Identification of novel potent HIV-1 inhibitors by exploiting the tolerant regions of the NNRTIs binding pocket

Article abstract of DOI:10.1016/j.ejmech.2021.113204

With our previously identified potent NNRTIs 25a and HBS-11c as leads, series of novel thiophene[3,2-d]pyrimidine and thiophene[2,3-d]pyrimidine derivatives were designed via molecular hybridization strategy. All the target compounds were evaluated for their anti-HIV-1 activity and cytotoxicity in MT-4 cells. Compounds 16a1 and 16b1 turned out to be the most potent inhibitors against WT and mutant HIV-1 strains (L100I, K103N, and E138K), with EC₅₀ values ranging from 0.007 μM to 0.043 μM. Gratifyingly, 16b1 exhibited significantly reduced cytotoxicity (CC₅₀ > 217.5 μM) and improved water solubility (S = 49.3 μg/mL at pH 7.0) compared to the lead 25a (S < 1 μg/mL at pH 7.0, CC₅₀ = 2.30 μM). Moreover, molecular docking was also conducted to rationalize the structure-activity relationships of these novel derivatives and to understand their key interactions with the binding pocket.

Full text of DOI:10.1016/j.ejmech.2021.113204

European Journal of Medicinal Chemistry 214 (2021) 113204
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Identification of novel potent HIV-1 inhibitors by exploiting the
tolerant regions of the NNRTIs binding pocket
Yanying Sun a, c, Dongwei Kang a d , Feng Da a , Tao Zhang , Pei Li , Baodan Zhang ,
,
,
*
,
c
a
,
c
a
a
b
b
a,
c
,
**
, Xinyong Liu a
, c, ***
Erik De Clercq , Christophe Pannecouque , Peng Zhan
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of
Medicine, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
a
b
Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U. Leuven, Herestraat 49 Postbus 1043 (09.A097), B-3000 Leuven,
Belgium
c
China-Belgium Collaborative Research Center for Innovative Antiviral Drugs of Shandong Province, 44 West Culture Road, 250012 Jinan, Shandong, PR
China
d
Suzhou Research Institute, Shandong University, Room522, Building H of NUSP, NO.388 Ruoshui Road, SIP, Suzhou, 215123 Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 August 2020
Received in revised form
With our previously identified potent NNRTIs 25a and HBS-11c as leads, series of novel thiophene[3,2-d]
pyrimidine and thiophene[2,3-d]pyrimidine derivatives were designed via molecular hybridization
strategy. All the target compounds were evaluated for their anti-HIV-1 activity and cytotoxicity in MT-
30 November 2020
4
cells. Compounds 16a1 and 16b1 turned out to be the most potent inhibitors against WT and mutant
HIV-1 strains (L100I, K103N, and E138K), with EC50 values ranging from 0.007 M to 0.043 M. Grati-
fyingly, 16b1 exhibited significantly reduced cytotoxicity (CC50 > 217.5 M) and improved water solu-
g/mL at pH 7.0) compared to the lead 25a (S < 1 M).
g/mL at pH 7.0, CC50 ¼ 2.30
Accepted 12 January 2021
Available online 24 January 2021
m
m
m
bility (S ¼ 49.3
m
m
m
Keywords:
HIV-1
DAPY
Moreover, molecular docking was also conducted to rationalize the structure-activity relationships of
these novel derivatives and to understand their key interactions with the binding pocket.
© 2021 Elsevier Masson SAS. All rights reserved.
NNRTIs
NNIBP
Tolerant regions
1. Introduction
transcriptase (RT), an RNA-dependent DNA polymerase that uses
single-stranded RNA templates to synthesize double-stranded viral
Human immunodeficiency virus (HIV) could enter the host cell
and eventually lead to significant weakening of the host immune
system, which was identified as the causative agent for the ac-
quired immune deficiency syndrome (AIDS) [1,2]. HIV-1 reverse
DNA, is a validated important target in the discovery and devel-
opment of novel anti-HIV drugs with the advantage of providing
well characterized biochemical mechanisms and abundant struc-
tural information [3,4]. Amongst the HIV-1 RT inhibitors, non-
nucleoside reverse transcriptase inhibitors (NNRTIs) have ach-
ieved significant attraction due to their remarkable antiviral ac-
tivity, excellent specificity and reduced toxicity in antiretroviral
therapy [5e7]. Up to now, there are six NNRTIs approved by the U.S.
Food and Drug Administration (FDA), including the first generation
nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), the second
generation etravirine (ETR), rilpivirine (RPV), and doravirine (DOR)
*
Corresponding author. Department of Medicinal Chemistry, Key Laboratory of
Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Cheeloo College of Medicine, Shandong University, 44 West Culture Road, 250012
Jinan, Shandong, PR China.
** Corresponding author. Department of Medicinal Chemistry, Key Laboratory of
Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Cheeloo College of Medicine, Shandong University, 44 West Culture Road, 250012
Jinan, Shandong, PR China.
[
7,8]. However, drug resistance remains to be a severe problem with
the first- and second-generation HIV-1 NNRTIs. K103N and Y181C
have limited the clinical use of the first-generation NNRTIs NVP and
EFV [9]. ETR and RPV are members of the diarylpyrimidine (DAPY)
family (Fig. 1), which could effectively inhibit most RT-resistant
mutations caused by the first-generation NNRTIs, but they gener-
ally failed to suppress the most refractory mutations E138K, while
*** Corresponding author. Department of Medicinal Chemistry, Key Laboratory of
Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Cheeloo College of Medicine, Shandong University, 44 West Culture Road, 250012
Jinan, Shandong, PR China.
E-mail addresses: kangdongwei@sdu.edu.cn (D. Kang), zhanpeng1982@163.com
(
P. Zhan), xinyongl@sdu.edu.cn (X. Liu).
https://doi.org/10.1016/j.ejmech.2021.113204
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
(NNIBP) was also confirmed by the co-crystal structures of HIV-1
wild-type (WT) RT in complex with 25a for the first time [25].
However, 25a suffered from higher cytotoxicity (CC50 ¼ 2.30 M)
and poor water solubility (<1 g/mL at pH 7.0). Interestingly,
m
m
another research in our lab culminated in the identification of
potent NNRTIs 4 (HBS-11c) with improved water solubility (¼
30.92 mg/mL at pH 7.0) by exploiting the tolerant region I of NNIBP,
indicating that the solvent-exposed regions could be regarded as
favorable sites for substantial modifications of existing NNRTIs
without serious loss of activity [14].
Encouraged by these interesting findings and in attempt to yield
HIV-1 NNRTIs with more potent activity, lower cytotoxicity and
favorable solubility, series of novel thiophene[3,2-d]pyrimidine and
thiophene[2,3-d]pyrimidine derivatives were designed based on
the co-crystal structure of 25a/WT RT and molecular hybridization
strategy (Fig. 2). The target compounds incorporated the common
left wing of 25a and HBS-11c, right wing of HBS-11c and central
core of 25a according to the DAPY’s “four-point phaemacophore”
model. Meanwhile, 1-phenylpiperazine and 1-phenylpiperidin-3-
amine motifs were introduced to replace the 1-benzylpiperazine
motif of the lead to further investigate the structure-activity rela-
tionship (SAR) of the tolerant region I. These newly synthesized
compounds were evaluated for their anti-HIV-1 activities and
cytotoxicity in MT-4 cells. In addition, molecular docking studies
were undertaken to guide further investigation.
Fig. 1. Chemical structures of diarylpyrimidine (DAPY)-based HIV-1 NNRTIs approved
by U.S. FDA.
V106A and F227L are the two most prevalent mutations selected by
DOR [10e12]. Additionally, the patients treated with second-
generation NNRTIs were frequently reported with hypersensitiv-
ity reactions and other adverse effects [13,14]. Therefore, the
development of new NNRTIs with greater potency, improved drug-
resistance profiles, and less toxicity is urgently needed [14e17].
With ETR and RPV as leads, our previous efforts have led to the
development of some novel and potent HIV-1 NNRTIs [18e23],
including piperidine-substituted thiophene[3,2-d]pyrimidine de-
rivative 3 (25a) as potent HIV-1 NNRTI (Fig. 2) [24]. The “four-point
pharmacophore” model (including the hydrophobic channel,
hydrogen bonding domain, tolerant region I, and tolerant region II)
of diarylpyrimidine (DAPY) derivatives in the NNRTI binding pocket
2. Results and discussion
2.1. Chemistry
The synthetic protocols for the novel designed compounds are
illustrated in Schemes 1-2. First, the treatment of 1-fluoro-4-
Fig. 2. Design of novel NNRTIs by exploiting the tolerant regions of NNIBP.
2

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
Scheme 1. Synthesis of the intermediates 7, 9 and 12a.
nitrobenzene (5) with tert-butyl piperazine-1-carboxylate and tert-
butyl piperidin-3-ylcarbamate gave intermediates and 8,
respectively, which provided intermediates 7 and 9 via reduction in
the presence of Pd/C and H in excellent yields. 4-Nitrobenzyl
resulting free amines were treated with methanesulfonyl chloride
or sulfamoyl chloride in the presence of triethylamine to yield the
target compounds.
6
2
bromide (10) reacted with tert-butyl piperazine-1-carboxylate via
a nucleophilic substitution reaction afforded compound 11, which
by reduction gave the intermediate 12 (Scheme 1).
2.2. In vitro anti-HIV-1 activity and cytotoxicity
For all novel chemical entities, antiviral activity, expressed as
EC50 (anti-HIV-1 potency), CC50 (cytotoxicity), and SI (selectivity
index, CC50/EC50 ratio), was measured against WT HIV-1 (IIIB) and a
panel of NNRTIs-resistant single and double mutant strains,
including L100I, K103N, Y181C, Y188L, E138K, F227L þ V106A, and
RES056 (K103N þ Y181C). The approved drugs NVP, EFV, ETR and
RPV were selected as controls.
a
ꢀ
Reagents and conditions: i) DMSO, 120 C; ii) Pd/C, H
r.t.; iii) tert-butyl piperazine-1-carboxylate, K CO , DMF, r.t.
(dba) , Cs CO
SO Cl or NH
2
, MeOH,
2
3
a
Reagents and conditions: i) BINAP, Pd
2
3
2
3
, 1,4-
SO Cl,
ꢀ
dioxane, 90 C, N
2
; ii) TFA, DCM, r.t.; iii) CH
3
2
2
2
Et
3
N, DCM, r.t.
As described in Scheme 2, the previously prepared in-
termediates 13a and 13b in our lab were selected as starting ma-
terials [22,24], which were treated with the obtained intermediates
As shown in Table 1, in the case of the HIV-1 IIIB, nine com-
pounds (15b, 16a1, 16b1, 16b2, 19a1, 19a2, 19b1, 22a1 and 22b1)
exhibited higher potency with EC50 values ranging from 0.007 to
7
,
9
and 12 to prepare 14a-b, 17a¡b and 20a¡b via
BuchwaldꢁHartwig reaction. Subsequent removal of the tert-
0.126
(EC50 ¼ 0.150
(EC50 ¼ 0.008
m
M, which are superior to that of the reference drug NVP
M). Among them, 16a1 (EC50 ¼ 0.007 M) and 16b1
M) turned out to be the most potent inhibitors,
butyloxycarbonyl (Boc) protecting group using trifluoroacetic acid
m
m
(
TFA) afforded free amines 15a-b, 18a¡b and 21a¡b, and the
m
3

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
Scheme 2. Synthesis of the target compoundsa.
being comparable to that of the reference drug ETR
compared to those of 15a-b and 21a-b. A SO
region seems essential for high antiviral potency; 16a1, 16b1, and
19b1 with SO NH substituent turned out to be more effective in-
hibitors with EC50 values of 0.007e0.028 M. Moreover, the com-
pounds had higher cytotoxicity when R was H (15a-b, 18a-b and
21a-b, CC50 ¼ 3.817 e 5.732 M), while most compounds with
SO NH and SO CH substituent in R position afforded advanta-
geous features of decreased cytotoxicity with high therapeutic in-
dex, with the exception of 16a2 (CC50 ¼ 20.70 M) and 19a2
(CC50 ¼ 11.54 M).
2 2
NH substituent in this
(
EC50 ¼ 0.004
m
M) and RPV (EC50 ¼ 0.001
mM). Gratifyingly, 16b1
(
CC50 > 217.5
mM, SI > 26923) exhibited much lower cytotoxicity
2
2
compared to RPV (CC50 ¼ 3.98
m
M, SI ¼ 3989), which contribute to
m
1
its higher selectivity index value toward HIV-1 IIIB.
The preliminary SAR indicated that the thiophene[3,2-d]py-
rimidine and thiophene[2,3-d]pyrimidine center rings have little
effect on the anti-HIV-1 activity of the compounds. However, the
scaffold substitution of the right wing has great influence on their
potency. Compounds of subseries 16 and subseries 19 showed
better activity than those of subseries 22, i.e., 16a1
m
1
2
2
2
3
m
m
Furthermore, the target compounds 16a1-2, 16b1-2, 19a1-2,
19b1-2, 22a1-2, and 22b1-2 were evaluated for their activity
against a panel of NNRTI-resistance mutations, including L100I,
K103N, Y181C, Y188L, E138K, F227L þ V106A, and RES056. As dis-
played in Table 2 and Table S1, all the target inhibitors exhibited
moderate to excellent potency against single-mutant strains and
double-mutant strain F227L þ V106A, but some inhibitors lost their
activity against the double-mutant strain RES056. Especially, 16a1
and 16b1 turned out to be the most potent inhibitors among all the
tested compounds. As for mutant HIV-1 strain L100I, 16a1 and 16b1
(
(
(
(
(
EC50
EC50
EC50
EC50
EC50
¼
¼
¼
¼
¼
0.007
0.059
0.028
0.036
1.431
m
m
m
M)
>
19a1 (EC50
¼
0.027
0.008
m
M)
>
>
22a1
19b1
M); 16b1 (EC50
M)
¼
mM)
>
>
22b1 (EC50
19b2 (EC50
¼
0.126
0.172
mM); 16b2
mM)
¼
mM)
>
22b2
m
M). These results suggested that the 1-
benzylpiperazine motif could contribute more to the activity than
that of the 1-phenylpiperazine and 1-phenylpiperidin-3-amine
motif. Next, we turned our attention to the SAR of the R substit-
1
uent of the right ring. Replacement the H atom of compounds 15a-
b, 18a-b and 21a-b with SO
the activity of the compound; i.e., 16a2 and 22b2 showed weaker
activity, while the activity of 16b2 and 22a2 increased slightly
2
CH
3
group has an uncertain effect on
yielded EC50 values of 0.043
were comparable to that of EFV (EC50 ¼ 0.031
K103N mutant HIV-1 strain, both 16a1 (EC50
m
M and 0.015
m
M, respectively, which
M). In the case of
0.007 M,
m
¼
m
4

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
Table 1
2.3. Inhibition of HIV-1 RT
Activity and cytotoxicity against HIV-1 IIIB strain.
Compds
R1
EC50
(
m
M)a (IIIB)
CC50
(
m
M)b
SIc
Next, all prepared compounds were assayed in vitro for their
ability to inhibit recombinant WT to validate their binding target.
Similarly, the FDA-approved drug NVP, EFV, ETR and RPV were
selected as the controls. The results of this evaluation are sum-
marized in Table 3.
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
5a
5b
H
H
0.216 ± 0.052
0.042 ± 0.008
0.007 ± 0.002
0.391 ± 0.240
0.008 ± 0.002
0.036 ± 0.012
0.387 ± 0.125
0.584 ± 0.146
0.027 ± 0.009
0.027 ± 0.008
0.028 ± 0.012
0.172 ± 0.045
0.815 ± 0.259
0.461 ± 0.178
0.059 ± 0.029
0.450 ± 0.211
0.126 ± 0.045
1.431 ± 0.307
0.150 ± 0.036
0.028 ± 0.0007
0.004 ± 0.0007
0.001 ± 0.0003
3.970 ± 1.113
4.030 ± 0.883
145.3 ± 51.92
20.70 ± 4.010
>217.5
131.1 ± 2.028
5.732 ± 0.215
4.737 ± 0.512
>222.9
11.54 ± 1.984
>222.9
>222.5
3.817 ± 1.166
4.613 ± 0.611
>217.5
18
94
6a1
6a2
6b1
6b2
8a
SO
SO
SO
SO
H
2
2
2
2
NH
2
18979
53
>26923
3557
15
CH
3
NH
2
The results demonstrated that all compounds (IC50 ¼ 0.103 e
CH
3
4
.790 mM) displayed lower binding-affinity to WT HIV-1 RT
8b
H
8
compared to that of EFV (IC50 ¼ 0.008
m
M), ETR (IC50 ¼ 0.012
mM)
9a1
9a2
9b1
9b2
1a
SO
SO
SO
SO
H
2
2
2
2
NH
2
>8310
428
>7790
>1295
5
and RPV (IC50 ¼ 0.015 M). Furthermore, the data clearly showed
m
CH
3
that the antiviral activities of these compounds were inconsistent
with their enzyme inhibitory potencies. As observed in most NNRTI
series, the differences between cell activity and enzyme activity are
considered to be caused by template-specific variation in the
relationship between HIV-RT-RNA binding affinity and polymerase
processivity [12]. Although the most potent inhibitors 16a1
NH
2
CH
3
1b
H
10
2a1
2a2
2b1
2b2
SO
SO
SO
SO
e
2
2
2
2
NH
2
>3632
>434
>1726
78
>100
>2268
>1105
3989
CH
3
>217.1
>217.5
111.8 ± 15.11
>15.02
>6.335
NH
2
(
IC50 ¼ 0.961
lower activity than that of ETR, their activity was comparable to
M), which confirmed that those newly
m
M) and 16b1 (IC50 ¼ 0.295
mM) exhibited much
CH
3
NVP
EFV
ETR
e
that of NVP (IC50 ¼ 0.575
m
e
>4.594
3.98
designed compounds could inhibit the activity of HIV-1 RT and
acted as typical HIV-1 NNRTIs.
d
RPV
e
a
EC50: Concentration of compound required to achieve 50% protection of MT-
cell cultures against HIV-1-induced cytopathic effect, as determined by the MTT
Furthermore, the preliminary SAR of enzyme activity was
concluded. Generally, subseries 22 exhibited the highest IC50 values
4
method.
b
CC50: Concentration required to reduce the viability of mock-infected cell cul-
tures by 50%, as determined by the MTT method.
c
SI: the ratio of CC50/EC50
Used for comparison. Data were reported in Ref. 22.
.
Table 3
d
Inhibitory activity against WT HIV-1 RT.
Compds
IC50
(
m
M)a
Compds
IC50 (m
M)a
SI ¼ 202134) and 16b1 (EC50 ¼ 0.007
mM, SI > 28409) exhibited
single-digit nanomolar activity and higher SI values, being far more
1
1
1
1
5a
5b
6a1
6a2
0.707 ± 0.230
0.151 ± 0.020
0.961 ± 0.121
0.139 ± 0.038
0.299 ± 0.047
0.103 ± 0.020
0.945 ± 0.184
0.849 ± 0.111
0.214 ± 0.044
0.211 ± 0.021
0.713 ± 0.045
19b2
21a
21b
0.239 ± 0.006
1.582 ± 0.065
0.956 ± 0.131
2.411 ± 0.389
0.775 ± 0.040
4.790 ± 0.005
1.015 ± 0.030
0.575 ± 0.177
0.008 ± 0.002
0.012 ± 0.002
0.015 ± 0.001
potent than NVP (EC50
¼
3.992
89), and comparable to ETR
M, SI ¼ 3045).
mM, SI > 4) and EFV
(
EC50
¼
0.071
mM, SI
>
22a1
22a2
22b1
22b2
NVP
EFV
(
EC50 ¼ 0.003
m
M, SI > 1417) and RPV (EC50 ¼ 0.002
m
16b1
1
1
1
1
6b2
8a
8b
9a1
Morevoer, 16b2, 19a1, 19a2 and 19b1 also showed comparable
antiviral activity (EC50 ¼ 0.021 e 0.056 M) to EFV. In the case of
Y181C and E138K, 16b1 (EC50 ¼ 0.045 and 0.025 M, SI > 4836 and
636, respectively) exhibited the most active potency, while 16a1
exhibited the greatest potency to Y188L and F227L þ V106A
EC50 ¼ 0.544 and 0.175 M, SI ¼ 267 and 830, respectively).
However, both of them exhibited significantly reduced inhibition of
m
m
b
8
19a2
19b1
ETR
RPVb
(
m
a
IC50: inhibitory concentration of test compounds required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into WT HIV-1 RT by 50%.
b
The data were obtained from the same laboratory with the same method (Prof.
RES056 mutant strain (EC50 ¼ 1.911 and 1.384
mM, respectively)
Erik De Clercq, Rega Institute for Medical Research, KU Leuven, Belgium). Data were
reported in Ref. 22.
compared to other mutant strains.
Table 2
Anti-HIV-1 activity against HIV-1 mutant strains.
Compds
EC50
L100I
(m
M)a
K103 N
Y181C
Y188L
E138K
F227L þ V106A
RES056
1
1
1
1
1
1
1
1
2
2
2
2
6a1
6a2
6b1
6b2
9a1
9a2
9b1
9b2
2a1
2a2
2b1
2b2
0.043 ± 0.006
1.560 ± 0.868
0.015 ± 0.008
0.387 ± 0.187
0.767 ± 0.318
0.938 ± 0.224
0.409 ± 0.100
3.322 ± 1.902
1.660 ± 0.575
6.823 ± 3.870
2.329 ± 0.571
8.519 ± 4.286
0.622 ± 0.276
0.031 ± 0.008
0.008 ± 0.002
0.002 ± 0.000
0.007 ± 0.002
0.356 ± 0.076
0.007 ± 0.001
0.054 ± 0.018
0.021 ± 0.002
0.056 ± 0.019
0.031 ± 0.004
0.219 ± 0.099
0.102 ± 0.022
0.461 ± 0.176
0.450 ± 0.160
1.358 ± 0.524
3.922 ± 0.675
0.071 ± 0.020
0.003 ± 0.0008
0.002 ± 0.0004
0.154 ± 0.021
5.636 ± 0.485
0.045 ± 0.006
1.270 ± 0.239
2.249 ± 0.703
4.531 ± 1.526
1.360 ± 0.274
42.73 ± 19.03
3.640 ± 0.444
43.40 ± 8.064
7.532 ± 1.462
14.01 ± 0.410
5.053 ± 0.921
0.005 ± 0.001
0.015 ± 0.004
0.005 ± 0.0005
0.544 ± 0.185
>20.70
0.026 ± 0.016
0.983 ± 0.157
0.025 ± 0.012
0.162 ± 0.023
0.043 ± 0.015
0.088 ± 0.038
0.050 ± 0.014
0.405 ± 0.119
0.123 ± 0.075
1.285 ± 0.070
0.621 ± 0.244
4.272 ± 0.778
0.168 ± 0.098
0.005 ± 0.001
0.009 ± 0.004
0.006 ± 0.0001
0.175 ± 0.071
2.198 ± 0.316
0.369 ± 0.237
1.638 ± 0.851
0.203 ± 0.111
0.285 ± 0.065
0.331 ± 0.150
1.952 ± 0.274
0.381 ± 0.082
16.78 ± 6.917
3.639 ± 1.661
13.78 ± 1.554
8.041 ± 3.532
0.380 ± 0.247
0.025 ± 0.016
0.081 ± 0.021
1.911 ± 0.791
>20.70
1.384 ± 0.323
18.78 ± 3.517
>222.9
0.877 ± 0.162
9.414 ± 1.192
23.11 ± 17.73
5.114 ± 1.368
10.14 ± 4.023
68.43 ± 17.40
6.754 ± 1.750
150.0 ± 30.48
88.20 ± 18.47
16.17 ± 1.591
9.074 ± 2.406
0.260 ± 0.117
0.018 ± 0.006
0.079 ± 0.0008
>11.54
195.6 ± 13.06
175.4 ± 31.02
>217.5
>217.1
>217.5
16.53 ± 2.884
>15.02
0.335 ± 0.149
0.060 ± 0.034
0.010 ± 0.008
NVP
EFV
ETR
RPVb
a
EC50: Concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytopathic effect, as determined by the MTT method.
Used for comparison. Data were reported in Ref. 22.
b
5

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
than that of subseries 16 and subseries 19, indicating that 1-
phenylpiperidin-3-amine motif located in the right wing would
result in a decreased inhibitory activity compared to that of 1-
the right wing could still form the critical hydrogen bond with
Val104, the classical horseshoe conformation was changed, and the
shift of the left wing causing decreased
p-p interactions with
phenylpiperazine and 1-benzylpiperazine motif. Additionally, the
Tyr181 and Tyr188, may account for its reduced antiviral potency.
In the binding mode of 16a1 and 19a1 with HIV-1 RES056 RT
(Fig. 4), the Lys103 to Asn103 substitution in RT brings the dramatic
changes of the NNIBP, and results in the disappearance of the
hydrogen-bonding interaction disappeared between Lys101 and
thiophene[2,3-d]pyrimidine compared to that of the lead 25a
1
compounds containing SO
2
CH
3
substituent on R position offered
higher RT inhibitory activities in each subseries than those with
SO NH substituent, exemplified by these pairwise comparisons:
6a1 vs 16a2, 16b1 vs 16b2, 19a1 vs 19a2, 19b1 vs 19b2, 22a1 vs
2
2
1
2
2a2, and 22b1 vs 22b2.
(Fig. 4A); the Tyr181 to Cys181 substitution leads to the greatly
reduced stacking interactions between the left wing of in-
hibitors and NNIBP. The above two reasons lead to the compounds
p p
ꢁ
2.4. Water solubility measurement
significant decreased activity against RES056. Compared to 19a1
Moreover, the most potent inhibitors 16a1 and 16b1 were
(Figs. 4C), 16a1 (Fig. 4B)is able to establish hydrogen bonding with
evaluated for their water solubility at different pH values (2.0, 7.0,
and 7.4) [14]. As depicted in Table 4, both compounds possessed
desirable solubility at different pH values. Especially, 16a1 and 16b1
exhibited significantly enhanced solubility at pH 7.0 (S ¼ 49.7 and
the main chain of Lys104 by varying the position of the piperazine-
linked sulfonamide group; the cyan group in the left wing could
develop additional hydrogen-bonding interactions with Lys223,
which account for its better resistance profiles than 19a1.
4
9.3
m
g/mL, respectively) and pH 7.4 (S ¼ 39.5 and 38.9
mg/mL,
respectively), which were much greater than that of 25a and ETR.
3. Conclusion
2.5. Molecular modeling analysis
To discover novel HIV-1 NNRTIs with more potent activity, lower
cytotoxicity and favorable solubility, series of novel thiophene[3,2-
d]pyrimidine and thiophene[2,3-d]pyrimidine derivatives were
designed applying by four-point pharmacophore model and
fragment-based molecular hybridization strategy. We report the
synthesis and biological evaluation of these novel analogues. The
antiviral activity results demonstrated that compounds 16a1 and
To fully understand the binding mode of the newly designed
compounds to the NNIBP, the molecular docking for the selected
compounds 15a, 16a1, 19a1 and 22a1 was carried out using the
software SurflexeDock SYBYL-X 2.0 by following a previously re-
ported methodology. Co-crystal structure of WT HIV-1 RT (PDB
code: 6c0n) and RES056 HIV-1 RT (PDB code: 6c0r) were used as
the input structures for docking calculations [25]. Docking results
were visualized with PyMOL and illustrated in Figs. 3 and 4.
As shown in Fig. 3, compounds 15a, 16a1, and 19a1 adopted a
horseshoe conformation in the NNIBP, as is typically found for most
NNRTIs (Fig. 3A) in the DAPY family. Especially, the most potent
compound 16a1 (Fig. 3B) exhibited a series of well-known in-
teractions with NNIBP: (i) the surface-positioned sulfonamide
group arched into the tolerant region I and formed double-
hydrogen bonding with the backbone carbonyl oxygen and nitro-
gen of Val106; (ii) the N atom of the thiophene[3,2-d]pyrimidine
and the amine group linking the central core and the piperidine
ring interacts with the main-chain backbone of Lys101, developing
double hydrogen bond observed in numerous of NNRTIs/RT com-
plexes, which is important for maintaining effective antiviral ac-
tivity; (iii) the left wing projected into the tunnel lined by Tyr181,
1
6b1 afforded the most potent activity, exhibiting nanomole po-
tency against WT, L100I, K103N, and E138K, with EC50 values
ranging from 0.007 M to 0.043 M. Moreover,16b1 also turned out
to be a 0.045 M inhibitor of Y181C. Furthermore, 16b1 exhibited
significantly improved water solubility (S ¼ 49.3 g/mL at pH 7.0)
and reduced cytotoxicity (CC50 > 217.5 M) compared to the lead
5a (S < 1 M). In addition, the mo-
g/mL at pH 7.0, CC50 ¼ 2.30
lecular docking results indicated that double hydrogen bonding
with the backbone of Lys101 and Val106, interactions with
m
m
m
m
m
2
m
m
p-p
Tyr181 and Tyr188 are responsible for the promising in vitro ac-
tivity of these novel compounds.
4. Experimental section
4.1. Chemistry
Tyr188, Phe227, and Trp229, forming
p-p interactions with these
All reactions were routinely monitored by thin layer chroma-
residues. However, the change of the right wing of these inhibitors
result in the key water-mediated hydrogen bonding with the
backbone Lys103 disappeared, which responsible for their reduced
activity toward HIV-1 IIIB.
Additionally, compared with 16a1, the 1-phenylpiperazine
structure of the right wing of 19a1 (Fig. 3C) causes the sulfon-
amide group to deviate from Val106 and resulted in the disap-
pearance of the hydrogen bond; while 15a (Fig. 3E) cannot develop
the hydrogen-bonding interaction with Val106. Although com-
pound 22a1 (Fig. 3D) with 1-phenylpiperidin-3-amine scaffold in
tography on Silica Gel GF254 (Merck). Flash column chromatog-
raphy was performed on columns packed with Silica Gel (200e300
mesh, Qingdao Haiyang Chemical Company). Solvents were puri-
fied and dried by standard methods. All melting points were
determined on a micro melting point apparatus (RY-1G, Tianjin
1
13
TianGuang Optical Instruments). H NMR and C NMR spectra
were recorded in DMSO‑d on a Bruker AV-400 spectrometer with
tetramethylsilane (TMS) as the internal standard. Chemical shifts
are reported in values (ppm) from TMS and coupling constants are
6
d
given in hertz; signals are abbreviated as s (singlet), d (doublet),
and m (multiplet). The mass spectra were measured in A G1313A
Standard LC Autosampler (Agilent).
Table 4
The solubility of compounds 16a1 and 16b1.
4
.1.1. Preparation of tert-butyl 4-(4-aminophenyl)piperazine-1-
Compds
Water solubility (
m
g/mL)
carboxylate (7)
pH ¼ 2.0
pH ¼ 7.0
pH ¼ 7.4
A mixture of 1-fluoro-4-nitrobenzene (5, 1.41 g, 10 mmol) and
tert-butyl piperazine-1-carboxylate (1.86 g, 10 mmol) in DMSO
(20 mL) was heated at 120 C overnight. Then the solution was
cooled to ambient temperature and 100 mL of ice water was added.
Stirring was continued for another 15 min, and the resulting
1
1
2
6a1
6b1
5a
73.1
64.9
84.7
127
49.7
49.3
5.26
<1
39.5
38.9
<1
ꢀ
ETR
<1
6

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
Fig. 3. The cocrystal structure of 25a/WT RT (A) and predicted binding modes of 16a1 (B), 19a1 (C), 22a1 (D) and 15a (E) with the HIV-1 WT RT (PDB code: 6c0n). The hydrogen
bonds are indicated with yellow dashed lines. Nonpolar hydrogen atoms are not shown for clarity.
Fig. 4. The cocrystal structure of 25a/RES056RT (A) and predicted binding modes of 16a1 (B) and 19a1 (C) with the HIV-1 RES056 RT (PDB code: 6c0r). The hydrogen bonds are
indicated with yellow dashed lines. Nonpolar hydrogen atoms are not shown for clarity.
precipitate was collected by filtration and dried to give crude in-
termediate 6, which was used directly in the next step without
further purification. To a solution of intermediate 6 (0.31 g,
white solid. The crude intermediate 7 was used in the next step
þ
without further purification. ESI-MS: m/z 278.5 [Mþ1] .
15 23 3 2
C H N O (277.18).
1.0 mmol) in methanol (15 mL) was added 10% palladium on carbon
(
wet) (0.21 g, 0.10 mmol), and the resulting mixture was stirred
4
.1.2. Preparation of tert-butyl (1-(4-aminophenyl)piperidin-3-yl)
under an atmosphere of hydrogen gas overnight. The reaction
mixture was filtered through a plug of Celite and the filtrate was
evaporated under reduced pressure to afford intermediate 7 as
carbamate (9)
The synthetic procedure was similar to that of 7, but the starting
material was 1-fluoro-4-nitrobenzene (5, 1.41 g, 10 mmol) and tert-
7

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
butyl piperidin-3-ylcarbamate (2.00 g,10 mmol). ESI-MS: m/z 292.4
118.77, 96.88, 61.61, 50.67, 49.42, 43.53, 29.04, 16.58. ESI-MS: m/z
þ
þ
[Mþ1] . C16
25
H N
3
O
2
(291.19).
28 6
497.7 [M þ 1] . C28H N OS (496.20).
4
.1.3. Preparation of tert-butyl 4-(4-aminobenzyl)piperazine-1-
carboxylate (12)
A mixture of 1-(bromomethyl)-4-nitrobenzene (10, 2.14 g,
0 mmol), tert-butyl piperazine-1-carboxylate (1.86 g, 10 mmol)
and K CO (1.52 g, 11 mmol) in DMF (40 mL) was stirred at ambient
4.1.7. (E)-3-(3,5-dimethyl-4-((2-((4-(piperazin-1-yl)phenyl)amino)
thieno[2,3-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (18b)
Recrystallized from EA/PE as a white solid, 67% yield, mp:
ꢀ
1
1
171e172 C. H NMR (400 MHz, DMSO‑d
J ¼ 11.9 Hz, 1H, C -thiophene-H), 7.70 (d, J ¼ 15.7 Hz, 1H, ArCH＝),
7.57 (s, 2H, C , C -thiophene-H),
-Ph’-H), 7.51 (d, J ¼ 11.9 Hz, 1H, C
7.50e7.37 (m, 2H, C , C -Ph’’-H), 7.23e7.18 (m, 2H C , C -Ph’’-H),
6.53 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 2.99 (d, J ¼ 7.1 Hz, 4H), 2.57e2.45
6
) d 9.45 (s, 1H, NH), 7.71 (d,
2
3
6
temperature for 5 h (monitored by TLC); then to the solution was
added 200 mL of ice water and it was stirred for another 15 min.
The resulting precipitate was collected by filtration and dried to
give crude intermediate 11 as yellow solid, which was used directly
in the next step without further purification. To a solution of in-
termediate 11 (0.32 g, 1.0 mmol) in methanol (15 mL) was added
2
6
5
2
6
3
5
13
(m, 4H), 2.14 (s, 6H), 1.34 (s, 1H, NH). C NMR (100 MHz, DMSO‑d
6
)
d
172.11,162.69, 156.91, 152.41, 150.62, 146.32, 132.98, 131.95, 131.83,
128.77, 116.12, 96.77, 49.12, 45.11, 29.06, 16.55. ESI-MS: m/z 483.5
þ
10% palladium on carbon (wet) (0.21 g, 0.10 mmol), and the
[M þ 1] . C27
26 6
H N OS (482.19).
resulting mixture was stirred under an atmosphere of hydrogen gas
overnight. The reaction mixture was filtered through a plug of
Celite and the filtrate was evaporated under reduced pressure to
4.1.8. (E)-3-(4-((2-((4-(3-aminopiperidin-1-yl)phenyl)amino)
thieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile
(21a)
þ
afford intermediate 12 as a white solid. ESI-MS: m/z 292.7 [Mþ1] .
C
16
25
H N
3
O
2
(291.19).
Recrystallized from EA/PE as a brown solid, 64% yield, mp:
ꢀ
1
1
66e167 C. H NMR (400 MHz, DMSO‑d
(d, J ¼ 16.6 Hz, 1H, ArCH＝), 7.56 (s, 2H, C
J ¼ 5.3 Hz, 1H, C -thiophene-H), 7.34 (d, J ¼ 5.3 Hz, 1H, C
phene-H), 6.97e6.84 (m, 2H, C , C -Ph’’-H), 6.80e6.71 (m, 2H, C
-Ph’’-H), 6.30 (d, J ¼ 16.7 Hz,1H, ＝CHCN), 3.24e3.12 (m, 4H), 2.74
(d, J ¼ 7.6 Hz, 1H), 2.12 (s, 6H), 1.69e1.55 (m, 2H), 1.50e1.45 (m, 2H),
6
)
d
9.29 (s, 1H, NH), 7.68
, C -Ph’-H), 7.45 (d,
-thio-
4
.1.4. General procedure for the preparation of intermediates 15a-b,
8a-b and 21aꢁb
In a schlenk-type flask, the starting materials 13a (or 13b, 0.34 g,
.0 mmol) and intermediate 7 (or 9, 12, 1.2 mmol) were dissolved in
dry dioxane (20 mL), and then Pd (dba) (0.09 g, 0.1 mmol), BINAP
2
6
1
6
7
2
6
3
,
1
C
5
2
3
1
3
(
0.06 g, 0.1 mmol) and Cs
2
CO
3
(0.97 g, 3.0 mmol) were added. The
2 6
1.33 (s, 2H, NH ). C NMR (100 MHz, DMSO‑d ) d 164.97, 162.89,
ꢀ
resulting mixture was stirred at 120 C under the atmosphere of
nitrogen for 10 h. Then the solvent was evaporated under reduced
pressure, and the residue was purified by flash column chroma-
tography to give intermediates 14a-b, 17a-b and 20a¡b. Then the
intermediates were dissolved in DCM (5 mL), which was followed
by addition of TFA (10 mmol) at room temperature. The solution
was stirred for another 6 h (monitored by TLC). The mixed solution
was alkalized to pH 9 with saturated NaHCO
DCM (3 ꢂ 10 mL). Then organic phase was dried over anhydrous
Na SO , filtered and purified by flash column chromatography.
159.62, 158.38, 151.33, 138.09, 137.38, 131.93, 131.55, 128.75, 128.20,
127.07, 124.31, 123.74, 120.05, 119.32, 117.31, 111.17, 96.98, 96.78,
54.55, 50.18, 47.24, 29.02, 22.57, 16.51, 16.38. ESI-MS: m/z 497.7
þ
[M þ 1] . C28
28 6
H N OS (496.20).
4.1.9. (E)-3-(4-((2-((4-(3-aminopiperidin-1-yl)phenyl)amino)
thieno[2,3-d]pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile
(21b)
3
and extracted with
Recrystallized from EA/PE as a white solid, 64% yield, mp:
ꢀ
1
2
4
205e206 C. H NMR (400 MHz, DMSO‑d
(d, J ¼ 16.5 Hz, 1H, ArCH＝), 7.49 (s, 2H, C
J ¼ 6.0 Hz, 1H, C -thiophene-H), 7.32 (d, J ¼ 5.9 Hz, 1H, C
phene-H), 7.11e7.08 (m, 2H, C , C -Ph’’-H), 6.52e6.48 (m, 2H, C
6
)
d
9.33 (s, 1H, NH), 7.62
, C -Ph’-H), 7.38 (d,
-thio-
, C
Recrystallized from ethyl acetate/petroleum ether to give in-
termediates 15a-b, 18a-b and 21a¡b, respectively.
2
6
6
5
2
6
3
5
-
4
.1.5. (E)-3-(3,5-dimethyl-4-((2-((4-(piperazin-1-ylmethyl)phenyl)
Ph’’-H), 6.45 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.30e3.21 (m, 4H),
amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (15a)
2.71e2.65 (m, 1H), 2.45e2.43 (m, 2H), 2.22e2.16 (m, 2H), 2.14 (s,
13
Recrystallized from EA/PE as a yellow solid, 67% yield, mp:
2 6
6H), 1.26 (s, 2H, NH ). C NMR (100 MHz, DMSO‑d ) d 172.13,
ꢀ
1
1
63e164 C. H NMR (400 MHz, DMSO‑d
J ¼ 16.7 Hz,1H, ArCH＝), 7.51e7.43 (m, 2H, C
, C -thiophene-H), 7.04 (d,
-Ph’ eH), 7.18 (d, J ¼ 4.2 Hz, 1H, C
-thiophene-H), 7.02e6.96 (m, 2H, C , C -Ph’’-H),
.72 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.00 (d, J ¼ 3.7 Hz, 2H, CH ),
.52e2.50 (m, 4H), 2.48e2.42 (m, 4H), 2.12 (s, 6H), 1.09e1.05 (m,
H, NH). 13C NMR (100 MHz, DMSO‑d
164.84, 158.26, 140.11,
37.55, 131.91, 131.57, 129.49, 128.26, 124.32, 118.63, 107.20, 61.75,
6
)
d
9.51 (s,1H, NH), 7.73 (d,
162.68, 156.96, 152.41, 150.58, 146.79, 132.37, 131.94, 131.82, 128.74,
120.47, 120.13, 119.39, 118.73, 116.49, 96.79, 60.23, 59.27, 49.92,
2
, C -Ph’’-H), 7.40 (s, 2H,
6
C
2
6
6
48.10, 33.88, 29.03, 24.08, 21.23, 16.56, 14.56. ESI-MS: m/z 497.7
þ
J ¼ 4.5 Hz, 1H, C
7
3
5
[M þ 1] . C28
28 6
H N OS (496.20).
6
2
1
2
4.1.10. General procedure for the preparation of final compounds
16a1-2, 16b1-2, 19a1-2, 19b1-2, 22a1-2, and 22b1-2
6
) d
1
Intermediates 15a (or 15b, 18a-b and 21a-b) were dissolved in
5
0.67, 50.13, 46.16, 43.97, 29.04, 16.60, 16.52. ESI-MS: m/z 497.7
anhydrous DCM (10 mL) in the presence of Et
3
N (1.2 eq), followed
þ
ꢀ
[M þ 1] . C28
H
28
N
6
OS (496.20).
by addition of the substituted sulfonyl chloride (1.2 eq) at 0 C. The
reaction mixture was stirred at room temperature for 2e5 h
(monitored by TLC), and to the reaction was added 10 mL DCM. The
organic phase was washed with saturated sodium chloride
4.1.6. (E)-3-(3,5-dimethyl-4-((2-((4-(piperazin-1-ylmethyl)phenyl)
amino)thieno[2,3-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (15b)
Recrystallized from EA/PE as a white solid, 54% yield, mp:
(3 ꢂ 5 mL), then dried over anhydrous Na
2 4
SO , filtered, and purified
ꢀ
1
1
(
(
＝
C
62e164 C. H NMR (400 MHz, DMSO‑d
dt, J ¼ 6.0, 4.1 Hz, 2H, C , C -Ph’’-H), 7.56 (s, 2H, C
dt, J ¼ 6.0, 4.1 Hz, 2H, C , C -Ph’’-H), 7.37 (d, J ¼ 16.5 Hz, 1H, ArCH
), 6.98 (d, J ¼ 5.1 Hz, 1H, C -thiophene-H), 6.72 (d, J ¼ 5.1 Hz, 1H,
-thiophene-H), 6.50 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.39 (s, 2H,
NeCH ), 3.08e3.05 (m, 4H), 2.57e2.40 (m, 4H), 2.13 (s, 6H), 1.34 (s,
H, NH). C NMR (100 MHz, DMSO‑d
52.26, 150.56, 139.73, 132.00, 131.82, 129.50, 128.74, 121.31, 119.42,
6
)
d
9.65 (s, 1H, NH), 7.71
by flash column chromatography. The product was recrystallized
from ethyl acetate/petroleum ether to afford the target compounds
16a1-2, 16b1-2, 19a1-2, 19b1-2, 22a1-2, and 22b1-2.
2
6
2
, C -Ph’-H), 7.48
6
3
5
6
5
4.1.11. (E)-4-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)thieno
[3,2-d]pyrimidin-2-yl)amino)benzyl)piperazine-1-sulfonamide
(16a1)
2
13
1
1
6
) d 171.87, 162.69, 156.77,
Recrystallized from EA/PE as a yellow solid, 44% yield, mp:
8

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
ꢀ
1
ꢀ
1
1
(
7
2
60e162 C. H NMR (400 MHz, DMSO‑d
d, J ¼ 16.7 Hz, 1H, ArCH＝), 7.95 (d, J ¼ 4.7 Hz, 1H, C
.73 (s, 2H, C , C -Ph’-H), 7.59e7.52 (m, 2H, C , C -Ph’’-H), 7.37 (s,
) 7.20 (d, J ¼ 5.2 Hz, 1H, C -thiophene-H), 7.03e6.96 (m,
6
)
d
9.52 (s, 1H, NH), 8.43
201e202 C. H NMR (400 MHz, DMSO‑d
(d, J ¼ 5.2 Hz,1H, C -thiophene-H), 7.63 (d, J ¼ 16.7 Hz,1H, ArCH＝),
7.49 (s, 2H, C , C -thiophene-H),
-Ph’-H), 7.27 (d, J ¼ 5.3 Hz, 1H, C
7.25 (d, J ¼ 8.2 Hz, 2H, C , C -Ph’’-H), 6.61 (d, J ¼ 8.1 Hz, 2H, C , C
Ph’’-H), 6.45 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.19e3.16 (m, 4H),
6
) d 9.23 (s, 1H, NH), 8.22
6
-thiophene-H),
6
2
6
2
6
2
6
7
H, SO
2
NH
2
7
2
6
3
5
-
2
H, C , C
3
5
-Ph’’-H), 6.40 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.50 (s, 2H,
13
CH
2
), 3.44e3.41 (m, 4H), 2.45e2.34 (m, 4H), 2.12 (s, 6H). ESI-MS: m/
3.05e3.00 (m, 4H), 2.86 (s, 3H, SO
(100 MHz, DMSO‑d 165.47, 162.96, 158.36, 152.07, 150.55, 145.51,
37.35, 133.95, 132.12, 131.93, 128.77, 123.75, 119.97, 119.35, 116.85,
2 3
CH ), 2.14 (s, 6H). C NMR
þ
z 576.7 [M þ 1] . C28
29
H N
7
O
3
S
2
(575.18).
6
) d
1
4
.1.12. (E)-3-(3,5-dimethyl-4-((2-((4-((4-(methylsulfonyl)
96.95, 49.39, 45.88, 34.27, 31.43, 22.53, 16.50, 14.43. ESI-MS: m/z
þ
piperazin-1-yl)methyl)phenyl)amino)thieno[3,2-d]pyrimidin-4-yl)
oxy)phenyl)acrylonitrile (16a2)
28 6 3 2
561.7 [M þ 1] . C28H N O S (560.17).
Recrystallized from EA/PE as a white solid, 52% yield, mp:
4.1.17. (E)-4-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)thieno
[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazine-1-sulfonamide
(19b1)
ꢀ
1
1
(
7
20e121 C. H NMR (400 MHz, DMSO‑d
d, J ¼ 17.0 Hz, 1H, ArCH＝), 8.32 (d, J ¼ 4.7 Hz, 1H, C
.57 (s, 2H, C , C -Ph’-H), 7.49e7.42 (m, 2H, C , C -Ph’’-H), 7.39 (d,
-thiophene-H), 7.03e6.96 (m, 2H, C , C -Ph’’-H),
), 3.10 (s, 3H,
6
)
d
9.50 (s, 1H, NH), 7.69
6
-thiophene-H),
2
6
2
6
Recrystallized from EA/PE as a white solid, 42% yield, mp:
ꢀ
1
J ¼ 5.2 Hz, 1H, C
7
3
5
161e163 C. H NMR (400 MHz, DMSO‑d
J ¼ 16.6 Hz,1H, ArCH＝), 7.60 (s, 2H, C , C
1H, C
-thiophene-H), 7.46 (d, J ¼ 6.0 Hz, 1H, C
(s, 2H, SO NH ), 7.32e7.18 (m, 2H, C , C -Ph’’-H), 6.82e6.64 (m, 2H,
, C
6
)
d
9.52 (s,1H, NH), 7.75 (d,
-Ph’-H), 7.51 (d, J ¼ 6.0 Hz,
-thiophene-H), 7.39
6
SO
.50 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.39 (s, 2H, NeCH
2
2
6
13
2
CH
3
), 2.92e2.85 (m, 4H), 2.53e2.48 (m, 4H), 2.12 (s, 6H).
165.32, 162.98, 158.21, 150.51, 139.97,
37.59, 132.17, 131.91, 129.70, 129.27, 128.75, 123.81, 118.62, 107.12,
C
6
5
NMR (100 MHz, DMSO‑d
6
)
d
2
2
2
6
1
9
[
C
3
5
-Ph’’-H), 6.57 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.88e3.62 (m, 4H),
þ
7.00, 61.63, 52.14, 45.93, 34.11, 29.00, 16.52. ESI-MS: m/z 575.5
3.16e3.07 (m, 4H), 2.15 (s, 6H). ESI-MS: m/z 562.5 [M þ 1] .
þ
M þ 1] . C29
H
30
N
6
O
S
3 2
(574.18).
27 27 7 3 2
C H N O S (561.16). (E)-3-(3,5-dimethyl-4-((2-((4-(4-(methyl-
sulfonyl)piperazin-1-yl)phenyl)amino)thieno[2,3-d]pyrimidin-4-
yl)oxy)phenyl)acrylonitrile (19b2).
4
.1.13. (E)-4-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)thieno
[
(
2,3-d]pyrimidin-2-yl)amino)benzyl)piperazine-1-sulfonamide
16b1)
Recrystallized from EA/PE as a yellow solid, 43% yield, mp:
Recrystallized from EA/PE as a white solid, 56% yield, mp:
ꢀ
1
218e220 C. H NMR (400 MHz, DMSO‑d
J ¼ 16.7 Hz,1H, ArCH＝), 7.56 (s, 2H, C , C
1H, C
-thiophene-H), 7.42 (d, J ¼ 5.9 Hz, 1H, C
7.25e7.20 (m, 2H, C , C -Ph’’-H), 6.74e6.68 (m, 2H, C
6.52 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.36e3.25 (m, 4H), 3.15e3.09 (m,
6
)
d
9.47 (s,1H, NH), 7.71 (d,
-Ph’-H), 7.47 (d, J ¼ 5.9 Hz,
-thiophene-H),
, C -Ph’’-H),
2
6
ꢀ
1
1
42e144 C. H NMR (400 MHz, DMSO‑d
d, J ¼ 16.7 Hz, 1H, ArCH＝), 7.57 (s, 2H, C
J ¼ 5.8 Hz, 1H, C -thiophene-H), 7.46 (d, J ¼ 5.9 Hz, 1H, C
phene-H), 7.43 (s, 2H, SO NH , C -Ph’’-H),
), 7.41 (d, J ¼ 6.4 Hz, 2H, C
.04e6.90 (m, 2H, C , C
6
)
d
9.65 (s, 1H, NH), 7.70
, C -Ph’-H), 7.49 (d,
-thio-
6
5
(
2
6
2
6
3
5
6
5
13
2
2
2
6
4H), 2.93 (s, 3H, SO
2 3 6
CH ), 2.11 (s, 6H). C NMR (100 MHz, DMSO‑d )
7
3
3
5
-Ph’’-H), 6.50 (d, J ¼ 16.7 Hz, 1H, ＝CHCN),
d
172.08, 162.69, 156.89, 152.38, 150.62, 133.44, 131.96, 131.84,
129.70, 128.76, 122.61, 120.66, 120.07, 118.73, 116.75, 99.99, 96.81,
.56 (s, NeCH
2
), 2.42e2.33 (m, 4H), 2.30e2.25 (m, 4H), 2.12 (s, 6H).
þ
ESI-MS: m/z 576.7 [M þ 1] . C28
29
H N
7
O
3
S
2
(575.18).
49.28, 48.54, 45.85, 45.68, 43.31, 34.27, 16.68, 16.55.ESI-MS: m/z
þ
5
61.5 [M þ 1] . C28
28 6 3 2
H N O S (560.17).
4
.1.14. (E)-3-(3,5-dimethyl-4-((2-((4-((4-(methylsulfonyl)
piperazin-1-yl)methyl)phenyl)amino)thieno[2,3-d]pyrimidin-4-yl)
oxy)phenyl)acrylonitrile (16b2)
4.1.18. (E)-N-(1-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)
thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)piperidin-3-yl)
sulfonamide (22a1)
Recrystallized from EA/PE as a white solid, 54% yield, mp:
ꢀ
1
1
49e151 C. H NMR (400 MHz, DMSO‑d
J ¼ 16.6 Hz,1H, ArCH＝), 7.50 (s, 2H, C , C
H, C , C
-Ph’’-H), 7.34 (d, J ¼ 4.4 Hz, 1H, C
J ¼ 8.3 Hz, 2H, C , C -Ph’’-H), 6.89 (d, J ¼ 4.5 Hz, 1H, C
H), 6.43 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.05e3.02 (m, 4H), 2.81 (s, 3H,
6
)
d
9.57 (s,1H, NH), 7.62 (d,
-Ph’-H), 7.41 (d, J ¼ 8.3 Hz,
-thiophene-H), 7.28 (d,
-thiophene-
Recrystallized from EA/PE as a white solid, 41% yield, mp:
ꢀ
1
2
6
147e148 C. H NMR (400 MHz, DMSO‑d
J ¼ 16.7 Hz, 1H, ArCH＝), 7.44 (d, J ¼ 5.4 Hz, 1H, NH), 7.35 (d,
J ¼ 8.6 Hz, 2H, C , C -Ph’’-H), 7.22 (s, 2H, C , C -Ph’-H), 6.87 (d,
J ¼ 8.9 Hz, 1H, C -thiophene-H), 6.85 (s, 2H, SO NH ), 6.75 (d,
J ¼ 9.1 Hz, 1H, C , C -Ph’’-
6
) d 9.42 (s,1H, NH), 7.45 (d,
2
2
6
6
3
5
5
2
6
2
6
6
2
2
1
3
SO
2
CH
3
), 2.35e2.32 (m, 4H), 2.15 (s, 6H), 1.87 (s, 2H, NeCH
162.66, 156.96, 150.53, 131.93, 131.82,
31.37, 128.73, 128.20, 127.41, 119.36, 118.74, 116.71, 96.82, 56.71,
2
).
C
7
-thiophene-H), 6.51 (d, J ¼ 8.7 Hz, 2H, C
3
5
NMR (100 MHz, DMSO‑d
6
)
d
H), 6.29 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.67e3.49 (m, 2H), 2.72e2.56
(m, 2H), 2.19e2.15 (m,1H), 2.14 (s, 6H),1.82e1.72 (m, 2H),1.64e1.49
(m, 2H). C NMR (100 MHz, DMSO‑d
151.33, 150.26, 148.37, 138.01, 133.87, 131.91, 131.66, 131.55, 128.20,
27.22, 124.32, 119.28, 116.16, 111.10, 96.75, 53.07, 49.30, 46.72,
1
5
5
1
3
0.64, 49.78, 49.68, 31.48, 29.03, 23.81, 16.58, 16.48. ESI-MS: m/z
75.7 [M þ 1] . C29
6
) d 164.97, 162.87, 159.64,
þ
30 6 3 2
H N O S (574.18).
1
þ
1] .
4.1.15. (E)-3-(3,5-dimethyl-4-((2-((4-(piperazin-1-yl)phenyl)
29.67, 23.59, 16.51, 16.37. ESI-MS: m/z 576.5 [M
(575.18).
þ
amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylonitrile (19a1)
28 29 7 3 2
C H N O S
Recrystallized from EA/PE as a white solid, 65% yield, mp:
ꢀ
1
1
(
7
7
57e159 C. H NMR (400 MHz, DMSO‑d
d, J ¼ 5.4 Hz, 1H, C -thiophene-H), 7.70 (d, J ¼ 16.7 Hz, 1H, ArCH＝),
.57 (s, 2H, C , C -thiophene-H),
-Ph’ eH), 7.34 (d, J ¼ 5.3 Hz, 1H, C
.32e7.25 (m, 2H, C , C -Ph’’-H), 6.70 (m, 2H, C , C -Ph’’-H), 6.53 (d,
6
)
d
9.28 (s, 1H, NH), 8.30
4.1.19. (E)-N-(1-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)
thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)piperidin-3-yl)
methanesulfonamide (22a2)
6
2
6
7
2
6
3
5
Recrystallized from EA/PE as a white solid, 56% yield, mp:
ꢀ
1
J ¼ 16.7 Hz, 1H, ＝CHCN), 2.70e2.65 (m, 4H), 2.36e2.32 (m, 4H),
122e124 C. H NMR (400 MHz, DMSO‑d ) d 9.18 (s,1H, NH), 8.21 (d,
6
þ
2
C
.12 (s, 6H), 1.24 (s, 1H, NH). ESI-MS: m/z 483.5 [M þ 1] .
J ¼ 5.3 Hz, 1H, NH), 7.60 (d, J ¼ 16.7 Hz, 1H, ArCH＝), 7.49 (s, 2H, C
2
,
H
27 26
N
6
OS (482.19).
C
C
6
-Ph’-H), 7.27 (d, J ¼ 7.1 Hz, 1H, C
, C
-Ph’’-H), 7.10 (d, J ¼ 7.1 Hz, 1H, C
, C
-Ph’’-H), 6.43 (d, J ¼ 16.7 Hz, 1H, ＝CHCN), 3.45e3.42
(m, 1H), 2.90 (s, 3H, SO CH ), 2.47e2.37 (m, 4H), 2.15 (s, 6H),
1.90e1.81 (m, 2H), 1.74e1.66 (m, 2H). C NMR (100 MHz, DMSO‑d
165.46, 162.92, 158.43, 152.03, 150.48, 146.01, 137.29, 133.24,
6
-thiophene-H), 7.26e7.11 (m, 2H,
2
6
7
-thiophene-H), 6.70e6.53
4
1
.1.16. (E)-3-(3,5-dimethyl-4-((2-((4-(4-(methylsulfonyl)piperazin-
-yl)phenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)
acrylonitrile (19a2)
(m, 2H, C
3
5
2
3
13
6
)
Recrystallized from EA/PE as a yellow solid, 51% yield, mp:
d
9

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
1
4
C
32.09, 131.91, 128.75, 123.76, 120.13, 119.34, 116.81, 96.96, 56.82,
4.3. HIV-1 RT inhibition assays
þ
1] .
9.81, 31.48, 23.84, 16.52. ESI-MS: m/z 575.5 [M
(574.18).
þ
29
30
H N
6
O
3
S
2
The HIV-1 reverse transcriptase (RT) assay kit (Roche) was used
to perform the RT inhibition assay. All the reagents for performing
the RT reaction were from the kit and the particular ELISA pro-
cedures were carried out following the description in the kit pro-
tocol [28]. Firstly, the reaction mixture containing the 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
4.1.20. (E)-N-(1-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)
thieno[2,3-d]pyrimidin-2-yl)amino)phenyl)piperidin-3-yl)
sulfonamide (22b1)
ꢀ
Recrystallized from EA/PE as a yellow solid, 45% yield, mp:
ꢀ
1
2
00e202 C. H NMR (400 MHz, DMSO‑d
d, J ¼ 16.7 Hz, 1H, ArCH＝), 7.48 (s, 2H, C
J ¼ 7.7 Hz, 1H, NH), 7.38 (d, J ¼ 5.9 Hz, 1H, C -thiophene-H), 7.33 (d,
J ¼ 6.0 Hz, 1H, C -thiophene-H), 7.28 (s, 2H, SO NH ), 7.18e7.07 (m,
H, C , C -Ph’’-H), 6.59e6.52 (m, 2H, C , C -Ph’’-H), 6.43 (d,
6
)
d
9.30 (s, 1H, NH), 7.61
ꢀ
(
2
, C -Ph’-H), 7.43 (d,
6
6
ensure that the retranscriptional cDNA chain that contained biotin-
labeled dNTPs was bound to streptavidin. Then un-bound dNTPs
were removed using washing buffer and anti-DIG-POD working
5
2
2
2
2
6
3
5
ꢀ
J ¼ 16.7 Hz,1H, ＝CHCN), 3.46e3.40 (m, 4H), 3.37e3.31 (m,1H), 2.14
solution was added. After incubation for another 1 h at 37 C, the
(
[
s, 6H), 1.94e1.84 (m, 2H), 1.71e1.64 (m, 2H). ESI-MS: m/z 576.7
M þ 1] . C28
DIG-labeled dNTPs incorporated into cDNA were bound to the anti-
DIG-POD antibody. The unbound anti-DIG-PODs were removed and
the peroxide substrate (ABST) solution was added to the MTPs. A
colored reaction proceeds during the cleavage of the substrate
catalyzed by POD. The absorbance of the sample was determined at
OD405 nm using a microtiter plate ELISA reader. The percentage
inhibitory activity of RT inhibitors was calculated using the formula
as given below:
þ
29 7 3 2
H N O S (575.18).
4.1.21. (E)-N-(1-(4-((4-(4-(2-cyanovinyl)-2,6-dimethylphenoxy)
thieno[2,3-d]pyrimidin-2-yl)amino)phenyl)piperidin-3-yl)
methanesulfonamide (22b2)
Recrystallized from EA/PE as a yellow solid, 57% yield, mp:
ꢀ
1
1
44e146 C. H NMR (400 MHz, DMSO‑d
d, J ¼ 15.8 Hz, 1H, ArCH＝), 7.48 (s, 2H, C
J ¼ 6.0, 2.1 Hz, 2H, C , C -Ph’’-H), 7.38 (d, J ¼ 5.9 Hz, 1H, C
-thiophene-H), 7.13e7.08 (m,
-Ph’’-H), 7.01 (s, 1H, NH), 6.74 (d, J ¼ 15.8 Hz, 1H, ＝CHCN),
.94e2.90 (m, 2H), 2.57e2.49 (m, 2H), 2.49e2.45 (m, 1H), 2.43 (s,
6
)
d
9.34 (s, 1H, NH), 7.67
, C -Ph’-H), 7.45 (dd,
-thio-
Inhibition (%) ¼ (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).
(
2
6
2
6
6
phene-H), 7.33 (d, J ¼ 5.9 Hz, 1H, C
5
2
2
3
H, C
3
, C
5
4.4. Water solubility measurement
13
H, SO
2
CH
3
), 2.14 (s, 6H), 1.92e1.85 (m, 2H), 1.28e1.24 (m, 2H).
162.66, 156.96, 150.53, 131.82, 131.37,
28.73, 128.20, 127.41, 118.74, 116.71, 96.82, 56.71, 50.64, 49.78,
C
Compound 16a1 or 16b1 (0.1, 1, 10 mg) was dissolved in
deionized water (pH ¼ 2.0, 7.0, and 7.4) until the solution was clear.
The assays were measured at least in duplicate [14].
6
NMR (100 MHz, DMSO‑d ) d
1
4
C
þ
9.68, 29.03, 23.81, 16.58, 16.48. ESI-MS: m/z 575.7 [M þ 1] .
29
30
H N
6
O
3
S
2
(574.18).
4.5. Molecular simulation studies
The molecular modelling study was performed by Sybyl-X 2.0
software. All the molecules were built using standard bond lengths
and angles from Sybyl-X 2.0/Base Builder and optimized using the
Tripos force field for 1000 generations, until the minimized con-
formers of the ligand were the same. The flexible docking method
(Surflex-Dock) docks the ligand automatically into the ligand-
binding site of the receptor with a protocol-based approach and
an empirically derived scoring function. The protocol is a compu-
tational representation of a putative ligand that binds to the
intended binding site and is a unique and essential element of the
docking algorithm. The scoring function in Surflex-Dock, contain-
ing hydrophobic, polar, repulsive, entropic, and solvation terms,
was trained to estimate the dissociation constant (Kd). The protein
was prepared by removing the ligand and other unnecessary small
molecules from the cocrystal structures (PDB code: 6c0n, 6c0r)),
polar hydrogen atoms and charges were added to the protein
before docking. 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 to move flexibly within the
tentative binding pocket. The atomic charges were recalculated
using the Kollman all-atom approach for the protein and the Gas-
teiger-Hückel approach for the ligand. The binding interaction en-
ergy was calculated, including van der Waals, electrostatic, and
torsional energy terms defined in the Tripos force field. The struc-
ture optimization was performed for 10,000 generations using a
genetic algorithm, and the 20-best-scoring ligand-protein com-
4
.2. In vitro anti-HIV-1 assay
Using MTT method, the anti-HIV-1 activity assay was performed
in MT-4 cells. Compounds stock solutions (10 ꢂ final concentration)
were added in 25 L volumes to two series of triplicate wells to
m
allow simultaneous evaluation of their effects on mock- and HIV-
infected cells [26,27]. Serial 5-fold dilutions of compounds were
made directly in flat-bottomed 96-well microtiter trays using a
Biomek 3000 robot (Beckman instruments, Fullerton, CA). Un-
treated HIV- and mock-infected cell samples were included as
controls. HIV stock (50
mL) at 100e300 CCID50 (50% cell culture
infectious doses) or culture medium was added to either the
infected or mock-infected cell wells of the microtiter tray. Mock-
infected cells were used to evaluate the effects of test compound
on uninfected cells to assess the cytotoxicity of the compounds.
Exponentially growing MT-4 cells were centrifuged for 5 min at
2
20 g and the supernatant was discarded. The MT-4 cells were
5
resuspended at 6 ꢂ 10 cells/mL and 50
mL volumes were trans-
ferred to the microtiter tray wells. Five days after infection, the
viability of mock-and HIV-infected cells was examined spectro-
photometrically using the MTT assay. The absorbances were read in
an eight-channel computer-controlled photometer (Infinite
M1000, Tecan), at two wavelengths (540 and 690 nm). All data were
calculated using the median absorbance value of three wells. The
5
0% cytotoxic concentration (CC50) was defined as the concentra-
tion of the compounds that reduced the absorbance (OD540) of the
mock-infected control sample by 50%. The concentration achieving
2
plexes were kept for further analysis. The -log (Kd) values of the
5
0% protection against the cytopathic effect of the virus in infected
20-best-scoring complexes, representing the binding affinities of
cells was defined as the 50% effective concentration (EC50).
ligand with RT, encompassed a wide range of functional classes
10

Y. Sun, D. Kang, F. Da et al.
European Journal of Medicinal Chemistry 214 (2021) 113204
10 2-10ꢁ9). The highest-scoring 3D structural model of ligand-
ꢁ
(
[10] C. Beyrer, A. Pozniak, HIV drug resistance - an emerging threat to epidemic
control, N. Engl. J. Med. 377 (2017) 1605e1607.
bound RT was chosen to define the binding interaction [25].
[
11] H. Xu, E.L. Asahchop, M. Oliveira, P.K. Quashie, Y. Quan, B.G. Brenner,
M.A. Wainberg, Compensation by the E138K mutation in HIV-1 reverse
transcriptase for deficits in viral replication capacity and enzyme processivity
associated with the M184I/V mutations, J. Virol. 85 (2011) 11300e11308.
12] D. Kang, Z. Fang, Z. Li, B. Huang, H. Zhang, X. Lu, H. Xu, Z. Zhou, X. Ding,
D. Daelemans, E. De Clercq, C. Pannecouque, P. Zhan, X. Liu, Design, synthesis,
and evaluation of thiophene 3,2-d pyrimidine derivatives as HIV-1 non-
nucleoside reverse transcriptase inhibitors with significantly improved drug
resistance profiles, J. Med. Chem. 59 (2016) 7991e8007.
Author contributions
[
All authors contributed to writing the manuscript. All authors
have given approval to the final version of the manuscript.
Notes
[13] M. Feng, D. Wang, J.A. Grobler, D.J. Hazuda, M.D. Miller, M.-T. Lai, Vitro
resistance selection with doravirine (MK-1439),
a novel nonnucleoside
reverse transcriptase inhibitor with distinct mutation development pathways,
Antimicrob. Agents Chemother. 59 (2015) 590e598.
The authors declare no competing financial interest.
Declaration of competing interest
[
14] B. Huang, W. Chen, T. Zhao, Z. Li, X. Jiang, T. Ginex, D. Vilchez, F.J. Luque,
D. Kang, P. Gao, J. Zhang, Y. Tian, D. Daelemans, E. De Clercq, C. Pannecouque,
P. Zhan, X. Liu, Exploiting the tolerant region I of the non-nucleoside reverse
transcriptase inhibitor (NNRTI) binding pocket: discovery of potent
diarylpyrimidine-typed HIV-1 NNRTIs against wild-type and E138K mutant
virus with significantly improved water solubility and favorable safety pro-
files, J. Med. Chem. 62 (2019) 2083e2098.
15] D. Kang, F.X. Ruiz, D. Feng, A. Pilch, T. Zhao, F. Wei, Z. Wang, Y. Sun, Z. Fang,
E. De Clercq, C. Pannecouque, E. Arnold, X. Liu, P. Zhan, Discovery and char-
acterization of fluorine-substituted diarylpyrimidine derivatives as novel HIV-
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[
Acknowledgments
1
NNRTIs with highly improved resistance profiles and low activity for the
hERG ion channel, J. Med. Chem. 63 (2020) 1298e1312.
We acknowledge financial support from the National Natural
Science Foundation of China (NSFC Nos. 81973181, 81903453),
Shandong Provincial Key research and development project (Nos.
[
[
16] X. Zuo, Z. Huo, D. Kang, G. Wu, Z. Zhou, X. Liu, P. Zhan, Current insights into
anti-HIV drug discovery and development: a review of recent patent litera-
ture (2014-2017), Expert Opin. Ther. Pat. 28 (2018) 299e316.
17] Y. Sang, S. Han, C. Pannecouque, E. De Clercq, C. Zhuang, F. Che, Ligand-based
design of nondimethylphenyl-diarylpyrimidines with improved metabolic
stability, safety, and oral pharmacokinetic profiles, J. Med. Chem. 62 (2019)
11430e11436.
2
019JZZY021011), Natural Science Foundation of Jiangsu Province
(
(
BK2019041035), Shandong Provincial Natural Science Foundation
ZR2019BH011, ZR2020YQ61), China Postdoctoral Science Founda-
[
[
18] D. Kang, H. Zhang, Z. Wang, T. Zhao, T. Ginex, F.J. Luque, Y. Yang, G. Wu,
D. Feng, F. Wei, J. Zhang, E. De Clercq, C. Pannecouque, C.H. Chen, K.-H. Lee,
N.A. Murugan, T.A. Steitz, P. Zhan, X. Liu, Identification of dihydrofuro 3,4-
d pyrimidine derivatives as novel HIV-1 non-nucleoside reverse transcrip-
tase inhibitors with promising antiviral activities and desirable physico-
chemical properties, J. Med. Chem. 62 (2019) 1484e1501.
19] D. Kang, T. Zhao, Z. Wang, D. Feng, H. Zhang, B. Huang, G. Wu, F. Wei, Z. Zhou,
L. Jing, X. Zuo, Y. Tian, V. Poongavanam, J. Kongsted, E. De Clercq,
C. Pannecouque, P. Zhan, X. Liu, Discovery of piperidine-substituted thiazolo
tion (2019T120596), Young Scholars Program of Shandong Uni-
versity (YSPSDU No. 2016WLJH32), National Science and
Technology Major Projects for “Major New Drugs Innovation and
Development” (2019ZX09301126), the Taishan Scholar Program at
Shandong Province. The technical assistance of Mr. Kris Uyttersprot
and Mrs. Kristien Erven, for the HIV experiments is gratefully
acknowledged.
5,4-d pyrimidine derivatives as potent and orally bioavailable HIV-1 non-
nucleoside reverse transcriptase inhibitors, Commun. Chem. 2 (2019) 74.
20] D. Kang, D. Feng, L. Jing, Y. Sun, F. Wei, X. Jiang, G. Wu, E. De Clercq,
C. Pannecouque, P. Zhan, X. Liu, In situ click chemistry-based rapid discovery
of novel HIV-1 NNRTIs by exploiting the hydrophobic channel and tolerant
regions of NNIBP, Eur. J. Med. Chem. 193 (2020), 112237.
21] D. Kang, Y. Sun, N.A. Murugan, D. Feng, F. Wei, J. Li, X. Jiang, E. De Clercq,
C. Pannecouque, P. Zhan, X. Liu, Structure-activity relationship exploration of
NNIBP tolerant region I leads to potent HIV-1 NNRTIs, ACS Infect. Dis. 6 (2020)
Appendix A. Supplementary data
[
[
[
[
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113204.
References
2225e2234.
22] D. Kang, D. Feng, Y. Sun, Z. Fang, F. Wei, E. De Clercq, C. Pannecouque, X. Liu,
P. Zhan, Structure-based bioisosterism yields HIV-1 NNRTIs with improved
drug-resistance profiles and favorable pharmacokinetic properties, J. Med.
Chem. 63 (2020) 4837e4848.
23] D. Kang, D. Feng, T. Ginex, J. Zou, F. Wei, T. Zhao, B. Huang, Y. Sun, S. Desta,
E. De Clercq, C. Pannecouque, P. Zhan, X. Liu, Exploring the hydrophobic
channel of NNIBP leads to the discovery of novel piperidine-substituted
thiophene[3,2-d]pyrimidine derivatives as potent HIV-1 NNRTIs, Acta
Pharm. Sin. B. 10 (2020) 878e894.
[
1] D.M. Margolis, N.M. Archin, M.S. Cohen, J.J. Eron, G. Ferrari, V. Garcia, C.L. Gay,
N. Goonetilleke, S.B. Joseph, R. Swanstrom, A.-M.W. Turner, A. Wahl, Curing
HIV: seeking to target and clear persistent infection, Cell 181 (2020) 189e206.
2] R.J. Shattock, M. Warren, S. McCormack, C.A. Hankins, Turning the tide against
HIV, Science 333 (2011) 42e43.
[
[
3] G. Bec, B. Meyer, M.-M. Gerard, J. Steger, K. Fauster, P. Wolff, D. Burnouf,
R. Micura, P. Dumas, E. Ennifar, Thermodynamics of HIV-1 reverse transcrip-
tase in action elucidates the mechanism of action of non-nucleoside in-
hibitors, J. Am. Chem. Soc. 135 (2013) 9743e9752.
[
24] D. Kang, Z. Fang, B. Huang, X. Lu, H. Zhang, H. Xu, Z. Huo, Z. Zhou, Z. Yu,
Q. Meng, G. Wu, X. Ding, Y. Tian, D. Daelemans, E. De Clercq, C. Pannecouque,
P. Zhan, X. Liu, Structure-based optimization of thiophene 3,2-d pyrimidine
derivatives as potent HIV-1 non-nucleoside reverse transcriptase inhibitors
with improved potency against resistance-associated variants, J. Med. Chem.
[
[
[
4] C. Zhuang, C. Pannecouque, E. De Clercq, F. Chen, Development of non-
nucleoside reverse transcriptase inhibitors (NNRTIs): our past twenty years,
Acta Pharm. Sin. B. 10 (2020) 961e978.
5] P. Zhan, X. Chen, D. Li, Z. Fang, E. De Clercq, X. Liu, HIV-1 NNRTIs: structural
diversity, pharmacophore similarity, and impliations for drug design, Med.
Res. Rev. 33 (2013) E1eE72.
6] L. Wang, S.G. Sarafianos, Z. Wang, Cutting into the substrate dominance:
pharmacophore and structure-based approaches toward inhibiting human
immunodeficiency virus reverse transcriptase-associated ribonuclease H, Ac-
counts Chem. Res. 53 (2020) 218e230.
60 (2017) 4424e4443.
[
25] Y. Yang, D. Kang, L.A. Nguyen, Z.B. Smithline, C. Pannecouque, P. Zhan, X. Liu,
T.A. Steitz, Structural basis for potent and broad inhibition of HIV-1 RT by
thiophene 3,2-d pyrimidine non-nucleoside inhibitors, elife 7 (2018), e36340
pii.
[
[
26] C. Pannecouque, D. Daelemans, E. De Clercq, Tetrazolium-based colorimetric
assay for the detection of HIV replication inhibitors: revisited 20 years later,
Nat. Protoc. 3 (2008) 427e434.
27] A. Hombrouck, A. Voet, B. Van Remoortel, C. Desadeleer, M. De Maeyer,
Z. Debyser, M. Witvrouw, Mutations in human immunodeficiency virus type 1
integrase confer resistance to the naphthyridine L-870,810 and cross-
resistance to the clinical trial drug GS-9137, Antimicrob. Agents, Chemother
[
7] V. Namasivayam, M. Vanangamudi, V.G. Kramer, S. Kurup, P. Zhan, X. Liu,
J. Kongsted, S.N. Byrareddy, The journey of HIV-1 non-nucleoside reverse
transcriptase inhibitors (NNRTIs) from lab to clinic, J. Med. Chem. 62 (2019)
4851e4883.
[
[
8] P. Zhan, C. Pannecouque, E. De Clercq, X. Liu, Anti-HIV drug discovery and
development: current innovations and future trends, J. Med. Chem. 59 (2016)
2849e2878.
52 (2008) 2069e2078.
9] D.A. Lehman, D.C. Wamalwa, C.O. McCoy, F.A. Matsen, A. Langat, B.H. Chohan,
S. Benki-Nugent, R. Custers-Allen, F.D. Bushman, G.C. John-Stewart,
J. Overbaugh, Low-frequency nevirapine resistance at multiple sites may
predict treatment failure in infants on nevirapine-based treatment, Jaids-J.
Acq. Imm. Def. 60 (2012) 225e233.
[
28] K. Suzuki, B.P. Craddock, N. Okamoto, T. Kano, R.T. Steigbigel, Poly A-linked
colorimetric microtiter plate assay for HIV reverse transcriptase, J. Virol.
Methods 44 (1993) 189e198.
11