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

Article abstract of DOI:10.1039/c9ob00032a

Enlightened by our previous efforts to modify diarylpyrimidines as HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) and the reported crystallographic studies, we designed and synthesized novel 1,2,3-triazole-derived diarylpyrimidine der

Full text of DOI:10.1039/c9ob00032a

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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
Zhongxia Zhou,1 Tao Liu,1 Gaochan Wu,1 Dongwei Kang,1Zhipeng Fu,1 Zhao Wang,1
Erik De Clercq,2 Christophe Pannecouque,2,* Peng Zhan,1,* and Xinyong Liu1,*
1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, 250012 Jinan, Shandong, PR China
2Rega Institute for Medical Research, K.U.Leuven, Minderbroedersstraat 10, B-3000
Leuven, Belgium
Abstract:
Enlightened by our previous efforts to modify diarylpyrimidines as HIV-1
non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) and the reported
crystallographic studies, we designed and synthesized novel 1,2,3-triazole-derived
diarylpyrimidine derivatives via CuAAC "click reaction", to make additional
interactions with the hydrophobic channel in the NNRTI binding pocket. The newly
synthesized compounds were evaluated for anti-HIV potency in MT-4 cells. All the
compounds showed favorable activity against wild-type HIV-1 strain with EC50 of
0.013-5.62 μM. Interestingly, some compounds displayed remarkable potency
inhibiting K103N mutant virus, a key drug-resistant mutant to the NNRTIs. Among
them, meta-methylbenzoate (ZL2, EC50(IIIB)=0.020 μM, EC50(K103N) = 0.043 μM, CC50
> 241.52 μM), para-methylbenzoate (ZL3, EC50(IIIB) = 0.013 μM, EC50 (K103N) = 0.022
μM, CC50 > 241.52 μM) and para-phenol (ZL7, EC50(IIIB) = 0.014 μM, EC50 (K103N)
=
0.054 μM, CC50 = 2.1 μM) derivatives are the three most promising compounds
which are superior to the first-line antiretroviral drug efavirenz (EC50(IIIB) = 0.003 μM,
EC50
= 0.11 μM, CC50 >6.34 μM) against K103N mutant strain. More
(K103N)
encouragingly, ZL2 and ZL3 exhibited much lower cytotoxicity and high selection

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index of >10,000 compared with all the control drugs (AZT, 3TC, NVP, EFV, ETV).
Detailed structure-activity relationship (SAR), enzymatic inhibitory activity and
docking study of the representative compounds are also discussed. Furthermore, the
preliminary physicochemical properties and the early metabolic stability of
representative compounds were examined to evaluate their drug-like properties.
KEYWORDS: HIV-1; NNRTIs; triazole; click chemistry; diarylpyrimidines;
hydrophobic channel.
1. Introduction
HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are
essential components of highly active antiretroviral therapy (HAART) due to its good
tolerability and low toxicity.1-4 The non-competitive binding of NNRTIs with an
allosteric site (non-nucleoside inhibitors binding pocket, NNIBP) in HIV-1 RT
induces conformational changes of the catalytic active site and finally leads to
inhibitory of DNA polymerase activity.5 There are currently six approved drugs that
target the NNIBP. Nevirapine (NVP), delavirdine (DLV) are considered first
generation NNRTIs and show a dramatic loss of activity with single mutations in the
NNIBP. Efavirenz (EFV) is effective against many mutations except for two of the
most prevalent NNRTI resistance-associated mutations, K103N and Y181C.6 The
second-generation NNRTIs contain etravirine (ETV) and rilpivirine (RPV), belonging
to diarylpyrimidine (DAPY) NNRTIs, possess broad-spectrum of anti-viral activity
against clinically relevant HIV-1 mutant strains and are considered to be the most
successful NNRTIs currently.7 Recently, a new NNRTI, doravirine was approved in
the United States. However, because of the high mutation rate of HIV-1 RT and the
lack of intrinsic exonucleolytic proofreading activity, new resistance strains with
reduced susceptibility to ETV and RPV have emerged and are also being observed in
patients.8,9 In addition, adverse effects such as hepatotoxicity, severe rash and central
nervous system side effects appeared successively in clinical application.10

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Consequently, novel NNRTI drugs with different molecular scaffolds and high
potency are still needed to overcome current drug resistance regimes and adverse
effects associated with the second-generation NNRTI-based anti-HIV drugs.11-13
O
NH
N
O
S O
NH
HN
CF3
O
Cl
N
HN
N
N
N
N
N
O
H
O
Nevirapine
Delavirdine
Efavirenz
CN
N
CN
CN
Cl
CN
O
O
F
N
N
O
N
NH
HN
N
HN
N
NH
N
F
F
Br
O
N
NH2
Etravirine
Rilpivirine
Doravirine
Figure 1. Chemical structures of NNRTIs approved by the U.S. FDA.
The binding mode of RPV with the NNIBP in RT is well described by the
crystallography studies6,14: the central pyrimidine ring and the NH linker connecting
to its 2-position form two hydrogen bonds with the chain of K101; two aromatic
wings of the molecule fit into two adjacent grooves composed mainly of lipophilic
amino acid residues; the acrylonitrile protrudes through an opening hydrophobic
channel formed by residues W229, Y188, F227, and L234.6 Thereinto, W229, F227,
L234 were identified as highly conserved amino acids among those lining the NNIBP.
Especially, it was never been observed that the mutation of the key W229 in
combination with other mutations, suggesting that compensatory mutations to restore
RT activity of the W229 mutated enzyme will not easily occur.6 The discovery of
some highly conserved residues in the NNIBP opens up the possibility of finding
inhibitors of anti-HIV-1 mutant strains and furnishes useful information for the
rational design of new anti-HIV drugs.15,16 Accordingly, we focused our efforts
mainly on identifying those structural requirements responsible for high activity
toward an extensive range of HIV-1 variants in order to develop potential drugs with
improved resistance profiles for chronic use in anti-HIV combination therapy. The

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design approach discussed in this paper was mainly based on the assumption that
targeting the conserved residues in the hydrophobic channel for more extensive
interactions can lead to discover effective NNRTI with less sensitivity to resistance
mutations.17
Figure 2. The binding modes of Z10 and Z13 (Our previous work).
In our previous work, we have conducted a preliminary exploration of the
conserved amino acids in the hydrophobic channel of the NNIBP.18 In addition, two
compounds, Z10 and Z13 turned out to be the most potent HIV-1 inhibitors with the
same EC50 value of 3.0 nM against wild-type (WT) HIV-1 strain (Figure 2). Docking
studies indicated that the substituted alkyne group (red) in the left wing of the
inhibitors could extend to protein-solvent interface by the hydrophobic channel of
NNIBP. Inspired by this, we recognized that the hydrophobic channel represented an
area that had remained largely unexplored, thus, it makes sense to further explore the
structure-activity relationship on the left-wing. Unique properties of 1,2,3-triazole,
such as rigidity, stability and good solubility in vivo, hydrogen bonding capability,
and dipole moment, are considered as decisive factors for their improved biological
activity. For example, some 1,2,3-triazoles synthesized in our group showed a broad
spectrum of antiviral activities.19-22 Due to the importance and implementability of
1,2,3-triazole compounds, the triazole was used as a linker in the structure to provide
potential additional interactions with the surrounding residues6,23 (Figure 3). Besides,
the application of the Cu(I)-catalyzed azide and alkyne 1,3-dipolar cycloaddition
(CuAAC), commonly known as the "click reaction", a powerful and versatile
synthetic tool in medicinal chemistry, make it easy to form the 1,4-disubstituted
triazole ring.20 The newly designed compounds were then synthesized and evaluated

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for their anti-HIV activities against WT and multiple mutant strains. These synthetic
compounds were also tested for their ability to inhibit the RT of HIV-1. Besides, the
preliminary physicochemical properties and the early metabolic stability of
representative compounds were examined to evaluate their drug-like properties.
Herein, the result of evaluation, preliminary structure-activity relationships (SARs),
modeling analysis and the drug-like properties of the most potent inhibitors will be
discussed in this article.
Hydrophobic channel
Targeting conserved amino acids
W229
R'
R
F227
N
N
N
CN
NH
Structure-based
drug design
CN
NH
O
N
N
O
N
N
K101
PDB code: 2ZD1
R'= propan-1-ol; EC50=3nM
R'= 3-pyridine; EC50=3nM
R = substituted aromatic rings,
linear alkanes and heterocycles
The Crystal Structure of Rilpivirine
Figure 3. Illustration of the chemical modification strategy and structures of the newly designed
DAPY derivatives in this study.
2. Results and discussion
2.1. Chemistry
Scheme 1. The synthetic route of 1,2,3-triazole-derived diarylpyrimidines.
I
H
H
N
H
H
I
CN
Cl
N
N
O
S
O
N
N
N
OH
POCl3
N
N
N
O
N
N
c
a
b
d
CN
CN
H
4
2
3
1
Si
N
N
N
CN
CN
R
N
CN
N
N
e
O
N
N
f
O
N
N
H
O
N
N
H
H
5
6
ZL1 - ZL21
Reagents and conditions: (a) 4-aminobenzonitrile, 180℃, 8h; (b) POCl3, reflux, 0.5h;
(c) K2CO3, DMF, 100℃, 10h; (d) Trimethylsilylacetylene,
bis(triphenylphosphine)palladium dichloride, cuprous iodide, triethylamine, THF,
25℃, 10h. (e) NaOH, MeOH, rt; (f) CuSO4·5H2O, L-ascorbic acid sodium salt,

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THF/H2O (V:V = 1:1), rt, 4-6 h.
The general synthetic steps adopted to obtain the target compounds ZL1~21
were straightforwardly outlined in Scheme 1. These target compounds were
synthesized
from
the
commercially
available
starting
was reacted
material
with
2-(methylthio)pyrimidin-4(3H)-one
(1). Firstly,
1
4-aminobenzonitrile at 180℃ by melting method to afford intermediate
4-((4-oxo-1,4-dihydropyrimidin-2-yl)amino) benzonitrile (2) which followed by
chlorination with POCl3 to generate 4-((4-chloropyrimidin-2-yl)amino)-benzonitrile
(3).24 Secondly, 4-iodo-2,6-dimethylphenol pass nucleophilic substitution reactions
with intermediate
3
in the presence of K2CO3 and DMF afforded
4-((4-(4-iodo-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzonitrile (4).25 Thirdly,
intermediate 4 underwent sonogashira coupling reaction with trimethylsilylacetylene,
catalyzed by cuprous iodide, trimethylamine and bis(triphenylphosphine)palladium(II)
dichloride in anhydrous THF and oxygen-free conditions to provide
4-((4-(2,6-dimethyl-4-((trimethylsilyl)ethynyl)phenoxy)pyrimidin-2-yl)amino)benzon
itrile (5).18 De-trimethylsilane with NaOH at room temperature in methanol afforded
the 4-((4-(4-ethynyl-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzonitrile (6).26
Then the desired compounds were generated by CuAAC “click chemistry” reaction
between the substituted-azides and intermediate 6.
2.2. Biological activity
The distinct synthesized DAPYs derivatives were evaluated for their anti-HIV
activities in MT4 cell against WT HIV-1 strain (IIIB), a panel of NNRTIs resistant
strains with five single mutations (L100I, K103N, E138K, Y181C, Y188L) and two
double mutations (K103N/Y181C (RES056) and F227L/V106A) in RT respectively.
Besides, the inhibitory activity against HIV-2 strain (ROD) was also evaluated to test
the selectivity of these NNRTIs, and zidovudine (AZT), lamivudine (3TC), nevirapine
(NVP), efavirenz (EFV), etravirine (ETV) were selected as the reference drugs. The
biological results are expressed as EC50, CC50 and SI (selectivity index, i.e.,
CC50/EC50 ratio) and summarized in Table 1 and Table 2.

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Table 1. Activity and cytotoxicity of ZL1~21 against HIV-1 (IIIB) and HIV-2 (ROD) strains in
MT-4 cells.
R
N N
N
CN
O
N
NH
N
EC50 (μM)a
SIc
Compds
ZL1
R
CC50 (μM)b
33.17±7.94
>241.52
IIIB
ROD
IIIB
ROD
HO
O
0.065±0.023
>33.17
511
<1
O
O
ZL2
0.020±0.010
0.013±0.0038
0.072±0.024
0.064±0.015
137.41±2.36
>241.52
>4.78
>11830
>18473
66
>2
X1
<1
<1
O
O
ZL3
ZL4
ZL5
>241.52
4.78±1.84
O
O
O
O
>128.07
128.07±56.91
1998
CN
ZL6
ZL7
ZL8
0.076±0.013
0.014±0.0054
0.049±0.047
>7.17
>2.10
7.18±1.59
2.10±0.50
13.84±8.55
94
<1
<1
<1
OH
147
284
H2N
O
>13.84

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NH2
O
S
O
ZL9
0.029±0.015
>81.64
81.64±71.65
3004
<1
ZL10
ZL11
0.048±0.018
0.075±0.038
>7.22
7.22±1.21
150
<1
O2N
O2N
>101.03
≥101.03
≥1343
<orX1
NO2
ZL12
ZL13
ZL14
ZL15
ZL16
0.045±0.018
0.049±0.022
0.16±0.051
0.135±0.070
0.056±0.014
>159.69
>8.55
>5.69
>5.53
>6.36
159.69±43.27
8.55±1.43
5.69±0.48
5.53±0.23
6.36±1.69
3514
175
36
<1
<1
<1
<1
<1
S
41
O
S
O
113
HN
Br
O
ZL17
5.62±0.35
>19.78
19.78±20.19
4
<1
HN
F
O
ZL18
ZL19
ZL20
1.66±0.032
2.32±0.34
0.27±0.12
>1.41
>10.35
>23.76
1.41±1.27
10.35±2.78
23.76±8.71
1
4
<1
<1
<1
HN
O
O
O
O
O
87

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O
O
ZL21
0.18±0.066
>20.15
20.15±9.59
112
<1
AZT
3TC
NVP
EFV
ETV
-
0.012±0.002
7.20±4.13
0.118±0.067
0.003±0002
0.003
0.013±0.002
22.03±3.46
>15.02
>7.48
>87.24
>15.02
>6.34
>604
>12
-
-
-
-
>51
-
-
-
>6.34
>1205
>913
>4.59
>4.59
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures
against HIV-1-induced cytotoxicity, as determined by the MTT method.
bCC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as
determined by the MTT method.
cSI: selectivity index, the ratio of CC50/EC50.
As shown in Table 1, the biological results clearly showed that most of the
newly synthesized compounds exhibited anti-HIV-1 activity from nanomolar to
sub-micromolar ranges. Notably, compounds ZL2, ZL3 and ZL7 turned out to be the
most potent HIV-1 inhibitors with the EC50 value of 20 nM, 13 nM and 14 nM against
WT HIV-1 strain, which were superior to those reference drugs NVP (EC50 = 0.118
μM), 3TC (EC50 = 7.2 μM) and comparable to AZT (EC50 = 0.012 μM). Besides, all
other compounds exhibited considerable potency against the WT strain with EC50
values in the range of 0.029 μM-5.62 μM, which were superior to 3TC. In addition,
most of the synthesized compounds displayed decreased cytotoxicity compared with
EFV and ETV. Particularly, the two most active compounds exhibit minimal
cytotoxicity (ZL2 and ZL3, both CC50 > 241.52 μM) and high selectivity index values
(SI >11830, SI >18473, respectively).
For the initial exploration, the phenyl group was regarded as the rational
hydrophobic group for the purpose of extending to the left hydrophobic channel of the
NNIBP, and three compounds ZL1 - ZL3 with different substituted phenyl moieties
were designed. As expected, all of the three compounds displayed potent anti-HIV-1
activity at nanomolar scale. The preliminary SARs of these novel NNRTIs indicated

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that the substituents in the triazole ring have great influence on the anti-HIV activity.
For instance, three compounds (ZL1, ZL2, ZL3) with benzene ring directly
connected to the triazole showed better inhibitory activity, to be specific, 4-methyl
benzoate derivative (ZL3) is slightly better than 3-methyl benzoate derivative (ZL2),
both of them are better than 4-benzoic acid derivative (ZL1, EC50=65 nM).
To evaluate our hypothesis concerning that some flexible linker between the
phenyl and the triazole ring may disturb the rigid structure of phenyl-triazole, and will
benefit the “induce-fit” mechanism in the binding of small-molecular inhibitor and
NNIBP, compounds ZL4-ZL13 with the benzyl moiety were designed, synthesized
and evaluated for anti-HIV-1 potency. In addition, ZL4-ZL12 with substituted benzyl
groups showed similar potency to the unsubstituted benzyl derivative ZL13 (EC50=49
nM) against WT HIV-1, with the exception of 4-hydroxyl derivative ZL7 (EC50=14
nM) and 4-methanesulfonamide derivative ZL9 (EC50=29 nM), indicating that the
hydrogen-bonding donors/acceptor groups have their advantages in potency.
The position of the substituents in the phenyl ring has some influence on the
anti-HIV activity. For instance, introduction of a methyl formate group at the C3
position yielded a 72 nM inhibitor ZL4, while the methyl formate group at C4
position increased the potency to 64 nM. It also indicated that the C2 or C4 positions
was more favorable for nitryl group than C3, as ZL10 (2-nitryl, EC50 = 48 nM) and
ZL12 (4-nitryl, EC50 = 45 nM) were more potent than ZL11 (3-nitryl, EC50 = 75 nM).
Moreover, the potency is strongly dependent on the nature of the substituents in
the phenyl ring. The activity of compounds with the para-substitution at the phenyl
ring suggested that some polar groups with suitable polarities and electronic nature at
the C4 position might enhance anti-HIV activity by inserting into the hydrophobic
channel of NNIBP and making more interactions with the protein-solvent interface.
For instance, the hydroxyl-substituted compound ZL7 (EC50 = 14 nM) was more
potent than the methylformate-substituted compound ZL6 (EC50 = 76 nM). However,
some substituent groups at C3 position might show slight influence on the potency.
For instance, the 3-methyl formate derivative ZL4 (EC50 = 72 nM) displayed similar
potency to the 3-nitryl derivative ZL11 (EC50=75 nM).

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The activity was also influenced by the length and structure of the connecting
chain between the aryl moiety and the triazole ring, for example, introduction of a
benzyl at the N4- position yielded a 49 nM inhibitor ZL13, while ethylbenzene at this
position reduced the potency to 0.16 μM (ZL14). Subsequently, the flexible-chain
was replaced by a rigid skeleton chain to explore the plastically of the hydrophobic
channel, resulting in three derivatives with the acetylamino linker between the
benzene ring and the triazole ring. The substituent groups at C4 position have great
influence on the activity and the order of activity of the para-substitution at the
phenyl ring was as follows: -SO2CH3 (ZL16, EC50 = 56 nM) > -F (ZL18, EC50 = 1.66
μM) >-Br (ZL17, EC50 = 5.62 μM), indicating that this position is preferred over
groups with polar groups such as methyl sulfonate and fluorine et al.
In addition to the phenyl and substituted phenyl derivatives, other hydrophobic
groups were also considered, the thienyl derivative ZL15 (EC50= 0.135 μM) showed
slight enhanced activity compared to its phenyl analogue ZL14 (EC50= 0.16 μM).
Moreover, the potency was reduced when the phenyl substituent connecting to the
triazole moiety was replaced by the methyl formate moiety. For instance, compound
ZL19 (EC50 = 2.32 μM) and ZL20 (EC50 = 0.27 μM) showed about 47- and 1.7-fold
less potent than the corresponding phenyl derivatives. Moreover, the activity was
greatly influenced by the length of the connecting chain between the methyl formate
moiety and the triazole ring, the potency was increased when the linker lengths are
longer in a suitable range (1-3 atoms were explored in this paper). In particular, the
ethyl-linker derivative ZL20 and the propyl-linker derivative ZL21 (EC50= 0.18 μM)
were 8.6-fold and 12.9-fold more potent than the methene-linker derivative ZL19.
Furthermore, it is noteworthy that most compounds except ZL7 and ZL18
showed extremely low cytotoxicity compared to reference drug ETV. The
cytotoxicity is also strongly dependent on the nature of the substituents attached to the
triazole. For instance, ZL2 and ZL3 with –COOCH3 substitution at C3 and C4
positions in the phenyl ring showed no cytotoxicity (CC50) up to the maximum tested
concentration of 241 μM, while addition of a -COOH group to phenyl ring sharply
increased the cytotoxicity to 33.17 μM ( >7.3-fold). The cytotoxicity of substituted

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benzyl derivatives (ZL4-ZL13) show significant change ranging from 2.10
μM-159.69 μM. Besides, only compound ZL2 (EC50 = 137.41 μM) showed faint
cellular activity against HIV-2 (ROD) in MT-4 cells
Table2. Activity of ZL1~21 against HIV-1 mutant strains.
EC50 (μM)a
Compds
L100I
0.77±0.018
0.47±0.094
1.04±0.69
>4.78
K103N
0.23±0.10
Y181C
0.84±0.13
0.46±0.028
0.34±0.055
>4.78
Y188L
6.22±0.19
>241.52
>241.52
>4.78
E138K
0.43±0.045
0.10±0.01
0.16±0.017
0.96±0.12
0.61±0.0044
0.56±0.14
0.091±0.010
0.12±0.021
0.12±0.0028
0.50±0.11
0.51±0.010
0.33±0.0095
0.24±0.098
≥2.05
F227L+V106A
2.73±0.76
≥180.4.3
>241.52
≥2.267
RES056
3.27±0.069
45.24±37.08
>241.52
>4.78
ZL1
ZL2
0.043±0.019
0.022±0.003
0.36±0.036
0.35±0.0054
0.42±0.094
0.054±0.009
0.068±0.028
0.10±0.0005
0.31±0.022
0.38±0.050
0.17±0.0054
0.22±0.057
0.42±0.012
0.37±0.018
1.04±0.12
ZL3
ZL4
ZL5
1.86±0.11
>7.18
2.11±0.48
>7.18
≥9.09
≥9.22
≥37.66
ZL6
>7.18
>7.18
>7.18
ZL7
≥0.35
0.53±0.40
0.56±0.032
0.53±0.020
>7.22
>2.10
>2.10
>2.10
ZL8
0.63±0.11
0.61±0.090
>7.22
1.33±0.13
≥3.56
>13.84
>13.84
ZL9
>86.44
>86.44
ZL10
ZL11
ZL12
ZL13
ZL14
ZL15
ZL16
ZL17
ZL18
ZL19
ZL20
ZL21
AZT
3TC
>7.22
>7.22
>7.22
9.45±7.02
1.52±0.19
1.11±0.10
>5.66
4.44±0.22
1.42±0.97
1.42±0.39
>5.66
4.54±0.43
≥36.49
>101.03
33.83±0.98
>8.55
>101.03
>159.69
>8.55
>8.55
>5.66
>5.66
>5.66
>5.53
>5.53
>5.53
0.61±0.059
1.23±0.099
>19.78
>5.53
>5.53
>6.36
>6.36
>6.36
>6.36
>6.36
>19.78
>19.78
>19.78
>19.78
>19.78
>19.78
>1.41
>1.41
>1.41
>1.41
>1.41
>1.41
>1.41
>10.35
6.64±1.43
>10.35
>10.35
>10.35
>10.35
>10.35
5.06±0.43
7.10±0.49
0.006±0.001
2.46±0.30
2.22±1.31
0.046±0.009
0.006±0.002
0.75±0.0096
0.79±0.26
3.37±0.23
≥7.16
>23.76
1.03±0.048
1.68±0.24
0.016±0.005
5.54±1.86
0.18±0.039
0.005±0.001
0.008±0.003
>23.76
>23.76
>20.15
>20.15
>20.15
0.019±0.013
3.76±1.23
0.008±0.001
4.40±1.60
>15.02
0.007±0.001
4.06±0.75
>15.02
0.007±0.002
2.74±0.78
>15.02
0.02±0.007
5.56±0.70
>15.02
NVP
EFV
ETV
12.35±0.88
0.11±0.035
0.003±0.0004
0.006±0.001
0.012±0.003
0.307±0.054
0.017±0.006
0.269±0.049
0.014±0.005
0.265±0.048
0.043±0.015
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against
HIV-1-induced cytotoxicity, as determined by the MTT method.
Table 3. Resistance folds for the K103N mutant strain of selected compounds in MT-4 cells.

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Resistance Foldsa
Compds
K103N
ZL2
ZL3
ZL7
ZL8
EFV
2.2
1.7
3.9
1.4
36.7
aResistance Fold: RF, ratio of EC50 against mutant strain/EC50 against WT strain.
The activity of the newly synthesized compounds was further evaluated against a
panel of clinical relevant HIV-1 mutant strains, including L100I, K103N, Y181C,
Y188L, E138K, F227L+V106A, and RES056, in MT-4 cells. In the case of the
mutant HIV-1 strains (Table 2), most compounds displayed potent activity against
single mutant HIV-1 strains, especially to K103N and E138K. As for K103N, which
is the most common mutation emerging in HIV patients treated with EFV, five
compounds provided double-digit nanomolar activity. In particular, ZL3 (EC50
=
0.022 μM) was notably more active than 3TC, NVP and EFV and compared to AZT.
Regarding the resistance fold (Table 3), compounds ZL2, ZL3, ZL7, ZL8 showed no
significant decrease in activity (RF = 2-6) for K103N while EFV had the RF of 36.7.
Furthermore, compounds ZL1, ZL2, ZL8 and ZL9 showed higher activity towards
mutant HIV-1 strain L100I (EC50 = 0.77, 0.47, 0.63, 0.61 and 0.57 μM, respectively),
being far more potent than 3TC (EC50 = 2.46 μM) or NVP (EC50 = 2.22 μM). Other
seven compounds (ZL3, ZL5, ZL11-13, ZL20-21) showed moderate activity.
However, the remaining nine compounds lacked activity towards this mutant strain.
Intriguingly, except for ZL17 and ZL18, all the target compounds displayed good
activity against K103N with EC50 values in the range of 0.022-10.85 μM.
Furthermore, ZL3 and ZL8 respectively showed the most potent activity against the
single mutants Y181C and Y188L with EC50 = 0.34 μM and 0.675 μM. Interestingly,
all the target compounds displayed good activity against E138K with EC50 values in
the range of 0.091-1.68 μM, and the inhibitory activity of most compounds is at the
submicromolar level. Moreover, ZL1 and ZL12 were effective inhibitors of the
double-mutant strain F227L+V106A with EC50 values of 2.73 and 33.83 μM,
respectively. ZL1 also show the best inhibitory activity towards RES056 with EC50 of

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3.27 μM. However, the other compounds were inactive towards these double mutant
strains except ZL2 (EC50 = 45.24 μM).
2.3. Inhibition of HIV-1 RT
To validate the binding target of these novel NNRTIs, all compounds were
further tested for their ability to inhibit recombinant WT HIV-1 RT enzymes; the
results are shown in Table 3. In the enzymatic assay, all the tested compounds
exhibited potent inhibitory activities toward WT HIV-1 RT with IC50 values ranging
from 0.02 μM to 0.76 μM.
Table 4. Inhibitory activity of compounds against HIV-1 WT RT
Compds
ZL1
IC50 (μM)a
0.02
ZL2
0.05
ZL3
0.06
ZL4
0.22±0.01
0.24±0.04
0.40±0.12
0.04±0.01
0.06±0.02
0.11±0.06
0.15±0.01
0.24±0.04
0.15±0.02
0.18
ZL5
ZL6
ZL7
ZL8
ZL9
ZL10
ZL11
ZL12
ZL13
ZL14
ZL15
ZL16
ZL17
ZL18
ZL19
ZL20
ZL21
NVP
EFV
ETV
0.62±0.07
0.44±0.11
0.29±0.05
0.76±0.10
0.36±0.03
0.39±0.05
0.14±0.01
0.21±0.04
0.49±0.26
0.007±0.002
1.35±0.35
aIC50: Inhibitory concentration of test compound required to inhibit biotin deoxyuridine
triphosphate (biotin-dUTP) incorporation into HIV-1 WT RT by 50%.

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The result showed that all compounds exhibited potent activity toward WT RT
with IC50 values ranging from 0.02 to 0.76 μM, and most of the compounds were
superior to NVP (IC50 = 0.49 μM) and ETV (IC50 = 1.35 μM) (Table 4). The three
most potent compounds ZL1, ZL2 and ZL7 exhibited higher enzymatic inhibition
activity (IC50 = 0.02, 0.05 and 0.04 μM, respectively). Among them, 4-benzoic acid
derivative ZL1 displayed the most potent activity toward WT RT, indicating
carboxylic acid moiety has better enzyme inhibitory activity and this phenomenon has
been identified in our previous research. However, the markedly decreased potency
against WT HIV-1 of ZL1 in cell culture may be due to its negative charged form
under experimental conditions influencing its cell membrane permeability. On the
whole, all these novel derivatives are RT and they all acted as classical NNRTIs.
It is noteworthy that the antiviral activity of some compounds in this study was
inconsistent with its enzyme-inhibitory potency to some extent. These differences
may be explained by template-specific variation in the relationship between
HIV-RT-RNA binding affinity and polymerase processivity, which has been observed
in most NNRTI series.27 Besides, there is a high probability of formation of cocrystals
or salts due to the multiple hydrogen bond donor and acceptor sites in these
compounds.
2.4. Molecular modeling analysis
In order to obtain further insight into the allosteric binding of
1,2,3-triazole-derived diarylpyrimidines to the NNIBP of RT, by means of software
SYBYL-X 2.0, the most potent three compounds ZL2, ZL3 and ZL7 in the
enzymatic assay was conducted further docking studies utilizing the structures of
cocrystal of WT RT (PDB code: 2ZD1 and 3MEG). Our docking results were
visualized by PyMOL. And the docking protocol was illustrated in the computational
section.
The docking simulations of these molecules with HIV-1 WT RT revealed that
the binding mode resembles those classical NNRTI drugs (Figures 4): the left wing of
these compounds occupies the hydrophobic channel formed by aromatic amino acid
residues Y181, Y188, F227, and W229, exhibiting π-π interaction with these residues

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and the substituent group in the triazole ring further stretch into protein-solvent
interface and can develop extensive interactions with surrounding residues
or water molecule, thereby improving the stability of the RT-inhibitor complex; Of
particular note is that both ZL3 and ZL7 form “water bridge” hydrogen bonds with
L228, which explain the excellent anti-HIV-1 activity. Besides, the pyrimidine
heterocycle and the NH linker connecting the central pyridine ring maintained the
“signature” double hydrogen bonds with the backbone of K101; The right
4-cyanoaniline was located in another pocket and interacted with residues V106, L234,
P236 and Y318.
(a)
(b)
(c)
Figure 4. Predicted binding modes of ZL2, ZL3 and ZL7 with the crystal structures of HIV-1
WT RT (PDB: 2ZD1). The hydrogen bonds between the inhibitors and amino acid residues are
indicated with dashed lines (yellow). Ligand carbon atoms are shown in cyan, and protein carbon
atoms in green and magenta. The water molecules are shown in red ball. Nonpolar hydrogen
atoms are hid for clarity.
Although these compounds are still able to maintain a good U-shaped
conformation, the activity is reduced compared to the lead compounds. From the
superposition of compound ZL7 and RPV in molecular docking (Figure 5), the left
benzene ring of ZL7 was shift away from the benzene ring of RPV despite of the
well-superimposition in right wing, causing the disappearance of π-π interaction
between the left benzene ring and Y181. Further, ZL7 develops water-mediated
hydrogen bonds with L228 instead of the hydrogen bond connecting hydroxyl- and
cyano group on RPV, compensating for the decline in compounds inhibitory activity
to some extent.

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Figure 5. Superimposition of ZL7 (cyan) and RPV (hot pink) with HIV-1 WT RT (PDB: 2ZD1).
The hydrogen bonds between the inhibitors and amino acid residues are indicated with dashed
lines (yellow). Ligand carbon atoms are shown in cyan, and protein carbon atoms in green. The
water molecules are shown in red ball. Nonpolar hydrogen atoms are hid for clarity.
When docking into the K103N mutant RT, ZL2 and ZL3 cannot enter the
binding pocket of RT by the original mode (Figure 6a, 6b), causing the decreased
activity. However, the distorted structure stretches into the entrance channel and
formed new hydrogen bond with N103, compensating the affinity loss with
sub-pocket in NNIBP to some extent, and this may be the reason why ZL2 and ZL3
possessed the most potent activity against K103N mutant HIV-1 strain. Regarding to
the binding mode of ZL7 in the NNIBP, it still maintained a hydrogen bond with
K101 and furthermore formed new hydrogen bond with Y186. As we all know, Y186
plays an important role in the catalytic active site of RT, this directed us to modify
targeting the active site next step.
(d)
(f)
(e)
Figure 6. Predicted binding modes of ZL2, ZL3 and ZL7 with the crystal structures of HIV-1
K103N RT (PDB: 3MEG). The hydrogen bonds between the inhibitors and amino acid residues
are indicated with dashed lines (yellow). Ligand carbon atoms are shown in cyan, and protein
carbon atoms in green. Nonpolar hydrogen atoms are hidden for clarity.

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2.5. Water solubility and other drug-like properties
Aqueous solubility of the compounds was the important physicochemical
property which could make a huge impact on the biological evaluation. So the water
solubility of the representative compounds ZL2, ZL3 and ZL7 were tested. As
revealed in Table 5, all three compounds possessed significantly improved water
solubility than that of ETV at different pH values, which may due to the good water
solubility of 1,2,3-triazole moiety. Furthermore, the preliminary physicochemical
properties of representative compounds ZL2, ZL3 and ZL7 were examined to
evaluate their drug-like properties by utilizing free online molinspiration software
(http://www.molinspiration.com/). Lipophilic parameter ligand efficiency (LE) was
also calculated. The results (Table 5) suggested that parameters like molecular weight
(MW), hydrogen bond acceptors (nON), molinspiration predicted LogP (miLogp),
and ligand efficiency (LE) for ZL2 and ZL3 were inconsistent with the Lipinski’s
rule of five. In addition, ZL7 showed the slightest deviation for miLogp and LE for
all tested compounds. Therefore, it is supposed that ZL7 may have the acceptable
physicochemical properties. Besides, the topological polar surface area (tPSA), which
is characterized by the absorption and membrane permeability of molecules, showed
the three compounds had a value ranging from 121.78 to 127.86 Å2 , confirming their
advantage for intestinal absorption (<140 Å2) and inability to penetrate the
blood-brain barrier, averting the central nervous system toxicity (>60 Å2).
Table 5. Water solubility and physicochemical properties of representative compounds and ETV.
Parameter itemsa
ZL2
ZL3
ZL7
ETV
Water solubility (pH=2, μg/ml)
Water solubility (pH=7, μg/ml)
Water solubility (pH=7.4, μg/ml)
18.2±0.09
36.5±0.31
30.8±0.15
25.0±0.08
36.3±0.17
43.3±0.11
23.0±0.30
38.7±0.13
30.8±0.10
<<1
natoms
39
517.55
10
39
517.55
10
36
475.51
9
28
435.29
7
MW (< 500 Da)
nON (< 10)
nOHNH (< 5)
Nrotb (<10)
1
8
1
8
2
6
3
4
tPSA (<140 A2)
MV
127.86
127.86
121.78
120.65
454.32
454.32
417.81
335.95

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miLog p (< 5)
5.93
5.74
5.09
5.03
nViol
2
2
1
1
LE (>0.3)
0.27
0.28
0.30
0.42
a nViol = number of violations; natoms = no. of atoms; miLogP = molinspiration predicted LogP;
MW= molecular weight; nON = no. of hydrogen bond acceptors; nOHNH = no. of hydrogen bond
donors; nrotb = no. of rotatable bonds; TPSA = topological polar surface area; MV = molar
volume; LE = calculated by the formula −ΔG/HA (non‑hydrogen atom), in which normalizing
binding energy Δ G = − 2.303RT log Kd, presuming Kd ≈ EC50(IIIB); R= 1.987 × 10–3
kcal/K/mol, T = 298 K.
2.6 In Vitro Effects of ZL3 on CYP Enzymatic Inhibitory Activity
As is well known, metabolizing enzymes in the liver (e.g. cytochrome P450,
CYP) are responsible for the majority of drug metabolism in vivo. CYP enzymes are
considered to be one of the most important enzyme families for drug-drug interaction
(DDI) assessments due to the involvement of these enzymes in the metabolic
clearance of the majority of prescribed drugs. The evaluation of cytochrome CYP
enzyme inhibition/inactivation development remains an important factor in overall
risk assessment and compound progression to the clinic. In addition, three main
subtypes CYP3A, CYP2D6 and CYP2C are involved in metabolism of about 90% of
the marketed drugs. Therefore, metabolism-mediated DDIs may occur when
co-administrating different drugs, leading to decreased therapeutic effect or adverse
reaction profile. According to reported study, etravirine is an inducer of CYP3A4 and
inhibitor of CYP2C9 and CYP2C19, and it can result in many drug-drug interactions
when co-administrated with other anti-HIV-1 drugs like protease inhibitors, integrase
strand inhibitors and CCR5 antagonists etc, which influence the use of concomitant
antiretrovirals. We performed the study of inhibitory activity of compound ZL3 on
main CYP drug metabolizing enzymes, with selective inhibitors of these enzymes as
positive controls. The results in Table 6 indicated that ZL3 displayed no CYP
enzymatic inhibition of CYP1A2, CYP2C19, CYP2D6 and CYP3A4M, however, it
exhibits strong inhibitory activity against CY2C9.
Table 6. Inhibitory activity on CYP1A2, CY2C9, CYP2C19, CYP2D6 and CYP3A4M of
compound ZL3 and positive controls

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Inhibitory
InhiDbOitIo: 1r0y.1039/C9OB00032A
CYP Isozyme
Standard Inhibitor
activity
Compd
activity
(IC50, μM)
(IC50, μM)
1A2
2C9
α-Naphthoflavone
Sulfaphenazole
(+)-N-3-benzylnirvanol
Quinidine
0.289
0.585
0.179
0.136
0.0423
ZL3
ZL3
ZL3
ZL3
ZL3
>50
0.113
>50
2C19
2D6
3A4
>50
Ketoconazole
>50
3. Conclusion
In general, a series of novel triazole derivatives targeting the hydrophobic
channel of NNIBP were rationally designed based on structure-guided approach,
synthesized and evaluated for their inhibitory activity against HIV-1 (including IIIB,
L100I, K103N, E138K, Y181C, Y188L, F227L+V106A, K103N+Y181C and HIV-2
(ROD) strains) in MT-4 cells. All compounds showed excellent to good activity
against wild-type HIV-1 strain with EC50 ranging from 0.013 μM to 5.97 μM. It is
noteworthy that, regarding the inhibitory activity against K103N, the major drug
resistant mutant to the new generation HIV-1 NNRTIs, three promising compounds
ZL2, ZL3, and ZL7 are superior to the first-line antiretroviral drugs efavirenz. More
encouragingly, ZL2 and ZL3 exhibited much lower cytotoxicity and high selection
index of >10,000 compared with all the reference drugs (AZT, 3TC, NVP, EFV,
ETV). In addition, the inhibition of HIV RT indicated that most of these compounds
could effectively inhibit the activity of HIV-1 RT at low micromolar concentrations,
confirming their target is HIV-1 RT. Furthermore, detailed SAR and docking study of
the representative compounds help us to understand the binding modes of the
compounds in the NNIBP and guide future optimization, which is ongoing in our lab
aimed at improving drug resistance profiles and reducing the cytotoxicity and will be
reported in due course. Subsequently, the preliminary physicochemical properties and
the early metabolic stability of representative compounds were examined to evaluate
their drug-like properties. Three tested compounds ZL2, ZL3 and ZL7 showed
significantly improved water solubility than that of ETV. In the CYP enzymatic
inhibitory assay, ZL3 displayed no CYP enzymatic inhibition of CYP1A2, CYP2C19,

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CYP2D6 and CYP3A4M but strong inhibitory activity against CY2C9, which should
be considered in further drug development.
To sum up, the biological activity and docking studies indicated that the
hydrophobicꢀchannel of NNIBP can accommodate various substituents and are worth
further exploring. In addition, it is essential to choose the appropriate linker moiety
and try to make the substituent groups structurally diverse to explore the left
hydrophobic channel.
4. Experimental section
4.1. Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer
using solvents as indicated (DMSO-d6). Chemical shifts were reported in δ values
(ppm) with tetramethylsilane as the internal reference, and J values were reported in
hertz (Hz). Mass spectra were performed on a LC Autos ampler Device: Standard
G1313A instrument. Melting points (m.p.) were determined on a micromelting point
apparatus (Tian Jin Analytical Instrument Factory, Nankai, Tianjin, China). TLC was
performed on Silica Gel GF254 for TLC (Merck) and spots were visualized by
irradiation with UV light (λ=254 nm). Flash column chromatography was performed
on column packed with Silica Gel60 (200-300 mesh). Thin layer chromatography was
performed on pre-coated HUANGHAI_HSGF254, 0.15-0.2 mm TLC-plates. Solvents
were of reagent grade and were purified and dried by standard methods when
necessary. Concentration of the reaction solutions involved the use of rotary
evaporator at reduced pressure.
4.1.1. 4-((6-oxo-1,6-dihydropyrimidin-2-yl)amino)benzonitrile (2).
2-(Methylthio)pyrimidin-4(3H)-one (1) (3 g, 0.021 mol) was reacted with
4-aminobenzonitrile (2.99 g, 0.025 mol) at 180℃ by melting method for 8h. After
cooling, CH3CN (20 mL) was added to dissolve the mixture by ultrasound treatment.
Thereafter, the reaction mixture was filtered and washed with CH3CN until no
residual 4-aminobenzonitrile was detected by TLC. It was then dried in vacuo to give
the product 4-((4-oxo-1,4-dihydropyrimidin-2-yl)amino)benzonitrile (2) as a light
yellow solid, yield 73.6%, ESI-MS: m/z 213.3 [M+H] +, C11H8N4O (212.12).

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4.1.2. 4-((4-chloropyrimidin-2-yl)amino)benzonitrile (3).
A mixture of 4-((4-oxo-1,4-dihydropyrimidin-2-yl)amino)benzonitrile (2) (0.80 g,
0.0038 mol) in POCl3 (5 mL) was refluxed for 0.5 h. Then, the mixture was slowly
poured into icy water (50 mL) vigorously stirring. Subsequently, the mixture was
filtered to give a filtrate cake, which was then again added into water (10 mL) and
was neutralized to pH = 7 with sodium hydroxide. The product was obtained by
filtration
and
dried
under
vacuo
to
provide
4-((4-chloropyrimidin-2-yl)amino)benzonitrile (3) as pale yellow solids with the yield
of 71.3%; 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H,
C6-pyrimidine-H), 7.87 (dd, 4H, Ph-H), 7.13 (d, J = 5.2 Hz, 1H, C6-pyrimidine-H);
ESI-MS: m/z 231.2 [M+H]+, C11H7ClN4 (230.04).
4.1.3. 4-((4-(4-iodo-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzonitrile (4).
In
a
schlenk-type
flask,
intermediates
3
(1.0
equiv),
4-((4-(4-iodo-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzonitrile (1.0 equiv)
and K2CO3 (1.2 equiv) were dissolved in DMF stirring for 10h at 100℃. The solution
was cooled to room temperature, and water was added to it. The resulting precipitate
was purified on silica gel gave intermediates 4.
4.1.4. 4-((4-(2,6-dimethyl-4-((trimethylsilyl)ethynyl)phenoxy)pyrimidin-2-yl)amino)
benzonitrile (5)
Trimethylsilylacetylene (3.0 equiv) was added to a solution of intermediates 4
(1.0 equiv), cuprous iodide (0.10 equiv), bis(triphenylphosphine) palladium(II)
dichloride (0.05 equiv) and trimethylamine (3 equiv) in anhydrous THF. The mixture
was stirred at room temperature under nitrogen atmosphere and monitored by TLC
until its completion. After cooling, the solvent was filtered using diatomaceous earth
and then evaporated. The residue was purified by flash column chromatography using
ethyl acetate/petroleum ether as eluent to obtain intermediate 5.
4.1.4. 4-((4-(4-ethynyl-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)benzonitrile (6)
In a schlenk-type flask, 5 (1.0 equiv) and NaOH (1.2 equiv) were dissolved in
methanol stirring for 10h at room temperature. Purification on silica gel gave the
important intermediate 6.

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4.1.5. General procedure for synthesis of target compounds (ZL1-ZL21).
The key intermediate 6 (1.0 eq), azide substituents (1.1 eq), ascorbic acid sodium
(0.6 eq) and CuSO4·5H2O (0.3 eq) were dissolved in the solution of
tetrahydrofuran/water (V:V= 1:1). The resulting mixture was stirred at rt for 4-6 h.
Then the reaction mixture was extracted with ethyl acetate (3 × 10 mL), and the
combined organic phase was washed with saturated salt water (3 × 10 mL), dried over
anhydrous NaSO4, filtered, and concentrated under reduced pressure to give the
corresponding crude target product, which was purified by flash column
chromatography to afford product ZL1-21.
4-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-1,2,3
-triazol-1-yl)benzoic acid (ZL1).
Recrystallized from EA/PE as a white solid, 57.3% yield, m.p. 303 − 306 °C; 1H
NMR (400 MHz, DMSO-d6) δ 13.18 (s, 1H, COOH), 10.08 (s, 1H, NH), 9.41 (s, 1H,
triazole-H), 8.42 (d, J = 5.7 Hz, 1H, C6-pyrimidine-H), 8.25 – 7.99 (m, 4H, Ph-H),
7.75 (s, 2H, Ph-H), 7.59 (d, J = 8.4 Hz, 2H, Ph-H), 7.39 (d, J = 8.4 Hz, 2H, Ph-H),
6.60 (d, J = 5.7 Hz, 1H, C5-pyrimidine-H), 2.10 (s, 6H, 2CH3). 13C NMR (100 MHz,
DMSO-d6) δ 168.96, 166.91, 160.79, 159.69, 149.95, 147.67, 145.07, 139.97, 133.06,
131.66, 131.64, 128.08, 126.40, 120.07, 119.83, 118.73, 102.92, 99.43, 16.69;
ESI-MS: m/z 504.4 [M+H]+. C28H21N7O3 (503.17).
Methyl 3-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)
-1H-1,2,3-triazol-1-yl)benzoate (ZL2).
1
Recrystallized from EA/PE as a white solid, 62.4% yield, m.p.: 232-234℃; H
NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H, NH), 9.53 (s, 1H, triazole-H), 8.56 – 8.47
(m, 2H, Ph-H, C6-pyrimidine-H), 8.31 (dd, J = 7.9, 2.2 Hz, 1H, Ph-H), 8.09 (d, J = 7.8
Hz, 1H, Ph-H), 7.83 (d, J = 6.8 Hz, 3H, Ph-H), 7.67 (d, J = 8.4 Hz, 2H, Ph-H), 7.47 (d,
J = 8.5 Hz, 2H, Ph-H), 6.67 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 3.95 (s, 3H, CH3),
2.18 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 168.97, 165.77, 160.77,
159.69, 149.89, 147.62, 145.07, 137.35, 133.06, 131.81, 131.61, 131.13, 129.52,
128.15, 126.36, 124.71, 120.41, 119.82, 118.71, 102.90, 99.42, 53.07, 16.69. ESI-MS:
m/z 518.4 [M+H]+. C29H23N7O3 (517.19).

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Methyl 4-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)
-1H-1,2,3-triazol-1-yl)benzoate(ZL3).
Recrystallized from EA/PE as a white solid, 62.3% yield, m.p. 252 − 254 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, NH), 9.52 (s, 1H, triazole-H), 8.53 (t, J =
1.9 Hz, 1H, Ph-H), 8.49 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 8.30 (d, J = 7.9, 2.3 Hz,
1H, Ph-H), 8.09 (d, 1H, Ph-H), 7.87 – 7.78 (m, 3H, Ph-H), 7.68 (d, J = 8.5 Hz, 2H,
Ph-H), 7.47 (d, J = 8.5 Hz, 2H, Ph-H), 6.67 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H),
3.95 (s, 3H,CH3), 2.18 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 168.98,
165.77, 160.77, 159.69, 149.89, 147.62, 145.07, 137.35, 133.05, 131.82, 131.60,
131.12, 129.52, 128.16, 126.37, 124.71, 120.43, 119.81, 118.73, 102.92, 99.42, 53.06,
16.68. ESI-MS: m/z 518.4 [M+H]+. C29H23N7O3 (517.19).
Methyl 3-((4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)
-1H-1,2,3-triazol-1-yl)methyl)benzoate (ZL4).
Recrystallized from EA/PE as a white solid, 71.8% yield, m.p. 197 − 198 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H, NH), 8.61 (s, 1H, triazole-H), 8.39 (d, J
= 5.6 Hz, 1H, C6-pyrimidine-H), 7.94 – 7.84 (m, 2H, Ph-H), 7.64 (s, 2H, Ph-H), 7.61
– 7.46 (m, 4H, Ph-H), 7.36 (d, J = 8.4 Hz, 2H, Ph-H), 6.56 (d, J = 5.6 Hz, 1H,
C5-pyrimidine-H), 5.72 (s, 2H, CH2), 3.79 (s, 3H, CH3), 2.05 (s, 6H, 2CH3). 13C NMR
(100 MHz, DMSO-d6) δ 168.99, 166.32, 160.75, 159.67, 149.59, 146.82, 145.07,
137.21, 133.22, 133.03, 131.45, 130.62, 129.90, 129.42, 129.07, 128.64, 126.18,
122.07, 119.80, 118.69, 102.88, 99.37, 53.02, 52.73, 16.60. ESI-MS: m/z 532.4
[M+H]+. C30H25N7O3 (531.20).
Methyl 4-((4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)
-1H-1,2,3-triazol-1-yl)methyl)benzoate (ZL5).
Recrystallized from EA/PE as a light yellow solid, 69.7% yield, m.p. 221 − 223
1
°C; H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, NH), 8.69 (s, 1H, triazole-H),
8.47 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 8.01 (d, J = 8.2 Hz, 2H, Ph-H), 7.72 (s, 2H,
Ph-H), 7.62 (d, J = 8.5 Hz, 2H, Ph-H), 7.47 (d, J = 8.1 Hz, 2H, Ph-H), 7.42 (d, J = 8.5
Hz, 2H, Ph-H), 6.64 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.81 (s, 2H, CH2), 3.86 (s,
3H, CH3), 2.12 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 168.97, 166.32,

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160.80, 159.64, 149.63, 146.82, 145.07, 141.71, 133.02, 131.47, 130.18, 129.82,
128.63, 128.46, 126.18, 122.25, 119.82, 118.68, 102.84, 99.35, 53.06, 52.69, 16.60.
ESI-MS: m/z 532.3 [M+H]+. C30H25N7O3 (531.20).
4-((4-(4-(1-(4-cyanobenzyl)-1H-1,2,3-triazol-4-yl)-2,6-dimethylphenoxy)pyrimidin-2-
yl)amino)benzonitrile (ZL6).
1
Recrystallized from EA/PE as a white solid, 65.3% yield, m.p.: 225-227℃; H
NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H, NH), 8.68 (s, 1H, triazole-H), 8.47 (d, J
= 5.6 Hz, 1H, C6-pyrimidine-H), 7.95 (dd, J = 7.7, 1.3 Hz, 1H, Ph-H), 7.85 – 7.68 (m,
3H, Ph-H), 7.69 – 7.54 (m, 3H, Ph-H), 7.44 (dd, J = 12.1, 8.2 Hz, 3H, Ph-H), 6.64 (d,
J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.91 (s, 2H, CH2), 2.13 (s, 6H, 2CH3). 13C NMR
(100 MHz, DMSO-d6) δ 168.98, 160.77, 159.66, 149.64, 146.68, 145.07, 139.19,
134.36, 133.93, 133.03, 131.48, 129.88, 129.72, 128.54, 126.19, 122.49, 119.81,
118.68, 117.40, 111.69, 102.88, 99.38, 51.77, 40.21, 16.60; ESI-MS: m/z 499.4
[M+H]+. C29H22N8O (498.19).
4-((4-(4-(1-(4-hydroxybenzyl)-1H-1,2,3-triazol-4-yl)-2,6-dimethylphenoxy)pyrimidin-
2-yl)amino)benzonitrile (ZL7).
Recrystallized from EA/PE as a white solid, 66.4% yield, m.p. 255 − 258 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H, NH), 9.26 (s, 1H, triazole-H), 8.41 (d, J
= 5.6 Hz, 1H, C6-pyrimidine-H), 7.87 (d, J = 8.2 Hz, 2H, Ph-H), 7.74 (s, 2H, Ph-H),
7.59 (d, J = 8.5 Hz, 2H, Ph-H), 7.51 (d, J = 8.2 Hz, 2H, Ph-H), 7.38 (d, J = 8.5 Hz, 2H,
Ph-H), 6.59 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.32 (t, J = 5.7 Hz, 1H, OH), 4.54
(d, J = 5.7 Hz, 2H, CH2), 2.10 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ
168.98, 160.79, 159.68, 149.82, 147.34, 145.07, 143.77, 135.73, 133.06, 131.58,
128.37, 128.14, 126.34, 120.16, 119.96, 119.83, 118.72, 102.90, 99.42, 62.70, 16.69.
ESI-MS: m/z 490.4 [M+H]+. C28H23N7O2 (489.19).
4-((4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-1,2,
3-triazol-1-yl)methyl)benzamide (ZL8).
Recrystallized from EA/PE as a white solid, 75.3% yield, m.p. 289 − 290 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H, NH), 8.67 (s, 1H, triazole-H), 8.47 (d, J
= 5.6 Hz, 1H, C6-pyrimidine-H), 7.98 (s, 1H, Ph-H), 7.90 (d, J = 8.2 Hz, 2H, Ph-H),

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7.71 (s, 2H, NH2), 7.64 (d, J = 8.5 Hz, 2H, Ph-H), 7.49 – 7.39 (m, 5H, Ph-H), 6.63 (d,
J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.76 (d, J = 5.3 Hz, 2H, CH2), 2.12 (s, 6H, 2CH3).
13C NMR (101 MHz, DMSO) δ 168.99, 167.88, 160.76, 159.67, 149.59, 146.79,
145.07, 139.43, 134.60, 133.04, 131.45, 128.68, 128.47, 128.13, 126.19, 122.10,
119.82, 118.70, 102.87, 99.37, 53.16, 16.61. ESI-MS: m/z 517.5 [M+H]+. C29H24N8O2
(516.20).
4-((4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-1,2,
3-triazol-1-yl)methyl)benzenesulfonamide (ZL9).
Recrystallized from EA/PE as a white solid, 73.5% yield, m.p. 290 − 292 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H, NH), 8.62 (s, 1H, triazole-H), 8.40 (d, J
= 5.7 Hz, 1H, C6-pyrimidine-H), 7.79 (d, J = 8.2 Hz, 2H, Ph-H), 7.64 (s, 2H), 7.56 (d,
J = 8.5 Hz, 2H, Ph-H), 7.46 (d, J = 8.1 Hz, 2H, Ph-H), 7.35 (m, 4H, NH2, Ph-H), 6.56
(d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.70 (d, J = 11.9 Hz, 2H, CH2), 2.05 (s, 6H,
13
2CH3). C NMR (100 MHz, DMSO-d6) δ 169.12, 160.78, 159.66, 149.63, 146.85,
145.08, 144.34, 140.21, 133.03, 131.47, 128.78, 128.64, 126.68, 126.21, 122.20,
119.84, 118.70, 102.86, 99.37, 52.94, 16.61; ESI-MS: m/z 553.3 [M+H]+.
C28H24N8O3S (552.17).
4-((4-(2,6-dimethyl-4-(1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl)phenoxy)pyrimidin-2-yl)a
mino)benzonitrile (ZL10).
Recrystallized from EA/PE as a white solid, 68.3% yield, m.p. 221 − 222 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, NH), 8.73 (s, 1H, triazole-H), 8.47 (d, J
= 5.7 Hz, 1H, C6-pyrimidine-H), 8.31 – 8.20 (m, 2H, Ph-H), 7.82 (d, J = 7.7 Hz, 1H,
Ph-H), 7.77 – 7.69 (m, 3H, Ph-H), 7.63 (d, J = 8.4 Hz, 2H, Ph-H), 7.41 (d, J = 8.4 Hz,
2H, Ph-H), 6.64 (d, J = 5.7 Hz, 1H, C5-pyrimidine-H), 5.88 (s, 2H, CH2), 2.13 (s, 6H,
13
2CH3). C NMR (100 MHz, DMSO-d6) δ 168.98, 160.78, 159.66, 149.65, 148.43,
146.88, 145.06, 138.52, 135.09, 133.00, 131.49, 130.97, 128.58, 126.19, 123.66,
123.28, 122.22, 119.78, 118.68, 102.87, 99.36, 52.51, 16.61. ESI-MS: m/z 519.3
[M+H]+. C28H22N8O3 (518.18).
4-((4-(2,6-dimethyl-4-(1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)phenoxy)pyrimidin-2-yl)a
mino)benzonitrile (ZL11).

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Recrystallized from EA/PE as a light yellow solid, 65.6% yield, m.p. 226 − 228
1
°C; H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, NH), 8.63 (s, 1H, triazole-H),
8.47 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 8.18 (d, J = 8.1 Hz, 1H, Ph-H), 7.80 (t, J =
7.5 Hz, 1H, Ph-H), 7.73 (s, 2H, Ph-H), 7.72 – 7.61 (m, 3H, Ph-H), 7.43 (d, J = 8.5 Hz,
2H, Ph-H), 7.18 (d, J = 7.8 Hz, 1H, Ph-H), 6.64 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H),
6.06 (s, 2H, CH2), 2.13 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 168.97,
160.82, 159.65, 149.70, 147.76, 146.90, 145.07, 143.89, 133.00, 131.51, 129.41,
128.57, 126.19, 124.47, 122.37, 119.82, 118.67, 102.83, 99.34, 52.66, 16.61. ESI-MS:
m/z 519.3 [M+H]+. C28H22N8O3 (518.18).
4-((4-(2,6-dimethyl-4-(1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)phenoxy)pyrimidin-2-yl)a
mino)benzonitrile (ZL12).
Recrystallized from EA/PE as a light yellow solid, 62.7% yield, m.p. 266 − 268
1
°C; H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, NH), 8.72 (s, 1H, triazole-H),
8.47 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 8.28 (d, J = 8.3 Hz, 2H, Ph-H), 7.73 (s, 2H,
Ph-H), 7.60 (t, J = 7.4 Hz, 4H, Ph-H), 7.40 (d, J = 8.4 Hz, 2H, Ph-H), 6.65 (d, J = 5.6
Hz, 1H, C5-pyrimidine-H), 5.89 (s, 2H, CH2), 2.13 (s, 6H, 2CH3). 13C NMR (100
MHz, DMSO-d6) δ 168.97, 160.82, 159.65, 149.70, 147.76, 146.90, 145.07, 143.89,
133.00, 131.51, 129.41, 128.57, 126.19, 124.47, 122.37, 119.82, 118.67, 102.83,
99.34, 52.66, 16.61. ESI-MS: m/z 519.3 [M+H]+. C28H22N8O3 (518.18).
4-((4-(4-(1-benzyl-1H-1,2,3-triazol-4-yl)-2,6-dimethylphenoxy)pyrimidin-2-yl)amino)
benzonitrile (ZL13)
Recrystallized from EA/PE as a white solid, 57.2% yield, m.p. 217 − 219 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H, NH), 8.63 (s, 1H, triazole-H), 8.40 (d, J
= 5.7 Hz, 1H, C6-pyrimidine-H), 7.65 (d, J = 9.7 Hz, 4H, Ph-H), 7.44 (d, J = 8.5 Hz,
2H, Ph-H), 7.29 (t, J = 7.4 Hz, 2H, Ph-H), 7.23 (d, J = 7.0 Hz, 3H, Ph-H), 6.57 (d, J
= 5.7 Hz, 1H, C5-pyrimidine-H), 5.76 (s, 2H, CH2), 2.06 (s, 6H, 2CH3). 13C NMR
(100 MHz, DMSO-d6) δ 168.98, 160.79, 159.65, 149.67, 146.88, 145.07, 141.93,
133.28, 133.01, 131.49, 129.09, 128.58, 126.19, 122.32, 119.83, 118.67, 111.45,
102.85, 99.34, 52.92, 16.60. ESI-MS: m/z 474.5 [M+H]+. C28H23N7O (473.20).

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4-((4-(2,6-dimethyl-4-(1-phenethyl-1H-1,2,3-triazol-4-yl)phenoxy)pyrimidin-2-yl)ami
no)benzonitrile (ZL14).
Recrystallized from EA/PE as a white solid, 64.6% yield, m.p. 167 − 168 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, NH), 8.55 (s, 1H, triazole-H), 8.47 (d, J
= 5.6 Hz, 1H, C6-pyrimidine-H), 7.65 (d, J = 9.7 Hz, 4H, Ph-H), 7.44 (d, J = 8.5 Hz,
2H, Ph-H), 7.29 (t, J = 7.4 Hz, 2H, Ph-H), 7.23 (d, J = 7.0 Hz, 3H, Ph-H), 6.65 (d, J =
5.6 Hz, 1H, C5-pyrimidine-H), 4.69 (t, J = 7.2 Hz, 2H, CH2), 3.25 (t, J = 7.2 Hz, 2H,
CH2), 2.13 (s, 6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 168.99, 160.74, 159.67,
149.48, 146.20, 145.09, 138.08, 133.02, 131.42, 129.15, 128.90, 127.08, 126.04,
121.73, 119.83, 118.70, 102.87, 99.42, 51.18, 36.09, 16.63. ESI-MS: m/z 488.4
[M+H]+. C29H25N7O (487.21).
4-((4-(2,6-dimethyl-4-(1-(2-(thiophen-2-yl)ethyl)-1H-1,2,3-triazol-4-yl)phenoxy)pyrim
idin-2-yl)amino)benzonitrile (ZL15).
Recrystallized from EA/PE as a light yellow crystal solid, 56.2% yield, m.p.
1
188−189 °C; H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, NH), 8.54 (s, 1H,
triazole-H), 8.47 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 7.65 (d, J = 13.7 Hz, 4H,
Ph-H), 7.51 – 7.38 (m, 3H, thiophene-H, Ph-H), 7.24 (d, J = 2.9 Hz, 1H, thiophene-H),
7.01 (dd, J = 4.9, 1.2 Hz, 1H, thiophene-H), 6.65 (d, J = 5.6 Hz, 1H,
C6-pyrimidine-H), 4.69 (t, J = 7.2 Hz, 2H, CH2), 3.27 (t, J = 7.2 Hz, 2H, CH2), 2.13 (s,
6H, 2CH3). 13C NMR (100 MHz, DMSO-d6) δ 169.00, 160.74, 159.67, 149.49, 146.22,
145.08, 138.28, 133.03, 131.42, 128.88, 128.66, 126.69, 126.05, 122.58, 121.70,
119.85, 118.69, 102.87, 99.41, 50.54, 30.73, 30.48, 16.64; ESI-MS: m/z 494.4
[M+H]+. C27H23N7OS (493.17).
2-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-1,2,3
-triazol-1-yl)-N-(4-(methylsulfonyl)phenyl)acetamide (ZL16).
Recrystallized from EA/PE as a light yellow solid, 67.5% yield, m.p. 285 − 287
°C; 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H, CONH), 10.13 (s, 1H, NH), 8.63
(s, 1H, triazole-H), 8.48 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 7.89 (q, J = 8.7 Hz, 4H,
Ph-H), 7.74 (s, 2H, Ph-H), 7.67 (d, J = 8.4 Hz, 2H, Ph-H), 7.45 (d, J = 8.5 Hz, 2H,
Ph-H), 6.65 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.50 (s, 2H, CH2), 3.19 (s, 3H,

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13
CH3), 2.15 (s, 6H, 2CH3). C NMR (100 MHz, DMSO-d6) δ 165.53, 160.74, 159.69,
150.13, 149.49, 146.34, 145.09, 143.36, 135.75, 133.06, 131.48, 128.83, 126.17,
123.51, 119.63, 118.70, 102.91, 94.64, 52.95, 44.23, 16.65. ESI-MS: m/z 595.4
[M+H]+. C30H26N8O4S (594.18).
N-(4-bromophenyl)-2-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimet
hylphenyl)-1H-1,2,3-triazol-1-yl)acetamide (ZL17).
Recrystallized from EA/PE as a white solid, 58.4% yield, m.p. 239 − 241 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H, CONH), 10.14 (s, 1H, NH), 8.62 (s, 1H,
triazole-H), 8.48 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 7.74 (s, 2H, Ph-H), 7.68 (d, J
= 8.5 Hz, 2H, Ph-H), 7.64 – 7.50 (m, 4H, Ph-H), 7.46 (d, J = 8.5 Hz, 2H, Ph-H), 6.64
13
(d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.43 (s, 2H, CH2), 2.15 (s, 6H, 2CH3). C
NMR (100 MHz, DMSO-d6) δ 169.01, 164.85, 160.73, 159.70, 149.53, 146.32,
145.09, 138.26, 133.06, 132.24, 131.46, 128.78, 126.16, 123.48, 121.68, 119.84,
118.70, 115.92, 102.90, 99.41, 52.91, 16.65. ESI-MS: m/z 595.3 [M+H]+.
C29H23BrN8O2 (594.11).
2-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-1,2,3
-triazol-1-yl)-N-(4-fluorophenyl)acetamide (ZL18).
Recrystallized from EA/PE as a white solid, 53.6% yield, m.p. 283 − 286 °C; 1H
NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H, CONH), 10.07 (s, 1H, NH), 8.55 (s, 1H,
triazole-H), 8.40 (d, J = 5.6 Hz, 1H, C6-pyrimidine-H), 7.67 (s, 2H, Ph-H), 7.64 –
7.53 (m, 4H, Ph-H), 7.39 (d, J = 8.5 Hz, 2H, Ph-H), 7.13 (t, J = 8.8 Hz, 2H, Ph-H),
6.57 (d, J = 5.6 Hz, 1H, C5-pyrimidine-H), 5.35 (s, 2H, CH2), 2.08 (s, 6H, 2CH3). 13C
NMR (100 MHz, DMSO-d6) δ 169.01, 164.57, 160.73, 159.92, 159.69, 157.54,
149.52, 146.31, 145.09, 135.29, 133.06, 131.46, 128.78, 126.16, 123.48, 121.59,
121.51, 119.85, 118.69, 116.12, 115.89, 102.89, 99.42, 52.83, 16.65. ESI-MS: m/z
535.3 [M+H]+. C29H23FN8O2 (534.19).
Methyl 2-(4-(4-((2-((4-cyanophenyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)
-1H-1,2,3-triazol-1-yl)acetate (ZL19).
Recrystallized from EA/PE as a light yellow solid, 66.4% yield, m.p.: 221-224℃;
1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H, NH), 8.59 (s, 1H, triazole-H), 8.47 (d,