Discovery of novel diarylpyrimidines as potent HIV NNRTIs via a structure-guided core-refining approach

Article abstract of DOI:10.1016/j.ejmech.2014.04.036

Guided by crystal structures of HIV-1 RT/DAPY complex and molecular modeling studies, a series of novel DAPY derivatives were rationally designed, synthesized and evaluated for their anti-HIV activities. Among them, 16 compounds significantly inhibited HIV-1 IIIB replication with EC values lower than 66 nM. Particularly, compound 7a was the most potent inhibitor against HIV-1 wild-type and double RT mutant HIV-1 strain K103N/Y181C, with an EC value of 2.5 nM (SI = 13,740) and 0.33 μM (SI = 107), respectively. Unexpectedly, compound 8c was found to show moderate anti-HIV-2 potency (EC = 5.57 μM). Preliminary structure-activity relationships (SARs) and molecular modeling of these new analogues were also discussed in detail.

Full text of DOI:10.1016/j.ejmech.2014.04.036

European Journal of Medicinal Chemistry 80 (2014) 112e121
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel diarylpyrimidines as potent HIV NNRTIs via a
structure-guided core-refining approach
,
c
Xiao Li a, Wenmin Chen a, Ye Tian a, Huiqing Liu b, Peng Zhan a , Erik De Clercq ,
*
,
Christophe Pannecouque c, Jan Balzarini c, Xinyong Liu a
*
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University,
44 West Culture Road, 250012 Jinan, Shandong, PR China
b Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, PR China
c Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 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:
Guided by crystal structures of HIV-1 RT/DAPY complex and molecular modeling studies, a series of
novel DAPY derivatives were rationally designed, synthesized and evaluated for their anti-HIV activ-
ities. Among them, 16 compounds significantly inhibited HIV-1 IIIB replication with EC50 values lower
than 66 nM. Particularly, compound 7a was the most potent inhibitor against HIV-1 wild-type and
Received 3 December 2013
Received in revised form
8 April 2014
Accepted 11 April 2014
Available online 13 April 2014
double RT mutant HIV-1 strain K103N/Y181C, with an EC50 value of 2.5 nM (SI ¼ 13,740) and 0.33
(SI ¼ 107), respectively. Unexpectedly, compound 8c was found to show moderate anti-HIV-2 potency
(EC50 ¼ 5.57 M). Preliminary structureeactivity relationships (SARs) and molecular modeling of these
new analogues were also discussed in detail.
mM
m
Keywords:
HIV-1
HIV-2
Ó 2014 Elsevier Masson SAS. All rights reserved.
DAPY
Anti-HIV activities
Structureeactivity relationships
Molecular modeling
1. Introduction
mutated strains has always been a very active research field in the
past two decades [6e9]. Especially, considerable efforts have been
devoted to the structural modification of diarylpyrimidines
(DAPYs), a family of NNRTIs with remarkable anti-HIV-1 activity,
leading to the discovery of two approved drugs, etravirine
(TMC125, 1) and rilpivirine (TMC278, 2) (Fig. 1) and other highly
potent compounds against both HIV-1 wild-type and mutant
strains, which provide additional options for HAART regimen
[10,11].
Currently, with attempts to increase the inhibitory efficacy
against HIV-1 and to further explore the structural features
required for anti-HIV-1 activity, different types of modifications
have been carried out in the DAPY scaffold, including the left wing
(L) that linked to the 4-position of the pyrimidine core, the right
wing (R), the central pyrimidine ring (C), and the linker [12e14].
Moreover, the disclosed crystal structures of the HIV-1 RT/DAPY
complex and/or molecular modeling studies have pointed out that
the presence of three key chemical features was closely related to
the RT inhibition, i.e., the left aryl wing, extending into the NNRTI
binding pocket (NNIBP) to form an important hydrophobic inter-
action, and a para-substituted aniline moiety in the right wing,
Acquired immunodeficiency syndrome (AIDS) mainly caused by
human immunodeficiency virus type-1 (HIV-1), remains a world-
wide health threat, with approximately 34 million people living
currently with HIV and more than 25 million deaths since it was
first identified [1]. Despite the dramatically decreased in morbidity
and mortality from HIV-1 infection by introducing the highly active
antiretroviral therapy (HAART), a definitive cure of AIDS by che-
motherapeutics still has a long way to go [2,3].
HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors
(NNRTIs) with unique antiviral potency, high specificity and low
cytotoxicity have played an indispensable role in HAART [4,5]. The
development of novel chemical entities with high inhibition for the
Abbreviations: AIDS, acquired immunodeficiency syndrome; HIV-1, human
immunodeficiency virus type-1; HAART, highly active antiretroviral therapy;
NNRTIs, non-nucleoside reverse transcriptase inhibitors; DAPYs, diarylpyrimidines;
SARs, structureeactivity relationships; NNIBP, NNRTI binding pocket.
* Corresponding authors.
E-mail addresses: zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn
(X. Liu).
interacting with an aminoacid residue by
a dipoleedipole
http://dx.doi.org/10.1016/j.ejmech.2014.04.036
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
113
CN
CN
CN
CN
NH
L
R
O
N
NH
C
HN
N
N
Br
N
NH2
Rilpivirine (TMC278)
Etravirine (TMC125,ETV)
2
1
Fig. 1. The structures of two DAPY NNRTIs drugs. For convenience, we refer to the
structural components of DAPYs as the left wing (L), the right wing (R), the central
pyrimidine ring (C), bridge LeC (O or NH linker), and bridge CeR (NH linker).
interaction, as well as the NH linker, forming a key hydrogen bond
with the backbone of K101, which are crucial for the drug resistance
profile (Fig. 2) [15,16]. Initial molecular modeling suggested that the
substitution at the position of the N1 pyrimidine could potentially
provide a vector to access consecutive lysine residues (Lys101e
Lys104) in the NNIBP (Fig. 2), and the overall conformation of the
inhibitor should be largely preserved.
Supported by these findings and with the aim to further explore
untapped chemical space in NNIBP and obtaining more potent
back-up series of DAPY derivatives, in this study, we designed and
synthesized new DAPYanalogues in which the positions of nitrogen
atoms in the central pyrimidine ring were changed, and nitro,
amino and other nitrogen-containing groups were introduced at
the primary position, supposing that the additional hydrogen
bonds with surrounding residues could be formed by extended
different types of nitrogen. Meanwhile, the two phenyl rings in the
left and right wings and the NH linker were maintained in view of
their paramount importance in parent drugs (Fig. 3). Thus, this
study will highlight the synthesis, biological evaluation of novel
DAPY derivatives, and also, SARs will be discussed according to the
antiviral activity and molecular docking results.
Fig. 2. Mode of TMC125 (red) at the NNIBP of wt RT (PDB code: 3MEC). Ligand is
shown in red. Hydrogen bonds are depicted as yellow dashes. The figure was generated
using PyMol (www.pymol.org). The design of new DAPY derivatives was guided by a
model of interaction between the lead compound TMC125 and the NNIBP. (For inter-
pretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
with wild-type HIV-1 strain (IIIB), double mutant HIV-1 strain
RES056 (K103N þ Y181C), and HIV-2 strain (ROD), respectively, in
parallel with their cytotoxicities. FDA-approved nevirapine (NVP)
and delavidine (DLV) were used as reference drugs for comparison.
The biological results are represented as EC50 values (anti-HIV ac-
tivity), CC50 values (cytotoxicity) and SI values (selectivity index,
given by the CC50/EC50 ratio).
As shown in Table 1, the biological results clearly indicated that
most derivatives proved to be highly effective in inhibiting HIV-1
IIIB replication at double-digit nanomolar ranges. Notably, com-
pounds 7a, 7b were the most potent inhibitors against HIV-1 IIIB
replication with an EC50 value of 2.5 nM and 7.2 nM, respectively,
which were superior to those of the reference drugs NVP, DLV, EFV,
AZT and 3 TC. Besides, some other compounds including 6a, 6b, 6e,
7c, 7d, 7e, 7f, 7g, 8a, 8b, 8c, 8d and 8e, also exhibited favorable
potency against the HIV-1 IIIB strain with EC50 values in the range
of 23e66 nM, which were more potent than that of NVP, DLV and
3TC.
2. Results and discussion
2.1. Chemistry
To achieve the synthesis of the target compounds 6e9, the steps
outlined in Scheme 1 were adopted. Briefly, intermediate 5 was
synthesized from two commercially available materials, 2,4-
dichloro-5-nitropyrimidine (3) and 4-aminobenzonitrile (4), by
a nucleophilic substitution reaction [17,18]. Treatment of 5 with the
different substituted phenols and potassium carbonate under at
60 ꢀC in DMF gave the target compounds 5-nitro-pyrimidine series
6aeg [19,20]. The nitro group on the central ring was treated with a
mild reductant, stannous chloride (6c, 6d, 6e, 6f) or with hydro-
genation catalyzed by Pd/C (5ꢁ10%) in anhydrous ethanol (6a, 6b,
6g) to afford the 5-amino-pyrimidine series 7aeg, which were then
converted into the trifluoroacetamide series 8aeg and acetamide
9aeg, by acylation reaction with trifluoroacetic anhydride and
acetylchloride, respectively [21,22]. The newly synthesized com-
pounds were characterized by physicochemical and spectral
means, and both analytical and spectral data of all the compounds
were found in full agreement with the proposed structures.
Besides the excellent activity against the HIV-1 wild-type strain,
7a was also the most active compound against HIV-1 double-
mutant strain K103N/Y181C (RES056) with EC50 value of 0.33 mM
and SI of 107. The potency of 7a against mutant HIV-1 in this assay
was higher than that of NVP, DLV, and 3TC, and similar to that of
EFV. In addition, it is interesting to note that, except for 2,6-
disubstituted phenoxyl derivatives (7f, 8f), other compounds with
R2 substituted with NH2 or NHCOCF3 (7a, 8a, 7b, 8b, 7c, 8c, 7d, 8d,
7e, 8e, 7g, 8g) displayed remarkable activities against HIV-1 RES056
strain with EC50 values in micromolar concentration ranges.
As can be seen in Table 1, the anti-HIV activity (IIIB and RES056
strains) is dramatically depended on the nature of the substituents
at C-5 of the pyrimidine ring. Significant difference in antiviral
activity against HIV-1 IIIB and RES056 strains can be observed, with
the general order: 7aeg series (R2
¼
NH2) > 8aeg series
2.2. Biological activity
(R2 ¼ NHCOCF3) > 6aeg series (R2 ¼ NO2) >> 9aeg series
(R2 ¼ NHCOCH3), indicating amino group as hydrogen bond donor
was the preferred substituent compared with hydrogen bond
acceptor group nitro, while introduction of NHCOCH3 severely
The newly prepared DAPY derivatives (compounds 6e9) were
evaluated for their anti-HIV activity in MT-4 cell cultures infected

114
X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
Fig. 3. Schematic diagram of the binding mode of TMC125 with NNIBP and design of novel DAPY derivatives via a structure-guided core-refining approach.
Regarding the potency against double-mutant strain RES056,
only the 7aeg series and 8aeg series exhibited the antiviral activ-
ities. The active sequence of the substitutions at the C2 position of
the pyrimidine core was as follows: 2,6-Me-4-CN > 2,4,6-Me > 2,6-
Me-4-CHO > 2,6-Cl-4-H. Analogues with the 2,6-Cl-4-H-phenoxy
substituent were found to be the worst inhibitors against the
RES056 strain. Therefore, it is further confirmed that the nature of
the substituents with a suitable volume and high hydrophobicity in
the left wing is crucial for these novel DAPY analogues to have
improved drug resistance profiles.
To the best of our knowledge, most NNRTIs are specific for HIV-1
and only a few might exert some minimal inhibition against HIV-2.
Unexpectedly, two trifluoroacylating compounds 8c and 8e have
moderate activity against HIV-2, in particular, 8c presented with an
EC50 value of 5.57 mM, suggesting that a potential interaction may
exist between these inhibitors and HIV-2 RT or other targets.
2.3. Inhibition of HIV-1 RT
With the aim to further confirm the drug target or other targets,
two most active compounds 7a, 7b were selected to be evaluated in
an enzymatic assay against highly purified recombinant HIV-1 RT
Scheme 1. The synthetic route of target compounds. Reagents and conditions: (a) 4-
aminobenzonitrile (4), THF, Et3N, 0 ꢀC. (b) DMF, K2CO3, 60 ꢀC. (c) SnCl2$2H2O, EtOH.
(d) EtOH, Pd/C, rt. (e) THF, CH2Cl2, Et3N, CF3COOCOCF3 or CH3COCl.
.
using hybrid poly (A) oligo (dT)15 (9 A260 nm/ml), lyophilizate as
template primer. As shown in Table 2, compounds 7a, 7b exhibited
high inhibition of enzymatic activity with an IC50 value of 2.1
5.2 M, respectively, which were more active than that of TMC125
(ETV, IC50 ¼ 6.5 M).
mM,
m
impaired the activity, which might reflect a spatial restriction in the
target site of the HIV RT. Besides, it was clearly revealed that
although trifluoroacetylation of NH2 group (8aeg) makes anti-HIV
activity (IIIB and RES056) reduced to a certain extent, while still
keep on the same active order of magnitudes as amino deriva-
tives(7aeg), which is in sharp contrast to other two series(6aeg,
9aeg).
In comparison with the substituent effect at the C2-position of
the pyrimidine ring, SAR analysis of the left ring moiety was
summarized as the following: 2,6-Me-4-CN-phenoxy substitution
was found to be the best functional group conferring high potency
against HIV-1 IIIB and RES056 strains. With regard to the activity
against the HIV-1 IIIB strain, substitution at 4-position of the 2,6-
Me-phenoxy dominated the antiviral potency and a declining
trend was found (7aeg series, 8aeg series, and 9aeg series) with
the following different groups at the 4-position: 4-CN > 4-Me > 4-
Br > 4-CHO. Compounds in the 6aeg series have a slightly different
in the activity sequence: 4-Me > 4-CN > 4-Br > 4-CHO. Among the
four series of compounds, only compounds in 7aeg series gave a
parallel order of activity against HIV-1 IIIB and RES056 strains (4-
CN > 4-Me > 4-Br > 4-CHO).
m
2.4. Molecular modeling analysis
To shed light on the preferred interaction mode in the NNIBP
and rationalize SARs of these derivatives, a molecular docking study
of the most active compound 7a was performed using software
SYBYL-X. The theoretical binding mode to the NNIBP is shown in
Fig. 4.
As expected, compound 7a had nearly the same binding mode in
wt HIV-1 RT as etravirine (ETV). Detailed analysis of the docking
results revealed the following notable features: (1) the left wing of
compound 7a fitted into the important hydrophobic sub-pocket
comprised of aromatic residues Y181, Y188, F227 and W229,
exhibiting pep interaction with these residues, while cyano group
at para-position pointed to conservative amino acid W229 to form a
dipoleedipole interaction. (2) Two hydrogen bonds were formed
with backbone carbonyl and backbone amino of residue K101,
improving the stability of RT-inhibitor complex and the affinity
between RT and inhibitor. (3) The right aniline ring extended to the

X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
115
Table 1
Activity against HIV-1 and cytotoxicity in MT-4 cells.
CN
R
4
O
N
NH
R2
2
N
5
b
Compds
R
R2
HIV-1IIIB
RES056
EC50 M)
ROD
EC50
CC50 (m)
a
EC50
(m
)
SIc
(
m
SI
(
m
M)
SI
6a
7a
8a
9a
6b
7b
8b
9b
6c
7c
8c
9c
6d
7d
8d
9d
6e
7e
8e
9e
6f
7f
8f
9f
6g
7g
8g
9g
NVP
3TC
AZT
EFV
DLV
ETVd
2,6-Me, 4-CN
2,6-Me, 4-CN
2,6-Me, 4-CN
2,6-Me, 4-CN
2,4,6-Me
2,4,6-Me
2,4,6-Me
2,4,6-Me
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Cl
2,4,6-Br
2,4,6-Br
2,4,6-Br
2,4,6-Br
2,6-Me, 4-Br
2,6-Me, 4-Br
2,6-Me, 4-Br
2,6-Me, 4-Br
2,6-Cl
2,6-Cl
2,6-Cl
2,6-Cl
2,6-Me, 4-CHO
2,6-Me, 4-CHO
2,6-Me, 4-CHO
2,6-Me, 4-CHO
NO2
NH2
NHCOCF3
NHCOCH3
NO2
0.062 ꢂ 0.008
0.002 ꢂ 0.001
0.037 ꢂ 0.007
1.8 ꢂ 0.7
>4473
13740
>7470
>169
ꢃ5480
1432
6254
>35
ꢃ3
ꢃ10.3.
><27
107
>55
><1
><1
14
>274
><1
<1
><1
><1
><1
<1
ꢃ274
0.33 ꢂ 0.00
5.0 ꢂ 0.8
>313
>35.1
>276
>313
>252
>10
>242
>322
>25
35.1 ꢂ 4
>276
>313
0.042 ꢂ 0.010
0.007 ꢂ 0.005
0.039 ꢂ 0.004
9.3 ꢂ 6.7
>252
ꢃ252
NH2
0.75 ꢂ 0.17
32 ꢂ 9
>322
10 ꢂ 9.8
242 ꢂ 14
>322
NHCOCF3
NHCOCH3
NO2
8
<1
><1
><1
9
>11
<1
<1
8
6
<1
><1
6
>23
<1
><1
<1
<1
><1
><1
<1
>45
<1
<1
<1
<1
<1
><1
<1
>7
<1
><1
<1
<1
<1
<1
><1
><1
<1
><1
><1
>7716
><1
><1
<1
7.4 ꢂ 1.0
>25.
ꢃ25
NH2
0.012 ꢂ 0.001
0.064 ꢂ 0.034
1.9 ꢂ 0.6
2311
>6968
17
2.8 ꢂ 1.8
24 ꢂ 12
>33
>26
26 ꢂ 3
NHCOCF3
NHCOCH3
NO2
5 ꢂ 1
>248
>33
33 ꢂ 1
20 ꢂ 2
37 ꢂ 25
17.0 ꢂ 1
118 ꢂ 8
ꢃ202
3.9 ꢂ 0.3
5
750
484
6
>20
>20
>37
NH2
0.050 ꢂ 0.017
0.035 ꢂ 0.002
20 ꢂ 1.2
4.9 ꢂ 0.6
2.9 ꢂ 0.4
>118
>202.3
4.8 ꢂ 1.7
11 ꢂ 7
>147
>206
>33
>190
>193
NHCOCF3
NHCOCH3
NO2
>17.0
>118
>202.3
>31
36 ꢂ 2
>147
>206
>33
0.063 ꢂ 0.025
0.046 ꢂ 0.002
0.053 ꢂ 0.012
32 ꢂ 5
ꢃ3199
NH2
702
31 ꢂ 3
>246
NHCOCF3
NHCOCH3
NO2
>4647
5
ꢃ65
1410
1355
6
147 ꢂ 8
3.2 ꢂ 0.3
ꢃ206
NH2
0.023 ꢂ 0.001
0.14 ꢂ 0.01
32 ꢂ 4.4
33 ꢂ 3
190 ꢂ 46
193 ꢂ 15
37 ꢂ 2
ꢃ34
NHCOCF3
NHCOCH3
NO2
NH2
NHCOCF3
NHCOCH3
>190
>193
>37
<1
<1
0.61 ꢂ 0.31
0.066 ꢂ 0.00
0.48 ꢂ 0.28
29 ꢂ 2
59
>37
ꢃ551
ꢃ441
8
>67
>23
>12235
>1133
>72
12884
8.4 ꢂ 0.5
35 ꢂ 15
>236
4.0 ꢂ 3.7
21 ꢂ 9
0.015 ꢂ 0.010
0.31 ꢂ 0.22
>36
ꢃ4
>34
ꢃ6
>216
>236
>15
ꢃ216
<1
236 ꢂ 15
0.22 ꢂ 0.06
3.7 ꢂ 2.2
>4
>4
>5959
>21
><1
1817
>15
>87
>87
>93
>6.3
>36
0.007 ꢂ 0.003
0.006 ꢂ 0.000
0.507 ꢂ 0.506
0.002 ꢂ 0.0004
0.011 ꢂ 0.01
>6.3
>36
>27
0.034 ꢂ 0.005
27 ꢂ 11
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against HIV-1-induced cytotoxicity, as determined by the MTT method.
CC50: concentration required to reduce the viability of mock-infected cell cultures by 50%, as determined by the MTT method.
SI: selectivity index, the ratio of CC50/EC50
b
c
.
d
ETV: etravirine. The data were obtained from the same laboratory (Rega Institute for Medical Research, KU Leuven, Belgium).
Table 2
bond was formed with easily mutated amino acid N103, providing
additional affinity with mutant HIV-1 RT. However, 7a still dis-
played less potent activity against K103N/Y181C resistant mutant
RT strains, which might be due to the mutation at the Y181 side
chain that led to partial loss of the dependent pep interaction and
finally resulted in decreased anti-HIV activity.
RT Inhibitory activity of compounds 7a and 7b.
Compd.
7a
7b
TMC125
6.5
IC50
(m
M)a
2.1
5.2
a
IC50: Inhibitory concentration of tested compounds required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.
Based on the above analysis of molecular modeling, further
optimization strategy can be concluded that (1) keeping hydrogen
bonds with the backbone of residue K101 as well as mutant amino
acid such as N103 is indispensable for obtaining improved activity
against both HIV-1 IIIB and RES056 strains. (2) a hydrophobic
flexible substituent on the left wing of DAPY might insert into the
narrow tunnel on the NNRTI binding site and interact with the
conserved W229 to enhance antiviral potency against resistant
viral strains.
surface of solvent and protein, and the para-substituted CN inter-
acted with amino acid residue H235. Overall, all of these molecular
interaction analyses were in accordance with the biological results.
Obviously, the novel compounds showed excellent activity against
wt HIV-1 because of specific interactions with key amino acid
residues in NNIBP (Fig. 4a).
In addition, the moderate potency of 7a against K103N/Y181C
double mutant HIV-1 was rationally explained by molecular
modeling results as well. As shown in Fig. 4b, two crucial hydrogen
bonds between C5eNH2, NH linker and peptidic carbonyl oxygen of
K101 were maintained. Even more surprising, a potential hydrogen
3. Conclusion
In summary, through structure-guided rescaffolding (core
refining) and side-chain optimization of the approved drug

116
X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
results were used to rationalize these biological data, which will be
beneficial to further design more potent anti-HIV-1 agents.
4. Experimental section
4.1. Chemistry
4.1.1. Synthetic procedures and analytical datas for all new
compounds
Mass spectrometry was performed on an API 4000 triple-
quadrupole mass spectrometer (Applied Biosystems/MDS Sciex,
Concord, ON, Canada). 1H NMR and 13C NMR spectra were recorded
on a Bruker AV-400 spectrometer (Bruker BioSpin, Switzerland),
using solvents as indicated (CDCl3 or DMSO-d6). Chemical shifts
were reported in
d values (ppm) with tetramethylsilane as the in-
ternal reference, and J values were reported in hertz (Hz). Melting
points (mp) were determined on a micromelting point apparatus
(Tian Jin Analytical Instrument Factory, Nankai, Tianjin, China).
Flash column chromatography was performed on columns packed
with silica gel 60 (200e300 mesh) (Qingdao waves silica gel
desiccant co., Ltd, Qingdao, China). Thin layer chromatography was
performed on pre-coated HUANGHAIÒ HSGF254, 0.15e0.2 mm TLC-
plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai,
Shandong, China).
4.1.2. General procedure for the synthesis of 4-(2-chloro-5-
nitropyrimidin-4-ylamino)benzonitrile (5)
To a solution of 2,4-dichloro-5-nitropyrimidine 3 (0.39 g,
2 mmol) in THF (15 mL) at 0 ꢀC was slowly added THF solution of 4-
aminobenzonitrile 4 (0.24 g, 2 mmol) and triethylamine (0.22 g,
2.2 mmol). The resulting mixture was stirred at 0 ꢀC for 3 h
(monitored by TLC). The solvent was removed under reduced
pressure and water (20 mL) was added and extracted with CH2Cl2
(3 ꢄ 10 mL). Combined organic phase was washed with brine, dried
over anhydrous Na2SO4, filtered and concentrated. Purification on
silica gel (EA:PE ¼ 1:8) gave 5 as a yellow solid.
4.1.3. General procedure for the synthesis of target compounds
(6aeg)
Crude intermediate 5 was dissolved in anhydrous DMF (10 mL)
in the presence of anhydrous K2CO3 (2 equiv) at room temperature
followed by addition of appropriate substituted phenols (1.2 equiv).
The reaction mixture was stirred at 60 ꢀC for 5 h (monitored by
TLC). The solvent was removed under reduced pressure, and water
(20 mL) was added. Extracted with EA (3 ꢄ 10 mL), and the organic
phase was washed with saturated sodium chloride (2 ꢄ 10 mL), and
dried over anhydrous Na2SO4 to give the corresponding crude
products, which were purified by flash column chromatography
using ethyl acetate-petroleum ether system to afford the target
compounds 6aeg, respectively.
Fig. 4. Predicted binding modes between the new compounds 7a and NNIBP of (a) wt
HIV-1 RT (PDB code: 3MEC) and (b) K103N/Y181C double mutant RT structure (PDB
code 3BGR).
4.1.4. General procedure for the synthesis of target compounds
(7aeg)
TMC125, a series of novel DAPY derivatives were designed, syn-
thesized and evaluated for their antiviral activity against wt HIV-1,
a double-mutated HIV-1 strain (K103N/Y181C) and HIV-2 (ROD) in
MT-4 cells, as well as against HIV-1 RT activity of some selected
compounds. It was demonstrated that most compounds showed
excellent activity against wt HIV-1 with EC50 values in the low
nanomolar concentration range (7a, EC50 ¼ 2.5 nM, SI ¼ 13740; 7b,
EC50 ¼ 7.2 nM, SI ¼ 1432) and moderate activity against resistant
mutant strain K103N/Y181C of HIV-1 with EC50 values in the sub-
Method A: The reaction system containing relevant compounds
6a, 6b, 6g (1 equiv), Pd/C (0.1 equiv) and 50 mL anhydrous EtOH was
vacuumed and filled with excess hydrogen gas. The reaction
mixture was kept for 10 h and then filtered out Pd/C, the solvent
was evaporated under reduced pressure. The obtained crude
product was washed by PE to give purified compound 7a, 7b, 7g.
Method B: Compounds 6c, 6d, 6e, 6f obtained from last step
(1 equiv) was dissolved in anhydrous EtOH (10 mL) in the presence
of the reducing reagent stannous chloride (5 equiv). This reaction
mixture was stirred at room temperature for 10 h (monitored by
TLC). The solvent was removed under reduced pressure, and water
(20 mL) was added. Extracted with EA (3 ꢄ 10 mL), and the organic
micromolar concentration range (7a, EC50 ¼ 0.33 M, SI ¼ 107). A
m
brief discussion of the SARs expatiated that the nature of the sub-
stituent on left ring of the central pyrimidine ring had a significant
impact on the antiviral activity. In addition, molecular docking

X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
117
phase was washed with saturated sodium chloride (2 ꢄ 10 mL), and
dried over anhydrous Na2SO4 to give the corresponding crude
products, which were purified by flash column chromatography
using methyl alcoholedichloromethane system to afford the target
compounds 7c, 7d, 7e, 7f, respectively.
d6, ppm) d: 170.26 (C]O), 160.65 (C2-pyrimidine), 156.80 (C4-py-
rimidine),156.80 (C1-Ph-O),155.83 (C6-pyrimidine), 154.31 (C1-Ph0-
N), 143.59, 133.08 (3 ꢄ C), 133.01 (2 ꢄ C), 120.67 (2 ꢄ C), 119.50
(2 ꢄ CN), 119.09, 115.07, 108.53, 104.83, 23.84, 16.21 (2 ꢄ CH3). ESI-
MS: m/z 399.3 (Mþ1), 416.5 (Mþ18), C22H18N6O2 [398.15].
4.1.5. General procedure for the synthesis of target compounds
(8aeg)
4.1.11. 4-(2-(Mesityloxy)-5-nitropyrimidin-4-ylamino)benzonitrile
(6b)
Trifluoroacetic anhydride (1.2 equiv) was added dropwise under
stirring to a solution of 7aeg (1.0 equiv) and triethylamine
(2.0 equiv) in THF (20 mL) at 0 ꢀC and stirred for 5 h (monitored by
TLC). The solvent was removed under reduced pressure and water
(20 mL) was added, then extracted with EA (3 ꢄ 10 mL). Combined
organic phase was washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated. Purification on silica gel by using
methyl alcoholedichloromethane system gave target compounds
8aeg.
Yellow crystals, yield: 65.8%. mp: 186e189 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 10.44 (s, 1H, NH), 9.34 (s, 1H, C6-
pyrimidine-H), 7.43 (s, 4H, Ph0-H), 6.99 (s, 2H, Ph-H), 2.40 (s, 3H,
CH3), 2.07 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
d:
165.70 (C2-pyrimidine), 160.41 (C4-pyrimidine), 154.37 (C6-pyrim-
idine), 147.91 (C1-Ph0-N), 140.52 (C1-Ph-O), 135.83, 132.99 (2 ꢄ C),
129.69 (2 ꢄ C), 129.56 (2 ꢄ C), 124.39, 121.77, 121.37, 118.47, 108.21,
20.89 (CH3), 16.24 (2 ꢄ CH3). ESI-MS: m/z 376.4 (Mþ1), C20H17N5O3
[375.13].
4.1.6. General procedure for the synthesis of target compounds
(9aeg)
The procedure for the synthesis of target compounds (9aeg) is
similar to 8aeg, except replacing trifluoroacetic anhydride with
acetylchloride.
4.1.12. 4-(5-Amino-2-(mesityloxy)pyrimidin-4-ylamino)
benzonitrile (7b)
White powder, yield: 75.5%. mp: 207e209 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 8.99 (s, 1H, NH), 7.72 (s, 1H, C6-py-
rimidine-H), 7.68 (d, 2H, J ¼ 8.84 Hz, Ph0-H), 7.49 (d, 2H, J ¼ 8.84 Hz,
2H, Ph0-H), 6.94 (s, 2H, Ph-H), 4.90 (s, 2H, NH2), 2.30 (s, 3H, CH3),
4.1.7. 4-(2-(4-Cyano-2,6-dimethylphenoxy)-5-nitropyrimidin-4-
ylamino)benzonitrile (6a)
1.98 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
d: 156.55
(C2-pyrimidine), 150.30 (C4-pyrimidine), 148.93 (C1-Ph’-N), 144.83
(C6-pyrimidine), 142.02 (C1-Ph-O), 133.94, 132.97 (2 ꢄ C), 130.53
(2 ꢄ C), 129.39 (2 ꢄ C), 125.03, 119.80, 119.52 (2 ꢄ C), 103.39, 20.80
(CH3), 16.28 (2 ꢄ CH3). ESI-MS: m/z 346.3 (Mþ1), 368.2 (Mþ23),
Yellow crystals, yield: 62.2%. mp: 227e230 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 10.50 (s, 1H, NH), 9.31 (s, 1H, C6-py-
rimidine-H), 7.53 (m, 6H, Ph-H, Ph0-H), 2.19 (s, 6H, 2 ꢄ CH3). 13C
NMR (100 MHz, DMSO-d6, ppm)
d
: 164.69 (C2-pyrimidine), 160.27
C20H19N5O [345.16].
(C4-pyrimidine), 154.60 (C1-Ph0-N), 153.09 (C6-pyrimidine), 140.08
(C1-Ph-O), 133.13 (2 ꢄ C), 132.75 (2 ꢄ C), 132.35 (2 ꢄ C), 124.99,
121.61 (2 ꢄ CN), 118.11 (2 ꢄ C), 110.33, 109.00, 16.31 (2 ꢄ CH3). ESI-
MS: m/z 387.4 (Mþ1), 404.5 (Mþ18), C20H14N6O3 [386.11].
4.1.13. N-(4-(4-Cyanophenylamino)-2-(mesityloxy)pyrimidin-5-
yl)-2,2,2-trifluoro-acetamide (8b)
White powder, yield: 52.2%. mp: 204e208 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 9.21 (s, 1H, C6-pyrimidine-H), 8.18 (d,
4.1.8. 4-(5-Amino-2-(4-cyano-2,6-dimethylphenoxy)pyrimidin-4-
ylamino)benzonitrile (7a)
J ¼ 8.40 Hz, 2H, Ph0-H), 7.94 (d, J ¼ 8.40 Hz, 2H, Ph0-H), 6.91 (s, 2H,
Ph-H), 2.24 (s, 3H, CH3), 1.97 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz,
White powder, yield: 70.4%. mp: 218e221 ꢀC. 1H NMR
DMSO-d6, ppm) d: 162.37 (C2-pyrimidine), 162.34 (C4-pyrimidine),
(400 MHz, DMSO-d6, ppm)
d
: 8.93 (s, 1H, NH), 7.72 (s, 3H, C6-py-
156.09 (C6-pyrimidine), 153.31 (C1-Ph0-N), 148.25 (C1-Ph-O), 136.71,
134,77, 134.23 (2 ꢄ C), 130.00 (2 ꢄ C), 129.75 (2 ꢄ C), 129.58, 128.71,
119.82, 118.37, 117.11, 113.81, 20.79 (CH3), 16.45 (2 ꢄ CH3). ESI-MS:
m/z 424.4 (C22H20F2N5Oþ2 ), C22H18F3N5O2 [441.14].
rimidine-H, Ph-H), 7.63 (d, 2H, J ¼ 8.80 Hz, 2H, Ph0-H), 7.56 (d, 2H,
J ¼ 8.80 Hz, 2H, Ph0-H), 4.93 (s, 2H, NH2), 2.08 (s, 6H, 2 ꢄ CH3). 13C
NMR (100 MHz, DMSO-d6, ppm) d: 155.66 (C2-pyrimidine), 154.96
(C4-pyrimidine), 150.36 (C1-Ph0-N), 144.51 (C6-pyrimidine), 141.55
(C1-Ph-O), 133.30 (2 ꢄ C), 133.09 (2 ꢄ C), 132.95 (2 ꢄ C), 125.71,
119.72, 119.58 (2 ꢄ CN), 119.24, 107.99, 103.77, 16.28 (2 ꢄ CH3). ESI-
MS: m/z 357.4 (Mþ1), 474.4 (Mþ18) C20H16N6O [356.14].
4.1.14. N-(4-(4-Cyanophenylamino)-2-(mesityloxy)pyrimidin-5-yl)
acetamide (9b)
White powder, yield: 49.9%. mp: 222e226 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 8.62 (s, 1H, NH), 8.35 (s, 1H, C6-py-
4.1.9. N-(2-(4-Cyano-2,6-dimethylphenoxy)-4-(4-
rimidine-H), 8.20 (s, 1H, NH), 7.39 (dd, J1 ¼ 32.00 Hz, J2 ¼ 8.40 Hz,
cyanophenylamino)pyrimidin-5-yl)-2,2,2-trifluoroacetamide (8a)
4H, Ph0-H), 6.93 (s, 2H, Ph-H), 2.35 (s, 3H, CH3), 2.30 (s, 3H, CH3),
White powder, yield: 56.5%. mp: 255e257 ꢀC. 1H NMR
2.07 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
d: 171.13
(400 MHz, DMSO-d6, ppm)
d
: 9.25 (s, 1H, C6-pyrimidine-H), 8.18 (d,
(C]O), 161.89 (C2-pyrimidine), 155.60 (C4-pyrimidine), 153.41 (C1-
Ph-O), 148.04 (C6-pyrimidine), 142.74 (C1-Ph0-N), 135.09, 132.78
(2 ꢄ C), 130.23 (2 ꢄ C), 129.32 (2 ꢄ C), 119.92 (2 ꢄ C), 119.23, 113.76,
105.50, 42.26 (CH3), 23.61, 16.37 (2 ꢄ CH3). ESI-MS: m/z 388.4
(Mþ1), C20H15N5O4 [387.17].
J ¼ 8.44 Hz, 2H, Ph0-H), 7.94 (d, J ¼ 8.44 Hz, 2H, Ph0-H), 7.67 (s, 2H,
Ph-H), 2.13 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
d:
161.82 (C2-pyrimidine), 155.38 (C4-pyrimidine), 153.50 (C1-Ph-O),
153.37 (C6-pyrimidine), 142.33 (C1-Ph0-N), 141.94, 135.64, 133.84
(2 ꢄ C), 132.71 (2 ꢄ C), 132.43 (2 ꢄ C), 128.73, 128.31 (2 ꢄ CN),
118.53, 117.26, 114.88, 109.83, 16.41 (2 ꢄ CH3). ESI-MS: m/z 435.5
(C22H17F2N6Oþ2 ), 452.4 (Mþ18), C22H15F3N6O2 [452.12].
4.1.15. 4-(5-Nitro-2-(2,4,6-trichlorophenoxy)pyrimidin-4-ylamino)
benzonitrile (6c)
Yellow crystals, yield: 66.9%. mp: 150e153 ꢀC. 1H NMR
4.1.10. N-(2-(4-Cyano-2,6-dimethylphenoxy)-4-(4-
cyanophenylamino)pyrimidin-5-yl) acetamide (9a)
White powder, yield: 54.2%. mp: 278e281 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 10.54 (s, 1H, NH), 9.29 (s, 1H, C6-py-
rimidine-H), 7.89 (s, 2H, Ph-H), 7.70 (d, J ¼ 8.68 Hz, 2H, Ph0-H), 7.53
(d, J ¼ 8.68 Hz, 2H, Ph0-H). 13C NMR (100 MHz, DMSO-d6, ppm)
d:
(400 MHz, DMSO-d6, ppm)
d
: 9.42 (s, 1H, NH), 9.22 (s, 1H, C6-py-
163.35 (C2-pyrimidine), 160.51 (C4-pyrimidine), 154.76 (C6-pyrim-
idine), 144.39 (C1-Ph0-N), 141.09 (C1-Ph-O), 132.87 (2 ꢄ C), 132.00,
129.49 (2 ꢄ C), 129.31 (2 ꢄ C), 126.87, 126.65 (2 ꢄ C), 118.99, 108.01.
rimidine-H), 8.17 (s, 1H, NH), 7.74 (s, 2H, Ph-H), 7.64 (s, 4H, Ph0-H),
2.10 (s, 6H, 2 ꢄ CH3), 2.09 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-

118
X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
ESI-MS: m/z 436.3 (Mþ1), 438.3 (Mþ3), 440.4 (Mþ5), C17H8Cl3N5O3
Ph-H), 7.97 (d, J ¼ 8.60 Hz, 2H, Ph0-H), 7.05 (d, J ¼ 8.40 Hz, 2H, Ph0-
[434.97].
H). 13C NMR (100 MHz, DMSO-d6, ppm)
d: 164.32 (C2-pyrimidine),
163.44 (C4-pyrimidine), 158.55 (C6-pyrimidine), 150.66, 148.13 (C1-
Ph0-N), 146.08 (C1-Ph-O), 136.45 (2 ꢄ C), 132.89 (2 ꢄ C), 127.77,
125.32 (2 ꢄ C), 122.42, 119.97, 118.71 (2 ꢄ C), 110.21, 107.95. ESI-MS:
m/z 538.0 (C17H11Br3N5Oþ), 540.0 (C17H10Br3N5Oþ þ2), 633.8
(Mþ1), 636.0 (Mþ3), 637.8 (Mþ5), C19H9Br3F3N5O2 [632.83].
4.1.16. 4-(5-Amino-2-(2,4,6-trichlorophenoxy)pyrimidin-4-
ylamino)benzonitrile (7c)
White powder, yield: 56.9%. mp: 150e153 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm)
7.74 (s, 1H, C6-pyrimidine-H), 7.65 (m, 4H, Ph0-H), 5.00 (s, 2H,
NH). 13C NMR (100 MHz, DMSO-d6, ppm)
: 154.92 (C2-pyrimidine),
d: 9.01 (s, 1H, NH), 7.87 (s, 2H, Ph-H),
d
4.1.22. N-(4-(4-Cyanophenylamino)-2-(2,4,6-tribromophenoxy)
pyrimidin-5-yl)aceta-mide (9d)
150.21 (C4-pyrimidine), 145.78 (C6-pyrimidine), 144.34 (C1-Ph0-N),
141.33 (C1-Ph-O), 133.10 (2 ꢄ C), 130.68, 130.26 (2 ꢄ C), 129.29
(2 ꢄ C), 126.32, 119.81 (2 ꢄ C), 119.65, 104.05. ESI-MS: m/z 406.4
(Mþ1), 408.4 (Mþ3), 410.4 (Mþ5), C17H10Cl3N5O [405.00].
White powder, yield: 45.6%. mp: 178e180 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm)
(s, 1H, C6-pyrimidine-H), 8.12 (s, 2H, Ph-H), 7.61 (s, 4H, Ph0-H), 2.09
(s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
: 170.26 (C]O),
d: 9.39 (s, 1H, NH), 9.27 (s, 1H, NH), 8.20
d
4.1.17. N-(4-(4-Cyanophenylamino)-2-(2,4,6-trichlorophenoxy)
pyrimidin-5-yl)-2,2,2-trifluoroacetamide (8c)
159.93 (C2-pyrimidine), 156.68 (C4-pyrimidine), 155.96 (C1-Ph-O),
147.78 (C6-pyrimidine), 143.41 (C1-Ph0-N), 135.33 (2 ꢄ C), 133.02
(2 ꢄ C), 120.93 (2 ꢄ C), 119.54 (2 ꢄ C), 119.50, 119.45, 115.10, 105.06,
23.85 (CH3). ESI-MS: m/z 579.9 (Mþ1), 582.0 (Mþ3), 583.9 (Mþ5),
586.0 (Mþ7), C19H12Br3N5O2 [578.85].
Brown oily solid, yield: 41.2%.1H NMR (400 MHz, DMSO-d6, ppm)
d
: 9.31 (s,1H, C6-pyrimidine-H), 8.20 (d, J ¼ 8.48 Hz, 2H, Ph0-H), 7.98
(d, J ¼ 8.48 Hz, 2H, Ph0-H) 7.87 (s, 2H, Ph-H). 13C NMR (100 MHz,
DMSO-d6, ppm) d: 160.73 (C2-pyrimidine), 156.07 (C4-pyrimidine),
153.28(C6-pyrimidine), 144.89 (C1-Ph0-N), 142.85 (C1-Ph-O), 136.51,
134.26 (2 ꢄ C), 131.53, 129.74 (2 ꢄ C), 129.64 (2 ꢄ C), 129.46 (2 ꢄ C),
118.32, 116.99, 114.28, 113.97. ESI-MS: m/z 484.2 (C19H11Cl3F2N5Oþ2 ),
586.2 (C19H11Cl3F2N5Oþ2 þ2), C19H9Cl3F3N5O2 [500.98].
4.1.23. 4-(2-(4-Bromo-2,6-dimethylphenoxy)-5-nitropyrimidin-4-
ylamino)benzonitrile (6e)
Yellow crystals, yield: 70.0%. mp: 190e195 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 10.44 (s, 1H, NH), 9.35 (s, 1H, C6-py-
rimidine-H), 7.49 (d, J ¼ 8.68 Hz, 2H, Ph-H), 7.39 (d, J ¼ 8.68 Hz, 2H,
4.1.18. N-(4-(4-Cyanophenylamino)-2-(2,4,6-trichlorophenoxy)
pyrimidin-5-yl) acetamide (9c)
Ph0-H), 7.26 (s, 2H, Ph0-H), 2.12 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz,
DMSO-d6, ppm) d: 165.25 (C2-pyrimidine), 160.44 (C4-pyrimidine),
White powder, yield: 40.5%. 1H NMR (400 MHz, DMSO-d6, ppm)
154.37 (C6-pyrimidine), 149.17 (C1-Ph0-N), 140.27 (C1-Ph-O), 133.12
(2 ꢄ C), 132.99, 132.50, 131.75 (2 ꢄ C), 124.67, 121.43 (2 ꢄ C), 119.16,
118.29, 108.71, 16.17 (2 ꢄ CH3). ESI-MS: m/z 440.4 (Mþ1), 442.4
(Mþ3), C19H14BrN5O3 [439.03].
d
: 9.40 (s, 1H, NH), 9.29 (s, 1H, NH), 8.20 (s, 1H, C6-pyrimidine-H),
7.90 (s, 2H, Ph-H) 7.63 (m, 4H, Ph0-H), 2.09 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6, ppm) : 170.29 (C]O), 160.01 (C2-pyrimi-
d
dine), 156.83 (C4-pyrimidine), 155.90 (C1-Ph-O), 145.20 (C6-pyrim-
idine), 143.36 (C1-Ph0-N), 133.04 (2 ꢄ C), 131.26, 129.92 (2 ꢄ C),
128.99 (2 ꢄ C), 121.03 (2 ꢄ C), 119.44, 115.66, 105.16, 23.84 (CH3).
ESI-MS: m/z 448.2 (Mþ1), 450.2 (Mþ3), 452.2 (Mþ5),
4.1.24. 4-(5-Amino-2-(4-bromo-2,6-dimethylphenoxy)pyrimidin-
4-ylamino)benzonitrile (7e)
White powder, yield: 55.8%. mp: 160e164 ꢀC. 1H NMR
C
19H12Cl3N5O2 [447.01].
(400 MHz, DMSO-d6, ppm) d: 8.88 (s, 1H, NH), 7.73 (s, 1H, C6-py-
rimidine-H), 7.64 (d, J ¼ 8.88 Hz, 2H, Ph0-H), 7.53 (d, J ¼ 8.88 Hz, 2H,
4.1.19. 4-(5-Nitro-2-(2,4,6-tribromophenoxy)pyrimidin-4-ylamino)
benzonitrile (6d)
Ph0-H), 7.26 (s, 2H, Ph-H), 4.87 (s, 2H, NH2), 2.02 (s, 6H, CH3). 13C
NMR (100 MHz, DMSO-d6, ppm) d: 156.09 (C2-pyrimidine), 150.32
Yellow crystals, yield: 66.4%. mp: 183e187 ꢀC. 1H NMR
(C4-pyrimidine), 150.03 (C6-pyrimidine), 144.65 (C1-Ph0-N), 141.92
(C1-Ph-O), 133.49 (2 ꢄ C), 130.30 (2 ꢄ C), 128.91, 128.29 (2 ꢄ C),
125.31, 119.73, 119.57 (2 ꢄ C), 103.62, 16.83 (2 ꢄ CH3). 410.4 (Mþ1),
412.4 (Mþ3), C19H16BrN5O [409.05].
(400 MHz, DMSO-d6, ppm) d: 10.52 (s, 1H, NH), 9.31 (s, 1H, C6-py-
rimidine-H), 8.12 (s, 2H, Ph-H), 7.67 (d, J ¼ 8.60 Hz, 2H, Ph0-H), 7.15
(d, J ¼ 8.40 Hz, 2H, Ph0-H). 13C NMR (100 MHz, DMSO-d6, ppm)
d:
163.32 (C2-pyrimidine), 160.54 (C4-pyrimidine), 154.56 (C6-pyrim-
idine), 147.03 (C1-Ph0-N), 141.08 (C1-Ph-O), 135.43 (2 ꢄ C), 132.87
(2 ꢄ C), 126.67, 124.42 (2 ꢄ C), 120.32, 118.99, 118.79 (2 ꢄ C), 107.94.
ESI-MS: m/z 568.1 (Mþ1), 570.1 (Mþ3), 572.1 (Mþ5), C17H8Br3N5O3
[566.82].
4.1.25. N-(2-(4-Bromo-2,6-dimethylphenoxy)-4-(4-
cyanophenylamino)pyrimidin-5- yl)-2,2,2-trifluoroacetamide (8e)
Brown oily solid, yield: 50.6%. 1H NMR (400 MHz, DMSO-d6,
ppm) d: 10.34 (s, 1H, NH), 10.11 (s, 1H, NH), 9.25 (s, 1H, C6-pyrimi-
dine-H), 7.60 (d, J ¼ 8.68 Hz, 2H, Ph-H), 7.48 (d, J ¼ 8.68 Hz, 2H, Ph0-
4.1.20. 4-(5-Amino-2-(2,4,6-tribromophenoxy)pyrimidin-4-
ylamino)benzonitrile (7d)
H), 7.34 (s, 2H, Ph-H), 2.32 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz,
DMSO-d6, ppm) d: 166.26 (C2-pyrimidine), 161.14 (C4-pyrimidine),
White powder, yield: 55.8%. mp: 160e164 ꢀC. 1H NMR
155.35 (C6-pyrimidine), 150.67, 148.27 (C1-Ph0-N), 147.28 (C1-Ph-O),
133.32 (2 ꢄ C), 132.09, 132.51, 130.85 (2 ꢄ C), 125.66, 124.47 (2 ꢄ C),
119.36, 118.00, 110.22, 109.71, 16.74 (2 ꢄ CH3). ESI-MS: m/z 588.3
(C21H17BrF2N5Oþ2 ), 590.3 (C21H17BrF2N5O2þ þ2), C21H15BrF3N5O2
[505.04].
(400 MHz, DMSO-d6, ppm)
7.74 (s, 1H, C6-pyrimidine-H), 7.63 (dd, J1 ¼ 18.24 Hz, J2 ¼ 8.96 Hz,
4H, Ph0-H), 4.97 (s, 2H, NH). 13C NMR (100 MHz, DMSO-d6, ppm)
d: 8.98 (s, 1H, NH), 8.09 (s, 2H, Ph-H),
d:
154.84 (C2-pyrimidine), 150.08 (C4-pyrimidine), 148.30 (C6-pyrim-
idine), 144.38 (C1-Ph0-N), 141.41 (C1-Ph-O), 135.23 (2 ꢄ C), 133.09
(2 ꢄ C), 126.18, 119.95 (2 ꢄ C), 119.75 (2 ꢄ C), 119.68, 118.93, 103.94.
ESI-MS: m/z 538.1 (Mþ1), 540.1 (Mþ3), 542.0 (Mþ5), 544.1 (Mþ7),
4.1.26. N-(2-(4-Bromo-2,6-dimethylphenoxy)-4-(4-
cyanophenylamino)pyrimidin-5-yl)acetamide (9e)
C
17H10Br3N5O [536.84].
White powder, yield: 59.7%. mp: 145e148 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 9.36 (s, 1H, NH), 9.14 (s, 1H, NH), 8.16
4.1.21. N-(4-(4-Cyanophenylamino)-2-(2,4,6-tribromophenoxy)
pyrimidin-5-yl)-2,2,2-trifluoroacetamide (8d)
(s, 1H, C6-pyrimidine-H), 7.63 (dd, J1 ¼ 24.92 Hz, J2 ¼ 8.92 Hz, 4H,
Ph0-H), 7.29 (s, 2H, Ph-H), 2.09 (s, 3H, CH3), 2.05 (s, 6H, 2 ꢄ CH3). 13C
Brown oily solid, yield: 40.7%. 1H NMR (400 MHz, DMSO-d6,
NMR (100 MHz, DMSO-d6, ppm) d: 170.29 (C]O), 161.06 (C2-py-
ppm)
d: 10.42 (s, 1H, NH), 9.21 (s, 1H, C6-pyrimidine-H), 8.38 (s, 2H,
rimidine), 156.73 (C4-pyrimidine), 156.02 (C1-Ph-O), 149.50 (C6-

X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
119
pyrimidine), 143.74 (C1-Ph0-N), 133.17 (2 ꢄ C), 132.96 (2 ꢄ C),
4.1.32. 4-(5-Amino-2-(4-formyl-2,6-dimethylphenoxy)pyrimidin-
4-ylamino)benzonitrile (7g)
White powder, yield: 78.2%. mp: 293e297 ꢀC. 1H NMR
129.42, 128.56 (2 ꢄ C), 120.59 (2 ꢄ C), 119.55, 114.72, 104.62, 23.85
(CH3), 16.33 (2 ꢄ CH3). ESI-MS: m/z 452.3 (Mþ1), 454.3 (Mþ3),
C
21H18BrN5O2 [451.06].
(400 MHz, DMSO-d6, ppm) d: 10.02 (s, 1H, CHO), 9.17 (s, 1H, NH),
7.79 (m, 3H, C6-pyrimidine-H, Ph0-H), 7.68 (d, 2H, J ¼ 8.80 Hz, Ph-H),
7.48 (d, 2H, J ¼ 8.80 Hz, Ph-H), 5.07 (s, 2H, NH2). 13C NMR (100 MHz,
4.1.27. 4-(2-(2,6-Dichlorophenoxy)-5-nitropyrimidin-4-ylamino)
benzonitrile (6f)
DMSO-d6, ppm) d: 192.81 (CHO), 156.10 (C2-pyrimidine), 155.76
(C4-pyrimidine), 150.26 (C1-Ph0-N), 144.68 (C6-pyrimidine), 141.47
(C1-Ph-O), 133.59, 133.00 (2 ꢄ C), 132.48 (2 ꢄ C), 132.29, 130.45
(2 ꢄ C) 125.75, 126.21, 119.73, 119.75 (2 ꢄ C), 103.56. ESI-MS: m/z
360.4 (Mþ1), 382.5 (Mþ23), C20H17N5O2 [359.14].
Yellow powder, yield: 62.2%. mp: 193e196 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 10.45 (s, 1H, NH), 9.30 (s, 1H, C6-py-
rimidine-H), 7.60 (s, 4H, Ph0-H), 7.32 (t, J ¼ 8.48 Hz, 1H, C4-Ph-H),
6.85 (d, J ¼ 8.52 Hz, 2H, Ph-H). 13C NMR (100 MHz, DMSO-d6, ppm)
d
: 164.54 (C2-pyrimidine), 160.76 (C4-pyrimidine), 154.59 (C6-py-
rimidine), 139.95 (C1-Ph0-N), 133.17 (4 ꢄ C), 133.00, 125.67, 122.69
(4 ꢄ C), 121.84, 118.29, 109.25. ESI-MS: m/z 402.4 (Mþ1), 404.4
(Mþ3), C17H9Cl2N5O3 [401.01].
4.1.33. N-(4-(4-Cyanophenylamino)-2-(4-formyl-2,6-
dimethylphenoxy)pyrimidin-5-yl)- 2,2,2-trifluoroacetamide (8g)
White powder, yield: 54.4%. mp: 179e182 ꢀC. 1H NMR (400 MHz,
DMSO-d6, ppm) d: 9.94 (s, 1H, HeC]O), 9.13 (s, 1H, C6-pyrimidine-
H), 8.47 (b, 1H, NH) 7.93 (d, J ¼ 8.44 Hz, 2H, Ph0-H), 7.67 (s, 2H, Ph-
4.1.28. 4-(5-Amino-2-(2,6-dichlorophenoxy)pyrimidin-4-ylamino)
benzonitrile (7f)
H), 2.18 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
d:
192.27 (CHO), 161.82 (C2-pyrimidine), 154.07 (C4-pyrimidine),
153.47 (C6-pyrimidine), 135.47 (C1-Ph-O), 133.86 (2 ꢄ C), 133.83
(4 ꢄ C), 131.89 (2 ꢄ C), 130.74 (2 ꢄ C), 128.40, 128.26 (2 ꢄ C), 117.16,
114.61, 16.51 (2 ꢄ CH3). ESI-MS: m/z 438.4 (C22H18F2N5Oþ3 ),
White crystals, yield: 78.2%. mp: 114e117 ꢀC. 1H NMR (400 MHz,
DMSO-d6, ppm) d: 8.95 (s, 1H, NH), 7.76 (s, 1H, C6-pyrimidine-H),
7.64 (m, 6H, Ph-H, Ph0-H), 7.41 (t, 1H, J ¼ 8.80 Hz, C4-Ph-H), 4.96 (s,
2H, NH2). 13C NMR (100 MHz, DMSO-d6, ppm)
d: 155.11 (C2-py-
C22H16F3N5O3 [455.12].
rimidine), 150.04 (C4-pyrimidine), 146.36 (C1-Ph0-N), 144.51 (C6-
pyrimidine), 141.34 (C1-Ph-O), 133.08 (2 ꢄ C), 129.64 (2 ꢄ C), 129.33
(2 ꢄ C), 127.68, 126.21, 119.73, 119.56 (2 ꢄ C), 103.75. ESI-MS: m/z
372.2 (Mþ1), 374.2 (Mþ3), 376.3 (Mþ5), 378.3 (Mþ7),
4.1.34. N-(4-(4-Cyanophenylamino)-2-(4-formyl-2,6-
dimethylphenoxy)pyrimidin-5-yl) acetamide (9g)
White crystals, yield: 53.3%. mp: 167e170 ꢀC. 1H NMR (400 MHz,
C
17H11Cl2N5O [371.03].
DMSO-d6, ppm) d: 10.00 (s, 1H, NH), 9.94 (s, 1H, NH), 9.81 (s, 1H,
CHO), 9.23 (s, 1H, C6-pyrimidine-H), 7.45 (s, 2H, Ph-H), 7.64 (s, 4H,
4.1.29. N-(4-(4-Cyanophenylamino)-2-(2,6-dichlorophenoxy)
pyrimidin-5-yl)-2,2,2-trifluoroacetamide (8f)
Ph0-H), 2.15 (s, 6H, 2 ꢄ CH3), 2.00 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6, ppm) d: 193.88 (HeC]O), 166.37 (C2-pyrimidine), 161.44
Brown oily solid, yield: 58.3%. mp: 205e208 ꢀC. 1H NMR
(C4-pyrimidine), 160.34 (C1-Ph-O), 155.20 (C6-pyrimidine), 144.29
(C1-Ph0-N), 135.46, 133.58 (2 ꢄ C), 132.51 (2 ꢄ C), 131.66 (2 ꢄ C),
127.28, 125.14, 122.25 (2 ꢄ C), 120.22, 20.35 (CH3), 16.31 (2 ꢄ CH3).
ESI-MS: m/z 402.3 (Mþ1), 424.4 (Mþ23), C22H19N5O3 [401.15].
(400 MHz, DMSO-d6, ppm) d: 9.09 (s, 1H, C6-pyrimidine-H), 7.91 (d,
J ¼ 8.60 Hz, 2H, Ph0-H), 7.64 (d, J ¼ 8.60 Hz, 2H, Ph0-H), 7.41 (d,
J ¼ 8.08 Hz, 2H, Ph-H), 7.21 (t, J ¼ 8.08 Hz, 1H, C4-Ph-H). 13C NMR
(100 MHz, DMSO-d6, ppm) d: 161.51 (C2-pyrimidine), 155.05 (C4-
pyrimidine), 153.45 (C6-pyrimidine), 145.59 (C1-Ph-O), 142.24 (C1-
Ph0-N), 135.72, 133.70 (2 ꢄ C), 129.16 (2 ꢄ C), 128.52 (2 ꢄ C), 127.09
(2 ꢄ C), 128.31 (2 ꢄ CN), 119.26, 117.36, 114.61. ESI-MS: m/z 450.2
4.2. Biological activity evaluation
4.2.1. In vitro anti-HIV assay
(C19H12Cl2F2N5O2þ
þ1),
19H10Cl2F3N5O2 [467.02].
452.2
(C19H12Cl2F2N5O2þ
þ2),
Evaluation of the antiviral activity of the compounds against
HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4 cells was per-
formed using the MTT assay as previously described [23]. Stock
solutions (10 ꢄ final concentration) of test compounds were added
C
4.1.30. N-(4-(4-Cyanophenylamino)-2-(2,6-dichlorophenoxy)
pyrimidin-5-yl)acetamide (9f)
in 25-mL volumes to two series of triplicate wells so as to allow
White crystals, yield: 60.7%. mp: 158e161 ꢀC. 1H NMR
simul-taneous evaluation of their effects on mock- and HIV-
infected cells at the beginning of each experiment. Serial fivefold
dilutions of test compounds were made directly in flat-bottomed
96-well microti-ter trays using a Biomek 3000 robot (Beckman
instruments, Fuller-ton, CA). Untreated control HIV- and mock-
infected cell samples were included for each sample.
(400 MHz, DMSO-d6, ppm) d: 9.38 (s, 1H, NH), 9.22 (s, 1H, NH), 8.20
(s, 1H, C6-pyrimidine-H), 7.67 (d, J ¼ 8.0 Hz, 2H, Ph-H), 7.59 (s, 4H,
Ph0-H), 7.44 (t, J ¼ 8.40 Hz, 1H, C4-Ph-H), 2.09 (s, 3H, CH3) 13C NMR
(100 MHz, DMSO-d6, ppm) d: 170.33 (C]O), 160.28 (C2-pyrimi-
dine), 156.79 (C4-pyrimidine), 156.06 (C1-Ph-O), 145.77 (C6-pyrim-
idine), 143.45 (C1-Ph0-N), 133.09 (2 ꢄ C), 129.76 (2 ꢄ C), 128.98
(2 ꢄ C), 128.23, 120.66 (2 ꢄ C), 119.49, 115.43, 104.93, 23.86 (CH3).
ESI-MS: m/z 414.3 (Mþ1), 416.3 (Mþ3), C19H13Cl2N5O2 [413.04].
HIV-1 (IIIB) [24] or HIV-2 (ROD) [25] stock (50 mL) at 100e300
CCID50 (cell culture infectious dose) or culture medium was added
to either the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test com-
pound on uninfected cells in order to assess the cytotoxicity of
the test compound. Exponentially growing MT-4 cells were cen-
trifuged for 5 min at 1000 rpm and the supernatant was dis-
carded. The MT-4 cells were resuspended at 6 ꢄ 105 cells/mL, and
4.1.31. 4-(2-(4-Formyl-2,6-dimethylphenoxy)-5-nitropyrimidin-4-
ylamino)benzonitrile (6g)
Yellow crystals, yield: 66.9%. mp: 171e173 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 10.46 (s, 1H, NH), 10.01 (s, 1H, CHO),
9.29 (s, 1H, C6-pyrimidine-H), 7.76 (s, 2H, Ph-H), 7.64 (s, 4H, Ph0-H),
50-mL volumes were transferred to the microtiter tray wells. Five
days after infection, the viability of mock- and HIV-infected cells
was examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial
dehydro-genase of metabolically active cells to a blue-purple
2.15 (s, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6, ppm)
d
: 192.68
(HeC]O), 164.27 (C2-pyrimidine), 160.45 (C4-pyrimidine), 154.84
(C1-Ph-O), 154.54 (C6-pyrimidine), 141.39 (C1-Ph0-N), 134.44, 132.58
(2 ꢄ C), 131.81 (2 ꢄ C), 130.56 (2 ꢄ C), 126.28, 125.12, 123.65 (2 ꢄ C),
119.02, 16.31 (2 ꢄ CH3). ESI-MS: m/z 390.3 (Mþ1), 407.5 (Mþ17),
C
20H15N5O4 [389.11].

120
X. Li et al. / European Journal of Medicinal Chemistry 80 (2014) 112e121
formazan that can be measured spectrophotometrically. The
absorbances were read in an eight-channel computer-controlled
photometer (Multi-scan Ascent Reader, Labsystems, Helsinki,
Finland), at two wave-lengths (540 and 690 nm). All data were
calculated using the median OD (optical density) value of tree
wells. The 50% cytotoxic concentration (CC50) was defined as the
concentration of the test compound that reduced the absorbance
(OD540) of the mock-in-fected control sample by 50%. The con-
centration achieving 50% protection from the cytopathic effect of
the virus in infected cells was defined as the 50% effective con-
centration (EC50).
secondary structure of RT is shown in cartoons, and only the key
residues for interactions with the inhibitors were shown in sticks
and labeled. The potential hydrogen-bondings were presented by
dashed lines.
Notes
The authors declare no competing financial interest.
Acknowledgments
We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the HIV assays. The financial support from the
National Natural Science Foundation of China (NSFC No. 81273354,
No. 81102320, No. 30873133, No. 30772629, No. 30371686), Key
Project of NSFC for International Cooperation (No. 30910103908),
Research Fund for the Doctoral Program of Higher Education of
China (Nos. 20110131130005, 20110131120037), Natural Science
Foundation of Shandong Province (ZR2009CM016) and KU Leuven
(GOA 10/014).
4.2.2. HIV-1 RT inhibition assay
A RT assay kit produced by Roche was used for the RT inhibition
assay. All the reagents for performing the RT reaction are contained
in the kit and the ELSIA procedures for RT inhibition assay was
performed as described in the kit protocol [26e28].
Briefly, the reaction mixture containing HIV-1 reverse tran-
scriptase (RT) enzyme, reconstituted template and viral nucleotides
(digoxigenin (DIG)-dUTP, biotin-dUTP and dTTP) in the incubation
buffer with or without inhibitors was incubated for 1 h at 37 ꢀC.
Then, the reaction mixture was transferred to a streptavidine-
coated microtiter plate (MTP) and incubated for another 1 h at
37 ꢀC. The biotin-labeled dNTPs that was incorporated in cDNA
chain in the presence of RT were bound to streptavidine. The un-
bound dNTPs were removed by rinsing using washing buffer fol-
lowed by the addition of anti-DIG-POD working solution into the
MTPs. After incubation for 1 h at 37 ꢀC, the DIG-labeled dNTPs
incorporated in cDNA were bound to the anti-DIG-POD antibody.
The unbound anti-DIG-PODs were removed and the peroxide
substrate (ABST) solution was added to the MTPs. Incubation of the
reaction mixture at 25 ꢀC until a green color was sufficiently
developed for detection. The absorbance of the sample was deter-
mined at O.D. 405 nm using microtiter plate ELISA reader. The
percentage inhibitory activity of RT inhibitors was calculated by
formula as given below:
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.036.
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