds were also evaluated for their potency against
K103N+Y181C double mutant HIV-1 strain (RES056) and HIV-2 strain (ROD). The
biological results are represented as EC50 values (anti-HIV activity), CC50 values
(
cytotoxicity) and SI values (selectivity index, CC50/EC50), and are summarized in
6

Table 1, with lamivudine (3TC), didanosine (ddI), RS-80, ETR and RPV as
references.
In total, 9 compounds exhibited moderate anti-HIV-1 (IIIB) activity in the micromolar
range (EC50 = 1.48 – 48.9 μM) with selectivity index (SI) values ranging from 2 to 80.
Among them, 3a and 7a were found to be the two most active inhibitors possessing
EC50 values of 1.48 μM and 1.61 μM respectively, which were much lower than the
reference drugs ddI (EC50 = 76.0 μM) and comparable to 3TC (EC50 = 2.54 μM),
while much higher than for RS-80 (EC50 = 0.0026 μM), ETR (EC50 = 0.0040 μM) and
RPV (EC50 = 0.0013 μM) against IIIB strain. Compound 7a also exhibited the highest
selectivity index (SI = 80). Additionally, none of the tested target compounds was
effective against the double mutant HIV-1 RES056 strain and HIV-2 strain.
The contribution of the core ring of these new hybrids to the antiviral activities was
firstly assessed. Compared to 3a (EC50 = 1.48 μM) or 3c (EC50 = 4.92 μM) with the
same CH or tert-butyl substituents at 4-position of the left phenyl ring respectively,
3
compounds 7a (EC50 = 1.61 μM) and 7c (EC50 = 7.52 μM) displayed comparable or
slightly inferior potency. On the other hand, more than half of the compounds in 7a-k
sub-series (6/11) possessed mild activities against IIIB strain, which is better than the
performance of compounds in 3a-k sub-series (3/11). Considering these facts, the
pyridine ring was an equivalent scaffold for these 4-sulfamide inhibitors to exhibit
anti-HIV-1 activity as the pyrimidine ring. The results were in accordance with those
of previously reported DAPYs [24-26].
Next, 4-position substituents, varying in size and electronic nature, were introduced
on the left phenyl ring of these 4-sulfamide analogues. It was found that compounds
3
a and 7a both bearing a 4-methyl-substituent on the left ring showed the best
activities. We also observed that the sequences of potency of these 4-position
substituted compounds in the two sub-series indicated the similar declining trend:
methyl > tert-butyl > CF
3
in 3a-e (3a vs 3c vs 3e), and methyl > tert-butyl > cyano >
CF in 7a-e (7a vs 7c vs 7b vs 7e). Some derivatives bearing electron-withdrawing
3
groups even lost activity (like 3b, 3e, 7d and 7e). It was therefore concluded that
electron-donating groups at the 4-position on the left phenyl ring were more favorable
7

for the activity than the electron-withdrawing groups.
As the 3,5-dimethylphenyl moiety and 2,4,6-trimethylphenyl moiety have been
proved to be the optimal or “privileged” sub-structures in IASs and DAPYs
respectively, combined with the existing results, 3,5-dimethyl and 2,4,6-trimethyl
groups and their bioisosteres were further introduced on the left phenyl ring (3f-g
and 7f-g). However, decreased or even lost anti-HIV-1 activity were obtained for
these analogues. Certainly, some crucial clues can be gained from the results:
3
2
2
,5-dimethyl > 3,5-di-F > 3,5-di-CF (7f vs 7g vs 7h), while 2,4,6-trimethyl >
3
,4,6-tri-isopropyl (3i vs 3j). The 3,5-dimethylphenyl moiety and
,4,6-trimethylphenyl moiety were still the best in the sub-series. Finally, the bigger
naphth-2-yl group was introduced to investigate its influence on the antiviral profiles.
The unsatisfactory biological results revealed it was not favorable for activity
(
EC50 >30.0 μM for 3k, EC50 = 48.9 μM for 7k).
Taken together, our preliminary SAR analysis of these novel DAPY-IAS hybrid
derivatives demonstrated the key structural fragments to obtain better activity against
wt HIV-1: (i) pyridine ring was an equivalent scaffold as the pyrimidine ring. ii)
electron-donating groups at the 4-position on the left phenyl ring were more favorable
than the electron-withdrawing groups. iii) 3,5-dimethyl and 2,4,6-trimethyl groups on
the left phenyl ring were not beneficial, distinct from those of IASs and DAPYs. iv)
the bigger naphth-2-yl group hampered the anti-HIV activity.
Table 1. In vitro anti-HIV-1 (IIIB) activity and cytotoxicity of target compounds (3a-k, 7a-k).
a
b
c
Compd
Ar
EC50 (μM)
CC50 (μM)
SI
3a
4-CH
3
-Ph
1.48 ± 0.27
14.0 ± 3.82
9
8

3
3
3
3
3
3
3
3
3
3
7
7
7
7
7
7
7
7
7
7
b
c
d
e
f
4-CN-Ph
>93.4
93.4 ± 21.0
29.3 ± 0.76
6.12 ± 0.39
28.0 ± 1.39
5.06 ± 0.31
28.6 ± 2.11
62.2 ±17.7
22.2 ± 3.55
11.5 ± 7.89
30.0 ± 0.84
130 ± 88.7
159 ± 6.96
30.3 ± 1.27
36.6 ± 2.74
54.7 ± 20.0
173 ± 14.7
93.9 ± 41.3
115 ± 5.62
4.40 ± 0.48
17.4 ± 0.44
77.1 ± 29.7
>87.2
<1
6
4-(tert-butyl)-Ph
4-F-Ph
4.92 ± 0.51
>6.12
<1
<1
<1
<1
<1
4
4-CF
3,5-di-CH
3,5-di-F-Ph
3,5-di-CF -Ph
2,4,6-tri-CH -Ph
3
-Ph
>28.0
3
-Ph
>5.06
g
h
i
>28.6
3
>62.2
3
6.08± 0.23
>11.5
j
2,4,6-tri-isopropyl-Ph
Naphth-2-yl
<1
<1
80
4
k
a
b
c
d
e
f
>30.0
4-CH
3
-Ph
1.61 ± 0.15
39.4 ± 3.95
7.52 ± 0.53
≥9.05
4-CN-Ph
4-(tert-butyl)-Ph
4-F-Ph
4
≤4
<1
28
2
4-CF
3,5-di-CH
3,5-di-F-Ph
3,5-di-CF -Ph
2,4,6-tri-CH -Ph
3
-Ph
>54.7
3
-Ph
6.21 ± 0.51
47.0 ± 2.92
>115
g
h
i
3
<1
<1
<1
2
3
>4.40
j
2,4,6-tri-isopropyl-Ph
Naphth-2-yl
>17.4
7
3
k
48.9 ± 15.2
TC
2
7
0
0
0
.54 ± 0.55
>34
>3
4260
541
3490
ddI
6.0 ± 18.3
>212
RS-80
ETR
RPV
.0026 ± 0.0007
.0040 ± 0.0003
.0013 ± 0.0004
11.0 ± 5.29
2.15 ± 0.11
4.38 ± 1.42
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cells against
HIV-1-induced cytopathicity, as determined by the MTT method.
9

b
CC50: concentration required to reduce the viability of mock-infected cells by 50%, as
determined by the MTT method.
c
SI: selectivity index (CC50/EC50).
Regarding the cytotoxicity, it can be observed that 7f possessed the lowest
cytotoxicity (CC50 = 173 μM) and the second highest safe profile (SI = 28) among all
the componds. It is noteworthy that the cytotoxicities of sub-series 7a-k were lower
than those of subseries 3a-k, except for 7i (CC50 = 4.40 μM for 7i, CC50 = 22.2 μM
for 3i). In addition, compounds 3b and 7b bearing a 4-CN substituent on the left
phenyl ring were found to be least toxic and second least toxic in their sub-series,
indicating this group was more “safe” than others.
In brief, in spite of the fact that these DAPY-IAS hybrids did not exhibit improved
antiviral potency in comparison with the parent ones, they represented a novel class of
anti-HIV agents with great potential for further modifications. Most importantly, the
new DAPY-IAS hybrid analogues can be readily derivatized, facilitating more
extensive SAR studies in the future.
2
.3. Inhibition of HIV-1 RT (wt)
To better understand the action mechanism of the newly synthesized inhibitors, the
two most potent analogues 3a and 7a were preliminarily tested in recombinant HIV-1
RT inhibitory assays which use poly (A) × oligo (dT)15 as template/primer (RT kit,
Roche). As listed in Table 2, compound 3a exhibited a very weak activity against
HIV-1 RT with a inhibition rate 5.7% at a concentration of 50 μM, while 7a showed a
moderate anti-RT potency with a inhibition rate 41.5%. Although the HIV-1 RT
inhibitory activities of 3a and 7a were inferior to those of control drugs (for NVP,
8
8.0% and for ETR, 93.3%), the results demonstrated that the new compounds could
probably act as potential HIV-1 NNRTIs, but their binding abilities with HIV-1 RT
were unsatisfactory which needed to be improved by further optimizations.
a
Table 2. Inhibitory activity of compounds 3a, 7a, NVP and ETR against HIV-1 RT .
Compd
Inhibition
3a
7a
NVP
88.0
ETR
93.3
b
%
5.7
41.5
10

a
The RT kit was commercially available and supplied by Roche, and data were obtained by
standard ELISA.
b
The inhibition rates of the tested compounds inhibiting biotin deoxyuridine triphosphate
(
biotin-dUTP) incorporation into the HIV-1 RT at a concentration of 50 μM.
2
.4. Molecular modeling analysis
Molecular modeling studies were performed to further provide the possible clues for
biological activities. The representative compounds 3a, 3i and 7f were selected to be
docked into the NNIBP by Surflex-Dock module of Sybyl-X 1.1 software, and the
docking results were shown by PyMOL. Default parameters were used as described in
the SYBYL-X 1.1 manual unless otherwise specified. PDB 3MEE of the wt RT/RPV
complex was used due to the high structural similarity between RPV and the newly
designed hybrid compounds.
As shown in Fig. 4a, the detailed binding pattern of compound 3a can be depicted as
follows: i) The overall conformation of compound 3a in the NNBS was a classical
“U” shape, which was highly similar to DAPYs. ii) The 4-methylbenzene moiety
fitted into a hydrophobic sub-pocket surrounded by the aromatic side chains of Y181,
Y188, F227, and W229, generating potential Van der Waals interactions. Particularly,
it was parallel to the side-chain phenyl group of Y188. iii) 1’-N of the pyridine ring
and the NH linker in 3a formed dual hydrogen bonds with backbone α-amino and
carbonyl of residue K101 as expected. iv) Moreover, the right benzonitrile moiety
extended to the solvent exposure region, which was surrounded by residues V106 and
P236. On the whole, the binding conformation of 3a revealed a fair superposition to
that of RPV, giving rise to its moderate anti-HIV potency.
Besides, the conformation of 3i bearing a 2,4,6-trimethylphenyl group in the NNBS
was shown in overlap with its analogue 3a (Fig. 4b). Obviously, 3i developed dual
key hydrogen bonds with K101 like 3a. However, 3i adopted an irregular “U”
conformation, which an angle existed between the 2,4,6-trimethylphenyl group and
the 4-methylbenzene moiety of 3a. It was conjectured that steric hindrance of the
bigger trisubstituted functional groups pushed 3i to move slightly towards the bottom
11

of the binding site compared to 3a, disrupting positive Van der Waals interactions
between the 2,4,6-trimethylphenyl group and the aromatic-rich sub-pocket, thus
leading to a 4-fold reduced antiviral potency (for 3a, EC50 = 1.48 μM; for 3i, EC50
.08 μM).
The binding mode of the inhibitor 7f was also studied as it possesses a
,5-dimethylphenyl moiety as IASs. It's important to note that this moiety solely
=
6
3
exists in potent IASs other than DAPYs. As revealed in Fig. 4c, 7f adopted a minor
twisted “U” conformation in the NNBS. Two hydrogen bonds also formed between
the 1’-N of the pyrimidine core ring and the NH linker in 7f and K101. Surprisingly,
one oxygen atom of SO formed an extra hydrogen bond with backbone NH of Y181,
2
which was uncommon in IASs and DAPYs. This special hydrogen bonding
interaction may play an important role in contributing to the protein-ligand binding
affinity.
Figure 4. (a) Proposed binding model of 3a docked into wt RT; (b) Superimposition of the docked
12

conformations of 3i (yellow) and 3a (magenta) in wt RT; (c) Proposed binding model of 7f docked
into wt RT. PDB code for molecular docking: 3MEE. Hydrogen bonds are indicated with dashed
lines in red, and all the non-polar hydrogen atoms are omitted for clarity. The docking results were
performed by SYBYL-X 1.1, shown by PyMOL.
Moreover, the obtained docking scores of 3a, 3i and 7f were shown in Table 3. All
three compounds have moderate to excellent total scores, but the total scores
displayed a reverse trend to the anti-HIV-1 activities. Some valuable information
could be also derived if just considering the scores: for 3a and 3i, the differences of
the “Polar” item maybe contribute to the different total scores. In addition, the
absolute value of the ‘Crash’ item of 7f is 2-fold less than that of 3i, which was mostly
responsible for the increased total score of 7f compared to that of 3i.
a
Table 3. Docking scores of 3a, 3i and 7f binding into wt RT (3MEE )
Compd T_S
C
P
S
D_S
P_S
G_S
CHS
CS G_C
3
3
7
a
i
5.52
7.32
9.58
-1.83
-1.68
-0.85
0.65
2.23
2.20
0.63
0.68
0.69
-144.34
-152.49
-155.57
-72.63
-86.37
-102.56
-212.01
-256.43
-262.00
-38.77
-45.67
-42.72
3
3
5
3
3
5
f
a
T_S = Total_score; C = Crash; P = Polar; S = Similarity; D_S = D_SCORE; P_S = PMF_SCORE;
G_S = G_SCORE; CHS = CHEMSCORE, CS = CSCORE, G_S = GLOBAL_CSCOR.
Overall, the molecular modeling studies indicated the designed compounds could
adopt similar conformations in NNBS as those of the leads DAPYs and IASs, and
keep the crucial binding interactions with RT as well. However, it’s a dynamic process
of mutual induction when NNRTIs bind with RT, which could likely be severely
influenced by the minor change of the molecular bulk properties. Also, it’s quite
difficult to describe the real induced-fit process and ultimate binding state between
NNRTIs and RT by current molecular simulation techniques. These maybe are the
reasons why some deviations appear between the obtained results and the expected
ones.
2
.5. In silico calculation of physicochemical properties
13

Furthermore, some physicochemical properties of the active HIV-1 inhibitors 3a, 7a,
and the lead compounds RS-80 and RPV (as control) were predicted using the free
on-line molinspiration software (http://www.molinspiration.com/). The calculated
results were summarized in Table 4, which suggested that the two compounds 3a, 7a
and the lead RS-80 meet the Lipinski’s ‘rule of five’ well, but the former ones could
not possess the striking antiviral potency as RS-80. It was therefore speculated that
there would be other factors, instead of physicochemical properties, which possibly be
responsible for their decreased inhibitory activities.
a
Table 4. Prediction of physicochemical properties of 3a, 7a, RS-80 and RPV.
Compd
EC50
nViol n.a miLogP MW
nON nOHNH nrotb TPSA
MV
(
μM) (wt)
Accepted
range
2
<
5
<500 Da <10
<5 ≤10 < 140 Å
3
7
a
a
1.48
1.61
0.0026
0.0013
0
0
0
1
26 3.95
26 3.42
24 3.47
28 5.46
364.42
365.41
362.83
366.43
6
7
5
6
2
2
3
2
5
94.88
107.77
93.03
97.42
312.36
308.20
292.79
337.61
5
3
5
RS-80
RPV
a
nViol = number of violations; n.a = no. of atoms; miLogP = molinspiration predicted LogP;
MW= molecular weight; nON = no. of hydrogen bond acceptors; nOHNH = no. of hydrogen bond
donors; nrotb = no. of rotatable bonds; TPSA = topological polar surface area; MV = molar
volume.
3
. Conclusion
Briefly, based on the crystallographic overlay studies and pharmacophoric analysis of
IASs and DAPYs-based NNRTIs, which showed similar binding modes and
pharmacophoric features, a novel series of DAPY-IAS hybrid derivatives were
designed using molecular hybridization based on crystallographic overlays. Some
target compounds exhibited moderate activities against HIV-1 IIIB strain, among
which the two most potent inhibitors 3a and 7a possessed EC50 values of 1.48 μM and
1
7
.61 μM, respectively. They were much potent than the reference drug ddI (EC50
=
6.0 μM) and comparable to 3TC (EC50 = 2.54 μM). Compound 7a also exhibited the
favorable selectivity index (SI = 80). Preliminary SARs and structure-cytotoxicity
relationships of these new inhibitors, in silico calculation of physicochemical
properties of compounds 3a and 7a as well, have been well discussed. Molecular
14

simulation studies also provided important information of binding modes of the new
inhibitors in NNBS and crucial clues for next-step modifications of this series of
derivatives.
4
. Experimental
4
.1. Chemistry
All melting points (mp) were determined on a micromelting point apparatus and are
uncorrected. Mass spectra were performed on a LC Autosampler Device: Standard
1
13
G1313A instrument by electrospray ionization. H NMR and C NMR spectra were
obtained on a Bruker AV-400 spectrometer (Bruker BioSpin, Fällanden, Switzerland)
in the indicated solvent DMSO-d . Chemical shifts were expressed in δ units (ppm),
6
using TMS as an internal standard, and J values were reported in hertz (Hz). TLC was
performed on Silica Gel GF254. Spots were visualized by irradiation with UV light (λ
2
54 nm). Flash column chromatography was carried out on columns packed with
silica gel 60 (200–300 mesh). Solvents were of reagent grade and, if needed, were
purified and dried by standard methods. The key reagents were purchased from
commercial suppliers and with no further purification when used. Rotary evaporators
were served in concentration of the reaction solutions under reduced pressure.
4
.1.1. General procedure for the synthesis of target compounds 3a-k
Preparation of 4-((4-aminopyridin-2-yl)amino)benzonitrile (2): A mixture of the
starting material 2-chloropyridin-4-amine
-aminobenzonitrile (1.84 g, 15.56 mmol), Cs CO
1
(2.00 g, 15.56 mmol),
(10.14 g, 31.12 mmol), Pd(OAc)
4
2
3
2
(
0.17 g, 0.78 mmol) and Xantphos (0.45 g, 0.78 mmol) was dissolved in 40 mL
,4-dioxane, and then the solution was refluxed at 100°C for 5h under N atmosphere.
1
2
After cooling to room temperature, the solution was filtered and concentrated under
reduced pressure. Finally, the residue was further purified by silica gel
chromatography with methanol/dichloromethane = 1/50 and recrystallized from
absolute methanol to give 2 (2.03 g) as a white solid. Yield: 62%. mp: 177.5-180.0°C.
+
ESI-MS: m/z 211.2 [M+1] , C12
H
10
4
N (210.09).
Preparation of target compounds 3a-k: Intermediate 2 (0.1 g) and arylsulfonyl
chloride (1 equiv) were dissolved in 3 mL dry pyridine at 0°C, and the reaction
15

mixture was stirred overnight at room temperature. The reaction was quenched by
adding water and acidifying the solution with 3 mL concentrated HCl. The product
was filtered and washed with water, or was extracted with ethyl acetate and dried over
Na
2
SO , and the solvent was evaporated. If needed, further purification was achieved
4
by recrystallization from absolute methanol, affording target compounds 3a-k.
4
.1.1.1. N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-4-methylbenzenesulfonamide (3a)
1
White solid, yield: 29%. mp: 237.5-239.5°C. H NMR (400 MHz, DMSO-d
6
) δ: 10.93
(
s, 1H, SO
2
NH), 9.63 (s, 1H, NH), 7.99 (d, J= 5.72 Hz, 1H, pyridine-H), 7.82 (d, J=
.84 Hz, 2H, Ph-H), 7.78 (d, J= 8.28 Hz, 2H, Ph-H), 7.65 (d, J= 8.80 Hz, 2H, Ph-H),
.41 (d, J= 8.08 Hz, 2H, Ph-H), 6.74 (d, J= 1.52 Hz, 1H, pyridine-H), 6.58 (dd, J
8
7
5
1
=
1
3
.68 Hz, J
2
= 1.80 Hz, 1H, pyridine-H), 2.35 (s, 3H, CH
3
). C NMR (100 MHz,
DMSO-d
6
) δ: 156.15, 148.70, 146.82, 146.37, 144.40, 136.76, 133.48 (Ph-C × 2),
1
9
4
30.41 (Ph-C × 2), 127.21 (Ph-C × 2), 120.25, 117.85 (Ph-C × 2), 106.52, 101.45,
-
9.07, 21.45 (CH
3
). ESI-MS: m/z 363.4 [M-1] , C19
H
16
N
4
2
O S (364.10).
.1.1.2. 4-cyano-N-(2-((4-cyanophenyl)amino)pyridin-4-yl)benzenesulfonamide (3b)
1
White solid, yield: 27%. mp: 263.9-265.2°C. H NMR (400 MHz, DMSO-d
6
) δ: 11.28
(
s, 1H, SO
Ph-H, pyridine-H), 7.81 (d, J= 8.40 Hz, 2H, Ph-H), 7.66 (d, J= 8.80 Hz, 2H, Ph-H),
.75 (d, J= 1.64 Hz, 1H, pyridine-H), 6.60 (dd, J = 5.72 Hz, J = 1.88 Hz, 1H,
) δ: 156.27, 148.99, 146.22, 143.56,
36.09, 134.25 (Ph-C × 2), 133.52 (Ph-C × 2), 130.76, 127.84 (Ph-C × 2), 120.21,
2
NH), 9.66 (s, 1H, NH), 8.12 (d, J= 8.52 Hz, 2H, Ph-H), 8.05-8.02 (m, 3H,
6
1
2
1
3
pyridine-H). C NMR (100 MHz, DMSO-d
6
1
1
-
17.94 (Ph-C × 2), 116.37, 106.62, 101.64, 99.42. ESI-MS: m/z 374.3 [M-1] ,
S (375.08).
.1.1.3. 4-(tert-butyl)-N-(2-((4-cyanophenyl)amino)pyridin-4-yl)benzenesulfonamide
3c)
White solid, yield: 43%. mp: 214.9-216.0°C. H NMR (400 MHz, DMSO-d
C
19
H
13
N
5
O
2
4
(
1
6
) δ: 10.99
NH), 9.66 (s, 1H, NH), 8.01 (d, J= 5.72 Hz, 1H, pyridine-H), 7.83 (d, J=
.24 Hz, 4H, Ph-H), 7.66-7.63 (m, 4H, Ph-H), 6.79 (d, J= 1.64 Hz, 1H, pyridine-H),
(
s, 1H, SO
2
8
6
1
3
.61 (dd, J
1
= 5.72 Hz, J
2
= 1.84 Hz, 1H, pyridine-H), 1.27 (s, 9H, CH × 3). C NMR
3
(
100 MHz, DMSO-d
6
) δ: 156.94, 156.20, 148.76, 146.37, 136.99, 133.48 (Ph-C × 2),
16

1
1
27.02 (Ph-C × 2), 126.89 (Ph-C × 2), 125.76, 120.25, 117.87 (Ph-C × 2), 106.39,
-
01.47, 98.92, 35.41 (C), 31.15 (CH
3
× 3). ESI-MS: m/z 405.6 [M-1] , C22
H
22
N
4
2
O S
(
406.15).
4
.1.1.4. N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-4-fluorobenzenesulfonamide (3d)
1
White solid, yield: 49%. mp: 229.9-232.0°C. H NMR (400 MHz, DMSO-d
6
) δ: 11.05
(
s, 1H, SO
2
NH), 9.66 (s, 1H, NH), 8.02 (d, J= 5.68 Hz, 1H, pyridine-H), 7.99-7.94 (m,
H, Ph-H), 7.83 (d, J= 8.84 Hz, 2H, Ph-H), 7.66 (d, J= 8.88 Hz, 2H, Ph-H), 7.51-7.45
= 5.72 Hz, J = 1.88
) δ: 165.07 (d, JC–F = 250.80 Hz,
C, Ph-C), 156.20, 148.81, 146.59, 146.31, 135.99 (d, JC–F = 2.91 Hz, 1C, Ph-C),
2
(
m, 2H, Ph-H), 6.76 (d, J= 1.68 Hz, 1H, pyridine-H), 6.60 (dd, J
1
2
1
3
Hz, 1H, pyridine-H). C NMR (100 MHz, DMSO-d
6
1
1
1
33.49 (Ph-C × 2), 130.26 (d, JC–F = 9.72 Hz, 2C, Ph-C), 120.24, 117.88 (Ph-C × 2),
17.29 (d, JC–F = 22.76 Hz, 2C, Ph-C), 106.57, 101.53, 99.24. ESI-MS: m/z 367.3
-
[
M-1] , C18
H13FN
4
2
O S (368.07).
4
.1.1.5.
N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-4-(trifluoromethyl)benzenesulfonamide
3e)
White solid, yield: 48%. mp: 209.9-211.8°C. H NMR (400 MHz, DMSO-d
(
1
6
) δ: 11.27
NH), 9.67 (s, 1H, NH), 8.11-8.03 (m, 5H, Ph-H, pyridine-H), 7.82 (d, J=
.76 Hz, 2H, Ph-H), 7.66 (d, J= 8.84 Hz, 2H, Ph-H), 6.77 (d, J= 1.60 Hz, 1H,
(
s, 1H, SO
2
8
1
3
pyridine-H), 6.62 (dd, J
1
= 5.72 Hz, J = 1.84 Hz, 1H, pyridine-H). C NMR (100
2
MHz, DMSO-d
6
) δ: 156.16, 148.84, 146.24, 143.60, 133.50 (Ph-C × 2), 133.43 (q,
JC–F = 32.24 Hz, 2C, Ph-C), 128.10, 127.35 (q, JC–F = 3.70 Hz, 1C, Ph-C), 125.11,
-
1
22.40, 120.21, 117.94 (Ph-C × 2), 106.67, 101.62, 99.43. ESI-MS: m/z 417.4 [M-1] ,
C
19
H
13
F
3
N
4
O
2
S (418.07).
4
.1.1.6. N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-3,5-dimethylbenzenesulfonamide
1
(
3f) White solid, yield: 39%. mp: 240.0-240.6°C. H NMR (400 MHz, DMSO-d
6
) δ:
1
0.93 (s, 1H, SO NH), 9.65 (s, 1H, NH), 8.01 (d, J = 5.72 Hz, 1H, pyridine-H), 7.83
2
(
d, J = 8.76 Hz, 2H, Ph-H), 7.65 (d, J = 8.76 Hz, 2H, Ph-H), 7.52 (s, 2H, Ph-H), 7.29
s, 1H, Ph-H), 6.74 (d, J= 1.32 Hz, 1H, pyridine-H), 6.60 (dd, J = 5.72 Hz, J = 1.64
× 2). C NMR (100 MHz, DMSO-d ) δ:
(
1
2
1
3
Hz, 1H, pyridine-H), 2.33 (s, 6H, CH
3
6
17

1
1
56.17, 148.72, 146.72, 146.39, 139.55, 139.53 (Ph-C × 2), 135.26, 133.47 (Ph-C × 2),
24.61 (Ph-C × 2), 120.24, 117.88 (Ph-C × 2), 106.42, 101.47, 98.99, 21.21 (CH × 2).
3
-
ESI-MS: m/z 377.4 [M-1] , C20
H
18
N
4
2
O S (378.12).
4
.1.1.7. N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-3,5-difluorobenzenesulfonamide
3g)
White solid, yield: 38%. mp: 240.0-241.2°C. H NMR (400 MHz, DMSO-d
(
1
6
) δ: 11.22
NH), 9.65 (s, 1H, NH), 8.04 (d, J = 5.48 Hz, 1H, pyridine-H), 7.82 (d, J =
.80 Hz, 2H, Ph-H), 7.70-7.64 (m, 3H, Ph-H), 7.59 (d, J = 4.40 Hz, 2H, Ph-H), 6.75
= 5.72 Hz, J = 1.76 Hz, 1H, pyridine-H).
) δ: 162.70 (dd, JC–F = 250.68 Hz, JC–F = 12.30 Hz, 2C,
(
s, 1H, SO
2
7
(
d, J = 0.88 Hz, 1H, pyridine-H), 6.62 (dd, J
1
2
1
3
C NMR (100 MHz, DMSO-d
6
Ph-C), 156.27, 149.00, 146.20, 142.76, 133.51 (Ph-C × 2), 120.18, 117.98 (Ph-C × 2),
1
11.12 (t, JC–F = 11.75 Hz, 1C, Ph-C), 111.06 (d, JC–F = 28.15 Hz, 2C, Ph-C), 109.95
-
(
t, JC–F = 23.72 Hz, 1C, Ph-C), 106.70, 101.67, 99.52. ESI-MS: m/z 385.2 [M-1] ,
C
18
H
12
F
2
N
4
2
O S (386.06).
4
.1.1.8.
N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-3,5-bis(trifluoromethyl)benzenesulfonamid
e (3h)
1
White solid, yield: 43%. decomposed at 235.0°C. H NMR (400 MHz, DMSO-d
6
) δ:
1
1.30 (s, 1H, SO
2
NH), 9.70 (s, 1H, NH), 8.52 (s, 1H, Ph-H), 8.43 (s, 2H, Ph-H), 8.04
(
d, J = 4.72 Hz, 1H, pyridine-H), 7.78 (s, 2H, Ph-H), 7.66 (d, J = 8.72 Hz, 2H, Ph-H),
1
3
6
.80 (s, 1H, pyridine-H), 6.62 (dd, J
) δ: 155.15, 146.06, 133.56, 132.56, 132.22, 131.89,
28.08, 128.06, 127.77, 124.25, 121.53, 120.12, 118.05, 101.80, 99.69. ESI-MS: m/z
1
= 5.80 Hz, J
2
= 1.84 Hz, 1H, pyridine-H). C
NMR (100 MHz, DMSO-d
6
1
4
4
-
85.4 [M-1] , C20
H
12
F
6
N
4
2
O S (486.06).
.1.1.9.
N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-2,4,6-trimethylbenzenesulfonamide
(3i)
1
White solid, yield: 32%. mp: 204.1-204.7°C. H NMR (400 MHz, DMSO-d
6
) δ: 11.04
(
s, 1H, SO
2
NH), 9.79 (s, 1H, NH), 7.96 (d, J = 5.68 Hz, 1H, pyridine-H), 7.83 (d, J =
.72 Hz, 2H, Ph-H), 7.62 (d, J = 8.80 Hz, 2H, Ph-H), 7.05 (s, 2H, Ph-H), 6.60 (d, J =
.24 Hz, 1H, pyridine-H), 6.50 (dd, J = 5.72 Hz, J = 1.72 Hz, 1H, pyridine-H), 2.63
8
1
1
2
18

1
3
(
s, 6H, CH
3
× 2), 2.23 (s, 3H, CH
3
). C NMR (100 MHz, DMSO-d
6
) δ: 156.10,
1
48.62, 146.74, 146.50, 142.99, 139.16 (Ph-C × 2), 133.97, 133.37 (Ph-C × 2), 132.42
(
(
Ph-C × 2), 120.29, 117.93 (Ph-C × 2), 105.69, 101.31, 98.59, 22.88 (CH × 2), 20.87
3
-
CH
3
). ESI-MS: m/z 391.4 [M-1] , C21
H
20
N
4
2
O S (392.13).
4
.1.1.10.
N-(2-((4-cyanophenyl)amino)pyridin-4-yl)-2,4,6-triisopropylbenzenesulfonamide (3j)
1
White solid, yield: 14%. mp: 217.0-218.5°C. H NMR (400 MHz, DMSO-d
6
) δ: 10.92
(
s, 1H, SO
2
NH), 9.61 (s, 1H, NH), 7.98 (d, J = 5.64 Hz, 1H, pyridine-H), 7.78 (d, J =
.60 Hz, 2H, Ph-H), 7.63 (d, J= 8.76 Hz, 2H, Ph-H), 7.26 (s, 2H, Ph-H), 6.65 (s, 1H,
= 5.68 Hz, J = 1.44 Hz, 1H, pyridine-H), 4.31-4.21 (m, 2H,
CH), 2.94-2.87 (m, 1H, CH), 1.20-1.17 (m, 18H, CH
DMSO-d ) δ: 156.09, 153.51, 150.45 (Ph-C × 2), 148.62, 146.96, 146.41, 133.48
Ph-C × 2), 132.74, 124.46 (Ph-C × 2), 120.23, 117.86 (Ph-C × 2), 106.05, 101.46,
8
pyridine-H), 6.48 (dd, J
1
2
1
3
3
× 6). C NMR (100 MHz,
6
(
9
4
4
9.20, 33.75 (CH), 29.25 (CH × 2), 25.03 (CH
3
× 4), 23.76 (CH × 2). ESI-MS: m/z
3
-
75.5 [M-1] , C27
H
32
N
4
2
O S (476.22).
.1.1.11. N-(2-((4-cyanophenyl)amino)pyridin-4-yl)naphthalene-2-sulfonamide (3k)
1
White solid, yield: 46%. mp: 226.0-227.2°C. H NMR (400 MHz, DMSO-d
6
) δ: 11.11
(
s, 1H, SO
2
NH), 9.63 (s, 1H, NH), 8.62 (d, J = 1.16 Hz, 1H, Naph-H), 8.19-8.14 (m,
H, Naph-H), 8.03 (d, J = 7.72 Hz, 1H, Naph-H), 7.98 (d, J = 5.72 Hz, 1H,
= 8.72 Hz, J = 1.88 Hz, 1H, Naph-H), 7.79 (d, J = 8.84 Hz,
H, Ph-H), 7.74-7.67 (m, 2H, Naph-H), 7.62 (d, J = 8.84 Hz, 2H, Ph-H), 6.79 (d, J=
2
pyridine-H), 7.86 (dd, J
1
2
2
1
1
3
.64 Hz, 1H, pyridine-H), 6.64 (dd, J
) δ: 156.13, 148.73, 146.68, 146.32, 136.54, 134.91,
33.45 (Ph-C × 2), 131.96, 130.31, 129.77, 129.76, 128.74, 128.39, 122.19, 120.22,
1
= 5.76 Hz, J = 1.88 Hz, 1H, pyridine-H). C
2
NMR (100 MHz, DMSO-d
6
1
1
-
17.87 (Ph-C × 2), 106.51, 101.48, 99.18. ESI-MS: m/z 399.3 [M-1] , C22
H
16
N
4
2
O S
(
400.10).
4
.1.2. General procedure for the synthesis of target compounds 7a-k
Preparation
of
4-((4-oxo-4,5-dihydropyrimidin-2-yl)amino)benzonitrile
(5):
2
4
-Methylthio-4-pyrimidone 4 (3.00 g, 21.10 mmol) was well mixed with
-aminobenzonitrile (2.98 g, 25.22 mmol). The reaction mixture was heated to 110°C
19

to melt the solid, and was then allowed to be heated at 150°C under N for 3h until the
2
solid reformed. The mixture was cooled down before adding DMF to smash the solid
under ultrasonic conditions. The product 5 was obtained by filtration and was then
dried in vacuo as a white solid. Yield: 54%. mp: 193.0-193.6°C. ESI-MS: m/z 213.3
+
+
[
M+1] , 235.3 [M+23] , C11
Preparation of 4-((4-chloropyrimidin-2-yl)amino)benzonitrile (6): Intermediate 5
2.41 g, 11.36 mmol) was added to 20 mL POCl and then refluxed at 100 °C for 5h.
H
8
N
4
O (212.07).
(
3
Excess solvent was removed under reduced pressure and the residue was then slowly
added to icy water with vigorously stirring. The resulting solid was precipitated and
filtrated to give a filtrate cake, which was then again added into 20 mL acetonitrile
and stirred for 2h. The product was obtained by filtration and dried under vacuo to
provide the key intermediate 6 as a pale yellow solid. Yield: 81%. mp: 200.8-201.2°C.
+
+
ESI-MS: m/z 231.2 [M+1] , 253.2 [M+23] , C11
H
7
ClN (230.04).
4
Preparation of substituted arylsulfamides: Substituted arylsulfonyl chlorides was
suspended in 3 mL ammonia water, and then the solution was refluxed at 100°C for
1
h. The mixture was cooled down to room temperature. Substituted arylsulfamides
were obtained by filtration, washing with water and were then dried in vacuo
excepted for the commercially available 4-methylbenzenesulfonamide).
(
Preparation of target compounds 7a-k: A mixture of intermediate 6 (0.20 g),
substituted arylsulfamides (1 equiv) and NaH (60% in oil, 0.0411 g, 1.2 equiv) was
dissolved in 4 mL DMSO at room temperature. The reaction was heated at 130°C
until its completion checked by TLC. The reaction was quenched by adding 150 mL
water and acidifying the solution with 3 mL concentrated HCl. The aqueous solution
was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with
saturated brine (40 mL) and dried over anhydrous Na
2
SO , filtered and concentrated
4
under reduced pressure. Purification by flash column chromatography and
recrystallization from absolute methanol of the residue provided target compounds
7
a-k.
4
.1.2.1.
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-4-methylbenzenesulfonamide
(7a)
20

1
White solid, yield: 10%. mp: 274.0-275.2°C. H NMR (400 MHz, DMSO-d
6
) δ: 11.62
(
s, 1H, SO NH), 9.96 (s, 1H, NH), 8.26 (d, J= 5.56 Hz, 1H, pyrimidine-H), 7.97 (d,
2
J= 8.72 Hz, 2H, Ph-H), 7.88 (d, J= 8.16 Hz, 2H, Ph-H), 7.70 (d, J= 8.72 Hz, 2H,
Ph-H), 7.36 (d, J= 8.04 Hz, 2H, Ph-H), 6.50 (d, J= 5.56 Hz, 1H, pyrimidine-H), 2.33
1
3
(
s, 3H, CH
3
). C NMR (100 MHz, DMSO-d
6
) δ: 161.42, 159.04, 158.53, 145.05,
1
1
44.35, 137.35, 133.29 (Ph-C × 2), 130.16 (Ph-C × 2), 127.60 (Ph-C × 2), 120.06,
+
18.98 (Ph-C × 2), 103.00, 100.37, 21.43 (CH ). ESI-MS: m/z 366.4 [M+1] , 388.4
3
+
[
M+23] , C18
H
15
N
5
2
O S (365.09).
4
.1.2.2.
4-cyano-N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)benzenesulfonamide
(7b)
1
White solid, yield: 22%. decomposed at 262.0°C. H NMR (400 MHz, DMSO-d
6
) δ:
1
1.96 (s, 1H, SO
2
NH), 9.98 (s, 1H, NH), 8.28 (d, J= 5.60 Hz, 1H, pyrimidine-H), 8.17
(
d, J= 8.48 Hz, 2H, Ph-H), 8.06 (d, J= 8.52 Hz, 2H, Ph-H), 7.90 (d, J= 8.84 Hz, 2H,
13
Ph-H), 7.71 (d, J= 8.84 Hz, 2H, Ph-H), 6.50 (d, J= 5.64 Hz, 1H, pyrimidine-H). C
NMR (100 MHz, DMSO-d ) δ: 158.60, 144.74, 144.44, 133.90 (Ph-C × 2), 133.33
Ph-C × 2), 128.27 (Ph-C × 2), 119.98 (Ph-C × 2), 119.07 (Ph-C × 2), 117.96, 116.11,
6
(
+
+
1
03.33, 100.89. ESI-MS: m/z 377.0870 [M+1] , 399.0686 [M+23] , C18
H
12
N
6
2
O S
(
376.0742).
4
.1.2.3.
4
-(tert-butyl)-N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)benzenesulfonamide (7c)
1
White solid, yield: 6.8%. mp: 236.2-237.0°C. H NMR (400 MHz, DMSO-d
6
) δ:
1
1.65 (s, 1H, SO
2
NH), 9.99 (s, 1H, NH), 8.28 (d, J= 5.48 Hz, 1H, pyrimidine-H),
7
.93-7.91 (m, 4H, Ph-H), 7.67 (d, J= 8.72 Hz, 2H, Ph-H), 7.60 (d, J= 8.48 Hz, 2H,
1
3
Ph-H), 6.54 (d, J= 5.56 Hz, 1H, pyrimidine-H), 1.25 (s, 9H, CH × 3). C NMR (100
3
MHz, DMSO-d
6
) δ: 159.26, 159.12, 158.50, 156.87, 145.01, 137.53, 133.28 (Ph-C ×
2
3
4
), 127.28 (Ph-C × 2), 126.67 (Ph-C × 2), 120.03, 118.95 (Ph-C × 2), 102.96, 100.35,
+
5.36 (C), 31.14 (CH
3
× 3). ESI-MS: m/z 408.1499 [M+1] , C21
H
21
N
5
2
O S (407.1416).
.1.2.4.
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-4-fluorobenzenesulfonamide
(7d)
21

1
White solid, yield: 9.3%. decomposed at 255.4°C. H NMR (400 MHz, DMSO-d
6
) δ:
NH), 9.98 (s, 1H, NH), 8.28 (d, J= 5.48 Hz, 1H, pyrimidine-H),
.10-8.06 (m, 2H, Ph-H), 7.94 (d, J= 8.84 Hz, 2H, Ph-H), 7.70 (d, J= 8.80 Hz, 2H,
1
8
1.74 (s, 1H, SO
2
13
Ph-H), 7.44-7.39 (m, 2H, Ph-H), 6.50 (d, J= 5.56 Hz, 1H, pyrimidine-H). C NMR
100 MHz, DMSO-d ) δ: 165.03 (d, JC–F = 250.75 Hz, 1C, Ph-C), 159.25, 158.37,
44.96, 136.58 (d, JC–F = 2.14 Hz, 1C, Ph-C), 133.58, 133.32 (Ph-C × 2), 130.73 (d,
C–F = 9.60 Hz, 2C, Ph-C), 120.03, 118.97 (Ph-C × 2), 116.97 (d, JC–F = 22.75 Hz, 1C,
(
6
1
J
+
+
Ph-C), 103.11, 100.47. ESI-MS: m/z 370.4 [M+1] , 392.3 [M+23] , C17
369.07).
.1.2.5.
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-4-(trifluoromethyl)benzenesulfonamide
7e)
White solid, yield: 20%. mp: 274.5-276.0°C. H NMR (400 MHz, DMSO-d
s, 1H, SO
J= 8.12 Hz, 2H, Ph-H), 7.97 (d, J= 8.20 Hz, 2H, Ph-H), 7.89 (d, J= 8.68 Hz, 2H,
H12FN
5
O
2
S
(
4
(
1
6
) δ: 11.97
(
2
NH), 10.01 (s, 1H, NH), 8.30 (d, J= 5.32 Hz, 1H, pyrimidine-H), 8.23 (d,
13
Ph-H), 7.70 (d, J= 8.68 Hz, 2H, Ph-H), 6.52 (d, J= 5.52 Hz, 1H, pyrimidine-H). C
NMR (100 MHz, DMSO-d ) δ: 158.60, 144.77, 144.30, 133.30 (Ph-C × 2), 133.28 (q,
C–F = 32.57 Hz, 1C, Ph-C), 128.47, 127.83, 127.00 (q, JC–F = 3.57 Hz, 2C, Ph-C),
6
J
1
25.12, 122.41, 119.97, 119.02 (Ph-C × 2), 103.24, 100.76. ESI-MS: m/z 420.0736
+
[
M+1] , C18
H
12
F
3
N
5
2
O S (419.0664).
4
.1.2.6.
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-3,5-dimethylbenzenesulfonamide
(7f)
) δ:
1
White solid, yield: 9.4%. mp: 208.0-208.8°C. H NMR (400 MHz, DMSO-d
6
1
7
7
1.62 (s, 1H, SO
.97 (d, J = 8.64 Hz, 2H, Ph-H), 7.73 (d, J = 8.64 Hz, 2H, Ph-H), 7.60 (s, 2H, Ph-H),
.22 (s, 1H, Ph-H), 6.46 (d, J = 5.52 Hz, 1H, pyrimidine-H), 2.23 (s, 6H, CH × 2).
) δ: 159.13, 158.91, 158.45, 144.97, 139.94, 139.11
Ph-C × 2), 135.15, 133.31 (Ph-C × 2), 125.17 (Ph-C × 2), 120.02, 119.10 (Ph-C × 2),
2
NH), 10.01 (s, 1H, NH), 8.26 (d, J = 5.52 Hz, 1H, pyrimidine-H),
3
1
3
C NMR (100 MHz, DMSO-d
6
(
+
+
1
02.99, 100.37, 20.97 (CH
S (379.11).
3
× 2). ESI-MS: m/z 380.5 [M+1] , 402.5 [M+23] ,
C
19
H
17
N
5
O
2
22

4
.1.2.7 N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-3,5-difluorobenzenesulfonamide
7g)
White solid, yield: 15%. mp: 245.0-246.5°C. H NMR (400 MHz, DMSO-d
s, 1H, SO NH), 10.04 (s, 1H, NH), 8.28 (d, J = 5.28 Hz, 1H, pyrimidine-H), 7.91 (d,
J= 8.76 Hz, 2H, Ph-H), 7.73-7.57 (m, 5H, Ph-H), 6.50 (d, J = 5.64 Hz, 1H,
(
1
6
) δ: 11.93
(
2
1
3
pyrimidine-H). C NMR (100 MHz, DMSO-d
6
) δ: 162.70 (dd, JC–F = 250.68 Hz, JC–F
=
12.30 Hz, 2C, Ph-C), 156.27, 149.00, 146.20, 142.76, 133.51 (Ph-C × 2), 120.18,
1
2
3
4
17.98 (Ph-C × 2), 111.12 (t, JC–F = 11.75 Hz, 1C, Ph-C), 111.06 (d, JC–F = 28.15 Hz,
C, Ph-C), 109.95 (t, JC–F = 23.72 Hz, 1C, Ph-C), 106.70, 101.67, 99.52. ESI-MS: m/z
+
+
88.3 [M+1] , 410.4 [M+23] , C17
.1.2.8.
H
11
F
2
N
5
2
O S (387.06).
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-3,5-bis(trifluoromethyl)benzenesulfona
mide (7h)
1
White solid, yield: 11%. decomposed at 202.3°C. H NMR (400 MHz, DMSO-d
6
) δ:
8
.50 (s, 1H, NH), 8.11 (s, 1H, imidazole-H), 7.84 (s, 1H, pyrazine-H), 7.68 (s, 1H,
imidazole-H), 7.05-6.96 (m, 6H, OPh-H, PhH), 3.87 (s, 2H, CH
2
), 2.32 (s, 3H, CH ),
3
1
3
2
1
1
.06 (s, 6H, CH
3
×2). C NMR (100 MHz, DMSO-d ) δ: 150.86, 147.90, 142.58,
6
39.43, 135.01, 134.46, 130.40, 129.99, 129.62, 128.70, 121.90, 120.02, 117.20,
+
+
16.73, 99.43, 22.05, 20.84, 16.48. ESI-MS: m/z 488.4 [M+1] , 510.4 [M+23] ,
C
19
H
11
F
6
N
5
O
2
S (487.05).
4
.1.2.9.
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-2,4,6-trimethylbenzenesulfonamide (7i)
1
White solid, yield: 15%. mp: 234.5-235.0°C. H NMR (400 MHz, DMSO-d
6
) δ: 11.64
(
s, 1H, SO
2
NH), 9.86 (s, 1H, NH), 8.26 (d, J = 5.52 Hz, 1H, pyridine-H), 7.85 (d, J =
8
5
.68 Hz, 2H, Ph-H), 7.62 (d, J = 8.64 Hz, 2H, Ph-H), 7.06 (s, 2H, Ph-H), 6.39 (d, J =
1
3
.56 Hz, 1H, pyridine-H), 2.62 (s, 6H, CH
) δ: 159.20, 159.12, 158.51, 145.06, 142.97, 138.91 (Ph-C × 2),
34.50, 133.22 (Ph-C × 2), 132.35 (Ph-C × 2), 120.04, 118.85 (Ph-C × 2), 102.91,
3
× 2), 2.23 (s, 3H, CH ). C NMR (100
3
MHz, DMSO-d
6
1
1
+
+
00.08, 22.74 (CH
3
× 2), 20.86 (CH ). ESI-MS: m/z 394.3 [M+1] , 416.5 [M+23] ,
3
C
20
H
19
N
5
2
O S (393.13).
23

4
.1.2.10.
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)-2,4,6-triisopropylbenzenesulfonamide
7j)
White solid, yield: 26%. mp: 222.5-223.0°C. H NMR (400 MHz, DMSO-d
(
1
6
) δ: 11.61
(
s, 1H, SO
2
NH), 9.81 (s, 1H, NH), 8.26 (d, J = 5.24 Hz, 1H, pyridine-H), 7.91 (d, J =
8
5
.80 Hz, 2H, Ph-H), 7.62 (d, J = 8.76 Hz, 2H, Ph-H), 7.25 (s, 2H, Ph-H), 6.39 (d, J =
.60 Hz, 1H, pyridine-H), 4.34-4.28 (m, 2H, CH), 2.94-2.87 (m, 1H, CH), 1.19-1.15
1
3
(
m, 18H, CH
3
). C NMR (100 MHz, DMSO-d
53.46, 150.36 (Ph-C × 2), 145.14, 133.24 (Ph-C × 2), 124.29 (Ph-C × 2), 119.99,
18.74 (Ph-C × 2), 102.83, 100.40, 33.78, 29.21 (CH × 2), 24.92 (CH × 4), 23.74
× 2). ESI-MS: m/z 478.2323 [M+1] , 500.2122 [M+23] , C26
477.2198).
.1.2.11.
7k)
6
) δ: 159.14, 159.02, 158.74, 153.53,
1
1
3
+
+
(
(
CH
3
H
31
N
5
2
O S
4
N-(2-((4-cyanophenyl)amino)pyrimidin-4-yl)naphthalene-2-sulfonamide
(
1
White solid, yield: 12%. decomposed at 176.0°C. H NMR (400 MHz, DMSO-d
6
) δ:
1
8
1.83 (s, 1H, SO NH), 9.98 (s, 1H, NH), 8.26 (d, J = 5.60 Hz, 1H, pyrimidine-H),
2
.16-7.90 (m, 7H, Naph-H, Ph-H), 7.71 (d, J = 8.72 Hz, 2H, Ph-H), 7.69-7.63 (m, 2H,
+
Naph-H), 6.53 (d, J = 5.60 Hz, 1H, pyrimidine-H). ESI-MS: m/z 402.5 [M+1] , 424.4
+
[
M+23] , C21
H
15
N
5
2
O S (401.09).
4
4
.2. Biological activity evaluation
.2.1. In vitro anti-HIV assay
Evaluation of the antiviral activity of the newly synthesized compounds against
HIV-1 wt strain (IIIB), double mutant strain (RES056) and HIV-2 strain (ROD) was
performed using the MTT method as previously described[27, 28]. 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 five-fold
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.
24

HIV-1 wt strain (IIIB), HIV-1 double mutant strain (RES056) or HIV-2 strain (ROD)
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 its cytotoxicity. Exponentially growing MT-4 cells were
centrifuged for 5 min at 1000 rpm and the supernatant was discarded. The MT-4 cells
5
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 method. The
5
0% cytotoxic concentration (CC50) was defined as the concentration of the tested
compound that reduced the viability of the mock-infected MT-4 cells 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).
4
.2.2. HIV-1 RT inhibition assay
Inhibition of HIV-1 RT was performed by using homopolymer template/primer linked
to a microtiter plate, biotin-dUTP and RT with detection by ELISA for quantifying
HIV-1 RT expression[29]. The incorporated quantities of the biotin-dUTP into the
template represented the activity of HIV-1 RT. The procedure for RT inhibition assay
was performed as depicted in details by the kit (Roche) protocol. The percentage
inhibitory values were calculated by the following formula:
%
Inhibition = [O.D. value without inhibitors (with RT) -O.D. value with RT and
inhibitors]/[O.D. value without inhibitors (with RT)- O.D. value without RT and
inhibitors].
4
.3. Molecular simulations
The molecules (3a, 3i and 7f) for docking were optimized for 2000-generations until
the maximum derivative of energy became 0.005 kcal/(mol*Å), using the Tripos force
field. Charges were computed and added according to Gasteiger-Huckel parameters.
The published 3D crystal structures of wt RT with RPV complexes (PDB code: 3MEE)
[
30, 31] was retrieved from the Protein Data Bank [32] and was used for the docking
studies by means of surflex-dock module of SYBYL-X 1.1. The protein was prepared
by using the Biopolymer application accompanying SYBYL according to SYBYL-X
1
.1 manual: The bound ligand was extracted from the complexes, water molecules
25

were removed, hydrogen atoms were automatically added (after which the resulting
structures haven’t been optimized), and charges and atom types were assigned
according to AMBER99. After the protomol was generated, which referred to a
computational representation of the intended binding site to which putative ligands
are aligned, the optimized compounds 3a, 3i and 7f were docked into NNIBP, with the
relevant parameters set as defaults. The original ligand (RPV) of the coordinates
(
3MEE) was used as reference molecule to calculate the RMSD values. The docking
scores related to binding affinities were calculated based on hydrophobic, polar, and
repulsive interactions as well as entropic effects and solvation. Top-scoring poses of
compounds were shown by the software of PyMOL version 1.5
(
http://www.pymol.org/). Only the key residues for interactions with the inhibitors
were labeled and shown in sticks. The potential hydrogen bonds were presented by
dashed lines in red.
Acknowledgements
Financial support from the National Natural Science Foundation of China (NSFC No.
8
3
2
1273354), Key Project of NSFC for International Cooperation (Nos. 81420108027,
0910103908), Young Scholars Program of Shandong University (YSPSDU No.
016WLJH32, to P.Z.), 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 anti-HIV assays.
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