Fused heterocycles bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 3: Optimization of [1,2,4]triazolo[1,5-a]pyrimidine core via structure-based and physicochemical property-driven approaches

Article abstract of DOI:10.1016/j.ejmech.2015.01.042

In our arduous efforts to develop new potent HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs), novel piperidine-linked [1,2,4]triazolo[1,5-a]pyrimidine derivatives were designed, synthesized and evaluated for their antiviral activities

Full text of DOI:10.1016/j.ejmech.2015.01.042

European Journal of Medicinal Chemistry 92 (2015) 754e765
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 3: Optimization of [1,2,4]triazolo[1,5-a]pyrimidine core
via structure-based and physicochemical property-driven approaches
Boshi Huang a, Cuicui Li a, Wenmin Chen a, Tao Liu a, Mingyan Yu b, Lu Fu a, Yueyue Sun a,
,
*
Huiqing Liu c, Erik De Clercq d, Christophe Pannecouque d, Jan Balzarini d, Peng Zhan a
,
,
*
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 Ji'nan, Shandong, PR China
b Shandong Institute for Food and Drug Control, 2749 Xinluo Street, 250101 Ji'nan, Shandong, PR China
c Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road, 250012 Ji'nan, Shandong, PR China
d 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:
In our arduous efforts to develop new potent HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors
(NNRTIs), novel piperidine-linked [1,2,4]triazolo[1,5-a]pyrimidine derivatives were designed, synthe-
sized and evaluated for their antiviral activities in MT-4 cell cultures. Biological results showed that all of
the title compounds displayed moderate to excellent activities against wild-type (wt) HIV-1 strain (IIIB)
with EC50 values ranging from 8.1 nM to 2284 nM in a cell-based assay. Among them, the most promising
analog 7d possessed an EC50 value of 8.1 nM against wt HIV-1, which was much more potent than the
reference drugs DDI, 3 TC, NVP and DLV. Additionally, 7d demonstrated weak activity against the double
mutant HIV-1 strain (K103N þ Y181C), and was more efficient than NVP in a RT inhibition assay. Besides,
some measured and calculated physicochemical properties of 7d, like log P and water solubility, as well
as the structureeactivity relationships (SARs) analysis have been discussed in detail. Furthermore, the
binding mode of the active compound 7d was rationalized by molecular simulation studies.
© 2015 Elsevier Masson SAS. All rights reserved.
Received 20 September 2014
Received in revised form
21 January 2015
Accepted 21 January 2015
Available online 22 January 2015
Keywords:
Triazolopyrimidines
Structure-based drug design
Biological activity
HIV-1 RT
Physicochemical properties
Molecular simulations
1. Introduction
antiviral activity, high specificity and low cytotoxicity. However,
during the clinical use of the first generation NNRTIs (nevirapine,
Acquired immune deficiency syndrome (AIDS) caused by human
immunodeficiency virus (HIV), is still a significant pandemic
worldwide. Highly active antiretroviral therapy (HAART) has
become the most standard and efficient treatment regimen for HIV
infection, which generally involves HIV-1 protease inhibitors (PIs)
and reverse transcriptase (RT) inhibitors [1]. Among currently
available RT inhibitors, non-nucleoside RT inhibitors (NNRTIs) can
directly interfere with RT through binding to an allosteric site called
NNRTI-binding pocket (NNIBP), which is distinct from, but located
at about 10 Å distance from the substrate site [2]. NNRTIs are an
important component of the HAART regimen, owing to their unique
delavirdine and efavirenz), rapid emergence of mutant viral strains
severely compromised their application [3e6].
In arduous efforts to explore the resilience of novel NNRTIs to
the highly mutable HIV-1, two most representative diary-
lpyrimidine derivatives (DAPYs) of the second generation of
NNRTIs, Etravirine (TMC125, ETR) and Rilpivirine (TMC278, RPV)
(Fig. 1) were approved by the US FDA for anti-HIV therapy in 2008
and 2011, respectively, because of their excellent drug resistance
profiles [7]. Even so, DAPYs suffer from the severe issues of low oral
bioavailability, poor water solubility, cross-resistance, and virologic
failure. Thus, the development of new NNRTI drugs with efficient
anti-HIV activity against both wild-type (wt) and mutant HIV
strains, low cytotoxicity, as well as good pharmacokinetic proper-
ties is still urgently required for the successful application of
NNRTIs in the drug combination regimens [7].
* Corresponding authors.
E-mail addresses: zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn
(X. Liu).
http://dx.doi.org/10.1016/j.ejmech.2015.01.042
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
755
Fig. 1. Graphical representation of our previous work in the structure modifications of DAPY NNRTIs, and optimization strategies for newly designed target compounds in the
present work.
Even though confronted with the issues mentioned above,
DAPYs, owing to their excellent pharmacological profiles, have still
encouraged several research groups to develop next-generation
NNRTIs for the treatment of AIDS [8]. As illustrated in Fig. 1, pre-
vious research in our laboratory using structure-based bioisosteric
replacement, and substituent decoration resulted in the discovery
value of 50 nM, which was much better than Nevirapine (NVP)
(EC50 ¼ 150 nM), and was slightly better than Delavirdine mesylate
(DLV) (EC50 ¼ 70 nM) and its isostere pyrazolo[1,5-a]pyrimidine YT-
5a (EC50 ¼ 70 nM). These encouraging results prompted us to
evaluate these bicyclic scaffolds during the course of our lead effort
for the NNRTI project.
of
a
collection of six-membered heterocyclic DAPY analogs
According to the X-ray crystallographic structures of DAPY-RT
complexes and the molecular modeling studies [18,19], the bind-
ing conformation of DAPYs resembled a characteristic horseshoe or
“U” shape in the NNIBP of RT, rather than the prototypical butterfly-
like binding shape of the first-generation NNRTIs. The DAPYanalogs
generally contained three pharmacophoric moieties [2,7]:
substituted benzene (A) or substituted piperidine in the protein/
solvent interface interaction domain, a heterocycle moiety con-
taining a hydrogen bond donor and/or acceptor (B) and a hydro-
phobic group (C) (Fig. 1). In particular, the six-membered or bicyclic
heterocycle (B) located in the center of the NNIBP, not only anchors
the functional groups for well engaging with the residues around
NNIBP, but also serves as key hydrogen bond acceptor and donor
(NH linker) to make key hydrogen bonds with the backbone of
K101. These hydrogen bonds are critical for binding affinity of
DAPYs especially in the cases of the appearance of K101 mutant
[18].
(exemplified by pyridazine DL-8g, nitropyridine JW-7a2 and py-
rimidine XL-7a) [9e11], and piperidine-substituted triazine/aniline
derivatives (exemplified by XC-6b3 and LZ-5a6) [12e14]. These
representative compounds were found to dramatically inhibit the
replication of wt HIV-1 strain at low or double-digit nanomolar
concentrations (Fig. 1), accompanied with good activity against
HIV-1 double mutant strain RES056 (K103N þ Y181C), which made
them promising leads for further optimization [9e14].
Furthermore, to discover highly potent DAPYs with novel
structural motifs that are easier to synthesize or to avoid existing
patent art, further modifications were focused in our lab on the
replacement of the pyrimidine core in DAPYs with an array of
privileged bicyclic scaffolds bearing bridgehead nitrogen [15], such
as pyrazolo[1,5-a]pyrimidine YT-5a and [1,2,4]triazolo[1,5-a]py-
rimidine LW-7c, characterized by
a 2,4,6-trimethyl group
substituted on the C ring and a para-cyano group substituted on the
A ring [16,17]. Interestingly, [1,2,4]triazolo[1,5-a]pyrimidine LW-7c
demonstrated striking potency against wt HIV-1 strain with an EC50
More importantly, crystal structures analysis of piperidine-
substituted DAPYs analogs showed that, besides the existing K101

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B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
backbone hydrogen bond interactions, the piperidine nitrogen
could probably make an additional hydrogen bond with the K103
backbone, which has been considered to be an important factor for
the design of inhibitors with resilience to mutations [19]. Moreover,
as substituted piperidine points toward the solvent-exposed re-
gion, introducing suitable groups into the right-wing piperidine
should be beneficial for the modulation of the aqueous solubility,
physicochemical and pharmacokinetics properties of the NNRTIs.
On the basis of these analysis and in connection with our pre-
vious studies on fused heterocycles bearing bridgehead nitrogen,
we continued to explore the privileged and synthetically feasible
[1,2,4]triazolo[1,5-a]pyrimidines as more potent anti-HIV agents
via structure-based molecular hybridization combined with phys-
icochemical property-driven scaffold re-evolution approaches [20].
Concretely, as shown in Fig. 1, the preferred N-(piperidin-4-yl)
amine group was installed at the right-wing portion of the [1,2,4]
triazolo[1,5-a]pyrimidine core. Meanwhile, different substituents,
varying in size and electronic nature were linked to piperidine to
further probe the chemical space of the right wing and to establish
SARs (Fig. 1).
Chlorination of 2 with phosphorous oxychloride at 95 ꢀC produced
5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidine (3) [17]. Coupling of
the intermediate 3 with 2,4,6-trimethylphenol using potassium
carbonate as the base in DMF at 40 ꢀC gave the intermediate 5-
chloro-7-(mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidine (4) [21].
Then the 5-Cl of compound 4 underwent a facile nucleophilic
substitution reaction with 4-amino-1-Boc-piperidine in DMF at
60 ꢀC to afford 5 exclusively [12]. Subsequently, intermediate 5 was
treated with trifluoroacetic acid (TFA) in dichloromethane solvent
at room temperature for 0.5 h to yield the key intermediate 6
[13,14]. Finally, alkylation at the N-1 atom of the piperidinyl group
afforded the title compounds 7aew, 8a and 8b [14]. These target
compounds were characterized by physicochemical and spectral
means, and both analytical and spectral data of these title com-
pounds were in full agreement with their proposed structures.
2.2. Anti-HIV activity in MT-4 cells
All the newly synthesized compounds were evaluated for their
in vitro anti-HIV activity and cytotoxicity in MT-4 cells infected with
the wild-type HIV-1 (strain IIIB), K103N þ Y181C double mutant
type HIV-1 (strain RES056), as well as HIV-2 (strain ROD). The
biological results, interpreted as EC50 values, CC50 values and
selectivity index (SI) values are summarized in Table 1 and Table 2.
Zidovudine (AZT), Didanosine (DDI), Lamivudine (3TC), Nevirapine
(NVP), Delavirdine mesylate (DLV), Efavirenz (EFV) and Etravirine
(ETR) were used as reference drugs.
Herein we report the synthesis, antiviral activity, some
measured and calculated physicochemical properties like log P and
water solubility of these novel piperidine-linked [1,2,4]triazolo[1,5-
a]pyrimidine derivatives. The structureeactivity relationships
(SARs) are also discussed in detail to gain further insights into this
series of analogs.
2. Results and discussion
As listed in Table 1, the antiviral activity in the cell-based assay
against HIV-IIIB strain of all the newly synthesized compounds
were superior to the reference drugs DDI, 3TC and DLV with the
exception of compound 8b (EC50 ¼ 2284 nM), and half of these
compounds performed better than the lead compound LW-7c
(EC50 ¼ 50 nM). The active derivatives showed moderate to excel-
lent activities with EC50 values ranging from 8.1 nM to 2284 nM and
SI values in the range of 39e2650. Most notably, compound 7d was
found to be the most potent analog possessing an EC50 value of
8.1 nM, much more effective than NVP (EC50 ¼ 312 nM) and in the
2.1. Chemistry
Newly designed triazolopyrimidine derivatives were expedi-
tiously synthesized via a short and efficient route at low cost. As
shown in Scheme 1, the starting material 2H-1,2,4-triazol-3-amine
(1) was first cyclized with the diethyl malonate in dimethyl form-
amide (DMF), then the reaction mixture was acidified to form the
intermediate
[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol
(2).
Scheme 1. Reagents and conditions: (a) Diethyl malonate, NaH, DMF, 80 ꢀC, 2 h; (b) POCl3, 95 ꢀC, 2 h. (c) 2,4,6-trimethylphenol, K2CO3, DMF, 40 ꢀC, 2.5 h. (d) tert-butyl 4-
aminopiperidine-1-carboxylate, K2CO3, DMF, 60 ꢀC, overnight. (e) TFA, CH2Cl2, r.t., 0.5 h. (f) K2CO3, DMF, RCH2X (X ¼ Cl, Br), r.t., overnight. (g) ClCH2CONHR', K2CO3, DMF, r.t.,
overnight.

B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
757
Table 1
Table 2
Anti-HIV-1 activity and cytotoxicity of the novel [1,2,4]triazolo[1,5-a]pyrimidine
compounds in MT-4 cells infected with HIV-1 strain IIIB.
Anti-HIV activity and cytotoxicity of the novel [1,2,4]Triazolo[1,5-a]pyrimidine
compounds in MT-4 cells infected with HIV RES056 strain.
FRa
CC50
(
m
M)
SI
O
Compd
EC50
(
m
M)
R'
R
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
8a
>13
6.4 3.2
>25
13 13
22 9.9
>10
>18
>24
>18
>30
>50
13 6.5
44 18
25 0.68
20 9.3
49 30
10 3.7
18 2.6
24 4.0
18 9.2
30 9.4
20 8.9
14 9.9
25 1.9
27 3.2
24 1.8
ꢁ25
<1
7
<1
2
N
385
>708
1650
838
H
N
N
2
>686
>522
>1176
>2055
>320
>1643
>689
>1324
>356
>424
ꢁ372
>520
>679
>590
>255
>346
>243
>57
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
ꢂ1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
>9149
ND
ND
>orX2
X1
>41
277
O
N
NH
O
N
NH
N
N
N
N
>20
>14
>25
>27
N
N
7a-w
8a,8b
>24
ꢁ39
>25
25 1.9
49 32
24 2.3
23 1.6
23 1.8
24 0.96
25 2.1
86 22
89 14
163.77 62.84
>94
>212
>87
>15
>36
>6.3
4.4 0.84
>49
>24
>23
>23
>24
>25
>86
>89
Compd
R/R0
EC50 (nM)a
CC50
(
m
M)b
SIc
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
8a
Cyclohexyl
Pyridin-4-yl
Phenyl
245 22
17 9.5
36 11
8.1 0.8
27 1.9
15 4.6
35 16
21 10
8.7 1.9
92 59
12 1.3
20 10
19 4.1
77 56
56 8.7
104 96
48 11
72 24
42 6.6
90 65
67 2.1
98 19
439 188
277 35
2284 624
50 40
7.1 2.9
23,198 7570
2239 826
312 56
540 515
6.3 1.7
1.8 0.5
13 6.5
44 18
25 0.7
20 9.3
49 30
10 3.7
18 2.6
24 4.0
18 9.2
30 9.4
20 8.9
14 9.9
25 1.9
27 3.2
24 1.8
ꢁ25
50
2650
708
2548
1890
686
4-SO2Me-Ph
4-SO2NH2-Ph
4-COOMe-Ph
4-NO2-Ph
4-CONH2-Ph
4-COCH3-Ph
4-Br-Ph
>315
>39
>3198
8b
522
LW-7c [17]
AZT
DDI
3TC
NVP
DLV
EFV
ETR
>163.77
0.010 0.0092
ND
1176
2055
320
1643
689
1324
356
424
1.4
ND
ND
ND
3-Cl-Ph
4-CN-Ph
3-CN-Ph
2-CN-Ph
4-F-Ph
3-F-Ph
2-F-Ph
4-CH3-Ph
ꢁ7.6
ꢁ24
>36
>67
0.16 0.015
0.016 0.0066
25
8.9
a
Fold resistance (FR): ratio of EC50 value against K103N þ Y181C double mutant
ꢁ238
mutant
wt
type HIV-1 over EC50 value against wild-type HIV-1 (EC
/EC ); data represent
50
50
25 1.9
49 32
24 2.3
23 1.6
23 1.8
24 1.0
25 2.1
86 22
89 14
520
the mean SD of at least two separate experiments performed in triplicate.
679
590
255
346
2-CH3-Ph
2,4-di-F-Ph
3,4-di-F-Ph
2,5-di-F-Ph
2,6-di-F-Ph
2-Cl-4-COOMe-Ph
2-Br-4-COOH-Ph
three analogs 7b (EC50 ¼ 6.4
7d (EC50 ¼ 13 M with a 1650-fold resistance ratio) and 7e
(EC50 ¼ 22 M with a 838-fold resistance ratio) displayed weak
potency against this mutant strain, which were more active than
the reference drug DLV (EC50 > 36 M, fold resistance ratio > 67)
and lead compound LW-7c (EC50 > 163.77 M, fold resistance ra-
tio > 3198), though still less potent than AZT, EFV and ETR. These
acceptable results demonstrated that the replacement of
piperidine-substituted groups for benzonitrile in the right wing of
the [1,2,4]triazolo[1,5-a]pyrimidine leads did not seriously impair
the HIV inhibitory potency and provided new opportunities for
further optimization to improve drug resistance profiles. Addi-
tionally, all the title compounds were screened for their inhibition
against HIV-2 (strain ROD), but like other NNRTIs [9e17], none of
them exhibited inhibitory activity at subtoxic concentrations,
indicating that this novel series of piperidine-linked [1,2,4]triazolo
[1,5-a]pyrimidine derivatives were specific for HIV-1 RT.
mM with a 385-fold resistance ratio),
243
m
57
m
315
8b
39
LW-7cd
AZT
DDI
3 TC
NVP
DLV
EFV
ETR
163.8 62.8
>94
3198
>13,144
>9
>39
>48
>67
>1014
2459
m
m
>212
>87
>15
>36
>6.3
4.4 0.8
a
Compound concentration required to achieve 50% protection of MT-4 cells
against HIV-1-induced cytopathic effect, as determined by the MTT method; values
are the mean SD of at least two parallel tests.
Concentration required to reduce the viability of uninfected cells by 50%, as
determined by the MTT method; values were averaged from at least four inde-
b
pendent experiments.
c
Selectivity index: CC50/EC50
.
d
Reference lead used for comparison [17].
Based on the results of the biological assays (Table 1), pre-
liminary SARs conclusions could be summarized as follows:
An aromatic ring located at the terminal portion of the right
wing of the inhibitors (R substitution) was more beneficial than an
aliphatic ring (7b, 7c versus 7a), and 7b with a pyridin-4-yl group
(EC50 ¼ 17 nM) was 2.1 times more active than compound 7c with a
phenyl group (EC50 ¼ 36 nM).
same order of magnitude as those attained for the reference drugs
AZT, EFV and ETR. Moreover, 7d also exhibited an acceptable SI
value (2548) in vitro. The above biological results demonstrated
that introducing a piperidinyl group into [1,2,4]triazolo[1,5-a]py-
rimidine precursor compound LW-7c was acceptable and may
improve antiviral potencies.
What is more, these compounds were also assayed against the
frequently encountered K103N þ Y181C double mutant HIV-1
(strain RES056) in the clinic. As the data indicated in Table 2,
Next, we focused our attention on SAR of the para-substituents
on the phenyl ring (R). Hydrophilic groups were more favorable
than hydrophobic groups at the para position of the phenyl ring, of
which the order ranked as the following: hydrophilic groups
[SO2CH3 (7d, EC50 ¼ 8.1 nM) > COCH3 (7i, EC50 ¼ 8.7 nM) > COOCH3

758
B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
(7f, EC50 ¼ 15 nM) > CN (7l, EC50 ¼ 20 nM) z CONH2 (7h,
EC50 ¼ 21 nM) > SO2NH2 (7e, EC50 ¼ 27 nM)] > H (7c,
EC50 ¼ 36 nM) > hydrophobic groups [F (7o, EC50 ¼ 56 nM) > CH3
(7r, EC50 ¼ 72 nM) > Br (7j, EC50 ¼ 92 nM)], except for 7g with a
synthesized compounds and RT, and to rationalize the results of
SAR studies, molecular modeling was carried out to investigate the
binding mode of target compounds. The representative compound
7d and the lead compound LW-7c were docked into the NNIBP of
HIV-1 RT (PDB code: 3M8Q [19,23]) by Surflex-Dock module of
Sybyl-X 1.1 software [24] and the docking results were shown by
PyMOL 1.5. Default parameters were used as described in the
SYBYL-X 1.1 manual unless otherwise specified.
hydrophobic NO2 group (EC50
¼
35 nM) similar to 7c
(EC50 ¼ 36 nM). The antiviral activity of 7i was nearly as high as the
most potent compound 7d bearing SO2CH3 as a classical bioisostere
of COCH3, suggesting that the sulfuryl/carbonyl group with
appropriate polarity and hydrophilicity can better accommodate
the chemical environment in this region of RT and provide potential
interactions with surrounding amino acids. This conclusion was in
agreement with the previously reported results in the piperidine-
substituted series.
As illustrated in Fig. 2aec, the binding mode of compound 7d is
similar to that of LW-7c, as observed by a fair superimposition of
Moreover, the influence of substituents at the ortho or meta
position of the phenyl ring (R) on the antiviral activity was inves-
tigated, but no distinct features were exhibited in this case (7l
versus 7m versus 7n; 7o versus 7p versus 7q; 7r versus 7s).
Considering the fact that the introduction of a fluorine atom to
bioactive molecules could significantly affect on pharmaceutical
profiles such as activity, metabolism and physicochemical proper-
ties including lipophilicity and solubility. Thus, multiple fluorine
atoms were also introduced to the benzyl moiety, which resulted in
compounds 7te7w. Unfortunately, the four di-F-substituted anal-
ogous merely showed moderate anti-HIV-1 profiles, and did not
demonstrate remarkable advantages over the single fluorine-
containing compounds 7oeq.
Finally, to further probe the chemical space of the right wing in
the protein/solvent interface of RT, the substituted acetanilide
motif, a dominating element in arylazolyl(azinyl)thioacetanilide-
typed NNRTIs [22], was linked to the piperidine-NH, resulting in
compounds 8a and 8b. Unfortunately, both of them displayed
significantly impaired anti-HIV-1 activity, which were inferior to all
of the derivatives in series 7 with the exception of 7w. This result
indicated that the length and size of the linker between piperidine-
NH and the terminal phenyl ring should be appropriate, to make
compounds well adopted in the NNIBP of HIV-1 RT. In brief, the
detailed SAR concluded above provides valuable information for
further rational design of this type of HIV-1 NNRTIs.
2.3. Inhibition of HIV-1 RT
To further confirm that the title compounds target HIV-1 RT, the
most active compound 7d was tested in recombinant HIV-1 RT
inhibitory assays which use poly (A) ꢃ oligo (dT)15 as template/
primer (RT kit, Roche). As shown in Table 3, compound 7d exhibited
moderate activity against HIV-1 RT with an IC50 value of 0.50
which was comparable to that of NVP (IC50 ¼ 0.58 M), while
M). The results further indi-
mM,
m
inferior to that of ETR (IC50 ¼ 0.089
m
cated that the newly synthesized compounds, represented by
compound 7d, could effectively inhibit the activity of HIV-1 RT, thus
acting as typical HIV-1 NNRTIs.
2.4. Molecular modeling analysis
To better understand the interactions between the newly
Table 3
Inhibitory activity of compound 7d, NVP and ETR against HIV-1 RT.a
Compd
7d
NVP
0.58
ETR
IC50
(m
M)b
0.50
0.089
Fig. 2. (a) Model of 7d docked into RT wt (PDB code: 3M8Q); (b) and (c) Superim-
position of the docked conformations of 7d (magenta) and lead LW-7c (cyan) in RT wt
(PDB code: 3M8Q). Hydrogen bonds are indicated with dashed lines in red, and all the
hydrogen atoms are omitted for clarity. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
a
The RT kit was commercially available and supplied by Roche, and data were
obtained by standard ELISA.
b
The inhibitory concentration of the tested compounds required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.

B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
759
their docked conformation. Particularly, their common binding
pattern can be described as follows: 1) The left wing of the com-
pounds fits into the aromatic-rich binding sub-pocket, which is
formed by the aromatic side chains of Tyr188, Tyr181, Phe227, and
Trp229. The 2,4,6-trimethyl-substituted phenoxyl group is parallel
to the side-chain phenyl group of Tyr181, exhibiting positive Van
der Waals interactions. 2) 30-N and 50-N of [1,2,4]triazolo[1,5-a]
representative compound 7d in parallel with the reference drug
ETR, as shown in Table 4. The results of physicochemical properties
revealed that 7d and ETR agreed well with the Lipinski's rule of five.
The experimental data indicated that 7d has a favorable log P value.
More importantly, 7d was much more water-soluble than ETR.
Thus, compound 7d has a better balance of favorable activity
against wt HIV-1 and reasonable drug-like properties, which seems
to make it a promising lead for further optimization.
pyrimidine core in 7d form two hydrogen bonds with backbone a-
amino, besides, NH forms another one with backbone carbonyl of
residue Lys101, whereas lead compound LW-7c only forms two
hydrogen bonds with residue Lys101. 3) The extra piperidinyl group
incorporated to enhance the binding affinity and to optimize the
inhibitors' water solubility (will be evidenced by measurement in
the next section) locates around amino acids Tyr318, Leu234 and
Val106, which can be well adopted in the NNIBP. Additionally, the
polar hydrophilic substituent eSO2Me group resides between
Val106 and Pro236 and is oriented directly toward a solvent
exposure region (Fig. 2c). Therefore, 7d probably reserves a tighter
interaction with RT than LW-7c, which may contribute to the
higher anti-HIV-1 activity of 7d as compared to compound LW-7c.
Overall, the binding model analysis confirmed our original
design hypothesis that introducing a piperidinyl group into the
right-wing portion of [1,2,4]triazolo[1,5-a]pyrimidines could make
terminal phenyl ring of new compounds to extend more closer to
the protein/solvent interface of HIV-1 RT. These aspects will be
taken into account for further structural optimization.
3. Conclusions
Briefly, based on the structure of the previously reported DAPY-
like lead compound LW-7c with a bridgehead nitrogen 1,2,4-
triazolo[1,5-a]pyrimidine core, we designed and synthesized a se-
ries of novel NNRTIs by employing structure-based molecular hy-
bridization combined with physicochemical property-driven
scaffold re-evolution approaches. Intriguingly, most of the target
compounds showed moderate to excellent anti-HIV-1 potency. Half
of these newly synthesized inhibitors exhibited antiviral profiles
superior to that of LW-7c and much better than those of DDI, NVP
and DLV. Among them, 7d was identified as the most promising
compound in inhibiting wt HIV-1 (IIIB) replication with an EC50
value of 8.1 nM, which was prior to LW-7c and much better than
those of the reference drugs DDI (EC50 ¼ 23,198 nM), 3TC
（EC50
¼
2239 nM）, NVP (EC50
¼
312 nM) and DLV
(EC50 ¼ 540 nM). In addition, 7d showed weak activity against the
double mutant HIV-1 strain RES056 (K103N þ Y181C), and could
inhibit HIV-1 RT more efficient than NVP. Furthermore, the pre-
liminary SAR and molecular modeling studies were carried out to
assess the interactions between these inhibitors and HIV-1 RT.
Some physicochemical properties like log P and water solubility of
7d were also measured. These valuable information provide us
deeper insight into these promising structures, and further efforts
to improve the antiviral activity of these piperidine-linked de-
rivatives will be reported in due course.
2.5. In silico calculation of physicochemical properties and
measured log P and water solubility
Because of hydrophobic nature of the NNRTIs binding site, the
high biological potency of current DAPY derivatives (including the
approved TMC125 and TMC278) is often compromised by poor
water solubility. Poor aqueous solubility often leads to inconclusive
biological assay results, undesirable ADME properties, such as
limited molecular absorption and low oral bioavailability, high
dosages, and difficulties in formulation, which hinder further drug
development. Therefore, molecular water solubility was considered
as a major factor evaluating the quality of a drug candidate [25]. To
investigate whether the additional piperidine group could enhance
aqueous solubility of the new compounds, we further investigated
water solubility and other physichemical properties for the
4. Experimental section
4.1. Chemistry
All melting points were measured with a micromelting point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
obtained on a Bruker AV-400 spectrometer (Bruker BioSpin,
Switzerland), or Bruker AVANCE-300 spectrometer (Bruker Bio-
Spin, Switzerland), using tetramethylsilane (TMS) as internal
reference. The solvent used was DMSO-d6 unless otherwise indi-
Table 4
Physicochemical propertiesa and water solubility of 7d and ETR.
cated. Chemical shifts were expressed in d units (ppm) and J values
Items of physicochemical properties
Acceptable range
7d
8.1
37
520.65
ETR
were presented in hertz (Hz). Mass spectrometry was performed on
an LC Autosampler Standard G1313A instrument (Applied Bio-
systems/MDS Sciex, Concord, ON, Canada). TLC was performed on
pre-coated HUANGHAI HSGF254, 0.15e0.2 mm TLC-plates (Yantai
Jiangyou Silica Gel Development Co., Ltd, Yantai, Shandong, PR
EC50(nM)
natoms
MW
1.8
28
435.28
7
<500 Da
<10
nON
9
nOHNH
nrotb
tPSA
MV
Log P
nViol
<5
1
7
3
4
China), and spots were visualized by irradiation with UV light (
l
ꢂ10
<140 Å2
101.732
465.592
1.91b
1
120.65
335.95
>5c
1
254 nm). Flash column chromatography was performed on col-
umns packed with silica gel 60 (200e300 mesh) (Qingdao waves
silica gel desiccant co., Ltd, Qingdao, PR China). Solvents were of
reagent grade, and if necessary, were purified and dried by standard
methods. The key reagents were purchased from commercial
suppliers and without further purification when used. Rotary
evaporators served in concentration of the reaction solutions at
reduced pressure.
<5
Solubility in water (pH ¼ 7) (
m
g/mL)
software
10.31d
<<1c
a
Using
free
on-line
(http://www.molinspiration.com/);
natoms ¼ number of atoms; MW ¼ molecular weight; nON ¼ no. of hydrogen bond
acceptors; nOHNH ¼ no. of hydrogen bond donors; nrotb ¼ no. of rotatable bonds;
tPSA
¼
topological polar surface area, as a prediction of oral bioavailability;
MV ¼ molar volume; log P ¼ logarithm of partition coefficient; nViol ¼ no. of
violations.
4.1.1. General procedure for the synthesis of 7-(mesityloxy)-N-
(piperidin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-5-amine (6)
[1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol (2): NaH (5.71 g,
b
Values were measured at pH 7 by HPLC method in duplicate.
See Ref. [26].
Measured in water at pH 7 by HPLC method in duplicate.
c
d

760
B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
238 mmol) was added in portions to a solution of 1H-1,2,4-triazol-
5-amine (1) (10.00 g, 119 mmol), diethyl malonate (22.86 g,
143 mmol) in redistilled DMF (50 mL) at room temperature. The
mixture was stirred at 80 ꢀC for 2 h, and the solution was partially
solidified. Then, H2O (150 mL) was added, and the mixture was
washed with EtOAc (3 ꢃ 30 mL). The aqueous layer was separated,
and then adjusted to pH 1.0 with 8N HCl in an ice bath. The
resultant solid was filtered, washed with water, and dried under
vacuum to give [1,2,4]triazolo[1,5-a]pyrimidine-5,7-diol (2) as a
white solid. Yield: 77%, mp: 238e240 ꢀC. ESI-MS: m/z 151.1 (Mꢄ1).
5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidine (3): Intermediate
2 (1.00 g, 6.6 mmol) was added to phosphorous oxychloride (POCl3)
(6.2 mL, 66 mmol) and refluxed at 95 ꢀC for 2 h in a round bottom
flask, during which the solution turned dark. Excess POCl3 was
removed under reduced pressure and the residue was treated with
triturated ice. Potassium carbonate (K2CO3) was slowly added into
this ice-water solution to adjust the pH to 8.0 in an ice bath. The
mixture was extracted with dichloromethane (CH2Cl2) (3 ꢃ 20 mL).
Then the combined organic layers were washed with saturated
brine (30 mL), subsequently dried over anhydrous Na2SO4, filtered
and concentrated to give the crude product 5,7-dichloro-[1,2,4]
triazolo[1,5-a]pyrimidine (3) as a yellow solid. 3 could be used in
the next step without further purification. Yield: 64%,
mp:126e130 ꢀC. ESI-MS: m/z 189.3 (Mþ1), 191.3 (Mþ3), 193.2
(Mþ5).
chromatography and subsequently recrystallized with anhydrous
methanol to afford pure title compounds 7ae7w and 8a, 8b.
4.1.2.1. N-(1-(cyclohexylmethyl)piperidin-4-yl)-7-(mesityloxy)-
[1,2,4]triazolo[1,5-a]pyrimidin-5-amine (7a). White solid, yield:
46%. mp: 249e250 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.16 (s,
1H, triazole-H), 7.49 (d, J ¼ 7.50 Hz, 1H, NH), 7.07 (s, 2H, OPh-H),
5.31 (s, 1H, pyrimidine-H), 3.78e3.76 (m, 1H, piperidine-H), 2.71
(d, J ¼ 11.70 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3), 2.09 (s, 6H,
CH3 ꢃ2), 2.06e2.03 (m, 2H, piperidine-H), 2.00e1.93 (m, 2H, hex-
H), 1.84 (d, J ¼ 11.10 Hz, 2H, CH2), 1.72e1.62 (m, 5H, piperidine-H,
hex-H), 1.44e1.40 (m, 1H, hex-H), 1.39e1.28 (m, 2H, piperidine-
H), 1.27e1.05 (m, 3H, hex-H), 0.85e0.74 (m, 2H, hex-H). 13C NMR
(75 MHz, DMSO-d6, ppm) d: 160.08, 157.29, 153.98, 152.67, 145.93,
136.15, 129.99, 129.49, 79.19, 64.98, 52.34, 47.49, 34.67, 31.30, 26.36,
25.52, 20.29, 15.24. ESI-MS: m/z 449.5 (Mþ1), 450.6 (Mþ2),
C26H36N6O (448.60).
4.1.2.2. 7-(Mesityloxy)-N-(1-(pyridin-4-ylmethyl)piperidin-4-yl)-
[1,2,4]triazolo[1,5-a] pyrimidin-5-amine (7b). White solid, yield:
34%. mp: 257e258 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 8.50
(dd, J ¼ 4.52 Hz,1.36 Hz, 2H, pyridine-30,50-H), 8.18 (s, 1H, triazole-
H), 7.55 (d, J ¼ 7.20 Hz, 1H, NH), 7.31 (d, J ¼ 5.76 Hz, 2H, pyridine-
20, 60-H), 7.08 (s, 2H, PhH), 5.32 (s, 1H, pyrimidine-H), 3.83e3.82 (m,
1H, piperidine-H), 3.49 (s, 2H, CH2), 2.72 (d, J ¼ 11.46 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.15e2.12 (m, 2H, piperidine-H),
2.09 (s, 6H, CH3ꢃ2), 1.88 (d, J ¼ 10.40 Hz, 2H, piperidine-H),
1.44e1.36 (m, 2H, piperidine-H). 13C NMR (100 MHz, DMSO-d6,
tert-butyl-4-((7-(mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-
yl)amino)piperidine-1-carboxylate (5): K2CO3 (1.46 g, 10.58 mmol)
was slowly added to a solution of 2,4,6-trimethylphenol (0.72 g,
5.3 mmol) in redistilled DMF (10 mL). The mixture was stirred at
room temperature for 10 min, then intermediate 3 (1.00 g,
5.3 mmol) was added. The reaction was heated at 40 ꢀC for 2.5 h
until its completion. After that, 4-amino-1-Boc-piperidine (1.17 g,
5.8 mmol), K2CO3 (0.73 g, 5.3 mmol) and redistilled DMF (4.0 mL)
were added into above solution, and then the reaction mixture was
stirred at 60 ꢀC overnight. Then, DMF was evaporated under
reduced pressure and H2O (30 mL) was added to the residue. The
aqueous solution was extracted with EtOAc (3 ꢃ 30 mL). The
combined organic layer was washed with saturated brine (40 mL)
and dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. Purification by flash column chromatography
and recrystallization of the residue gave intermediate 5 as a white
solid. Two-step total yield: 34%, mp: 240e242 ꢀC. ESI-MS: m/z
453.6 (Mþ1), 475.5 (Mþ23).
ppm) d: 160.57, 157.77, 154.50, 153.19, 149.98, 148.24, 146.41, 136.67,
130.50, 130.00, 124.10, 79.68, 61.24, 52.23, 47.62, 31.68, 20.80, 15.75.
ESI-MS: m/z 444.6 (Mþ1),466.5 (Mþ23), C25H29N7O (443.54).
4.1.2.3. N-(1-benzylpiperidin-4-yl)-7-(mesityloxy)-[1,2,4]triazolo
[1,5-a]pyrimidin-5-amine (7c). White solid, yield: 48%. mp:
236.5e237 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17 (s, 1H,
triazole-H), 7.52 (d, J ¼ 7.20 Hz, 1H, PhH), 7.34e7.21 (m, 5H, NH and
PhH), 7.07 (s, 2H, OPh-H), 5.32 (s, 1H, pyrimidine-H), 3.82e3.80 (m,
1H, piperidin-H), 3.45 (s, 2H, CH2), 2.73 (d, J ¼ 11.10 Hz, 2H,
piperidin-H), 2.30 (s, 3H, CH3), 2.09e2.04 (m, 8H, piperidin-H, CH3),
1.86 (d, J ¼ 10.20 Hz, 2H, piperidin-H), 1.42e1.32 (m, 2H, piperidin-
H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 160.08, 157.29, 153.98,
152.69, 145.94, 138.56, 136.15, 129.99, 129.49, 128.69, 128.09,
126.78, 79.21, 62.16, 51.66, 47.31, 31.23, 20.29, 15.23. ESI-MS: m/z
443.7 (Mþ1), C26H30N6O (442.56).
7-(mesityloxy)-N-(piperidin-4-yl)-[1,2,4]triazolo[1,5-a]pyr-
imidin-5-amine (6): To a solution of 5 (1.05 g, 2.3 mmol) in CH2Cl2
(3.0 mL), trifluoroacetic acid (TFA, 3.0 mL) was added dropwise. The
reaction mixture was stirred for 0.5 h at room temperature. After
removal of the excess solvent under reduced pressure, saturated
K2CO3 aqueous solution (10 mL) was added. The resultant precip-
itate was filtered, washed by water and dried in vacuum to give the
key intermediate 6 as a white solid. Yield: 86%, decomposed (below
the mp). ESI-MS: m/z 353.5 (Mþ1), 375.4 (Mþ23).
4.1.2.4. 7-(Mesityloxy)-N-(1-(4-(methylsulfonyl)benzyl)piperidin-4-
yl)-[1,2,4]triazolo [1,5-a]pyrimidin-5-amine (7d). White solid, yield:
45%. mp: 258e259 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 8.17 (s,
1H, triazole-H), 7.88 (d, J ¼ 8.28 Hz, 2H, PhH), 7.57 (d, J ¼ 8.24 Hz,
2H, PhH), 7.54 (d, J ¼ 7.24 Hz, 1H, NH), 7.08 (s, 2H, OPh-H), 5.32 (s,
1H, pyrimidine-H), 3.83e3.82 (m, 1H, piperidine-H), 3.57 (s, 2H,
CH2), 3.20 (s, 3H, SO2CH3), 2.73 (d, J ¼ 11.36 Hz, 2H, piperidine-H),
2.30 (s, 3H, CH3), 2.16e2.13 (m, 2H, piperidine-H), 2.09 (s, 6H,
CH3 ꢃ2), 1.88 (d, J ¼ 10.36 Hz, 2H, piperidine-H), 1.43e1.35 (m, 2H,
4.1.2. General procedure for the synthesis of target compounds
7ae7w and 8a, 8b
piperidin-H). 13C NMR (100 MHz, DMSO-d6, ppm)
d: 160.57, 157.77,
At room temperature, key intermediate 6 (0.20 g, 0.57 mmol)
and anhydrous K2CO3 (0.16 g, 1.14 mmol) were added to anhydrous
DMF (5 mL), followed by the addition of the appropriate RCH2X
(X ¼ Cl, Br) or ClCH2CONHR'(0.63 mmol). Then the reaction mixture
was stirred overnight at room temperature. The solvent was
evaporated under reduced pressure, and H2O (30 mL) was added.
After extraction with CH2Cl2 or EtOAc (3 ꢃ 30 mL), the combined
organic phase was washed with saturated brine (40 mL), dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. Then the residue was further purified by flash column
154.50, 153.20, 146.42, 145.45, 139.83, 136.68, 130.50, 130.00,
129.79, 127.40, 79.69, 61.88, 52.21, 47.67, 44.06, 31.70, 20.80, 15.75.
ESI-MS: m/z 521.5 (Mþ1), 543.5 (Mþ23), C27H32N6O3S (520.65).
4.1.2.5. 4-((4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
amino)piperidin-1-yl)methyl)benzenesulfonamide (7e). White solid,
yield: 38%. mp: 319e319.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d:
8.17 (s, 1H, triazole-H), 7.78 (d, J ¼ 7.80 Hz, 2H, PhH), 7.53 (d,
J ¼ 7.50 Hz, 1H, NH), 7.48 (d, J ¼ 8.10 Hz, 2H, PhH), 7.29 (s, 2H,
SO2NH2), 7.08 (s, 2H, OPh-H), 5.32 (s, 1H, pyrimidine-H), 3.82 (m,

B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
761
1H, piperidine-H), 3.53 (s, 2H, CH2), 2.72 (d, J ¼ 10.80 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.14e2.09 (m, 8H, piperidine-H,
CH3), 1.87 (d, J ¼ 10.50 Hz, 2H, piperidine-H), 1.43e1.33 (m, 2H,
4.1.2.10. N-(1-(4-bromobenzyl)piperidin-4-yl)-7-(mesityloxy)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7j). White solid, yield: 42%.mp:
249.5e251.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.16 (s, 1H,
piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 160.59, 157.78,
triazole-H), 7.51e7.49 (m, 3H, NH, PhH), 7.25 (d, J ¼ 8.10 Hz, 2H,
154.49, 153.21, 146.44, 143.34, 143.17, 136.67, 130.50, 130.00, 129.45,
126.07, 79.71, 61.95, 52.16, 47.72, 31.69, 20.79, 15.74. ESI-MS: m/z
522.5 (Mþ1), 544.5 (Mþ23), C26H31N7O3S (521.63).
PhH), 7.07 (s, 2H, OPh-H), 5.31 (s, 1H, pyrimidine-H), 3.80 (m, 1H,
piperidine-H), 3.41 (s, 2H, CH2), 2.71 (d,
piperidine-H), 2.30 (s, 3H, CH3), 2.09 (m, 8H, piperidine-H, CH3),
J
¼
11.40 Hz, 2H,
1.86 (d, J ¼ 9.60 Hz, 2H, piperidine-H), 1.41e1.31 (m, 2H, piperidine-
H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 160.07, 157.27, 153.99,
4.1.2.6. Methyl-4-((4-((7-(mesityloxy)-[1,2,4]triazolo[1,5-a]pyr-
imidin-5-yl)amino) piperidin-1-yl)methyl)benzoate (7f). White
solid, yield: 35%. mp: 240.5e241 ꢀC. 1H NMR (300 MHz, DMSO-d6,
152.69, 145.92, 136.17, 131.01, 130.83, 130.00, 129.49, 119.77, 79.19,
61.23, 51.59, 47.21, 31.20, 20.29, 15.24. ESI-MS: m/z 522.5 (Mþ1),
523.4 (Mþ2), C26H29BrN6O (521.45).
ppm)
d
: 8.17 (s,1H, triazole-H), 7.92 (d, J ¼ 8.10 Hz, 2H, PhH), 7.52 (d,
J ¼ 6.90 Hz, 1H, NH), 7.45 (d, J ¼ 8.10 Hz, 2H, PhH), 7.08 (s, 2H, OPh-
H), 5.32 (s, 1H, pyrimidine-H), 3.84 (s, 4H, COOCH3 and piperidine-
H), 3.53 (s, 2H, CH2), 2.72 (d, J ¼ 11.10 Hz, 2H, piperidine-H), 2.30 (s,
3H, CH3), 2.15e2.09 (m, 8H, piperidine-H, CH3), 1.87 (d, J ¼ 10.20 Hz,
2H, piperidine-H), 1.44e1.33 (m, 2H, piperidine-H). 13C NMR
4.1.2.11. N-(1-(3-chlorobenzyl)piperidin-4-yl)-7-(mesityloxy)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7k). White solid, yield: 39%. mp:
240e242.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17 (s, 1H,
triazole-H), 7.52 (d, J ¼ 7.20 Hz, 1H, NH), 7.37e7.24 (m, 4H, PhH),
7.08 (s, 2H, OPh-H), 5.32 (s, 1H, pyrimidine-H), 3.82e3.80 (m, 1H,
piperidine-H), 3.46 (s, 2H, CH2), 2.72 (d, J ¼ 11.40 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.13e2.09 (m, 8H, piperidine-H,
CH3), 1.87 (d, J ¼ 10.20 Hz, 2H, piperidine-H), 1.42e1.32 (m, 2H,
(75 MHz, DMSO-d6, ppm) d: 166.11, 160.08, 157.28, 153.99, 152.70,
145.94, 144.51, 136.16, 129.99, 129.49, 129.08, 128.80, 128.22, 79.20,
61.63, 51.97, 51.71, 47.21, 31.20, 20.29, 15.23. ESI-MS: m/z 501.4
(Mþ1), 523.5 (Mþ23), C28H32N6O3 (500.59).
piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 160.09, 157.28,
153.99, 152.70, 145.94, 141.37, 136.16, 132.87, 129.99, 129.49, 128.26,
127.26, 126.77, 79.21, 61.29, 51.60, 47.22, 31.20, 20.29, 15.23. ESI-MS:
m/z 477.4 (M), 499.5 (Mþ22), C26H29ClN6O (477.00).
4.1.2.7. 7-(Mesityloxy)-N-(1-(4-nitrobenzyl)piperidin-4-yl)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7g). Yellow solid, yield: 58%. mp:
decomposed. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.20 (s, 1H,
triazole-H), 8.17 (d, J ¼ 1.50 Hz, 2H, PhH), 7.59 (d, J ¼ 8.70 Hz, 2H,
PhH), 7.53 (d, J ¼ 7.20 Hz, 1H, NH), 7.08 (s, 2H, OPh-H), 5.32 (s, 1H,
pyrimidine-H), 3.84e3.82 (m, 1H, piperidine-H), 3.60 (s, 2H, CH2),
2.73 (d, J ¼ 11.70 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3), 2.18e2.14
(m, 2H, piperidine-H), 2.09 (s, 6H, CH3 ꢃ2), 1.88 (d, J ¼ 10.20 Hz, 2H,
piperidine-H), 1.46e1.35 (m, 2H, piperidine-H). 13C NMR (75 MHz,
4.1.2.12. 4-((4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
amino)piperidin-1-yl)methyl)benzonitrile (7l). White solid, yield:
61%. mp: 250e251 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17 (s,
1H, triazole-H), 7.78 (d, J ¼ 8.40 Hz, 2H, PhH), 7.53 (d, J ¼ 6.60 Hz,
1H, NH), 7.50 (d, J ¼ 8.10 Hz, 2H, PhH), 7.08 (s, 2H, OPh-H), 5.32 (s,
1H, pyrimidine-H), 3.83e3.81 (m, 1H, piperidine-H), 3.55 (s, 2H,
CH2), 2.71 (d, J ¼ 11.70 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3),
2.16e2.12 (m, 2H, piperidine-H), 2.09 (s, 6H, CH3 ꢃ2), 1.87 (d,
DMSO-d6, ppm) d: 160.09, 157.28, 153.99, 152.71, 147.08, 146.50,
145.93, 136.17, 129.99, 129.55, 129.49, 123.31, 79.20, 61.13, 51.70,
47.15, 31.20, 20.29, 15.23. ESI-MS: m/z 488.5 (Mþ1), 510.5 (Mþ23),
J ¼ 10.20 Hz, 2H, piperidine-H), 1.44e1.33 (m, 2H, piperidine-H). 13
C
C
26H29N7O3 (487.55).
NMR (75 MHz, DMSO-d6, ppm) d: 160.09, 157.28, 153.99, 152.71,
145.94, 144.81, 136.16, 132.10, 129.99, 129.49, 129.40, 118.86, 109.59,
79.20, 61.44, 51.67, 47.16, 31.20, 20.28, 15.23. ESI-MS: m/z 468.4
(Mþ1), 490.6 (Mþ23), C27H29N7O (467.57).
4.1.2.8. 4-((4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
amino)piperidin-1-yl)methyl)benzamide (7h). White solid, yield:
32%. mp: decomposed. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17
(s,1H, triazole-H), 7.90 (s, 1H, CONH2), 7.82 (d, J ¼ 8.40 Hz, 2H, PhH),
7.52 (d, J ¼ 7.20 Hz, 1H, NH), 7.36 (d, J ¼ 8.40 Hz, 2H, PhH), 7.28 (s,
1H, CONH2), 7.08 (s, 2H, OPh-H), 5.31 (s, 1H, pyrimidine-H), 3.80 (m,
1H, piperidine-H), 3.50 (s, 2H, CH2), 2.72 (d, J ¼ 11.40 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.13e2.09 (m, 8H, piperidine-H,
CH3), 1.86 (d, J ¼ 9.60 Hz, 2H, piperidine-H), 1.43e1.32 (m, 2H,
4.1.2.13. 3-((4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
amino)piperidin-1-yl)methyl)benzonitrile (7m). White solid, yield:
55%. mp: decomposed. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17
(s, 1H, triazole-H), 7.72e7.70 (m, 2H, PhH), 7.65 (d, J ¼ 7.80 Hz, 1H,
NH), 7.56e7.51 (m, 2H, PhH), 7.08 (s, 2H, OPh-H), 5.32 (s, 1H,
pyrimidine-H), 3.83e3.81 (m, 1H, piperidine-H), 3.52 (s, 2H, CH2),
2.72 (d, J ¼ 11.40 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3), 2.15 (m,
2H, piperidine-H), 2.09 (s, 6H, CH3 ꢃ2), 1.87 (d, J ¼ 10.20 Hz, 2H,
piperidine-H), 1.43e1.33 (m, 2H, piperidine-H). 13C NMR (75 MHz,
piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 167.74, 160.08,
157.28, 153.99, 152.69, 145.94, 142.01, 136.16, 132.95, 129.99, 129.50,
128.36, 127.37, 79.21, 61.70, 51.68, 47.25, 31.22, 20.29, 15.23. ESI-MS:
m/z 486.5 (Mþ1), 487.5 (Mþ2), 488.5 (Mþ3), 508.4 (Mþ23),
DMSO-d6, ppm) d: 160.09, 157.28, 153.99, 152.70, 145.94, 140.44,
C
27H31N7O2 (485.58).
136.16, 133.54, 131.97, 130.70, 129.99, 129.49, 129.40, 118.84, 111.17,
79.20, 60.97, 51.56, 47.18, 31.18, 20.29, 15.23. ESI-MS: m/z 468.4
(Mþ1), 490.6 (Mþ23), C27H29N7O (467.57).
4.1.2.9. 1-(4-((4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-
yl)amino) piperidin-1-yl)methyl)phenyl)ethanone (7i). White solid,
yield: 43%. mp: >320 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.16
4.1.2.14. 2-((4-((7-(Mesityloxy)-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl)
(s, 1H, triazole-H), 7.91 (d, J ¼ 8.10 Hz, 2H, PhH), 7.52 (d, J ¼ 7.20 Hz,
1H, NH), 7.44 (d, J ¼ 8.10 Hz, 2H, PhH), 7.08 (s, 2H, OPh-H), 5.31 (s,
1H, pyrimidine-H), 3.80 (m, 1H, piperidine-H), 3.53 (s, 2H, CH2),
2.72 (d, J ¼ 11.10 Hz, 2H, piperidine-H), 2.56 (s, 3H, COCH3), 2.30 (s,
3H, CH3), 2.14e2.09 (m, 8H, piperidine-H, CH3), 1.87 (d, J ¼ 9.60 Hz,
2H, piperidine-H), 1.43e1.33 (m, 2H, piperidine-H). 13C NMR
amino)piperidin-1-yl)methyl)benzonitrile (7n). White solid, yield:
57%. mp: 252.5e253.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d:
8.17 (s, 1H, triazole-H), 7.80 (dd, J ¼ 7.50 Hz, J ¼ 0.90 Hz, 1H, PhH),
7.67 (td, J ¼ 7.50 Hz, J ¼ 1.20 Hz, 1H, PhH), 7.56 (d, J ¼ 7.80 Hz, 1H,
PhH), 7.53 (d, J ¼ 7.50 Hz, 1H, NH), 7.46 (td, J ¼ 7.50 Hz, J ¼ 0.90 Hz,
1H, PhH), 7.08 (s, 2H, OPh-H), 5.32 (s, 1H, pyrimidine-H), 3.85e3.84
(m, 1H, piperidine-H), 3.63 (s, 2H, CH2), 2.73 (d, J ¼ 11.70 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.24e2.18 (m, 2H, piperidine-H),
2.09 (s, 6H, CH3 ꢃ2), 1.87 (d, J ¼ 9.90 Hz, 2H, piperidine-H),
1.44e1.32 (m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6,
(75 MHz, DMSO-d6, ppm) d: 197.49, 160.08, 153.99, 152.70, 145.93,
144.34, 136.17, 135.63, 130.00, 129.49, 128.76, 128.16, 79.21, 61.65,
51.72, 47.21, 31.21, 26.63, 20.29, 15.24. ESI-MS: m/z 485.5 (Mþ1),
507.5 (Mþ23), C28H32N6O2 (484.59).

762
B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
ppm)
d
: 160.09, 157.28, 153.99, 152.70, 145.94, 142.40, 136.16,
4.1.2.19. 7-(Mesityloxy)-N-(1-(2-methylbenzyl)piperidin-4-yl)-[1,2,4]
132.95, 132.93, 129.99, 129.49, 127.86, 117.62, 112.01, 79.22, 59.85,
51.53, 47.04, 31.08, 20.28, 15.23. ESI-MS: m/z 468.4 (Mþ1), 469.5
(Mþ2), 470.6 (Mþ3), 490.6 (Mþ23), C27H29N7O (467.57).
triazolo[1,5-a] pyrimidin-5-amine (7s). White solid, yield: 67%. mp:
255e257.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.16 (s, 1H,
triazole-H), 7.50 (d, J ¼ 7.50 Hz, 1H, NH), 7.48 (d, J ¼ 8.10 Hz, 2H,
PhH), 7.21e7.19 (m, 1H, PhH), 7.14e7.10 (m, 3H, PhH), 7.07 (s, 2H,
OPh-H), 5.31 (s, 1H, pyrimidine-H), 3.83e3.78 (m, 1H, piperidine-
H), 3.40 (s, 2H, CH2), 2.72 (d, J ¼ 11.70 Hz, 2H, piperidine-H), 2.30
(s, 3H, CH3), 2.29 (s, 3H, CH3), 2.14e2.09 (m, 8H, piperidine-H, CH3),
1.87 (d, J ¼ 10.50 Hz, 2H, piperidine-H), 1.39e1.28 (m, 2H,
4.1.2.15. N-(1-(4-fluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7o). White solid, yield: 54%. mp:
249e249.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17 (s, 1H,
triazole-H), 7.52 (d, J ¼ 7.20 Hz, 1H, NH), 7.32 (dd, JHeH ¼ 8.40 Hz,
JHeF ¼ 5.70 Hz, 2H, PhH), 7.12 (dd, JHeH ¼ 6.90 Hz, JHeF ¼ 8.90 Hz,
2H, PhH), 7.07 (s, 2H, OPh-H), 5.31 (s, 1H, pyrimidine-H), 3.82e3.78
(m, 1H, piperidine-H), 3.43 (s, 2H, CH2), 2.71 (d, J ¼ 11.70 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.09e2.04 (m, 8H, piperidine-H,
CH3), 1.86 (d, J ¼ 9.90 Hz, 2H, piperidine-H), 1.42e1.30 (m, 2H,
piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 160.08, 157.28,
153.98, 152.68, 145.93, 136.91, 136.62, 136.16, 129.99, 129.49, 129.40,
126.77, 125.34, 79.20, 60.16, 51.79, 47.35, 31.30, 20.29, 18.75, 15.23.
ESI-MS: m/z 457.6 (Mþ1), 479.5 (Mþ23), C27H32N6O (456.58).
piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d
: 161.16 (d,
4.1.2.20. N-(1-(2,4-difluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-
[1,2,4]triazolo[1,5-a] pyrimidin-5-amine (7t). White solid, yield:
JCeF ¼ 239.25 Hz, 1C, Ph-C), 160.08, 157.28, 153.98, 152.69, 145.94,
136.16, 134.71 (d, JCeF ¼ 3.00 Hz, 1C, Ph-C), 130.47 (d, JCeF ¼ 7.50 Hz,
2C, Ph-C),129.99,129.49,114.79 (d, JCeF ¼ 21.00 Hz, 2C, Ph-C), 79.20,
61.20, 51.55, 47.30, 31.21, 20.28, 15.23. ESI-MS: m/z 461.4
(Mþ1),483.4 (Mþ23), C26H29FN6O (460.55).
64%. mp: 249e249.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17
(s, 1H, triazole-H), 7.51 (d, J ¼ 7.20 Hz, 1H, NH), 7.43 (dd,
JHeF ¼ 15.30 Hz, JHeH ¼ 8.40 Hz, 1H, PhH), 7.18 (td, JHeF ¼ 9.90 Hz,
JHeH ¼ 2.40 Hz, 2H, PhH), 7.07e7.04 (m, 3H, OPh-H, PhH), 5.31 (s,
1H, pyrimidine-H), 3.81e3.79 (m, 1H, piperidine-H), 3.48 (s, 2H,
CH2), 2.72 (d, J ¼ 11.70 Hz, 2H, piperidine-H), 2.29 (s, 3H, CH3),
2.15e2.09 (m, 8H, piperidine-H, CH3), 1.86 (d, J ¼ 10.20 Hz, 2H,
piperidine-H), 1.42e1.29 (m, 2H, piperidine-H). 13C NMR (75 MHz,
4.1.2.16. N-(1-(3-fluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7p). White solid, yield: 52%. mp:
246.5e247 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17 (s, 1H,
triazole-H), 7.52 (d, J ¼ 7.20 Hz, 1H, NH), 7.39e7.32 (m, 1H, PhH),
7.15e7.04 (m, 5H, PhH, OPh-H), 5.32 (s, 1H, pyrimidine-H), 3.81 (m,
1H, piperidine-H), 3.47 (s, 2H, CH2), 2.73 (d, J ¼ 10.80 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.13e2.09 (m, 8H, piperidine-H,
CH3), 1.87 (d, J ¼ 10.50 Hz, 2H, piperidine-H), 1.43e1.33 (m, 2H,
DMSO-d6, ppm) d: 163.05, 162.21, 160.08, 158.93, 157.28, 153.98,
152.70, 145.93, 136.16, 132.64, 132.56, 132.43, 129.98, 129.49, 121.33,
121.28, 121.13, 121.08, 111.30, 111.25, 111.02, 103.86, 103.51, 103.16,
79.20, 54.11, 51.35, 47.13, 31.14, 20.28, 15.22. ESI-MS: m/z 479.4
(Mþ1), 501.4 (Mþ23), C26H28F2N6O (478.54).
piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 162.20 (d,
JCeF ¼ 242.25 Hz, 1C, Ph-C), 160.09, 157.28, 153.99, 152.70, 145.94,
141.84, 136.16, 129.99, 129.49, 124.54, 115.01 (d, JCeF ¼ 21.00 Hz, 2C,
Ph-C), 113.54 (d, JCeF ¼ 20.25 Hz, 2C, Ph-C), 79.21, 61.37, 51.62, 47.23,
31.21, 20.28, 15.23. ESI-MS: m/z 461.4 (Mþ1), C26H29FN6O (460.55).
4.1.2.21. N-(1-(3,4-difluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-
[1,2,4]triazolo[1,5-a] pyrimidin-5-amine (7u). White solid, yield:
48%. mp: 253e256 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17 (s,
1H, triazole-H), 7.52 (d, J ¼ 7.20 Hz, 1H, NH), 7.40e7.29 (m, 2H,
PhH), 7.15e7.11 (m, 1H, PhH), 7.08 (s, 2H, OPh-H), 5.32 (s, 1H,
pyrimidine-H), 3.82e3.80 (m, 1H, piperidine-H), 3.44 (s, 2H, CH2),
2.72 (d, J ¼ 11.70 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3), 2.13e2.06
(m, 8H, piperidine-H, CH3), 1.87 (d, J ¼ 10.20 Hz, 2H, piperidine-H),
1.42e1.32 (m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
4.1.2.17. N-(1-(2-fluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7q). White solid, yield: 56%. mp:
248.5e251.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.16 (s, 1H,
triazole-H), 7.51 (d, J ¼ 7.20 Hz, 1H, NH), 7.39 (ddd, JHeH ¼ 7.50 Hz,
JHeF ¼ 5.70 Hz, JHeH ¼ 1.50 Hz, 1H, PhH), 7.35e7.27 (m, 1H, PhH),
7.19e7.12 (m, 2H, PhH), 7.07 (s, 2H, OPh-H), 5.31 (s, 1H, pyrimidine-
H), 3.81e3.79 (m, 1H, piperidine-H), 3.50 (s, 2H, CH2), 2.74 (d,
J ¼ 11.70 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3), 2.16e2.09 (m, 8H,
piperidine-H, CH3), 1.86 (d, J ¼ 9.90 Hz, 2H, piperidine-H),1.42e1.30
d: 160.08, 157.27, 153.98, 152.69, 145.93, 136.62, 136.58, 136.56,
136.51, 136.16, 129.99, 129.49, 125.17, 125.13, 125.08, 125.04, 117.31,
117.12, 116.90, 79.20, 60.73, 51.51, 47.22, 31.19, 20.28, 15.23. ESI-MS:
m/z 479.4 (Mþ1), 501.4 (Mþ23), C26H28F2N6O (478.54).
(m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
d: 160.72
4.1.2.22. N-(1-(2,5-difluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-
[1,2,4]triazolo[1,5-a] pyrimidin-5-amine (7v). White solid, yield:
(d, JCeF ¼ 243.00 Hz, 1C, Ph-C), 160.07, 157.27, 153.99, 152.69, 145.93,
136.16, 131.44 (d, JCeF ¼ 4.50 Hz, 1C, Ph-C), 129.99, 129.49, 128.92 (d,
JCeF ¼ 7.50 Hz, 1C, Ph-C), 124.86 (d, JCeF ¼ 14.25 Hz, 1C, Ph-C), 124.11
(d, JCeF ¼ 3.00 Hz, 1C, Ph-C), 115.07 (d, JCeF ¼ 22.50 Hz, 1C, Ph-C),
79.19, 54.61, 51.49, 47.15, 31.16, 20.29, 15.23. ESI-MS: m/z 461.4
(Mþ1), C26H29FN6O (460.55).
63%. mp: 244.5e245 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.17
(s, 1H, triazole-H), 7.52 (d, J ¼ 7.20 Hz, 1H, NH), 7.26e7.20 (m, 2H,
PhH), 7.18e7.10 (m, 1H, PhH), 7.07 (s, 2H, OPh-H), 5.32 (s, 1H,
pyrimidine-H), 3.82e3.80 (m, 1H, piperidine-H), 3.51 (s, 2H, CH2),
2.74 (d, J ¼ 11.40 Hz, 2H, piperidine-H), 2.30 (s, 3H, CH3), 2.19e2.09
(m, 8H, piperidine-H, CH3), 1.87 (d, J ¼ 10.20 Hz, 2H, piperidine-H),
1.44e1.33 (m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6, ppm)
4.1.2.18. 7-(Mesityloxy)-N-(1-(4-methylbenzyl)piperidin-4-yl)-[1,2,4]
triazolo[1,5-a] pyrimidin-5-amine (7r). White solid, yield: 65%. mp:
d: 160.09, 159.61, 158.34, 157.28, 156.46, 155.16, 153.98, 152.70,
249.5e250.5 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d
: 8.16 (s, 1H,
145.93, 136.15, 129.98, 129.49, 127.35, 127.25, 127.12, 127.02, 117.27,
117.20, 116.95, 116.88, 116.79, 116.68, 116.46, 116.34, 115.38, 115.26,
115.06, 114.94, 79.21, 54.23, 51.45, 47.07, 31.15, 20.27, 15.22. ESI-MS:
m/z 479.4 (Mþ1), 501.4 (Mþ23), C26H28F2N6O (478.54).
triazole-H), 7.51 (d, J ¼ 7.20 Hz, 1H, NH), 7.17e7.09 (m, 4H, PhH),
7.07 (s, 2H, OPh-H), 5.31 (s, 1H, pyrimidine-H), 3.81e3.79 (m, 1H,
piperidine-H), 3.39 (s, 2H, CH2), 2.71 (d, J ¼ 11.40 Hz, 2H,
piperidine-H), 2.30 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.09e1.99 (m, 8H,
piperidine-H, CH3), 1.85 (d, J ¼ 12.30 Hz, 2H, piperidine-H),
1.40e1.30 (m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6,
4.1.2.23. N-(1-(2,6-difluorobenzyl)piperidin-4-yl)-7-(mesityloxy)-
[1,2,4]triazolo[1,5-a] pyrimidin-5-amine (7w). White solid, yield:
ppm)
d
: 160.08, 157.28, 153.98, 152.69, 145.94, 136.15, 135.79,
51%. mp: 254.5e256 ꢀC. 1H NMR (300 MHz, DMSO-d6, ppm)
d: 8.16
135.45, 129.99, 129.49, 128.66, 79.19, 61.91, 51.59, 47.33, 31.22,
(s, 1H, triazole-H), 7.49 (d, J ¼ 7.20 Hz, 1H, NH), 7.45e7.35 (m, 1H,
PhH), 7.12e7.07 (m, 4H, PhH, OPh-H), 5.30 (s, 1H, pyrimidine-H),
3.78e3.76 (m, 1H, piperidine-H), 3.55 (s, 2H, CH2), 2.73 (d,
20.63, 20.29, 15.23. ESI-MS: m/z 457.5 (Mþ1), 479.4 (Mþ23),
C
27H32N6O (456.58).

B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
763
J ¼ 11.70 Hz, 2H, piperidine-H), 2.29 (s, 3H, CH3), 2.18e2.08 (m, 8H,
piperidine-H, CH3), 1.84 (d, J ¼ 10.20 Hz, 2H, piperidine-H),
1.39e1.27 (m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6,
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 examined spectrophotometrically by the MTT method.
The 50% cytotoxic concentration (CC50) was defined as the con-
centration of the test compound that reduced the viability of the
mock-infected MT-4 cells by 50%. The concentration achieving 50%
protection from the cytopathic effect of the virus in infected cells
was defined as the 50% effective concentration (EC50).
ppm)
d
: 161.35 (dd, JCeF ¼ 245.25 Hz, JCeF ¼ 8.25 Hz, 2C, Ph-C),
160.06, 157.27, 153.98, 152.69, 145.92, 136.15, 129.98, 129.91 (t,
JCeF ¼ 10.50 Hz, 1C, Ph-C), 129.49, 113.03 (t, JCeF ¼ 19.50 Hz, 1C, Ph-
C), 111.33 (d, JCeF ¼ 25.50 Hz, 2C, Ph-C), 79.19, 51.13, 48.35, 46.96,
31.07, 20.27, 15.22. ESI-MS: m/z 479.4 (Mþ1), 501.4 (Mþ23),
C
26H28F2N6O (478.54).
4.3. HIV-1 RT inhibition assay
4.1.2.24. Methyl-3-chloro-4-(2-(4-((7-(mesityloxy)-[1,2,4]triazolo
[1,5-a]pyrimidin-5-yl)amino)piperidin-1-yl)acetamido)benzoate
(8a). White solid, yield: 61%. mp: 262.5e264 ꢀC. 1H NMR
The inhibition assay of HIV-1 RTwt was performed by utilizing
the template/primer hybrid poly(A) ꢃ oligo(dT)15, digoxigenin- and
biotin-labeled nucleotides, an antibody to digoxigenin which was
conjugated 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 implemented by using an RT
assay kit (Roche), and the procedures for assaying RT inhibition was
done as described in the kit protocol [29,30]. The tested compound
7d and two control drugs NVP and ETR was used at different con-
(400 MHz, DMSO-d6, ppm)
d: 10.33 (s, 1H, CONH), 8.52 (d,
J ¼ 8.64 Hz,1H, PhH), 8.18 (s, 1H, triazole-H), 8.02 (d, J ¼ 1.88 Hz,1H,
PhH), 7.95 (dd, J ¼ 8.64 Hz, J ¼ 1.80 Hz, 1H, PhH), 7.57 (d, J ¼ 7.28 Hz,
1H, NH), 7.09 (s, 2H, OPh-H), 5.30 (s,1H, pyrimidine-H), 3.89 (m,1H,
piperidine-H), 3.85 (s, 3H, COOCH3), 3.23 (s, 2H, CH2), 2.87 (d,
J ¼ 11.56 Hz, 2H, piperidine-H), 2.47e2.41 (m, 2H, piperidine-H),
2.31 (s, 3H, CH3), 2.10 (s, 6H, CH3 ꢃ2), 1.96 (d, J ¼ 10.76 Hz, 2H,
piperidine-H), 1.55e1.50 (m, 2H, piperidine-H). 13C NMR (100 MHz,
DMSO-d6, ppm)
d
: 169.69, 165.19, 160.58, 157.79, 154.53, 153.26,
centration gradient (1.040, 0.208, 0.0416, 0.00832, 0.001664 M for
m
146.44, 138.95, 136.70, 130.50, 130.40, 130.00, 129.77, 125.78,
122.23, 120.02, 79.73, 61.73, 52.77, 52.34, 46.89, 32.12, 20.80, 15.75.
ESI-MS: m/z 578.5 (M), 580.4 (Mþ2), 600.5 (Mþ22), C29H32ClN7O4
(578.06).
7d; 100, 20, 4, 0.8, 0.16 M for NVP; 1, 0.2, 0.04, 0.008, 0.0016 mM for
m
ETR). Concretely, the reaction mixture consisted of template/primer
complex, 20-deoxy-nucleotide-50-triphosphates (dNTPs) and RT
enzyme in the lysis buffer with or without inhibitors. After incu-
bation for 1 h at 37 ꢀC, the reaction mixture was transferred to
streptavidin-coated microtiter plate (MTP). The biotin labeled
dNTPs that are incorporated in the template due to presence of RT
were bound to streptavidine. The unbound 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 also washed elaborately for 5 times by using wash
buffer and finally, the peroxide substrate (ABST) was added to the
MTP. A colored reaction product emerged during the cleavage of the
substrate catalyzed by a peroxide enzyme. The absorbance of the
sample was determined at OD450 using microtiter plate ELISA
reader. The percentage inhibitory activity of RT inhibitors was
calculated by comparison with a sample lacking an inhibitor. The
resulting color intensity is directly proportional to RT activity. The
percentage inhibitory values were calculated by the following for-
4.1.2.25. 3-Bromo-4-(2-(4-((7-(mesityloxy)-[1,2,4]triazolo[1,5-a]pyr-
imidin-5-yl)amino) piperidin-1-yl)acetamido)benzoic acid (8b).
White solid, yield: 25%. mp: >320 ꢀC. 1H NMR (300 MHz, DMSO-d6,
ppm)
d: 13.00 (brs, 1H, COOH), 10.31 (s, 1H, CONH), 8.50 (d,
J ¼ 8.40 Hz, 1H, PhH), 8.18 (s, 1H, triazole-H), 8.14 (d, J ¼ 1.80 Hz, 1H,
PhH), 7.95 (dd, J ¼ 8.70 Hz, J ¼ 1.50 Hz, 1H, PhH), 7.54 (d, J ¼ 7.20 Hz,
1H, NH), 7.09 (s, 2H, OPh-H), 5.30 (s, 1H, pyrimidine-H), 3.91e3.85
(m, 1H, piperidine-H), 3.21 (s, 2H, CH2), 2.87 (d, J ¼ 11.70 Hz, 2H,
piperidine-H), 2.45e2.42 (m, 2H, piperidine-H), 2.30 (s, 3H, CH3),
2.10 (s, 6H, CH3 ꢃ2), 1.96 (d, J ¼ 10.20 Hz, 2H, piperidine-H),
1.63e1.53 (m, 2H, piperidine-H). 13C NMR (75 MHz, DMSO-d6,
ppm)
d: 169.23, 165.63, 160.06, 157.29, 154.03, 152.76, 145.92,
139.21, 136.20, 133.26, 130.01, 129.89, 129.50, 119.36, 111.84, 79.20,
61.28, 51.86, 31.52, 20.30, 15.25. ESI-MS: m/z 608.3 (M), 630.4
(Mþ22), C28H30BrN7O4 (608.49).
mula: %Inhibition
¼
[O.D. value without inhibitors (with
4.2. In vitro anti-HIV assay
RT) ꢄ O.D.value with RT and inhibitors]/[O.D. value with RT and
inhibitors-O.D. value without RT and inhibitors]. The IC50 value
corresponded to the concentration of the tested compound
required to inhibit the incorporation of the labeled dUTP into RT by
50%.
Evaluation of the antiviral activity and cytotoxicity of the syn-
thesized compounds was performed using the MTT assay as pre-
viously described [27,28]. Stock solutions (10ꢃ final concentration)
of test compounds were added in 25 mL volumes to two series of
triplicate wells to allow simultaneous evaluation of their effects on
mock- and HIV-infected cells at the beginning of each experiment.
Serial five-fold dilutions of test compounds were made directly in
4.4. Molecular simulations
The molecules (7d and the lead compound LW-7c) for docking
were optimized for 2000-generations until the maximum deriva-
tive of energy became 0.005 kcal/(mol*Å), using the Tripos force
field. Charges were computed and added according to Gastei-
gereHuckel parameters. The published 3D crystal structures of wt
RT complexes (PDB code: 3M8Q [19]) was retrieved from the Pro-
tein Data Bank [23] and was used for the docking studies by means
of surflex-dock module of SYBYL-X 1.1 [24]. The protein was pre-
pared by using the Biopolymer application accompanying SYBYL
according to SYBYL-X 1.1 manual: The bound ligand was extracted
from the complexes, water molecules were removed, hydrogen
atoms were automatically added (after which the resulting struc-
tures haven't been optimized), and charges and atom types were
assigned according to AMBER99. After the protomol was generated,
flat-bottomed 96-well microtiter trays by adding 100
the 25 L stock solution and transferring 25- L of this solution to
another well that contained 100 L medium using a Biomek 3000
mL medium to
m
m
m
robot (Beckman instruments, Fullerton, CA). Untreated control HIV-
and mock-infected cell samples were included for each sample.
HIV-1 wt strain (IIIB), HIV-1 double mutant strain (RES056) or HIV-
2 strain (ROD) stock (50 mL) at 100e300 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 its cytotoxicity. Exponentially growing MT-4
cells were centrifuged for 5 min at 1000 rpm and the supernatant
was discarded. The MT-4 cells were resuspended at 6 ꢃ 105 cells/

764
B. Huang et al. / European Journal of Medicinal Chemistry 92 (2015) 754e765
which referred to a computational representation of the intended
binding site to which putative ligands are aligned, the optimized
compounds 7d and LW-7c were docked into NNIBP, with the
relevant parameters set as defaults. The original ligand of the co-
ordinates (3M8Q) was used as reference molecule to calculate the
RMSD values. The docking scores related to binding affinities were
calculated based on hydrophobic, polar, and repulsive interactions
as well as entropic effects and solvation. Top-scoring poses of
compounds were shown by the software of PyMOL version 1.5
(http://www.pymol.org/). The secondary structure of RT was
shown in cartoons, and only the key residues for interactions with
the inhibitors were labeled and shown in sticks. The potential
hydrogen bonds were presented by dashed lines.
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Conflict of interest
The authors declare no conflict of interest.
Acknowledgment
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The financial support from the Key Project of NSFC for Inter-
national Cooperation (No. 81420108027, 30910103908), National
Natural Science Foundation of China (NSFC No. 81102320,
81273354), Research Fund for the Doctoral Program of Higher Ed-
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of Shandong Province (ZR2009CM016) and KU Leuven (GOA 10/
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