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

Article abstract of DOI:10.1016/j.bmc.2014.02.029

In our continuous efforts to identify novel potent HIV-1 NNRTIs, a novel class of 5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives were rationally designed, synthesized and evaluated for their anti-HIV activities in MT4 cell cultures. Biological results showed that most of the tested compounds displayed excellent activity against wild-type HIV-1 with a wide range of EC₅₀ values from 5.98 to 0.07 μM. Among the active compounds, 5a was found to be the most promising analogue with an EC₅₀ of 0.07 μM against wild-type HIV-1 and very high selectivity index (SI, 3999). Compound 5a was more effective than the reference drugs nevirapine (by 2-fold) and delavirdine (by 2-fold). In order to further confirm their binding target, an HIV-1 RT inhibitory assay was also performed. Furthermore, SAR analysis among the newly synthesized compounds was discussed and the binding mode of the active compound 5a was rationalized by molecular modeling studies.

Full text of DOI:10.1016/j.bmc.2014.02.029

Bioorganic & Medicinal Chemistry 22 (2014) 2052–2059
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
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
c
Ye Tian a, Deping Du a, Diwakar Rai a, Liu Wang a, Huiqing Liu b, Peng Zhan a, , Erik De Clercq ,
⇑
Christophe Pannecouque c, Xinyong Liu a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Educational Ministry of China), School of Pharmaceutical Sciences, Shandong University, No. 44
Wenhuaxi Road, Jinan 250012, China
b Institute of Pharmacology, School of Medicine, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, 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:
In our continuous efforts to identify novel potent HIV-1 NNRTIs, a novel class of 5,7-disubstituted pyraz-
olo[1,5-a]pyrimidine derivatives were rationally designed, synthesized and evaluated for their anti-HIV
activities in MT4 cell cultures. Biological results showed that most of the tested compounds displayed
Received 15 December 2013
Revised 18 February 2014
Accepted 22 February 2014
Available online 1 March 2014
excellent activity against wild-type HIV-1 with a wide range of EC50 values from 5.98 to 0.07
l
M. Among
the active compounds, 5a was found to be the most promising analogue with an EC50 of 0.07
lM against
wild-type HIV-1 and very high selectivity index (SI, 3999). Compound 5a was more effective than the ref-
erence drugs nevirapine (by 2-fold) and delavirdine (by 2-fold). In order to further confirm their binding
target, an HIV-1 RT inhibitory assay was also performed. Furthermore, SAR analysis among the newly
synthesized compounds was discussed and the binding mode of the active compound 5a was rationalized
by molecular modeling studies.
Keywords:
HIV-1
RT
NNRTIs
Heterocycle
Pyrazolo[1,5-a]pyrimidine
Synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
Activity assay
Molecular modeling
1. Introduction
second-generation NNRTIs, DAPYs demonstrate extremely excel-
lent potency against both the wild-type (WT) virus and mutant
Up to now, five HIV-1 non-nucleoside reverse transcriptase
inhibitors (NNRTIs) have already been launched on the market
and are widely used for the treatment of AIDS as one important
component of highly active antiretroviral therapy (HAART). How-
ever, the usage of existing drugs, especially the first generation
NNRTIs (nevirapine, delavirdine and efavirenz), has been compro-
mised by the emergence of drug-resistant viral strains which have
common mutants in the binding pocket of RT.1 This has driven
medicinal chemists to further develop novel NNRTIs with high
potency and improved anti-resistance profiles.
strains.2–5 So far, two drugs belonging to this series TMC125 (etra-
virine, ETV) and TMC278 (rilpivirine), have been approved by FDA6
(Fig. 1). Over the past few years, considerable efforts have been
devoted to the structural modification of DAPYs, leading to the dis-
covery of highly potent drug candidates. Recently, pyrrolopyrimi-
dine derivatives RDEA427 and RDEA640 have been disclosed7–9
to possess excellent HIV antiviral activity against wild-type (with
EC50 values of 0.9 and 0.8 nM, respectively) and a broad panel of
NNRTI-resistant mutant viruses (e.g., the fold-changes in EC50
against K103N-Y181C mutant strains was 11 and 3.8, respectively)
with low potential for human cytochrome P450 (CYP) induction
and large selectivity index (SI >50,000). It is worth noting that
RDEA427 has better metabolic stability and lower covalent binding
potential than RDEA640 or TMC278 and is undergoing preliminary
clinical evaluation (Fig. 1).
Diarylpyrimidine (DAPY) analogues was proved to be one of the
most important and promising class of HIV-1 NNRTIs. As the
⇑
Corresponding authors. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail addresses: zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn
(X. Liu).
http://dx.doi.org/10.1016/j.bmc.2014.02.029
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Tian et al. / Bioorg. Med. Chem. 22 (2014) 2052–2059
2053
N
2. Results and discussion
2.1. Chemistry
CN
O
CN
NH
N
The synthetic route of target compounds pyrazolo[1,5-a]pyrim-
idine derivatives is depicted in Scheme 1.
N
For the preparation of the designed series, commercially avail-
able 1H-pyrazol-5-amine (1) was used as the starting material that
reacted with diethyl malonate to form the pyrazolo[1,5-a]pyrimi-
dine system (2). Chlorination of 2 with POCl3 afforded the key
intermediate 5,7-dichloropyrazolo[1,5-a]pyrimidine (3) by the re-
ported method.14 Then different phenols were connected to the
relatively active 7-position15,16 of the fused pyrazolo[1,5-a]pyrim-
idine ring by a nucleophilic aromatic substitution reaction under
mild reaction condition, providing intermediates 4–9. At last, in
the presence of Pd(OAc)2 and Xantphos catalytic system
(Buchwald–Hartwig reaction), 4–9 were reacted with various
anilines and yielded 18 title compounds (4a–g, 5a–g, 6a, 7a, 8a,
9a). The synthesized compounds were characterized by physico-
chemical and spectral means and the IR, MS, 1H NMR, 13C NMR
spectral data were found in agreement with the assigned
molecular structures.
HN
N
NH
N
Br
N
NH2
Etravirine (2008)
Rilpivirine (2011)
CN
O
CN
NH
CN
O
CN
NH
N
N
N
N
HN
RDEA640
NH
RDEA427 (preclinical)
Figure 1. Representative analogues of the DAPY series.
2.2. Anti-HIV-1 (IIIB) activity
As part of our research interest on these DAPY-like NNRTIs,10–13
encouraged by the promising results of pyrrolopyrimidine deriva-
tives, we conducted molecular modifications by core refinements
and substituents’ optimization for obtaining improved anti-HIV
activity and pharmacokinetic profiles (Fig. 2). According to the bio-
isosterism of drug design, we employed the bridgehead nitrogen
heterocycle pyrazolo[1,5-a]pyrimidine as the central ring and
adopted various groups of two ‘wings’ of the molecule in order to
explore the steric, electrical and hydrophobic effects of the nucleus
and substituents, while keeping the overall molecular conforma-
tion of DAPYs which is required for the anti-RT potency. To achieve
our goal, novel 5,7-disubstituted pyrazolo[1,5-a]pyrimidine deriv-
atives were designed, synthesized and evaluated for their anti-HIV
activities in cell line and enzyme inhibition.
All the synthesized compounds were evaluated for their antivi-
ral activity based on their capacity to inhibit the replication of
wild-type HIV-1 (IIIB), mutated HIV-1 (RES056) and HIV-2 (ROD)
strains in MT4 cell cultures, using the MTT assay. The FDA ap-
proved drugs nevirapine (NVP) and delavirdine (DLV) were used
as controls. The reported activity of etravirine (ETV) was also
included as reference standard. The bioactivity results of 18
compounds are presented in Table 1.
It is worth noting that, except for compound 8a, all the newly
synthesized compounds demonstrated moderate to excellent
anti-HIV-1 activity with EC50 ranging from 5.98 to 0.07 lM, while
none of them were active against HIV-2. Among them, compound
5a was identified as the most active derivative against wild-type
HIV-1 (EC50 = 0.07 lM and SI of 3999). In comparison with the ref-
erence drugs, compound 5a was two-fold more potent than NVP
and DLV against HIV-1 IIIB strain, but was still inferior to the
O
N
O
Cl
Cl
CN
O
CN
NH
H
N
NH2
N
NH
N
N
N
N
(i)
(ii)
2
1
3
N
R2
N
HN
RDEA640
CN
R2
R1
R1
R1
O
Cl
O
NH
Core refinements
CN
NH
N
N
O
N
NH
N
N
(iii)
(iv)
N
N
N
N
4 - 9
4a-g, 5a-g, 6a, 7a, 8a, 9a
O
N
4
5
6
7
8
9
: R1 = 2,6-dimethyl-4-cyano
: R1 = 2,4,6-trimethyl
: R1 = 2,6-dimethyl-4-bromo
: R1 = 2,4,6-trifluoro
N
NH
: R1 = 2,6-dimethyl
RDEA427
: R1 = 3,5-dimethyl
Scheme 1. The synthetic route for pyrazolo[1,5-a]pyrimidine derivatives. Reagents
and conditions: (i) Diethyl malonate, Na, EtOH; (ii) POCl3, Dimethylaniline; (iii)
ArOH, K2CO3, DMF; (iv) ArOH, Pd(OAc)2, Xantphos, Cs2CO3, 1,4-Dioxane.
Figure 2. Structure-based biosterism design of 5,7-disubstituted pyrazolo[1,5-
a]pyrimidine derivatives as HIV-1 NNRTIs.

2054
Y. Tian et al. / Bioorg. Med. Chem. 22 (2014) 2052–2059
Table 1
Inhibitory action of compounds 4a–g, 5a–g, 6a, 7a, 8a, 9a against HIV replication and cytotoxicity in MT-4 cells
R2
R1
O
N
NH
N
N
Pyrazolo[1,5-a]pyrimidine series
a
b
Compd
R1
R2
EC50
(l
M)
HIV-2 ROD
CC50
(
lM)
SIc
HIV-1 IIIB
HIV-1 IIIB
HIV-2 ROD
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
5g
6a
7a
8a
9a
NVP
DLV
2,6-Dimethyl-4-Cyano
2,6-Dimethyl-4-Cyano
2,6-Dimethyl-4-Cyano
2,6-Dimethyl-4-Cyano
2,6-Dimethyl-4-Cyano
2,6-Dimethyl-4-Cyano
2,6-Dimethyl-4-Cyano
2,4,6-Trimethyl
2,4,6-Trimethyl
2,4,6-Trimethyl
2,4,6-Trimethyl
2,4,6-Trimethyl
2,4,6-Trimethyl
2,4,6-Trimethyl
2,6-Dimethyl-4-Bromo
2,4,6-Trifluoro
4-Cyano
4-Bromo
4-Chloro
4-Fluoro
4-Methyl
4-Methoxy
4-Nitro
4-Cyano
4-Bromo
4-Chloro
4-Fluoro
4-Methyl
4-Methoxy
4-Nitro
4-Cyano
4-Cyano
4-Cyano
4-Cyano
—
1.52 0.17
1.60 0.65
1.13 0.11
3.09 0.72
0.46 0.15
0.91 0.43
4.04 0.50
0.07 0.01
0.14 0.09
0.17 0.08
0.73 0.55
0.17 0.12
0.28 0.09
0.69 0.42
0.49 0.23
5.98 2.34
>188
>181
>8.51
>13.3
>33.6
>10.8
>280
>229
>276
>122
>139
>289
>157
>334
>321
>266
>45.4
>188
>47.7
—
181 26.74
8.51 8.08
13.3 10.8
33.6 2.16
10.8 10.7
P280
229 23.1
276 53.7
122 15.3
139 26.8
289 59.8
157 23.9
>334
120
5
12
11
23
<1
<1
<1
<1
<1
<orX1
<1
<1
<1
<1
<1
<1
X1
X1
<1
<1
<1
<1
—
P308
57
3999
854
814
397
950
>1178
>466
544
8
<1
9
>89
>277
>1537
>321
266 47.5
45.4 7.65
188 11.4
47.7 9.87
>14.99
2,6-Dimethyl
3,5-Dimethyl
—
—
5.11 2.53
0.17 0.06
0.16 0.15
0.003
—
—
—
0.011
>43.81
>4.6
—
>412
d,14
ETV
—
a
b
c
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.
SI: selectivity index (CC50/EC50), and the SI values: X1 stands for P1 or <1.
d
The data were obtained from the same lab of the Rega Institute for Medical Research, KU Leuven, Belgium.
clinically used etravirine (3 nM) (as a reference here, the data were
obtained from the same lab of the Rega Institute for Medical Re-
search, KU Leuven, Belgium).17 Besides, other three compounds
series. The data suggested that the substitution on the right ring
played a significant role in the antiviral activity profiles.
To illuminate the SAR of the left wing, the EC50 values of com-
pounds 4a, 5a, 6a, 7a, 8a and 9a were compared, which suggested
the para-substituent of phenol was pivatal for the anti-HIV-1 po-
tency. We found that compounds 5a–g with the 2,4,6-trimethyl
substituent exhibited 3 ꢁ 20-fold better potency than their corre-
sponding analogues in 4a–g, which suggested the more favorable
property of 2,4,6-trimetyl substituent than 2,6-dimethyl-4-cyano
of the left phenol of these molecule. Besides, 7a (trifluoro-) and
9a (3,5-dimethyl) have only moderate activity, and 8a totally lost
the activity with the SI <1. So, the order of other substituents based
on the potency was: 2,4,6-trimethyl (5a) > 2,6-dimethyl-4-bromo
(6a) > 2,6-dimethyl-4-cyano (4a) > 3,5-dimethyl (9a) > trifluoro
(7a).
(5b, EC50 = 0.14
showed better or comparable potency than the control drugs
(NVP, EC50 = 0.17 M; DLV, EC50 = 0.16 M). Although these pyraz-
lM; 5c, EC50 = 0.17 lM; 5e, EC50 = 0.17 lM) also
l
l
olo[1,5-a]pyrimidines did not provide the improved activity in
comparison with ETV, they represent a novel class of antiviral
agents with great potential for further investigation. Preliminary
structure–activity relationship (SAR) derived from these results
was analyzed as follows.
First of all, the nature of the 4-substitutions of anilines connect-
ing to the right wing was investigated by introducing a series of
different groups in this site while keeping other positions un-
changed. In the 4a–g subseries with a 2,6-dimethyl-4-cyano sub-
stituent in the left wing, compounds 4a (–CN) and 4b (–Br)
showed similar potency, while the presence of chloride atom
slightly increased the activity (4c). The introduction of a methoxyl
Unfortunately, the bioactivity results also demonstrated that all
the title compounds were inactive against mutated HIV-1 strain
RES056 bearing both the K103N and Y181C mutations.
group afforded 4f with further improved EC50 value of 0.91
and the 4e bearing 4-methyl substituent displayed the best activity
(EC50 = 0.46
M) in this subseries, whereas the Fꢀ or NO2ꢀ obviously
decreased their effectiveness against HIV-1. On the whole, the ef-
fect of diverse functional groups of 5a–g on anti-HIV-1 activity is
similar to that of 4a–g, but there are also some differences. Com-
pound 5a was found to be the most promising compound with
the lowest EC50 and the highest selective index (SI = 3999) of this
l
M,
Regarding to the cytotoxicity, compounds bearing various
substituents were roughly ranked in the following sequence:
MeO < NO2 < CN ꢂ F < Me ꢂ Cl < Br (substituents on the right
aniline); 2,4,6-trimethyl < 2,6-dimethyl-4-bromo < 3,5-dimethyl ꢂ
2,6-dimethyl < 2,6-dimethyl-4-cyano (substituents on left phenol).
Among them, compound 4b exhibited the greatest toxicity with an
l
CC50 of 8.51
lM while 5f was the least toxic one with a CC50 higher
than 334 M.
l

Y. Tian et al. / Bioorg. Med. Chem. 22 (2014) 2052–2059
2055
Table 2
Activity of pyrazolo [1,5-a] pyrimidine derivative 5a against HIV-1 RT
2.4. Molecular modeling study
To understand the binding mode of these novel pyrazolo[1,5-
a]pyrimidine derivatives, a docking study was performed using
the Surflex-Dock module of Sybyl-X 1.1 software. The most active
molecule 5a was chosen as a representative to be docked into the
non-nucleoside binding site (NNBS) of HIV-1 RT (taken from the
crystal structure of RT/TMC125, PDB code: 3MEC). And the lead
compounds TMC125 and RDEA640 were also studied as references.
Surflex-Dock GeomX (SFXC) mode was applied and the obtained
scores are shown in Table 3. All three compounds have excellent
total scores. Most of these figures of the tested compound 5a and
two reference molecules are very similar, while the absolute value
of the ‘Crash’ item of 5a is significantly larger than these of the oth-
ers. This indicated that an inappropriate targeting of 5a with the
protein might explain why 5a demonstrated relatively lower
anti-HIV-1 activity. Additionally, the binding mode analysis sug-
gested that compound 5a binds to HIV-1 RT in an U-shape and
shares a close similar conformation with TMC125 and RDEA640
(Fig. 3a). Their resulting structures can overlap very well including
the original ligand contained in the crystal. According to the mode
of action (Fig. 3b), the N atom at 4 position of the fused ring and the
NH group linked to the 5-position of the core could make hydrogen
bonds with Lys101. Further, the mesityloxy of 5a points to the aro-
matic-rich binding pocket surrounded by the aromatic Tyr181,
Compound
5a
NVP
0.55
ETV
a
IC50
2.26
lM
l
M
0.083 lM
a
IC50: inhibitory concentration required to inhibit biotin deoxyuridine triphos-
phate (biotin-dUTP) incorporation into poly [A] ꢃ oligo [dT]15 by the HIV-1 RT by
50% (RT kit, Roche.
2.3. HIV-RT inhibitory activity evaluation
To further characterize the mechanism of action of the newly
designed compounds, the most active 5a as a representative com-
pound was chosen to evaluate the HIV-RT inhibitory activity using
the HIV-RT kit assay, together with the marketed drugs of NVP and
ETV as the controls.18 As listed in Table 2, the data revealed that
compound 5a exhibited its inhibitory activity with an IC50 value
of 2.25
l
M, which is about 5 times and 28 times lower than that
M) and ETV (IC50 = 0.08 M),
of reference drug NVP (IC50 = 0.55
l
l
respectively. The results seemed to indicate that this type of 5,7-
disubstitutedpyrazolo[1,5-a]pyrimidine derivatives acted as HIV-
1 NNRTIs. However, the inhibitory activity against HIV-RT did
not correspond to the anti-HIV efficiency in the MTT cell assay,
which is probably due to the physicochemical properties (e.g.,
membrane permeability) or target promiscuity (potential to bind
to other targets) of the tested compounds demonstrating different
impact on the cellular and enzymatic assays.
Tyr188, Phe227 and Trp229 through
p–p interactions, while the
4-cyanoaniline ring parallels the Tyr318 position. However, the
Table 3
Docking scoresa of TMC125, RDEA640 and 5a by Surflex-Dock
Compd
T_S
C
P
R
S
D_S
P_S
G_S
CHS
CS
G_C
TMC125
RDEA640
5a
10.15
10.11
8.99
ꢀ0.84
ꢀ1.19
ꢀ2.22
1.64
1.39
1.30
0.47
1e+006
1e+006
0.96
0.80
0.83
ꢀ175.69
ꢀ175.64
ꢀ173.76
ꢀ103.11
ꢀ101.15
ꢀ82.34
ꢀ321.527
ꢀ326.61
ꢀ323.61
ꢀ36.02
ꢀ37.62
ꢀ39.38
5
5
5
5
5
5
a
T_S = Total_ score; C = Crash; P = Polar; R = RMSD_ISO; S = Similarity; D_S = D_SCORE; P_S = PMF_SCORE; G_S = G_SCORE; CHS = CHEMSCORE, CS = CSCORE,
G_S = GLOBAL_CSCOR.
Figure 3. Docking of 5a is shown by Pymol (version: 0.99rc6): (a) Molecular superposition of 5a (carbon atoms in yellow) with TMC125 (original ligand extracted from 3MEC,
carbon atoms in gray; newly docked molecule as the reference, carbon atoms in pink) and RDEA640 (carbon atoms in salmon) in the binding site; (b) Molecular model of 5a in
the RT non-nucleoside binding site (PDB code: 3MEC).

2056
Y. Tian et al. / Bioorg. Med. Chem. 22 (2014) 2052–2059
Table 4
Physicochemical propertiesa of 5a, ETV and NVP
Compd
EC50
(
l
M)
nViol
natoms
miLogP
MW
nON
<10
nOHNH
<5
nrotb
TPSA
MV
0
Acceptable range
(<5)
(<500 Da)
(<140 ÅA)
75.25
120.65
63.58
5a
ETV
NVP
0.07
1
1
0
28
28
20
5.741
5.027
1.385
369.43
435.28
266.30
6
7
5
1
3
1
4
4
1
335.87
335.95
236.62
0.003b,14
0.17
a
nViol = number of violations; natoms = no. of atoms; miLogP = molinspiration predicted LogP; MW = molecular weight; nON = no. of hydrogen bond acceptors;
nOHNH = no. of hydrogen bond donors; nrotb = no. of rotatable bonds; TPSA = topological polar surface area; MV = molar volume.
b
The data were obtained from the Rega Institute for Medical Research, KU Leuven, Belgium).
lack of one polar interaction between the pyrazole part of 5a and
Glu138 may be another reason for the inferior potency of 5a. The
computational modeling study has helped in understanding the
interactions between these inhibitors and the HIV-1 RT.
13C NMR spectra were recorded on a Brucker Avance-100 NMR-
spectrometer and are reported accordingly. TLC was performed
on Silica Gel GF254 for TLC and spots were visualized by irradiation
with UV light (254 nm). Flash column chromatography was per-
formed on a column packed with Silica Gel 60 (200–300 mesh).
Solvents were reagent grade and, when necessary, were purified
and dried by standard methods. Concentration of the reaction solu-
tion involved the use of rotary evaporator at reduced pressure.
2.5. Physicochemical properties study
Furthermore, the physicochemical properties (logP, molecular
weight, topological polar surface area, molecular volume etc.) of
5a (as representative structures), ETV and NVP (as control) were
determined using free on-line molinspiration software (http://
www.molinspiration.com/) for their compliance to the Lipinski’s
rule of five.19 The physicochemical results (Table 4) suggested that
compounds 5a, nevirapine and TMC125 agreed well with the
Lipinski’s rule of five. Additionally, pyrazolo[1,5-a]pyrimidine
derivatives and ETV share very similar properties. Their physico-
chemical properties would not be mainly responsible for their
moderate activity against HIV-1.
4.2. General synthetic procedure for compounds 5-chloro-7-
subtitutedphenoxypyrazolo[1,5-a]pyrimidines (4–9)
3-Aminopyrazole (1) (20 mmol, 1 equiv) and diethyl malonate
(22 mmol, 1.1 equiv) were added to a freshly made solution of so-
dium metal (40 mmol, 2 equiv) in ethanol (60 mL). The mixture
was heated to 80 °C and stirred for 5 h. After the reaction, the solu-
tion was cooled to room temperature and filtered. The obtained so-
lid was washed with ethanol and dissolved in water (30 mL). Then
the solution was acidified to pH 1 ꢁ 2 using concentrated HCl in an
ice-bath. The precipitate was filtered, washed with water and dried
to give light yellow powder (2, yield 73.2%, EI-MS: 152.2 [M+H],
174.3 [M+Na]) that was directly used in next step.
Pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (2) (20 mmol,
1 equiv) was added to the mixture of POCl3 (440 mmol, 22 equiv)
and dimethylaniline (60 mmol, 3 equiv) in an ice-bath under nitro-
gen protection. The solution was stirred overnight at 80 °C and
then cooled and concentrated. The obtained residue was dissolved
in methylene chloride (50 mL) and poured into ice-water. After
being stirred for 10 min, it was adjusted to pH 8 ꢁ 9 using solid
NaHCO3. The organic layer was then separated, washed (water),
dried (MgSO4), filtered and concentrated. The crude product was
purified by flash chromatographic column to afford 5,7-dichloro-
pyrazolo[1,5-a]pyrimidine (3) with a yield of 61.2% (EI-MS: 188.3
[M+H], 190.4 [M+H]).
5,7-Dichloropyrazolo[1,5-a]pyrimidine (3, 2.5 mmol, 1 equiv)
and appropriate substituted phenol (2.5 mmol, 1 equiv) and potas-
sium carbonate (5 mmol, 2 equiv) were added to DMF, and then
stirred for 12 h at room temperature. After the reaction was ended,
water was added to the mixture to give more precipitate. The crude
product 5-chloro-7-subtitutedphenoxypyrazolo[1,5-a]pyrimidine
(4–9) was obtained by filtration and desiccation with good yield
(4: yield 98.1%, EI-MS: 299.4 [M+H], 301.3 [M+H]; 5: yield 99.5%,
EI-MS: 288.2 [M+H], 290.3 [M+H]; 6: yield 96.9%, EI-MS: 352.2
[M+H], 354.1 [M+H]; 7: yield 93.6%, EI-MS: 300.3 [M+H], 302.3
[M+H]; 8: yield 89.6%, EI-MS: 274.3 [M+H], 276.4 [M+H]; 9: yield
96.5%, EI-MS: 274.3 [M+H], 276.3 [M+H]).
3. Conclusion
In brief, based on the bioisosterism and core refinements strate-
gies of drug design, with convenient and efficient synthesis,
biological evaluation, a novel series of 5,7-disubstituted pyrazol-
o[1,5-a]pyrimidine derivatives was identified as potent HIV-1
inhibitors. Most of these compounds exhibited good activity against
HIV-1 with EC50 values ranging from 70 nM to micromolar concen-
trations. Among them, compound 5a was identified as the most
promising candidate in inhibiting HIV-1 replication with EC50 value
of 0.070
lM and the SI of 3999, which were much better than those
of NVP (EC50 = 0.17
l
M) and DLV (EC50 = 0.16 M). Although
l
compound 5a was less active than ETV, these promising results
demonstrated that the isosteric replacement of pyrazolo[1,5-
a]pyrimidine for pyrimidine in the central ring of DAPY compounds
was gratifying and afforded a series of potent bridgehead nitrogen
heterocycle-derived NNRTIs. Further, preliminary SAR information
has been summarized. The results of in vitro HIV-RT kit assay and
docking study for compound 5a indicates its possible mechanisms
of action. These valuable information and readily accessible
synthetic methods greatly encourage us to further optimize the
promising structures, which will be reported in due course.
4. Materials and methods
4.1. Chemistry
All melting points (mp) were determined on a micromelting
point apparatus and are uncorrected. Mass spectra were taken on
a LC Autosampler Device: Standard G1313A instrument by electro-
spray ionization. 1H NMR spectra were obtained on a Brucker
Avance-400 NMR-spectrometer in the indicated solvents. Chemical
shifts are reported in d units and TMS as an internal reference. The
4.3. General synthetic procedure for target compounds (4a–g,
5a–g, 6a, 7a, 8a, 9a)
Pd(OAc)2 (0.025 mmol, 0.05 equiv) and Xantphos (0.05 mmol,
0.1 equiv) were added to 1,4-dioxane (3 mL), after stirring for

Y. Tian et al. / Bioorg. Med. Chem. 22 (2014) 2052–2059
2057
15 min at room temperature, 5-chloro-7-subtitutedphenoxypyraz-
4.3.6. 4-(5-(4-Methoxyphenylamino)pyrazolo[1,5-a]pyrimidin-
olo[1,5-a]pyrimidine (0.5 mmol, 1 equiv) (4–9), 4-substitued ani-
line (0.6 mmol, 1.2 equiv) and Cs2CO3 (0.75 mmol, 1.5 equiv),
were added to the mixture. The reaction mixture was allowed to
stir overnight at 100 °C under N2 protection, and then the solution
was cooled and concentrated. Further, water (20 mL) and CH2Cl2
(20 mL) were added to the residue for extraction, the obtained
organic phase was dried (MgSO4), filtered and concentrated under
reduced pressure. Flash chromatography and recrystallization
using proper solvent finally give the pyrazolo[1,5-a]pyrimidine
derivatives (4a–g, 5a–g, 6a, 7a, 8a, 9a).
7-yloxy)-3,5-dimethylbenzonitrile (4f)
White powder, yield: 19.3%. Mp: >300 °C; 1H NMR (DMSO-d6)
dppm: 2.22 (s, 6H, CH3), 3.72 (s, 3H, CH3), 5.36 (s, 1H, ACH@),
6.22 (d, 1H, J = 2.1 Hz, ACH@), 6.87–7.62 (m, 4H, benzene), 7.89
(s, 2H, benzene), 7.98 (d, 1H, J = 2.1 Hz, ACH@), 9.14 (s, 1H, NH);
13C NMR (DMSO-d6) dppm: 15.57 (ACH3 ꢃ2), 55.65 (ACH3),
118.78 (ACN), Ar-C (ꢃ18): 79.71, 93.36, 110.32, 114.40 (ꢃ2),
120.86 (ꢃ2), 133.02 (ꢃ2), 133.98 (ꢃ3), 145.19 150.30, 152.26
(ꢃ2), 154.80 (ꢃ2); EI-MS: 386.4 [M+H], 408.4 [M+Na].
4.3.7. 4-(5-(4-Nitrophenylamino)pyrazolo[1,5-a]pyrimidin-7-
yloxy)-3,5-dimethylbenzonitrile (4g)
4.3.1. 4-(5-(4-Cyanophenylamino)pyrazolo[1,5-a]pyrimidin-7-
yloxy)-3,5-dimethylbenzonitrile (4a)
Greenyellow crystal, yield: 37.3%. Mp: >300 °C; 1H NMR
(DMSO-d6) dppm: 2.23 (s, 6H, CH3), 5.53 (s, 1H, ACH@), 6.45 (d,
1H, J = 2.1 Hz, ACH@), 7.91 (s, 2H, benzene), 7.96–8.24 (m, 4H, ben-
zene), 8.13 (d, 1H, J = 2.1 Hz, ACH@), 10.02 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 15.56 (ACH3 ꢃ2), 118.58 (ACN), Ar-C (ꢃ18):
80.55, 94.75, 110.51, 118.21 (ꢃ2), 125.64 (ꢃ2), 133.03 (ꢃ2),
134.07 (ꢃ2), 141.17, 145.77, 147.08, 149.52, 152.08, 152.74,
154.00; EI-MS: 401.4 [M+H], 423.4 [M+Na].
White crystal, yield: 22.6%. Mp: >300 °C; 1H NMR (DMSO-d6)
dppm: 2.22 (s, 6H, CH3), 5.49 (s, 1H, ACH@), 6.42 (d, 1H,
J = 2.1 Hz, ACH@), 7.74–7.94 (m, 4H, benzene), 7.91 (s, 2H, ben-
zene), 8.10 (d, 1H, J = 2.1 Hz, ACH@), 9.81 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 15.56 (ACH3 ꢃ2), 118.60 (ACN), 119.90 (ACN),
Ar-C (ꢃ18): 80.37, 94.54, 103.40, 110.47, 118.74, 133.03 (ꢃ2),
133.71 (ꢃ2), 134.07 (ꢃ2), 145.67, 145.67, 149.62, 152.08, 152.64,
154.19; EI-MS: 381.4 [M+H], 403.4 [M+Na].
4.3.8. 4-(7-(Mesityloxy)pyrazolo[1,5-a]pyrimidin-5-
ylamino)benzonitrile (5a)
4.3.2. 4-(5-(4-Bromophenylamino)pyrazolo[1,5-a]pyrimidin-7-
yloxy)-3,5-dimethylbenzonitrile (4b)
Cream white crystal, yield: 28.1%. Mp: >300 °C; 1H NMR
(DMSO-d6) dppm: 2.12(s, 6H, CH3), 2.32(s, 3H, CH3), 5.50 (s, 1H,
ACH@), 6.39 (d, 1H, J = 2.1 Hz, ACH@), 7.11 (s, 2H, benzene),
7.73–7.95 (m, 4H, benzene), 8.08 (d, 1H, J = 2.1 Hz, ACH@), 9.83
(s, 1H, NH); 13C NMR (DMSO-d6) dppm: 15.71 (ACH3 ꢃ2), 20.82
(ACH3), 119.91 (ACN), Ar-C (ꢃ18): 79.95, 94.28, 103.27, 118.74
(ꢃ2), 130.12 (ꢃ2), 130.57 (ꢃ2), 133.62 (ꢃ2), 136.71, 145.16,
145.42, 146.50, 149.61, 153.83, 154.45; EI-MS: 370.4 [M+H],
392.4 [M+Na].
White crystal, yield: 52.9%. Mp: >300 °C; 1H NMR (DMSO-d6)
dppm: 2.22 (s, 6H, CH3), 5.42 (s, 1H, ACH@), 6.32 (d, 1H,
J = 2.1 Hz, ACH@), 7.46–7.73 (m, 4H, benzene), 7. 89 (s, 2H, ben-
zene), 8.04 (d, 1H, J = 2.1 Hz, ACH@), 9.45 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 15.56 (ACH3 ꢃ2), 118.59 (ACN), Ar-C (ꢃ18):
80.07, 93.95, 110.42, 113.66, 120.49 (ꢃ2), 131.91 (ꢃ2),133.02
(ꢃ2), 134.01 (ꢃ2), 140.16, 145.42, 149.95, 152.19, 152.44,
154.50; EI-MS: 434.3 [M+H], 436.3 [M+H].
4.3.3. 4-(5-(4-Chlorophenylamino)pyrazolo[1,5-a]pyrimidin-7-
yloxy)-3,5-dimethylbenzonitrile (4c)
4.3.9. N-(4-Bromophenyl)-7-(mesityloxy)pyrazolo[1,
5-a]pyrimidin-5-amine (5b)
White crystal, yield: 63.2%. Mp: 285–287 °C; 1H NMR (DMSO-
d6) dppm: 2.22 (s, 6H, CH3), 5.42 (s, 1H, ACH@), 6.32 (d, 1H,
J = 2.1 Hz, ACH@), 7.34–7.78 (m, 4H, benzene), 7. 89 (s, 2H, ben-
zene), 8.04 (d, 1H, J = 2.1 Hz, ACH@), 9.45 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 15.56 (ACH3 ꢃ2), 118.61 (ACN), Ar-C (ꢃ18):
80.00, 93.94, 110.40, 120.46 (ꢃ2), 125.73, 129.04 (ꢃ2), 133.02
(ꢃ2), 134.03 (ꢃ2), 139.75, 145.42, 149.93, 152.16, 152.41,
154.50; EI-MS: 390.3 [M+H], 392.3 [M+H].
Light yellow crystal, yield: 49.2%. Mp: 286–287 °C; 1H NMR
(DMSO-d6) dppm: 2.12(s, 6H, CH3), 2.32(s, 3H, CH3), 5.42 (s, 1H,
ACH@), 6.29 (d, 1H, J = 2.1 Hz, ACH@), 7.10 (s, 2H, benzene),
7.44–7.74 (m, 4H, benzene), 8.01 (d, 1H, J = 2.1 Hz, ACH@), 9.47
(s, 1H, NH); 13C NMR (DMSO-d6) dppm: 15.74 (ACH3 ꢃ2), 20.84
(ACH3), Ar-C (ꢃ18): 79.56, 93.75, 113.46, 120.83 (ꢃ2), 130.12
(ꢃ2), 130.54 (ꢃ2), 131.87 (ꢃ2), 136.62, 140.33, 145.18, 146.49,
149.87, 153.54, 154.73; EI-MS: 423.3 [M+H], 425.3 [M+H].
4.3.4. 4-(5-(4-Fluorophenylamino)pyrazolo[1,5-a]pyrimidin-7-
yloxy)-3,5-dimethylbenzonitrile (4d)
4.3.10. N-(4-Chlorophenyl)-7-(mesityloxy)pyrazolo[1,
5-a]pyrimidin-5-amine (5c)
White powder, yield: 59.0%. Mp: 251–252 °C; 1H NMR (DMSO-
d6) dppm: 2.22 (s, 6H, CH3), 5.40 (s, 1H, ACH@), 6.28 (d, 1H,
J = 2.1 Hz, ACH@), 7.12–7.76 (m, 4H, benzene), 7.89 (s, 2H, ben-
zene), 8.02 (d, 1H, J = 2.1 Hz, ACH@), 9.34 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 15.57 (ACH3 ꢃ2), 118.62 (ACN), Ar-C (ꢃ18):
79.80, 93.72, 110.37, 115.61, 115.83, 120.73 (ꢃ2), 133.02 (ꢃ2),
134.02 (ꢃ2), 137.18 (ꢃ2), 145.37, 150.05, 152.19 (ꢃ2), 154.64;
EI-MS: 374.3 [M+H].
White crystal, yield: 40.6%. Mp: 274–275 °C; 1H NMR (DMSO-
d6) dppm: 2.12 (s, 6H, CH3), 2.32 (s, 3H, CH3), 5.43 (s, 1H, ACH@),
6.29 (d, 1H, J = 2.1 Hz, ACH@), 7.10 (s, 2H, benzene), 7.32–7.80
(m, 4H, benzene), 8.02 (d, 1H, J = 2.1 Hz, ACH@), 9.47 (s, 1H, NH);
13C NMR (DMSO-d6) dppm: 15.73 (ACH3 ꢃ2), 20.83 (ACH3), Ar-C
(ꢃ18): 79.54, 93.71, 120.42 (ꢃ2), 125.55, 128.98 (ꢃ2), 130.12
(ꢃ2), 131.54 (ꢃ2), 136.62, 139.93, 145.17, 146.50, 149.89, 153.55,
154.75; EI-MS: 379.4 [M+H], 381.4 [M+H].
4.3.5. 4-(5-(4-Methylphenylamino)pyrazolo[1,5-a]pyrimidin-7-
yloxy)-3,5-dimethylbenzonitrile (4e)
4.3.11. N-(4-Fluorophenyl)-7-(mesityloxy)pyrazolo[1,
5-a]pyrimidin-5-amine (5d)
Light orange crystal, yield: 31.2%. Mp: 280–282 °C; 1H NMR
(DMSO-d6) dppm: 2.22 (s, 6H, CH3), 2.24 (s, 3H, CH3), 5.41 (s, 1H,
ACH@), 6.26 (d, 1H, J = 2.1 Hz, ACH@), 7.09–7.62 (m, 6H, benzene),
7. 89 (s, 2H, benzene), 8.00 (d, 1H, J = 2.1 Hz, ACH@), 9.21 (s, 1H,
NH); 13C NMR (DMSO-d6) dppm: 15.56 (ACH3 ꢃ2), 20.87 (ACH3),
110.34 (ACN), Ar-C (ꢃ18): 93.55, 118.61, 119.22 (ꢃ2), 129.56
(ꢃ3), 131.24, 133.02 (ꢃ2), 133.99 (ꢃ2), 138.25, 145.25, 150.22,
152.25 (ꢃ2), 154.77; EI-MS: 368.3 [MꢀH].
White crystal, yield: 32.4%. Mp: 252–253 °C; 1H NMR (DMSO-
d6) dppm: 2.13 (s, 6H, CH3), 2.32 (s, 3H, CH3), 5.40 (s, 1H, ACH@),
6.25 (d, 1H, J = 2.1 Hz, ACH@), 7.10–7.77 (m, 6H, benzene), 8.00
(d, 1H, J = 2.1 Hz, ACH@), 9.37 (s, 1H, NH); 13C NMR (DMSO-d6)
dppm: 15.74 (ACH3 ꢃ2), 20.83 (ACH3), Ar-C (ꢃ18): 79.33, 93.49,
115.54, 115.76, 120.59, 120.66, 130.12 (ꢃ2), 130.53 (ꢃ2), 136.59,
145.08, 146.51, 150.01, 153.46, 154.88, 156.44, 158.80; EI-MS:
363.4 [M+H], 385.4 [M+Na].

2058
Y. Tian et al. / Bioorg. Med. Chem. 22 (2014) 2052–2059
4.3.12. 7-(Mesityloxy)-N-p-tolylpyrazolo[1,5-a]pyrimidin-5-
amine (5e)
4.3.18. 4-(7-(3,5-Dimethylphenoxy)pyrazolo[1,5-a]pyrimidin-5-
ylamino)benzonitrile (9a)
Cream white crystal, yield: 41.3%. Mp: 251–253 °C; 1H NMR
(DMSO-d6) dppm: 2.12 (s, 6H, CH3), 2.24 (s, 3H, CH3), 2.32 (s, 3H,
CH3), 5.41 (s, 1H, ACH@), 6.23 (d, 1H, J = 2.1 Hz, ACH@), 7.09 (s,
2H, benzene), 7.07–7.62 (m, 4H, benzene), 7.97 (d, 1H, J = 2.1 Hz,
ACH@), 9.24 (s, 1H, NH); 13C NMR (DMSO-d6) dppm: 15.75
(ACH3 ꢃ2), 20.84 (ACH3), 20.88 (ACH3), Ar-C (ꢃ18): 79.42,
93.35, 119.15 (ꢃ2), 129.51 (ꢃ2), 130.13 (ꢃ2), 130.51 (ꢃ2),
131.02, 136.53, 138.43, 145.00, 146.54, 150.16, 153.35, 155.01;
EI-MS: 359.4 [M+H], 381.4 [M+Na].
Cream white crystal, yield: 24.6%. Mp: 270–272 °C; 1H NMR
(DMSO-d6) dppm: 2.17 (s, 6H, CH3), 5.50 (s, 1H, ACH@), 6.41 (d,
1H, J = 2.1 Hz, ACH@), 7.26–7.34 (m, 3H, benzene), 7.73–7.96 (m,
4H, benzene), 8.09 (d, 1H, J = 2.1 Hz, ACH@), 9.86 (s, 1H, NH); 13C
NMR (DMSO-d6) dppm: 15.77 (ACH3 ꢃ2), 119.93 (ACN), Ar-C
(ꢃ18): 80.00, 94.33, 103.26, 118.73 (ꢃ2), 127.57, 130.15 (ꢃ2),
130.58 (ꢃ2), 133.64 (ꢃ2), 145.13, 145.47, 148.63, 149.60, 153.59,
154.40; EI-MS: 356.3 [M+H], 378.4 [M+Na].
4.4. In vitro anti-HIV assay
4.3.13. 7-(Mesityloxy)-N-(4-methoxyphenyl)pyrazolo[1,
5-a]pyrimidin-5-amine (5f)
The evaluation of target compounds for their activity against
HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells was performed
using the MTT method as previously described.20 Stock solutions
(10 ꢃ final concentration) of test compounds were added in 25-
White crystal, yield: 29.2%. Mp: 213–215 °C; 1H NMR (DMSO-
d6) dppm: 2.12 (s, 6H, CH3), 2.31 (s, 3H, CH3), 3.71 (s, 3H, CH3),
5.36 (s, 1H, ACH@), 6.19 (d, 1H, J = 2.1 Hz, ACH@), 7.09 (s, 2H, ben-
zene), 6.86–7.63 (m, 4H, benzene), 7.96 (d, 1H, J = 2.1 Hz, ACH@),
9.17 (s, 1H, NH); 13C NMR (DMSO-d6) dppm: 15.75 (ACH3 ꢃ2),
20.84 (ACH3), 55.62 (ACH3), Ar-C (ꢃ18): 79.21, 93.14, 114.33
(ꢃ2), 120.76 (ꢃ2), 130.13 (ꢃ2), 130.50 (ꢃ2), 134.10, 136.51,
144.94, 146.54, 150.24, 153.30, 154.81, 155.04; EI-MS: 375.4
[M+H].
l
l 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 flat-bottomed 96-well microti-
ter trays by adding 100
and transferring 25 l of this solution to another well that con-
tained 100 l medium using a Biomek 3000 robot (Beckman
ll medium to the 25 ll stock solution
l
l
4.3.14. 7-(Mesityloxy)-N-(4-nitrophenyl)pyrazolo[1,
instruments, Fullerton, CA). Untreated control HIV- and mock-in-
5-a]pyrimidin-5-amine (5g)
fected cell samples were included for each sample.
Yellow needle crystal, yield: 33.4%. Mp: 283–284 °C; 1H NMR
(DMSO-d6) dppm: 2.13 (s, 6H, CH3), 2.33 (s, 3H, CH3), 5.54 (s, 1H,
ACH@), 6.42 (d, 1H, J = 2.1 Hz, ACH@), 7. 12 (s, 2H, benzene),
8.10 (d, 1H, J = 2.1 Hz, ACH@), 7.97–8.22 (m, 4H, benzene), 9.37
(s, 1H, NH); 13C NMR (DMSO-d6) dppm: 15.71 (ACH3 ꢃ2), 20.83
(ACH3), Ar-C (ꢃ18): 80.12, 94.50, 118.18 (ꢃ2), 125.57 (ꢃ2),
130.12 (ꢃ2), 130.58 (ꢃ2), 136.75, 141.04, 145.53, 146.49, 147.28,
149.50, 153.93, 154.24; EI-MS: 390.3 [M+H], 412.4 [M+Na].
HIV-1 (IIIB)21 or HIV-2 (ROD)22 stock (50
ll) at 100–300 CCID50
(50% cell culture infectious dose-50%) or culture medium was
added to either the infected or mock-infected wells of the microti-
ter plate. Mock-infected cells were used to evaluate the effects of
test compounds on uninfected cells in order to assess the cytotox-
icity of the test compounds. 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/mL,
and 50-ll volumes were transferred to the microtiter tray wells.
4.3.15. 4-(7-(4-Bromo-2,6-dimethylphenoxy)pyrazolo[1,
5-a]pyrimidin-5-ylamino)benzonitrile (6a)
Five days after infection, the viability of mock- and HIV-infected
cells was examined spectrophotometrically by the MTT assay.
The 50% cytotoxic concentration (CC50) was defined as the concen-
tration 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).
White crystal, yield: 43.9%. Mp: >300 °C; 1H NMR (DMSO-d6)
dppm: 2.17(s, 6H, CH3), 5.52 (s, 1H, ACH@), 6.41 (d, 1H,
J = 2.1 Hz, ACH@), 7. 58 (s, 2H, benzene), 7.74–7.95 (m, 4H, ben-
zene), 8.09 (d, 1H, J = 2.1 Hz, ACH@), 9.81 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 15.55 (ACH3 ꢃ2), 119.91 (ACN), Ar-C (ꢃ18):
80.13, 94.42, 103.33, 118.71 (ꢃ2), 119.87, 132.55 (ꢃ2), 133.47
(ꢃ2), 133.68 (ꢃ2), 145.05, 145.55, 147.94, 149.62, 153.19,
154.32; EI-MS: 434.4 [M+H], 436.4 [M+H].
4.5. HIV-RT inhibition assay
Inhibition of HIV-1 RT was performed by using homopolymer
template/primer linked to a microplate, biotin-dUTP and RT with
detection by ELISA for quantifying expression. The incorporated
quantities of the biotin-dUTP into the template represented the
activity of HIV-1 RT. IC50 values corresponded to the concentration
of target compound required to inhibit biotin-dUTP incorporation
by 50%. The procedure for assaying RT inhibition was performed
as described in the kit (Roche) protocol.18 A total of 4 ng recombi-
nant HIV-1-RT was added per well. The tested compound 5a and
two control drugs NVP, ETV was used at different concentration
4.3.16. 4-(7-(2,4,6-Trifluorophenoxy)pyrazolo[1,5-a]pyrimidin-
5-ylamino)benzonitrile (7a)
Pink crystal, yield: 43.5%. Mp: 247–250 °C; 1H NMR (DMSO-d6)
dppm: 5.88 (s, 1H, ACH@), 6.45 (d, 1H, J = 2.1 Hz, ACH@), 7.73 (t,
2H, benzene, J = 8.7 Hz), 7.77–7.96 (m, 4H, benzene), 8.13 (d, 1H,
J = 2.1 Hz, ACH@), 9.90 (s, 1H, NH); 13C NMR (DMSO-d6) dppm:
119.81 (ACN), Ar-C (ꢃ18): 80.64, 94.64, 103.04, 103.28, 103.55,
103.74, 118.95 (ꢃ3), 133.70 (ꢃ3), 144.77, 145.95, 149.68, 153.43,
153.98; EI-MS: 382.4 [M+H], 404.4 [M+Na].
gradient (25, 5, 1, 0.2, 0.04
lg/mL for 5a; 10, 2, 0.4, 0.08,
4.3.17. 4-(7-(2,6-Dimethylphenoxy)pyrazolo[1,5-a]pyrimidin-5-
ylamino)benzonitrile (8a)
0.016 g/mL for NVP; 1, 0.2, 0.04, 8.0, 1.6 ng/mL for ETV). The per-
l
centage inhibition was calculated by formula as given below: inhi-
bition rate = [OD (with RT only)ꢀOD (with RT and inhibitor)]/[OD
(with RT only)ꢀOD (without RT and inhibitor)].
White crystal, yield: 15.7%. Mp: 270–273 °C; 1H NMR (DMSO-
d6) dppm: 2.35(s, 6H, CH3), 5.64 (s, 1H, ACH@), 6.38 (d, 1H,
J = 2.1 Hz, ACH@), 7. 09 (s, 3H, benzene), 7.73–7.96 (m, 4H, ben-
zene), 8.05 (d, 1H, J = 2.1 Hz, ACH@), 9.87 (s, 1H, NH); 13C NMR
(DMSO-d6) dppm: 21.26 (ACH3 ꢃ2), 119.91 (ACN), Ar-C (ꢃ18):
81.54, 94.24, 103.29, 118.78 (ꢃ2), 119.04 (ꢃ2), 128.95, 133.62
(ꢃ2), 140.81 (ꢃ2), 145.18 (ꢃ2), 149.57, 152.20, 154.26, 155.64;
EI-MS: 356.4 [M+H], 378.4 [M+Na].
Acknowledgments
The financial support from the National Natural Science Foun-
dation of China (NSFC Nos. 81273354, 81102320, 30873133,
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Supplementary data associated with this article can be found, in
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These data include MOL files and InChiKeys of the most important
compounds described in this article.
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