Design and synthesis of a new series of modified CH-diarylpyrimidines as drug-resistant HIV non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.05.059

This article reports the design, synthesis and antiviral evaluation of a new series of non-nucleoside reverse transcriptase inhibitors (NNRTIs). The basic skeleton of these target 18 molecules is diarylpyrimidine featuring a substituted amino group between the pyrimidine scaffold and the aryl wing. All of the new compounds have been characterized by spectra analysis. The entire target molecules were evaluated for their in vitro anti-HIV activity with controlling group of FDA approved drugs. Most of them showed good to potent activities against wild-type (WT) HIV-1 with IC₅₀ values in the range of 0.0175-69.21 μM. 2-(4-Cyanophenylamino)-4-(2- cyanovinylphenylhydrazonomethyl)pyrimidine (1d) displayed potent anti-HIV-1 activity against WT HIV-1 with a selectivity index (SI) of 106367 and an IC ₅₀ value of 1.75 nM, which was 47 fold lower than that of AZT. Compound 1d also showed a broad-spectrum inhibitory activity, with an IC ₅₀ value of 5.33 μM and 5.05 μM against both HIV-1 double-mutated (K103N/Y181C) strain and HIV-2 strain, respectively. The preliminary structure-activity relationship (SAR) was also investigated. The binding modes with HIV-1 RT for both the wild type and mutant type have also been discussed.

Full text of DOI:10.1016/j.ejmech.2014.05.059

Accepted Manuscript
Design and synthesis of a new series of modified CH-diarylpyrimidines as drug-
resistant HIV non-nucleoside reverse transcriptase inhibitors
Ge Meng , Yang Liu , Aqun Zheng , Fener Chen , Wenxue Chen , Erik De Clercq ,
Christophe Pannecouque , Jan Balzarini
PII:
S0223-5234(14)00483-8
10.1016/j.ejmech.2014.05.059
EJMECH 7017
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 February 2014
Revised Date: 11 May 2014
Accepted Date: 25 May 2014
Please cite this article as: G. Meng, Y. Liu, A. Zheng, F. Chen, W. Chen, E. De Clercq, C. Pannecouque,
J. Balzarini, Design and synthesis of a new series of modified CH-diarylpyrimidines as drug-resistant
HIV non-nucleoside reverse transcriptase inhibitors, European Journal of Medicinal Chemistry (2014),
doi: 10.1016/j.ejmech.2014.05.059.
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ACCEPTED MANUSCRIPT
Graphic abstract
Design and synthesis of a new series of modified CH-diarylpyrimidines as drug
-resistant HIV non-nucleoside reverse transcriptase inhibitors
Ge Menga,*, Yang Liua, Aqun Zhengb, Fener Chenc,*, Wenxue Chenc, Erik De Clercqd, Christophe Pannecouqued, Jan Balzarinid
a School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China
b School of Science, Xi’an Jiaotong University, Xi’an, Shaanxi, 710049, P. R. China
c Department of Chemistry, Fudan University, Shanghai, 200433, P. R. China
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
A series of new diarylpyrimidine analogues featuring a functional substituted amino group between the pyrimidine scaffold and the
aryl wing have been designed and synthesized, which were also evaluated as anti-drug-resistant HIV reverse transcriptase inhibitors.

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
18 new compounds were designed, synthesized as the anti-drug resistant HIV NNRTIs.
All the new molecules were evaluated for their biological activities against HIV.
Compound 1d displayed anti HIV-1 activities 47 fold than that of AZT.
Compound 1d also showed activities against double mutated HIV-1 and HIV-2 strain.
SAR study including the docking analysis was also investigated deeply.

ACCEPTED MANUSCRIPT
Design and synthesis of a new series of modified CH-diarylpyrimidines as
drug-resistant HIV non-nucleoside reverse transcriptase inhibitors
Ge Menga,*, Yang Liua, Aqun Zhengb, Fener Chenc,*, Wenxue Chenc, Erik De Clercqd, Christophe
Pannecouqued, Jan Balzarinid
a School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China
b School of Science, Xi’an Jiaotong University, Xi’an, Shaanxi, 710049, P. R. China
c Department of Chemistry, Fudan University, Shanghai, 200433, P. R. China
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Article history:
Received
Revised
Accepted
Available online
This article reports the design, synthesis and antiviral evaluation of a new series of non-nucleoside reverse
transcriptase inhibitors (NNRTIs). The basic skeleton of these target 18 molecules is diarylpyrimidine featur-
ing a substituted amino group between the pyrimidine scaffold and the aryl wing. All of the new compounds
have been characterized by spectra analysis. The entire target molecules were evaluated for their in vitro
anti-HIV activity with controlling group of FDA approved drugs. Most of them showed good to potent activi-
ties against wild-type (WT) HIV-1 with IC50 values in the range of 0.0175-69.21 ꢀM.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylhydrazonomethyl)pyrimidine (1d) displayed potent an-
ti-HIV-1 activity against WT HIV-1 with a selectivity index (SI) of 106367 and an IC50 value of 1.75 nM,
which was 47 fold lower than that of AZT. Compound 1d also showed a broad-spectrum inhibitory activity,
with an IC50 value of 5.33 ꢀM and 5.05 ꢀM against both HIV-1 double-mutated (K103N/Y181C) strain and
HIV-2 strain, respectively. The preliminary structure-activity relationship (SAR) was also investigated. The
binding modes with HIV-1 RT for both the wild type and mutant type have also been discussed.
Keywords:
Non-nucleoside HIV RT inhibitors
Diarylpyrimidines
Cyanide group
Double mutant HIV-1
Docking analysis
SAR
@ 2014 Elsevier Masson SAS. All rights reserved.
rylpyrimidine
(DAPY)
analogs[18],
represented
by
1.
Introduction1
TMC125[19-21] and TMC278[22-24], which have been cor-
roborated by many crystallographic data[25-27].
The crystal structures of DAPY/HIV-1 RT complexes and
the relative molecular modeling results have revealed some
important features of enzyme-ligand interactions, which are
crucial for maintaining the antiviral activity against a wide
range of resistance mutations[27, 28]. DAPYs shared the similar
pharmacophore with the first-generation NNRTIs, including the
hydrophobic center, the hydrogen bond donor and acceptors, the
Human immunodeficiency virus type 1 reverse transcrip-
tase (HIV-1 RT) is one of the key enzymes in the HIV life cy-
cle[1], which represents one of the main targets for designing
selective agents for the treatment of HIV/AIDS[2].
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
against HIV-1 are the main and important drugs to combating
with the plague of AIDs[3]. Among the classes of compounds to
which the US FDA-approved RT inhibitors belong [4], the three
most important main types of NNRTIs are aromatic hetero-
cylclic compounds, including HEPTs[5, 6], DABOs[7, 8], dia-
rylpyrimidines (DAPYs) [9-11], etc[12]. All these therapeutic
agents shared the pyrimidine ring as the main structural skeleton
(Fig. 1) [6], which could directly bind with the allosteric site,
located 10-15 Å away from the catalytic site of RT [13-15].
While the emergence of RT mutations (such as Y181C, K103N,
etc.) rapidly confers resistance to the first-generation NNRTIs,
highly potent second-generation NNRTIs have also been identi-
fied[1, 16, 17], such as the Y181C mutation-associated dia-
active binding conformation of DAPYs resembled
a
horseshoe[29] or “U” shape when bound in the non-nucleoside
inhibitor binding pocket (NNIBP)[25, 27]. For example,
TMC125 could be taken as an example to delineate this interac-
tion mechanism in details as following: the ether and amino
linkages of the two cyanophenyl groups of the TMC125 could
provide sufficient flexibility to allow strong π-π stacking inter-
actions with the aromatic ring of Y181, Y188, F227 and
W229[27, 30], respectively. It was also suggested that DAPY
NNRTIs could bind to RT through torsional flexibility (“Wig-
gling”) and repositioning flexibility (“Jiggling”)[25]. Based on
the above concepts, a general strategy for designing flexible
drugs against a variety of drug-resistant mutants HIV-1 strains
has been proposed.
*
Abbreviations: NNRTIs, non-nucleoside reverse transcriptase inhibitors; HEPTs,
Considerable efforts made on the structural modifications
of the linker group of CH2-DAPYs might end up with various
effects on the biological activity via the assistance of the differ-
ent substituted groups on both sides of the aromatic rings[31,
32]. It has already been shown that there are still some possibili-
ties to achieve the anti-drug resistance effects by modifying the
linker group between the left benzene ring and the central py-
rimidine ring[33]. In order to identify more potent DAPYs as
possible NNRTIs, we have modified DAPY derivatives with
cyclopropylamino groups attached to the CH2- linker between
the left benzene ring and the central pyrimidine ring[34]. The
results have shown that both the cyano and the cyanovinyl
groups are beneficial for enhancement of the anti-HIV activity.
1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio) thymines; DABOs, 3,4-dihydro-2-alkyl-
6-benzyl-pyrimidin-4(3H)-ones; DAPYs, diarylpyrimidine derivatives; NVP, nevirapine;
DLV, delavirdine; HIV-1 RT, Human immunodeficiency virus type 1 reverse transcrip-
tase; US FDA, United States Food and Drug Administration; NNRTIs, non-nucleoside
RT inhibitors; EFV, efavirenz; TMC125, etravirine; TMC278, rilpivirine; NNIBP,
nonnucleoside inhibitor binding pocket; wt, wild-type; BPs, benzophenone derivatives;
SI, selectivity index; AZT, zidovudine; ESI, electrospray ionization; TMS, tetrame-
thylsilane; MTT, 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
CCID50, 50% cell culture infectious dose; CC50, 50% cytotoxic concentration; EC50, 50%
effective concentration; Kd, dissociation constant; IC50, 50% inhibitory concentration.
*Corresponding author. Tel./fax: +86 29 82656327-801.
E-mail address: mengge@mail.xjtu.edu.cn (G. Meng),
rfchen@fudan.edu.cn (F.-E. Chen).
1

ACCEPTED MANUSCRIPT
We therefore proposed that it might be a short cut to start from
also been proven to be very beneficial for improving the antivi-
ral activity, especially increasing the drug resistance activities,
which have been also verified by many biological assays[37].
fixing the cyano or cyanovinyl group while changing the at-
tached group on the CH2 linker. On the other hand, either a hy-
drazine[35] (2, Fig. 2) or hydroxyl[36, 37] (3, Fig. 2) groups has
Fig. 1. Structure of some important NNRTIs including HEPT, DABCO and DAPYs.
Guided by the mechanism of interaction between DAPY
and HIV-1 RT as well as the related SAR analysis of these ana-
logues, we wanted to find more drug candidates against the
drug-resistant HIV-1 strains. In this paper, we therefore have
designed and synthesized another series of new
4-(substituted-amino)arylmethyl DAPYs derivatives (1, Fig. 2)
accordingly. The target molecules shared the pyrimidine skele-
ton and the hydrazine or the hydroxyl-CH2 linker, as well as the
cyano and the cyanovinyl groups on both of the aromatic rings.
The chemical structures of all the new target molecules have
been characterized by spectra analysis data. The anti-HIV
screening tests show that almost all of the compounds show
moderate to excellent activity against HIV, of which compound
1d is the best one. The relative SAR discussions was also inves-
tigated together with the comparative docking analysis.
Fig. 2. Molecular design of new substituted CH-DAPYs.
2

ACCEPTED MANUSCRIPT
H
N
H
O
S
O
N
S
O
N
N
a
b
c
NH
NH
NH
77.2%
CN
65.8%
78.4%
5
6
4
CN
CN
CN
NH
R
R
e
d
9a R = 2-Cl
9b R = 4-Br
9c R = 4-CN
9d R = 4-C2H2CN
N
NH
24-65%
O
N
NH
Cl
N
NC
N
N
N
9a-9d
9b R = 4-Br
8a R = 2-Cl
8b R = 4-Br
f
7
9c R = 4-CN
without being isolated
g
9d R = 4-C2H2CN
CN
CN
R
R
h
i
N
NH
N
24.6-72.3%
N
NH
HN
R'
R = 2-Cl, 4-CN, 4-C2H2CN
R' = Me, Et, n-propyl, i-propyl,
-OH, -NH2, -CH2CH(OH)CH2
R'
N
N
10a-10r
without being isolated
1a-1r
10, 1
R
R’
10, 1
R
R’
Me
Et
10, 1
R
2-Cl
R’
a
b
c
d
e
f
4-CN
OH
OH
g
h
i
2-Cl
4-CN
m
n
o
n-Pr
4-CH=CHCN
4-CN
4-CN
i-Pr
i-Pr
NH2-NH
NH2-NH
Me
4-CH=CHCN
2-Cl
Et
4-CH=CHCN
2-Cl
4-CH=CHCN
4-CN
j
Et
p
q
r
i-Pr
k
l
4-CN
n-Pr
n-Pr
4-CN
2-Hydroxylpropyl
2-Hydroxylpropyl
4-CH=CHCN
Me
4-CH=CHCN
2-Cl
Scheme 1. Synthetic route of the new DAPYs derivatives (1a-1r). Reagents and conditions: (a) MeI, NaOH, H2O, r. t.; (b) 4-cyanoaniline, 180 ℃; (c) POC13,
reflux; (d) R (R=2-Cl or 4-Br)-phenylacetonitrile, 60 % NaH, Ar, DMF, -20℃ to r. t., 48-72 h; (e) NaH, air, DMF, r. t., 36-72 h; (f) CuCN, DMF, 150℃, 10h; (g)
acrylonitrile, Pd(OAc)2, P(o-Tol)3, NaOAc, DMA, 150℃, overnight; (h) R’-NH2, EtOH, Na2SO4, reflux, 4-8 h; (i) NaBH3CN, EtOH, 60 ℃, 4 h
DMF gave the corresponding 4-cyano intermediate 9c[34]. The
acrylonitrile derivative 9d was prepared via the coupling reac-
tion, which was conducted on 9b with classical Heck conditions
using palladium (II) diacetate as a catalyst in the presence of
sodium acetate in DMA[34]. Imino compounds 10a-10r were
synthesized by refluxing 9a-9d with various substituted amines
in ethanol with the assistance of small amount of acetic acid and
anhydrous sodium sulfate, respectively. Reductions of 10a-10r
with sodium cyanoborohydride in ethanol provided the target
compounds 1a-1r (Scheme 1).
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds 1a-1r was shown in
Scheme 1. Starting from commercially available material
2-thioxo-2,3-dihydropyrimidin-4(1H)-one (4) and iodomethane,
2-(methylthio)-2,3-dihydropyrimidin-4(1H)-one (5) were ob-
tained via methylation. Methylthio ether 5 was transformed into
4-((4-oxo-1, 2, 3, 4-tetrahydropyrimidin-2-yl)amino) benzo-
nitrile (6) through the reaction with excess amount of
4-cyanoniline at high temperature of 180℃. The purification of
6 was achieved by washing with organic solvent. The important
intermediates 4-(4-chloropyrimidin-2-yl-amino) benzonitriles (7)
was easily prepared from intermediate 6 via refluxing with
POC13. The key intermediates, oxo-CH2-DAPYs (9a-9d) were
conveniently synthesized from benzonitriles 7 in two steps
through our previously reported modified methods[31, 38]. The
cyanation of 9b with CuCN at 150 ℃ for 10 h in anhydrous
2.2. Biological activity
The activity and cytotoxicity of these newly synthesized
DAPY analogues were evaluated in MT-4 cells for their ability
to inhibit HIV-1 and HIV-2 induced cytopathogenicities. The
results, expressed as the cytotoxicity (CC50), half maximal (50%)
inhibitory concentration IC50 and selective index (SI
=
CC50/IC50), which indicates the specificity of the antiviral
effect, are illustrated in Table 1 together with those of the five
3

ACCEPTED MANUSCRIPT
FDA-approved drugs: Nevirapine, Zidovudine, Efavirenz,
Delavirdine and Etravirine as the reference standards. The cells
mutant (K103N/ Y181C) strain derived from wild-type LAI,
and HIV-2 strain (ROD).
were infected with HIV-1 wild-type virus (LAI) or double RT
Table 1
Anti-HIV activities and cytotoxicity of compounds 1a-1r MT-4 cells.
IC50 (µM)
Compd
R
R’
CC50
SI
IIIB
7.90±1.45
0.0163±0.0012
0.234±0.019
0.00175
RES056
>218.73
>160.946
5.95
ROD
>218.73
35.66±7.20
2.92±1.45
5.05±0.79
>189.13
>41.414
>6.09
1a
1b
1c
1d
1e
1f
4-CN
4-C2H2CN
4-CN
218.73±23.01
160.946±40.217
40.175±4.907
186.308±15.58
189.13±6.99
41.414±11.741
36.09±1.873
157.52±11.34
37.324±1.955
39.44±0.91
34.94±3.19
33.970±3.457
68.476±17.664
69.21±55.00
32.322±2.589
35.462±0.779
>65.03
28
9891
172
106367
12
4-C2H2CN
4-CN
5.33
15.17±7.29
0.108±0.049
0.063±0.015
21.06±9.16
0.192±0.041
0.35±0.01
≥9.45
>189.13
>41.414
>36.09
>157.52
>37.324
>39.44
>34.94
>37.324
4-C2H2CN
2-Cl
385
571
7
1g
1h
1i
4-CN
>157.52
>37.324
>39.44
4-C2H2CN
2-Cl
194
113
1j
1k
1l
4-CN
>34.94
≤4
4-C2H2CN
2-Cl
0.107±0.063
0.048±0.003
>69.21
>37.324
>68.476
>69.21
319
1422
na*
1m
1n
1o
1p
1q
>68.476
4-CN
>69.21
>32.322
>35.462
>65.03
>34.66
>34.71
>56.108
>37.89
0.90
4-C2H2CN
2-Cl
0.080±0.003
0.040±0.012
23.87±1.45
0.33±0.04
>34.71
>32.322
>35.462
>65.03
405
879
>3
4-CN
1r
2-Cl
>34.66
34.66±1.35
34.71±1.08
56.108±9.788
37.89±1.98
>15.021
105
<1
*
4-CN
>34.71
**
4-C2H2CN
2-Cl
0.196±0.03
0.12±0.01
0.101±0.015
3.89±2.89
0.0066±0.0006
0.0226
>56.108
>37.89
287
317
>148
>22
>14124
>280
>54
***
Nevirapine
Lamivudine
Zidovudine
Efavirenz
15.53±11.17
0.0017
>87.24
0.0077
0.16
>93.5489
6.3356
Delavirdine
0.81
43.84
*na: The data are not available or calculated.
*
**
,
*** : The data for the preparation of the compounds have been described in another manuscript.
,
In general, most of the tested compounds were active
activity to that of Efavirenz. From all the tested compounds,
2-(4-cyanophenylamino)-4-(2-cyanovinylphenylhydrazonometh
yl)pyrimidine (1d) was found to be the most promising one with
the lowest IC50 of 1.75 nM, 4 time as the reference compound
AZT. The toxicity of compound 1d is also very low with the SI
against the wild type of HIV-1 infected cell lines except for the
compounds with 4-cyanophenyl as the left wing of DAPYs. The
activities of these compounds are comparable to that of Nevi-
rapine. Some of the compounds show the similar biological
4

ACCEPTED MANUSCRIPT
of 1d being 106367 = 7 times than that of AZT.
tance of the 2-substituted groups on the aromatic rings. The
introduction of the cyanovinyl group could enhance the biolog-
ical potency against wild-type virus strain, while the same group
could also decrease the cytotoxicity, which again verified the
cyanovinyl group at 4-position to be superior to other substitu-
ents.
In addition, all the title compounds were also evaluated for
their capability to inhibit the HIV-2 (ROD) replication in MT-4
cells. The result showed that compound 1b, 1c, 1g also have
some activity against HIV-2, with the potency less than that of
AZT (Table 1). It is also interesting to note that the 4-cyano
group was the best one in keeping potency against HIV-2. This
similar sequence in enhancing ability of the different groups
might imply some similar mechanism in the different circum-
stances of both the HIV-1 and HIV-2. These findings showed
that this new series of DAPY derivatives was active against both
HIV-1 and HIV-2, which might be lead to a new way to broaden
the anti-virus spectrum.
As shown in Table 1, the substitution pattern on the phenyl
ring of DAPYs might play a determinant role in their HIV-1
inhibitory potency. In the case of the 4-cyanovinyl or 2-chloro
substituted analogues (1b, 1d, 1f, 1g, 1i, 1j, 1l, 1m, 1o, 1p and
1r), all of the compounds show very high activity. But as for the
4-cyano substitution, the majority of these compounds are inac-
tive against HIV-1 with an IC50 above 10 ꢀM, except for the
compound (1c) with a hydrazine group on the methylene linker.
The activity of 1c was not as high as what has been expected
from the SAR analysis. The reasons for the ordinary activity of
these compounds with similar substitutions can be explained as
follows.
It was well-known that the introduction of a cyano group
into DAPYs could elevate both the biological activity and the
anti-drug resistance ability via an interaction with the conserved
amino acid W229 in NNIBP. This kind of interaction can be
assisted by the 2-substituted group on the phenyl ring of DAPYs
with increased π-π stacking between the ligand and Y181 or
Y188 in the hydrophobic pocket of NNIBP by the restriction on
the flexible rotation of the left phenyl ring of DAPYs[39]. It was
also observed that most of the active DAPY derivatives shared
both 4-cyano group and 2-substituted groups. For example,
TMC125 contains two methyl groups on the 2, 6-positions of
the left phenyl ring (Fig. 1)[30].
The possibility for the insertion of 4-substituted group into
the W229-containing narrow pocket of NNIBP is depended on
both the physical properties (including the steric effects etc.) of
the 4-substituted group and the synergistic effect of the
C2-sustituted groups on the phenyl ring. In some circumustance,
the function of the C2-sustituted groups might exert much more
assisting effect than the 4-substituted group via fixing the suita-
ble conformations of the 4-substituted group. The 4-cyanovinyl
could adjust its conformation to adapt with the hydrophobic
channel by σ-bond rotation, while the cyano group could exert a
similar effect only by the assistance of the 2-substituted groups.
The biological result of the former series of DAPYs with cyclo-
propyl attached to the linker also supported these
suggestions[34].
Replacing cyclopropyl with a smaller or more flexible al-
kyl amino group could inevitably result in the enhancement of
the biological activities to 2-8 folds as the mother skeletons
including the less active cyano substituted molecules. It is quite
interesting that even the sequence of the activity for different
substitution groups are quite similar in the more active
4-cyanovinyl and 2-chloro series, which could be in the follow-
ing order: iso-propylamino > n-propylamino > methylamino >
ethylamino ≈ cyclopropylamino. The result also showed that the
flexibility of these substituted groups can play a very important
role in enhancing the activity together with the steric effects.
These alkyl groups are less effective than the hydrazino group,
which is probably related to the fact that the unsubstituted NH2
group could easily form an H-bond with the V179 residue,
while the substituted NH structures are less beneficial for the
H-bond formation, although the alkyl group could occupy more
space in the V179 pocket.
3.
Molecular docking analysis
The docking analysis about the possible binding mode has
been compared between the most potential compound 1d and
TMC278 with HIV-1 RTwt and double mutant HIV-1
RTK103N/Y181C, respectively. The results are shown in Fig. 3. The
following features of the docking models are worthy to be out-
lined in detail.
The torsion angles of different HIV-1 RTIs are very im-
portant parameters and correlated with the beneficial interaction
between the ligand and the HIV-1 RT, which is supposed to be
further related with the anti-HIV-1 biological activities[40].
Therefore, the torsion angles in the binding conformations of
TMC278 and the most potent compound 1d were calculated and
are further shown in Table 2 for comparison.
As shown in Fig. 3 (b), the binding mode between com-
pound 1d and wild type RT is illustrated as follow. The pyrimi-
dine ring of compound 1d is located in the space between L100
and V179. The torsional rotation initiated by the methylene
group could lead the pyrimidine ring of 1d not to be paralleled
with the pyrimidine plane of TMC278. While moving upward
for 0.5Å, the pyrimidine ring of 1d might get close to E138 for
about 0.5Å. Formed by both the K101 main chain carbonyl and
the NH group, the two hydrogen bonds in the crystal structure of
TMC278-RTwt (PDB ID: 2ZD1[25], Fig.3 (a)) still exist be-
tween 1d and K101 in the RT-1d docking model as a similar
fashion to that of TMC278, while the bond lengths of both of
the two hydrogen bond are 0.1Å longer than that of TMC278.
The right cyanophenyl rings of both compound 1d and TMC278
are almost overlapping with each other by comparison of (a) and
(b) in Fig. 3. Although the methylene group between the left
wing and pyrimidine ring moves over a distance from the NH of
TMC278 on account of the variation in τ, but the distance be-
tween the CH2 linker and E138 did not change markedly.
It is quite interesting to note that another three hydrogen
bonds are also formed between the NH2 of the hydrazino group
of 1d and the oxygen of Y188 (2.547 Å, 2.734 Å), the oxygen of
V179 (2.477Å), respectively. While the left phenyl ring of 1d
might rotate to some extent comparing with that of TMC278
with the variations in τ1 and τ3, it could still adopt the parallel
position to the phenyl ring of Y181 so as to keep the π-π stack-
ing interactions. Because of the remarkable changes in τ5, the
cyanovinyl group of compound 1d moved closer to W229 for
0.3 Å more than that of TMC278, which could explain the en-
hanced biological activity.
Interesting results were also observed in considering the
hydrazino group substitution on the NH-linker. There are two
compounds showed a micromolar activity on double mutant
strains (K103N/Y181C), comparable to Nevirapine but less
active than Efavirenz and Zidovudine. Anyway, the anti-drug
resistant activities of these two compounds were still similar to
that of the hydroxyl or hydrazino substituted DAPY derivatives
even if the cyanovinyl group was introduced into the DAPY
skeletons. This result might be explained by the disappearance
of the preferred fixing conformation due to lacking the assis-
5

ACCEPTED MANUSCRIPT
Fig. 3. Comparison of the binding modes between the representative potential compound 1d and TMC278 in NNRTBP of wild type of HIV-1 RT (PDB code:
2ZD1[25]) and K103N/Y181C double mutated HIV-1 RT (PDB code: 3BGR[25]). (a) TMC278/RTWT, (b) 1d/RTWT, (c) TMC278/RTK103N/Y181C, (d)
1d/RTK103N/Y181C The figures are generated by the software of SYBYL-X 2.0. (For interpretation of the references to color in these figures legend, the
.
reader is referred to the web version of this article.) Possible hydrogen bonds are indicated with dashed lines in red. The amino acid residues are shown in
liner frame and the docked conformations of the small molecules are shown in ball and stick both for 1d and TMC278, respectively.
Table 2
Comparison of the torsion angles of the binding conformations of TMC278 and compound 1d.
Torsion angles (°)
Compound and RT complex
τ1
τ2
τ3
τ4
-12.0
0.6
τ5
TMC278/RTWT
1d/ RTWT
85.0
-35.1
-98.4
16.1
28.0
16.88
-11.5
-48.9
-6.0
-49.6
-150.4
-49.2
TMC278/RTK103N/Y181C
-9.6
1d/RTK103N/Y181C
-79.6
37.0
-33.7
0.65
-67.5
TMC278
The K103N mutant and Y181C mutant are two of the most
commonly known mutants relevant to clinical NNRTI-resistance.
Generally speaking, the mutation of aromatic tyrosine to nonaro-
matic cysteine would cause a dramatic change from a hydrophobic
environment to a hydrophilic environment of the binding pocket,
so to abolish most of the possible hydrophobic contacts[41]. The
IC50 of TMC278 against K103N, Y181C and K103N+Y181C
double mutant strains are less than 1 nm. The cocrystal structure
for the complex of TMC278 and K103N/Y181C double mutant RT
is therefore retrieved from PDB (PDB ID: 3BGR[25]) and is
shown in Fig. 3 (c). The interaction mode show that the confor-
mation of TMC278 in mutant RT complex did not change signifi-
cantly compared with that in the wild type RT-complex. The tor-
sion angle τ3 in TMC278 might change by a slight outside angle of
5° away from C181. The mutation would lead to the disappearance
of the aromatic interaction between the phenyl ring and the aro-
matic ring of the primary Y181. At this point, the Y183MDD se-
quence at the active site of this polymerase would move for a dis-
tance of 1.5 Å, so as to lead the amino residue Y183 moving close
to the cyanovinyl group of TMC278, inducing another new kind of
6

interaction to compensate the lose in π-interaction caused by
These results of this paper might provide the useful indicators for
guiding further rational design of new DAPYs analogues as novel
HIV-1 NNRT inhibitors that are more active against drug resistant
HIV-strains.
Y181C. Although this kind of compensation eAffeCct CcaEusPedTbEyD MANUSCRIPT
Y183 is serendipitous, it could still be relied on in the further de-
signing new potent HIV-1 RT inhibitors since the YMDD is a
highly conserved sequence. This kind of new interaction with
Y183 in mutant strain of HIV-1 RT is caused by slight change in
the conformation of TMC278, which is contributed by rotation of
the cyanovinyl group.
5.
Experimental section
5.1. Chemistry
Chemical reagents and solvents, purchased from commercial
As for another mutant site N103, the interaction mode did not
change very much compared to that of K103 in wild type of RT.
N103 is situated below both the pyrimidine and phenyl ring,
forming a hydrogen bond (3.0 Å) with the carbonyl group of the
main chain of K101, without hampering the formation of the hy-
drogen bond between K101 and TMC278.
As shown in Fig. 3 (d), generally speaking, the interaction
mode of 1d with mutant type of HIV-1 RT is similar to that of
TMC278, but the whole skeleton of the molecule of 1d moved
distinctively upward. The pyrimidine ring moved for 1.5 Å, the
distances between E138 and V179 increased for about 1 Å and 0.3
Å, respectively. These changes would inevitably lead to the result
that the original conserved hydrogen bond in the TMC278-mutant
RT complex could not be formed between the N1 of 1d and K101.
The bond length of the hydrogen bond between the secondary
sources, were of analytical grade and were used without further
purification. Melting points were measured on a SGW X-1 micro-
scopic melting point apparatus. TLC analyses were carried on
precoated silica gel G plates at 254 nm under a UV lamp using a
variety of solvent systems. Column chromatography separations
1
were performed with silica gel (300-400 mesh). H NMR and 13C
NMR spectra were recorded on a Bruker AV400 MHz spectrome-
ter in DMSO-d6. Chemical shifts are reported in δ (ppm) units
relative to the internal standard tetramethylsilane (TMS). Mass
spectra were obtained on a Waters Quattro Micromass instrument
using electrospray ionization (ESI) techniques.
5.1.1. Preparation of 1-aminopropan-2-ol
Propylene epoxide (5.80 g, 100 mmol) was added slowly into
a concentrated ammonium hydroxide (50.0 mL) with stirring in an
ice bath. The reaction mixture was kept under 0℃ for 1 h, and the
evaporated under vacuo to get rid of the water to give the product
as the colorless oil (6.2 g), the GC-MS analysis showed that the
content percentage of 1-amino-2-propanol is 35.0 %, which could
be stoichiometrically used in the final step in the synthesis of the
target compounds (1q and 1r).
amine of 1d and K101 is 0.6
Å longer than that on
TMC278-complex, the result of which is not beneficial for en-
hancing the biological activity. The cyano group on the right wing
of 1d moved upward for about 0.8 Å, which might be beneficial
for the biological activity. The bond lengths of the hydrogen bonds
between the hydrazino group of 1d and Y188 and V179 are 2.070
Å and 2.571 Å, respectively.
The effect of Y181C mutation on this skeleton of 1d shall be
discussed as follows. The losing of π-π stacking effect in the in-
teractions might be crucial for drug resistance. TMC278 show the
high anti-drug resistance effect for the newly formed interaction
with Y183 instead of the original π-π stacking in wild type. The
changing in torsion angle τ5 could lead the cyanovinyl group of 1d
to get deepen into the hydrophobic channel composed of W229,
Y188 and L234 and get much closer to Y188. From theoretical
speculation, compound 1d should have excellent anti-drug re-
sistance, but the experimental result show that 1d is only a moder-
ate drug-resistant inhibitor. The result could be explained by the
different interaction with other positions of the molecule of 1d,
especially by the disappearance of the conserved hydrogen bond
between the pyrimidine ring of 1d and K101, which might influ-
ence the biological activity significantly. Quite similar to that of
TMC278, the variation in the interaction between 1d with N103 is
not very distinct, for most of the DAPYs derivatives are active
against the K103N mutant strain.
5.1.2. Synthesis of 2-(methylthio)-2,3-dihydropyrimidin-4(1H)
-one(5)
NaOH (12.6 g, 315 mmol) was dissolved in water (300 mL),
to which 2-thiouracil (3, 38.4 g, 300 mmol) was added with stir-
ring to make the materials to dissolve completely. Methyl iodide
(53.3 g, 375 mmol) was then added to the above solution. The
reaction mixture was then kept stirring at r. t. for 24 h with TLC
monitoring. A large amount of white solid appeared from the reac-
tion mixture while being adjusted the pH to 7 with acetic acid. The
product was obtained by filtration and washed with small amount
of water, dried in vacuo to afford the intermediate 5 with the yield
of 77.2%.
5.1.3. Synthesis of 2-(4-cyanophenylamino)pyrimidin-4(1H)
-one(6)
Intermediate 5 (28.4 g, 200 mmol) was mixed well together
with 4-aminobenzonitrile (59.0 g, 500 mmol). The reaction tem-
perature was rise to 160 ℃ to melt the solids, and was then al-
lowed to be raised to 180-190 ℃ with stirring for 10 h. The reac-
tion mixture was cooled down a little bit before adding acetonitrile
to smash the solid with ultrasonic conditions. The product was
obtained by filtration and washed with a small amount of acetoni-
trile and methylene chloride. It was then dried in vacuo to give the
product 6 as yellow solid with the yield of 65.8%.
4.
Conclusions
In summary, a series of new 4-(substituted-amino) arylmethyl
DAPYs were successfully synthesized as anti-HIV RT agents
against the drug resistant virus strains. Biological evaluation indi-
cated that most of these derivatives showed good to potent activi-
ties against wild-type (WT) HIV-1 with IC50 values in the range of
69.21-0.0175 ꢀM. Compound 1d displayed potent anti-HIV-1
activity against WT HIV-1 with an IC50 value of 1.75 nM and a SI
of 106367, 47 fold lower than that of AZT. The compound 1d also
showed a broad-spectrum inhibitory activity, with an IC50 value of
5.33 ꢀM and 5.05 ꢀM against HIV-1 double -mutated
(K103N/Y181C) strain and HIV-2 strain, respectively. These at-
tributes earmark compound 1d as a potential new lead for devel-
oping more therapeutically applicable inhibitors against both
HIV-1 and HIV-2 with better resistance profile. A preliminary
SAR analysis of these target molecules highlighted a preference
for the smaller or more flexible groups substitution in the linker
between the left ring and the pyrimidine skeleton for enhancing
the anti-HIV activity. It was also demonstrated that 4-cyano group
substitution was also the best one in keeping potency against
HIV-2. The subsequent studies undertaken to evaluate the different
interaction modes of the most potential compound 1d with both
the wild type and mutant type of HIV-1 RT have identified the
similar interaction modes with the FDA-approved drug TMC278.
5.1.4. Synthesis of 2-(4-cyanophenylamino)-4-chloropyrimidine
(7)
Compound 6 (32.1 g, 150 mmol) was mixed with phosphorus
oxychloride (40.0 mL) and then heating to refluxing for 0.5 h. The
reaction mixture was then slowly added to icy water (250 mL)
with vigorously stirring. The yellow solid was precipitated and
filtrated to give a filtrate cake, which was then again added into
water (50.0 mL) and was adjusted to pH = 7 with sodium hydrox-
ide. The solid was obtained by filtration and dried under vacuo to
provide the intermediate 7 as pale yellow solids with the yield of
78.4%.
5.1.5. General procedure for the synthesis of 2-(4-cyano phe-
nylamino)-4-(2-chlorobenzoyl)pyrimidine (9a) and 2-(4- cyano-
phenylamino)-4-(4-bromobenzoyl)pyrimidine (9b)
To dried DMF (30.0 mL) was added the intermediate 7 (2.00
7

mmol) and the halogenated phenylacetonitrile (a, R = 2-Cl, 755
mg; b, 4-Br, 975 mg; 5.00 mmol). The reaction mixture was
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H, NH), 8.55 (t, J =
4.5 Hz, 1H, pyrimidine H6), 7.88 (d, J = 8.5 Hz, 2H, ArH3,5),
7.80-7.33 (m, 7H, Ar’H+alkenyl H+ ArH2,6), 7.18 (d, J = 5.1 Hz,
1H, pyrimidine H5), 6.53-6.26 (m, 2H, alkenyl H+OH), 5.63 (d, J
= 4.1 Hz, 1H, CH); 13C NMR (100 MHz, DMSO-d6) δ 173.41,
159.41, 159.20, 150.75, 146.57, 145.34, 133.48, 133.36 (2C),
128.14 (2C), 127.78 (2C), 120.05, 119.28, 118.74 (2C), 110.03,
102.77, 97.08, 74.95; MS (ESI+) m/z 354 (M+H)+; HR-MS: m/z
Calcd for C21H15N5O: 353.1277. Found: 353.1285.
ACCEPTED MANUSCRIPT
cooled down to -20 ℃ within an ethanol cooling bath. NaH
(60.0 %, 184 mg, 4.60 mmol) was added under nitrogen atmos-
phere and was continued with stirring for 1 h. The reaction mix-
ture was raised to r. t. for 24-72 h with the monitoring by TLC.
The intermediate 8 (8a or 8b) was then obtained without further
purifications. The reaction mixture was then kept for another
24-72 h without N2 protection until the TLC analysis showed the
disappearance of compound 8. The reaction mixture was then
poured into water (300 mL) and acidified with HCl to pH = 7. The
obtained mixture was extracted with ethyl acetate (150 mL × 3),
dried over anhydrous sodium sulfate, and the solvent was evapo-
rated to give the solid, which was recrystallized with acetonitrile to
afford the important intermediate 9a or 9b, with a yield of 24-65%,
respectively.
2) General procedure for the synthesis of hydrazino substituted
CH2-DAPYs target molecules (1c, 1d)
The intermediate 9 (9c, 975 mg; or 9d, 1.05 g; 3.00 mmol)
and the hydrazine hydrochloride (411 mg, 6.00 mmol) were dis-
solved in ethanol (30.0 mL), to which pyridine (1.94 mL, 1.90 g,
d= 0.98 g/mL, 24.0 mmol) was added. The reaction mixture was
refluxed for 2 h before cooling down to r. t.. The obtained mixture
was then poured into water (100 mL) and extracted with ethyl
acetate. The extraction was dried over anhydrous sodium sulfate
and was then concentrated under vacuo to get rid of the excess
solvent. The residue was purified with column chromatography to
give the pure product 1c and 1d, respectively. The yields, the
physical properties and the related spectra data of these two com-
pounds were as follows:
5.1.6. Synthesis of 2-(4-cyanophenylamino)-4-(4-cyanobenzoyl)
pyrimidine (9c)
A mixture of 9b (756 g, 2.00 mmol), CuCN (178 mg, 2.00
mmol) and DMF (2.00 mL) was heated at 150℃ for 10 h with
stirring, then cooled. The suspension was a solution of iron (III)
chloride (1.00 g), concentrated HCl (0.20 mL), and water (20.0
mL). The mixture was stirred at 70℃ for 20 min and extracted
with CH2Cl2 (20.0 mL×3). The combined organic layers were
dried over anhydrous MgSO4, filtered and concentrated in vacuo.
Purification by silica gel column chromatography (silica gel;
CH2Cl2) gave 9c.
2-(4-Cyanophenylamino)-4-(4-cyanophenylhydrazonomethyl)p
yrimidine (1c) Yield: 49.0%; yellow solid; 1H NMR (400 MHz,
DMSO-d6) δ 9.89 (s, 1H, NH), 8.40 (d, J = 5.4, 1H, pyrimidine
H6), 8.04 (d, J = 8.0, 2H, ArH3,5), 7.59 (s, 2H, NH2), 7.54-7.44 (d,d,
4H, ArH2,6+ Ar’H2,6), 7.43-7.32 (m, 3H, Ar’H+ pyrimidine H5);
13C NMR (100 MHz, DMSO-d6) δ 164.54, 159.24, 157.75, 145.42,
138.96, 138.38, 133.34 (2C), 132.85 (2C), 131.44 (2C), 120.04,
119.37, 118.41 (2C), 111.48, 107.74, 102.27; MS (ESI+) m/z 340
(M+H)+; HR-MS: m/z Calcd for C19H13N7: 339.1232. Found:
339.1245.
5.1.7. Synthesis of 2-(4-cyanophenylamino)-4-(2-cyanovinyl)
benzoyl)pyrimidine (9d)
A mixture of 9b (756 mg, 2.00 mmol.), acrylonitrile (1.06 g,
20.0 mmol), Pd(OAc)2 (89.8 mg, 400 mmol), tri-O-tolylphosphine
(0.60 g, 2.00 mmol) and sodium acetate (656 mg, 8.00 mmol.) in
anhydrous DMA (30.0 mL) was stirred in a sealed tube at 150℃
overnight. The reaction mixture was hydrolyzed with water and
extracted with CH2Cl2 (20.0 mL×3). The organic layer was dried
over anhydrous MgSO4 and concentrated under reduced pressure.
The crude product 9d was recrystallized from acetonitrile to pro-
vide the pure product, which could be used directly in the next
step.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylhydrazonomet
1
hyl)pyrimidine (1d) Yield: 38.2%; yellow solid; H NMR (400
MHz, DMSO-d6) δ 9.87 (s, 1H, NH), 8.39 (d, J = 5.3 Hz, 1H, py-
rimidine H6), 8.16-7.22 (m, 12H, ArH +Ar’H +pyrimidine
H5+NH2+alkenyl H), 6.60 (d, J = 16.8 Hz, 1H, alkenyl H); 13C
NMR (100 MHz, DMSO-d6) δ 164.83, 159.26, 157.70, 150.84,
145.44, 140.16, 135.71, 134.19, 132.86 (2C), 130.80 (2C), 128.75
(2C), 120.04, 119.31, 118.46 (2C), 107.91, 102.19, 97.67; MS
(ESI+) m/z 366 (M+H)+; HR-MS: m/z Calcd for C21H15N7:
365.1389. Found: 365.1401.
5.1.8. Synthesis of target compounds 1a-1r
1) General procedure for the synthesis of hydroxyl substituted
CH2-DAPYs target molecules (1a, 1b)
3) General procedure for the synthesis of rest target molecules
(1e-1r)
The intermediate 9 (9c, 975 mg; or 9d, 1.05 mg; 3.00 mmol)
was dissolved in methanol (30.0 mL). KBH4 (108 mg, 2.00 mmol)
was then added into the above solution while cooling down in an
ice bath. The reaction mixture was kept stirring at this temperature
for 2 h before pouring into water (100 mL) under stirring. The
mixture was extracted with ethyl acetate (50.0 mL × 3), dried over
anhydrous Na2SO4, and was then concentrated to evaporate the
excess solvent. The residue was purified with column chromatog-
raphy (EtOAc/petroleum ether = 1 : 4) to give the pure product 1a
and 1b, respectively. The yields, the physical properties and the
related spectra data of these two compounds were as follows:
To a mixture of 9 (1.00 mmol), Na2SO4 (213 mg, 1.50 mmol)
and ethanol (20.0 mL) was added various substituted amine (5.00
mmol) and 5 drops of acetic acid. The mixture was heated to re-
flux for 4-8 h monitored by TLC, then cooled. Sodium cyanobo-
rohydride was added at r. t. and subsequently the mixture was
heated at 60 ℃ for 4 h, then poured into saturated sodium bicar-
bonate and extracted with CH2Cl2 (20.0 mL × 3). The organic lay-
ers were combined together, dried over anhydrous MgSO4, and
concentrated under vacuo. The residue was then purified by silica
gel column chromatography (silica gel; EtOAc/petroleum ether,
1:5 to 1:3). The yields, the physical properties and the related
spectra data of these compounds were as follows:
2-(4-Cyanophenylamino)-4-(4-cyanophenylhydroxymethyl)
pyrimidine (1a) Yield: 67.6%; gray white solid; mp
1
177.9-179.7 ℃; H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H,
NH), 8.56 (d, J = 4.9 Hz, 1H, pyrimidine H6), 7.85 (d, d, J = 8.6
Hz, 4H, 2-ArH3,5+ 4-Ar’H3’,5’), 7.66 (d, d, J = 8.3 Hz, 4H,
2-ArH2,6+ 4-Ar’H2’,6’), 7.18 (d, J = 4.9 Hz, 1H, pyrimidine H5),
6.53 (d, J = 4.1 Hz, 1H, OH), 5.69 (d, J = 3.9 Hz, 1H, CH); 13C
NMR (100 MHz, DMSO-d6) δ 172.85, 159.59, 159.22, 148.98,
145.27, 133.38 (2C), 132.71 (2C), 128.18 (2C), 120.03, 119.28,
118.75 (2C), 110.69, 110.11, 102.84, 74.64; MS (ESI+) m/z 328
(M+H)+; HR-MS: m/z Calcd for C19H13N5O: 327.1120. Found:
327.1129.
2-(4-Cyanophenylamino)-4-(4-cyanophenylmethylaminomethy
l)pyrimidine (1e) Yield: 24.6%; gray yellow solid; mp
71.0-72.4 ℃; 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H, NH),
8.50 (d, J = 5.0, 1H, pyrimidine H6), 7.85 (d, J = 8.6, 2H, ArH3,5),
7.80 (d, J = 8.1, 2H, Ar’H), 7.71-7.56 (d, d, 4H, Ar’H2’,6’ +,
ArH2,6), 7.10 (d, J = 5.0, 1H, pyrimidine H5), 4.75 (s, 1H, CH),
2.25 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 171.61,
159.53, 159.36, 147.77, 145.21, 133.42 (2C), 132.83 (2C), 129.14
(2C), 120.05, 119.26, 118.79 (2C), 111.42, 110.58, 102.83, 68.98,
34.57; MS (ESI+) m/z 341 (M+H)+; HR-MS: m/z Calcd for
C20H16N6: 340.1436. Found: 340.1449.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylhydroxymethy
l)pyrimidine (1b) Yield: 58.7%; yellow solid; mp 193.5-195.1 ℃;
8

2H, CH2), 1.55-1.37 (m, 2H, CH2), 0.84 (t, J = 7.1 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ 172.08, 159.55, 159.24, 148.33,
145.29, 133.38 (2C), 132.79 (2C), 129.09 (2C), 120.03, 119.26,
118.76 (2C), 111.46, 110.52, 102.83, 67.26, 49.73, 23.16, 12.21;
MS (ESI+) m/z 369 (M+H)+; HR-MS: m/z Calcd for C22H20N6:
368.1749. Found: 368.1761.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenyAlmCethCy1lHamNinMoR
EPTED MANUSCRIPT
methyl)pyrimidine (1f) Yield: 26.7%; yellow solid;
(400 MHz, DMSO-d6) δ 10.12 (s, 1H, NH), 8.50 (t, J = 6.0 Hz, 1H,
pyrimidine H6), 7.89 (d, J = 9.0 Hz, 2H, ArH3,5), 7.83-7.45 (m, 7H,
ArH2,6+Ar’H+alkenyl H), 7.11 (dd, J = 9.8, 4.6 Hz, 1H, pyrimi-
dine H5), 6.10 (dd, J = 14.5 Hz, 1H, alkenyl H), 4.79-4.52 (m, 1H,
CH), 2.26 (d, J = 5.2 Hz, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 172.58, 159.56, 159.48, 150.72, 145.66, 145.35,
133.37 (2C), 133.34, 128.61 (2C), 128.28 (2C), 120.05, 119.32,
118.77 (2C), 111.36, 102.75, 96.95, 67.25, 32.8; MS (ESI+) m/z
367 (M+H)+; HR-MS: m/z Calcd for C22H18N6: 366.1593. Found:
366.1607.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylpropylaminom
ethyl)pyrimidine (1l) Yield: 34.0%; yellow solid; mp
60.7-61.5 ℃; 1H NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H, NH),
8.46 (d, J = 4.7 Hz, 1H, pyrimidine H6), 7.89 (d, J = 8.5 Hz, 2H,
ArH3,5), 7.79-7.40 (m, 7H, ArH3,5+ Ar’H+ alkenyl H), 7.08 (d, J =
7.0 Hz, 1H, pyrimidine H5), 6.07 (dd, J = 14.5 Hz, 1H, alkenyl H),
4.78 (s, J = 8.6 Hz, 1H, CH), 2.45-2.34 (m, J = 7.0 Hz, 2H, CH2),
1.44 (dd, J = 14.3, 7.2 Hz, 2H, CH2), 0.81 (t, J = 7.3 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ 172.46, 159.49, 159.05, 150.69,
145.67, 145.30, 133.36 (2C), 133.30, 128.62 (2C), 128.28 (2C),
120.05, 119.29, 118.76 (2C), 111.39, 102.77, 96.91, 67.33, 49.71,
23.09, 12.18; MS (ESI+) m/z 395 (M+H)+; HR-MS: m/z Calcd for
C24H22N6: 3394.1906. Found: 394.1918.
2-(4-Cyanophenylamino)-4-(2-chlorophenylmethylaminometh
yl)pyrimidine (1g) Yield: 78.5%; earth yellow solid; mp
1
130.5-131.6 ℃; H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H,
NH), 8.52 (d, J = 4.8 Hz, 1H, pyrimidine H6), 7.84 (d, J = 8.3 Hz,
2H, ArH3,5), 7.61 (d, J = 8.3 Hz, 2H, ArH2,6), 7.56 (d, J = 7.6 Hz,
1H, Ar’H), 7.44 (d, J = 7.9 Hz, 1H, Ar’H), 7.37 (t, J = 7.4 Hz, 1H,
Ar’H), 7.29 (t, J = 7.5 Hz, 1H, Ar’H), 7.08 (d, J = 4.9 Hz, 1H,
pyrimidine H5), 5.08 (s, 1H, CH), 2.28 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6) δ 171.42, 159.55, 159.14, 145.33, 139.46,
133.66, 133.26 (2C), 129.81, 129.76, 129.35, 127.84, 120.04,
118.70 (2C), 111.85, 102.70, 65.59, 34.86; MS (ESI+) m/z 350
(M+H)+; HR-MS: m/z Calcd for C19H16ClN5: 349.1094. Found:
349.1109.
2-(4-Cyanophenylamino)-4-(2-chlorophenylpropylaminomethy
l)pyrimidine (1m) Yield: 72.3%; light yellow solid; mp
1
125.5-126.8 ℃; H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H,
NH), 8.52 (d, J = 5.0 Hz, 1H, pyrimidine H6), 7.84 (d, J = 8.8 Hz,
2H, ArH3,5), 7.61 (d, J = 8.7 Hz, 2H, ArH2,6), 7.59-7.55 (m, 1H,
Ar’H), 7.44 (d, J = 7.7 Hz, 1H, Ar’H), 7.36 (d, J = 7.4 Hz, 1H,
Ar’H), 7.33-7.26 (m, 1H, Ar’H), 7.09 (d, J = 5.0 Hz, 1H, pyrimi-
dine H5), 5.20 (s, 1H, CH), 2.45 (t, J = 7.0 Hz, 2H, CH2),
1.55-1.37 (m, J = 7.2 Hz, 2H, CH2), 0.85 (t, J = 7.4 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ 171.68, 159.53, 159.12, 145.35,
139.81, 133.57, 133.27 (2C), 129.90, 129.76, 129.32, 127.86,
120.03, 118.69 (2C), 111.84, 102.70, 63.68, 49.87, 23.15, 12.23;
MS (ESI+) m/z 378 (M+H)+; HR-MS: m/z Calcd for C21H20ClN5:
377.1407. Found: 377.1419.
2-(4-Cyanophenylamino)-4-(4-cyanophenylethylaminomethyl)
1
pyrimidine (1h) Yield: 49.6%; yellow solid; mp 51.0-52.8 ℃; H
NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H, NH), 8.50 (d, J = 5.1
Hz, 1H, pyrimidine H6), 7.88 (d, J = 8.8 Hz, 2H, ArH3,5), 7.80 (d, J
= 8.2 Hz, 2H, Ar’H), 7.73-7.59 (d, d, 4H, Ar’H2’6’+ArH2,6), 7.12
(d, J = 5.1 Hz, 1H, pyrimidine H5), 4.87 (s, 1H, CH), 2.49-2.40 (m,
2H, CH2), 1.05 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 172.06, 159.54, 159.25, 148.29, 145.28, 133.39 (2C),
132.80 (2C), 129.09 (2C), 120.04, 119.27, 118.76 (2C), 111.43,
110.51, 102.82, 67.15, 42.02, 15.52; MS (ESI+) m/z 355 (M+H)+;
HR-MS: m/z Calcd for C23H23N5O: 354.1593. Found: 354.1605.
2-(4-Cyanophenylamino)-4-(4-cyanophenylisopropylaminomet
hyl)pyrimidine (1n) Yield: 46.8%; yellow solid; mp 60.9-62.6 ℃;
1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, NH), 8.50 (d, J =
4.8 Hz, 1H, pyrimidine H6), 7.88 (d, J = 8.2 Hz, 2H, ArH3,5), 7.80
(d, J = 8.0 Hz, 2H, Ar’H), 7.72-7.62 (d, d, 4H, Ar’H2’,6’+ArH2,6),
7.14 (d, J = 4.8 Hz, 1H, pyrimidine H5), 5.00 (s, 1H, CH),
2.66-2.56 (m, 1H, CH), 1.01 (d, J = 5.7 Hz, 6H, 2CH3); 13C NMR
(100 MHz, DMSO-d6) δ 172.22, 159.54, 159.15, 148.58, 145.29,
133.38 (2C), 132.77 (2C), 129.12 (2C), 120.02, 119.27, 118.76
(2C), 111.74, 110.45, 102.84, 64.31, 46.25, 23.37, 23.10; MS
(ESI+) m/z 369 (M+H)+; HR-MS: m/z Calcd for C22H20N6:
368.1749. Found: 368.1763.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylethylaminome
thyl)pyrimidine (1i) Yield: 50.7%; yellow solid; mp 68.2-69.9 ℃;
1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H, NH), 8.48 (d, J =
5.1 Hz, 1H, pyrimidine H6), 7.90 (d, J = 8.7 Hz, 2H, ArH3,5), 7.66
(d, J = 8.7 Hz, 2H, ArH2,6), 7.63-7.54 (m, 3H, Ar’H + alkenyl H),
7.49 (d, J = 8.1 Hz, 2H, Ar’H), 7.10 (d, J = 5.1 Hz, 1H, pyrimidine
H5), 6.38 (d, J = 16.7 Hz, 1H, alkenyl H), 4.80 (s, 1H, CH),
2.49-2.42 (m, J = 7.3 Hz, 2H, CH2), 1.04 (t, J = 7.0 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ 172.48, 159.50, 159.08, 150.71,
145.63, 145.32, 133.39 (2C), 133.33, 128.62 (2C), 128.29 (2C),
120.06, 119.30, 118.76 (2C), 111.35, 102.76, 96.96, 67.24, 42.03,
15.45; MS (ESI+) m/z 381 (M+H)+; HR-MS: m/z Calcd for
C23H20N6: 380.1749. Found: 380.1755.
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylisopropyl
aminomethyl)pyrimidine (1o) Yield: 39.8%; yellow solid; mp
1
58.7-59.0 ℃; H NMR (400 MHz, DMSO-d6) δ 10.09 (d, J = 8.0
Hz, 1H, NH), 8.60-8.32 (m, 1H, pyrimidine H6), 7.87 (t, J = 8.7
Hz, 2H, ArH3,5), 7.78-7.39 (m, 7H, ArH3,5+ Ar’H+ alkenyl H),
7.10 (dd, J = 8.8, 5.1 Hz, 1H, pyrimidine H5), 6.16 (dt, J = 9.0 Hz,
1H, alkenyl H), 4.93 (d, J = 8.8 Hz, 1H, CH), 2.59 (dt, J = 12.2,
6.0 Hz, 1H, CH), 0.99 (d, J = 6.0 Hz, 6H, 2CH3); 13C NMR (100
MHz, DMSO-d6) δ 172.55, 159.49, 158.96, 150.67, 145.85,
145.29, 133.35 (2C), 133.23, 128.63 (2C), 128.27 (2C), 120.04,
119.28, 118.77 (2C), 111.68, 102.80, 96.89, 64.39, 46.19, 23.29,
22.97; MS (ESI+) m/z 395 (M+H)+; HR-MS: m/z Calcd for
C24H22N6: 394.1906. Found: 394.1921.
2-(4-Cyanophenylamino)-4-(2-chlorophenylethylaminomethyl)
pyrimidine (1j) Yield: 64.4%; gray white solid; mp
1
117.8-119.3 ℃; H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H,
NH), 8.50 (d, J = 5.0 Hz, 1H, pyrimidine H6), 7.83 (d, J = 8.6 Hz,
2H, ArH3,5), 7.58 (d, d, J = 8.1 Hz, 3H, ArH2,6+Ar’H2’,6’), 7.42 (d,
J = 7.8 Hz, 1H, Ar’H), 7.36 (t, J = 7.3 Hz, 1H, Ar’H), 7.29 (d, J =
7.3 Hz, 1H, Ar’H), 7.07 (d, J = 5.0 Hz, 1H, pyrimidine H5), 5.20 (s,
1H, CH), 2.54-2.46 (m, 2H, CH2), 1.04 (t, J = 7.0 Hz, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ 171.61, 159.51, 159.11, 145.31,
139.73, 133.53, 133.25 (2C), 129.85, 129.74, 129.32, 127.85,
120.04, 118.70 (2C), 111.83, 102.70, 63.47, 42.10, 15.48; MS
(ESI+) m/z 364 (M+H)+; HR-MS: m/z Calcd for C20H18ClN5:
363.1251. Found: 363.1259.
2-(4-Cyanophenylamino)-4-(2-chlorophenylisopropylaminomet
hyl)pyrimidine (1p) Yield: 64.3%; light yellow solid; mp
1
143.6-144.9 ℃; H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H,
NH), 8.51 (d, J = 5.1 Hz, 1H, pyrimidine H6), 7.84 (d, J = 8.7 Hz,
2H, ArH3,5), 7.61 (d, d, J = 8.2 Hz, 3H, Ar’H5’+ArH2,6), 7.45 (d, J
= 7.8 Hz, 1H, Ar’H2’), 7.36 (t, J = 7.2 Hz, 1H, Ar’H3’), 7.30 (t, 1H,
Ar’H4’), 7.10 (d, J = 5.1 Hz, 1H, pyrimidine H5), 5.34 (s, 1H, CH),
2.68-2.60 (m, 1H, CH), 1.02 (d, J = 5.8 Hz, 6H, 2CH3); 13C NMR
(100 MHz, DMSO-d6) δ 171.90, 159.49, 159.07, 145.33, 140.00,
133.48, 133.26 (2C), 130.01, 129.78, 129.30, 127.86, 120.02,
118.69 (2C), 111.87, 102.72, 60.91, 46.36, 23.57, 22.95; MS (ESI+)
2-(4-Cyanophenylamino)-4-(4-cyanophenylpropylaminomethyl
)pyrimidine (1k) Yield: 35.5%; yellow solid; mp 39.2-41.0 ℃; 1H
NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, NH), 8.51 (d, J = 4.4
Hz, 1H, pyrimidine H6), 7.89 (d, J = 8.3 Hz, 2H, ArH3,5), 7.81 (d, J
= 7.6 Hz, 2H, Ar’H), 7.75-7.56 (d, d, 4H, Ar’H2’,6’+ArH2,6), 7.12
(d, J = 4.5 Hz, 1H, pyrimidine H5), 4.85 (s, 1H, CH), 2.46-2.34 (m,
9

m/z 378 (M+H)+; HR-MS: m/z Calcd for C21H20ClN5: 377.1407.
Found: 377.1419.
fected cells was defined as the 50% effective concentration (EC50).
Reverse transcriptase assay: Recombinant wild type p66/p51
ACCEPTED MANUSCRIPT
HIV-1 RT was expressed and purified as the previously described
procedure[44]. The RT assay was performed with the EnzCheck
Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen), as
described by the manufacturer. The assay was based on the dsD-
NA quantitation reagent PicoGreen. This reagent showed a pro-
nounced increase in fluorescence signal upon binding to dsDNA or
RNA-DNA heteroduplexes. Single-stranded nucleic acids generate
only minor fluorescence signal enhancement when a sufficiently
high dye: base pair ratio was applied[45].
A poly(rA) template of approximately 350 bases long, and an
oligo(dT)16 primer, were annealed in a molar ratio of 1:1.2 (60 min,
at r. t.). 52 ng of the RNA/DNA was brought into each well of a
96-well plate in a volume of 20 mL polymerization buffer (60 mM
Tris-HCl, 60 mM KCl, 8 mM MgCl2, 13 mM DTT, 100 mM dTTP,
pH 8.1). 5.0 mL of RT enzyme solution, diluted to a suitable con-
centration in enzyme dilution buffer (50 mM Tris-HCl, 20% glyc-
erol, 2m MDTT, pH = 7.6), was added. The reaction mixture were
incubated at 25 ℃ for 40 min and then stopped by the addition of
EDTA (15 mM). Heteroduplexes were then detected by addition
of PicoGreen. Signals were read using an excitation wavelength of
490 nm and emission detection at 523 nm using a spectrofluorom-
eter (Safire2, Tecan). To test the activity of compounds against RT,
1.0 mL of compound in DMSO was added to each well before the
addition of RT enzyme solution. Control wells without compound
contained the same amount of DMSO. Results were expressed as
relative fluorescence, i. e. the fluorescence signal of the reaction
mixed with compound divided by the signal of the same reaction
mixed without compound.
2-(4-Cyanophenylamino)-4-(4-cyanophenyl-2-hydroxypropyla
minomethyl)pyrimidine (1q) Yield: 38.5%; brown solid; mp
1
48.6-50.0 ℃; H NMR (400 MHz, DMSO-d6) δ 10.15 (d, J = 2.6
Hz, 1H, NH), 8.50 (d, J = 5.0 Hz, 1H, pyrimidine H6), 7.89 (d, J =
8.7, 2H, ArH3,5), 7.81 (d, J = 8.2, 2H, Ar’H3’,5’), 7.63 (d, d, J = 8.4
Hz, 4H, Ar’H2’,6’ +ArH2,6), 7.09 (dd, J = 5.0 Hz, 1H, pyrimidine
H5), 4.89 (d, J = 5.3 Hz, 1H, CH), 4.62 (d, J = 7.6 Hz, 1H, OH),
3.74 (s, 1H, CH), 2.44-2.33 (m, 2H, CH2), 1.04 (d, J = 6.0 Hz, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) δ 171.80, 159.59 (d, J =
5.5 Hz), 159.33, 148.16, 145.25, 133.39 (2C), 132.84 (2C, d, J =
2.9 Hz), 129.12 (2C), 120.03, 119.26, 118.82 (2C, d, J = 3.9 Hz),
111.60 (d, J = 16.9 Hz), 110.56, 102.84, 67.04, 65.99 (d, J = 18.6
Hz), 55.55 (d, J = 28.4 Hz), 21.95 (d, J = 4.7 Hz); MS (ESI+) m/z
385 (M+H)+; HR-MS: m/z Calcd for C22H20N6O: 384.1699. Found:
384.1707.
2-(4-Cyanophenylamino)-4-(2-chlorophenyl-2-hydroxypropyla
minomethyl)pyrimidine (1r) Yield: 41.8%; brown solid; mp
1
54.6-56.0 ℃; H NMR (400 MHz, DMSO-d6) δ 10.14 (d, J = 6.2
Hz, 1H, NH), 8.51 (t, J = 4.0 Hz, 1H, pyrimidine H6), 7.85 (t, J =
8.4 Hz, 2H, ArH3,5), 7.68-7.51 (d, d, 3H, Ar’H5’+ArH2,6),
7.49-7.34 (m, 2H, Ar’H), 7.30 (t, J = 7.4 Hz, 1H, Ar’H), 7.05 (dd,
J = 4.9 Hz, 1H, pyrimidine H5), 5.21 (d, J = 13.7 Hz, 1H, CH),
4.61 (s, 1H, OH), 3.74 (d, J = 5.2 Hz, 1H, CH), 2.47-2.35 (m, J =
8.1 Hz, 2H, CH2), 1.04 (d, J = 4.7 Hz, 3H, CH3); 13C NMR (100
MHz, DMSO-d6) δ 171.39 (d, J = 17.0 Hz), 159.58 (d, J = 6.6 Hz),
159.20, 145.31, 139.59, 133.52 (d, J = 9.1 Hz), 133.27 (2C),
129.82 (d, J = 4.8 Hz), 129.79, 129.41, 127.92, 120.04, 118.74 (2C,
d, J = 4.7 Hz), 111.90 (d, J = 11.1 Hz), 102.75, 65.99 (d, J = 20.3
Hz), 63.78 (d, J = 16.8 Hz), 55.80 (d, J = 26.7 Hz), 21.97; MS
(ESI+) m/z 394 (M+H)+; HR-MS: m/z Calcd for C21H20ClN5O:
393.1356. Found: 393.1368.
5.3. Molecular simulation
Molecular modeling was carried out with the Tripos molecu-
lar modeling packages Sybyl-X2.0. All the molecules for docking
were built using standard bond lengths and angles from
Sybyl-X2.0/base Builder and were then optimized using the Tripos
force field for 2000 generations two times or more, until the min-
imized conformers of the ligand were the same. The flexible
docking method, called Surflex-Dock[46], docks the ligand auto-
matically into the ligand binding site of the receptor by using a
protocol-based approach and an empirically derived scoring func-
tion[47]. The protocol is a computational representation of a puta-
tive ligand that binds to the intended binding site and is a unique
and essential element of the docking algorithm[48]. The scoring
function in Surflex-Dock, which contains hydrophobic, polar,
repulsive, entropic, and salvation terms, was trained to estimate
the dissociation constant (Kd) expressed in -log (Kd)2.
Prior to docking, the protein was prepared by removing water
molecules, the ligands (TMC278), and other unnecessary small
molecules from the crystal structure of the ligands-HIV-1 RT
complex (PDB code: 2ZD1[25] and 3BGR[25]); and then the hy-
drogen atoms were added to the protein also. 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 resi-
due side chains inside the defined RT binding pocket were re-
garded as rotatable or flexible, and the ligand was allowed to ro-
tate on all single bonds and move flexibly within the tentative
binding pocket. The atomic charges were recalculated using the
Kollman all-atom approach for the protein and the Gasteiger
Hückel approach for the ligand. The binding interaction energy
was calculated to include van der Waals, electrostatic, and torsion-
al energy terms defined in the Tripos force field. The structure
optimization was performed for 20,000 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 affini-
ties of ligand with RT, ranged over a wide scope of functional
classes (10-2~10-9). Therefore, only the highest-scoring 3D struc-
tural model of the ligand-bound RT was chosen to define the
7.61 (d, d, J = 8.2 Hz, 3H, Ar’H5’+ArH2,6),
5.2. Anti-HIV activity assay
The anti-HIV activity and cytotoxicity of the compounds
1a-1r, were evaluated against wild-type HIV-1 strain IIIB, a dou-
ble 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-diphenyltetrazolium bromide (MTT)
method[42, 43]. Briefly, stock solutions (10 × final concentration)
of test compounds were added in 25 mL 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-and
mock-infected cell samples were included for each sample. Virus
stock (50.0 mL) at 100-300 CCID50 (50.0 % cell culture infectious
dose) or culture medium was added to either the virus-infected or
mock-infected wells of the microtiter tray. Mock-infected cells
were used to evaluate the effect of test compounds on uninfected
cells in order to 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 6×105 cells/mL and 50.0 mL volumes were trans-
ferred to the microtiter tray wells. Five days after infection, the
viability of mock-and HIV-infected cells was examined spectro-
photometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored
MTT by mitochondrial dehydrogenase activity of metabolically
active cells to a blue-purple formazan that can be measured spec-
trophotometrically. The absorbances were read in an eight-channel
computer-controlled photometer (Infinite M1000, Tecan), at two
wavelengths (540 and 690 nm). All data were calculated using the
median OD (optical density) values of tree wells. The 50% cyto-
toxic concentration (CC50) was defined as the concentration of the
test compound that reduced the absorbance (OD540) of the
mock-infected control sample by 50%. The concentration achiev-
ing 50% protection from the cytopathic effect of the virus in in-
10

binding interaction[49].
safety of TMC125 (etravirine) in treatment-experienced HIV-1-infected patients in DUET-2:
24-week results from a randomised, double-blind, placebo-controlled trial, Lancet, 370 (2007)
ACCEPTED MANUSCRIPT
Acknowledgments
39-48.
[20] J.V. Madruga, P. Cahn, B. Grinsztejn, R. Haubrich, J. Lalezari, A. Mills, G. Pialoux, T. Wilkin,
M. Peeters, J. Vingerhoets, G. de Smedt, L. Leopold, R. Trefiglio, B. Woodfall, Efficacy and safety
of TMC125 (etravirine) in treatment-experienced HIV-1-infected patients in DUET-1: 24-week
results from a randomised, double-blind, placebo-controlled trial, Lancet, 370 (2007) 29-38.
[21] A.M. Geretti, Shifting paradigms: the resistance profile of etravirine, The Journal of
antimicrobial chemotherapy, 62 (2008) 643-647.
The authors are grateful to the Scientific Research Founda-
tion for the Returned Overseas Chinese Scholars, State Education
Ministry, P. R. China (2011), the Shaanxi Province Science and
Technology Research and Development Program of China, Inter-
national Cooperation (2013KW31-04) and the National Natural
Science Foundation of China (Grant No. 81172918).
[22] X. Chen, P. Zhan, D. Li, E. De Clercq, X. Liu, Recent advances in DAPYs and related
analogues as HIV-1 NNRTIs, Curr. Med. Chem., 18 (2011) 359-376.
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A list of captions should be provided as the following:
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Fig. 1. Structure of some important NNRTIs including HEPT, DABCO and DAPYs.
Fig. 2. Molecular design of new substituted CH-DAPYs.
Fig. 3. Comparison of the binding modes between the representative potential compound 1d and TMC278 in NNRTBP of wild type of
HIV-1 RT (PDB code: 2ZD1[25]) and K103N/Y181C double mutated HIV-1 RT (PDB code: 3BGR[25]). (a) TMC278/RTWT, (b) 1d/RTWT
,
(c) TMC278/RTK103N/Y181C, (d) 1d/RTK103N/Y181C The figures are generated by the software of SYBYL-X 2.0. (For interpretation of the
.
references to color in these figures legend, the reader is referred to the web version of this article.) Possible hydrogen bonds are indicated
with dashed lines in red. The amino acid residues are shown in liner frame and the docked conformations of the small molecules are shown
in ball and stick both for 1d and TMC278, respectively.
Scheme 1. Synthetic route of the new DAPYs derivatives (1a-1r). Reagents and conditions: (a) MeI, NaOH, H2O, r. t.; (b) 4-cyanoaniline,
180 ℃; (c) POC13, reflux; (d) R (R=2-Cl or 4-Br)-phenylacetonitrile, 60 % NaH, Ar, DMF, -20℃ to r. t., 48-72 h; (e) NaH, air, DMF, r. t.,
36-72 h; (f) CuCN, DMF, 150℃, 10h; (g) acrylonitrile, Pd(OAc)2, P(o-Tol)3, NaOAc, DMA, 150℃, overnight; (h) R’-NH2, EtOH, Na2SO4,
reflux, 4-8 h; (i) NaBH3CN, EtOH, 60 ℃, 4 h
Table 1 Anti-HIV activities and cytotoxicity of compounds 1a-1r MT-4 cells.
Table 2 Comparison of the torsion angles of the binding conformations of TMC278 and compound 1d.
13

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Supplementary material for the following article
Design and synthesis of a new series of modified CH-diarylpyrimidines as
the drug resistant HIV non-nucleoside reverse transcriptase inhibitors
Ge Menga*, Yang Liua, Aqun Zhengb, Fener Chen*c, Wenxue Chenc, Erik De Clercqd, Christophe
Pannecouqued, Jan Balzarinid
a School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China
b School of Science, Xi’an Jiaotong University, Xi’an, Shaanxi, 710049, P. R. China
c Department of Chemistry, Fudan University, Shanghai, 200433, P. R. China
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
1
13
The representative H and C NMR spectra and MS of the new compounds reported in this
article are shown as follow.
2-(4-Cyanophenylamino)-4-(4-cyanophenylhydroxymethyl) pyrimidine (1a)
CN
CN
4
4'
5
3
2
5'
6'
3'
2'
6
1
1'
N3 NH
4
HN
2
OH
N
5
1
6
Fig. 1. 1H NMR spectrum of 1a

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Fig. 2.13C NMR spectrum of 1a
Fig. 3. MS spectrum of 1a
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylhydroxymethyl)pyrimidine (1b)
Fig. 4. 1H NMR spectrum of 1b

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Fig. 5. 13C NMR spectrum of 1b
2-(4-Cyanophenylamino)-4-(4-cyanophenylhydrazonomethyl)pyrimidine (1c)
Fig. 6. 1H NMR spectrum of 1c

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Fig. 7. 13C NMR spectrum of 1c
Fig. 8. MS spectrum of 1c
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylhydrazonomethyl)pyrimidine (1d)
Fig. 9. 1H NMR spectrum of 1d

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Fig. 10. 13C NMR spectrum of 1d
Fig. 11. MS spectrum of 1d
2-(4-Cyanophenylamino)-4-(4-cyanophenylmethylaminomethyl)pyrimidine (1e)
Fig. 12. 1H NMR spectrum of 1e
Fig. 13. 13C NMR spectrum of 1e

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2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylmethylaminomethyl)pyrimidine (1f)
Fig. 14. 1H NMR spectrum of 1f
Fig. 15. MS spectrum of 1f
2-(4-Cyanophenylamino)-4-(2-chlorophenylmethylaminomethyl)pyrimidine (1g)

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Fig. 16. 1H NMR spectrum of 1g
Fig. 17. 13C NMR spectrum of 1g
Fig. 18. MS spectrum of 1g
2-(4-Cyanophenylamino)-4-(4-cyanophenylethylaminomethyl)pyrimidine (1h)

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Fig. 19. 1H NMR spectrum of 1h
Fig. 20. 13C NMR spectrum of 1h
Fig. 21. MS spectrum of 1h
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylethylaminomethyl)pyrimidine (1i)

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Fig. 22. 1H NMR spectrum of 1i
Fig. 23. 13C NMR spectrum of 1i
Fig. 24. MS spectrum of 1i
2-(4-Cyanophenylamino)-4-(2-chlorophenylethylaminomethyl)pyrimidine (1j)

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Fig. 25. 1H NMR spectrum of 1j
Fig. 26. 13C NMR spectrum of 1j
Fig. 27. MS spectrum of 1j
2-(4-Cyanophenylamino)-4-(4-cyanophenylpropylaminomethyl)pyrimidine (1k)
Fig.28. 1H NMR spectrum of 1k

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Fig. 29. 13C NMR spectrum of 1k
Fig. 30. MS spectrum of 1k
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylpropylaminomethyl)pyrimidine (1l)
Fig. 31. 1H NMR spectrum of 1l

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Fig. 32. 13C NMR spectrum of 1l
Fig. 33. MS spectrum of 1l
2-(4-Cyanophenylamino)-4-(2-chlorophenylpropylaminomethyl)pyrimidine (1m)
Fig. 34. 1H NMR spectrum of 1m

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Fig. 35. 13C NMR spectrum of 1m
Fig. 36. MS spectrum of 1m
2-(4-Cyanophenylamino)-4-(4-cyanophenylisopropylaminomethyl)pyrimidine (1n)
Fig. 37. 1H NMR spectrum of 1n

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Fig. 38. 13C NMR spectrum of 1n
Fig. 39. MS spectrum of 1n
2-(4-Cyanophenylamino)-4-(2-cyanovinylphenylisopropyl aminomethyl)pyrimidine (1o)
Fig. 40. 1H NMR spectrum of 1o