Synthesis and biological evaluation of CHX-DAPYs as HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.03.020

A series of new diarylpyrimidines (DAPYs) characterized by a halogen atom on the methylene linker between wing I and the central pyrimidine ring was synthesized and evaluated for their anti-HIV activity in MT-4 cell cultures. The two most promising compou

Full text of DOI:10.1016/j.bmc.2014.03.020

Accepted Manuscript
Synthesis and biological evaluation of CHX-DAPYs as HIV-1 non-nucleoside
reverse transcriptase inhibitors
Zi-Hong Yan, Hai-Qiu Wu, Wen-Xue Chen, Yan Wu, Hu-Ri Piao, Qiu-Qin He,
Fen-Er Chen, Erik De Clercq, Christophe Pannecouque
PII:
S0968-0896(14)00193-X
http://dx.doi.org/10.1016/j.bmc.2014.03.020
BMC 11467
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 February 2014
8 March 2014
11 March 2014
Please cite this article as: Yan, Z-H., Wu, H-Q., Chen, W-X., Wu, Y., Piao, H-R., He, Q-Q., Chen, F-E., De Clercq,
E., Pannecouque, C., Synthesis and biological evaluation of CHX-DAPYs as HIV-1 non-nucleoside reverse
transcriptase inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
2014.03.020
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Synthesis and biological evaluation of CHX-
DAPYs as HIV-1 non-nucleoside reverse
transcriptase inhibitors
Leave this area blank for abstract info.
Zi-Hong Yan a , Hai-Qiu Wu a , Wen-Xue Chen a , Yan Wu b, Hu-Ri Piao b, Qiu-Qin He a, ∗ , Fen-Er
Chen a, c, ∗ , Erik De Clercq d , and Christophe Pannecouque d
a Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
b College of Pharmacy, Yanbian University,Yanji 133000, People’s Republic of China
c Institute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
d
Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium

Bioorganic & Medicinal Chemistry
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Synthesis and biological evaluation of CHX-DAPYs as HIV-1 non-nucleoside reverse
transcriptase inhibitors
Zi-Hong Yan a , Hai-Qiu Wu a , Wen-Xue Chen a , Yan Wu b, Hu-Ri Piao b, Qiu-Qin He a, ∗ , Fen-Er Chena,
c, ∗
, Erik De Clercq d , and Christophe Pannecouque d
a Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
bCollege of Pharmacy, Yanbian University,Yanji 133000, People’s Republic of China
c Institute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of new diarylpyrimidines (DAPYs) characterized by a halogen atom on the methylene
linker between wing I and the central pyrimidine ring was synthesized and evaluated for their
anti-HIV activity in MT-4 cell cultures. The two most promising compounds 7f and 7g showed
excellent activity against wild-type HIV-1 with low nanomolar EC50 values of 0.005 µM and
0.009 µM, respectively, which were comparable to or more potent than all the reference drugs
zidovudine (AZT), lamivudine (3TC), nevirapine (NEV), efavirenz (EFV), delaviridine (DLV)
and etravirine (ETV). In particular, 7g also displayed strong activity against the double mutant
strain 103N+181C with an EC50 value of 8.2 µM. The preliminary structure–activity relationship
(SAR) and molecular docking analysis of this new series of CHX-DAPYs were also
investigated.
Available online
Keywords:
HIV-1
NNRTIs
diarylpyrimidine
SAR
2009 Elsevier Ltd. All rights reserved.
Antiviral activity
1. Introduction
regarded as one of the most successful scaffolds for structural
modification to obtain more promising anti-HIV agents due to
their excellent potency and low cytotoxicity.4-9
The reverse transcriptase of human immunodeficiency virus
type 1 (HIV-1 RT) is an essential enzyme for viral replication
and represents one of the main targets for antiretroviral
chemotherapy.1 The inhibitors of this enzyme can be divided into
two broad classes: nucleoside/nucleotide reverse transcriptase
inhibitors (NRTIs) and non-nucleoside reverse transcriptase
inhibitors (NNRTIs). NRTIs inhibit viral replication by binding
directly to the polymerase active site, whereas NNRTIs
specifically bind to an allosteric hydrophobic site, called the non-
nucleoside reverse transcriptase inhibitor binding pocket
(NNIBP), which lies approximately 10Å away from the active
site.2,3 As indispensable components in highly active
antiretroviral therapy (HAART), NNRTIs have gained
a
definitive and important place in clinical use. NNRTIs currently
approved for AIDS therapy include nevirapine (NVP, 1, Figure
1), delavirdine (DLV, 2, Figure 1), efavirenz (EFV, 3, Figure 1),
etravirine (TMC125, ETV, 4, Figure 1) and rilpivirine
(TMC278, 5, Figure 1). Among numerous efforts to develop
structurally different NNRTIs, the diarylpyrimidine derivatives
(DAPYs) represented by etravirine and rilpivirine have been
Figure 1. Structures of currently FDA-approved clinical NNRTIs.
———
∗ Corresponding author. Tel.: +86 21 65643809; fax: +86 21 65643811;e-mail: qqhe@ fudan.edu.cn
∗ Corresponding author. Tel.: +86 21 65643809; fax: +86 21 65643811; e-mail: rfchen@fudan.edu.cn

In our previous DAPY project, we focused on structural
modification of the linker between wing I and the central
pyrimidine ring and identified CH(OH)-DAPYs (6, Figure 2) as
a new class of potent NNRTIs.10 Among these CH(OH)-DAPYs,
the most potent compound 6a (R1= R2=H, R3=2-Cl) displayed
anti-HIV-1 activity with an EC50 value of 0.009 µM. It indicates
that introduction of a small group at the CH2 linker is tolerable
for antiviral activity. On the basis of this result and hydrophobic
property of NNIBP, a halogen atom was introduced to CH2 linker
with the aim to enhance the ligand-protein binding affinity via
hydrophobic interaction between the added halogen atom and
NNIBP. Thus we report herein the design, synthesis, and anti-
HIV activity of these CHX-DAPYs (7, Figure 2).
Interestingly, the introduction of a methyl group at the para
position of the benzene ring of 7a resulted into 7b (EC50=0.069
µM), which appeared to be slightly less active than the
corresponding 7s (EC50=0.047 µM).
Next we examined the effect of introducing substituents into
the central pyrimidine ring. Compound 7d (X=Cl, R1=R2=H,
R3=4-t-butyl-phenyl) was as potent as 7l (X=Cl, R1=Me, R2=H,
R3=4-t-butyl-phenyl), but 10-fold more potent than 7o (X=Cl,
R1=H, R2=Me, R3=4-t-butyl-phenyl). As consistent with the
results obtained previously on the CHOH-DAPYs, compound 7h
(X=Cl, R1=R2=H, R3=4-F-phenyl) showed higher activity than
that of 7m (X=Cl, R1=Me, R2=H, R3=4-F-phenyl) and 7p (X=Cl,
R1=H, R2=Me, R3=4-F-phenyl), compound 7j (X=Cl, R1=R2=H,
R3=4-Br-phenyl) was more potent than 7n (X=Cl, R1=Me, R2=H,
R3=4-Br-phenyl) and 7q (X=Cl, R1=H, R2=Me, R3=4-Br-phenyl).
These results suggested that no substituent at pyrimidine ring
may be favorable for antiviral potency.
Finally we examined the effect of introducing substituents into
the benzene ring of wing I. The introduction of a methyl,
methyoxy or tert-butyl group at the para position of 7a led to
significantly decreased activity against wild-type HIV-1. The
introduction of a halogen group at the meta or para position of 7a
led to compounds 7e-j, whose inhibitory activity seemed to be
greatly influenced by the position of the introduced halogen
group. Compounds 7e-g with a halogen atom at meta position of
7a were more active than the corresponding para-position
halogenated compounds 7h-j. Moreover, chlorinated or
brominated compounds exhibited significantly stronger potency
than the corresponding fluorinated derivatives. For example, 7f
(3-Cl) and 7g (3-Br) were more active than 7e (3-F). Similarly, 7i
(4-Cl) and 7j (4-Br) were more potent than 7h (4-F). The
replacement of benzene ring of 7a with a bulky naphthyl group
led to 7k, which showed decreased antiviral activity.
Figure 2. Structures of CH(OH)-DAPYs and CHX-DAPYs.
2. Chemistry
The synthetic route to the target compounds 7a-s is depicted
in
Scheme
1.
The
key
intermediates
4-((4-
(hydroxy(phenyl)methyl)pyrimidin-2-yl)amino)benzonitrile 14a-
q were prepared from 8a-c according to our previously reported
protocol in 6 steps.10 Halogenation of 14a-q with thionyl chloride
or phosphorus tribromide in anhydrous dichloromethane afforded
the target compounds 7a-s in 45-78% yields.
3. Results and discussion
3.2. Molecular modeling analysis
3.1. Biological activity
With the aim to investigate the binding mode of CHX-DAPYs
in NNIBP of wild-type HIV-1 RT, molecular docking was
performed using Sybyl Surflex-Dock program. The crystal
structure of RT in complex with ETV (PDB ID: 3MEC) was used
for molecular docking and the representative compounds 7f and
7g were chosen for docking analysis. Compound 7f was the most
active one tested against wild-type HIV-1 among the newly
synthesized compounds, while 7g was the only one endowed
with inhibitory activity against double mutant strain 103N+181C.
The newly synthesized compounds 7a-s were tested in
infected MT-4 cells to evaluate their antiviral activity against
wild-type HIV-1 (LAI strain IIIB), double mutant HIV-1 strain
RES056 (K103N + Y181C) and HIV-2 strain ROD. AZT, 3TC,
NVP, EFV, DLV and ETV were used as reference drugs in the
test. The results, expressed as EC50 (anti-HIV activity), CC50
(cytotoxicity) and SI (selectivity index, given by the CC50/EC50
ratio), are summarized in Table 1.
As shown in Figure 3a, both representative compounds
adopted very similar binding orientations and horseshoe
conformational shapes in NNIBP to that of ETV. Two typical
hydrogen bonds were clearly observed with all three compounds,
which are crucial for antiviral activity of DAPYs. One hydrogen
bond was between N-1 of pyrimidine ring and NH2 group of the
residue Lys101, another involved the NH linker and C=O group
of Lys101. The wing I of both 7f and 7g provided strong π-
stacking interaction with the residues Tyr181 and Tyr188.
However, the orientation of 7f moved downward compared to
those of 7g and ETV and no interaction with the residues Phe227
and Trp229 could be observed. This may determine the loss of
the inhibitory activity for 7f against mutant virus.
As shown in Table 1, all the compounds 7a-s showed
moderate to excellent inhibitory activity against wild-type HIV-1
with EC50 values ranging from 7.880 to 0.005 µM. The two most
potent compounds 7f and 7g had low nanomolar EC50 values of
0.005 µM and 0.009 µM, respectively, which were comparable to
or more active than all the reference drugs AZT, 3TC, NVP,
EFV, DLV and ETV. SI values of both 7f and 7g were >3000. In
particular, 7g also strongly inhibited the double mutant strain
103N+181C with an EC50 value of 8.2 µM. None of the tested
NNRTIs including the newly synthesized CHX-DAPYs and the
reference drugs NVP, EFV, DLV, ETV showed activity against
HIV-2 strain ROD.
To explore SAR of this new series of CHX-DAPYs, the
modifications are focused on CH2 linker, the central pyrimidine
ring as well as the wing I. First we examined the effect of
introducing different halogen atom into CH2 linker between wing
I and the central pyrimidine ring. The biological results showed
that 7a (X=Cl, EC50=0.016 µM) displayed approximately 10-fold
stronger antiviral activity than 7r (X=Br, EC50=0.200 µM).
From Figures 3b and 3c, it is important to note that the
chloride atom of the linker for 7f could form halogen bond with
C=O group of Tyr188 (d O···Cl=2.67 Å; angle O···Cl···C≈140o),
whereas 7g could not exhibit this halogen bond (d O···Cl=5.94
Å).11-16 It may explain why 7f displayed higher anti-HIV-1
activity than 7g.

To further explore the interaction of CHX-DAPYs with
mutated HIV-1 RT, compounds 7f and 7g were also docked into
NNIBP of mutant HIV-1 RT (K103N/Y181C) in complex with
TMC278 (PDB ID: 3BGR). As illustrated in Figure 4, 7g is
predicted to bind with NNBIP still in a horseshoe conformational
shape similar to that of TMC278 and it kept the two typical
hydrogen bonds with Lys101; however, 7f bound with NNBIP in
a totally different mode from 7g and TMC278. The orientation of
the horseshoe shape for 7f was downward and no direct
interactions between 7f and binding site could be observed. This
might reveal the reason why compound 7g could inhibit the
replication of double mutant strain 103N+181C, whereas 7f was
completely inactive, although these two compounds differ
structurally only in the identity of the substituents on the benzene
ring of wing I.
Scheme 1. Synthetic route to compounds7a-s. Reagents and conditions: a) MeI, NaOH, H2O, rt, 24 h; b) 4-cyanoaniline, 180-190 oC, 8 h; c) POCl3, reflux, 0.5 h;
d) substituted phenylactonitrile, 60% NaH, Ar, DMF, -20oC to rt, 48-72 h; e) air, rt, 48-72 h; f) KBH4, methanol, -20oC to rt, 2 h; g) SOCl2 or PBr3,
dichloromethane.
Table 1. Anti-HIV-1 activities and cytotoxicity of compounds 7a-s in MT-4 cellsa
EC50[µM]b
SId
compd
CC50[µM]c
WT(ШB)
WT(ШB)
K103N+Y181C
>37.8
HIV-2
>37.8
7a
7b
7c
7d
0.016 0.003
37.8
2336
0.069 0.006
0.219 0.022
>49.7
>33.6
>49.7
>33.6
49.7
713
154
≥33.6
0.170 0.092
>32.9
>32.9
32.9
193
7e
7f
1.358 0.472
0.005 0.003
0.009 0.001
1.830 0.177
>36.8
>24.3
8.2 0.05
>35.4
>36.8
>24.3
>30.4
>35.4
36.8
24.3
30.4
35.4
27
5162
3106
19
7g
7h
7i
0.068 0.014
0.038 0.013
0.202 0.024
>36.1
>32.5
>11.3
>36.1
>32.5
>11.3
36.1
32.5
11.3
545
875
56
7j
7k
7l
7m
0.174 0.070
5.839 1.729
>30.3
>31.1
>30.3
>31.1
30.3
31.1
173
5
7n
7o
0.435 0.230
1.791 0.716
>31.9
>28.7
>31.9
>28.7
31.9
28.7
75
16
7p
7.880 2.154
0.109 0.073
0.200 0.088
0.047 0.011
0.006 0.001
>30.8
>30.8
>10.3
>69.0
>31.5
30.8
10.3
69.0
31.5
4
7q
>10.3
95
7r
>69.0
348
672
>17045
7s
>31.5
AZT
0.007 0.001
0.005 0.000 >93.5

3TC
NVP
EFV
DLV
ETV
2.442 0.262
0.090 0.015
0.006 0.001
0.085 0.007
NDe
7.2 0.8
NDe
NDe
>87.2
15.0
>36
>15.0
>163
>1136
>511
7383
0.270 0.108
>43.8
>6.3
NDe
>43.8
14.25
0.002 0.0005 0.0147
>14.25
a
Data represent the mean of at least three separate experiments.
b Compound concentration required to inhibit MT-4 cells against viral cytopathogenicity by 50%.
c Compound concentration that decreases the uninfected MT-4 cell viability by 50%.
d Selectivity index:CC50/IC50 ratio.
e Not detected.
Fig. 3. Predicted binding mode of the representative compounds in NNIBP of wild type HIV-1 RT (PDB ID: 3MEC): a) compound 7f (blue) and 7g (red), ETV
(green) was superposed into the same pocket for comparison, b) halogen bond of compound 7f (blue), c) halogen bond of compound 7g (red). Possible hydrogen
bonds between the inhibitors and the residues are indicated with dashed lines (yellow).
EC50 values of 0.005 µM and 0.009 µM, respectively, which were
comparable to or more active than all the six reference drugs
AZT, 3TC, NVP, EFV, DLV and ETV. Moreover, 7g was also
active against the double mutant strain 103N+181C. The binding
modes with wide-type HIV-1 RT suggested that the added
halogen atom at CH2 linker for 7f could form halogen bond with
C=O group of Tyr188 and thus enhanced its binding affinity
resulting in an improved antiviral activity. The binding modes
with mutant HIV-1 RT showed that 7g still adopted a horseshoe
conformational shape in NNBIP and kept two hydrogen bonds
with Lys101; this may be the reason why 7g could remain the
activity against drug-resistant viral strain.
5. Material and methods
5.1. Chemistry
Melting points were measured on a SGW X-1 microscopic
1
melting-point apparatus. H NMR and 13C NMR spectra on a
Bruker AV 400 MHz spectrometer were recorded in DMSO-d6.
Chemical shifts are reported in δ (ppm) units relative to the
Fig. 4. Predicted binding mode of the representative compounds in NNIBP of
internal standard tetramethylsilane (TMS). Mass spectra were
double mutant (K103N/Y181C) (PDB code: 3BGR), compound 7f (blue) and
obtained on a Waters Quattro Micromass instrument using
7g (red), TMC278 (purple) was superposed into the same pocket for
electrospray ionization (ESI) techniques. Infrared spectra (IR)
comparison.
were recorded with a nexus FT/IR-4200 spectro-meter. All
chemicals and solvents used were of reagent grade and were
purified and dried by standard methods before use. All air-
sensitive reactions were run under a nitrogen atmosphere. All the
reactions were monitored by thin layer chromatography (TLC) on
pre-coated silica gel G plates at 254 nm under a UV lamp using
ethyl acetate/petroleum ether as eluent. Flash chromatography
separations were obtained on silica gel (300–400 mesh). All
yields are unoptimized and only represent one experiment’s
result.
4. Conclusion
In conclusion, a new series of new CHX-DAPYs featuring a
halogen atom at CH2 linker between wing I and the central
pyrimidine ring was synthesized and evaluated against wild-type
HIV-1, HIV-2 and drug-resistant viral strain in cellular assays.
All the target compounds displayed anti-HIV-1 activity with
micromolar to nanomolar EC50 values in infected MT-4 cells.
Compounds 7f and 7g exhibited the most antiviral activity with

5.2. General procedure for preparation of target compounds
7a-q
5.2.4. 4-((4-((4-(tert-butyl)phenyl)chloromethyl)pyrimidin-2-
yl)amino)benzonitrile (7d).
Yield 63%; light yellow solid; mp 143.5-144.0 oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 1.24 (s, 9 H, t-butyl), 6.32 (s, 1 H,
CH), 7.18 (d, J = 5.2 Hz, 1 H, pyrimidine H5), 7.41 (d, J = 8.4
Hz, 2 H, ArH3,5), 7.47 (d, J = 8.4 Hz, 2 H, ArH2,6), 7.69 (d, J =
8.8 Hz, 2 H, ArH2,6), 7.95 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.61 (d, J
= 4.8 Hz, 1 H, pyrimidine H6), 10.31 (s, 1 H, NH); 13C NMR
(100MHz, DMSO-d6) δ (ppm) 30.97, 34.34, 62.16, 102.63,
110.99, 118.45, 125.49, 127.79, 132.92, 135.96, 144.64, 151.20,
159.15, 159.73, 167.92; IR (KBr, cm-1) 2219 (CN); MS (ESI)
m/z 377 [M+H]+.
To
a
solution of 14a-q (1 mmol) in anhydrous
dichloromethane (10 mL) was added thionyl chloride (1.0 mL)
dropwise at -20 ℃. After being stirred for 2 h at room
temperature, the mixture was poured into ice water (20 mL) and
extracted with ethyl acetate (3×20 mL). The combined organic
layers were washed with 5% aqueous sodium hydrogen carbonate
and brine, dried over anhydrous sodium sulfate, and concentrated
in vacuo. The residue was purified by column chromatography
on silica gel (ethyl acetate /hexane, 1: 2) to give the desired
compounds.
5.2.5.
4-((4-(chloro(3-fluorophenyl)methyl)pyrimidin-2-
5.3. General procedure for preparation of target compounds 7r-
s
yl)amino)benzonitrile (7e).
Yield 59%; yellow solid; mp 109.6-110.5oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 6.43 (s, 1 H, CH), 7.19 (d, J = 4.8
Hz, 1 H, pyrimidine H5), 7.23 (s, 1 H, ArH2), 7.39-7.43 (m, 2 H,
ArH4,6), 7.45-7.50 (m, 1 H, ArH5), 7.68 (d, J = 8.4 Hz, 2 H,
ArH2,6), 7.91 (d, J = 8.4 Hz, 2 H, ArH3,5), 8.63 (d, J = 4.8 Hz, 1
H, pyrimidine H6), 10.33 (s, 1 H, NH); 13C NMR (100MHz,
DMSO-d6) δ (ppm) 61.07, 102.73, 111.16, 114.89, 115.11,
115.73, 118.46, 124.27, 124.30, 130.76, 130.84, 132.91, 144.58,
159.18, 159.98, 167.26; IR (KBr, cm-1) 2220 (CN); MS (ESI)
m/z 337 [M-H]-.
To
a
solution of 14a-b (1 mmol) in anhydrous
dichloromethane (10 mL) was added phosphorus tribromide
(1.35 g, 5 mmol ). After being stirred for 1.5 h at room
temperature, the mixture was poured into ice water (20 mL) and
extracted with ethyl acetate (3×20 mL). The combined organic
layers were washed with 5% aqueous sodium hydrogen carbonate
and brine, dried over anhydrous sodium sulfate, and concentrated
in vacuo. The residue was purified by column chromatography
on silica gel (ethyl acetate /hexane, 1: 2) to give the desired
compounds 7r-s.
5.2.6. 4-((4-(chloro(3-chlorophenyl)methyl)pyrimidin-2-
yl)amino)benzonitrile (7f).
5.2.1.
yl)amino)benzonitrile (7a).
4-((4-(chloro(phenyl)methyl)pyrimidin-2-
1
Yield 68%; light yellow solid; mp 130.1-130.6oC; H NMR
(400MHz, DMSO-d6) δ (ppm) 6.43 (s, 1 H, CH), 7.19 (d, J = 4.8
Hz, 1 H, pyrimidine H5), 7.24 (s, 1 H, ArH2), 7.39-7.43 (m, 2 H,
ArH4,6), 7.45-7.50 (m, 1 H, ArH5), 7.68 (d, J = 8.4 Hz, 2 H,
ArH2,6), 7.91 (d, J = 8.4 Hz, 2 H, ArH3,5), 8.63 (d, J = 4.8 Hz, 1
H, pyrimidine H6), 10.33 (s, 1 H, NH); 13C NMR (100MHz,
DMSO-d6) δ (ppm) 61.07, 102.73, 111.16, 114.89, 115.11,
115.73, 118.46, 124.27, 124.30, 130.76, 130.84, 132.91, 144.58,
159.18, 159.98, 167.26; IR (KBr, cm-1) 2218 (CN); MS (ESI)
m/z 354 [M-H]-.
Yield 51%; light yellow solid; mp 145.0-145.4oC；1H NMR
(400MHz, DMSO-d6) δ (ppm) 6.37 (s, 1 H, CH), 7.19 (d, J = 5.2
Hz, 1 H, pyrimidine H5), 7.35-7.40 (m, 1 H, ArH4), 7.43 (d, J =
7.6 Hz, 2 H, ArH3,5), 7.55 (d, J = 8.4 Hz, 2 H, ArH2,6), 7.69 (d, J
= 8.8 Hz, 2 H, ArH2,6), 7.91 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.62 (d,
J = 5.2 Hz, 1 H, pyrimidine H6), 10.31 (s, 1 H, NH); 13C NMR
(100MHz, DMSO-d6) δ (ppm) 62.23, 102.67, 111.12, 118.46,
128.12, 128.71, 128.75, 132.97, 138.94, 144.65, 159.17, 159.85,
167.87; IR (KBr, cm-1) 2220 (CN); MS (ESI) m/z 321 [M+H]+.
5.2.7.
yl)amino)benzonitrile (7g).
4-((4-((3-bromophenyl)chloromethyl)pyrimidin-2-
5.2.2. 4-((4-(chloro(p-tolyl)methyl)pyrimidin-2-
yl)amino)benzonitrile (7b).
1
Yield 78%; light yellow solid; mp 140.1-140.6oC; H NMR
(400MHz, DMSO-d6) δ (ppm) 6.42 (s,1 H, CH), 7.21 (d, J = 5.2
Hz, 1 H, pyrimidine H5), 7.37-7.41 (m, 1 H, ArH5), 7.55-7.59 (m,
2 H, ArH4,6), 7.69 (d, J = 8.8 Hz, 2 H, ArH2,6), 7.68 (s, 1 H,
ArH2), 7.89 (d, J = 8.8 Hz, 2 H, ArH3,5)，8.63 (d, J = 4.8 Hz, 1
H, pyrimidine H6), 10.33 (s, 1 H, NH); 13C NMR (100MHz,
DMSO-d6) δ (ppm) 60.92, 102.73, 111.15, 118.46, 121.70,
127.21, 130.79, 130.95, 131.57, 132.93, 141.41, 144.56, 58,
159.18, 160.05, 167.19; IR (KBr, cm-1) 2221 (CN); MS (ESI)
m/z 399 [M+H]+.
Yield 66%; yellow solid; mp 127.0-127.8oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 2.28 (s, 3 H, Me), 6.32 (s, 1 H,
CH), 7.16 (d, J = 5.2 Hz, 1 H, pyrimidine H5), 7.21 (d, J = 7.6
Hz, 2 H, ArH3,5), 7.43 (d, J = 8.0 Hz, 2 H, ArH2,6), 7.69 (d, J =
8.8 Hz, 2 H, ArH2,6), 7.94 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.61 (d, J
= 4.8 Hz, 1 H, pyrimidine H6), 10.32 (s, 1 H, NH); 13C NMR
(100MHz, DMSO-d6) δ (ppm) 20.73, 62.27, 102.65, 111.02,
118.46, 128.02, 129.25, 132.93, 132.97, 136.01, 138.19, 144.67,
159.16, 159.76, 167.99; IR (KBr, cm-1) 2219 (CN); MS (ESI)
m/z 335 [M+H]+.
5.2.8.
yl)amino)benzonitrile (7h).
4-((4-(chloro(4-fluorophenyl)methyl)pyrimidin-2-
5.2.3.
4-((4-(chloro(4-methoxyphenyl)methyl)pyrimidin-2-
yl)amino)benzonitrile (7c).
1
Yield 62%; light yellow solid; mp 161.2-162.0oC; H NMR
(400MHz, DMSO-d6) δ (ppm) 6.42 (s, 1 H, CH), 7.24-7.29 (m, 2
H, ArH2,6), 7.59-7.63 (m, 2 H, ArH3,5), 7.70 (d, J = 8.8 Hz, 2 H,
ArH2,6), 7.92 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.63 (d, J = 4.8 Hz, 1
H, pyrimidine H6), 10.33 (s, 1 H, NH); 13C NMR (100MHz,
DMSO-d6) δ (ppm) 61.31, 102.71, 111.08, 115.53, 115.75,
118.46, 119.53, 130.34, 130.42, 132.98, 144.61, 159.20, 159.96,
167.60; IR (KBr, cm-1) 2220 (CN); MS (EI) m/z 337 [M-H]-.
Yield 58%; yellow solid; mp 177.7-178.2oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 3.73 (s, 3 H, OMe), 6.30 (s, 1 H,
CH), 6.95 (d, J = 8.4 Hz, 2 H, ArH3,5), 7.15 (d, J = 5.2 Hz, 1 H,
pyrimidine H5), 7.47 (d, J = 8.4 Hz, 2 H, ArH2,6), 7.69 (d, J = 8.4
Hz, 2 H, ArH2,6), 7.97 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.60 (d, J =
5.2 Hz, 1 H, pyrimidine H6), 10.31 (s, 1 H, NH); 13C NMR
(100MHz, DMSO-d6) δ (ppm) 55.20, 62.29, 102.69, 110.92,
114.07, 118.48, 129.51, 130.90, 132.92, 144.68, 159.17, 159.44,
159.65, 168.05; IR (KBr,cm-1): 2221 (CN); MS (ESI) m/z 349
[M-H]-.
5.2.9.
yl)amino)benzonitrile (7i).
4-((4-(chloro(4-chlorophenyl)methyl)pyrimidin-2-

Yield 48%; brown solid; mp 156.3-156.7oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 6.40 (s, 1 H, CH), 7.16 (d, J = 5.2
Hz, 1 H, pyrimidine H5), 7.47 (d, J = 8.4 Hz, 2 H, ArH3,5), 7.56
(d, J = 8.4 Hz, 2 H, ArH2,6), 7.68 (d, J = 8.8 Hz, 2 H, ArH2,6),
7.90 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.61 (d, J = 5.2 Hz, 1 H,
pyrimidine H6), 10.33 (s, 1 H, NH); 13C NMR (100MHz, DMSO-
d6) δ (ppm) 61.25, 102.74, 111.07, 118.45, 118.47, 128.73,
130.00, 132.91, 133.41, 137.92, 144.59, 159.18, 159.90, 167.41;
IR (KBr, cm-1) 2215 (CN); MS (ESI) m/z 354 [M-H]-.
159.02, 166.98, 169.79; IR (KBr, cm-1) 2212 (CN); MS (ESI)
m/z 411 [M-H]-.
5.2.15.
4-((4-((4-(tert-butyl)phenyl)chloromethyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (7o).
Yield 62%; brown solid; mp 119.3-120.0 oC 1H NMR
(400MHz, DMSO-d6) δ (ppm) 1.25 (s, 9 H, t-butyl), 2.23 (s, 3 H,
Me), 6.55 (s, 1 H, CH), 7.43 (d, J = 8.4 Hz, 2 H, ArH2,6), 7.51 (d,
J = 8.4 Hz, 2 H, ArH3,5), 7.67 (d, J = 8.4 Hz, 2 H, ArH2,6), 7.95
(d, J = 8.8 Hz, 2 H, ArH3,5), 8.41 (s, 1 H, pyrimidine H6), 10.21
(s, 1 H, NH); 13C NMR (100MHz, DMSO-d6) δ (ppm) 14.04,
31.03, 34.38, 59.43, 102.04, 118.04, 119.39, 125.37, 128.10,
132.93, 134.98, 145.00, 151.07, 157.90, 160.28, 164.47; IR (KBr,
cm-1) 2210 (CN); MS (ESI) m/z 389 [M-H]-.
5.2.10.
4-((4-((4-bromophenyl)chloromethyl)pyrimidin-2-
yl)amino)benzonitrile (7j).
Yield 55%; yellow solid; mp 194.5-195.3oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 6.42 (s, 1 H, CH), 7.18 (d, J = 4.8
Hz, 1 H, pyrimidine H5), 7.51 (d, J = 8.0 Hz, 2 H, ArH3,5), 7.63
(d, J = 8.0 Hz, 2 H, ArH2,6), 7.69 (d, J = 8.4 Hz, 2 H, ArH2,6),
7.89 (d, J = 8.4 Hz, 2 H, ArH3,5), 8.62 (d, J = 5.2 Hz, 1 H,
pyrimidine H6), 10.33 (s, 1 H, NH); 13C NMR (100MHz, DMSO-
d6) δ (ppm) 61.25, 102.71, 111.12, 118.45, 122.00, 130.30,
131.68, 132.94, 138.34, 144.58, 159.16, 159.96, 167.35; IR (KBr,
cm-1) 2219 (CN); MS (ESI) m/z 417 [M+H2O]+.
5.2.16.
4-((4-(chloro(4-fluorophenyl)methyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (7p).
Yield 52%; ivory solid; mp 159.7-160.3 oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 2.25 (s, 3 H, Me), 6.63 (s, 1 H,
CH), 7.26-7.30 (m, 2 H, ArH2,6), 7.62-7.67 (m, 4 H, ArH3,5
+
ArH2,6), 7.88 (d, J = 8.4 Hz, 2H, ArH3,5), 8.43 (s, 1 H, pyrimidine
H6), 10.19 (s, 1 H, NH); 13C NMR (100MHz, DMSO-d6) δ (ppm)
13.97, 58.69, 102.13, 115.31, 115.53, 117.99, 119.58, 119.62,
130.60, 130.68, 132.91, 134.22, 144.91, 157.85, 160.45, 164.21;
IR (KBr, cm-1) 2216 (CN); MS (ESI) m/z 351 [M-H]-.
5.2.11.
4-((4-(chloro(naphthalen-1-yl)methyl)pyrimidin-2-
yl)amino)benzonitrile (7k).
Yield 55%; yellow solid; mp 174.8-175.3oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 7.17 (s, 1 H, CH), 7.19 (d, J = 4.8
Hz, 1 H, pyrimidine H5), 7.25-8.16 (m, 11 H, ArH4+NapH7), 8.64
(d, J = 5.2 Hz, 1 H, pyrimidine H6), 10.30 (s, 1 H, NH); 13C
NMR (100MHz, DMSO-d6) δ (ppm) 59.79, 102.60, 111.34,
118.35, 123.72, 125.48, 126.11, 126.76, 127.34, 128.89, 129.48,
130.06, 132.81, 133.56, 134.64, 144.57, 159.06, 159.77, 168.01;
IR (KBr, cm-1) 2215 (CN); MS (ESI) m/z 369 [M-H]-.
5.2.17.
4-((4-(1-(4-(tert-butyl)phenyl)-1-hydroxyethyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (7q).
Yield 45%; ivory solid; mp 202.8-203.3oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 2.26 (s, 3 H, Me), 6.62 (s, 1 H,
CH), 7.53 (d, J = 8.4 Hz, 2 H, ArH2,6), 7.62-7.66 (m, 4 H, ArH3,5
+ ArH2,6), 7.81 (d, J = 8.8 Hz, 2 H, ArH3,5), 7.43 (s, 1 H,
pyrimidine H6), 10.17 (s, 1 H, NH); 13C NMR (100MHz, DMSO-
d6) δ (ppm) 13.96, 58.74, 102.11, 117.96, 119.75, 121.80, 130.55,
131.43, 132.82, 137.36, 144.85, 157.76, 160.48, 164.03; IR (KBr,
cm-1) 2216 (CN); MS (ESI) m/z 411 [M-H]-.
5.2.12.
4-((4-((4-(tert-butyl)phenyl)chloromethyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (7l).
Yield 65%; ivory solid; mp 162.5-163.2oC; 1H NMR
(400MHz, DMSO-d6) δ (ppm) 1.23 (s, 9 H,t-butyl), 2.42 (s, 3 H,
Me), 6.26 (s, 1 H, CH), 7.09 (s, 1 H, pyrimidine H5), 7.40 (d, J =
8.4 Hz, 2 H, ArH2,6), 7.48 (d, J= 8.4 Hz, 2 H, ArH3,5), 7.67 (d, J
= 8.8 Hz, 2 H, ArH2,6), 7.97 (d, J = 8.8 Hz, 2 H, ArH3,5), 10.25
(s, 1 H, NH); 13C NMR (100MHz, DMSO-d6) δ (ppm) 23.78,
30.95, 34.30, 62.28, 102.38, 110.32, 118.31, 125.40, 127.75,
132.86, 136.09, 144.84, 151.09, 158.99, 167.52, 169.47; IR (KBr,
cm-1) 2216 (CN); MS (ESI) m/z 389 [M-H]-.
5.3.1.
4-((4-(bromo(phenyl)methyl)pyrimidin-2-
yl)amino)benzonitrile (7r).
Yield 72%; yellow solid; mp 120.2-121.0oC, 1H
NMR(400MHz, DMSO-d6) δ (ppm) 6.42 (s, 1 H, CH), 7.11 (d, J
= 4.8 Hz, 1 H, pyrimidine H5), 7.20 (d, J = 8.0 Hz, 2 H, ArH2,6),
7.51 (d, J = 8.0 Hz, 2 H, ArH3,5), 7.71 (d, J = 8.8 Hz, 2 H,
ArH2,6), 7.98 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.58 (d, J = 5.2 Hz, 1
H, pyrimidine H6), 10.32 (s, 1 H, NH); 13C NMR (100MHz,
DMSO-d6) δ (ppm) 61.17, 102.70, 111.11, 118.44, 118.49,
128.74, 130.00, 132.93, 133.35, 137.92, 144.57, 159.16, 159.96,
167.38; IR (KBr, cm-1) 2219 (CN); MS (ESI) m/z 363 [M-H]-.
5.2.13.
4-((4-(chloro(4-fluorophenyl)methyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (7m).
Yield 68%; light yellow solid；mp 127.8-128.2oC；1H NMR
(400MHz, DMSO-d6) δ (ppm) 2.42 (s, 3H, Me), 6.35 (s, 1H,
CH), 7.07 (s, 1 H, pyrimidine H5), 7.22-7.27 (m, 2 H, ArH2,6),
7.59-7.62 (m, 2 H, ArH3,5), 7.67 (d, J = 8.8 Hz, 2 H, ArH2,6), 7.93
(d, J = 8.8 Hz, 2 H, ArH3,5), 10.25 (s, 1 H, NH); 13C NMR
(100MHz, DMSO-d6) δ (ppm) 23.83, 61.49, 102.51, 110.45,
115.46, 115.67, 118.37, 119.60, 130.30, 130.39, 132.91, 135.40,
135.43, 159.07, 167.24, 169.73; IR (KBr, cm-1) 2213 (CN); MS
(ESI) m/z 351 [M-H]-.
5.3.2 .
4-((4-(bromo(p-tolyl)methyl)pyrimidin-2-
yl)amino)benzonitrile (7s).
Yield 70%; light yellow solid; mp 161.7-162.4oC; 1H
NMR(400MHz, DMSO-d6) δ (ppm) 2.28 (s, 3 H, Me), 6.42 (s, 1
H, CH), 7.12 (d, J = 4.8 Hz, 1 H, pyrimidine H5), 7.20 (d, J = 8.0
Hz, 2 H, ArH2,6), 7.51 (d, J = 8.0 Hz, 2 H, ArH3,5), 7.71 (d, J =
8.8 Hz, 2 H, ArH2,6), 7.98 (d, J = 8.8 Hz, 2 H, ArH3,5), 8.58 (d, J
= 5.2 Hz, 1 H, pyrimidine H6), 10.32 (s, 1 H, NH); 13C NMR
(100MHz, DMSO-d6) δ (ppm) 20.71, 53.01, 102.67, 111.50,
118.49, 128.69, 129.19, 132.96, 135.79, 138.22, 144.66, 159.24,
159.78, 167.85; IR (KBr, cm-1) 2220 (CN); MS (ESI) m/z 377
[M-H]-.
5.2.14.
4-((4-((4-bromophenyl)chloromethyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (7n).
1
Yield 65%; light yellow solid; mp 169.3-169.6oC; H NMR
(400MHz, DMSO-d6) δ (ppm) 2.43 (s, 3 H, Me), 6.37 (s, 1 H,
CH), 7.09 (s, 1 H, pyrimidine H5), 7.51 (d, J = 8.4 Hz, 2 H,
ArH2,6), 7.64 (d, J = 8.4 Hz, 2 H, ArH3,5), 7.68 (d, J = 8.8 Hz, 2
H, ArH2,6), 7.90 (d, J = 8.8 Hz, 2 H, ArH3,5), 10.27 (s, 1 H, NH);
13C NMR (100MHz, DMSO-d6) δ (ppm) 23.84, 61.36, 102.48,
110.54, 118.33, 121.93, 130.29, 131.64, 132.94, 138.49, 144.78,
5.4. Anti-HIV activity assay
The anti-HIV activity and cytotoxicity of the compounds 7a–s
were evaluated against wild type HIV-1 strain IIIB, a double RT

mutant (K103N + Y181C) HIV-1 strain and HIV-2 strain ROD in
MT-4 cell cultures using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) method.17,18 Briefly, stock
solutions (10 × final concentration) of test compounds were
added in 25 µl volumes to two series of triplicate wells so as to
allow simultaneous evaluation of their effects on mock-and HIV-
infected cells at the beginning of each experiment. Serial 5-fold
dilutions of test compounds were made directly in flat-bottomed
96-well microtiter trays using a Biomek 3000 robot (Beckman
instruments). Untreated control HIV-1 and mock-infected cell
samples were included for each sample. Virus stock (50 µl) at
100-300 CCID50 (50% cell culture infectious dose) or culture
medium was added to either the virus-infected or mock-infected
cells of the microtiter tray. Mock-infected cells were used to
evaluate the effect of test compounds on uninfected cells in order
to assess the cytotoxicity of the test compounds. Exponentially
growing MT-4 cells were centrifuged for 5 min at 220 g and the
supernatant was discarded. The MT-4 cells were resuspended at
6 × 10 cells/ml and 50 µL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of
mock-and HIV-infected cells was examined spectrophoto-
metrically by the MTT assay.
Molecular modeling was carried out with the Tripos molecular
modeling packages Sybyl-1.2. All the molecules for docking
were built using standard bond lengths and angles from Sybyl-
1.2/base Builder and then optimized using the Tripos force field
for 1000 generations two times or more, with an energy gradient
of 0.001 kcal/(molÅ) and Gasteiger–Hückel charges loaded, until
the minimized conformers of the ligand were the same. The X-
ray co-crystal structure of wild type RT in complex with ETV in
the Brookhaven Protein Data Bank (PDB entry code: 3MEC) was
used for docking. Prior to docking, the protein receptor was
preprocessed by removing the initial ligand. Then the free protein
was prepared by removing water molecules and other
unnecessary small molecules, and adding hydrogen atoms. The
atomic charges were recalculated using the Kollman all-atom
approach for the protein and the Gasteiger–Hückel approach for
the ligand. The geometry of the non-nucleoside binding site
(NNIBP) was defined by selecting all the residues within of the
initial inhibitor. Surflex-Dock default settings were used for other
parameters, such as the number of starting conformations per
molecule (set to 0), the size to expand search grid (set to 8Å), the
maximum number of rotatable bonds per molecule (set to 100),
and the maximum number of poses per ligand (set to 20). During
the docking procedure, all of the single bonds in residue side
chains inside the defined RT binding pocket were regarded as
rotatable or flexible, and the ligand was allowed to rotate on all
single bonds and move flexibly within the tentative binding
pocket. The binding interaction energy was calculated to include
van der Waals, electrostatic and torsional energy terms defined in
the Tripos force field. The structure optimization was performed
for 20000 generations using a genetic algorithm, and the 20 best
scoring ligand–protein complexes were kept for further analyses.
The –log(Kd)2 values of the 20 best scoring complexes, which
represented the binding affinities of ligand with RT, ranged over
a wide scope of functional classes. Therefore, only the highest
scoring 3D structural model of the ligand-bound RT was chosen
to define the binding interaction.
The MTT assay is based on the reduction of yellow colored
MTT (Acros Organics) by mitochondrial dehydrogenase activity
of metabolically active cells to a blue-purple formazan that can
be measured spectrophotometrically. The absorbances were read
in an eight-channel computer-controlled photometer (Infinite
M1000, Tecan) at two wavelengths (540 and 690 nm). All datas
were calculated using the median OD (optical density) values of
tree wells. The 50% cytotoxic concentration (CC50) was defined
as the concentration of the test compounds that reduced the
absorbance (OD540) of the mock-infected control sample by
50%. The concentration achieving 50% protection from the
cytopathic effect of the virus in infected cells was defined as the
50% effective concentration (EC50).
5.5. Molecular simulation
8. Chen, X.; Li, Y.; Ding, S.; Balzarini, J.; Pannecouque, C.; De
Clercq, E.; Liu, H.; Liu, X.; Chem. Med. Chem., 2013, 7, 1117-
1126.
Acknowledgments
9. Bailey, C. M.; Sullivan, T. J.; Iyidogan, P.; Tirado-Rives, J.;
Chung, R.; Ruiz-Caro, J.; Mohamed, E.; Jorgensen, W.; Hunter,
R.; Anderson, K. S.; J. Med. Chem., 2013, 10, 3889-3903.
10. Gu, S. X.; He, Q. Q.; Yang, S. Q.; Ma, X. D.; Chen, F. E.; De
Clercq, E.; Balzarini, J.; Pannecouque, C. Bioorg Med Chem.,
2011, 19, 5117-5124.
This research was financially supported by grants from
Chinese National Science and Technology Major Project (No.
2012ZX09103101-068), National Natural Science Foundation of
China (No. 81172918), Shanghai Municipal Natural Science
Foundation (No.13ZR1402200) and KU Leuven (GOA 10/014).
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12. Metrangolo, P.; Meyer, F.; Pilati, T.; Resnati, G.; Terraneo, G.;
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