Targeting dual tolerant regions of binding pocket: Discovery of novel morpholine-substituted diarylpyrimidines as potent HIV-1 NNRTIs with significantly improved water solubility

Article abstract of DOI:10.1016/j.ejmech.2020.112811

To address the intractable issues of drug resistance and poor solubility, a novel series of morpholine-substituted diarylpyrimidines targeting the tolerant region I and tolerant region II of NNIBP were rationally designed by utilizing the available crystallography studies. The biological evaluation results showed that four most promising compounds (14e1, 14g1, 14g2 and 14j2) displayed excellent potency against WT HIV-1 strain with EC₅₀ values ranging from 58 to 87 nM, being far more potent than NVP and comparable to ETV. Besides, some derivatives exhibited moderate activity in inhibiting the mutant HIV-1 strains. More encouragingly, 14d2 (RF = 0.4) possessed higher antiresistance profile than ETV (RF = 6.3) and K-5a2 (RF = 3.0) toward the double mutant strain F227L + V106A. The HIV-1 RT inhibition assay confirmed their binding target. The molecular docking studies were conducted and discussed in detail to rationalize the preliminary SARs. Further test indicated that morpholine could indeed promote the improvement of water solubility. Additionally, the in silico prediction of physicochemical properties and CYP enzymatic inhibitory ability were investigated to evaluate their drug-like features.

Full text of DOI:10.1016/j.ejmech.2020.112811

European Journal of Medicinal Chemistry 206 (2020) 112811
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Targeting dual tolerant regions of binding pocket: Discovery of novel
morpholine-substituted diarylpyrimidines as potent HIV-1 NNRTIs
with significantly improved water solubility
, c,
Zhao Wang a, Dongwei Kang a **, Da Feng a, Srinivasulu Cherukupalli a, Xiangyi Jiang a,
, c, ***
Zhipeng Fu a, Erik De Clercq b, Christophe Pannecouque b, Xinyong Liu a
,
, c, *
Peng Zhan a
a 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
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
a r t i c l e i n f o
a b s t r a c t
Article history:
To address the intractable issues of drug resistance and poor solubility, a novel series of morpholine-
substituted diarylpyrimidines targeting the tolerant region I and tolerant region II of NNIBP were
rationally designed by utilizing the available crystallography studies. The biological evaluation results
showed that four most promising compounds (14e1, 14g1, 14g2 and 14j2) displayed excellent potency
against WT HIV-1 strain with EC50 values ranging from 58 to 87 nM, being far more potent than NVP and
comparable to ETV. Besides, some derivatives exhibited moderate activity in inhibiting the mutant HIV-1
strains. More encouragingly, 14d2 (RF ¼ 0.4) possessed higher antiresistance profile than ETV (RF ¼ 6.3)
and K-5a2 (RF ¼ 3.0) toward the double mutant strain F227L þ V106A. The HIV-1 RT inhibition assay
confirmed their binding target. The molecular docking studies were conducted and discussed in detail to
rationalize the preliminary SARs. Further test indicated that morpholine could indeed promote the
improvement of water solubility. Additionally, the in silico prediction of physicochemical properties and
CYP enzymatic inhibitory ability were investigated to evaluate their drug-like features.
Received 9 August 2020
Received in revised form
1 September 2020
Accepted 1 September 2020
Available online 10 September 2020
Keywords:
HIV-1
NNRTIs
Tolerant region I
Tolerant region II
Morpholine
SARs
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Acquired immune deficiency syndrome (AIDS) is a pandemic
fatal disease that seriously threatens human health worldwide,
which was mainly caused by human immunodeficiency virus type
* 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.
*** 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: zhanpeng1982@sdu.edu.cn, zhanpeng1982@sdu.edu.cn
(P. Zhan).
1 (HIV-1) [1]. Reverse transcriptase (RT) plays a very crucial role in
the viral replication cycle, on account of its unique function of
reverse transcription of single-stranded RNA into double-stranded
DNA. Consequently, the HIV-1 RT has become one of the important
targets for designing potent anti-HIV agents for the treatment of
AIDS [2,3]. Non-nucleoside reverse transcriptase inhibitors
(NNRTIs) can occupy an allosteric site (NNRTI binding pocket,
NNIBP) about 10 Å distant from the RT polymerase active site,
resulted in the distortion of enzyme’s active site and eventually
disrupting the normal functions of RT [4]. Nowadays, NNRTIs have
achieved significant attraction as the most powerful and successful
https://doi.org/10.1016/j.ejmech.2020.112811
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
component of highly active antiretroviral therapy (HAART) regi-
mens, owing to their promising antiviral potency, modest toxicity,
high specificity, and favorable pharmacokinetics profiles [5,6].
Up to now, there are six NNRTIs approved by the U.S. Food and
Drug Administration (FDA) for the treatment of AIDS (Fig. 1). Ne-
virapine (1, NVP), delavirdine (2, DLV), and efavirenz (3, EFV) are
first generation NNRTIs, and their clinical application was
compromised by the rapid emergence of drug-resistant mutants. In
particular, the K103N and Y181C are the two most prevalent single
mutations selected by NVP and EFV, which displayed significantly
reduced susceptibility to the first generation NNRTIs. Etravirine (4,
ETV) and rilpivirine (5, RPV) approved by U.S. FDA in 2008 and 2011
respectively comprise the second generation NNRTIs, and both of
them belong to the diarylpyrimidine (DAPY) family [7e9]. Besides,
another new NNRTI called as doravirine (6, DOR) has appeared on
the market in 2018 [10].
However, attributing to the low genetic barrier of resistant
strains and high mutation ability of RT, new mutants with
decreased susceptibility to recently approved NNRTIs have
emerged, thus increasing the probability of failure of anti-HIV drug
therapy. Notably, the V106A is a non-polymorphic mutation
selected by NVP and DOR, which confers about 50-fold less sus-
ceptible to NVP and 5-fold less susceptible to EFV. Although V106A
alone has no major impact on DOR sensitivity, it tends to cause
high-level resistance to DOR in combination with other DOR-
Our previous research work on the modifications of DAPY-typed
NNRTIs has led to the identification of novel potent drug candidate
K-5a2 [18e23]. Although K-5a2 exhibited better antiresistance
profiles compared to ETV, it still has the disadvantage of poor sol-
ubility (0.05 mg/mL at pH 7.0). Therefore, it is imperative to dis-
covery novel compounds with maintained or enhanced potency as
well as improved solubility. The subsequently reported crystallo-
graphic studies of K-5a2/RT complexes have illustrated some
important structural features, which can provide valuable insights
for further structure-based optimization [24]. These features are
summarized as follows: 1) the left-wing of K-5a2 is positioned in
the hydrophobic tunnel composed by highly conserved residues
W229, Y188, F227, and L234 and forms hydrophobic interactions
and aryl-aryl interactions with these residues; 2) the thiophene
[3,2-d]pyrimidine central ring points to the tolerant region II and
exhibits additional nonpolar interactions with the alkyl chain of
E138 while maintaining hydrophobic interactions with V179 and
L100; 3) the substituted benzyl-linked piperidin-4-yl-amino moi-
eties stretches into the tolerant region I, a rather plastic groove of
the binding pocket, providing extensive Van der Waals contacts and
hydrogen-bonding interactions with the surrounding residues
K103, V106, P225, F227, P236, and Y318; 4) the NH linker and ni-
trogen atom of central pyrimidine ring can develop conserved
hydrogen bond or water-mediated hydrogen bond with the main
chain backbone of K101.
associated mutations. F227L is
a
non-polymorphic mutation
Inspired by the binding modes between the K-5a2 and NNIBP,
we recognized that the two solvent-exposed regions, namely the
tolerant region I and tolerant region II, providing an insufficiently
explored chemical space for the design of novel DAPY derivatives.
Besides, these two broad regions could well accommodate struc-
turally diverse chemical moieties and involve in forming various
interactions, making them potential sites worthy of more in-depth
modifications. Morpholine is a privileged fragment featured in
numerous approved drugs and bioactive molecules due to its su-
perior physicochemical, biological, and easily accessible properties.
There are many research examples emphasizing that morpholine
not only has the potential of multiple therapeutic properties, but
also can effectively modulate the drug-like parameters [25,26].
Most importantly, the non-planar structure of the alicyclic ring
selected by NVP and EFV that usually appears with V106A. And the
combined occurrence of F227L and V106A will lead to significant
reductions in NVP and EFV susceptibility [11e13]. Also, adverse
effects including hepatotoxicity and hypersensitivity reactions have
been observed after long-term clinical treatment. More impor-
tantly, the two DAPY-typed drugs (ETV: ≪1 mg/mL at pH 7.0; RPV:
20 ng/mL at pH 7.0) are suffering from extremely poor solubility
due to their structural rigidity, leading to the undesirable phar-
macokinetic properties [14,15]. In view of these current obstacles,
unremitting efforts are still urgently required to develop new
NNRTIs with structural diversity and improved safety profile to
address the intractable issues of drug resistance and low solubility
[16,17].
Fig. 1. Chemical structures of NNRTIs approved by the U.S. FDA.
2

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
morpholine could change the crystal packing of the original con-
jugated aromatic ring as well as reduce the lattice energy, thereby
improving the water solubility of the molecule [27]. Recently,
morpholine pharmacophore has been widely applied in drug dis-
covery for different antiviral targets including HBV capsid assembly
inhibitor GLS4 and HIV-1 NNRTIs, especially for the successful
introduction of morpholine into solvent-exposed tolerant region II
(7) or “NNRTI Adjacent” binding site (8) in the optimization process
of NNRTIs (Fig. 2) [28e30]. More encouragingly, the previous work
of our group has obtained compound 9 by introducing morpholine
into the right wing of DAPYs, which exhibited significantly
improved water solubility while maintaining excellent activity
against the WT HIV-1 strain [31].
14a1-2 were synthesized from the commercially available starting
material 2,4-dichloropyrimidine (10a). Initially, 10a was treated
with 3,5-dimethyl-4-hydroxybenzonitrile to obtain the interme-
diate 11a through nucleophilic substitution reaction. Then, 11a was
reacted with 4-(tert-butoxycarbonyl)-aminopiperidine to generate
the intermediate 12a. Next, subsequent deprotection with TFA
provided the key intermediate 13a. Finally, treatment of 13a with 4-
(2-chloroethyl)morpholine or 4-(2-chloroacetyl)morpholine in the
presence of Cs2CO3 at 60 ꢀC yielded the final products 14a1-2. In an
analogous way, other series 14b-l were prepared [33,34]. All the
newly synthesized compounds were fully characterized by means
of physicochemical and spectral, and their spectral data (ESI-MS, 1H
NMR and 13C NMR) were found to be in full agreement with the
assigned molecular structures.
In the present work, we designed a series of novel DAPYs
characterized by morpholine in the right-wing and different aro-
matic or alicyclic fused pyrimidine rings in the central scaffold via
the substituent decorating and scaffold hopping strategies, for the
purpose of targeting the tolerant region I and tolerant region II
simultaneously (Fig. 3). Concretely, considering the cyanovinyl
group of RPV may be acted as a “Michael acceptor” that covalently
modifies nucleic acids, proteins, and other biological entities, so we
kept the 2,6-dimethyl-4-cyanophenyl group in the left-wing un-
changed to avoid potential toxicity [32]. Meanwhile, based on our
previously reported studies, the privileged piperidin-4-yl-amino
moiety bearing morpholine substituent was installed at the right-
wing portion [31]. We expected that the introduction of hydro-
philic morpholine group towards the tolerant region I may be
beneficial for the improvement of solubility of new DAPYs. The
function of the polar group is essentially to increase the compound
hydration and to facilitate the thermodynamic process of dissolu-
tion. Simultaneously, in order to investigate the chemical space of
tolerant region II, we have employed a set of fused pyrimidine rings
to replace the thienopyrimidine platform, anticipating that it could
effectively occupy this open area and generate additional in-
teractions with the surrounding residues, which will contribute to
the enhancement of potency against WT and resistance-associated
HIV-1 strains.
2.2. Anti-HIV activity evaluation
All the newly synthesized DAPYs derivatives were evaluated for
their anti-HIV activities in MT-4 cell cultures infected with WT HIV-
1 strain (IIIB) and double mutant strain K103N þ Y181C (RES056) by
using the MTT method. Among them, eleven compounds were
selected for further evaluation of their inhibitory activities toward a
panel of NNRTI resistant strains including five single mutant strains
L100I, K103N, Y181C, Y188L, E138K, and double mutant strain
F227L þ V106A. Zidovudine (AZT), lamivudine (3TC), nevirapine
(NVP), efavirenz (EFV), etravirine (ETV) were selected as the
reference drugs. The biological results, expressed as EC50 (anti-HIV
potency), CC50 (cytotoxicity) and SI (selectivity index, CC50/EC50
ratio) are illustrated in Table 1 and Table 2. Additionally, the values
of RF (fold-resistance factor, EC50 against the mutant strain/EC50
against the WT strain ratio) are summarized in Table 3.
As shown in Table 1, the biological results clearly showed that all
the novel DAPYs displayed moderate to excellent potency at
inhibiting WT HIV-1 strain replication with an EC50 value ranging
from 0.058 to 2.18
were significantly better than that of reference drug 3TC
(EC50 ¼ 5.02 M). Among them, 20 compounds exhibited promising
antiviral activity from nanomolar to sub-micromolar ranges
(EC50 ¼ 0.058e0.597 M), being more potent or equipotent
compared to NVP (EC50 ¼ 0.164 M). Especially, compounds 14e1,
mM and SI values in the range of 52e1237, which
m
Herein, we report the synthesis, biological evaluation, modeling
simulation analysis of the newly designed diarylpyrimidine de-
rivatives. Moreover, the results of preliminary structure-activity
relationships (SARs), solubility measurement and other drug-like
properties assessment are also be discussed comprehensively.
m
m
14g1, 14g2 and 14j2 turned out to be the four effective inhibitors
against WT HIV-1 with a double-digit nanomolar EC50 value of
87 nM, 65 nM, 82 nM and 58 nM respectively, which were much
superior to that of NVP and reached the considerable level of AZT
2. Results and discussion
(EC50 ¼ 0.019
mM). More importantly, compound 14j2 was identi-
2.1. Chemistry
fied as the most active derivative possessing an extremely low EC50
value and high SI value, which was found to be up to 87-fold more
potent than 3 TC and 2.8-fold more potent than NVP, while slightly
inferior to that of AZT. Moreover, most of the compounds
The general synthetic routes adopted to obtain series 14a-l are
straightforwardly depicted in Scheme 1. The target compounds
Fig. 2. Chemical structures of morpholine-containing antiviral lead compounds.
3

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
Fig. 3. Illustration of the optimization strategies for the newly designed DAPY derivatives in the present work (PDB code: 6C0J).
Scheme 1. Reagents and conditions: (i) 3,5-dimethyl-4-hydroxybenzonitrile, DMF, K2CO3, r. t.; (ii) N-(tert-butoxycarbonyl)-4-aminopiperidine, DMF, K2CO3, 100 ꢀC; (iii) TFA, DCM, r.
t.; (iv) 4-(2-chloroethyl)morpholine or 4-(2-chloroacetyl)morpholine, DMF, Cs2CO3, 60 ꢀC.
demonstrated dramatically reduced cytotoxicity compared with
those reference drugs. Unfortunately, none of the target com-
pounds showed effective inhibitory potency against the double
mutant strain RES056 at the maximum tested concentration. All the
compounds lost the activity toward the HIV-2 strain (ROD) as ex-
pected, which means that they are all only specific for HIV-1 and act
as classical HIV-1 NNRTIs (data was not shown in the article).
Based on the anti-HIV-1 (IIIB) assay results, preliminary SARs
analysis can be concluded as follows. First of all, we focused our
attention on the modifications of the central scaffold and thus
investigated the contribution of ring substituents varying in elec-
tronic feature and bulk to the anti-HIV activities. It is apparent that
the antiviral potency is strongly dependent on the nature of the
central core. With the K-5a2 as lead, the initial part of our explo-
ration introduced diverse aromatic-fused pyrimidine rings for the
purpose of extending to the “tolerant region II” of NNIBP to enhance
the resistance profiles, and 16 compounds (series 14a-h) were
designed. Just consistent with the SARs established in our previous
studies, the thiophene[3,2-d]pyrimidine (14e1-2) and thiophene
[2,3-d]pyrimidine (14g1-2) proved to be more favorable for potency
than other rings. It is also worth noting that the compounds 14e1-2,
which possess the same central core as the lead K-5a2, exhibited
prominent activities among the analogues. Besides, pairwise
comparison of the activities, namely 14e1 (EC50 ¼ 0.087
14g1 (EC50 ¼ 0.065 M), 14e2 (EC50 ¼ 0.101 M) and 14g2
M), indicated that the sulphur atom position of
mM) and
m
m
(EC50 ¼ 0.082
m
thiophene ring has some influence on the potency. Replacement of
the central core of 14g1-2 with thiazolo[5,4-d]pyrimidine (14h1,
EC50 ¼ 0.291
m
M; 14h2, EC50 ¼ 0.140
mM) resulted in slightly
reduced antiviral activity against WT HIV-1, which underlined
again the critical role of thiophene ring in maintaining activity. The
anti-HIV activity is also strongly dependent on the type of atom
incorporated in the aromatic fused ring, exemplified by compounds
14f1 (EC50 ¼ 1.10
m
M) and 14f2 (EC50 ¼ 0.327
mM). And the results
seems to indicate that the sulphur atom appeared to be more
beneficial for the potency than the oxygen atom, probably because
it can develop an extensive electrostatic interaction with Val179.
Removing the fused ring yielded the pyrimidine analogues 14a1
4

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
Table 1
Antiviral activity against HIV-1 IIIB and RES056 strains and cytotoxicity of the target compounds in MT-4 cells.
Compd
14a1
Central Scaffold
R
EC50
IIIB
(
m
M)a
CC50
(
m
M)b
SIc
RES056
IIIB
219
RES056
1.12 0.42
>246
246 16.6
143 26.2
26.0 4.47
<1
14a2
0.141 0.036
0.502 0.23
>143
>26.0
1016
52
<1
<1
14b1
14b2
14c1
0.597 0.66
0.241 0.061
>42.6
>16.9
42.6 15.8
16.9 8.48
71
70
<1
<1
14c2
14d1
0.577 0.33
1.79 0.60
>16.4
>121
41.1 14.7
121 6.41
71
68
<2.51
<1
14d2
2.18 0.67
>211
211 15.4
97
<1
14e1
14e2
14f1
14f2
0.087 0.021
0.101 0.045
1.10 0.78
>36.8
>41.9
>138
>125
36.8 10.7
41.9 14.6
138 8.71
125 15.9
423
416
125
383
<1
<1
<1
<1
0.327 0.13
(continued on next page)
5

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
Table 1 (continued)
Compd
Central Scaffold
R
EC50
IIIB
(
m
M)a
CC50
(
m
M)b
SIc
RES056
IIIB
579
RES056
14g1
0.065 0.021
0.082 0.043
0.291 0.089
0.140 0.015
>37.9
37.9 23.7
36.3 11.3
88.6 22.0
129 5.43
<1
14g2
14h1
14h2
>36.3
>88.6
>129
442
305
919
<1
<1
<1
14i1
14i2
14j1
14j2
14k1
0.188 0.065
0.106 0.027
0.114 0.029
0.058 0.017
0.173 0.096
>49.1
>73.5
>43.0
>38.6
>28.5
49.1 21.0
73.5 14.3
43.0 14.4
38.6 11.7
28.5 7.59
261
696
376
663
164
<1
<1
<1
<1
<1
14k2
14l1
14l2
0.154 0.078
0.366 0.12
0.149 0.049
>64.6
>140
>184
64.6 37.8
140 6.38
184 3.75
420
<1
<1
<1
383
1237
AZT
3TC
NVP
EFV
e
e
e
e
e
e
e
e
e
e
e
e
0.019 0.006
5.02 1.93
0.164 0.10
0.002 0.001
0.003 0.001
0.0014 0.0004
0.024 0.007
8.35 2.88
>15.0
0.314 0.148
0.038 0.021
0.0306 0.012
>7.48
>87.2
>15.0
>6.34
>4.59
>228
>397
>17
>92
>2824
>1638
>159,101
>311
>10
X1d
>20
>122
>53,533
ETV
K-5a2e
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method.
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
b
c
SI: selectivity index, the ratio of CC50/EC50
.
d
e
X1: ꢁ1 or <1.
K-5a2: the activity data were quoted from our published paper [18].
and 14a2 (EC50 ¼ 1.12
m
M and 0.141
m
M) with sharply decreased
the improvement of activity. Conversely, the elaboration with
methoxy group at the C6 and C7 position were detrimental to the
antiviral potency; to be specific, both of 14d1 and 14d2
potency against WT HIV-1, which may be due to the disappearance
of interactions with the surrounding residues of tolerant region II.
What’s more, compounds 14b1 and 14b2 (EC50 ¼ 0.502
m
M and
(EC50 ¼ 1.79
mM and 2.18 mM) showed markedly weaker activity
0.597 M) featuring the quinazoline central core showed accept-
m
than 14b1-2, supposing that the 6,7-dimethoxyquinazoline ring
can not fit the binding pocket well due to its larger size and rigid
construction.
able potency toward HIV-1 IIIB; the introduction of fluorine atom at
the C7 position afforded more potent inhibitors 14c1 and 14c2 with
EC50 values of 0.241 and 0.577
fact that the hydrogen bond donor/acceptor atom is conducive to
m
M, which might be attributed to the
Meanwhile, in continuation of our exploration, considering that
disrupting the planar conformation of the molecule can decrease its
6

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
Table 2
Antiviral activity against several HIV-1 mutant strains of selected compounds in MT-4 cells.
Compd
EC50
L100I
(m
M)a
K103N
Y181C
Y188L
E138K
F227L þ V106A
14c1
14d2
14e1
14e2
14g1
14g2
14h2
14i2
14j2
14k1
14k2
AZT
1.24 0.24
>211
0.522 0.21
24.8 5.85
1.04 0.20
1.38 0.41
0.61 0.11
1.62 0.30
3.74 1.32
2.54 0.35
1.28 0.20
1.96 0.45
3.73 0.99
0.019 0.005
3.75 1.13
5.10 1.00
0.094 0.025
0.003 0.001
0.0029
1.35 0.24
>211
2.40 0.19
>211
0.963 0.18
>211
2.92 0.29
0.974 0.19
7.14 0.43
ꢁ17.9
8.70 0.90
5.17 1.18
14.7 14.3
3.67 1.13
15.1 1.79
15.9 15.5
6.12 1.73
16.6 2.68
24.4 16.3
0.006 0.002
1.62 0.56
1.03 0.54
0.040 0.016
0.005 0.001
0.0034 0.0006
8.35 1.92
5.27 0.63
6.90 0.75
3.64 1.69
3.06 0.21
10.6 2.62
5.45 1.10
13.2 3.90
13.4 6.51
0.016 0.006
4.79 1.73
6.69 1.89
0.006 0.001
0.014 0.004
0.0032 0.0004
9.39 2.08
4.92 1.20
45.2 2.89
7.18 2.83
12.1 1.46
6.48 1.38
5.21 1.48
13.8 0.56
7.45 0.88
0.009 0.003
3.36 0.93
ꢁ7.70
0.437 0.10
0.171 0.05
0.586 0.11
0.334 0.06
0.703 0.08
1.12 0.13
1.31 0.23
1.18 0.16
0.627 0.08
0.027 0.010
6.64 1.83
0.191 0.064
0.005 0.001
0.007 0.003
0.0029
18.7 21.7
13.8 2.21
35.9 5.26
27.0 7.54
>38.6
11.8 2.08
41.2 7.10
0.009 0.003
2.77 0.75
ꢁ5.41
3TC
NVP
EFV
0.262 0.080
0.014 0.005
0.003 0.0001
0.365 0.130
0.018 0.005
0.0042 0.0012
ETV
K-5a2b
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method.
K-5a2: the activity data were quoted from our published paper [18].
b
Table 3
activities was assessed and concluded that the inhibitory activity
was not directly related to the type of R substituent. There was no
doubt that the given conclusions are in agreement with the
aforementioned SARs.
Resistance folds for several HIV-1 mutant strains of selected compounds in MT-
4 cells.
Compd
Resistance foldsa
Based on the preliminary anti-HIV-1 (IIIB and RES056) activity
results, we further selected some potent compounds to evaluate
their ability to inhibit a variety of NNRTI-resistance mutations,
including L100I, K103N, Y181C, Y188L, E138K, and F227L þ V106A.
Table 2 demonstrated that all the tested compounds (except 14d2)
maintained acceptable activity against the whole mutant strains,
especially against the K103N and E138K. With regard to the K103N
mutant strain, which represented the most prevalent resistance-
associated mutation conferring susceptibility to NVP and EFV
greatly, all these compounds displayed highly potent inhibitory
activities against K103N with low EC50 values ranging from 0.522 to
L100I
K103N
Y181C
Y188L
E138K
F227L þ V106A
14c1
14d2
14e1
14e2
14g1
14g2
14h2
14i2
14j2
14k1
14k2
AZT
3TC
NVP
EFV
ETV
K-5a2
5.1
2.2
5.6
10.0
>96.8
107.9
48.8
690.6
87.5
86.0
61.4
89.4
79.7
48.4
0.5
4.0
>96.8
5.0
1.7
9.0
4.1
5.0
10.6
22.6
6.8
4.1
1.4
1.3
1.2
2.3
2.6
2.0
12.1
0.4
81.9
>96.8
99.9
51.4
224.5
44.8
107.7
150.4
105.0
95.6
158.7
0.3
0.3
6.3
17.8
1.9
2.4
11.4
11.9
13.7
9.3
19.8
26.7
24.1
21.9
11.3
24.2
1.0
>96.8
95.8
52.4
105.5
44.3
21.8
100.8
93.5
76.2
86.9
0.9
1.0
40.7
2.5
4.9
2.3
ꢁ178.1
285.5
168.0
256.1
256.0
>662.9
68.4
267.3
0.5
0.6
3.74
14c1 provided the highest potency toward K103N, which was about
10 times and 8 times more active than NVP (EC50 ¼ 5.10 M) and
3TC (EC50 3.75 M), respectively. Moreover, 14c1
(EC50(L100I) ¼ 1.24 M)
mM, except for 14d2 (EC50 ¼ 24.8
mM). In particular, compound
0.7
0.7
31.1
41.9
1.0
ꢁ46.9
116.7
4.9
ꢁ32.9
162.5
6.3
m
¼
m
2.0
2.1
3.0
m
M; EC50(Y181C) ¼ 1.35
m
M; EC50(Y188L) ¼ 2.40
m
a
Resistance fold: RF, ratio of EC50 against the mutant strain/EC50 against the WT
showed the best inhibitory activities against other mutant strains
L100I, Y181C, and Y188L, which were superior to or at the same
strain.
magnitude as 3TC (EC50(L100I) ¼ 1.62
m
M; EC50(Y181C) ¼ 4.79
m
m
M;
M;
EC50(Y188L)
¼
3.36
m
M) and NVP (EC50(L100I) 1.03
¼
lattice energy, we further introduced some alicyclic rings to the
central core with the aim of improving the compounds’ aqueous
solubility. Among these four subseries derivatives, compounds 14j1
and 14j2 bearing 6,7-dihydrothieno[3,2-d]pyrimidine possessed
EC50(Y181C) ¼ 6.69
mM; EC50(Y188L) ꢁ 7.70
mM), suggesting that these
mutants are most susceptible to 14c1. In the case of the E138K
strain, the most common mutant emerging in patients failed RPV-
containing regimens, ten compounds exhibited a prominent EC50
the relatively best activity, with EC50 values of 0.114 and 0.058 mM.
The position of sulphur atom in the tetrahydrothiophene ring had
some impact on the potency. For instance, compared with 14j1 and
value below 1.31
activity at the submicromolar level, being far more potent than 3TC
M). As for
the double mutant strain F227L þ V106A, most of the compounds
displayed weak or lost efficacy compared to those control drugs.
Interestingly, although 14d2 was inactive towards many single
mutant viruses; however, it displayed exceptionally potent activity
mM, and more than half of them showed inhibitory
(EC50 ¼ 6.64
m
M) and equivalent to NVP (EC50 ¼ 0.191
m
14j2, 14k1 and 14k2 (EC50 ¼ 0.173
mM and 0.154 mM) obtained by
changing the position of the sulphur atom showed a slightly
decreased potency. Removing the sulphur atom from the central
core also led to the reduced activity, exemplified by 14i1
(EC50 ¼ 0.188
incorporation of oxygen atom instead of sulphur atom impaired the
M),
mM) and 14i2 (EC50 ¼ 0.106
mM). Analogously,
against the F227L þ V106A with an EC50 value of 0.974
was much better than 3TC (EC50 2.77 M) and NVP
(EC50 ꢁ 5.41 M).
mM, which
¼
m
anti-HIV potency (14l1, EC50 ¼ 0.366 M; 14l2, EC50 ¼ 0.149
m
m
m
M) and was comparable to EFV (EC50 ¼ 0.365
m
which suggested that the activity is susceptible to the kind of the
atom. These observations revealed that C5 position is very optimal
for prominent potency and the sulphur atom is more suitable at
tolerant region II. On the other hand, the effect of R groups
substituted on the piperidinamine of right-wing on anti-HIV
In addition, the resistance fold of the tested compounds were
calculated in Table 3. To our delight, all the compounds exhibited no
obvious decline in activity when it comes to K103N (RF ¼ 2.2e26.7),
because of their lower RF values compared to EFV (RF ¼ 41.9).
7

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
Surprisingly, compound 14c1 had relatively low RF values
(RF ¼ 2.2e12.1) toward these HIV-1 mutant strains. It is speculated
that the bulkier fluoroquinazoline ring of 14c1 may help to bind
tightly to the RT, so that the inhibitory potency will not decrease
significantly once individual amino acid mutations occurred. It was
particularly noteworthy that the RF value of compound 14d2
(RF ¼ 0.4) for F227L þ V106A was less than 1, that means 14d2
possesses higher inhibitory ability against the F227L þ V106A
mutant strain than against the WT strain. These findings may imply
that 14d2 has subtle interactions with the amino acids in the RT
containing F227L and V106A. The above mentioned biological re-
sults and detailed SARs analysis verified that our original design
hypothesis that introducing a morpholine group into the right-
wing portion was reasonable and will be helpful to the further
rational design.
notable features were consistent with our previous investigations
and could be described as follows: i) the left 4-cyano-2,6-
dimethylphenyl moiety occupied the hydrophobic sub-pocket
formed by aromatic amino acid residues Tyr181, Tyr188 and Phe
227, exhibiting extensive
pep stacking interactions with these
aromatic-rich residues; ii) the central dihydrothiophene ring was
oriented toward the NNIBP entrance channel, forming favorable
nonpolar interactions with the alkyl chain of Glu138 and electro-
static interactions with Val179; iii) the pyrimidine N atom and NH
linker developed typical double hydrogen bonds with the backbone
of Lys 101 that are essential for maintaining binding affinity; iv) the
right morpholine ring connected by the flexible aminopiperidine
motif stretched into the proteinesolvent interface and it was worth
noting that the carbonyl group involves in the hydrogen bonding
interactions with Lys103 and Pro236 mediated by the water
molecule. Therefore, all these factors reasonably explain the
excellent potency of 14j2 against the WT HIV-1 strain.
2.3. Inhibition of HIV-1 RT
From the simulation results between K103N RT and compound
14c1, which exhibited the most promising activity against the
K103N mutant strain (Fig. 4b), it was observed that the nitrogen
atom of the piperidine ring established water-mediated hydrogen
bonds with the new Asn103 and Pro236 to compensate for the
interactions loss caused by the Lys103 mutation. Similarly, com-
pound 14e2 with the best anti-E138K variant activity was docked
into the E138K RT (Fig. 4c). Although a certain degree of confor-
mational distortion occurred at the terminal morpholine ring, the
crucial hydrogen bonds formed by the piperidine ring with Lys103
and Pro236 through a water molecule continued to be retained.
In addition, by comparing the binding modes of 14j2 with WT
RT and RES056 RT (Fig. 4d), the Y181C mutation significantly
With the aim to further validate the binding target of these
newly designed morpholine-substituted diarylpyrimidine de-
rivatives, four representative compounds 14e1, 14g1, 14g2 and 14j2
with superior antiviral activities against WT HIV-1 strain, were
selected to evaluate their ability to inhibit recombinant WT HIV-1
RT enzymes. ETV was chose as the reference drug in this assay. As
shown in Table 4, all the target compounds displayed moderate
inhibitory potencies toward WT HIV-1 RT with IC50 values ranging
from 1.49
mM to 3.37
mM, being slightly inferior to that of ETV
(IC50 ¼ 0.059
mM). Certainly, the order of enzymatic activity of these
analogues was not completely consistent with the SARs of their
cellular activity, which can be considered as the difference in
membrane permeability, the cellular metabolism of molecules, or
components of the assay system. In addition, the IC50 values
(micromolar level) of tested compounds including ETV are gener-
ally quite different from the EC50 values (nanomolar level), prob-
ably attributing to the dependence on the test conditions or higher
affinity for the full-length cellular enzyme than the recombinant RT
enzyme. On the whole, the results were able to illustrate that these
newly synthesized derivatives exhibited high affinity to HIV-1 RT
and they all acted as typical NNRTIs.
weakened the original
pep stacking interactions. On the other
hand, the shift of 14j2 in the NNIBP and the conformational
inversion of the carbonyl group led to the disappearance of the
hydrogen bonds with Asn103 and Pro236, which provided a
reasonable explanation for the sharply decreased potency against
double mutant strain RES056.
Generally speaking, certain mutations of RT amino acid residues
almost always result in a significant decrease in the antiviral ac-
tivity of NNRTIs, especially for the double mutant strains. Intrigu-
ingly, the potency of 14d2 against the F227L þ V106A strain was
about 2.24-fold more potent than against WT strain. As shown in
Fig. 4e and Fig. 4f, the comparative analysis of the binding modes of
14d2 with WT RT and F227L/V106A RT indicated that the much
smaller side chains of Leu 227 and Ala 106 allow the chemical space
of NNIBP to be further enlarged, which is more conducive to the
expansion of bulky molecule to fully occupy the pocket. Further-
more, the multiple water-mediated hydrogen bonds formed by the
methoxy group of the central ring with Glu138 and the nitrogen
atom of piperidine ring with Lys103 and Pro236 may also
contribute to the improvement of activity.
To some extent, the molecular modeling results accurately
elucidated the theoretical binding modes as well as validated our
original design hypothesis that introducing the privileged hetero-
cycle morpholine into the solvent-exposed region to establish more
interactions with the binding pocket, which may provide valuable
clues for the follow-up structural optimization.
2.4. Molecular modeling analysis
With the aim to better rationalize the anti-HIV potency of these
newly designed compounds and to obtain further insights into
their binding modes in the NNIBP of RT, the molecular docking
studies of four representative compounds (14j2, 14c1, 14e2 and
14d2) were carried out by utilizing the software SurflexeDock
SYBYL-X 2.0. X-ray crystal structure of HIV-1 WT RT (PDB code:
6C0J), K103N RT (PDB code: 6C0K), E138K RT (PDB code: 6C0L),
RES056 RT (PDB code: 6C0R) together with F227L/V106A RT (PDB
code: 6DUF) were selected as the input structures for docking
calculations. The docking results were visualized by PyMOL 1.5. And
the docking protocol was described in the computational section.
As shown in Fig. 4a, the binding mode of compound 14j2 with
WT RT was similar to other classical DAPY-typed NNRTIs as a
common horseshoe conformation in the NNIBP. Concretely, several
Table 4
Inhibitory activity of compounds 14e1, 14g1, 14g2, 14j2 and ETV against HIV-1 WT RT.
Compd
14e1
14g1
14g2
14j2
ETV
IC50
(
m
M)a
2.53 0.95
2.36 1.15
1.49 0.07
3.37 1.57
0.059 0.010
a
IC50: inhibitory concentration of test compounds required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into HIV-1 WT RT by 50%.
8

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
Fig. 4. (a) Predicted binding mode of 14j2 (cyan) with the HIV-1 WT RT (PDB code: 6C0J). (b) Predicted binding mode of 14c1 (yellow) with the HIV-1 K103N RT (PDB code: 6C0K).
(c) Predicted binding mode of 14e2 (magenta) with the HIV-1 E138K RT (PDB code: 6C0L). (d) Predicted binding mode of 14j2 (cyan) with the HIV-1 RES056 RT (PDB code: 6C0R). (e)
Predicted binding mode of 14d2 (white) with the HIV-1 WT RT (PDB code: 6C0J). (f) Predicted binding mode of 14d2 (white) with the HIV-1 F227L/V106A RT (PDB code: 6DUF). The
hydrogen bonds between the inhibitor and amino acid residues are indicated with dashed lines (yellow) and protein carbon atoms in green. The water molecules are indicated with
red balls. Nonpolar hydrogen atoms are not shown for clarity.
2.5. Water solubility measurement
solubilizing group was commonly adopted in the optimization of
the pharmacokinetic profiles of lead compounds, typically repre-
sented by the approved drug gefitinib.
It is well known that water solubility was considered as an
important physicochemical property of small molecules and
occupied a major position in the early evaluation of oral drug
candidates. Good water solubility often leads to accelerated ab-
sorption and thus promotes the therapeutic efficacy of the drugs.
Therefore, the water solubility of compounds 14e1, 14g1, 14g2 and
14j2 were determined under different pH values (7.4, 7.0, and 2.0).
As depicted in Table 5, all the selected compounds possessed
2.6. In silico prediction of physicochemical properties
To further investigate the drug-like properties of representative
compounds 14e1, 14g1, 14g2 and 14j2, a preliminary assessment of
other physicochemical properties were conducted by utilizing free
online software (http://www.molinspiration.com/) [36]. As shown
in Table 6, the results indicated that various parameters of
desirable solubility at pH 2.0 (Sol. ¼ 207e248
much greater than that of ETV (Sol. ¼ 127 g/mL) and equivalent to
g/mL). Compared with K-5a2, compound
mg/mL), which were
m
that of K-5a2 (Sol. ¼ 190
m
14e1 bearing the same scaffold showed an overall improved water
solubility. Among them, 14j2 displayed the optimal water solubility
Table 6
Physicochemical properties of compounds 14e1, 14g1, 14g2, 14j2 and ETV.
(Sol. ¼ 248
m
g/mL, pH ¼ 2.0; Sol. ¼ 35.2 g/mL, pH ¼ 7.0; Sol. ¼
m
Parameter itemsa Acceptable 14e1
14g1
35
14g2
36
14j2
36
ETV
28
29.1
m
g/mL, pH ¼ 7.4). The obtained results revealed that our pro-
natoms
MW
nON
nOHNH
nrotb
tPSA
e
35
posed design concept of improving the water solubility by intro-
ducing appropriate polar groups into the proteinesolvent interface
was completely feasible. Similarly, the morpholine as the dominant
<500 Da
<10
<5
ꢂ10
<140 Å2
e
492.65 492.65 506.63 508.65 435.29
8
1
7
8
1
7
9
1
6
9
1
6
7
3
4
86.55
86.55
103.62 103.62 120.65
MV
450.93 450.93 453.11 459.30 335.95
Table 5
miLog p
nViol
<5
e
4.58
0
4.58
0
3.90
1
3.47
1
5.03
1
Water solubility of compounds 14e1, 14g1, 14g2, 14j2, K-5a2 and ETV.
a
Compd
14e1
14g1
14g2
14j2
K-5a2 [35]
ETV [35]
natoms ¼ number of non-hydrogen atoms; MW ¼ molecular weight; nON ¼
number of hydrogen bond acceptors; nOHNH ¼ number of hydrogen bond donors;
nrotb ¼ number of rotatable bonds; tPSA ¼ topological polar surface area; MV ¼
molar volume; miLog P ¼ molinspiration predicted Log P; nViol ¼ number of
violations.
pH ¼ 7.4 (
pH ¼ 7.0 (
pH ¼ 2.0 (
m
m
m
g/mL)
g/mL)
g/mL)
15.6
24.4
207
19.7
29.3
243
27.6
34.2
228
29.1
35.2
248
≪1
0.05
190
<1
<1
127
9

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
compounds 14e1 and 14g1 including molecular weight (MW),
hydrogen bond acceptors (nON), hydrogen bond donors (nOHNH),
rotatable bonds (nrotb) and miLog p were precisely consistent with
the “Lipinski’s rule of five” within a definitive level. Regrettably,
compounds 14g2 and 14j2 cannot meet the typical requirements
due to slight deviations of their MW values. Inspiringly, all the test
compounds displayed an acceptable miLog p, while the ETV go
beyond the normal criteria. In terms of the topological polar surface
area (tPSA), which reflects the absorption and membrane perme-
ability of molecules, the values of all compounds were in the range
of 86.55 to 103.62 Å2, confirming their advantage for intestinal
absorption (<140 Å2) and impossibility to pass through the blood-
brain barrier (>60 Å2), avoiding the adverse effect on the central
nervous system [37].
structural biology information of K-5a2/RT complexes. Encourag-
ingly, all the newly synthesized derivatives were found to be active
against WT HIV-1 strain with EC50 values in the range of
0.058ꢃ2.18
14e1, 14g1, 14g2 and 14j2 provided double-digit nanomolar activ-
ities (EC50 ¼ 58e87 nM), being far more potent than NVP (EC50
164 nM) and comparable to ETV (EC50 ¼ 3 nM). In terms of the HIV-
1 mutant strains, compounds 14c1 (EC50(K103N) ¼ 0.522 M) and
M) maintained prominent activity
mM. Among them, four most promising compounds
¼
m
14e2 (EC50(E138K) ¼ 0.171
m
against K103N and E138K mutant strain at the sub-micromolar
scale, respectively. Furthermore, compound 14d2 exhibited mod-
erate potency toward the F227L þV106A double mutant strain with
an EC50 value of 0.974 mM. It is noteworthy that the resistance fold
(RF) value of 14d2 for F227L þV106A was only 0.4, which was much
lower than that of ETV (RF ¼ 6.3) and K-5a2 (RF ¼ 3.0). In the
enzyme inhibition assay, all the selected compounds exhibited high
affinity for WT HIV-1 RT and thus acted as classical NNRTIs. Mo-
lecular docking studies were performed to investigate the theo-
retical binding modes within the NNIBP and rationalize the
preliminary SARs. Most importantly, four tested compounds 14e1,
14g1, 14g2 and 14j2 possessed significantly improved water solu-
bility compared to ETV under different pH conditions. Besides,
some valuable drug-like characteristics including physicochemical
properties and CYP enzymatic inhibitory ability were also predicted
comprehensively by using online software.
Taken together, all these results confirmed our initial design
assumption that the introduction of the privileged fragment mor-
pholine into the solvent-exposed region could achieve an optimal
balance between antiviral efficacy and drug-like parameters.
Currently, further optimization focused on improving the inhibi-
tory activity toward HIV-1 mutant strains is ongoing and will be
disclosed in due course.
2.7. In silico predicted effects of representative compounds on
inhibition of CYP enzyme
According to the published clinical data, certain drugs can alter
the activities or expression levels of hepatic drug metabolic en-
zymes, especially the cytochrome P450 (CYP), thus may causing
metabolism-mediated drugedrug interactions (DDI). CYP enzymes
play an essential role in the process of detoxification and bio-
activation and widely involves the metabolism of more than half of
the current drugs in vivo. In addition, three main subtypes CYP2C,
CYP2D6 and CYP3A contribute to the majority of the CYP-mediated
drug metabolism. The induction and inhibition of those CYP en-
zymes could lead to changes in the pharmacokinetic profile of co-
administered drugs. Relevant studies have shown that ETV exhib-
iting inhibitory (CYP2C9, CYP2C19) and inductive (CYP3A4) prop-
erties, and it will produce DDI when co-administrating with other
antiretrovirals [38]. Therefore, it is necessary to evaluate the effect
of chemical molecules on the expression of CYP isozymes. In this
work, we assessed the inhibitory ability of four representative
compounds on the main CYP drug-metabolizing enzymes through
online software (http://admet.scbdd.com/calcpre/index/) [39]. As
described in Table 7, compounds 14e1 and 14g1 demonstrated no
significant CYP enzymatic inhibition of CYP1A2, CYP2C9, CYP2C19
and CYP3A4 apart from CYP2D6. Although compound 14g2 did not
inhibit CYP2D6, it displayed potential inhibitory ability against
CYP2C19 as well as CYP3A4. Besides, compound 14j2 only inhibited
CYP2C19 among the five CYP isozymes, while the lead compound
K-5a2 and ETV had an inhibitory effect on three different CYP
enzymes.
4. Experimental section
4.1. Chemistry
All melting points were determined on a micromelting point
apparatus (Tian Jin Analytical Instrument Factory, Nankai, Tianjin,
China) and were uncorrected. 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance-400 spectrometer (Bruker BioSpin,
Switzerland) using DMSOd6 as the solvent and tetramethylsilane
(TMS) as the internal standard. Chemical shifts are expressed as
d
units (ppm) and J values are presented in hertz (Hz). Signals are
abbreviated as s (singlet), d (doublet), t (triplet), q (quartet) or m
(multiplet). Mass spectrometry were obtained on a LC Autosampler
Device: Standard G1313A instrument. TLC was performed on Silica
Gel GF254 for TLC (Merck) and spots were visualized by iodine
3. Conclusion
In summary, to fully explore the chemical space around the
tolerant region I and tolerant region II of NNIBP, a novel series of
DAPY-typed HIV-1 NNRTIs bearing the morpholine moiety were
rationally designed and synthesized guided by the available
vapors or by irradiation with UV light (
l
¼ 254 nm). Flash column
chromatography was performed on column packed with Silica Gel
60 (200e300 mesh) (Qing Dao Haiyang Chemical Company, Qing-
dao, China). Solvents were of reagent grade, and if necessary, were
purified and dried by standard methods. Concentration of the re-
action solutions involved the use of rotary evaporator at reduced
pressure.
Table 7
Inhibitory ability on CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 of compounds
14e1, 14g1, 14g2, 14j2, K-5a2 and ETV.
CYP isozyme
14e1
14g1
14g2
14j2
K-5a2
ETV
4.1.1. General procedure for the synthesis of intermediates 11a-l
a
1A2
2C9
2C19
2D6
3A4
ꢃ
ꢃ
ꢃ
ꢃ
þ
ꢃ
ꢃ
ꢃ
þ
ꢃ
þ
ꢃ
ꢃ
þ
ꢃ
ꢃ
ꢃ
þ
ꢃ
þ
þ
þ
þ
þ
ꢃ
ꢃ
A reaction mixture of 4-hydroxy-3,5-dimethylbenzonitrile
ꢃ
ꢃ
(0.15 g, 1.0 mmol) and potassium carbonate (0.17 g, 1.2 mmol) in
3 mL of DMF was stirred at room temperature for 15 min. Then the
different starting material 10a-l (1.0 mmol) was added to it and
stirring was continued for an additional 2 h. After completion of the
reaction (monitored by TLC), the solvent was evaporated under
reduced pressure, then ethyl acetate (30 mL) was added and
washed with water (3 ꢄ 5 mL) and saturated brine (3 ꢄ 5 mL). The
b
þ
ꢃ
a
Non-inhibitor: the probability of predicted category is less than or equal to 0.5
(minimum value is 0).
b
Inhibitor:bthe probability of predicted category is greater than 0.5 (maximum
value is 1).
10

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
combined organic layer was dried over anhydrous Na2SO4, filtered
and concentrated, and finally recrystallized from ethyl acetate/pe-
troleum ether (1:10) to provide the corresponding intermediates
11a-l as a white solid.
4.1.1.10. 4-((2-chloro-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)-
3,5-dimethylbenzonitrile (11j). White solid, yield: 85.7%, mp:
272ꢃ274 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 7.67 (s, 2H, C3,C5-Ph-H),
3.37e3.35 (m, 2H, C7-dihydrothienopyrimidine-2H), 3.14 (t, J ¼
8.2 Hz, 2H, C6-dihydrothienopyrimidine-2H), 2.06 (s, 6H, CH3 ꢄ 2).
ESI-MS: m/z 318.2 [MþH]þ. C15H12ClN3OS (317.04).
4.1.1.1. 4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile
(11a). White solid, yield: 86.1%, mp: 126ꢃ128 ꢀC. 1H NMR
(400 MHz, DMSOd6)
d
8.70 (d, J ¼ 5.6 Hz,1H, C6-pyrimidine-H), 7.75
4.1.1.11. 4-((2-chloro-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)-
(s, 2H, C3,C5-Ph-H), 7.31 (d, J ¼ 5.6 Hz, 1H, C5-pyrimidine-H), 2.10 (s,
3,5-dimethylbenzonitrile (11k). White solid, yield: 87.6%, mp:
6H, CH3 ꢄ 2). ESI-MS: m/z 260.1 [MþH]þ. C13H10ClN3O (259.05).
268ꢃ270 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 7.72 (s, 2H, C3,C5-Ph-H),
4.12 (s, 2H, C5-dihydrothienopyrimidine-H), 4.07 (s, 2H, C7-dihy-
drothienopyrimidine-H), 2.10 (s, 6H, CH3 ꢄ 2). ESI-MS: m/z 318.2
[MþH]þ. C15H12ClN3OS (317.04).
4.1.1.2. 4-((2-chloroquinazolin-4-yl)oxy)-3,5-dimethylbenzonitrile
(11b). White solid, yield: 84.5%, mp: 180ꢃ182 ꢀC. 1H NMR
(400 MHz, DMSOd6)
d
8.48 (dd, J ¼ 8.3, 1.4 Hz, 1H, C8-quinazoline-
H), 8.14 (ddd, J ¼ 8.5, 7.0, 1.5 Hz, 1H, C5-quinazoline-H), 8.00 (d, J ¼
8.4 Hz, 1H, C7-quinazoline-H), 7.86 (ddd, J ¼ 8.2, 7.0, 1.1 Hz, 1H, C6-
quinazoline-H), 7.78 (s, 2H, C3,C5-Ph-H), 2.14 (s, 6H, CH3 ꢄ 2). ESI-
MS: m/z 310.6 [MþH]þ. C17H12ClN3O (309.07).
4.1.1.12. 4-((2-chloro-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)-3,5-
dimethylbenzonitrile (11l). White solid, yield: 82.1%, mp:
180ꢃ182 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 7.69 (s, 2H, C3,C5-Ph-H),
4.97 (s, 2H, C5-dihydrofuropyrimidine-H), 4.80 (s, 2H, C7-dihy-
drofuropyrimidine-H), 2.10 (s, 6H, CH3 ꢄ 2). ESI-MS: m/z 302.1
[MþH]þ. C15H12ClN3O2 (301.06).
4.1.1.3. 4-((2-chloro-7-fluoroquinazolin-4-yl)oxy)-3,5-
dimethylbenzonitrile (11c). White solid, yield: 82.9%, mp:
245ꢃ247 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.59 (dd, J ¼ 8.9, 6.2 Hz,
4.1.2. General procedure for the synthesis of intermediates 13a-l
1H, C5-fluoroquinazoline-H), 7.85 (d, J ¼ 9.6 Hz, 1H, C8-fluo-
roquinazoline-H), 7.79 (s, 3H, C3,C5-Ph-H, C6-fluoroquinazoline-H),
2.14 (s, 6H, CH3 ꢄ 2). ESI-MS: m/z 328.1 [MþH]þ. C17H11ClFN3O
(327.06).
A
solution of compound 11a-l (1.0 mmol), N-Boc-4-
aminopiperidine (0.24 g, 1.2 mmol), and potassium carbonate
(0.29 g, 2.0 mmol) in DMF was stirred overnight at 100 ꢀC. The
solution was cooled to room temperature, and ice water (30 mL)
was added to it. The precipitated white solid was collected by
filtration and dried to give crude 12a-l, which was used directly in
the next step without further purification. Subsequently, the in-
termediates 12a-l (1.0 mmol) and TFA (0.75 mL, 10 mmol) were
dissolved in DCM (5 mL) at room temperature, and the reaction
mixture was stirred until completion monitored by TLC. Then the
resulting liquid was alkalized to pH 9 with saturated sodium bi-
carbonate solution and the aqueous phase was extracted with DCM
(3 ꢄ 10 mL). The combined organic phase was dried over anhydrous
Na2SO4, filtered and concentrated. The crude residue was further
purified by flash column chromatography with methanol/
dichloromethane (1:10) as eluent and recrystallized from ethyl
acetate/petroleum ether to give the key intermediates 13a-l as a
white solid.
4.1.1.4. 4-((2-chloro-6,7-dimethoxyquinazolin-4-yl)oxy)-3,5-
dimethylbenzonitrile (11d). White solid, yield: 79.3%, mp:
256ꢃ258 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 7.77 (s, 2H, C3,C5-Ph-H),
7.66 (s, 1H, C8-dimethoxyquinazoline-H), 7.49e7.37 (m, 1H, C5-
dimethoxyquinazoline-H), 4.00 (d, J ¼ 2.5 Hz, 6H, OCH3 ꢄ 2), 2.14 (s,
6H, CH3 ꢄ 2). ESI-MS: m/z 370.2 [MþH]þ. C19H16ClN3O3 (369.09).
4.1.1.5. 4-((2-chlorothieno[3,2-d]pyrimidin-4-yl)oxy)-3,5-
dimethylbenzonitrile (11e). White solid, yield: 89.1%, mp:
258ꢃ260 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.61 (d, J ¼ 8.6 Hz, 1H,
C6-thienopyrimidine-H), 7.79 (s, 2H, C3,C5-Ph-H), 7.70 (d, J ¼ 7.7 Hz,
1H, C7-thienopyrimidine-H), 2.14 (s, 6H, CH3 ꢄ 2). ESI-MS: m/z
316.3 [MþH]þ. C15H10ClN3OS (315.02).
4.1.1.6. 4-((2-chlorofuro[3,2-d]pyrimidin-4-yl)oxy)-3,5-
4.1.2.1. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)pyrimidin-4-yl)
dimethylbenzonitrile (11f). White solid, yield: 86.7%, mp:
oxy)benzonitrile (13a). White solid, yield: 74.2%, mp: 254ꢃ256 ꢀC.
225ꢃ227 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.66 (d, J ¼ 2.2 Hz, 1H,
1H NMR (400 MHz, DMSOd6)
d
8.22 (d, J ¼ 5.6 Hz, 1H, C6-pyrimi-
C6-furopyrimidine-H), 7.78 (s, 2H, C3,C5-Ph-H), 7.32 (d, J ¼ 2.2 Hz,
1H, C7-furopyrimidine-H), 2.14 (s, 6H, CH3 ꢄ 2). ESI-MS: m/z 300.2
[MþH]þ. C15H10ClN3O2 (299.05).
dine-H), 7.68 (s, 2H, C3,C5-Ph-H), 7.24 (s, 1H, NH), 6.27 (d, J ¼ 5.6 Hz,
1H, C5-pyrimidine-H), 3.19 (t, J ¼ 8.1 Hz, 3H, piperidine-H),
2.97e2.59 (m, 2H, piperidine-H), 2.09 (s, 6H, CH3 ꢄ 2), 1.93 (s, 1H,
piperidine-NH), 1.79e1.35 (m, 4H, piperidine-H). ESI-MS: m/z 324.1
[MþH]þ. C18H21N5O (323.17).
4.1.1.7. 4-((2-chlorothieno[2,3-d]pyrimidin-4-yl)oxy)-3,5-
dimethylbenzonitrile (11g). White solid, yield: 90.4%, mp:
269ꢃ271 ꢀC. ESI-MS: m/z 316.3 [MþH]þ. C15H10ClN3OS (315.02).
4.1.2.2. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)quinazolin-4-yl)
oxy)benzonitrile (13b). White solid, yield: 65.5%, mp: 119ꢃ121 ꢀC.
4.1.1.8. 4-((5-chlorothiazolo[5,4-d]pyrimidin-7-yl)oxy)-3,5-
1H NMR (400 MHz, DMSOd6)
d
8.14 (d, J ¼ 7.8 Hz, 1H, C8-quina-
dimethylbenzonitrile (11h). White solid, yield: 85.8%, mp:
zoline-H), 7.75 (s, 2H, C3,C5-Ph-H), 7.48 (d, J ¼ 8.5 Hz, 1H, C5-qui-
nazoline-H), 7.37 (t, J ¼ 8.6 Hz, 1H, C7-quinazoline-H), 7.30 (t, J ¼
7.6 Hz, 1H, C6-quinazoline-H), 4.09 (s, 1H, NH), 3.21e2.87 (m, 5H,
piperidine-H), 2.13 (s, 6H, CH3 ꢄ 2), 2.11 (s, 1H, piperidine-NH),
1.91e1.59 (m, 4H, piperidine-H). ESI-MS: m/z 374.5 [MþH]þ.
265ꢃ267 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 9.06 (s, 1H,
thiazolopyrimidine-H), 7.67 (s, 2H, C3,C5-Ph-H), 2.14 (s, 6H,
CH3 ꢄ 2). ESI-MS: m/z 317.1 [MþH]þ. C14H9ClN4OS (316.02).
4.1.1.9. 4-((2-chloro-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)
C22H23N5O (373.19).
oxy)-3,5-dimethylbenzonitrile (11i). White solid, yield: 93.1%, mp:
254ꢃ256 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
7.73 (s, 2H, C3,C5-Ph-H),
4.1.2.3. 4-((7-fluoro-2-(piperidin-4-ylamino)quinazolin-4-yl)oxy)-
3,5-dimethylbenzonitrile (13c). White solid, yield: 60.2%, mp:
3.04 (t, J ¼ 7.8 Hz, 2H, C5-cyclopentapyrimidine-H), 2.92 (t, J ¼
7.7 Hz, 2H, C7-cyclopentapyrimidine-H), 2.13 (p, J ¼ 7.7 Hz, 2H, C6-
cyclopentapyrimidine-H), 2.09 (s, 6H, CH3 ꢄ 2). ESI-MS: m/z 300.1
[MþH]þ. C16H14ClN3O (299.08).
143ꢃ145 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.18 (t, J ¼ 7.5 Hz, 1H,
C5-fluoroquinazoline-H), 7.73 (s, 2H, C3,C5-Ph-H), 7.26 (d, J ¼ 7.7 Hz,
1H, NH), 7.18 (d, J ¼ 10.8 Hz, 1H, C8-fluoroquinazoline-H), 7.12 (t, J ¼
11

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
8.9 Hz, 1H, C6-fluoroquinazoline-H), 4.01e3.87 (m, 1H, piperidine-
H), 3.14e2.80 (m, 2H, piperidine-H), 2.66 (t, J ¼ 12.2 Hz, 1H,
piperidine-NH), 2.12 (s, 6H, CH3 ꢄ 2), 2.00e1.73 (m, 2H, piperidine-
H), 1.59e1.34 (m, 3H, piperidine-H), 1.31e1.14 (m, 1H, piperidine-
H). ESI-MS: m/z 392.5 [MþH]þ. C22H22FN5O (391.18).
DMSOd6)
d
7.67 (s, 2H, C3,C5-Ph-H), 7.09 (s, 1H, NH), 3.36e3.35 (m,
3H, piperidine-H, C6-dihydrothienopyrimidine-H), 3.13 (t, J ¼
8.0 Hz, 2H, C7-dihydrothienopyrimidine-H), 2.71e2.62 (m, 2H,
piperidine-H), 2.06 (s, 6H, CH3 ꢄ 2), 1.83e1.45 (m, 6H, piperidine-
H). ESI-MS: m/z 382.2 [MþH]þ. C20H23N5OS (381.16).
4.1.2.4. 4-((6,7-dimethoxy-2-(piperidin-4-ylamino)quinazolin-4-yl)
4.1.2.11. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)-5,7-
oxy)-3,5-dimethylbenzonitrile (13d). White solid, yield: 57.4%, mp:
dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)benzonitrile
(13k).
159ꢃ161 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
7.72 (s, 2H, C3,C5-Ph-H),
White solid, yield: 63.8%, mp: 149ꢃ151 ꢀC. 1H NMR (400 MHz,
7.38 (s, 1H, C8-dimethoxyquinazoline-H), 6.88 (s, 1H, C5-dime-
thoxyquinazoline-H), 6.68 (s, 1H, piperidine-NH), 5.33 (s, 1H, NH),
3.88 (d, J ¼ 18.9 Hz, 6H, OCH3 ꢄ 2), 3.39 (d, J ¼ 7.6 Hz, 1H,
piperidine-H), 3.06e2.72 (m, 2H, piperidine-H), 2.12 (s, 6H,
CH3 ꢄ 2), 1.72 (d, J ¼ 43.0 Hz, 2H, piperidine-H), 1.24 (t, J ¼ 7.3 Hz,
4H, piperidine-H). ESI-MS: m/z 434.3 [MþH]þ. C24H27N5O3
(433.21).
DMSOd6) d 7.72 (s, 2H, C3,C5-Ph-H), 6.89 (s, 1H, NH), 4.12 (s, 2H, C5-
dihydrothienopyrimidine-H), 4.08 (s, 2H, C7-dihydrothienopyr-
imidine-H), 3.72e3.70 (m, 1H, piperidine-H), 2.74e2.72 (m, 2H,
piperidine-H), 2.10 (s, 6H, CH3 ꢄ 2), 1.97e1.75 (m, 4H, piperidine-
H), 1.47e1.42 (m, 2H, piperidine-H). ESI-MS: m/z 382.2 [MþH]þ.
C
20H23N5OS (381.16).
4.1.2.12. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)-5,7-dihydrofuro
4.1.2.5. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)thieno[3,2-d]pyr-
imidin-4-yl)oxy)benzonitrile (13e). White solid, yield: 84.2%, mp:
[3,4-d]pyrimidin-4-yl)oxy)benzonitrile (13l). White solid, yield:
60.7%, mp: 122ꢃ124 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 7.67 (s, 2H,
114ꢃ116 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.20 (d, J ¼ 5.4 Hz, 1H,
C3,C5-Ph-H), 7.08 (s, 1H, NH), 4.96 (s, 2H, C5-dihydrofuropyr-
imidine-H), 4.77 (s, 2H, C7-dihydrofuropyrimidine-H), 3.63e3.61
(m, 1H, piperidine-H), 2.67 (s, 2H, piperidine-H), 2.09 (s, 6H,
CH3 ꢄ 2), 1.97e1.32 (m, 6H, piperidine-H). ESI-MS: m/z 366.2
[MþH]þ. C20H23N5O2 (365.19).
C6-thienopyrimidine-H), 7.72 (s, 2H, C3,C5-Ph-H), 7.26 (s, 1H, C7-
thienopyrimidine-H), 6.92 (s, 1H, NH), 3.78 (s, 1H, piperidine-H),
2.89 (s, 2H, piperidine-H), 2.12 (s, 6H, CH3 ꢄ 2), 1.74ꢃ1.78 (m, 2H,
piperidine-H), 1.23ꢃ1.28 (m, 4H, piperidine-H). ESI-MS: m/z 380.5
[MþH]þ. C20H21N5OS (379.15).
4.1.3. General procedure for the synthesis of target compounds 14a-
l1 and 14a-l2
4.1.2.6. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)furo[3,2-d]pyr-
imidin-4-yl)oxy)benzonitrile (13f). White solid, yield: 70.3%, mp:
The key intermediates 13a-l (1.0 mmol) were dissolved in
anhydrous DMF (5 mL) in the presence of anhydrous cesium car-
bonate (0.39 g, 1.2 mmol), followed by the addition of corre-
sponding 4-(2-chloroethyl)morpholine (0.17 g, 1.1 mmol) or 4-(2-
chloroacetyl)morpholine (0.18 g, 1.1 mmol). The reaction solution
was stirred at 60 ꢀC and monitored by TLC until its completion.
After cooling to room temperature, the solvent was removed under
vacuum and water (20 mL) was added to it, followed by extraction
with ethyl acetate (3 ꢄ 10 mL). The collected organic phase was
washed with saturated brine (3 ꢄ 5 mL), and then dried over
anhydrous Na2SO4, filtered and concentrated to obtain the crude
product. Finally, the crude residue was further purified by flash
column chromatography with methanol/dichloromethane (1:5) as
eluent and recrystallized from ethyl acetate/petroleum ether to
afford the target compounds 14a-l1 and 14a-l2.
123ꢃ125 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.23 (d, J ¼ 2.1 Hz, 1H,
C6-furopyrimidine-H), 7.78 (d, J ¼ 8.0 Hz, 2H, C3,C5-Ph-H), 6.87 (s,
1H, C7-furopyrimidine-H), 6.77 (s, 1H, NH), 3.62 (s, 1H, piperidine-
H), 2.73e2.72 (m, 2H, piperidine-H), 2.12 (s, 6H, CH3 ꢄ 2), 1.72e1.53
(m, 3H, piperidine-H),1.40e1.24 (m, 3H, piperidine-H). ESI-MS: m/z
364.1 [MþH]þ. C20H21N5O2 (363.17).
4.1.2.7. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)thieno[2,3-d]pyr-
imidin-4-yl)oxy)benzonitrile (13g). White solid, yield: 66.8%, mp:
131ꢃ133 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
7.35 (d, J ¼ 5.5 Hz, 1H,
C6-thienopyrimidine-H), 7.31 (s, 2H, C3,C5-Ph-H), 7.27 (d, J ¼ 5.9 Hz,
1H, C5-thienopyrimidine-H), 3.77 (s, 1H, NH), 3.72ꢃ3.74 (m, 1H,
piperidine-H), 2.69ꢃ2.71 (m, 2H, piperidine-H), 2.10 (s, 6H,
CH3 ꢄ 2), 1.70ꢃ1.86 (m, 2H, piperidine-H), 1.18ꢃ1.48 (m, 4H,
piperidine-H). ESI-MS: m/z 380.2 [MþH]þ. C20H21N5OS (379.15).
4.1.3.1. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
4.1.2.8. 3,5-dimethyl-4-((5-(piperidin-4-ylamino)thiazolo[5,4-d]pyr-
imidin-7-yl)oxy)benzonitrile (13h). White solid, yield: 87.9%, mp:
amino)pyrimidin-4-yl)oxy)benzonitrile (14a1). White solid, yield:
31.2%, mp: 138ꢃ140 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.20 (d, J ¼
130ꢃ132 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
9.06 (s, 1H,
5.6 Hz, 1H, C6-pyrimidine-H), 7.68 (s, 2H, C3,C5-Ph-H), 7.19 (d, J ¼
123.3 Hz, 1H, NH), 6.25 (d, J ¼ 5.6 Hz, 1H, C5-pyrimidine-H), 4.09 (t,
J ¼ 5.8 Hz, 2H, morpholine-H), 3.88 (s, 2H, morpholine-H), 3.55 (t,
J ¼ 4.7 Hz, 4H, morpholine-H), 2.99 (d, J ¼ 123.9 Hz, 2H, piperidine-
H), 2.55e2.49 (m, 3H, piperidine-H), 2.42e2.37 (m, 4H, NeCH2 ꢄ 2),
2.09 (s, 6H, CH3 ꢄ 2), 1.68 (d, J ¼ 74.3 Hz, 2H, piperidine-H),
1.33e1.13 (m, 2H, piperidine-H). 13C NMR (100 MHz, DMSOd6)
thiazolopyrimidine-H), 8.42 (d, J ¼ 7.2 Hz, 1H, NH), 7.67 (s, 2H,
C3,C5-Ph-H), 3.65e3.63 (m, 1H, piperidine-H), 2.79e2.78 (m, 2H,
piperidine-H), 2.10 (s, 6H, CH3 ꢄ 2), 1.86e1.82 (m, 2H, piperidine-
H), 1.70e1.52 (m, 4H, piperidine-H). ESI-MS: m/z 381.1 [MþH]þ.
C
19H20N6OS (380.14).
4.1.2.9. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)-6,7-dihydro-5H-
cyclopenta[d]pyrimidin-4-yl)oxy)benzonitrile (13i). White solid,
d 168.38, 133.10, 132.94, 119.07, 108.66, 66.32, 53.38, 52.84, 50.81,
45.71, 16.30. ESI-MS: m/z 437.20 [MþH]þ. C24H32N6O2 (436.26).
yield: 67.2%, mp: 135ꢃ137 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 7.64 (s,
2H, C3,C5-Ph-H), 6.90 (s, 1H, NH), 3.65 (s, 1H, piperidine-H),
2.76e2.72 (m, 4H, C5,C7-cyclopentapyrimidine-H), 2.69e2.59 (m,
2H, piperidine-H), 2.07 (s, 6H, CH3 ꢄ 2), 2.02e1.97 (m, 2H, C6-
cyclopentapyrimidine-H), 1.81e1.21 (m, 6H, piperidine-H). ESI-MS:
m/z 364.2 [MþH]þ. C21H25N5O (363.21).
4.1.3.2. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piperidin-
4-yl)amino)pyrimidin-4-yl)oxy)benzonitrile (14a2). White solid,
yield: 52.1%, mp: 187ꢃ189 ꢀC. 1H NMR (400 MHz, DMSOd6)
d 8.19
(d, J ¼ 5.7 Hz,1H, C6-pyrimidine-H), 7.67 (s, 2H, C3,C5-Ph-H), 7.08 (d,
J ¼ 110.5 Hz, 1H, NH), 6.22 (d, J ¼ 5.6 Hz, 1H, C5-pyrimidine-H), 3.53
(dt, J ¼ 8.6, 4.4 Hz, 6H, morpholine-H), 3.42 (d, J ¼ 4.7 Hz, 2H,
morpholine-H), 3.08 (s, 2H, NeCH2), 2.95e2.59 (m, 3H, piperidine-
H), 2.08 (s, 6H, CH3 ꢄ 2), 1.98e1.20 (m, 6H, piperidine-H). 13C NMR
4.1.2.10. 3,5-dimethyl-4-((2-(piperidin-4-ylamino)-6,7-
dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile
(13j).
White solid, yield: 58.4%, mp: 135ꢃ137 ꢀC. 1H NMR (400 MHz,
(100 MHz, DMSOd6) d 168.29, 168.18, 162.12, 160.59, 133.13, 132.93,
12

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
119.08, 108.60, 66.86, 66.71, 61.04, 52.52, 46.29, 42.13, 31.65, 16.29.
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 164.31,156.47,154.13,
ESI-MS: m/z 451.18 [MþH]þ. C24H30N6O3 (450.24).
151.05, 146.47, 133.17, 132.93, 119.11, 108.73, 105.22, 102.32, 94.78,
66.50, 56.30, 56.21, 53.77, 52.42, 52.08, 40.44, 29.51, 16.36. ESI-MS:
m/z 547.22 [MþH]þ. C30H38N6O4 (546.30).
4.1.3.3. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
amino)quinazolin-4-yl)oxy)benzonitrile (14b1). White solid, yield:
44.5%, mp: 116ꢃ118 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.13 (d, J ¼
4.1.3.8. 4-((6,7-dimethoxy-2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)quinazolin-4-yl)oxy)-3,5-dimethylbenzonitrile
(14d2). White solid, yield: 43.6%, mp: 127ꢃ129 ꢀC. 1H NMR
8.1 Hz, 1H, C8-quinazoline-H), 7.75 (d, J ¼ 7.1 Hz, 3H, C3,C5-Ph-H, C5-
quinazoline-H), 7.47 (d, J ¼ 8.5 Hz,1H, C7-quinazoline-H), 7.27 (t, J ¼
7.5 Hz,1H, C6-quinazoline-H), 7.08 (s,1H, NH), 3.57 (t, J ¼ 4.6 Hz, 6H,
morpholine-H), 3.14 (s, 3H, piperidine-H), 2.48e2.26 (m, 6H,
morpholine-H, NeCH2 ꢄ 2), 2.13 (s, 6H, CH3 ꢄ 2), 1.95 (d, J ¼
12.1 Hz, 2H, piperidine-H), 1.64 (s, 2H, piperidine-H), 1.23 (s, 2H,
(400 MHz, DMSOd6)
d 7.71 (s, 2H, C3,C5-Ph-H), 7.38 (s, 1H, C8-
dimethoxyquinazoline-H), 6.89 (s, 1H, C5-dimethoxyquinazoline-
H), 6.65 (s,1H, NH), 3.91 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.64e3.50
(m, 6H, morpholine-H), 3.44 (p, J ¼ 4.4 Hz, 2H, morpholine-H), 3.19
(d, J ¼ 16.2 Hz, 2H, NeCH2), 3.01e2.67 (m, 3H, piperidine-H), 2.12 (s,
6H, CH3 ꢄ 2), 1.97e1.16 (m, 6H, piperidine-H). 13C NMR (100 MHz,
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 165.32, 154.28,
135.01, 133.07, 129.23, 125.21, 123.97, 122.52, 119.05, 110.63, 108.97,
66.50, 53.79, 52.15, 29.91, 16.30. ESI-MS: m/z 487.21 [MþH]þ.
DMSOd6) d 156.45, 154.89, 150.19, 147.79, 146.38, 133.19, 132.90,
C
28H34N6O2 (486.27).
108.71,107.26,107.01,102.84, 66.94, 66.81, 66.67, 56.70, 56.31, 56.21,
52.67, 46.27, 42.15, 39.67, 31.87, 16.36. ESI-MS: m/z 561.18 [MþH]þ.
4.1.3.4. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piperidin-
C30H36N6O5 (560.27).
4-yl)amino)quinazolin-4-yl)oxy)benzonitrile (14b2). White solid,
yield: 40.8%, mp: 165ꢃ167 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.11
4.1.3.9. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
(d, J ¼ 8.1 Hz, 1H, C8-quinazoline-H), 7.74 (s, 2H, C3,C5-Ph-H),
7.68e7.62 (m, 1H, C5-quinazoline-H), 7.47 (d, J ¼ 8.6 Hz, 1H, C7-
quinazoline-H), 7.25 (t, J ¼ 7.5 Hz,1H, C6-quinazoline-H), 6.96 (s,1H,
NH), 3.59e3.50 (m, 6H, morpholine-H), 3.43 (q, J ¼ 5.5, 4.6 Hz, 2H,
morpholine-H), 3.14 (d, J ¼ 5.8 Hz, 2H, NeCH2), 2.99e2.59 (m, 3H,
piperidine-H), 2.12 (d, J ¼ 8.4 Hz, 6H, CH3 ꢄ 2), 2.01e1.26 (m, 6H,
amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile
(14e1).
White solid, yield: 61.7%, mp: 170ꢃ172 ꢀC. 1H NMR (400 MHz,
DMSOd6)
d
8.20 (d, J ¼ 5.5 Hz, 1H, C6-thienopyrimidine-H), 7.72 (s,
2H, C3,C5-Ph-H), 7.29 (dd, J ¼ 26.8, 5.1 Hz, 1H, C7-thienopyrimidine-
H), 6.88 (s, 1H, NH), 3.55 (t, J ¼ 4.6 Hz, 6H, morpholine-H), 2.84 (s,
3H, piperidine-H), 2.44e2.34 (m, 6H, morpholine-H, NeCH2 ꢄ 2),
2.12 (s, 6H, CH3 ꢄ 2), 1.97e1.10 (m, 6H, piperidine-H). 13C NMR
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 168.17,161.94, 154.67,
134.92, 133.58, 133.16, 133.07, 132.63, 126.60, 123.93, 123.73, 122.32,
119.05, 113.05, 108.94, 107.91, 66.86, 66.72, 61.00, 52.58, 48.70,
46.30, 42.14, 31.69, 31.25, 16.37, 16.31. ESI-MS: m/z 501.14 [MþH]þ.
(100 MHz, DMSOd6) d 162.49, 160.53, 156.96, 153.45, 136.73, 133.22,
132.96, 123.99, 119.00, 109.06, 66.63, 56.29, 55.35, 54.14, 54.09,
53.03, 48.30, 31.51, 16.25. ESI-MS: m/z 493.15 [MþH]þ.
C
28H32N6O3 (500.25).
C26H32N6O2S (492.23).
4.1.3.5. 4-((7-fluoro-2-((1-(2-morpholinoethyl)piperidin-4-yl)
4.1.3.10. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (14e2).
White solid, yield: 65.2%, mp: 174ꢃ176 ꢀC. 1H NMR (400 MHz,
amino)quinazolin-4-yl)oxy)-3,5-dimethylbenzonitrile
(14c1).
White solid, yield: 30.9%, mp: 169ꢃ171 ꢀC. 1H NMR (400 MHz,
DMSOd6)
d
8.22 (dd, J ¼ 8.8, 6.4 Hz, 1H, C5-fluoroquinazoline-H),
DMSOd6)
d
8.13 (d, J ¼ 5.5 Hz, 1H, C6-thienopyrimidine-H), 7.65 (s,
7.74 (s, 2H, C3,C5-Ph-H), 7.67 (s, 1H, NH), 7.17 (td, J ¼ 9.5, 3.4 Hz, 2H,
2H, C3,C5-Ph-H), 7.29e7.08 (m, 1H, C7-thienopyrimidine-H), 6.81 (s,
1H, NH), 3.47 (dd, J ¼ 10.9, 4.9 Hz, 6H, morpholine-H), 3.36 (d, J ¼
4.9 Hz, 2H, morpholine-H), 3.07 (d, J ¼ 28.7 Hz, 2H, NeCH2), 2.75 (d,
J ¼ 50.6 Hz, 3H, piperidine-H), 2.05 (s, 6H, CH3 ꢄ 2), 1.86e1.14 (m,
C6,C8-fluoroquinazoline-H), 3.60 (dt, 9.4, 4.5 Hz, 6H,
J
¼
morpholine-H), 3.06 (d, J ¼ 7.5 Hz, 3H, piperidine-H), 2.45 (s, 6H,
morpholine-H, NeCH2 ꢄ 2), 2.13 (s, 6H, CH3 ꢄ 2), 2.05e1.33 (m, 6H,
piperidine-H). ESI-MS: m/z 505.18 [MþH]þ. C28H33FN6O2 (504.26).
6H, piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 168.21, 162.47,
160.55, 156.97, 153.43, 136.80, 133.23, 132.96, 124.00, 119.01, 109.05,
66.86, 66.72, 61.05, 52.57, 46.30, 42.13, 31.73, 16.26. ESI-MS: m/z
507.12 [MþH]þ. C26H30N6O3S (506.21).
4.1.3.6. 4-((7-fluoro-2-((1-(2-morpholino-2-oxoethyl)piperidin-4-yl)
amino)quinazolin-4-yl)oxy)-3,5-dimethylbenzonitrile
(14c2).
White solid, yield: 34.1%, mp: 185ꢃ187 ꢀC. 1H NMR (400 MHz,
DMSOd6)
d
8.18 (t, J ¼ 7.6 Hz, 1H, C5-fluoroquinazoline-H), 7.73 (s,
4.1.3.11. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
2H, C3,C5-Ph-H), 7.66 (s, 1H, NH), 7.21e7.06 (m, 2H, C6,C8-fluo-
roquinazoline-H), 3.54 (dt, J ¼ 9.8, 5.0 Hz, 6H, morpholine-H), 3.42
(t, J ¼ 4.7 Hz, 2H, morpholine-H), 3.14 (s, 2H, NeCH2), 2.96e2.58 (m,
3H, piperidine-H), 2.12 (s, 6H, CH3 ꢄ 2), 1.98e1.22 (m, 6H,
amino)furo[3,2-d]pyrimidin-4-yl)oxy)benzonitrile
(14f1). White
solid, yield: 60.6%, mp: 231ꢃ233 ꢀC. 1H NMR (400 MHz, DMSOd6)
d
8.26 (d, J ¼ 2.2 Hz, 1H, C6-furopyrimidine-H), 7.72 (s, 2H, C3,C5-Ph-
H), 6.89 (s, 2H, C7-furopyrimidine-H, NH), 3.58 (t, J ¼ 4.6 Hz, 6H,
morpholine-H), 2.58 (s, 3H, piperidine-H), 2.43 (d, J ¼ 5.2 Hz, 6H,
morpholine-H, NeCH2 ꢄ 2), 2.13 (s, 6H, CH3 ꢄ 2), 2.03e1.22 (m, 6H,
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d
168.05 (JCF
¼
37.9 Hz), 165.18 (JCF ¼ 39.9 Hz), 158.72, 153.60, 133.06, 133.06,
132.66, 127.09 (JCF ¼ 10.9 Hz), 119.03, 111.66 (JCF ¼ 23.1 Hz), 109.39,
109.04, 107.89 (JCF ¼ 28.1 Hz), 66.85, 66.71, 60.95, 53.93, 52.56,
48.02, 46.30, 42.12, 31.62, 16.33. ESI-MS: m/z 519.21 [MþH]þ.
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 158.86, 153.25,
152.86, 152.06, 133.22, 132.97, 127.76, 119.00, 109.05, 107.27, 66.46,
53.68, 51.79, 29.16, 16.33. ESI-MS: m/z 477.28 [MþH]þ. C26H32N6O3
(476.25).
C
28H31FN6O3 (518.24).
4.1.3.7. 4-((6,7-dimethoxy-2-((1-(2-morpholinoethyl)piperidin-4-yl)
4.1.3.12. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)furo[3,2-d]pyrimidin-4-yl)oxy)benzonitrile (14f2).
White solid, yield: 57.2%, mp: 177ꢃ179 ꢀC. 1H NMR (400 MHz,
amino)quinazolin-4-yl)oxy)-3,5-dimethylbenzonitrile
(14d1).
White solid, yield: 40.5%, mp: 168ꢃ170 ꢀC. 1H NMR (400 MHz,
DMSOd6)
d
7.72 (s, 2H, C3,C5-Ph-H), 7.39 (s, 1H, C8-dimethox-
DMSOd6)
d
8.23 (d, J ¼ 2.2 Hz,1H, C6-furopyrimidine-H), 7.72 (s, 2H,
yquinazoline-H), 6.89 (s, 1H, C5-dimethoxyquinazoline-H), 6.79 (s,
1H, NH), 3.91 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.57 (t, J ¼ 4.7 Hz, 6H,
morpholine-H), 2.78 (s, 3H, piperidine-H), 2.48e2.36 (m, 6H,
morpholine-H, NeCH2 ꢄ 2), 2.13 (s, 6H, CH3 ꢄ 2), 2.02e1.18 (m, 6H,
C3,C5-Ph-H), 6.88 (s,1H, C7-furopyrimidine-H), 6.74 (s,1H, NH), 3.54
(p, J ¼ 5.0, 4.4 Hz, 6H, morpholine-H), 3.42 (t, J ¼ 4.7 Hz, 2H,
morpholine-H), 3.09 (s, 2H, NeCH2), 2.92e2.62 (m, 3H, piperidine-
H), 2.13 (s, 6H, CH3 ꢄ 2), 1.93e1.21 (m, 6H, piperidine-H). 13C NMR
13

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
(100 MHz, DMSOd6)
d
168.21, 158.97, 153.28, 152.72, 152.00, 133.23,
477.22 [MþH]þ. C27H36N6O2 (476.29).
132.92, 127.62, 119.00, 109.01, 107.27, 66.86, 66.71, 61.09, 52.61,
46.30, 42.13, 31.75, 16.32. ESI-MS: m/z 491.21 [MþH]þ. C26H30N6O4
(490.23).
4.1.3.18. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)
benzonitrile (14i2). White solid, yield: 56.1%, mp: 212ꢃ214 ꢀC. 1H
4.1.3.13. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
NMR (400 MHz, DMSOd6)
d
7.65 (s, 2H, C3,C5-Ph-H), 6.76 (d, J ¼
amino)thieno[2,3-d]pyrimidin-4-yl)oxy)benzonitrile
(14g1).
92.5 Hz, 1H, NH), 3.53 (dq, J ¼ 8.5, 4.4 Hz, 6H, morpholine-H), 3.41
(t, J ¼ 4.7 Hz, 2H, morpholine-H), 3.06 (s, 2H, NeCH2), 2.76 (dt, J ¼
20.5, 7.5 Hz, 7H, C5,C7-cyclopentapyrimidine-H, piperidine-H), 2.08
(s, 6H, CH3 ꢄ 2), 2.03 (d, J ¼ 7.9 Hz, 2H, C6-cyclopentapyrimidine-H),
1.94e1.08 (m, 6H, piperidine-H). 13C NMR (100 MHz, DMSOd6)
White solid, yield: 65.0%, mp: 153ꢃ155 ꢀC. 1H NMR (400 MHz,
DMSOd6)
d
7.65 (s, 2H, C3,C5-Ph-H), 7.29 (d, J ¼ 5.9 Hz, 1H, C6-
thienopyrimidine-H), 7.21 (d, J ¼ 5.9 Hz, 1H, C5-thienopyrimidine-
H), 7.05 (s, 1H, NH), 3.48 (t, J ¼ 4.6 Hz, 6H, morpholine-H), 2.86 (s,
3H, piperidine-H), 2.38e2.26 (m, 6H, morpholine-H, NeCH2 ꢄ 2),
2.04 (s, 6H, CH3 ꢄ 2), 1.91e0.99 (m, 6H, piperidine-H). 13C NMR
d
168.18, 164.31, 162.07, 154.29, 133.15, 132.59, 119.16, 108.30, 66.86,
66.70, 61.08, 52.50, 46.29, 42.12, 34.21, 31.72, 25.96, 22.05, 16.32.
(100 MHz, DMSOd6)
d
162.23, 159.14, 153.67, 133.14, 132.97, 119.31,
ESI-MS: m/z 491.21 [MþH]þ. C27H34N6O3 (490.27).
119.05, 118.64, 108.88, 66.59, 55.90, 54.00, 52.71, 30.97, 16.33. ESI-
MS: m/z 493.10 [MþH]þ. C26H32N6O2S (492.23).
4.1.3.19. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
amino)-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)benzonitrile
(14j1). White solid, yield: 45.7%, mp: 188ꢃ190 ꢀC. 1H NMR
4.1.3.14. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)thieno[2,3-d]pyrimidin-4-yl)oxy)benzonitrile (14g2).
White solid, yield: 61.9%, mp: 202ꢃ204 ꢀC. 1H NMR (400 MHz,
(400 MHz, DMSOd6) d 7.67 (s, 2H, C3,C5-Ph-H), 7.03 (s, 1H, NH), 3.56
(s, 6H, morpholine-H), 3.36 (t,
J
¼
8.2 Hz, 2H, C6-dihy-
8.1 Hz, 2H, C7-dihy-
DMSOd6)
d
7.72 (s, 2H, C3,C5-Ph-H), 7.36 (d, J ¼ 6.0 Hz, 1H, C6-
drothienopyrimidine-H), 3.15 (t, J
¼
thienopyrimidine-H), 7.27 (d, J ¼ 6.0 Hz, 1H, C5-thienopyrimidine-
H), 7.07 (s, 1H, NH), 3.54 (dt, J ¼ 9.1, 4.8 Hz, 6H, morpholine-H), 3.42
(t, J ¼ 4.7 Hz, 2H, morpholine-H), 3.11 (s, 2H, NeCH2), 2.94e2.60 (m,
3H, piperidine-H), 2.11 (s, 6H, CH3 ꢄ 2), 2.03e1.14 (m, 6H,
drothienopyrimidine-H), 2.93 (s, 3H, piperidine-H), 2.42 (d, J ¼
32.4 Hz, 6H, morpholine-H, NeCH2 ꢄ 2), 2.07 (s, 6H, CH3 ꢄ 2),
1.82e0.92 (m, 6H, piperidine-H). 13C NMR (100 MHz, DMSOd6)
d
162.01, 160.57, 153.94, 133.08, 132.74, 119.06, 108.66, 66.55, 53.89,
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d
168.15, 162.76,
52.34, 36.95, 30.60, 29.33, 16.25. ESI-MS: m/z 495.21 [MþH]þ.
162.21, 159.14, 153.69, 133.15, 132.96, 119.24, 119.05, 118.61, 108.86,
66.85, 66.70, 60.97, 52.51, 46.28, 42.13, 36.25, 31.64, 16.32. ESI-MS:
m/z 507.05 [MþH]þ. C26H30N6O3S (506.21).
C26H34N6O2S (494.25).
4.1.3.20. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl)oxy)benzo-
nitrile (14j2). White solid, yield: 50.8%, mp: 201ꢃ203 ꢀC. 1H NMR
4.1.3.15. 3,5-dimethyl-4-((5-((1-(2-morpholinoethyl)piperidin-4-yl)
amino)thiazolo[5,4-d]pyrimidin-7-yl)oxy)benzonitrile
(14h1).
(400 MHz, DMSOd6)
d
7.67 (s, 2H, C3,C5-Ph-H), 6.93 (d, J ¼ 80.6 Hz,
White solid, yield: 28.8%, mp: 160ꢃ162 ꢀC. 1H NMR (400 MHz,
1H, NH), 3.53 (q, J ¼ 5.3 Hz, 6H, morpholine-H), 3.41 (t, J ¼ 4.7 Hz,
2H, morpholine-H), 3.37 (d, J ¼ 8.2 Hz, 2H, NeCH2), 3.13 (dd, J ¼
22.3,14.2 Hz, 4H, C6,C7-dihydrothienopyrimidine-H), 2.94e2.59 (m,
3H, piperidine-H), 2.07 (s, 6H, CH3 ꢄ 2), 1.97e1.09 (m, 6H,
DMSOd6)
d
9.01 (d, J ¼ 43.5 Hz, 1H, thiazolopyrimidine-H), 7.70 (d,
J ¼ 22.9 Hz, 2H, C3,C5-Ph-H), 7.45 (d, J ¼ 90.3 Hz,1H, NH), 3.56 (q, J ¼
4.7 Hz, 6H, morpholine-H), 2.78 (d, J ¼ 86.1 Hz, 3H, piperidine-H),
2.41 (d, J ¼ 19.9 Hz, 6H, morpholine-H, NeCH2 ꢄ 2), 2.11 (d, J ¼
8.8 Hz, 6H, CH3 ꢄ 2), 1.97e1.14 (m, 6H, piperidine-H). ESI-MS: m/z
494.17 [MþH]þ. C25H31N7O2S (493.23).
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 168.09, 162.00,
160.61, 153.89, 133.08, 132.68, 119.07, 108.64, 66.84, 66.69, 60.96,
52.42, 46.26, 42.12, 36.92, 31.57, 29.33, 16.25. ESI-MS: m/z 509.20
[MþH]þ. C26H32N6O3S (508.23).
4.1.3.16. 3,5-dimethyl-4-((5-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)thiazolo[5,4-d]pyrimidin-7-yl)oxy)benzonitrile
(14h2). White solid, yield: 39.2%, mp: 195ꢃ197 ꢀC. 1H NMR
4.1.3.21. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
amino)-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)benzonitrile
(14k1). White solid, yield: 52.1%, mp: 119ꢃ121 ꢀC. 1H NMR
(400 MHz, DMSOd6)
d
9.01 (d, J ¼ 45.6 Hz, 1H, thiazolopyrimidine-
H), 7.70 (d, J ¼ 23.6 Hz, 2H, C3,C5-Ph-H), 7.62e7.23 (m, 1H, NH), 3.54
(dt, J ¼ 8.8, 4.3 Hz, 6H, morpholine-H), 3.43 (d, J ¼ 5.0 Hz, 2H,
morpholine-H), 3.21e3.01 (m, 2H, NeCH2), 2.97e2.59 (m, 3H,
piperidine-H), 2.12 (d, J ¼ 8.3 Hz, 6H, CH3 ꢄ 2), 2.06e1.09 (m, 6H,
(400 MHz, DMSOd6)
d
7.67 (s, 2H, C3,C5-Ph-H), 7.15 (d, J ¼ 97.8 Hz,
1H, NH), 4.11 (d, J ¼ 23.1 Hz, 4H, C5,C7-dihydrothienopyrimidine-H),
3.56 (t, J ¼ 4.6 Hz, 6H, morpholine-H), 2.95 (d, J ¼ 56.1 Hz, 3H,
piperidine-H), 2.47e2.26 (m, 6H, morpholine-H, NeCH2 ꢄ 2), 2.09
(s, 6H, CH3 ꢄ 2), 1.96e0.90 (m, 6H, piperidine-H). 13C NMR
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 168.17, 161.17, 160.33,
156.63, 154.41, 150.20, 148.54, 133.08, 133.04, 132.76, 128.28, 119.02,
108.98, 108.26, 66.85, 66.71, 60.96, 52.55, 48.35, 46.28, 42.12, 39.67,
31.56, 31.18, 16.28. ESI-MS: m/z 508.10 [MþH]þ. C25H29N7O3S
(507.21).
(100 MHz, DMSOd6) d 164.47, 162.14, 133.07, 132.79, 119.06, 108.67,
66.53, 53.87, 52.32, 30.95, 30.52, 16.28. ESI-MS: m/z 495.22
[MþH]þ. C26H34N6O2S (494.25).
4.1.3.22. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl)oxy)benzo-
nitrile (14k2). White solid, yield: 57.4%, mp: 167ꢃ169 ꢀC. 1H NMR
4.1.3.17. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
amino)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)oxy)benzoni-
trile (14i1). White solid, yield: 50.4%, mp: 184ꢃ186 ꢀC. 1H NMR
(400 MHz, DMSOd6)
d 7.67 (s, 2H, C3,C5-Ph-H), 7.33e6.84 (m, 1H,
(400 MHz, DMSOd6)
d
7.65 (s, 2H, C3,C5-Ph-H), 6.81 (d, J ¼ 77.9 Hz,
NH), 4.27e3.88 (m, 4H, C5,C7-dihydrothienopyrimidine-H),
3.57e3.48 (m, 6H, morpholine-H), 3.41 (t, J 4.7 Hz, 2H,
1H, NH), 3.55 (t, J ¼ 4.6 Hz, 6H, morpholine-H), 2.77 (dt, J ¼ 21.8,
7.5 Hz, 7H, C5,C7-cyclopentapyrimidine-H, piperidine-H),
2.44e2.31 (m, 6H, morpholine-H, NeCH2 ꢄ 2), 2.08 (s, 6H,
CH3 ꢄ 2), 2.03 (d, J ¼ 7.7 Hz, 2H, C6-cyclopentapyrimidine-H),
1.89e1.13 (m, 6H, piperidine-H). 13C NMR (100 MHz, DMSOd6)
¼
morpholine-H), 3.09 (d, J ¼ 19.3 Hz, 2H, NeCH2), 2.95e2.61 (m, 3H,
piperidine-H), 2.09 (d, J ¼ 3.4 Hz, 6H, CH3 ꢄ 2), 1.88e0.93 (m, 6H,
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 168.04, 164.46,
162.18, 133.08, 132.79, 119.07, 108.66, 66.84, 66.69, 60.86, 52.42,
d
164.33, 162.04, 154.30, 133.16, 132.63, 119.16, 108.32, 66.59, 55.87,
46.26, 42.12, 31.48, 30.99, 16.28. ESI-MS: m/z 509.19 [MþH]þ.
53.99, 52.69, 39.66, 34.22, 31.17, 25.95, 22.04, 16.32. ESI-MS: m/z
C
26H32N6O3S (508.23).
14

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
4.1.3.23. 3,5-dimethyl-4-((2-((1-(2-morpholinoethyl)piperidin-4-yl)
amino)-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)benzonitrile
(14l1). White solid, yield: 50.8%, mp: 173ꢃ175 ꢀC. 1H NMR
description in the kit protocol. In brief, the reaction mixture con-
taining template/primer complex, viral nucleotides (dNTPs) and
HIV-1 reverse transcriptase (RT) enzyme in the incubation buffer
with or without inhibitors was incubated for 1 h at 37 ꢀC. Subse-
quently, the reaction mixture was transferred to a streptavidine
coated microtiter plate (MTP) and incubated for another 1 h at 37 ꢀC
to ensure the retranscriptional cDNA chain that consisted biotin
labeled dNTPs bound to streptavidine. Then used washing buffer to
remove the unbound dNTPs and anti-DIG-POD working solution
was added. After incubation for 1 h at 37 ꢀC, the DIG-labeled dNTPs
incorporated in cDNA were bound to the anti-DIG-POD antibody.
The unbound anti-DIG-PODs were removed and the peroxidesub-
strate (ABST) solution was added to the MTPs. A colored reaction
proceeds during cleavage of the substrate catalyzed by POD. The
absorbance of the sample was determined at O.D. 405 nm using a
microtiter plate ELISA reader. The percentage inhibitory activity of
RT inhibitors was calculated by formula as given below:
(400 MHz, DMSOd6)
d
7.67 (s, 2H, C3,C5-Ph-H), 7.20 (d, J ¼ 82.9 Hz,
1H, NH), 4.97 (d, J ¼ 2.3 Hz, 2H, C5-dihydrofuropyrimidine-H), 4.78
(s, 2H, C7-dihydrofuropyrimidine-H), 3.55 (t, J ¼ 4.6 Hz, 6H,
morpholine-H), 2.87 (d, J ¼ 41.8 Hz, 3H, piperidine-H), 2.46e2.30
(m, 6H, morpholine-H, NeCH2 ꢄ 2), 2.10 (s, 6H, CH3 ꢄ 2), 1.98e1.11
(m, 6H, piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 162.95,
162.49, 133.14, 132.94, 119.05, 108.75, 69.39, 66.59, 55.86, 54.96,
54.01, 52.72, 39.64, 31.07, 16.27. ESI-MS: m/z 479.22 [MþH]þ.
C
26H34N6O3 (478.27).
4.1.3.24. 3,5-dimethyl-4-((2-((1-(2-morpholino-2-oxoethyl)piper-
idin-4-yl)amino)-5,7-dihydrofuro[3,4-d]pyrimidin-4-yl)oxy)benzoni-
trile (14l2). White solid, yield: 61.2%, mp: 184ꢃ186 ꢀC. 1H NMR
(400 MHz, DMSOd6)
d
7.67 (s, 2H, C3,C5-Ph-H), 7.16 (d, J ¼ 87.7 Hz,
1H, NH), 4.96 (s, 2H, C5-dihydrofuropyrimidine-H), 4.77 (s, 2H, C7-
dihydrofuropyrimidine-H), 3.60e3.48 (m, 6H, morpholine-H), 3.41
(t, J ¼ 4.7 Hz, 2H, morpholine-H), 3.08 (s, 2H, NeCH2), 2.98e2.60
(m, 3H, piperidine-H), 2.10 (s, 6H, CH3 ꢄ 2), 1.99e1.03 (m, 6H,
% Inhibition¼[O.D. value with RT but without inhibitorsꢃO.D.
value with RT and inhibitors]/[O.D. value with RT and inhib-
itorsꢃO.D. value without RT and inhibitors].
The IC50 values corresponded to the concentrations of the test
compounds required to inhibit the incorporation of biotin-dUTP
into RT by 50%.
piperidine-H). 13C NMR (100 MHz, DMSOd6)
d 168.15, 162.99,
162.49, 133.14, 132.88, 119.04, 108.74, 69.41, 66.85, 66.70, 61.00,
52.44, 46.28, 42.12, 31.59, 16.27. ESI-MS: m/z 493.15 [MþH]þ.
C
26H32N6O4 (492.25).
4.4. Molecular simulation
4.2. In vitro anti-HIV assay
Molecular modeling was performed with the Tripos molecular
modeling packages Sybyl-X 2.0. All the molecules (14j2, 14c1, 14e2
and 14d2) for docking were built using standard bond lengths and
angles from Sybyl-X 2.0/Base Builder and were optimized using the
Tripos force field for 1000-generations two times or more until the
maximum derivative of energy became 0.005 kcal/(mol*Å).
Charges were computed and added according to GasteigereHuckel
parameters. The published five dimensional crystal structures of RT
complexes {HIV-1 WT RT (PDB code: 6C0J), K103N RT (PDB code:
6C0K), E138K RT (PDB code: 6C0L), RES056 RT (PDB code: 6C0R)
and F227L/V106A RT (PDB code: 6DUF)} were retrieved from the
Protein Data Bank and were used for the docking experiment by
means of the surflex-docking module of Sybyl-X 2.0. The protein
was prepared by removing the ligand, water molecules, and other
unnecessary small molecules from the crystal structure of the
ligand HIV-1 RTcomplex before docking; polar hydrogen atoms and
charges were added to the protein. After the protomol was gener-
ated, which referred to a computational representation of the
intended binding site to which putative ligands are aligned, the
optimized compounds 14j2, 14c1, 14e2 or 14d2 was surflex-docked
into the NNIBP, with the relevant parameters set as defaults. The
original ligand of the coordinates was used as a reference molecule
to calculate the RMSD values. The docking scores related to binding
affinities were calculated based on hydrophobic, polar, and repul-
sive interactions as well as entropic effects and solvation. Top-
scoring pose was shown by the software of PyMOL version 1.5
(http://www.pymol.org/). The secondary structure of RT was
shown in cartoons, and only the key residues for interactions with
the inhibitor were labeled and shown in sticks. The potential
hydrogen bonds were presented by dashed lines.
Evaluation of the anti-HIV activity and cytotoxicity of the target
compounds was performed by utilizing the MTT method in MT-
4 cells described previously [40,41]. Stock solutions (10 ꢄ final
concentration) of test compounds were added in 25 mL volumes to
two series of triplicate wells to allow simultaneous evaluation of
their effects on mock- and HIV-infected cells at the beginning of
each experiment. Serial five-fold dilutions of test compounds were
prepared directly in flat-bottomed 96-well microtiter trays by
adding 100
mL medium to the 25
m
L stock solution and transferring
25 L of this solution to another well that contained 100
m
mL medium
using a Biomek 3000 robot (Beckman Instruments, Fullerton, CA).
Each sample includes untreated control HIV- and mock-infected
cell samples. HIV-1 WT strain (IIIB), HIV-1 drug-resistant strains
including K103N þ Y181C (RES056), L100I, K103N, Y181C, Y188L,
E138K and F227L þ V106A or HIV-2 strain (ROD) stock (50
mL) at
100e300 CCID50 (50% cell culture infectious dose) or culture me-
dium 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 compounds on uninfected cells in order to evaluate its
cytotoxicity. Exponentially growing MT-4 cells were centrifuged for
5 min at 1000 rpm and the supernatant was discarded. The MT-
4 cells were resuspended at 6 ꢄ 105 cells/mL, and then transferred
50 mL volumes to the microtiter tray wells. After infection five days,
the viability of mock- and HIV-infected cells was examined spec-
trophotometrically by the MTT assay. The 50% cytotoxic concen-
tration (CC50) was defined as the concentration of the test
compound that reduced the viability of the mock-infected MT-
4 cells by 50%. The 50% effective concentration (EC50) was defined
as the concentration of the test compound achieving 50% protec-
tion from the cytopathic effect of the virus in infected cells.
4.5. Water solubility measurement
4.3. HIV-1 RT inhibition assay
Water solubility was measured in phosphate buffer at three
different pH conditions. The compound was initially weighed in
three orders of magnitude, namely approximately 0.01 mg, 0.1 mg
and 1 mg, and then it was dissolved in the phosphate buffer of the
same pH value until the solution was clear [43]. The average value
of water solubility was calculated from the weight of the compound
A HIV-1 reverse transcriptase (RT) assay kit produced by Roche
was used for the RT inhibition assay [42]. All the reagents for per-
forming the RT reaction came from the kit and the ELSIA proced-
ures for RT inhibition assay were conducted following the
15

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
control, N. Engl. J. Med. 377 (2017) 1605e1607.
[16] Y. Wu, C. Tang, R. Rui, L. Yang, W. Ding, J. Wang, Y. Li, C.C. Lai, Y. Wang, R. Luo,
W. Xiao, H. Zhang, Y. Zheng, Y. He, Synthesis and biological evaluation of a
series of 2-(((5-akly/aryl-1H-pyrazol-3-yl)methyl)thio)-5-alkyl-6-(cyclo-
hexylmethyl)-pyrimidin-4(3H)-ones as potential HIV-1 inhibitors, Acta
Pharm. Sin. B 10 (2020) 512e518.
and the volume of the solution. The assays were measured at least
in duplicate.
Declaration of competing interest
[17] E. De Clercq, An Odyssey in antiviral drug development-50 years at the Rega
Institute: 1964-2014, Acta Pharm. Sin. B 5 (2015) 520e543.
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.
[18] 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.
[19] 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.
60 (2017) 4424e4443.
[20] 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
[5,4-d]pyrimidine derivatives as potent and orally bioavailable HIV-1 non-
nucleoside reverse transcriptase inhibitors, Commun. Chem. 2 (2019) 74.
[21] 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.
Acknowledgments
We gratefully acknowledge the financial support from the Na-
tional Natural Science Foundation of China (NSFC Nos. 81973181
and 81903453), the Key Project of NSFC for International Cooper-
ation (No. 81420108027), Shandong Provincial Natural Science
Foundation (ZR2019BH011), China Postdoctoral Science Foundation
(2019T120596), Shandong Provincial Key Research and Develop-
ment Project (Nos. 2017CXGC1401 and 2019JZZY021011), and the
Taishan Scholar Program at Shandong Province. We thank K. Erven,
K. Uyttersprot and C. Heens for technical assistance with the anti-
HIV assays.
[22] 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-
1 NNRTIs with highly improved resistance profiles and low activity for the
hERG ion channel, J. Med. Chem. 63 (2020) 1298e1312.
[23] 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.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112811.
References
[24] 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
[1] R.J. Shattock, M. Warren, S. McCormack, C.A. Hankins, AIDS. Turning the tide
against HIV, Science 333 (2011) 42e43.
[2] 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.
thiophene[3,2-d]pyrimidine non-nucleoside inhibitors, eLife
e36340.
7
(2018),
[25] A.P. Kourounakis, D. Xanthopoulos, A. Tzara, Morpholine as
a privileged
structure: a review on the medicinal chemistry and pharmacological activity
of morpholine containing bioactive molecules, Med. Res. Rev. 40 (2020)
709e752.
[3] N. Sluis-Cremer, Future of nonnucleoside reverse transcriptase inhibitors,
Proc. Natl. Acad. Sci. U.S.A. 115 (2018) 637e638.
[4] G. Bec, B. Meyer, M.A. 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.
[5] 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.
[6] J. Du, J. Guo, D. Kang, Z. Li, G. Wang, J. Wu, Z. Zhang, H. Fang, X. Hou, Z. Huang,
G. Li, X. Lu, X. Liu, L. Ouyang, L. Rao, P. Zhan, X. Zhang, Y. Zhang, New tech-
niques and strategies in drug discovery, Chin. Chem. Lett. 31 (2020)
1695e1708.
[26] X. Jiang, J. Yu, Z. Zhou, J. Kongsted, Y. Song, C. Pannecouque, E. De Clercq,
D. Kang, V. Poongavanam, X. Liu, P. Zhan, Molecular design opportunities
presented by solvent-exposed regions of target proteins, Med. Res. Rev. 39
(2019) 2194e2238.
[27] W. Wei, S. Cherukupalli, L. Jing, X. Liu, P. Zhan, Fsp3: a new parameter for
drug-likeness, Drug Discov. Today (2020), https://doi.org/10.1016/
j.drudis.2020.07.017.
[28] Q. Ren, X. Liu, Z. Luo, J. Li, C. Wang, S. Goldmann, J. Zhang, Y. Zhang, Discovery
of hepatitis
B virus capsid assembly inhibitors leading to a hetero-
aryldihydropyrimidine based clinical candidate (GLS4), Bioorg. Med. Chem. 25
(2017) 1042e1056.
[29] K. Jin, M. Liu, C. Zhuang, E. De Clercq, C. Pannecouque, G. Meng, F. Chen,
Improving the positional adaptability: structure-based design of biphenyl-
substituted diaryltriazines as novel non-nucleoside HIV-1 reverse transcrip-
tase inhibitors, Acta Pharm. Sin. B 10 (2020) 344e357.
[7] C. Flexner, HIV drug development: the next 25 years, Nat. Rev. Drug Discov. 6
(2007) 959e966.
[8] S.X. Gu, H.H. Lu, G.Y. Liu, X.L. Ju, Y.Y. Zhu, Advances in diarylpyrimidines and
related analogues as HIV-1 nonnucleoside reverse transcriptase inhibitors,
Eur. J. Med. Chem. 158 (2018) 371e392.
[9] L. Battini, M. Bollini, Challenges and approaches in the discovery of human
immunodeficiency virus type-1 non-nucleoside reverse transcriptase in-
hibitors, Med. Res. Rev. 39 (2019) 1235e1273.
[10] E.D. Deeks, Doravirine: first global approval, Drugs 78 (2018) 1643e1650.
[11] M. Feng, D. Wang, J.A. Grobler, D.J. Hazuda, M.D. Miller, M.T. Lai, In vitro
[30] Z. Huo, H. Zhang, D. Kang, Z. Zhou, G. Wu, S. Desta, X. Zuo, Z. Wang, L. Jing,
X. Ding, D. Daelemans, E. De Clercq, C. Pannecouque, P. Zhan, X. Liu, Discovery
of novel diarylpyrimidine derivatives as potent HIV-1 NNRTIs targeting the
“NNRTI Adjacent” binding site, ACS Med. Chem. Lett. 9 (2018) 334e338.
[31] B. Huang, W. Chen, T. Zhao, Z. Li, X. Jiang, T. Ginex, D. Vílchez, 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.
[32] S. Castellino, M.R. Groseclose, J. Sigafoos, D. Wagner, M. de Serres, J.W. Polli,
E. Romach, J. Myer, B. Hamilton, Central nervous system disposition and
metabolism of Fosdevirine (GSK2248761), a non-nucleoside reverse tran-
scriptase inhibitor: an LC-MS and Matrix-assisted laser desorption/ionization
imaging MS investigation into central nervous system toxicity, Chem. Res.
Toxicol. 26 (2013) 241e251.
[33] A. Basiri, B.M. Abd Razik, M.O. Ezzat, Y. Kia, R.S. Kumar, A.I. Almansour,
N. Arumugam, V. Murugaiyah, Synthesis and cholinesterase inhibitory activity
study of new piperidone grafted spiropyrrolidines, Bioorg. Chem. 75 (2017)
210e216.
[34] Z. Wang, D. Kang, M. Chen, G. Wu, D. Feng, T. Zhao, Z. Zhou, Z. Huo, L. Jing,
X. Zuo, D. Daelemans, E. De Clercq, C. Pannecouque, P. Zhan, X. Liu, Design,
synthesis, and antiviral evaluation of novel hydrazone-substituted thiophene
resistance selection with doravirine (MK-1439),
a novel nonnucleoside
reverse transcriptase inhibitor with distinct mutation development pathways,
Antimicrob. Agents Chemother. 59 (2015) 590e598.
[12] G. Sterrantino, V. Borghi, A.P. Callegaro, B. Bruzzone, F. Saladini, F. Maggiolo,
G. Maffongelli, M. Andreoni, M. De Gennaro, N. Gianotti, P. Bagnarelli,
A. Vergori, A. Antinori, M. Zazzi, M. Zaccarelli, ARCA Study Group, Prevalence
of predicted resistance to doravirine in HIV-1-positive patients after exposure
to non-nucleoside reverse transcriptase inhibitors, Int. J. Antimicrob. Agents
53 (2019) 515e519.
[13] C. Soulie, M.M. Santoro, C. Charpentier, A. Storto, D. Paraskevis, D. Di Carlo,
W. Gennari, G. Sterrantino, M. Zazzi, C.F. Perno, V. Calvez, D. Descamps,
F. Ceccherini-Silberstein, A.G. Marcelin, Rare occurrence of doravirine
resistance-associated mutations in HIV-1-infected treatment-naive patients,
J. Antimicrob. Chemother. 74 (2019) 614e617.
[14] P. Zhan, X. Chen, D. Li, Z. Fang, E. De Clercq, X. Liu, HIV-1 NNRTIs: structural
diversity, pharmacophore similarity, and implications for drug design, Med.
Res. Rev. 33 (2013) E1ꢃE72.
[15] C. Beyrer, A. Pozniak, HIV drug resistance - an emerging threat to epidemic
16

Z. Wang, D. Kang, D. Feng et al.
European Journal of Medicinal Chemistry 206 (2020) 112811
[3,2-d]pyrimidine derivatives as potent human immunodeficiency virus-1
inhibitors, Chem. Biol. Drug Des. 92 (2018) 2009e2021.
ADMETlab:
a platform for systematic ADMET evaluation based on a
comprehensively collected ADMET database, J. Cheminf. 10 (2018) 29.
[35] 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 transcriptase
inhibitors with promising antiviral activities and desirable physicochemical
properties, J. Med. Chem. 62 (2019) 1484e1501.
[36] D.E. Clark, S.D. Pickett, Computational methods for the prediction of ‘drug-
likeness’, Drug Discov. Today 5 (2000) 49e58.
[37] K. Palm, P. Stenberg, K. Luthman, P. Artursson, Polar molecular surface
properties predict the intestinal absorption of drugs in humans, Pharm. Res.
14 (1997) 568e571.
[40] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn,
J. Desmyter, E. De Clercq, Rapid and automated tetrazolium-based colori-
metric assay for the detection of anti-HIV compounds, J. Virol. Methods 20
(1988) 309e321.
[41] 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.
[42] 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.
[43] Z. Zhou, T. Liu, G. Wu, D. Kang, Z. Fu, Z. Wang, E. De Clercq, C. Pannecouque,
P. Zhan, X. Liu, Targeting the hydrophobic channel of NNIBP: discovery of
novel 1,2,3-triazole-derived diarylpyrimidines as novel HIV-1 NNRTIs with
high potency against wild-type and K103N mutant virus, Org. Biomol. Chem.
17 (2019) 3202e3217.
[38] J.P. Havens, A.T. Podany, K.K. Scarsi, C.V. Fletcher, Clinical pharmacokinetics
and pharmacodynamics of etravirine: an updated review, Clin. Pharmacoki-
net. 59 (2020) 137e154.
[39] J. Dong, N.N. Wang, Z.J. Yao, L. Zhang, Y. Cheng, D. Ouyang, A.P. Lu, D.S. Cao,
17