Synthesis and Anti-HIV Activity of Aryl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hydrazones as Potent Non-nucleoside Reverse Transcriptase Inhibitors

Article abstract of DOI:10.1002/cmdc.201100334

A series of novel diarylpyrimidines (DAPYs) with a ketone hydrazone substituent on the methylene linker between the pyrimidine nucleus and the aryl moiety at the C-4 position were synthesized, and their antiviral activity against human immunodeficiency virus (HIV)-1 in MT-4 cells was evaluated. Most compounds of this class exhibited excellent activity against wild-type HIV-1, with EC₅₀ values in the range of 1.7-13.2nM. Of these compounds, 2-bromophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hydrazone (9k) displayed the most potent anti-HIV-1 activity (EC₅₀=1.7±0.6nM), with excellent selectivity for infected over uninfected cells (SI=5762). In addition, the 4-methyl phenyl analogue 9d (EC₅₀=2.4±0.2nM, SI=18461) showed broad spectrum HIV inhibitory activity, with EC₅₀ values of 2.4±0.2nM against wild-type HIV-1, 5.3±0.4μM against HIV-1 double-mutated strain RES056 (K103N+Y181C), and 5.5μM against HIV-2 ROD strain. Furthermore, structure-activity relationship (SAR) data and molecular modeling results for these compounds are also discussed. Antiviral agents: A series of new diarylpyrimidine (DAPY) derivatives with a ketone hydrazine substituent on the CH₂ linker between the pyrimidine nucleus and the phenyl ring were synthesized, and their anti-HIV activity in MT-4 cells was evaluated. Most compounds of this class exhibited excellent activity against wild-type HIV-1, with EC₅₀ values in the range of 1.7-13.2nM.

Full text of DOI:10.1002/cmdc.201100334

DOI: 10.1002/cmdc.201100334
Synthesis and Anti-HIV Activity of Aryl-2-[(4-
cyanophenyl)amino]-4-pyrimidinone hydrazones as Potent
Non-nucleoside Reverse Transcriptase Inhibitors
Xiao-Dong Ma,[a] Shi-Qiong Yang,[a] Shuang-Xi Gu,[a] Qiu-Qin He,[a] Fen-Er Chen,*[a, b]
Erik De Clercq,[c] Jan Balzarini,[c] and Christophe Pannecouque[c]
A series of novel diarylpyrimidines (DAPYs) with a ketone hy-
drazone substituent on the methylene linker between the pyri-
midine nucleus and the aryl moiety at the C-4 position were
synthesized, and their antiviral activity against human immu-
nodeficiency virus (HIV)-1 in MT-4 cells was evaluated. Most
compounds of this class exhibited excellent activity against
wild-type HIV-1, with EC50 values in the range of 1.7–13.2 nm.
Of these compounds, 2-bromophenyl-2-[(4-cyanophenyl)ami-
no]-4-pyrimidinone hydrazone (9k) displayed the most potent
anti-HIV-1 activity (EC50 =1.7Æ0.6 nm), with excellent selectivity
for infected over uninfected cells (SI=5762). In addition, the 4-
methyl phenyl analogue 9d (EC50 =2.4Æ0.2 nm, SI=18461)
showed broad spectrum HIV inhibitory activity, with EC50
values of 2.4Æ0.2 nm against wild-type HIV-1, 5.3Æ0.4 mm
against HIV-1 double-mutated strain RES056 (K103N+Y181C),
and 5.5 mm against HIV-2 ROD strain. Furthermore, structure–
activity relationship (SAR) data and molecular modeling results
for these compounds are also discussed.
Introduction
Diarylpyrimidine (DAPY) analogues (1) represent a class of
highly potent non-nucleoside reverse transcriptase inhibitors
(NNRTIs), endowed with micromolar to nanomolar activity
against wild-type (WT) human immunodeficiency virus (HIV)-1
and clinically relevant mutant HIV-1 strains.[1–5] To identify more
potent DAPYs as NNRTIs, we made considerable efforts to
chemically modify the methylene linker between the pyrimi-
dine nucleus and phenyl ring I of the diarylpyrimidine, which
has given a number of highly active inhibitors, such as cyano-
CH2-DAPYs (2),[6] oxo-CH2-DAPYs (3),[7] and oxi-CH2-DAPYs (4).[8]
and Tyr188.[8–11] Herein, a series of aryl 2-[(4-cyanophenyl)ami-
no]-4-pyrimidinyl ketone hydrazones were synthesized, and
their anti-HIV activity against WT HIV-1 and HIV-2, as well as
against a double-mutated HIV-1 strain (K103N+Y181C) was
evaluated. In addition, preliminary structure–activity relation-
ship (SAR) data and molecular modeling results of these new
compounds are also discussed.
Results and Discussion
Chemistry
The synthesis of the ketone hydrazones 9a–t is shown in
Scheme 1. The key intermediates, oxo-CH2-DAPYs (8a–t), were
conveniently synthesized from 4-(4-chloropyrimidin-2-yl-ami-
no)benzonitriles (6) in two steps as previously described.[12,13]
Briefly, treatment of 6 with several substituted aryl acetonitriles
in the presence of 60% sodium hydride at room temperature
for 24–72 h under a N2 atmosphere afforded cyano-CH2-DAPYs
(7a–t). Further oxidation of 7a–t by bubbling air through the
reaction solution at room temperature yielded the oxo-CH2-
DAPYs 8a–t. Final compounds 9a–t were synthesized by reac-
[a] Dr. X.-D. Ma, S.-Q. Yang, S.-X. Gu, Q.-Q. He, Prof. F.-E. Chen
Department of Chemistry, Fudan University
220 Handan Road, Shanghai 200433 (P.R. China)
E-mail: rfchen@fudan.edu.cn
Encouragingly, the oxi-CH2-DAPYs were endowed with promis-
ing anti-HIV activity with EC50 values ranging from 0.569 to
0.005 mm. Therefore, it was of interest to introduce a hydrazine
group to the methylene linker, forming hyd-CH2-DAPYs (5), to
further improve the hydrogen-bonding and p–p stacking inter-
actions between inhibitors and amino acid residues, Tyr181
[b] Prof. F.-E. Chen
Institute of Biomedical Science, Fudan University
220 Handan Road, Shanghai 200433 (P.R. China)
[c] Prof. E. De Clercq, Prof. J. Balzarini, Prof. C. Pannecouque
Rega Institute for Medical Research, Katholieke Universiteit Leuven
10 Minderbroedersstraat, 3000 Leuven (Belgium)
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F.-E. Chen et al.
MED
derivatives tested proved to be more potent against HIV and
less toxic against uninfected cells than the corresponding oxi-
CH2-DAPYs.[8]
In terms of SAR, sterically small substituents, such as methyl
(9d), chloro (9h), and bromo (9k) in the ortho or para position,
which would reach the hydrophobic region adjacent to the
side chains of Tyr181 and Tyr188 of HIV-1 reverse transcriptase
(RT), were tolerated well, and these compounds were quite
active with EC50 values of 2.4Æ0.2, 2.6Æ1.2, and 1.7Æ0.6 nm
concentrations, respectively, against HIV-1 IIIB-infected MT-4
cells. In the case of di over mono-substitution of phenyl ring I,
2,4-dichloro-substituted 9o (EC50 =3.8Æ2.2 nm) did not show
a marked improvement on the ability to inhibit WT virus com-
pared with the 2-chloro (9h) and 4-chloro (9j) analogues
(EC50 =2.6Æ1.2 and 9.1Æ2.7 nm, respectively), whereas it had
a tenfold higher potency than 3,4-substituted analogue 9p
(EC50 =38.0Æ13.9 nm). In addition, to enhance the p–p stack-
ing interactions between phenyl ring I of the DAPYs and
amino acid residues Tyr181, Tyr188, and Trp229 within the
binding pocket of RT,[12, 1316–19] 1-naphthyl- and 2-naphthyl-sub-
stituted parent compounds 9s and 9t were synthesized and
determined to have EC50 values of 13.2Æ5.6 and 8.4Æ2.1 nm,
respectively. It is also noteworthy that 2-naphthyl analogue 9t
Scheme 1. Synthesis of ketone hydrazone analogues 9a–t. Reagents and
conditions: a) Ar-CH2CN, 60% NaH, DMF, room temperature, 24–72 h, N2;
b) air, DMF, room temperature, 48–72 h; c) NH2NH2·2HCl, pyridine, EtOH,
reflux, 2 h.
tion of 8a–t with hydrazine dihydrochloride in the presence of
pyridine and ethanol in yields of 28–45%.
Biological activity
According to the MTT method,[14,15] the anti-HIV activity of
compounds 9a–t was assayed in MT-4 cells infected with WT
HIV-1 strain IIIB, HIV-1 double-mutant strain RES056 (K103N+
Y181C) and HIV-2 strain ROD.
Five US Food and Drug Adminis-
tration (FDA)-approved agents,
Table 1. Anti-HIV-1 activity and cytotoxicity of compounds 9a–t in MT-4 cells.[a]
namely, zidovudine (AZT), nevira-
pine (NEV), delavirdine (DEV),
efavirenz (EFV), and etravirine
(ETV), were also included as ref-
erence drugs. The results, ex-
pressed as CC50 (cytotoxicity)
and EC50 (anti-HIV activity)
values, and the calculated selec-
tivity index (SI) given by the
CC50/EC50 ratio, are listed in
Table 1 (HIV-1) and Table 2 (HIV-
2).
Compd
Ar
EC50[b] [nm]
HIV-1 IIIB
EC50[b] [mm]
K103N+Y181C
CC50[c] [mm]
SI[d]
9a
9b
9c
9d
9e
9 f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
AZT[e]
DEV[e]
NEV[e]
EFV[e]
ETV[e]
Ph
11.2Æ7.0
6.5Æ4.2
8.6Æ1.3
6.2Æ2.3
5.0Æ0.5
5.3Æ0.4
>7.3
7.5Æ0.9
5.7Æ0.8
>6.9
24.8Æ13.1
34.7Æ12.9
38.0Æ9.0
44.2Æ3.9
7.3Æ0.9
21.0Æ8.3
39.5Æ2.6
6.9Æ1.0
20.7Æ9.9
> 358.4
9.7Æ4.1
35.8Æ1.8
39.4Æ5.1
35.4Æ4.5
10.2Æ6.5
38.4Æ3.0
6.3Æ1.2
144.6Æ41.0
42.5Æ2.5
128.8Æ70.3
>93.5
2210
5317
3662
18461
2140
917
1011
2673
1069
>39599
5762
3725
10980
250
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-F-Ph
3-F-Ph
4-F-Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
4-Br-Ph
4-MeO-Ph
4-tBu-Ph
2,4-Cl2Ph
3,4-Cl2Ph
2,5-F2Ph
3,4-F2Ph
1-Naphthyl
2-Naphthyl
–
10.4Æ3.3
2.4Æ0.2
3.4Æ1.9
22.9Æ10.7
39.1Æ17.8
2.6Æ1.2
Most of the compounds
showed
inhibitory
activity
19.4Æ3.6
9.1Æ2.7
5.2Æ0.03
12.7Æ1.3
ꢀ9.2
against WT HIV-1 (Table 1) with
EC50 values ranging from 1.7Æ
0.6 to 13.2Æ5.6 nm, which are
superior to those of reference
drugs NEV and DEV. In particular,
compound 9k displayed the
highest potency with an EC50
value of 1.7Æ0.6 nm against
HIV-1, and an SI value of 5762. It
is worth noting that several
compounds, such as 9d (EC50 =
2.4Æ0.2 nm, SI=18461) and 9h
1.7Æ0.6
9.6Æ2.1
>35.8
3.6Æ0.8
5.1Æ0.08
>35.4
>10.2
141.4Æ45.5
3.8Æ2.2
2660
1011
637
3288
38.0Æ13.9
9.8Æ4.2
ꢀ4.3
>6.3
44.0Æ2.9
13.2Æ5.6
8.4Æ2.1
7.1Æ1.3
>42.5
8.4Æ3.4
0.0066Æ0.0006
>4.4
3211
15407
>14988
>74
>107
>765
>1537
6.2Æ2.1
–
–
–
–
59.5Æ7.6
140.3Æ58.0
8.3Æ4.3
>4.4
>15.0
>6.3
>4.6
>15.0
0.49Æ0.02
0.0256Æ0.004
3.0Æ0.2
(EC50 =2.6Æ1.2 nm,
SI=2673),
also exhibited the same inhibito-
ry activity as AZT and ETV
against WT HIV-1. Additionally,
most of the ketone hydrazone
[a] Data represent the meanÆSD of at least three separate experiments. [b] Compound concentration required
to protect MT-4 cells against viral cytopathogenicity by 50%. [c] Compound concentration that decreases the
uninfected MT-4 cell viability by 50%. [d] Selectivity index: CC50/EC50 ratio (wild type). [e] Zidovudine (AZT), ne-
virapine (NEV), delavirdine (DEV), efavirenz (EFV), and etravirine (ETV).
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Pyrimidinone hydrazones as Potent NNRTIs
(EC50 =8.4Æ2.1 nm, SI=15407) showed a slightly higher activi-
ty and sevenfold lower cytotoxicity than the corresponding
phenyl analogue 9a, making it a potential lead for further
modifications.
Although the target of DAPYs is known to be HIV-1 RT,[1–5]
compounds 9d and 9k were also evaluated for their activity
against HIV-1 RT(WT) at a molecular level, using a poly(rA)/oli-
go(dT)15 homopolymer template with the HIV antigen detec-
tion ELISA for quantifying expression of HIV-1 RT(WT) in culture
medium,[20] and NVP as a reference compound (Table 3). The
results showed that the hyd-CH2-DAPYs inhibit HIV-1 RT(WT)
with IC50 values of 0.761 and 0.585 mm for 9d and 9k, respec-
tively.
The activity of compounds 9a–t against the known NNRTI-
resistant HIV-1 double-mutated (K103N+Y181C) strain RES056
was also determined (Table 1). In this assay, the majority of the
tested analogues were still rather active against this virus
strain within the 5.0 to 10 mm concentration range. Although
less active than reference drugs AZT, EFV, and ETV, almost all of
these derivatives displayed higher potency then NEV, and com-
pounds 9c (EC50 =5.0Æ0.5 mm), 9d (EC50 =5.3Æ0.4 mm), 9i
(EC50 =5.2Æ0.03 mm), and 9m (EC50 =5.1Æ0.08 mm) were
nearly as potent as DEV against the double-mutant strain.
Furthermore, the bioactivity of the synthesized compounds
was also tested against HIV-2 (strain ROD) in MT-4 cells
(Table 2). Interestingly, most of the derivatives, such as 9b
(EC50 =5.8Æ0.02 mm), 9c (EC50 =4.9Æ0.2 mm), 9d (EC50 =
5.5 mm), and 9 f (EC50 =5.8Æ0.1 mm), exhibited moderate activi-
ty at micromolar concentrations, indicating that the ketone hy-
drazone group on the methylene linker between the pyrimi-
dine nucleus and phenyl ring I of the CH2-DAPYs did enhance
interactions of the inhibitors with their target, HIV-2 reverse
transcriptase (RT). Generally, NNRTIs only inhibit HIV-1 replica-
tion; however, encouraged by the potency of these analogues
against HIV-2 ROD, it was promising to discover a new series
of NNRTIs, which targets both HIV-1 and HIV-2 RT, using hyd-
CH2-DAPY as a lead compound.
Table 3. Anti-HIV-1 RT(WT) activity of compound 9d and 9k.
Compd
IC50 [mm][a]
9d
9k
NVP
0.761
0.585
6.947
[a] Compound concentration required to inhibit HIV-1 RT(WT) activity by
50%.
Furthermore, the capacity of analogue 9d to inhibit the
CCR5-tropic and CXCR4-tropic virus strains, which exploit CCR5
and CXCR4, respectively, for viral entry due to the prominent
roles these receptors play in the fusion process, was performed
using the classical model of detecting calcium mobilization
based on co-expression of receptor and Ga16.[21–23] As seen in
Figure 1, compound 9d, which showed broad-spectrum anti-
HIV activity, does not effectively inhibit either CCR5 or CXCR4-
tropic HIV strains at concentrations greater than 10 mm. Refer-
ence compounds maraviroc (formerly UK-427857) and T140
were tested for comparison and gave IC50 values of 8.56 and
8.78 nm, respectively.
Table 2. Anti-HIV-2 ROD activity and cytotoxicity of compounds 9a–t in
MT-4 cells.[a]
Molecular simulation
Compd
EC50 [mm][b]
CC50 [mm][c]
SI[d]
To investigate the potential binding mode of the newly synthe-
sized compounds, molecular simulations were performed by
docking potent compounds 9e, 9 f, 9g, 9k, 9m, and 9n into
the non-nucleoside binding site (NNBS) of wild-type HIV-1 RT
(PDB code: 1S6Q),[10] considering their structural diversity and
potency. Of these compounds, 9e, 9 f, and 9g have the same
substituents at different positions of phenyl ring I, whereas 9g,
9m, and 9n are substituted by different groups at the same
position. In addition, the most potent compound 9k and the
least active compound 9n were also selected for docking anal-
ysis to compare two derivatives with vastly different activities
against WT HIV. Additionally, the binding mode of compound
4 in the NNBS, which is the most active oxi-CH2-DAPY previous-
ly reported by us,[8] was conducted for comparison according
to the same protocols. As illustrated in detail in the Experimen-
tal Section, the simulations were carried out on the basis of lit-
erature protocols,[24–26] using the Base-builder and Surflex-Dock
programs interfaced with SYBYL-1.2. The default SYBYL-1.2 pa-
rameters were used, and only the key steps and important pa-
rameters were interpreted according to the reported general
protocols of the Tripos molecular modeling packages Sybyl-X.
As shown in Figure 2, both compounds 9k and 4 are pre-
dicted to display a series of well-known interactions with RT:
9a
9b
9c
9d
9e
9 f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
7.2Æ0.5
5.8Æ0.02
4.9Æ0.2
5.5
24.8Æ13.1
34.7Æ12.9
38.0Æ9.0
44.2Æ3.9
7.3Æ0.9
3.4
6.0
7.8
8.0
<1.0
6.8
ꢁ3.1
<1.0
ꢁ4.6
>57.9
<1.0
<1.0
5.9
<1.0
<1.0
<1.0
ꢁ3.0
21.3
>7.3.1
5.8Æ0.1
39.5Æ2.6
21.0Æ8.3
6.9Æ1.0
ꢀ6.7
>6.7
ꢀ4.5
20.7Æ9.8
> 358.4
6.2Æ1.3
>9.7
9.7Æ4.1
>35.8
35.8Æ1.8
39.4Æ5.1
>35.4
6.7Æ1.5
>35.4
>10.2
>38.4
ꢀ2.1
10.2Æ6.3
38.4Æ3.0
6.3Æ1.2
6.8Æ0.2
144.6Æ41.0
42.5Æ2.5
128.8Æ70.3
>93.5
9s
9t
AZT[e]
ETV[e]
>42.5
<1.0
12.1
>22341.4
>412.4
10.7Æ5.0
0.0042Æ0.0021
0.011Æ0.00078
>4.6
[a] Data represent the meanÆSD of at least three separate experiments.
[b] Compound concentration required to protect MT-4 cells against viral
cytopathogenicity by 50%. [c] Compound concentration that decreases
the uninfected MT-4 cell viability by 50%. [d] Selectivity index (SI): CC50/
EC50 ratio. [e] Zidovudine (AZT), and etravirine (ETV).
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2227

F.-E. Chen et al.
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three hydrogen bonds (two between one hydrogen atom of
the NH2 group and the carbonyl of Val179, and another be-
tween the other hydrogen atom of the NH2 group and the car-
bonyl of Tyr188), while the hydroxy group of oxi-CH2-DAPY (4)
is predicted to only form two hydrogen bonds (two between
the NH2 group and the carbonyl groups of Val179 and Tyr188).
This binding model supports our initial design hypothesis that
introducing a hydrazine group might improve the hydrogen-
bonding interactions of these inhibitors with RT.
To further explore the interactions of newly synthesized
compounds with RT, several docking conformations of hyd-
CH2-DAPYs were studied. Similarly to 9k, compound 9e had
the same interactions with RT, except for the loss of one hydro-
gen bond between a hydrogen atom of the NH2 group and
the carbonyl of Val179. Moreover, both compounds, 9 f and
9g also lost a hydrogen bond with RT (9 f lost the hydrogen
bond with Val179; 9g lost the hydrogen bond with Lys101). In
addition, the two least active compounds, 9m and 9n, were
also docked to the NNBS of HIV-1 RT. The predicted binding
modes of compounds 9m and 9n are significantly different
from that predicted for compound 9k. Not only are all of the
hydrogen bonds with RT predicted to disappear, but these
compounds are also predicted to bind out of the NNBS. The
predicted poor binding of these compounds with RT is in
agreement with the observed significantly weaker anti-HIV
activity.
Overall, all these analyses were in accordance with the bio-
logical data. Apparently, as designed, the hydrazine group on
the methylene linker of hyd-CH2-DAPY enhanced the interac-
tion with wild-type HIV-1 RT. However, molecular modeling
predicts that most of these compounds lack contact with the
key mutant amino acid residue Asn103 in the NNBS, explaining
their observed high potency against wild-type HIV-1 but weak
activity against the double-mutant strain RES056 (K103N+
Y181C).
Conclusions
In summary, led by the oxi-CH2-DAPYs, a series of new DAPY
derivatives with a ketone hydrazine substituent on the methyl-
ene linker between the pyrimidine nucleus and phenyl ring I
were synthesized and evaluated in cellular assays. Evaluation
of their anti-HIV activity indicated that most of the compounds
possess antiviral potency superior to that of DEV and NEV
against HIV-1 and HIV-2, and equal to that of AZT, EFV, and ETV
against HIV-1. In particular, compounds 9d (EC50 =2.4Æ0.2 nm,
SI=18461), 9h (EC50 =2.6Æ1.2 nm, SI=2673), and 9k (EC50 =
1.7Æ0.6 nm, SI=5762) exhibited more potent activity than
ETV against wild-type HIV-1. Furthermore, the activity of com-
pounds 9d and 9k against HIV-1 RT(WT), CCR5-tropic HIV, and
CXCR4-tropic HIV indicates that the target of hyd-CH2-DAPYs is
wild-type RT only. In addition, compared with the oxi-CH2-
DAPYs, the newly synthesized derivatives exhibit more potent
activity and lower cytotoxicity against the chosen HIV strains
(HIV-1 IIIB, RES056, and HIV-2).
Figure 1. Antagonist models of calcium mobilization assay: a) dose response
for maraviroc on CHO/CCR5/Ga16 (regulated on activation, normal T-ex-
pressed and secreted (RANTES), 10 nm); b) dose response for 9d on CHO/
CCR5/Ga16 (RANTES, 10 nm); c) dose response for T140 on CHO/CXCR4/
Ga16 (SDF-1 30 nm); d) dose response of 9d on CHO/CXCR4/Ga16 (SDF-1,
30 nm).
a) van der Waals interactions between the cyano group at the
C-4 position of phenyl ring II and amino acid residues Leu100
and Lys101; b) p–p interactions between phenyl ring I and
Tyr181; c) hydrogen-bonding interactions between the NH
group that links the pyrimidine ring and 4-cyanophenyl group
with the backbone of Lys101. As well as these interactions, the
NH2 group of hyd-CH2-DAPY 9k is predicted to form another
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Pyrimidinone hydrazones as Potent NNRTIs
Figure 2. Predicted binding modes and docking poses between the representative compounds and the non-nucleoside binding site (NNBS) of wild-type HIV-
1 RT (PDB code: 1S6Q):[10] a) compound 4; b) compound 9k; c) compound 9e; d) compound 9 f; e) compound 9g; f) compound 9m; g) compound 9n. Hy-
drogen bonds are indicated with dashed lines in red. Hydrogen atoms are omitted for the sake of clarity, except those involved in bonding interactions.
General procedure for the preparation of aryl 2-[(4-cyanophen-
yl)amino]-4-pyrimidinone hydrazones (9a–t): A stirred mixture of
Experimental Section
Chemistry
8a–t (6.0 mmol) and hydrazine dihydrochloride (3.15 g, 30 mmol)
in EtOH (30 mL) was treated with pyridine (6.50 mL, 80 mmol) and
heated to reflux for 2 h then poured into H2O (100 mL) and extract-
ed with EtOAc (3ꢁ100 mL). The organic layers were combined,
washed with 2n HCl and brine, dried over Na2SO4, filtered and
concentrated in vacuo to obtain the crude product. The residue
was purified by column chromatography on silica gel (20% EtOAc/
hexane) to give the desired product as a yellow solid.
General: All chemicals used were purchased from commercial
sources. Reagents were of analytical grade and used without fur-
ther purification. Melting points (mp) were measured on a SGW
X-1 microscopic melting point apparatus and are uncorrected.
Mass spectra (MS) were obtained on a Waters Quattro Micromass
instrument using electrospray ionization (ESI). H NMR and 13C NMR
spectra were recorded on
1
a Bruker AV400spectrometer in
4-Methylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
[D6]DMSO. Chemical shifts (d) are reported in parts per million
(ppm) relative to the internal standard, tetramethylsilane (TMS). El-
emental analyses were performed on a Carlo Erba 1106 instrument,
and the results of elemental analyses for C, H, and N are within
Æ0.4% of the theoretical values. TLC analyses were run on silica
gel 60 F254 plates (Merck) using a variety of solvent systems and a
fluorescent indicator for visualization. Spots were visualized under
254 nm UV illumination. Flash chromatography was performed on
silica gel (300–400 mesh) using EtOAc and hexane as eluents.
drazone (9d): Yield: 47%; mp: 183.4–184.78C; 1H NMR (400 MHz,
[D6]DMSO): d=2.42 (s, 3H, CH3), 7.13–7.62 (m, 8H, PhH), 7.28 (s,
2H, NH2), 7.37 (d, 1H, J=4.8 Hz, CH), 8.37 (d, 1H, J=4.8 Hz, CH),
9.90 ppm (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=21.0,
101.7, 107.6, 118.1 (2C), 119.6, 129.0, 129.4 (2C), 129.6 (2C), 132.4
(2C), 137.6, 141.0, 145.1, 157.0, 158.9, 161.6 ppm; MS (ESI+): m/z
(%): 329 [M+H]+ (100); Anal. calcd for C19H16N6: C 69.50, H 4.91, N
25.59, found: C 69.53, H 4.87, N 25.60.
ChemMedChem 2011, 6, 2225 – 2232
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2229

F.-E. Chen et al.
MED
Phenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hydrazone
13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.5, 115.9 (2C, JCF
14.4 Hz), 118.0 (2C), 119.6, 128.4 (JCF =3.2 Hz), 131.9 (2C, JCF
8.3 Hz), 132.4 (2C), 139.6, 145.1, 157.1, 158.9, 162.1 (JCF =242.9 Hz),
164.5 ppm; MS (ESI+): m/z (%): 333 [M+H]+ (100); Anal. calcd for
C18H13FN6: C 65.05, H 3.94, N 25.29, F 5.92, found: C 65.03, H 3.93,
N 25.33, F 5.91.
=
=
(9a): Yield: 43%; mp: 177.8–178.98C; 1H NMR (400 MHz,
[D6]DMSO): d=7.26–7.61 (m, 9H, PhH), 7.37 (s, 2H, NH2), 7.38 (d,
1H, J=5.2 Hz, CH), 8.40 (d, 1H, J=5.2 Hz, CH), 9.93 ppm (s, 1H,
NH); 13C NMR (100 MHz, [D6]DMSO): d=101.6, 107.5, 118.0 (2C),
119.6, 128.4, 129.0 (2C), 129.5 (2C), 132.1, 132.4 (2C), 140.9, 145.1,
157.1, 158.9, 164.5 ppm; MS (ESI+): m/z (%): 315 [M+H]+ (100);
Anal. calcd for C18H14N6: C 68.78, H 4.49, N 26.74, found: C 68.75, H
4.52, N 26.74.
2-Chlorophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
1
drazone (9h): Yield: 37%; mp: 195.5–196.48C; H NMR (400 MHz,
[D6]DMSO): d=7.29–7.69 (m, 8H, PhH), 7.38 (d, 1H, J=5.2 Hz, CH),
7.51 (s, 2H, NH2), 8.40 (d, 1H, J=5.2 Hz, CH), 9.94 ppm (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): d=101.6, 106.9, 117.8 (2C), 119.6,
127.9, 129.7, 130.5, 131.6, 131.8, 132.4 (2C), 133.2, 137.9, 145.1,
157.2, 158.9, 163.8 ppm; MS (ESI+): m/z (%): 349 [M+H]+ (100),
351 [M+H+2]+ (32); Anal. calcd for C18H13ClN6: C 61.98, H 3.76, N
24.09, Cl 10.16, found: C 61.99, H 3.75, N 24.11, Cl 10.14.
2-Methylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9b): Yield: 41%; mp: 179.9–180.78C; 1H NMR (400 MHz,
[D6]DMSO): d=2.03 (s, 3H, CH3), 7.23 (s, 2H, NH2), 7.38 (d, 1H, J=
5.2 Hz, CH), 7.09–7.51 (m, 8H, PhH), 8.40 (d, 1H, J=5.2 Hz, CH),
9.93 ppm (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=19.0,
101.5, 107.2, 117.8 (2C), 119.6, 126.4, 128.6, 129.3, 130.2, 132.2,
132.4, 136.7 (2C), 141.1, 145.1, 157.2, 159.0, 164.3 ppm; MS (ESI+):
m/z (%): 329 [M+H]+ (100); Anal. calcd for C19H16N6: C 69.50, H
4.91, N 25.59, found: C 69.53, H 4.90, N 25.58.
3-Chlorophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9i): Yield: 35%; mp: 198.4–199.38C; 1H NMR (400 MHz,
[D6]DMSO): d=7.23–7.62 (m, 8H, PhH), 7.34 (s, 2H, NH2), 7.37 (d,
1H, J=5.2 Hz, CH), 8.40 (d, 1H, J=5.2 Hz, CH), 9.95 ppm (s, 1H,
NH); 13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.3, 117.9 (2C),
119.6, 128.3 (2C), 129.5, 130.9, 132.4 (2C), 133.7, 134.5, 138.8, 145.1,
157.2, 158.8, 164.2 ppm; MS (ESI+): m/z (%): 349 [M+H]+ (100),
351 [M+H+2]+ (32); Anal. calcd for C18H13ClN6: C 61.98, H 3.76, N
24.09, Cl 10.16, found: C 61.70, H 3.77, N 24.08, Cl 10.13.
3-Methylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9c): Yield: 44%; mp: 181.1–181.98C; 1H NMR (400 MHz,
[D6]DMSO): d=2.37 (s, 3H, CH3), 7.05–7.61 (m, 8H, PhH), 7.37 (d,
1H, J=5.6 Hz, CH), 7.38 (s, 2H, NH2), 8.40 (d, 1H, J=5.6 Hz, CH),
9.93 ppm (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=21.1,
101.7, 107.6, 118.0 (2C), 120.0, 126.5, 129.0, 129.82 (2C), 132.0 (2C),
132.4, 138.2, 141.1, 145.1, 157.1, 158.9, 164.6 ppm; MS (ESI+): m/z
(%): 329 [M+H]+ (100); Anal. calcd for C19H16N6: C 69.50, H 4.91, N
25.59, found: C 69.49, H 4.90, N 25.61.
4-Chlorophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9j): Yield: 36%; mp: 195.7–196.68C; 1H NMR (400 MHz,
[D6]DMSO): d=7.27–7.65 (m, 8H, PhH), 7.36 (d, 1H, J=5.2 Hz, CH),
7.46 (s, 2H, NH2), 8.38 (d, 1H, J=5.2 Hz, CH), 9.90 ppm (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.4, 118.1 (2C), 119.6,
129.1 (2C), 131.3, 131.7 (2C), 132.4 (2C), 133.1, 139.4, 145.1, 157.2,
158.8, 164.3 ppm; MS (ESI+): m/z (%): 349 [M+H]+ (100), 351 [M+
H+2]+ (32); Anal. calcd for C18H13ClN6: C 61.98, H 3.76, N 24.09, Cl
10.16, found: C 61.71, H 3.77, N 24.07, Cl 10.14.
4-Methylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9d): Yield: 47%; mp: 183.4–184.78C; 1H NMR (400 MHz,
[D6]DMSO): d=2.43 (s, 3H, CH3), 7.14–7.63 (m, 8H, PhH), 7.29 (s,
2H, NH2), 7.36 (d, 1H, J=5.6 Hz, CH), 8.38 (d, 1H, J=5.6 Hz, CH),
9.91 ppm (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=21.0,
101.7, 107.6, 118.1 (2C), 119.6, 129.0, 129.4 (2C), 129.6 (2C), 132.4
(2C), 137.6, 141.0, 145.1, 157.0, 158.9, 164.6 ppm; MS (ESI+): m/z
(%): 329 [M+H]+ (100); Anal. calcd for C19H16N6: C 69.50, H 4.91, N
25.59, found: C 69.53, H 4.87, N 25.60.
2-Bromophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9k): Yield: 45%; mp: 197.9–198.78C; 1H NMR (400 MHz,
[D6]DMSO): d=7.28–7.70 (m, 8H, PhH), 7.51 (s, 2H, NH2), 7.38 (d,
1H, J=5.2 Hz, CH), 8.40 (d, 1H, J=5.2 Hz, CH), 9.93 ppm (s, 1H,
NH); 13C NMR (100 MHz, [D6]DMSO): d=102.1, 109.4, 117.8 (2C),
119.5, 122.2, 128.4, 131.0, 131.7, 132.3, 132.5 (2C), 133.0, 144.6,
145.1, 158.7, 159.0, 161.7 ppm; MS (ESI+): m/z (%): 393 [M+H]+
(100), 395 [M+H+2]+ (90); Anal. calcd for C18H13BrN6: C 54.98, H
3.33, Br 20.32, N 21.37, found: C 54.96, H 3.34, Br 20.34, N 21.36.
2-Fluorolphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9e): Yield: 38%; mp: 209.9–210.78C; 1H NMR (400 MHz,
[D6]DMSO): d=7.29–7.65 (m, 8H, PhH), 7.36 (d, 1H, J=5.2 Hz, CH),
7.38 (s, 2H, NH2), 8.40 (d, 1H, J=5.2 Hz, CH), 9.94 ppm (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.0, 116.2 (JCF
=
20.9 Hz), 117.8 (2C), 119.6, 119.7 (JCF =19.0 Hz), 125.1 (JCF =3.2 Hz),
131.1 (JCF =8.5 Hz), 131.6 (JCF =4.2 Hz), 132.4 (2C), 135.0, 145.1,
157.3, 159.0, 160.1 (JCF =243.0 Hz), 164.1 ppm; MS (ESI+): m/z (%):
333 [M+H]+ (100); Anal. calcd for C18H13FN6: C 65.05, H 3.94, N
25.29, F 5.92, found: C 65.04, H 3.95, N 25.32, F 5.91.
4-Bromophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9l): Yield: 49%; mp: 199.1–200.38C; 1H NMR (400 MHz,
[D6]DMSO): d=7.21–7.79 (m, 8H, PhH), 7.36 (d, 1H, J=5.6 Hz, CH),
7.52 (s, 2H, NH2), 8.39 (d, 1H, J=5.6 Hz, CH), 9.92 ppm (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.3, 118.0 (2C), 119.6,
121.7 (2C), 131.7, 132.0 (3C), 132.3 (2C), 139.2, 145.0, 157.1, 158.8,
164.3 ppm; MS (ESI+): m/z (%): 393 [M+H]+ (100), 395 [M+H+
2]+ (90); Anal. calcd for C18H13BrN6: C 54.98, H 3.33, Br 20.32, N
21.37, found: C 54.97, H 3.34, Br 20.33, N 21.36.
3-Fluorolphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9 f): Yield: 28%; mp: 196.4–197.58C; 1H NMR (400 MHz,
[D6]DMSO): d=7.10–7.64 (m, 8H, PhH), 7.37 (d, 1H, J=5.6 Hz, CH),
7.51 (s, 2H, NH2), 8.40 (d, 1H, J=5.6 Hz, CH), 9.94 ppm (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.4, 115.2 (JCF
=
20.8 Hz), 116.5 (JCF =124.0 Hz), 118.0 (2C), 119.6, 125.8 (JCF =2.6 Hz),
131.1 (JCF =8.3 Hz), 132.4 (2C), 134.6 (JCF =7.0 Hz), 139.1, 145.1,
157.2, 158.8, 162.7 (JCF =243 Hz), 164.2 ppm; MS (ESI+): m/z (%):
333 [M+H]+ (100); Anal. calcd for C18H13FN6: C 65.05, H 3.94, N
25.29, F 5.92, found: C 65.07, H 3.92, N 25.30, F 5.93.
4-Methoxyphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hy-
drazone (9m): Yield: 32%; mp: 193.9–194.78C; H NMR (400 MHz,
1
[D6]DMSO): d=3.86 (s, 3H, OCH3), 7.13–7.66 (m, 8H, PhH), 7.31 (s,
2H, NH2), 7.35 (d, 1H, J=5.2 Hz, CH), 8.38 (d, 1H, J=5.2 Hz, CH),
9.91 ppm (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=55.2,
101.6, 107.7, 114.4 (2C), 118.1 (2C), 119.6, 123.8, 130.9 (2C), 132.4
(2C), 140.8, 145.1, 157.0, 158.9, 159.2, 164.7 ppm; MS (ESI+): m/z
(%): 345 [M+H]+ (100); Anal. calcd for C19H16N6O: C 66.27, H 4.68,
N 24.40, found: C 66.28, H 4.67, N 24.41.
4-Fluorolphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hy-
drazone (9g): Yield 32%; mp: 194.2–195.48C; 1H NMR (400 MHz,
[D6]DMSO): d=7.30–7.61 (m, 8H, PhH), 7.32 (d, 1H, J=5.6 Hz, CH),
7.43 (s, 2H, NH2), 8.39 (d, 1H, J=5.6 Hz, CH), 9.92 ppm (s, 1H, NH);
2230
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2225 – 2232

Pyrimidinone hydrazones as Potent NNRTIs
4-tButylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hydra-
zone (9n): Yield: 32%; mp: 179.0–180.18C; 1H NMR (400 MHz,
[D6]DMSO): d=1.38 (s, 9H, 3CH3), 7.20–7.63 (m, 8H, PhH), 7.27 (s,
2H, NH2), 7.37 (d, 1H, J=5.2 Hz, CH), 8.39 (d, 1H, J=5.2 Hz, CH),
9.93 ppm (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=31.1 (3C),
34.5, 101.5, 107.7, 118.1 (2C), 119.4, 125.7 (2C), 128.9, 129.1 (2C),
132.3 (2C), 140.8, 145.0, 150.5, 157.1, 158.9, 164.6 ppm; MS (ESI+):
m/z (%): 371 [M+H]+ (100); Anal. calcd for C22H22N6: C 71.33, H
5.99, N 22.69, found: C 71.31, H 5.60, N 22.70.
1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=101.4, 107.5, 117.9 (2C),
119.5, 126.2, 126.5, 127.2, 127.7, 128.2, 128.5, 129.0, 130.3, 132.1,
132.8 (2C), 133.3, 140.6, 145.0, 157.2, 158.8, 164.7 ppm; MS (ESI+):
m/z (%): 365 [M+H]+ (100); Anal. calcd for C22H16N6: C 72.51, H
4.43, N 23.06, found: C 72.50, H 4.44, N 23.05.
Biology
Anti-HIV assays: Anti-HIV activity (wild-type HIV-1 strain IIIB) and cy-
totoxicity were evaluated in MT-4 cells using the 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.[14,15]
Briefly, virus stocks were titrated in MT-4 cells and expressed as the
50% cell culture infective dose (CCID50). MT-4 cells were suspended
in culture medium at 1ꢁ105 cellsmLÀ1 and infected with HIV at a
multiplicity of infection value of 0.02. Immediately after viral infec-
tion, 100 mL of the cell suspension was placed in each well of a
flat-bottomed microtiter tray containing various concentrations of
the test compounds. Stock solutions of the test compounds were
dissolved in DMSO at 50 mm or higher. After incubation for four
days at 378C, the number of viable cells was determined using the
MTT method. Compounds were tested in parallel for cytotoxic
effects in uninfected MT-4 cells.
2,4-Dichlorophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hydrazone (9o): Yield: 42%; mp: 172.4–173.58C; 1H NMR
(400 MHz, [D6]DMSO): d=7.31–7.84 (m, 7H, PhH), 7.38 (d, 1H, J=
5.2 Hz, CH), 7.52 (s, 2H, NH2), 8.39 (d, 1H, J=5.2 Hz, CH), 9.94 ppm
(s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=101.8, 106.9, 117.9
(2C), 119.2, 128.3, 129.8, 131.3, 132.8 (2C), 133.2, 134.7, 136.4, 144.4,
145.1, 157.2, 159.2, 163.6 ppm; MS (ESI+): m/z (%): 383 [M+H]+
(100), 385 [M+H+2]+ (64); Anal. calcd for C18H12 Cl2N6: C 56.41, H
3.16, Cl 18.50, N 21.93, found: C 56.40, H 3.17, Cl 18.48, N 21.95.
3,4-Dichlorophenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hydrazone (9p): Yield: 40%; mp: 166.7–167.88C; 1H NMR
(400 MHz, [D6]DMSO): d=7.25–7.84 (m, 7H, PhH), 7.35 (d, 1H, J=
5.2 Hz, CH), 7.68 (s, 2H, NH2), 8.39 (d, 1H, J=5.2 Hz, CH), 9.93 ppm
(s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=101.7, 107.2, 118.0
(2C), 119.5, 130.3, 131.0, 131.2, 131.8, 131.9 (2C), 132.3, 133.4, 137.5,
145.1, 157.1, 158.8, 164.0 ppm; MS (ESI+): m/z (%): 383 [M+H]+
(100), 385 [M+H+2]+ (64); Anal. calcd for C18H12 Cl2N6: C 56.41, H
3.16, Cl 18.50, N 21.93, found: C 56.39, H 3.18, Cl 18.49, N 21.94.
RT inhibition assay: According to the method reported by Liu et
al.,[20] assays against HIV-1 RT(WT) were performed using a poly(rA)/
oligo(dT)15 homopolymer template with HIV antigen detection
ELISA for quantifying expression of HIV-1 RT in culture medium.
Oligo(dT) was immobilized through its 50 terminal phosphate to
Covalink-NH microtiter plates. The biotin-11-2’-deoxyuridin-5’-tri-
phosphate (biotin-11-dUTP) was incorporated by wild-type RT. The
reaction mixture contained 50 mm Tris·HCl (pH 8.3), 3 mm MgCl2,
75 mm KCl, 5 mm 1,4-dithio-d-threitol (DTT), 0.13 mgmLÀ1 bovine
serum albumin (BSA), 10 mgmLÀ1 poly(A), 0.75 mm biotin-11-dUTP,
and 1.5 mm deoxythymidine triphosphate (dTTP). After incubation
at 378C for 1 h, the plate was washed three times with a wash
buffer containing 50 mm Tris-HCl (pH 7.5), 0.15m NaCl, 0.05 mm
MgCl2, and 0.02% Tween-20. After addition of 1% BSA (100 mL), to
each well and incubation for 30 min at room temperature, the
plate was washed with the same buffer. Subsequently, 50 mL of al-
2,5-Difluorolphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
hydrazone (9q): Yield: 45%; mp: 205.1–205.98C; 1H NMR
(400 MHz, [D6]DMSO): d=7.20–7.58 (m, 7H, PhH), 7.82 (s, 2H, NH2),
7.36 (d, 1H, J=5.2 Hz, CH), 8.40 (d, 1H, J=5.2 Hz, CH), 9.97 ppm (s,
1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=101.8, 106.8, 117.3–
117.7 (m, 2C), 117.8 (2C), 117.9–118.2 (m, 1C), 119.6, 121.4–121.7 (m,
1C), 132.4 (2C), 133.2, 145.1, 154.8–157.4 (m, 2C), 157.3, 159.0,
163.8 ppm; MS (ESI+): m/z (%): 351 [M+H]+ (100); Anal. calcd for
C18H12F2N6: C 61.71, H 3.45, F 10.85, N 23.99, found: C 61.68, H
3.47, F 10.88, N 23.97.
kaline phosphatase streptavidin (SA-ALP) solution (100 ngmLÀ1
)
3,4-Difluorolphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone
1
hydrazone (9r): Yield: 42%; mp: 199.9–200.88C; H NMR (400 MHz,
was added per well, and the plates were then incubated for 1 h at
378C. The plate was washed as described above, and then 50 mL
of p-nitrophenyl phosphate disodium (PNPP) (1 mgmLÀ1, pH 9.5)
was added. After 30 min at 378C, the reaction was stopped by ad-
dition of 0.5m NaOH. The products were detected and quantified
using a colorimetric streptavidin alkaline phosphatase reporter
system.
[D6]DMSO): d=7.13–7.66 (m, 7H, PhH), 7.35 (d, 1H, J=5.2 Hz, CH),
7.64 (s, 2H, NH2), 8.39 (d, 1H, J=5.2 Hz, CH), 9.92 ppm (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): d=101.8, 107.3, 118.0 (2C), 118.2–
119.6 (m, 1C), 123.9 (m, 1C), 127.0 (m, 1C), 130.0 (m, 1C), 129.8,
132.3 (2C), 138.0, 145.1, 145.7–161.7 (m, 2C), 157.0, 158.8,
164.1 ppm; MS (ESI+): m/z (%): 351 [M+H]+ (100); Anal. calcd for
C18H12F2N6: C 61.71, H 3.45, F 10.85, N 23.99, found: C 61.69, H
3.47, F 10.86, N 23.98.
Molecular simulation
1-Naphthylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hy-
drazone (9s): Yield: 31%; mp: 218.9–219.78C; 1H NMR (400 MHz,
[D6]DMSO): d=7.21–7.79 (m, 11H, PhH, NaphH), 7.52 (s, 2H, NH2),
7.36 (d, 1H, J=5.6 Hz, CH), 8.39 (d, 1H, J=5.6 Hz, CH), 9.92 ppm (s,
1H, NH); 13C NMR (100 MHz, [D6]DMSO): d=101.4, 107.2, 117.6 (2C),
119.6, 124.9, 126.2 (2C), 126.6, 127.8, 128.5, 128.7, 130.7 (2C), 132.1
(2C), 133.7, 139.3, 144.9, 157.2, 158.9, 164.8 ppm; MS (ESI+): m/z
(%): 365 [M+H]+ (100); Anal. calcd for C22H16 N6: C 72.51, H 4.43, N
23.06, found: C 72.52, H 4.42, N 23.07.
Molecular modeling was carried out with the Tripos molecular
modeling packages Sybyl-1.2. All the molecules for docking were
built using standard bond lengths and angles from Sybyl-1.2/base
Builder and were then optimized using the Tripos force field for
2000 generations two times or more, until the minimized conform-
ers of the ligand were the same. The flexible docking method,
called Surflex-Dock, docks the ligand automatically into the ligand
binding site of the receptor by using a protocol-based approach
and an empirically derived scoring function.[24–26] The protocol is a
computational representation of a putative ligand that binds to
the intended binding site and is a unique and essential element of
the docking algorithm. The scoring function in Surflex-Dock, which
contains hydrophobic, polar, repulsive, entropic, and salvation
2-Naphthylphenyl-2-[(4-cyanophenyl)amino]-4-pyrimidinone hy-
drazone (9t): Yield: 36%; mp: 199.8–200.78C; 1H NMR(400 MHz,
[D6]DMSO): d=6.88–8.13 (m, 11H, PhH, NaphH), 7.51 (s, 2H, NH2),
7.43 (d, 1H, J=5.2 Hz, CH), 8.42 (d, 1H, J=5.2 Hz, CH), 9.88 ppm (s,
ChemMedChem 2011, 6, 2225 – 2232
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www.chemmedchem.org
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Acknowledgements
We are grateful to the National Natural Science Foundation of
China (grant nos. 30672536, 20872018 and 81172918) and the
Katholieke Universiteit Leuven (Belgium) (GOA no. 10/14) for the
financial support of this research. We thank Mrs. K. Erven and
Mr. K. Uyttersprot (Rega Institute for Medical Research, Katholieke
Universiteit Leuven) for excellent technical assistance.
Keywords: antiviral agents
· diarylpyrimidines · HIV-1 ·
NNRTIs · reverse transcriptase · structure–activity relationships
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Received: July 7, 2011
Published online on September 8, 2011
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