Fused heterocycles bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 2: Discovery of novel [1,2,4]Triazolo[1,5-a]pyrimidines using a structure-guided core-refining approach

Article abstract of DOI:10.1016/j.ejmech.2014.07.104

Guided by crystal structures of HIV-1 RT/DAPY complex and molecular modeling studies, a series of novel [1,2,4]triazolo[1,5-a]pyrimidine derivatives were rationally designed via structure-based core refining approach, synthesized through the readily acces

Full text of DOI:10.1016/j.ejmech.2014.07.104

European Journal of Medicinal Chemistry 85 (2014) 293e303
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Fused heterocycles bearing bridgehead nitrogen as potent HIV-1
NNRTIs. Part 2: Discovery of novel [1,2,4]Triazolo[1,5-a]pyrimidines
using a structure-guided core-refining approach*
,
,
,
Liu Wang a 1, Ye Tian a 1, Wenmin Chen a, Hong Liu a, Peng Zhan a *, Dongyue Li a,
Huiqing Liu b, Erik De Clercq c, Christophe Pannecouque 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
[1,2,4]triazolo[1,5-a]pyrimidine derivatives were rationally designed via structure-based core refining
approach, synthesized through the readily accessible synthetic methods and evaluated for their anti-HIV
activities in MT-4 cells. Preliminary biological evaluation indicated that most of the compounds exhibited
marked inhibitory activity against the wild-type HIV-1 IIIB. Particularly, compound 7n was the most
potent inhibitor against wild-type and K103N/Y181C double resistant mutant strain of HIV-1, possessing
Received 11 January 2014
Received in revised form
28 July 2014
Accepted 29 July 2014
Available online 30 July 2014
EC50 values of 0.02
(NVP, EC50 ¼ 0.15
m
M and 7.6
m
M, respectively, which were much better than or similar to nevirapine
M, >36 M). Besides, some other
Keywords:
DAPY
[1,2,4]Triazolo[1,5-a]pyrimidine
Bridgehead nitrogen heterocycle
anti-HIV activities
m
M, 2.9
m
M) and delavirdine (DLV, EC50 ¼ 0.07
m
m
compounds, 5b, 7c, 7e, 7f, and 7m, were also endowed with favorable anti-HIV-1 potency (EC50 ¼ 0.07,
0.05, 0.05, 0.07, and 0.05 mM, respectively), which were better than or similar to those of NVP and DLV,
suggesting a high potential to further develop this type of bridgehead nitrogen heterocycle as a novel
Structureeactivity relationships
Molecular modeling
class of NNRTIs with improved antiviral efficacy and resistance profile. The selected compound, 7i, was
found moderately inhibitory towards RT (IC50
(IC50 ¼ 0.56 M). Preliminary structureeactivity relationships (SARs) and molecular modeling of these
new analogues were detailed in this manuscript.
¼
0.39 mM), which was higher than for ETV
m
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
termed as the NNRTIs binding pocket (NNIBP), which is located
about 10 Å away from the RT catalytic site, [1]. HIV-1 NNRTIs are a
key component of the HAART regimen for the long-term manage-
ment of HIV infection because of their unique antiviral potency,
high specificity and low toxicity. However, during the clinical usage
of first generation NNRTIs (nevirapine, delavirdine and efavirenz),
significant resistance has been generated by the emergence of
mutant viral strains. Therefore, there is still need for discovery of
novel HIV-1 NNRTIs with an improved potency against the wild-
type (wt) and key clinically observed mutant virus strains [2e7].
The diarylpyrimidines (DAPYs) family, with two approved anti-
HIV-1 drugs etravirine (ETV, Intelence®, TMC125) and rilpivirine
(Edurant®, TMC278), has been regarded as one representative class
of the promising HIV-1 NNRTIs with robust anti-HIV-1 potency
against both the wt and drug-resistant strains carrying multiple
mutations [6,7]. However, the pharmacokinetic profiles of most
DAPY derivatives are not very satisfactory [8e10]. For example,
Human immunodeficiency virus type-1 (HIV-1) reverse tran-
scriptase (HIV-1 RT) is a key enzyme in the HIV replicative cycle,
which represents one of the main targets for the treatment of HIV/
AIDS [1]. Among RT inhibitors, non-nucleoside RT inhibitors
(NNRTIs) can directly inhibit RT by binding to an allosteric site,
*
Part 1: An accompanying article: Tian Y, Du D, Rai D, Wang L, Liu H, Zhan P, De
Clercq E, Pannecouque C, Liu X. Fused heterocyclic compounds bearing bridgehead
nitrogen as potent HIV-1 NNRTIs. Part 1: design, synthesis and biological evaluation
of novel 5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives. Bioorg Med Chem.
2014; 22(7):2052e9.
* Corresponding authors.
E-mail addresses: zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn
(X. Liu).
1
Liu Wang and Ye Tian contributed equally to this article.
http://dx.doi.org/10.1016/j.ejmech.2014.07.104
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

294
L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
etravirine is both a substrate and an inducer of cytochrome P450
3A4 (CYP3A4), while it is an inhibitor of CYP2C9 and CYP2C19.
Numerous drugedrug interactions must be considered when pre-
scribing etravirine. Therefore, many drugs are limited on that
occasion. This situation have encouraged several research groups to
explore next-generation NNRTI agents with better pharmacological
profiles and enhanced oral bioavailability for the treatment of HIV
infections.
Based on the X-ray crystal structures of DAPYs-RT complexes
and molecular modeling, the binding conformation of DAPYs
resembled a horseshoe or “U” shape when bound in the NNIBP in
contrast to the typical butterfly-like binding shape of the three
first-generation NNRTIs. The relative flexibility of the molecules
might allow DAPYs to better adapt to the plasticity of the NNIBP,
which appear to be critical for maintaining potency against wt and
a wide range of drug-resistant mutant HIV-1 RTs [5e7]. As shown in
Fig. 1, their molecular structure consists of three rings bound by
rotatable bonds with an essential NH linker between the right wing
(A ring) and the central ring (B ring). The DAPYs also shared a
similar pharmacophore as the first-generation NNRTIs, including
chemistry to obtain intellectual property and novel hits, as well as
to improve synthetic accessibility [6]. To discover highly potent
DAPY derivatives with better drug-like properties that are easier to
synthesize or to avoid existing patent art, further modifications
were focused on the replacement of the pyrimidine core with an
array of aromatic scaffolds [11].
In this regard, previous research in our laboratory using
structure-based drug design and isosteric principle resulted in the
discovery of a novel series of potent DAPYs back-up analogues, such
as pyridazines (DL-8g), nitropyridines (JW-7a2), and pyrimidines
(XL-7a), as RT inhibitors which proved to be highly effective in
inhibiting HIV-1 replication at low or double-digit nanomolar
concentrations [12e14]. According to the molecular modeling and
structureeactivity relationship (SAR) results, the central pyrimi-
dine ring of DAPYs is relatively tolerant.
Recently, a closely related scaffold type, pyrrolopyrimidine, has
been successfully investigated for the discovery of highly potent
NNRTIs against wild-type and single mutant strains. Especially,
RDEA427 (preclinical) and RDEA640 (Fig. 1) are superior to efavir-
enz against a batch of NNRTI-resistant strains and show weaker
binding affinity to serum proteins than rilpivirine and efavirenz.
RDEA640 and RDEA427 also demonstrate a low potential for hu-
man cytochrome P450 induction and a low cytotoxicity. The
favorable in vitro pharmacology profiles of these novel molecules
display great potential for improved performance over the existing
NNRTIs and warrants further clinical evaluation [15].
hydrophobic center able to participate in pep stacking interactions
(C ring), hydrogen bond donor and acceptor. The six-membered
heterocycle moiety not only stays in the center of the NNIBP,
anchoring the functional groups for optimal engagement with the
residues around NNIBP, but also serves as a critical hydrogen bond
acceptor (the pyrimidine nitrogen at 1-position) to make hydrogen
bond with the
a
-amino group of Lys101. Besides, the NH linker
Additionally, Riley et al. studied CYP3A4 inhibition of drugs that
contained pyridine and suggested general rules for reducing this
inhibition of nitrogen heterocycle-containing drugs [16]. They
concluded that the decreased lipophilicity (LogD) of the molecule
and increased steric hindrance to the heterocycle (which reduced
the interaction of the nitrogen lone pair with the 3A4 heme group)
were beneficial to reduce CYP3A4 inhibition. Other structur-
eemetabolism relationship studies have shown that incorporation
of one or more nitrogen atoms in the rings decreases the electron
connecting the central B ring and the right A wing can function as a
hydrogen bond donor able to interact with the backbone carbonyl
of Lys101 [5e7]. These conclusions provided critical insights for
further structural evolution or scaffold refining towards improving
their activity and drug resistance profiles.
Scaffold refining via the replacement of the central core in
bioactive molecules combined with introducing privileged sub-
stituents is
a common practice in contemporary medicinal
Fig. 1. Design of 1,2,4-triazolo[1,5-a]pyrimidine derivatives as DAPYs back-up series via a scaffold (core) refining approach.

L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
295
density on the carbons of aromatic ring, thereby decreasing P450-
mediated interaction [17]. Thus, the lower reactive rings with
multiple nitrogens improve the metabolic properties of these
prime molecules.
straightforward synthetic procedures are described in the experi-
mental section. Briefly, the commercially available material 2H-
1,2,4-triazol-3-amine (1) was condensed with the diethyl malonate
to form the [1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol (2) [19e21].
Chlorination with phosphorous oxychloride gave the 5,7-dichloro-
[1,2,4]triazolo[1,5-a]pyrimidine (3) [19e21]. Then the 7-Cl of
compound 3 underwent a facile nucleophilic aromatic substitution
reaction with 4-aminobenzonitrile in ethanol to afford 4-(5-chloro-
[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamino)benzonitrile (4) exclu-
sively [21e23]. Treatment of intermediate 4 with a range of
substituted phenols and potassium carbonate under 85 ꢀC in DMF
gave the target compounds 4-(5-substituted phenoxy-[1,2,4]tri-
azolo[1,5-a]pyrimidin-7-ylamino)benzonitriles (series 5). Alterna-
tively, replacement of the 7-chloro of compound 3 with the
corresponding ArOH (for compounds N-(4-substituted-phenyl)-7-
substituted phenoxy-[1,2,4]triazolo[1,5-a]pyrimidin-5-amines) or
As an extension of our investigations and with the aim to
generate novel NNRTIs with desirable potency and properties, we
focused on our interests on the bridgehead nitrogen heterocycles,
which were considered as privileged structures in numerous bio-
logically active compounds [17,18] (including drugs, like Zaleplon,
Zolpidem and Temozolomide) and conformed to the optimizing
strategies for better metabolic mentioned above. Therefore, a novel
series of 1,2,4-triazolo[1,5-a]pyrimidine derivatives were designed
based on the fused pyrrolopyrimidine series as the lead. In these
structures the pyrimidine ring in the original DAPY scaffold was
replaced with a synthetically accessible 1,2,4-triazolo[1,5-a]py-
rimidine heterocycle to ensure the optimal pharmacodynamic and
pharmacokinetic properties, while the NH linker connecting the
central ring and the right wing, and the privileged substitutions of
A and C rings were maintained in view of their paramount
importance in the previous series. Further, we substituted the A and
C rings with additional groups to investigate their influence on the
HIV inhibitory activity. General structure of such a new scaffold is
illustrated in Fig. 1.
a
ArNH2
(for compounds
N5-(4-substituted-phenyl)-N7-
substituted-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diamines),
in the presence of potassium carbonate in DMF, yielded the desired
intermediates 6(aeh) [21e23]. Compounds 7aeo were prepared by
further treatment of compounds 6(aeh) with the required amines.
The newly synthesized compounds were characterized by physi-
cochemical and spectral means and both analytical and spectral
data of all the compounds were found to be in full agreement with
the proposed structures.
Herein, we will further report the synthesis, anti-HIV evaluation
of [1,2,4]triazolo[1,5-a]pyrimidine derivatives against wt HIV-1 and
HIV-2 ROD, as well as against a double-mutated HIV-1 strain
RES056 (K103N þ Y181C). In addition, preliminary SAR data and
molecular modeling results of these new compounds are also dis-
cussed to provide deeper insight into their interactions with the
allosteric binding site.
2.2. Anti-HIV evaluation
All of the newly synthesized [1,2,4]triazolo[1,5-a]pyrimidines
derivatives together with the marketed drugs of NVP and DLV as
the controls were evaluated for their anti-HIV activity (against the
wt HIV-1 strain IIIB [24], the commonly encountered double mutant
strain RES056 (K103N þ Y181C) and HIV-2 strain ROD [25,26]) and
cytotoxicity in MT-4 cells. The methodology of the anti-HIV assay
has been previously described [27,28]. The results, expressed as
EC50 (anti-HIV activity), CC50 (cytotoxicity) and SI (selectivity index,
given by the CC50/EC50 ratio), are summarized in Table 1. The
2. Results and discussion
2.1. Chemistry
To achieve the synthesis of the target compounds 5(aee) and
7(aeo), the steps outlined in Scheme 1 were adopted. The
Scheme 1. Synthetic route to [1,2,4]triazolo[1,5-a]pyrimidine derivatives. Reagents and conditions: (i) Diethyl malonate, Na, EtOH, reflux; (ii) POCl3, 95 ꢀC; (iii) ArNH2, EtOH, rt; (iv)
ArOH, K2CO3, DMF, 85 ꢀC; (v) ArOH or ArNH2, K2CO3, DMF, 30 ꢀC; (vi) ArNH2, 150e160 ꢀC.

296
L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
Table 1
HIV inhibitory effecta and cytotoxicity of the title compounds.
Compd
R2 ¼ R6
R4
R
X
EC50
IIIB
(m
M)b
CC50
(
m
M)c
SId
IIIB
RES056
ROD
RES056
ROD
5a
5b
5c
5d
5e
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
Me
Me
Me
Cl
F
Me
Cl
Me
Me
Me
Me
Me
Me
Me
Me
Cl
H
CN
CN
CN
CN
CN
CN
CN
CN
NO2
Cl
Me
OMe
CN
CN
CN
CN
CN
CN
CN
CN
e
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
e
e
e
1.5 1.0
>253
ꢁ50
>253
>106
>128
>32
>174
>233
>150
>164
>33
>46
>47
>37
>136
>224
>104
>29
253 33
106 46
128 36
32 2.0
174 19
233 2.4
150 41
164 63
33 1.7
46 5.5
47 2.3
37 31
136 31
224 22
104 32
29 2.8
47 24
192 34
190 5.7
272 44
>15
170
<1
ꢂ2
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
25
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
e
Me
Br
Cl
F
H
0.07 0.06
0.30 0.09
0.30 0.09
>174
1.3 0.8
3.4 2.4
0.05 0.04
0.6 0.5
0.05 0.02
0.07 0.02
0.2 0.1
0.23 0.07
1.2 0.8
0.4 0.3
1449
427
109
<1
182
44
>128
>32
>174
>233
>150
>164
>33
>46
>47
>37
>136
>224
>104
>29
H
Me
Me
Me
Me
Me
Br
CN
Cl
3198
57
864
649
165
579
182
256
47
142
3618
8986
127
>14
>494
>1537
Cl
0.6 0.3
0.3 0.2
7l
7m
7n
Br
Br
Me
Me
Br
Me
Me
H
>47
>47
0.05 0.01
0.02 0.01
2.1 3.3
0.15 0.08
0.07 0.05
0.003 0.0002
>192
7.6 0.4
>272
2.9 1.8
>36
>192
>190
>272
e
7o
<1
>5
X1
>177
NVP
DLV
ETV [23],e
e
e
e
e
>36
>4.6
e
e
e
0.026 0.004
e
e
a
All of the title compounds were evaluated for their anti-HIV activity against the wt HIV-1 strain IIIB, the commonly encountered double mutant strain RES056
(K103N þ Y181C) and HIV-2 strain ROD.
b
EC50: concentration of compound required to achieve 50% protection of MT-4 cells against HIV-1-induced cytopathic effect, as determined by the MTT method.
CC50: concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
c
d
SI: selectivity index (CC50/EC50), and the SI values: X1 stands for ꢁ1 or <1.
e
Data were obtained from the same lab of the Rega Institute for Medical Research, KU Leuven, Belgium.
activities of etravirine (ETV) were also included as reference ma-
terials [29].
As seen from Table 1, the results indicated that the majority of
target compounds have displayed remarkable potency against wt
Based on the results of antiviral assay (Table 1), several impor-
tant structural features for conferring optimum HIV inhibition and
cytotoxicity were investigated and summarized as the following:
The data clearly demonstrated that the orientation of the central
[1,2,4]triazolo[1,5-a]pyrimidine ring does not significantly affect
the anti-HIV potency by comparing the activities of the counter-
parts between series 5 and 7 (5a vs 7a; 5b vs 7c; 5c vs 7h; 5d vs 7k),
indicating that the [1,2,4]triazolo[1,5-a]pyrimidine provided
similar conformational and electronic contributions to the binding
of the inhibitors with the NNIBP in both series.
Next we turned our attention to SAR of the left ring in 5 series. In
the case of 2,6-dimethylphenoxy analogues (5a, 5b, 5c), a clear
order of R4-substitution for anti-HIV activity was observed by direct
comparison: CH3 (5a) > Br (5c) > H (5b). In the case of 2,4,6-
trisubstituted-phenoxy derivatives (5b, 5d, 5e), we found the
HIV-1 (IIIB) with EC50 values between 0.02
for compound 5e (EC50 > 174 M). Most of them are of low toxicity
in MT-4 cells with CC50 values more than 100 M. Encouragingly,
the most effective compound 7n with an EC50 of 0.02 M against
wild-type HIV-1, was better than nevirapine (0.15 M) and DLV
(0.07 M), but it was inferior to the clinically used etravirine
(0.003 M) [29]. Moreover, 7n exhibited an excellent selectivity
index (SI against the wild-type HIV-1 was approximately 9000).
mM and 3.4 mM, except
m
m
m
m
m
m
Besides, five other compounds, 5b (EC50 ¼ 0.07
m
m
M, SI ¼ 1449), 7c
M, SI ¼ 864), 7f
M, SI ¼ 649) and 7m (EC50 ¼ 0.05 M, SI ¼ 3618),
(EC50 ¼ 0.05
(EC50 ¼ 0.07
m
M, SI ¼ 3198), 7e (EC50 ¼ 0.05
m
m
were more active than reference drugs NVP and DLV against wt
HIV-1(IIIB). These promising results demonstrated that the isosteric
replacement of [1,2,4]triazolo[1,5-a]pyrimidine for pyrimidine in
the central B-ring of DAPY compounds was gratifying and afforded
a series of potent [1,2,4]triazolo[1,5-a]pyrimidine NNRTIs.
antiviral potency in the order of 2,4,6-trimethyl > 2,4,6-
trichloro > 2,4,6-trifluoro.
As highlighted in Table 1, the potency of 7 series is also strongly
dependent on the nature of substituents at the R4 position of the C-
ring. In the case of the 2,6-dimethylphenoxy analogues with

L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
297
R ¼ CN, the order of R4-substition for anti-HIV activity ranked as
2.4. Molecular modeling analysis
follows: CH3 (7c) >> Br (7h) > Cl (7j) > CN (7i) > H (7a), which is
similar to that of the 5 series. This conclusion is in agreement with
the observation in 2,6-dichlorophenoxy analogues (Cl > H) and 2,6-
dibromophenoxy analogues (CH3 > Br). These results seemed to
indicate that R4 substituents with appropriate bulk are beneficial
for potency.
To understand the interactions between these inhibitors and the
target, the representative compounds 5b, 7c, 7i, 7n along with the
reference compounds ETV, RDEA427 and RDEA640 were docked
into the NNIBP of HIV-1 RT by Surflex-Dock module of Sybyl-X 1.1
software. X-ray crystal structure of HIV-1 wt RT (PDB code: 3MEC)
and K103N/Y181C mutant RT (PDB code: 3BGR) were used as the
input structures for docking calculations. Default parameters were
used as described in the Sybyl-1.1 manual unless otherwise speci-
fied. The theoretical binding modes of these compounds to the
NNIBP are shown in Fig. 2.
Comparatively speaking, the anti-HIV-1 activity has been less
affected by the nature of the substituents at the R2/R6 position than
at R4. Bearing the identical R4-CN substituent, the R2/R6-methyl
derivative 7c and R2/R6-bromo derivative 7m shared the same anti-
HIV activity (EC50 ¼ 0.05
0.04
m
M, EC50 ¼ 0.05
0.01 mM,
respectively). Moreover, the R2/R6-methyl derivative 7j showed
It is important to point out that the synthesized compounds
adopted a very similar conformation of interaction in the NNIBP as
that of the reference DAPY compound ETV. As illustrated in
Fig. 2aec, the common left wing of compounds 5b, 7c and 7n fits
into the aromatic-rich binding pocket, surrounded by the aromatic
side chains of Tyr188, Tyr181, Phe227, and Trp229. And right wing
in the pocket lined by Lys103, Val106, Leu234, Pro236 and Tyr318.
These compounds undergo one or two hydrogen bonds with the
backbone of residue Lys101 through the NH linker moiety with the
right wing or the nitrogen atom of the central ring, which is
important for the affinity between inhibitor and RT. Besides, the
fusing triazole part of the [1,2,4]triazolo[1,5-a]pyrimidine could
enhance the van der Waals interactions (for 7c and 7n) or the
hydrogen bonds (for 5b) between the inhibitors and the adjacent
amino acid residues (Glu138, Val179, and Lys101) within the
binding pocket of RT, explaining the observed nearly equal potency
against wt HIV-1 between 5aee and their counterparts in the 7
series. All the above interactions would favor the high binding af-
finity and increased antiviral activity.
Detailed analysis of the superimposed binding modes of 7n
(purple) and ETV (pink) in the HIV-1 RT showed that the fused
triazole part of the [1,2,4]triazolo[1,5-a]pyrimidine overlays with
the amino and bromine groups of ETV. Although the established
hydrogen bond between ETV and Glu138 was not observed in 7n,
the fused triazole part could interact favorably with the Glu138 side
chain, giving rise to compensatory van der Waals interactions,
which may explain that the representative compound 7i exhibited
similar activities with ETV against the wt RT.
activity in the low submicromolecular range with an EC50 value
(0.4
(EC50
trimethyl >> 2,4,6-tribromo
m
M) comparable to that of the R2/R6-chloro derivative 7k
¼
0.6
m
M). The substituents ranked as 2,4,6-
>
2,4,6-trichloro for 2,4,6-
trisubstituted-phenoxy derivatives (7c, 7l, 7k) in the order of
antiviral potency.
Regarding to SAR of the R substituent at the para position of the
A-ring, a declining trend of anti-HIV-1 (IIIB) potency was found for
the congeners with the 2,4,6-trimethylphenoxy group: 7c (CN) > 7e
(Cl) > 7f (Me) > 7g (OMe) > 7d (NO2). The R-cyano compound 7c
also showed the best toxicity profile. This implies that the para-
cyano moiety could be preferred for anti-HIV activity.
Furthermore, the replacement of the O linker connecting the
central B ring and the left C wing of 7c with NH linker culminated in
the identification of the most potent compound 7n. But this
improvement did not appear in the case of 7a and 7o.
Like for other typical NNRTIs, all the title compounds were
inactive against the HIV-2 strain. However, compound 7n
possessed potent activity against HIV-1 mutated strain RES056
bearing both K103N and Y181C mutations in MT-4 cells with an
EC50 value of 7.6
(EC50 > 63 M). Although compound 7n was still less potent than
M), it is a good starting
mM, which was lower than that for DLV
m
NVP (EC50 ¼ 2.9
m
M) and ETV (EC50 ¼ 0.026
m
point for further modifications.
2.3. Inhibition of HIV-1 RT
Compound 7n was also simulated with a K103N/Y181C mutant
RT binding pocket (PDB code: 3BGR). It is worth noting that this
mode of binding of 7n allows to keep the conformational flexibility,
which may compensate for the effects of resistance mutations.
Secondly, the result revealed that the potent compound retained
the hydrogen bond between NH and Asn103. This observation is in
agreement with the conclusion that the potent compounds are able
to accommodate themselves in the mutant binding pocket so as to
retain the key hydrogen bond and efficacy against HIV-1 mutant
strains.
To further study the reason why the designed compounds are
less active than pyrrolopyrimidine series, comparison between
these compounds for their binding comformations and docking
scores were conducted. As shown in Fig. 2eef, the designed com-
pounds share a closely similar conformation with RDEA427 and
RDEA640 both in wild and mutant RT. Then obtained docking
scores presented in Tables 3 and 4 display more detailed informa-
tion. All these compounds have excellent total scores. Although the
scores are not completely consistent with the biological assay, the
results reflect the difference between designed compounds and the
reference to a certain extent. Most of the figures of the tested
compounds and reference molecules are very close, while the ab-
solute values of the ‘Crash’ item of 5b, 7c, 7i and 7n are significantly
larger than these of the RDEA427 and RDEA640. This indicates that
the larger degree of inappropriate penetration by the designed
With the aim to further confirm the drug target of [1,2,4]triazolo
[1,5-a]pyrimidine derivatives, the selected title compound 7i (due
to the high degree of structural similarity with ETV) was tested in
enzymatic assays against highly purified recombinant HIV-1 RT
which use poly [A] ꢃ oligo [dT]15 as template/primer and digox-
igenin-/biotin-labeled dUTP as nucleotides in this assay [30,31]. The
detection and quantification of the synthesized DNA represented
the activity of HIV-1 RT. IC50 values correspond to the concentration
of the [1,2,4]triazolo[1,5-a]pyrimidine derivatives required to
inhibit biotin-dUTP incorporation by HIV-1 RT by 50%. As shown in
Table 2, compound 7i exhibited moderate inhibition of enzymatic
activity with an IC50 value of 0.39
mM, which was slightly lower
than that of ETV (0.56 M). The results show that these newly
m
synthesized derivatives, represented by compound 7i, are targeted
at HIV-1 RT, thus acting as genuine NNRTIs.
Table 2
Inhibitory activity of compound 7i against HIV-1 RT.
Compd.
7i
Etravirine
0.56
IC50 (
m
M)a
0.39
a
50% inhibitory concentration of tested compounds required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 (wt) RT by
50%.

298
L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
Fig. 2. (a) Predicted binding mode of compound 5b in the allosteric site of HIV-1 wt RT (PDB code: 3MEC); (b) Predicted binding mode of compound 7c in the allosteric site of HIV-1
wt RT (PDB code: 3MEC); (c) Superimposition of the docked conformations of 7n (purple) and ETV (pink) in the HIV-1 RT (PDB code: 3MEC); (d) Predicted binding mode of
compound 7n in the allosteric site of HIV-1 K103N/Y181C mutant RT (PDB code: 3BGR). The docking results are shown by PyMOL. Hydrogen bonds are indicated by dashed lines; (e)
& (f) Molecular superposition of 5b (orange), 7c (yellow), 7i (cyan), 7n (purple), RDEA427 (green), and RDEA640 (white) in the binding site of 3MEC and 3BGR, respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
299
Table 3
Docking scoresa of 5b, 7c, 7i, 7n, RDEA427 and RDEA640 binding into wild RT (3MEC).
Comp.
T_S
C
P
S
D_S
P_S
G_S
CHS
CS
G_C
RDEA640
RDEA427
5b
7c
7n
7i
10.25
9.94
8.95
8.87
8.71
8.69
ꢄ1.29
ꢄ1.35
ꢄ2.39
ꢄ2.16
ꢄ2.49
ꢄ1.93
1.38
1.36
1.86
1.30
1.28
1.32
0.80
0.89
0.79
0.81
0.78
0.85
ꢄ175.78
ꢄ177.54
ꢄ157.88
ꢄ167.00
ꢄ169.37
ꢄ167.94
ꢄ99.34
ꢄ94.16
ꢄ90.44
ꢄ92.09
ꢄ89.17
ꢄ86.01
ꢄ328.79
ꢄ322.15
ꢄ323.56
ꢄ321.22
ꢄ316.94
ꢄ320.96
ꢄ40.41
ꢄ38.68
ꢄ42.04
ꢄ38.97
ꢄ42.73
ꢄ36.12
5
5
5
5
5
5
5
5
5
5
5
5
a
T_S ¼ Total_score; C ¼ Crash; P ¼ Polar; S ¼ Similarity; D_S ¼ D_SCORE; P_S ¼ PMF_SCORE; G_S ¼ G_SCORE; CHS ¼ CHEMSCORE, CS ¼ CSCORE, G_S ¼ GLOBAL_CSCOR.
molecules into the RT and of interpenetration (self-clash) between
ligand atoms that are separated by rotatable bonds might explain
why this novel series demonstrated relatively lower anti-HIV-1
activity. These findings provide valuable information for the
rational design of effective inhibitors with better therapeutic pro-
files for the treatment of AIDS.
Standard G1313A instrument. TLC was performed on silica gel
GF254 for TLC (Merck) and spots were visualized by iodine vapors
or by irradiation with UV light (254 nm). Flash column chroma-
tography was performed on a column packed with silica gel 60
(230e400 mesh). Solvents were reagent grade and, when neces-
sary, were purified and dried by standard methods. Concentration
of the reaction solutions involved the use of rotary evaporator at
reduced pressure.
3. Conclusions
In this article, based on the structure-based core-refining
design, replacement of the heterocycle fragment in the previously
reported DAPYs lead structures with a bridgehead nitrogen 1,2,4-
triazolo[1,5-a]pyrimidine heterocycle led to the discovery of a
novel series of potent NNRTIs. Most of these new congeners
exhibited moderate to excellent activity against wild-type virus
4.1.1. General procedure for the synthesis of 5,7-dichloro-[1,2,4]
triazolo[1,5-a]pyrimidine (3)
A mixture of 1H-1,2,4-triazol-5-amine (1) (5.0 g, 59.5 mmol),
diethyl malonate(9.52 g, 59.5 mmol), and sodium ethoxide (4.05 g,
59.5 mmol) was heated under reflux in absolute ethanol (50 mL) for
12 h. After removal of the solvent, the residue was poured into ice
water, and extracted with ethyl acetate for 3 times. The water layer
was separated, the pH of liquid was adjusted to 1.0 with concen-
trated hydrochloric acid. After cooling in ice water, white solid was
formed, filtered, washed with water, and dried under vacuum to
give [1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol (2) 5.8 g with 65e70%
yield. Mp: 238e240 ꢀC. ESI-MS: m/z 151.1 (Mꢄ1); C5H4N4O2
(152.0334).
Compound 5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidine (3)
was synthesized as previously described. Concretely, compound
[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol (2) 6.08 g (40 mmol) was
added to 57 mL (600 mmol) of phosphorous oxychloride and
heated under reflux for 12 h in a round bottom flask, during which
the solid dissolved and hydrogen chloride was evolved. Excess
phosphorous oxychloride was removed by distillation at reduced
pressure in a steam-bath and the residue triturated with ice water.
The mixture was extracted with methylene chloride. Then the
organic layer was separated, washed with water, dried over anhy-
drous sodium sulfate, filtered and concentrated to give the crude
product 5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidine (3) as yellow
solid, 4.2 g, Yield: 50e60%. mp:126e130 ꢀC. ESI-MS: m/z
189.4(Mþ1), 191.3(Mþ3), 193.3(Mþ5); C5H2Cl2N4 (187.9657).
Compound 3 was used in the next step without further purification.
with an EC50 value ranging from 3.41
SAR was established. Among the newly synthesized compounds, 7n
M,
mM to 0.02 mM. Preliminary
was identified as the most active compound (EC50 ¼ 0.02
m
SI ¼ 8986) associated with moderate activity against the HIV-1
double mutant strain (K103N þ Y181C) with an EC50 value in
7.61
mM, suggesting a high potential to further develop these
compounds as a novel class of NNRTIs with improved antiviral ef-
ficacy and resistance profile. None of the compounds exhibited
activity against the HIV-2 ROD strain. Furthermore, the selected
1,2,4-triazolo[1,5-a]pyrimidine 7i showed moderate activity
against wt RT. Molecular modeling studies with representative
compounds were performed to gain insight into its binding mode
with the allosteric site of HIV-1 RT, and to provide the basis for
further structure-guided design of new NNRTIs candidates.
Ongoing studies involve extension of the illustrated SAR clues to
identify additional activity parameters in the bridgehead nitrogen
heterocycle scaffold to overcome drug-viral resistance.
4. Experimental section
4.1. Chemistry
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
with a Nexus 470FT-IR Spectrometer.1H NMR spectra were obtained
on a Brucker Avance-600 NMR-spectrometer in the indicated sol-
4.1.2. General procedure for the synthesis of 4-(5-substituted
phenoxy-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamino) benzonitriles
(5aee)
vents. Chemical shifts are expressed in
reference. Mass spectra were taken on an LC Autosampler Device:
d
units and TMS as internal
The 4-aminobenzonitrile (1.56 g, 13.2 mmol) was added to 5,7-
dichloro-[1,2,4]triazolo[1,5-a]pyrimidine (3) (2.5 g, 13.2 mmol) in
Table 4
Docking scores of 5b, 7c, 7i, 7n, RDEA427 and RDEA640 binding into mutant RT (3BGR).
Comp.
T_S
C
P
S
D_S
P_S
G_S
CHS
CS
G_C
RDEA640
RDEA427
5b
7i
7c
7n
10.38
10.21
10.03
9.74
9.38
8.46
ꢄ0.85
ꢄ0.66
ꢄ1.17
ꢄ0.97
ꢄ0.70
ꢄ1.14
1.86
1.70
2.13
1.77
1.59
1.51
0.65
0.64
0.69
0.66
0.70
0.76
ꢄ160.93
ꢄ161.36
ꢄ159.08
ꢄ164.92
ꢄ158.51
ꢄ163.88
ꢄ81.75
ꢄ86.20
ꢄ83.68
ꢄ66.78
ꢄ90.47
ꢄ97.48
ꢄ305.52
ꢄ301.19
ꢄ293.76
ꢄ298.22
ꢄ282.52
ꢄ282.91
ꢄ36.76
ꢄ34.59
ꢄ38.46
ꢄ31.93
ꢄ34.80
ꢄ39.10
5
5
5
5
5
5
5
5
5
5
5
5

300
L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
absolute ethanol (35 mL) and stirred at room temperature for 6 h.
The reaction mixture was filtered, washed with cold ethanol, and
dried under vacuum to give the crude product 4-((7-chloro-[1,2,4]
triazolo[1,5-a]pyrimidin-5-yl)amino)benzonitrile (4) as white
solid. 3.3 g, yield: 92%. ESI-MS: m/z 271.4(Mþ1), 273.4(Mþ3);
presence of anhydrous K2CO3 (about 5.4 mmol) at room tempera-
ture followed by addition of the intermediate 5,7-dichloro-[1,2,4]
triazolo[1,5-a]pyrimidine (3) (about 3 mmol). The reaction mixture
was stirred at 30 ꢀC for 6e12 h (monitored by TLC). Then some
water was added to the reaction mixture. The formed solid was
filtered, washed with water, and dried under vacuum to give the
desired intermediates 6(aeh), which were used in the following
step without further purification. Yields ranged from 50 to 90%.
The mixture of the corresponding intermediate 6(aeh) (about
1.5 mmol) and 4-aminobenzonitrile (about 5.25 mmol) was stirred
at 150 ꢀC for about 2 h (monitored by TLC) according to the reported
method. The mixture was cooled to room temperature, diluted with
DMF to get a clear solution, then concentrated with a balanced
amount of silica gel in vacuo. Products were purified by column
chromatography with ethyl acetate/petroleum ether (1:2) to afford
15 target compounds 7aeo, respectively. Yields ranged from 60 to
80%.
C
12H7ClN6 (270.0421).
The appropriate substituted phenols (1.2 equiv.) were dissolved
in anhydrous DMF (10 mL) in the presence of anhydrous K2CO3
(1 equiv.) at room temperature followed by addition of crude in-
termediate 4 (1.0 equiv.). The reaction mixture was stirred at 150 ꢀC
for 8e12 h (monitored by TLC). The solvent was cooled to room
temperature, and some water was added. The residue was extrac-
ted with ethyl acetate for several times, and the combined organic
phase was washed with water, and dried over anhydrous sodium
sulfate to give the corresponding crude products, which were pu-
rified by flash column chromatography (ethyl acetate: petroleum
ether ¼ 1:2) to afford five target compounds 5aee, respectively.
Yields ranged from 5 to 10%.
4.1.3.1. 4-((7-(2,6-Dimethylphenoxy)-[1,2,4]triazolo[1,5-a]pyrimidin-
4.1.2.1. 4-(5-(2,6-Dimethylphenoxy)-[1,2,4]triazolo[1,5-a]pyrimidin-
5-yl)amino)benzonitrile (7a). White solid, yield 50%. mp:
7-ylamino)benzonitrile (5a). White solid, yield 10%. mp:
290e293 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.14 (s, 1H, NH), 8.43
298e300 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.15 (s, 1H, NH), 8.43
(s,1H, triazole-H), 7.97 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.81 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.34 (m, 3H, OPh-H), 5.68 (s, 1H, pyrimidine-H),
2.18 (s, 6H, CH3). IR (KBr, cmꢄ1): 3295 (NH), 2224 (CN). ESI-MS:
m/z 375.3 (Mþ1); C20H16N6O (356.1386).
(s,1H, triazole-H), 7.97 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.81 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.32 (m, 3H, OPh-H), 5.68 (s, 1H, pyrimidine-H),
2.18 (s, 6H, CH3). IR (KBr, cmꢄ1): 3303 (NH), 2223(CN). ESI-MS: m/
z 357.4 (Mþ1); C20H16N6O (356.1386).
4.1.3.2. 4-((7-(2,6-Dichlorophenoxy)-[1,2,4]triazolo[1,5-a]pyrimidin-
4.1.2.2. 4-(5-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamino)
benzonitrile (5b). White solid, yield 10%. 1H NMR (DMSO-d6,
5-yl)amino)benzonitrile (7b). White solid, yield 36%. mp:
273e278 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.22 (s, 1H, NH), 8.48
400 MHz) d: 10.14 (s, 1H, NH), 8.43 (s, 1H, triazole-H), 7.98 (d, 2H,
(s, 1H, triazole-H), 7.97 (d, 2H, J ¼ 8.7 Hz, OPh-H), 7.86 (m, 4H, Ph-
H), 7.62 (d, 1H, J ¼ 8.8 Hz, OPh-H), 5.93 (s, 1H, pyrimidine-H). IR
(KBr, cmꢄ1): 3431 (NH), 2224 (CN). ESI-MS: m/z 397.3 (Mþ1), 401.3
(Mþ5); C18H10Cl2N6O (396.0293).
J ¼ 8.8 Hz, Ph-H), 7.81 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.12 (s,2H, OPh-H),
5.70 (s, 1H, pyrimidine-H), 2.33 (s, 3H, CH3), 2.14 (s, 6H, CH3). IR
(KBr, cmꢄ1): 3303 (NH), 2223 (CN). ESI-MS: m/z 371.4 (Mþ1);
C21H18N6O (370.1542).
4.1.2.3. 4-(5-(4-Bromo-2,6-dimethylphenoxy)-[1,2,4]triazolo[1,5-a]
4.1.3.3. 4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
amino)benzonitrile (7c). White solid, yield 70%. mp: 255e258 ꢀC. 1H
pyrimidin-7-ylamino)benzonitrile (5c). White solid, yield 10%. mp:
decomposed. 1H NMR (DMSO-d6, 400 MHz)
d: 10.13 (s, 1H, NH),
NMR (DMSO-d6, 400 MHz) d: 10.14 (s, 1H, NH), 8.42 (s, 1H, triazole-
8.44 (s,1H, triazole-H), 7.98 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.82 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.60 (s, 2H, OPh-H), 5.71 (s, 1H, pyrimidine-H),
2.18 (s, 6H, CH3). IR (KBr, cmꢄ1): 3358 (NH), 2218 (CN). ESI-MS:
m/z 435.4 (Mþ1), 437.4 (Mþ3); C20H15BrN6O (434.0491).
H), 7.98 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.81 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.12
(s,2H, OPh-H), 5.69 (s, 1H, pyrimidine-H), 2.33 (s, 3H, CH3), 2.14 (s,
6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d: 15.73 (eCH3 ꢃ 2), 20.82
(-CH3), 119.73 (-CN), AreC (ꢃ17): 81.85, 104.18, 119.38 (ꢃ2), 130.08
(ꢃ2), 130.63 (ꢃ2), 133.72 (ꢃ2), 137.03, 144.45, 146.33, 154.04,
155.33, 156.74, 158.44. IR (KBr, cmꢄ1): 3324 (NH), 2223 (CN). ESI-
MS: m/z 371.4 (Mþ1), 393.3 (Mþ23); C21H18N6O (370.1542).
4.1.2.4. 4-(5-(2,4,6-Trichlorophenoxy)-[1,2,4]triazolo[1,5-a]pyr-
imidin-7-ylamino)benzonitrile (5d). White solid, yield 8%. mp:
258e260 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.16 (s, 1H, NH), 8.48
(s,1H, triazole-H), 8.13 (s, 2H, OPh-H), 7.96 (d, 2H, J ¼ 8.8 Hz, Ph-H),
7.84 (d, 2H, J ¼ 8.8 Hz, Ph-H), 5.91 (s, 1H, pyrimidine-H). IR (KBr,
cmꢄ1): 3303 (NH), 2231 (CN). ESI-MS: m/z 431.3 (Mþ1), 433.4
(Mþ3), 435.3 (Mþ5), 437.3 (Mþ7); C18H9Cl3N6O (429.9903).
4.1.3.4. 7-(Mesityloxy)-N-(4-nitrophenyl)-[1,2,4]triazolo[1,5-a]pyr-
imidin-5-amine (7d). White solid, yield 54%. mp: 293e297 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) d: 10.33 (s, 1H, NH), 8.45 (s, 1H, triazole-
H), 8.27 (d, 2H, J ¼ 8.8 Hz, Ph-H), 8.03 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.13
(s,2H, OPh-H), 5.73 (s, 1H, pyrimidine-H), 2.33 (s, 3H, CH3), 2.14 (s,
4.1.2.5. 4-(5-(2,4,6-Trifluorophenoxy)-[1,2,4]triazolo[1,5-a]pyr-
imidin-7-ylamino)benzonitrile (5e). White solid, yield 5%. mp:
6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d
: 15.74 (eCH3 ꢃ 2), 20.83
(eCH3), AreC (ꢃ17): 82.08, 118.89 (ꢃ2), 125.61 (ꢃ2), 130.09 (ꢃ2),
130.65 (ꢃ2), 137.07, 141.72, 146.34, 146.54, 154.16, 155.43, 156.68,
158.31. IR (KBr, cmꢄ1): 2218 (CN), 1328 (NO2). ESI-MS: m/z 391.3
(Mþ1); C20H18N6O3 (390.144).
248e251 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.85 (s, 1H, NH), 8.53
(s, 1H, triazole-H), 7.96 (s, 2H, OPh-H), 7.75 (d, 2H, J ¼ 8.8 Hz, Ph-H),
7.50 (d, 2H, J ¼ 8.8 Hz, Ph-H), 6.54 (s, 1H, pyrimidine-H). IR (KBr,
cmꢄ1): 3434 (NH), 2229 (CN). ESI-MS: m/z 383.4 (Mþ1);
C
18H9F3N6O (382.079).
4.1.3.5. N-(4-Chlorophenyl)-7-(mesityloxy)-[1,2,4]triazolo[1,5-a]pyr-
4.1.3. General procedure for the synthesis of N-(4-substituted-
phenyl)-7-substituted phenoxy-[1,2,4]triazolo[1,5-a]pyrimidin-5-
amines (7a-m) and N5-(4-substituted- phenyl)-N7-substituted-
phenyl-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diamines (7n, 7o)
The appropriate substituted phenols or anilines (about
2.7 mmol) were dissolved in anhydrous DMF (15 mL) in the
imidin-5-amine (7e). White solid, yield 62%. mp: 200e205 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) d: 9.82 (s, 1H, NH), 8.36 (s,1H, triazole-
H), 7.81 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.40 (d, 2H, J ¼ 8.9 Hz, Ph-H), 7.11
(s,2H, OPh-H), 5.61 (s, 1H, pyrimidine-H), 2.32 (s, 3H, CH3), 2.13 (s,
6H, CH3). IR (KBr, cmꢄ1): 3305 (NH). ESI-MS: m/z 380.3 (Mþ1);
C20H18ClN5O (379.12).

L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
301
4.1.3.6. 7-(Mesityloxy)-N-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-
4.1.3.12. 4-((7-(2,4,6-Tribromophenoxy)-[1,2,4]triazolo[1,5-a]pyr-
amine (7f). White solid, yield 68%. mp: 260e263 ꢀC. 1H NMR
imidin-5-yl)amino)benzonitrile (7l). White solid, yield 36%. mp:
(DMSO-d6, 400 MHz)
d: 9.63 (s, 1H, NH), 8.33 (s,1H, triazole-H), 7.65
270e273 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.22 (s, 1H, NH), 8.49
(d, 2H, J ¼ 8.4 Hz, Ph-H), 7.15 (d, 2H, J ¼ 8.4 Hz, Ph-H), 7.11 (s,2H,
(s, 1H, triazole-H), 8.32 (s, 2H, OPh-H), 7.98 (d, 2H, J ¼ 8.7 Hz, Ph-H),
7.84 (d, 2H, J ¼ 8.7 Hz, Ph-H), 5.90 (s, 1H, pyrimidine-H). IR (KBr,
cmꢄ1): 3432 (NH), 2227 (CN). ESI-MS: m/z 563.0 (Mþ1), 565.1
(Mþ3), 357.1 (Mþ5), 569.0 (Mþ7); C18H9Br3N6O (561.8388).
OPh-H), 5.61 (s, 1H, pyrimidine-H), 2.32 (s, 3H, OPh-CH3), 2.27 (s,
3H, Ph-CH3), 2.13 (s, 6H, OPh-CH3). 13C NMR (DMSO-d6, 100 MHz)
d:
15.72 (eCH3 ꢃ 2), 20.80 (eCH3), 20.87 (eCH3), AreC (ꢃ17): 81.12,
118.87 (ꢃ2), 129.59 (ꢃ2), 130.07 (ꢃ2), 130.57 (ꢃ2), 132.05, 136.84,
137.65, 146.42, 153.54, 154.94, 157.21, 158.79. IR (KBr, cmꢄ1): 3301
4.1.3.13. 4-((7-(2,6-Dibromo-4-methylphenoxy)-[1,2,4]triazolo[1,5-a]
(NH). ESI-MS: m/z 360.4 (Mþ1), 382.5 (Mþ23);
C
21H21N5O
pyrimidin-5-yl)amino)benzonitrile (7m). White solid, yield 46%.
(359.1746).
mp: 278e282 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.21 (s, 1H, NH),
8.48 (s, 1H, triazole-H), 7.98 (d, 2H, J ¼ 8.3 Hz, Ph-H), 7.83 (m, 4H,
4.1.3.7. 7-(Mesityloxy)-N-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]
OPh-H, Ph-H), 5.90 (s, 1H, pyrimidine-H). 13C NMR (DMSO-d6,
pyrimidin-5-amine (7g). Brownish black solid, yield 32%. Decom-
100 MHz)
d
: 20.31 (eCH3 ꢃ 2), 119.62 (eCN), AreC (ꢃ17): 82.60,
posed (below the mp). 1H NMR (DMSO-d6, 400 MHz)
d
: 9.56 (s, 1H,
104.26, 116.55 (ꢃ2), 119.62 (ꢃ2), 133.80 (ꢃ2), 134.52 (ꢃ2), 141.77,
NH), 8.30 (s, 1H, triazole-H),7.65 (d, 2H, J ¼ 9 Hz, Ph-H), 7.11 (s,2H,
OPh-H), 6.93 (d, 2H, J ¼ 9 Hz, Ph-H), 5.56 (s, 1H, pyrimidine-H), 3.74
(s, 3H, OCH3), 2.32 (s, 3H, OPh-CH3), 2.14 (s, 6H, OPh-CH3). 13C NMR
143.25, 144.07, 152.67, 155.76, 156.74, 158.07. IR (KBr, cmꢄ1): 3440
(NH), 2226 (CN). ESI-MS: m/z 500.9 (Mþ3);
C19H12Br2N6O
(497.9439).
(DMSO-d6, 100 MHz)
d
: 15.73 (eCH3 ꢃ 2), 20.81 (eCH3), 55.64
(eCH3), AreC (ꢃ17): 80.85, 114.39 (ꢃ2), 121.56 (ꢃ2), 130.07 (ꢃ2),
130.56 (ꢃ2), 133.22, 136.83, 146.40, 153.45, 154.88, 155.47, 157.27,
158.76. IR (KBr, cmꢄ1): 3295 (NH). ESI-MS: m/z 376.4 (Mþ1), 398.4
(Mþ23); C21H21N5O2 (375.1695).
4.1.3.14. 4-((7-(Mesitylamino)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
amino)benzonitrile (7n). White solid, yield 33%. mp:275e278 ꢀC. 1H
NMR (DMSO-d6, 400 MHz) d: 9.79 (s, 1H, NH), 9.51 (s, 1H, NH), 8.36
(s, 1H, triazole-H), 7.97 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.75 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.06 (s, 2H, NHPh-H), 5.21 (s, 1H, pyrimidine-H),
4.1.3.8. 4-((7-(4-Bromo-2,6-dimethylphenoxy)-[1,2,4]triazolo[1,5-a]
2.31 (s, 3H, CH3), 2.16 (s, 6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d:
pyrimidin-5-yl)amino)benzonitrile (7h). White solid, yield 46%. mp:
17.91 (eCH3 ꢃ 2), 21.01 (-CH3), 119.98 (eCN), AreC (ꢃ17): 76.74,
102.97, 118.90 (ꢃ2), 129.70 (ꢃ2), 131.54, 133.54 (ꢃ2), 136.65 (ꢃ2),
137.69, 145.39, 147.06, 154.30, 155.74, 158.24. IR (KBr, cmꢄ1): 3354
(NH), 3251 (NH), 2219 (CN). ESI-MS: m/z 370.4 (Mþ1); C21H19N7
(369.1702).
285e288 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.11 (s, 1H, NH), 8.43
(s,1H, triazole-H),7.97 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.82 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.60 (s,2H, OPh-H), 5.71 (s,1H, pyrimidine-H), 2.19
(s, 6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d
: 15.57 (eCH3 ꢃ 2),
119.71 (eCN), AreC (ꢃ17): 82.08, 104.26, 119.38 (ꢃ2), 120.14, 132.62
(ꢃ2),133.45 (ꢃ2), 133.75 (ꢃ2),144.36,147.77,153.42,155.38,156.77,
158.35. IR (KBr, cmꢄ1): 3358 (NH), 2218 (CN). ESI-MS: m/z 435.6
(Mþ1), 437.6 (Mþ3); C20H15BrN6O (434.0491).
4.1.3.15. 4-((7-((2,6-Dimethylphenyl)amino)-[1,2,4]triazolo[1,5-a]
pyrimidin-5-yl)amino)benzonitrile (7o). White solid, yield 48%. mp:
>300 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 9.79 (s, 1H, NH), 9.59 (s,
1H, NH), 8.36 (s, 1H, triazole-H), 7.95 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.74
4.1.3.9. 4-((5-((4-Cyanophenyl)amino)-[1,2,4]triazolo[1,5-a]pyr-
(d, 2H, J ¼ 8.8 Hz, Ph-H), 7.26 (m, 3H, NHPh-H), 5.19 (s, 1H,
imidin-7-yl)oxy)-3,5-dimethylbenzonitrile (7i). White solid, yield
pyrimidine-H), 2.20 (s, 6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d:
60%. mp: 285e287 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d
: 10.10 (s, 1H,
17.99 (eCH3 ꢃ 2), 119.97 (-CN), AreC (ꢃ17): 76.78, 103.00, 118.92
(ꢃ2), 128.49, 129.14 (ꢃ2), 133.56 (ꢃ2), 134.17, 137.02 (ꢃ2), 145.36,
146.86, 154.33, 155.73, 158.22. IR (KBr, cmꢄ1): 3295 (NH), 3294
(NH), 2221 (CN). ESI-MS: m/z 356.4 (Mþ1), 378.5 (Mþ23); C20H17N7
(355.1545).
NH), 8.45 (s, 1H, triazole-H),7.97 (d, 2H, J ¼ 8.6 Hz, Ph-H), 7.92 (s,2H,
OPh-H), 7.82 (d, 2H, J ¼ 8.6 Hz, Ph-H), 5.67 (s, 1H, pyrimidine-H),
2.23 (s, 6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d: 15.59
(eCH3 ꢃ 2), 118.51 (eCN), 119.69 (eCN), AreC (ꢃ17): 82.31, 104.36,
110.74, 119.43 (ꢃ2), 133.03 (ꢃ2), 133.77 (ꢃ2), 134.13 (ꢃ2), 144.29,
151.82, 152.87, 155.46, 156.79, 158.27. IR (KBr, cmꢄ1): 3342 (NH),
2223 (CN). ESIMS: m/z 382.5 (Mþ1), 404.5 (Mþ23); C21H15N7O
(381.1338).
4.2. In vitro anti-HIV assay
Evaluation of the antiviral activity of the compounds was per-
formed using the MTT assay as previously described [27,28]. Stock
solutions (10ꢃ final concentration) of test compounds were added
4.1.3.10. 4-((7-(4-Chloro-2,6-dimethylphenoxy)-[1,2,4]triazolo[1,5-a]
pyrimidin-5-yl)amino)benzonitrile (7j). White solid, yield 52%. mp:
in 25 ml volumes to two series of triplicate wells so as to allow
285e286 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d
: 10.12 (s, 1H, NH), 8.43
simultaneous evaluation of their effects in mock-and HIV-infected
cells. Serial 5-fold dilutions of test compounds were made directly
in flat-bottomed 96-well microtiter trays using a Biomek 3000
robot (Beckman instruments, Fullerton, CA). Untreated control HIV-
and mock-infected cell samples were included for each sample.
(s, 1H, triazole-H), 7.98 (d, 2H, J ¼ 8.8 Hz, Ph-H), 7.81 (d, 2H,
J ¼ 8.8 Hz, Ph-H), 7.46 (s,2H, OPh-H), 5.71 (s,1H, pyrimidine-H), 2.19
(s, 6H, CH3). 13C NMR (DMSO-d6, 100 MHz)
d
: 15.67 (eCH3 ꢃ 2),
119.71 (-CN), AreC (ꢃ17): 82.08, 104.26, 119.38 (ꢃ2), 129.68 (ꢃ2),
131.67, 133.10 (ꢃ2), 133.74 (ꢃ2), 144.36, 147.26, 153.50, 155.38,
156.77, 158.36. IR (KBr, cmꢄ1): 3432 (NH), 2227 (CN). ESI-MS: m/z
391.3 (Mþ1), 413.5 (Mþ23); C20H15ClN6O (390.0996).
HIV-1 (IIIB), RES056 or HIV-2 (ROD) [24e26] stock (50 ml) at 100-
300 CCID50 (50% 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 compounds on uninfected cells in order to assess their
cytotoxicity. Exponentially growing MT-4 cells were centrifuged for
5 min at 1000 rpm and the supernatant was discarded. The MT-4
4.1.3.11. 4-((7-(2,4,6-Trichlorophenoxy)-[1,2,4]triazolo[1,5-a]pyr-
imidin-5-yl)amino)benzonitrile (7k). White solid, yield 45%. mp:
246e252 ꢀC. 1H NMR (DMSO-d6, 400 MHz)
d: 10.20 (s, 1H, NH), 8.49
(s, 1H, triazole-H), 8.13 (s, 2H, OPh-H), 7.97 (d, 2H, J ¼ 8.6 Hz, Ph-H),
7.84 (d, 2H, J ¼ 8.6 Hz, Ph-H), 5.93 (s, 1H, pyrimidine-H). IR (KBr,
cmꢄ1): 3323 (NH), 2231 (CN). ESI-MS: m/z 431.2 (Mþ1), 433.3
(Mþ3), 435.3 (Mþ5), 437.3 (Mþ7); C18H9Cl3N6O (429.9903).
cells were resuspended at 6 ꢃ 105 cells/ml, and 50
ml volumes
were transferred to the microtiter tray wells. Five days after
infection, the viability of mock-and HIV-infected cells was exam-
ined spectrophotometrically by the MTT assay.

302
L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
The MTT assay is based on the reduction of yellow colored 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
complexes with TMC278 (PDB code: 3BGR) were retrieved from the
Protein Data Bank and were used for the docking studies by means
of surflex-docking module of Sybyl-1.1. The protein was prepared
by using the Biopolymer application accompanying Sybyl: The
bound ligand was extracted from the complexes, water molecules
were removed, hydrogen atoms were added, and charges and atom
types were assigned according to AMBER99. After the protomol
was generated, the optimized compounds 5b, 7c, 7n and ETV were
surflex-docked into the binding pocket of NNRTIs, with the relevant
parameters set as defaults. The original ligand (ETV/TMC278) of the
coordinates (3MEC/3BGR) was used as reference molecule to
calculate the RMSD values. The docking scores related to binding
affinities were calculated based on hydrophobic, polar, and repul-
sive interactions as well as entropic effects and solvation. Top-
scoring poses were shown by the software of PyMOL version 1.5
(http://www.pymol.org/), in overlap with the bound ligand (ETV)
in the binding site of RT. The secondary structure of RT are shown in
cartoons, and only the key residues for interactions with the in-
hibitors were shown in sticks and labeled. The potential hydrogen-
bonds were presented by dashed lines.
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
genase of metabolically active cells to a blue-purple formazan that
can be measured spectrophotometrically. The absorbances were
read in an eight-channel computer-controlled photometer (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 two or three wells.
The 50% effective antiviral concentration (EC50) was defined as
the concentration of the tested compound achieving 50% protection
from viral cytopathicity. The 50% cytotoxic concentration (CC50
)
was defined as the compound concentration that reduced the
viability of mock-infected cells by 50%. The symbol ‘>’ was used to
indicate the highest concentration at which the compound was
tested and still found to be non-cytotoxic.
4.3. HIV-1 RT inhibition assay
Inhibition assay of HIV-1 RTwt was implemented by utilizing the
template/primer hybrid poly (A) ꢃ oligo (dT)15, digoxigenin- and
biotin-labeled nucleotides, an antibody to digoxigenin which con-
jugated to peroxidase (anti-DIG-POD), and the peroxidase substrate
ABTS. The incorporation quantities of the digoxigenin- and biotin-
labeled dUTP into DNA represented the activity of HIV-1 RT. The
HIV-RT inhibition assay was performed by using an RT assay kit
(Roche), and the procedure for assaying RT inhibition was per-
formed as described in the kit protocol [30,31]. The tested com-
pound 7i and the control drug ETV was used at different
Conflict of interest
The authors declare no conflict of interest.
Acknowledgment
The financial support from the National Natural Science Foun-
dation of China (NSFC No. 81273354, No. 81102320), Key Project of
NSFC for International Cooperation (No. 30910103908), Research
Fund for the Doctoral Program of Higher Education of China (No.
20110131130005, 20110131120037), Shandong Provincial Natural
Science Foundation, China (No. ZR2009CM016), and KU Leuven
(GOA 10/014) is gratefully acknowledged.
concentration gradient (0.1
mg/Ml, 1
mg/mL, 10
mg/mL, 100
mg/mL,
1 mg/mL for 7i; 0.01 g/mL, 0.1
m
mg/mL, 1
mg/mL, 10
m
g/mL, 100 m
g/
mL, 1 mg/mL for ETV). Concretely, the reaction mixture consisted of
template/primer complex, 20-deoxy-nucleotide-50-triphosphates
(dNTPs) and reverse transcriptase (RT) enzyme in the lysis buffer
with or without inhibitors. After 1 h incubation at 37 ꢀC the reaction
mixture was transferred to streptavidine-coated microtiter plate
(MTP). The biotin labeled dNTPs that are incorporated in the tem-
plate due to activity of RT were bound to streptavidine. The un-
bound dNTPs were washed using wash buffer and then
antidigoxigenin-peroxidase (anti-DIG-POD) was added in MTP.
The DIG-labeled dNTPs incorporated in the template was bound to
anti-DIG-POD antibody. The unbound anti-DIG-POD was washed
carefully by using buffer and the peroxide substrate (ABST) was
added to the MTP. A colored reaction product was produced during
the cleavage of the substrate catalyses by a peroxide enzyme. The
absorbance of the sample was determined at OD 405 nM using
microtiter plate ELISA reader. The resulting color intensity was
directly proportional to the actual RT activity. The percentage
inhibitory activity of RT inhibitors was calculated by comparing to a
sample that does not contain an inhibitor. The percentage inhibi-
tion was calculated by formula as given below: inhibition rate
% ¼ 100 ꢄ (OD 405 nm without inhibitor ꢄ OD 405 nm with in-
We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the anti-HIV assays.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.104. These data include MOL
files and InChiKeys of the most important compounds described in
this article.
References
[1] E. De Clercq, The design of drugs for HIV and HCV, Nat. Rev. Drug. Discov. 6
(2007) 1001e1018.
[2] P. Zhan, X. Liu, Novel HIV-1 non-nucleoside reverse transcriptase inhibitors: a
patent review (2005e2010), Expert Opin. Ther. Patents 21 (2011) 717e796.
[3] P. Zhan, X. Liu, Z. Li, Recent advances in the discovery and development of
novel HIV-1 NNRTI platforms: 2006e2008 update, Curr. Med. Chem. 16 (2009)
2876e2889.
[4] Y. Song, P. Zhan, X. Liu, Recent advances in the discovery and development of
novel HIV-1 NNRTI platforms (Part II): 2009e2013 update, Curr. Med. Chem.
21 (2013) 329e355.
[5] P. Zhan, X. Liu, Z. Li, C. Pannecouque, E. De Clercq, Design strategies of novel
NNRTIs to overcome drug resistance, Curr. Med. Chem. 16 (2009) 3903e3917.
[6] P. Zhan, X. Chen, D. Li, Z. Fang, E. De Clercq, X. Liu, HIV-1 NNRTIs: structural
diversity, pharmacophore similarity, and implications for drug design, Med.
Res. Rev. 33 (2013) E1eE72.
hibitor)/(OD 405 nm without inhibitor
ꢄ
OD 405 nm
background) ꢃ 100. IC50 values corresponded to the concentration
of the tested compounds required to inhibit biotin-dUTP incorpo-
ration into the HIV-1 RT by 50%.
4.4. Molecular modeling
[7] D. Li, P. Zhan, E. De Clercq, X. Liu, Strategies for the design of HIV-1 non-
nucleoside reverse transcriptase inhibitors: lessons from the development of
seven representative paradigms, J. Med. Chem. 55 (2012) 3595e3613.
[8] P. Sax, FDA approval: etravirine, AIDS Clin. Care 20 (2008) 17e18.
The molecules (5b, 7c, 7n and ETV) for docking were optimized
for 2000-generations until the maximum derivative of energy
became 0.05 kcal/(mol*Å), using the Tripos force field. Charges
were computed and added according to GasteigereHuckel pa-
rameters. The published three-dimensional crystal structures of wt
RT complexes with ETV (PDB code: 3MEC) and double mutant RT
€
[9] M. Scholler-Gyüre, T.N. Kakuda, G. De Smedt, H. Vanaken, M.P. Bouche,
M. Peeters, B. Woodfall, R.M. Hoetelmans, A pharmacokinetic study of etra-
virine (TMC125) co-administered with ranitidine and omeprazole in HIV-
negative volunteers, Br. J. Clin. Pharmacol. 66 (2008) 508e516.

L. Wang et al. / European Journal of Medicinal Chemistry 85 (2014) 293e303
303
[10] E. Seminari, A. Castagna, A. Lazzarin, Etravirine for the treatment of HIV
infection, Expert Rev. Anti-infective Ther. 6 (2008) 427e433.
[11] B.C. Johnson, G.T. Pauly, G. Rai, D. Patel, J.D. Bauman, H.L. Baker, K. Das,
J.P. Schneider, D.J. Maloney, E. Arnold, A comparison of the ability of rilpivirine
(TMC278) and selected analogues to inhibit clinically relevant HIV-1 reverse
transcriptase mutants, Retrovirology 9 (2012) 99.
[12] D. Li, P. Zhan, H. Liu, C. Pannecouque, J. Balzarini, E. De Clercq, X. Liu, Synthesis
and biological evaluation of pyridazine derivatives as novel HIV-1 NNRTIs,
Bioorg. Med. Chem. 21 (2013) 2128e2134.
[21] N. Zhang, S. Ayral-Kaloustian, T. Nguyen, J. Afragola, R. Hernandez, J. Lucas,
J. Gibbons, C. Beyer, Synthesis and SAR of [1,2,4] triazolo [1,5-a] pyrimidines, a
class of anticancer agents with a unique mechanism of tubulin inhibition,
J. Med. Chem. 50 (2007) 319e327.
[22] C.M. Richardson, D.S. Williamson, M.J. Parratt, J. Borgognoni, A.D. Cansfield,
P. Dokurno, G.L. Francis, R. Howes, J.D. Moore, J.B. Murray, A. Robertson,
A.E. Surgenor, C.J. Torrance, Triazolo [1,5-a] pyrimidines as novel CDK2 in-
hibitors: protein structure-guided design and SAR, Bioorg. Med. Chem. Lett. 16
(2006) 1353e1357.
[13] X. Li, W. Chen, Y. Tian, H. Liu, P. Zhan, C. Pannecouque, J. Balzarini, E. De Clercq,
X. Liu, Discovery of novel diarylpyrimidines as potent HIV NNRTIs via a
structure-guided core-refining approach, Eur. J. Med. Chem. 80 (2013)
112e121.
[23] Z. Zeng, Q. He, Y. Liang, X. Feng, F. Chen, E.D. Clercq, J. Balzarini,
C. Pannecouque, Hybrid diarylbenzopyrimidine non-nucleoside reverse
transcriptase inhibitors as promising new leads for improved anti-HIV-1
chemotherapy, Bioorg. Med. Chem. 18 (2010) 5039e5047.
[14] J. Wang, P. Zhan, Z. Li, E. De Clercq, C. Pannecouque, J. Balzarini, X. Liu, Syn-
thesis and biological evaluation of substituted nitropyridine derivatives as
potent HIV-1 non-nucleoside reverse transcriptase inhibitors, Eur. J. Med.
Chem. 76 (2013) 531e538.
[24] M. Popovic, M.G. Sarngadharan, E. Read, R.C. Gallo, Detection, isolation, and
continuous production of cytopathic retroviruses (HTLV-III) from patients
with AIDS and pre-AIDS, Science 224 (1984) 497e500.
ꢀ
[25] F. Barre-Sinoussi, J.-C. Chermann, F. Rey, M.T. Nugeyre, S. Chamaret, J. Gruest,
ꢀ
[15] A. Raney, R. Hamatake, W. Xu, J. Vernier, J. Girardet, P. Weingarten, D. Zhou,
H. Kim, R. Dick, L. Yeh, RDEA427 and RDEA640 are novel NNRTIs with potent
anti-HIV activity against NNRTI-resistant viruses, in: 15th Conference on
Retroviruses and Opportunistic Infections (CROI), 2008, pp. 3e6.
[16] E.H. Kerns, L. Di, Drug-like Properties: Concepts, Structure Design and
Methods: from ADME to Toxicity Optimization, Elsevier, London, 2008.
[17] Deepak Dalvie, Ping Kang, Cho-Ming Loi, Lance Goulet, S. Nair, Influence of
heteroaromatic rings on ADME properties of drugs, in: D.A. Smith (Ed.),
Metabolism, Pharmacokinetics and Toxicity of Functional Groups : Impact of
Chemical Building Blocks on ADMET, Royal Society of Chemistry, London,
2010, pp. 328e369.
[18] Y. Song, P. Zhan, Q. Zhang, X. Liu, Privileged scaffolds or promiscuous binders:
a glance of pyrrolo [2, 1-f][1, 2, 4] triazines and related bridgehead nitrogen
heterocycles in medicinal chemistry, Curr. Pharm. Des. 19 (2013) 1528e1548.
[19] R. Gujjar, F. El Mazouni, K.L. White, J. White, S. Creason, D.M. Shackleford,
X. Deng, W.N. Charman, I. Bathurst, J. Burrows, D.M. Floyd, D. Matthews,
F.S. Buckner, S.A. Charman, M.A. Phillips, P.K. Rathod, Lead optimization of aryl
and aralkyl amine-based triazolopyrimidine inhibitors of Plasmodium falcip-
arum dihydroorotate dehydrogenase with antimalarial activity in mice, J. Med.
Chem. 54 (2011) 3935e3949.
C. Dauguet, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, Isolation of a T-lym-
photropic retrovirus from a patient at risk for acquired immune deficiency
syndrome (AIDS), Science 220 (1983) 868e871.
[26] I. Miyoshi, H. Taguchi, I. Kubonishi, S. Yoshimoto, Y. Ohtsuki, Y. Shiraishi,
T. Akagi, Type C virus-producing cell lines derived from adult T cell leukemia,
Gann Monogr. 28 (1982) 19e228.
[27] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn,
J. Desmyter, E. De Clercq, Rapid and automated tetrazolium-based colori-
metric assay for the detection of anti-HIV compounds, J. Virol. Methods. 20
(1988) 309e321.
[28] C. Pannecouque, D. Daelemans, E. De Clercq, Tetrazolium-based colorimetric
assay for the detection of HIV replication inhibitors: revisited 20 years later,
Nat. Protoc. 3 (2008) 427e434.
[29] X.D. Ma, S.Q. Yang, S.X. Gu, Q.Q. He, F.E. Chen, E. De Clercq, J. Balzarini,
C. Pannecouque, Synthesis and anti-HIV activity of aryl-2-[(4-cyanophenyl)
amino]-4-pyrimidinone hydrazones as potent non-nucleoside reverse tran-
scriptase inhibitors, ChemMedChem 6 (2011) 2225e2232.
[30] K. Suzuki, B.P. Craddock, N. Okamoto, T. Kano, R.T. Steigbigel, Poly A-linked
colorimetric microtiter plate assay for HIV reverse transcriptase, J. Virol.
Methods. 44 (1993) 189e198.
[20] L. McKendry, Preparation of carbon-14 labeled N-(2, 6-dichloro-3-
methylphenyl)-5, 7-dimethoxy [1, 2, 4] triazolo [1,5-a] pyrimidine-2-
sulfonamide, J. Label. Compd. Radiopharm. 39 (1997) 231e240.
[31] Reverse Transcriptase Assay, Colorimetric kit, Roche Diagnostics GmbH,
Roche. Applied Science, Sandhofer Strasse 116, D-68305 Mannheim,
Germany.