Synthesis and biological evaluation of 4-(hydroxyimino)arylmethyl diarylpyrimidine analogues as potential non-nucleoside reverse transcriptase inhibitors against HIV

Article abstract of DOI:10.1016/j.bmc.2010.03.007

A series of novel diarylpyrimidine analogues featuring a hydroxyiminomethyl group between the pyrimidine scaffold and the aryl wing I have been synthesized and tested in MT-4 cells culture as non-nucleoside reverse transcriptase inhibitors against human immunodeficiency virus (HIV). Most of these new congeners exhibited moderate to excellent activity against wild-type virus with an EC₅₀ value ranging from 0.569 μM to 0.005 μM. 4-(4-((Hydroxyimino) (3-methoxyphenyl)methyl)pyrimidin-2-ylamino)benzonitrile (12n) was identified as the most active compound of this new series (EC₅₀ = 0.025 μM, SI >1223) associated with moderate activity against HIV-1 double mutant strains (K103N + Y181C) (EC₅₀ = 8.72 μM) in addition to its anti-HIV-2 activity with an EC₅₀ value of 8.31 μM. Preliminary structure-activity relationship (SAR) among the newly synthesized DAPYs was also investigated.

Full text of DOI:10.1016/j.bmc.2010.03.007

Bioorganic & Medicinal Chemistry 18 (2010) 2370–2374
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 4-(hydroxyimino)arylmethyl
diarylpyrimidine analogues as potential non-nucleoside reverse
transcriptase inhibitors against HIV
b
Xiao-Qing Feng a, Zhao-Sen Zeng a, Yong-Hong Liang a, Fen-Er Chen a, , Christophe Pannecouque ,
*
Jan Balzarini b, Erik De Clercq b
a Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, 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:
A series of novel diarylpyrimidine analogues featuring a hydroxyiminomethyl group between the pyrim-
idine scaffold and the aryl wing I have been synthesized and tested in MT-4 cells culture as non-nucle-
oside reverse transcriptase inhibitors against human immunodeficiency virus (HIV). Most of these new
congeners exhibited moderate to excellent activity against wild-type virus with an EC50 value ranging
Received 1 February 2010
Revised 3 March 2010
Accepted 4 March 2010
Available online 9 March 2010
from 0.569
no)benzonitrile (12n) was identified as the most active compound of this new series (EC50 = 0.025
SI >1223) associated with moderate activity against HIV-1 double mutant strains (K103N + Y181C)
(EC50 = 8.72 M) in addition to its anti-HIV-2 activity with an EC50 value of 8.31 M. Preliminary struc-
ture–activity relationship (SAR) among the newly synthesized DAPYs was also investigated.
Ó 2010 Elsevier Ltd. All rights reserved.
l
M
to 0.005
l
M. 4-(4-((Hydroxyimino) (3-methoxyphenyl)methyl)pyrimidin-2-ylami-
l
M,
Keywords:
HIV
NNRTIs
SAR
l
l
DAPYs
1. Introduction
activity and preliminary structure–activity relationship (SAR) of
these new DAPY congeners.
Progress in the last decade has been achieved by marked ad-
vances in the chemical modifications of pyrimidine scaffold, aryl
wing I and the linker between the pyrimidine nucleus and the aryl
moiety at C-4 position of the diarylpyrimidine analogues (DAPYs)
(Fig. 1).1–5 A panel of novel DAPY derivatives including S-DAPYs,
NH-DAPYs, and O-DAPYs were identified as non-nucleoside reverse
transcriptase inhibitors (NNRTIs) with strong potency against
wild-type and clinically mutant strains of human immunodefi-
ciency virus (HIV) (1–6, Fig. 1).1–5 In the course of our studies on
the development of new NNRTIs, we have recently synthesized a
series of 4-(4-(substituted-naphthalen-1or 2-yloxy)pyrimidin-2-
ylamino)benzonitriles in which the phenyl wing I was replaced
by a naphthalene ring (7, Fig. 1).6,7 This series of compounds exhib-
ited high activity and very low cytotoxicity against HIV-1. As part
of our ongoing efforts to identify potent and selective DAPY inhib-
itors, we decided to examine whether the introduction of a hydrox-
yimino group onto the CH2 linker between the pyrimidine nucleus
and the aryl wing I of the DAPYs could reflect on an improved bio-
logical activity against HIV-1 and offer a structurally novel scaffold
for the future development of HIV-1 inhibitors (8, Fig. 1). In this pa-
per, we describe the synthesis, biological evaluation of anti-HIV
2. Chemistry
The synthesis of the newly designed DAPYs 12a–s is depicted in
Scheme 1. The known 4-(4-chloropyrimidin-2-ylamino)benzonitr-
iles (9) were conveniently prepared by refluxing of 4-(4-hydroxy-
pyrimidin-2-ylamino)benzonitriles with POCl3 according to our
reported procedure.6 Treatment of 9 with the corresponding
arylacetonitriles in the presence of 60% NaH in anhydrous DMF at
room temperature for 24–72 h under N2 atmosphere afforded 4-
(4-(cyanoarylmethyl)pyrimidin-2-ylamino)benzonitriles (10a–s),
which were subjected to oxidation by bubbling air at room temper-
ature to yield the 4-(4-aroylpyrimidin-2-ylamino)benzonitriles
(11a–s).8 Oximation of 11a–s with hydroxylamine hydrochloride
in the presence of NaOH in EtOH and H2O provided the title
4-(4-((hydroxyimino)arylmethyl)pyrimidin-2-ylamino)benzonitr-
iles (12a–s) in 20–60% yield.9
3. Results and discussion
All the 4-(4-((hydroxyimino)arylmethyl)pyrimidin-2-ylami-
no)benzonitriles 12a–s were assayed according to the MTT method
in MT-4 cells for their biological activity against wild-type HIV-1
strain IIIB and HIV-2 strain ROD along with the double mutant
* Corresponding author. Tel./fax: +86 21 65643811.
E-mail address: rfchen@fudan.edu.cn (F.-E. Chen).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.007

X.-Q. Feng et al. / Bioorg. Med. Chem. 18 (2010) 2370–2374
2371
R3
R2
N
N
5
1
N
HN
N
X
HN
N
O
Br
HN
N
3
X
R2
R1
I
II
R1
CN
CN
CN
1
2
3
4
O
-DAPYs (X=O)
7
5 TMC125, R1=CN, R2=Br,
R3=NH2, X=O
6 TMC278, R1=(E)CH=CHCN,
R2=H, R3=H, X=NH
S-DAPYs (X=S)
NH-DAPYs (X=NH)
CH2-DAPYs (X=CH2)
N
NOH
N
HN
Ar
CN
8
Figure 1. Chemical structures of DAPYs.
strains RES056 (K103N + Y181C). The biological evaluation is
summarized in Table 1. Efavirenz (EFV), delavirdine (DEV), and
nevirapine (NEV), three drugs currently used in clinical treatment
of HIV-1 infection, were also tested as reference compounds.
Table 1 shows the results of anti-HIV activities of the newly
synthesized DAPY analogues containing hydroxyimino group be-
tween the pyrimidine scaffold and the aryl wing I. All the newly
synthesized compounds inhibited the replication of HIV-1 effec-
played slightly less potency than the compounds with correspond-
ing substituents at the meta or para position 12l (EC50 = 0.032 M)
and 12m (EC50 = 0.006 M). For the meta or para substitutions, in
general, para substituted compounds were more active than the
meta substituted ones except for the fact that varying the fluorine
atom from meta to para position dramatically decreased the activ-
l
l
ity from 0.225 lM to 0.827 lM. In light of the above findings that
introducing a methyl group at the para position of the phenyl ring
tively at lower micromolar concentration range from 0.827
0.005 M. Except for the compound 12d, all of the substituted-
phenyl DAPYs conferred more potent activity than the non-substi-
tuted parent compound 12a (EC50 = 0.569 M). It is worth noting
that striking variable SI values of these new DAPYs are observed
from 24.57 to 3791. In general, compound 12n was identified as
lM to
l
Table 1
l
Biological activities of compounds 12a–s against HIV-1 in MT-4 cellsa
NC
N
Ar
N
N
the most active compound of this series (EC50 = 0.025
>1223), along with moderate activity against double mutant
strains RES056 (K103N + Y181C) with an EC50 value of 8.72 M.
Differing from other typical NNRTIs, 12n still displayed moderate
activity against HIV-2 ROD strains (EC50 = 8.31 M). In comparison
lM, SI
H
NOH
Ar
EC50
(
lM)
CC50 (lM)
SIe
b
d
l
IIIB
RES056c
l
12a
12b
12c
12d
12e
12f
12g
12h
12i
Ph
0.569 0.404
0.053 0.007
0.225 0.011
0.827 0.124
0.013 0.003
0.298 0.095
0.083 0.007
0.005 0.003
0.362 0.198
0.127 0.058
0.048 0.001
0.032 0.019
0.006 0.005
0.025 0.006
0.013 0.005
0.144 0.074
5.60
13.99 9.51
5.67 0.66
15.30 18.17 67.87
>377.26
12.62 8.85
30.44 1.63
17.24 13.50 208.52
13.23 10.60 2501.20
23.54 2.97
26.01 5.62
29.89 3.98
19.96 12.76 626.26
13.27 7.31
30.51 1.43
24.57
106.37
with the reference drugs tested, compound 12n was 8-fold and 29-
fold more potent than NEV and DEV against HIV-1 IIIB strains,
respectively.
To explore the potential SAR among the newly designed ana-
logues, this series of DAPYs with various substituents on wing I
were synthesized and assayed for their in vitro anti-HIV bioactiv-
ity. As seen from Table 1, the introduction of a halogen atom onto
the ortho position of the phenyl ring resulted in compounds 12b,
2-F-Ph
3-F-Ph
4-F-Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Me-Ph
3-Me-Ph
>5.67
P6.70
13.85
>12.62
>14.92
P7.62
>13.23
>23.53
P7.21
P1.59
4.52
>456
997.16
102.10
64.97
204.87
619.30
12j
12k
12l
12e, and 12h with relatively excellent activity of 0.053
0.013 M, and 0.005 M, respectively. In contrast, compound 12k
M) with a methyl group at the ortho position dis-
lM,
l
l
12m 4-Me-Ph
P5.10
8.72
10.29
7.20
2394.60
1223.00
(EC50 = 0.048
l
12n
12o
12p
3-OMe-Ph
4-OMe-Ph
4-t-Butyl-
Ph
3,5-DiMe-
Ph
50.24 15.94 3791.21
33.66 7.35
35.88 2.91
233.57
756.17
NC
NC
NC
N
N
N
b
a
12q
0.047 0.005
7.89
Ar
CN
10 a-s
Ar
N
N
N
N
N
N
Cl
H
H
H
O
12r
1-Naphthyl
2-Naphthyl
0.039 0.009
0.047 0.011
>33.10
>14.28
33.09 1.41
31.64 2.26
843.86
672.51
>1843
>62
9
11 a-s
12s
EFV
DEV
NEV
0.003 0.0006 0.18 0.08 >6.34
0.72 0.07
0.20 0.11
>43.81
>15.02
>43.81
>15.02
>75
NC
N
c
a
All data represent mean values for at least two separate experiments.
EC50: effective concentration of compound required to protect the cell against
Ar
N
H
N
b
NOH
viral cytopathogenicity by 50% in MT-4 cells.
c
RES056: HIV-1 mutated strain bearing both K103N and Y181C mutations.
CC50: cytotoxic concentration of compound that reduces the uninfected MT-4
12 a-s
d
cell viability by 50%.
Scheme 1. Reagents and conditions: (a) Ar-CH2CN, 60% NaH, DMF, rt, 24–72 h, N2;
(b) air, DMF, rt, 48–72 h; (c) H2NOHÁHCl, NaOH, EtOH, H2O, reflux, 1 h.
e
SI: selectivity index: ratio CC50/EC50
.

2372
X.-Q. Feng et al. / Bioorg. Med. Chem. 18 (2010) 2370–2374
led to excellent activity with an EC50 value of 0.006
pounds 12o, 12p and 12q with para-OCH3, para-t-butyl and 3,5-
l
M, com-
at micromolar concentration (EC50 = 19.90
respectively).
lM, EC50 = 8.31 lM,
diMe group on the phenyl ring were synthesized and evaluated.
Compounds 12o (EC50 = 0.013
12q (EC50 = 0.047 M) afforded a slight loss of potency compared
with the parent compound 12m (EC50 = 0.006 M), indicating that
methyl might have the appropriate bulk to accommodate the
hydrophobic pocket formed by Y181, Y188 and W229 of the RT.
In our previous research, based on the hypothesis that replacing
the phenyl wing I of DAPYs with a naphthyl group might enhance
lM), 12p (EC50 = 0.144 lM) and
4. Conclusions
l
l
In this study, a series of novel diarylpyrimidine analogues (DA-
PYs) containing a hydroxyiminomethyl group between the pyrim-
idine scaffold and the aryl wing I have been synthesized and
evaluated for their in vitro bioactivity against human immunodefi-
ciency virus. Their preliminary structure–activity relationship was
also investigated. Among all the compounds (12a–s) examined,
12n was identified as the most active compound (EC50 = 0.025
SI >1223) associated with moderate activity against HIV-1 double
mutant strains (K103N + Y181C) (EC50 = 8.72 M) in addition to
its anti-HIV-2 activity with an EC50 value of 8.31 M. The bioactiv-
ity test results indicated that the linker between the pyrimidine
scaffold and the aryl wing I played an essential role in the interac-
tion between the inhibitors and RT so that it should deserve further
modification which might lead to even more potent compounds
against HIV.
p
ꢀ stacking interaction between the inhibitors and the amino acid
residues Tyr181, Tyr188, and Trp229 within the binding pocket of
RT,10 a panel of compounds endowed with excellent potency
against HIV were achieved.6–8,11,12 Then, promoted by the intrigu-
ing observations, compounds 12r and 12s were successfully syn-
thesized and assayed. The results indicated that they possessed
lM,
l
l
comparable activity (EC50 = 0.039 lM and EC50 = 0.047 lM, respec-
tively) with the substituted-phenyl DAPYs implying that they
might lead to another series of DAPYs with potent anti-HIV
activity.
In addition, we further evaluated the capability of the com-
pounds to inhibit the multiplication of HIV-1 strains (RES056) bear-
ing both K103N and Y181C mutations in cell culture (Table 1). It was
found that meta or para substituted-phenyl DAPYs displayed moder-
ate activity against double mutant strains in the low micromolar
range, while the ortho position substituted ones almost lost their
activity entirely. Among the tested compounds, 12n was the most
potent inhibitor exhibiting activity against the mutant strains at
5. Experimental
5.1. Chemistry
5.1.1. General
Chemical reagents and solvents, purchased from commercial
sources, were of analytical grade and were used without further
purification. Melting points were determined on a WRS-1 digital
melting point apparatus and are uncorrected. 1H NMR spectra were
recorded on a Brucker AV 400 MHz spectrometer were recorded in
DMSO-d6, and the chemical shifts are reported in d (ppm) units rel-
ative to the internal standard tetramethylsilane (TMS). EI Mass
spectra were obtained on an Agilent MS/5975 spectrometer. All
the progress of the reactions was monitored by thin-layer chroma-
tography (TLC) on pre-coated silica gel G plates at 254 nm under a
UV lamp using ethyl acetate/hexane as eluents. Flash chromatogra-
phy separations were performed on silica gel (300–400 mesh).
low micromolar concentration with an EC50 value of 8.72 lM.
Although less active than EFV, compound 12n was still superior to
DEV and NEV which disclosed a promising perspective for this series
of derivatives.
Furthermore, all of the newly synthesized compounds 12a–s
were tested for their bioactivity against HIV-2 (strain ROD) and
the results are listed in Table 2. Unfortunately, none of them exhib-
ited activity except for 12d and 12n displaying moderate potency
Table 2
Biological activities of compounds 12a–s against HIV-2 in MT-4 cellsa
5.1.2. General procedure for the preparation of 4-(4-aroylpyr-
imidin-2-ylamino)benzonitriles (11a–s)
NC
N
Ar
N
H
N
To a solution of 9 (1.4 g, 6 mmol) in anhydrous DMF (30 mL) was
added appropriate aryl acetonitriles (12 mmol). After stirred at
À10 °C for about 15 min, 60% NaH (0.48 g, 12 mmol) was added
portionwise under a nitrogen atmosphere and maintained at this
condition for 1 h. Then, the whole solution was warmed slowly to
room temperature and continued to react for 24–72 h. Air was
passed through the solution for 48–72 h. The resulting mixture
was poured into 300 ml H2O and neutralized with 3 N HCl. The pre-
cipitate was collected for the next step without further purification.
NOH
b
c
Ar
EC50
(
l
M) CC50 (lM)
SId
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
Ph
2-F-Ph
3-F-Ph
4-F-Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Br-Ph
P13.98
>5.67
P8.33
19.90
13.99 9.51
5.67 0.66
15.30 18.17
>377.26
<1
<1
62
>19
<1
64
62
<1
<1
65
<1
>12.62
P7.30
P7.33
>13.23
>23.53
P4.97
>29.90
P11.55
P6.54
8.31
P4.72
P33.67
P8.96
>33.10
>14.28
12.62 8.85
30.44 1.63
17.24 13.50
13.23 10.60
23.54 2.97
26.01 5.62
29.89 3.98
19.96 12.76
13.27 7.31
30.51 1.43
50.24 15.94
33.66 7.35
35.88 2.91
33.09 1.41
31.64 2.26
3-Br-Ph
4-Br-Ph
5.1.3. General procedure for the preparation of 4-(4-((hydrox-
yimino) arylmethyl) pyrimidin-2-ylamino)benzonitriles (12a–s)
To a violently stirred solution of 11a–s (6 mmol) in EtOH
(50 mL) and H2O (20 mL) was added NaOH (4.0 g, 100 mmol) and
hydroxylamine hydrochloride (2.1 g, 30 mmol). After refluxed for
1.5 h, the resulting solution was poured into 300 mL H2O and neu-
tralized with 3 N HCl. The filtrated precipitation was dried. The
product was purified by flash chromatography to give the pure tar-
get compounds 12a–s.
2-Me-Ph
3- Me-Ph
4-Me-Ph
3-OMe-Ph
4-OMe-Ph
4-t-Butyl-Ph-Ph
3,5-DiMe-Ph
1-Naphthyl
2-Naphthyl
<1
62
3.67
611
<1
64
<1
<1
a
All data represent mean values for at least two separate experiments.
EC50: effective concentration of compound required to protect the cell against
b
5.1.4. 4-(4-((Hydroxyimino)(phenyl)methyl)pyrimidin-2-
ylamino)benzonitrile (12a)
viral cytopathogenicity by 50% in MT-4 cells.
c
CC50: cytotoxic concentration of compound that reduces the normal uninfected
Yield 23.6%; light yellow solid, mp 208.8–212.3 °C; 1H NMR
(DMSO-d6) d: 7.42 (d, 1H, J = 5.2 Hz, CH), 7.37–7.67 (m, 9H, PhH),
MT-4 cell viability by 50%.
d
SI: selectivity index: ratio CC50/EC50
.

X.-Q. Feng et al. / Bioorg. Med. Chem. 18 (2010) 2370–2374
2373
8.61 (d, 1H, J = 5.2 Hz, CH), 10.16 (s, 1H, NH), 12.17 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 102.6, 110.1, 118.6 (2C), 120.0, 128.4 (2C),
128.9 (2C), 129.7, 132.5, 133.0 (2C), 145.2, 155.1, 159.1, 159.5,
163.2; MS (EI) m/z 315.1 (M+), calcd for C18H13N5O 315.3.
129.5, 130.7, 131.1, 132.1, 133.0 (2C), 135.2, 145.2, 154.7, 159.3,
159.5, 161.8; MS (EI) m/z 393.0 (M+), calcd for C18H12BrN5O 394.2.
5.1.12. 4-(4-((3-Bromophenyl)(hydroxyimino)methyl) pyrimi-
din-2-ylamino) benzonitrile (12i)
5.1.5. 4-(4-((2-Fluorophenyl)(hydroxyimino)methyl)pyrimidin-
2-ylamino) benzonitrile (12b)
Yield 36.5%; light yellow solid, mp 220.2–222.1 °C; 1H NMR
(DMSO-d6) d: 7.44 (d, 1H, J = 5.2 Hz, CH), 7.08–7.99 (m, 8H, PhH),
8.64 (d, 1H, J = 5.2 Hz, CH), 10.42 (s, 1H, NH), 12.38 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 103.4, 111.9, 118.9 (2C), 120.0, 125.8,
129.1, 132.0, 132.1, 133.4 (2C), 133.7, 137.1, 144.9, 157.6, 159.8,
160.6, 164.4; MS (EI) m/z 393.1 (M+), calcd for C18H12BrN5O 394.2.
Yield 28.7%; yellow solid, mp 205.6–206.1 °C; 1H NMR (DMSO-
d6) d: 7.47 (d, 1H, J = 5.2 Hz, CH), 7.35–7.66 (m, 8H, PhH), 8.62 (d,
1H, J = 5.2 Hz, CH), 10.18 (s, 1H, NH), 12.50 (s, 1H, NOH); 13C
NMR (DMSO-d6) d: 102.7, 109.1, 116.0, 118.5 (2C), 119.9,
124.7(2C), 131.3, 131.4, 133.0 (2C), 145.1, 151.0, 159.3, 159.6,
160.6, 162.1; MS (EI) m/z 333.1 (M+), calcd for C18H12FN5O 333.3.
5.1.13. 4-(4-((4-Bromophenyl)(hydroxyimino)methyl) pyrimi-
din-2-ylamino) benzonitrile (12j)
5.1.6. 4-(4-((3-Fluorophenyl)(hydroxyimino)methyl)pyrimidin-
2-ylamino)benzonitrile (12c)
Yield 21.3%; light yellow solid, mp 208.7–210.2 °C; 1H NMR
(DMSO-d6) d: 7.44 (d, 1H, J = 5.2 Hz, CH), 7.35–7.76 (m, 8H, PhH),
8.62 (d, 1H, J = 5.2 Hz, CH), 10.17 (s, 1H, NH), 12.34 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 102.7, 109.9, 118.7 (2C), 120.0, 125.4,
131.4 (2C), 131.7 (2C), 132.0, 133.0 (2C), 145.1, 154.2, 159.1,
159.4, 162.7; MS (EI) m/z 393.1 (M+), calcd for C18H12BrN5O 394.2.
Yield 19.8%; light yellow solid, mp 195.9–196.8 °C; 1H NMR
(DMSO-d6) d: 7.43 (d, 1H, J = 5.2 Hz, CH), 7.20–7.68 (m, 8H, PhH),
8.62 (d, 1H, J = 5.2 Hz, CH), 10.18 (s, 1H, NH), 12.35 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 102.7, 110.0, 115.7, 116.8, 118.6 (2C),
120.0, 126.0, 130.4, 133.0 (2C), 134.6, 145.1, 154.0, 159.1, 159.5,
162.7, 163.5; MS (EI) m/z 333.1 (M+), calcd for C18H12FN5O 333.3.
5.1.14. 4-(4-((Hydroxyimino)(o-tolyl)methyl)pyrimidin-2-ylami-
no)benzonitrile (12k)
5.1.7. 4-(4-((4-Fluorophenyl)(hydroxyimino)methyl)pyrimidin-
2-ylamino) benzonitrile (12d)
Yield 30.7%; light yellow solid, mp 190.1–191.5 °C; 1H NMR
(DMSO-d6) d: 2.11 (s, 3H, CH3), 7.46 (d, 1H, J = 5.2 Hz, CH), 7.13–
7.57 (m, 8H, PhH), 8.61 (d, 1H, J = 5.2 Hz, CH), 10.16 (s, 1H, NH),
12.18 (s, 1H, NOH); 13C NMR (DMSO-d6) d: 19.8, 102.5, 109.2,
118.5 (2C), 120.0, 125.9, 128.8 (2C), 130.0, 133.0 (2C), 133.7,
136.1, 145.2, 155.8, 159.2, 159.6, 162.6; MS (EI) m/z 329.1 (M+),
calcd for C19H15N5O 329.4.
Yield 24.6%; light yellow solid, mp 220.7–222.5 °C; 1H NMR
(DMSO-d6) d: 7.41 (d, 1H, J = 5.2 Hz, CH), 7.34–7.72 (m, 8H, PhH),
8.62 (d, 1H, J = 5.2 Hz, CH), 10.16 (s, 1H, NH), 12.26 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 102.7, 110.2, 115.4 (2C), 118.7 (2C), 120.0,
128.5, 132.3 (2C), 133.0 (2C), 145.2, 154.1, 159.1, 159.5, 161.3,
163.7; MS (EI) m/z 333.1 (M+), calcd for C18H12FN5O 333.3.
5.1.15. 4-(4-((Hydroxyimino)(m-tolyl)methyl)pyrimidin-2-yla-
mino)benzonitrile (12l)
5.1.8. 4-(4-((2-Chlorophenyl)(hydroxyimino)methyl)pyrimidin-
2-ylamino) benzonitrile (12e)
Yield 40.2%; light yellow solid, mp 198.5–200.4 °C; 1H NMR
(DMSO-d6) d: 2.36 (s, 3H, CH3), 7.38 (d, 1H, J = 5.2 Hz, CH), 7.14–
7.68 (m, 8H, PhH), 8.59 (d, 1H, J = 5.2 Hz, CH), 10.15 (s, 1H, NH),
12.11 (s, 1H, NOH); 13C NMR (DMSO-d6) d: 21.5, 102.6, 110.1,
118.6 (2C), 120.0, 126.8, 128.3, 129.5, 130.0, 132.5, 133.0 (2C),
137.5, 145.3, 155.2, 159.0, 159.5, 163.3; MS (EI) m/z 329.1 (M+),
calcd for C19H15N5O 329.4.
Yield 20.0%; light yellow solid, mp 186.9–188.0 °C; 1H NMR
(DMSO-d6) d: 7.44 (d, 1H, J = 5.2 Hz, CH), 7.32–7.63 (m, 8H, PhH),
8.58 (d, 1H, J = 5.2 Hz, CH), 10.15 (s, 1H, NH), 12.36 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 102.6, 108.9, 118.4 (2C), 120.0, 127.5,
129.5, 130.7, 131.1, 132.1, 133.0 (2C), 139.8, 145.2, 153.6, 159.3,
159.6, 161.9; MS (EI) m/z 349.1 (M+), calcd for C18H12ClN5O 349.8.
5.1.9. 4-(4-((3-Chlorophenyl)(hydroxyimino)methyl)pyrimidin-
2-ylamino) benzonitrile (12f)
5.1.16. 4-(4-((Hydroxyimino)(p-tolyl)methyl)pyrimidin-2-yla-
mino)benzonitrile (12m)
Yield 28.9%; light yellow solid, mp 189.5–191.0 °C; 1H NMR
(DMSO-d6) d: 7.45 (d, 1H, J = 5.2 Hz, CH), 7.08–8.00 (m, 8H, PhH),
8.64 (d, 1H, J = 5.2 Hz, CH), 10.42 (s, 1H, NH), 12.38 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 103.4, 111.9, 118.6 (2C), 119.9, 122.7,
129.1, 131.3, 132.1, 133.4 (2C), 135.7, 137.1, 144.9, 148.4, 159.8,
160.6, 164.4; MS (EI) m/z 349.1 (M+), calcd for C18H12ClN5O 349.8.
Yield 32.0%; light yellow solid, mp 236.5–238.2 °C; 1H NMR
(DMSO-d6) d: 2.42 (s, 3H, CH3), 7.38 (d, 1H, J = 5.2 Hz, CH), 7.26–
7.72 (m, 8H, PhH), 8.59 (d, 1H, J = 5.2 Hz, CH), 10.13 (s, 1H, NH),
12.09 (s, 1H, NOH); 13C NMR (DMSO-d6) d: 21.5, 102.7, 110.4,
118.7 (2C), 120.0, 128.9 (2C), 129.4 (2C), 129.8, 130.0 (2C), 138.4,
145.2, 154.9, 159.0, 159.5, 163.5; MS (EI) m/z 329.1 (M+), calcd
for C19H15N5O 329.4.
5.1.10. 4-(4-((4-Chlorophenyl)(hydroxyimino)methyl) pyrimi-
din-2-ylamino) benzonitrile (12g)
5.1.17. 4-(4-((Hydroxyimino)(3-methoxyphenyl)methyl) pyrim-
idin-2-ylamino) benzonitrile (12n)
Yield 30.5%; yellow solid, mp 221.9–223.1 °C; 1H NMR (DMSO-
d6) d: 7.42 (d, 1H, J = 5.2 Hz, CH), 7.41–7.69 (m, 8H, PhH), 8.61 (d,
1H, J = 5.2 Hz, CH), 10.16 (s, 1H, NH), 12.33 (s, 1H, NOH); 13C
NMR (DMSO-d6) d: 102.7, 110.0, 118.7 (2C), 120.0, 128.5 (2C),
131.3 (2C), 131.8, 133.0 (2C), 133.6, 145.2, 154.1, 159.1, 159.4,
162.8; MS (EI) m/z 349.1 (M+), calcd for C18H12ClN5O 349.8.
Yield 27.9%; light yellow solid, mp 213.7–214.4 °C; 1H NMR
(DMSO-d6) d: 2.51 (s, 3H, OCH3), 7.39 (d, 1H, J = 5.2 Hz, CH),
6.90–7.70 (m, 8H, PhH), 8.59 (d, 1H, J = 5.2 Hz, CH), 10.15 (s, 1H,
NH), 12.16 (s, 1H, NOH); 13C NMR (DMSO-d6) d: 55.6, 102.6,
110.0, 114.4, 115.3, 118.7 (2C), 120.0, 122.0, 129.6, 133.0 (2C),
133.8, 145.2, 154.9, 159.0, 159.4, 159.5, 163.1; MS (EI) m/z 345.1
(M+), calcd for C19H15N5O2 345.4.
5.1.11. 4-(4-((2-Bromophenyl)(hydroxyimino)methyl) pyrimi-
din-2-ylamino) benzonitrile (12h)
5.1.18. 4-(4-((Hydroxyimino)(4-methoxyphenyl)methyl) pyrim-
idin-2-ylamino) benzonitrile (12o)
Yield 26.9%; light yellow solid, mp 189.3–190.8 °C; 1H NMR
(DMSO-d6) d: 7.44 (d, 1H, J = 5.2 Hz, CH), 7.29–7.79 (m, 8H, PhH),
8.58 (d, 1H, J = 5.2 Hz, CH), 10.15 (s, 1H, NH), 12.33 (s, 1H, NOH);
13C NMR (DMSO-d6) d: 102.6, 109.0, 118.4 (2C), 120.0, 127.5,
Yield 35.4%; yellow solid, mp 185.2–187.8 °C; 1H NMR (DMSO-
d6) d: 3.78 (s, 3H, OCH3), 7.40 (d, 1H, J = 5.2 Hz, CH), 6.93–7.71 (m,
8H, PhH), 8.60 (d, 1H, J = 5.2 Hz, CH), 10.17 (s, 1H, NH), 12.18 (s, 1H,

2374
X.-Q. Feng et al. / Bioorg. Med. Chem. 18 (2010) 2370–2374
NOH); 13C NMR (DMSO-d6) d: 55.7, 102.7, 110.8, 113.7 (2C), 118.7
(2C), 120.0, 124.2, 131.6 (2C), 133.1 (2C), 145.3, 154.4, 158.9, 159.5,
159.8, 163.8; MS (EI) m/z 345.2 (M+), calcd for C19H15N5O2 345.4.
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.13,14
Briefly, virus stocks were titrated in MT-4 cells and expressed as
50% cell culture infective dose (CCID50). MT-4 cells were suspended
in culture medium at 1 Â 105 cells/mL and infected with HIV at a
multiplicity of infection of 0.02. Immediately after virus infection,
5.1.19. 4-(4-((4-tert-Butylphenyl)(hydroxyimino)methyl) pyr-
imidin-2-ylamino) benzonitrile (12p)
100 lLof thecellsuspensionwasbroughtintoeachwellofaflat-bot-
Yield 38.1%; light yellow solid, mp 192.3–193.2 °C; 1H NMR
(DMSO-d6) d: 1.04 (s, 9H, CH3), 7.36 (d, 1H, J = 5.2 Hz, CH), 7.29–
7.69 (m, 8H, PhH), 8.57 (d, 1H, J = 5.2 Hz, CH), 10.13 (s, 1H, NH),
12.09 (s, 1H, NOH); 13C NMR (DMSO-d6) d: 31.6 (3C), 35.0, 102.5,
110.4, 118.8 (2C), 119.9, 125.1 (2C), 129.4 (2C), 129.7, 133.0 (2C),
145.2, 151.3, 154.8, 159.0, 159.5, 163.4; MS (EI) m/z 371.2 (M+),
calcd for C22H21N5O 371.4.
tomed microtiter tray containing various concentrations of the test
compounds. The test compounds were dissolved in DMSO at
50 mM or higher. After a 4-day incubation at 37 °C, the number of
viable cells was determined using the MTT method. Compounds
were tested in parallel for cytotoxic effects in uninfected MT-4 cells.
Acknowledgements
5.1.20. 4-(4-((3,5-Dimethylphenyl)(hydroxyimino)methyl) pyr-
imidin-2-ylamino) benzonitrile (12q)
We are grateful to the National Natural Science Foundation of
China (No. 30672536) for the financial support of this research.
Yield 24.3%; light yellow solid, mp 209.8–210.2 °C; 1H NMR
(DMSO-d6) d: 2.33 (s, 6H, CH3), 7.37 (d, 1H, J = 5.2 Hz, CH), 6.96–
7.71 (m, 7H, PhH), 8.60 (d, 1H, J = 5.2 Hz, CH), 10.16 (s, 1H, NH),
12.09 (s, 1H, NOH); 13C NMR (DMSO-d6) d: 21.4 (2C), 102.7,
110.2, 118.6 (2C), 120.0, 127.2 (2C), 130.2, 132.5, 133.0 (2C),
137.4 (2C), 145.3, 155.2, 159.0, 159.5, 163.4; MS (EI) m/z 343.2
(M+), calcd for C20H17N5O 343.4.
References and notes
1. Heeres, J.; de Jonge, M. R.; Koymans, L. M. H.; Daeyaert, F. F. D.; Vinkers, M.; Van
Aken, K. J. A.; Arnold, E.; Das, K.; Kilonda, A.; Hoornaert, G. J.; Compernolle, F.;
Cegla, M.; Azzam, R. A.; Andries, K.; de Béthune, M.-P.; Azijn, H.; Pauwels, R.;
Lewi, P. J.; Janssen, P. A. J. J. Med. Chem. 2005, 48, 1910.
2. Tian, X.; Qin, B.; Lu, H.; Lai, W.; Jiang, S.; Lee, K. H.; Chen, C. H.; Xie, L. Bioorg.
Med. Chem. Lett. 2009, 19, 5482.
3. Guillemont, J.; Pasquier, E.; Palandjian, P.; Vernier, D.; Gaurrand, S.; Lewi, P. J.;
Heeres, J.; de Jonge, M. R.; Koymans, L. M. H.; Daeyaert, F. F. D.; Vinkers, M. H.;
Arnold, E.; Das, K.; Pauwels, R.; Andries, K.; de Béthune, M. P.; Bettens, E.;
Hertogs, K.; Wigerinck, P.; Timmerman, P.; Janssen, P. A. J. J. Med. Chem. 2005,
48, 2072.
4. Mordant, C.; Schmitt, B.; Pasquier, E.; Demestre, C.; Queguiner, L.; Masungi, C.;
Peeters, A.; Smeulders, L.; Bettern, E.; Hertogs, K.; Heeres, J.; Lewi, P.;
Guillemont, J. Eur. J. Med. Chem. 2007, 42, 567.
5. Ludovici, D. W.; De Corte, B. L.; Kukla, M. J.; Ye, H.; Ho, C. Y.; Lichtenstein, M. A.;
Kavash, R. W.; Andries, K.; de Béthune, M. P.; Azijn, H.; Pauwels, R.; Lewi, P. J.;
Heeres, J.; Koymans, L. M. H.; de Jonge, M. R.; Van Aken, K. J. A.; Daeyaert, F. F.
D.; Das, K.; Arnold, E.; Janssen, P. A. J. Bioorg. Med. Chem. Lett. 2001, 11, 2235.
6. Feng, X. Q.; Liang, Y. H.; Zeng, Z. S.; Chen, F. E.; Balzarini, J.; Pannecouque, C.; De
Clercq, E. ChemMedChem 2009, 4, 219.
5.1.21. 4-(4-((Hydroxyimino)(naphthalen-1-yl)methyl)pyrimi-
din-2-ylamino) benzonitrile (12r)
Yield 34.8%; light yellow solid, mp 220.2–222.1 °C; 1H NMR
(DMSO-d6) d: 7.57 (d, 1H, J = 5.2 Hz, CH), 7.19–8.13 (m, 11H, PhH,
NaphH), 8.64 (d, 1H, J = 5.2 Hz, CH), 10.07 (s, 1H, NH), 12.28 (s,
1H, NOH); 13C NMR (DMSO-d6) d: 102.4, 109.3, 118.3 (2C), 120.0,
125.8, 125.9, 126.5, 126.9 (2C), 128.8, 128.9, 130.7, 131.8, 132.7
(2C), 133.5, 145.0, 154.9, 159.3, 159.5,162.95; MS (EI) m/z 365.2
(M+), calcd for C22H15N5O 365.4.
7. Liang, Y. H.; Feng, X. Q.; Zeng, Z. S.; Chen, F. E.; Balzarini, J.; Pannecouque, C.; De
Clercq, E. ChemMedChem 2009, 4, 1537.
8. Ji, L.; Chen, F. E.; De Clercq, E.; Balzarini, J.; Pannecouque, C. J. Med. Chem. 2007,
50, 1778.
9. Jeong, T. S.; Kim, M. J.; Yu, H.; Kim, K. S.; Choi, J. K.; Kim, S. S.; Lee, W. S. Bioorg.
Med. Chem. Lett. 2005, 15, 1525.
10. Das, K.; Clarc, A. D., Jr.; Lewi, P. J.; Heeres, J.; De Jonge, M. R.; Koymans, L. M. H.;
Vinkers, H. M.; Daeyaert, F. F. D.; Ludovici, D. W.; Kukla, M. J.; De Corte, B. L.;
Kavash, R. W.; Ho, C. Y.; Ye, H.; Lichtenstein, M. A.; Andries, K.; Pauwels, R.; de
Béthune, M.-P.; Boyer, P. L.; Clark, P.; Hughes, S. H.; Janssen, P. A. J.; Arnold, E. J.
Med. Chem. 2004, 47, 2550.
5.1.22. 4-(4-((Hydroxyimino)(naphthalen-2-yl)methyl) pyrim-
idin-2-ylamino) benzonitrile (12s)
Yield 21%; white solid, mp 189.5–191.0 °C; 1H NMR (DMSO-d6)
d: 7.50 (d, 1H, J = 5.2 Hz, CH), 7.08–8.08 (m, 11H, PhH, NaphH), 8.65
(d, 1H, J = 5.2 Hz, CH), 10.14 (s, 1H, NH), 12.29 (s, 1H, NOH); 13C
NMR (DMSO-d6) d: 102.5, 110.1, 118.6 (2C), 119.9, 126.7, 127.1,
127.5, 127.7, 128.1, 128.7, 129.1, 130.3, 132.8, 133.0 (2C), 133.1,
145.1, 155.2, 159.1, 159.5, 163.2; MS (EI) m/z 365.1 (M+), calcd
for C22H15N5O 365.4.
11. He, Y. P.; Chen, F. E.; Sun, G. F.; Wang, Y. P.; De Clercq, E.; Balzarini, J.;
Pannecouque, C. Bioorg. Med. Chem. Lett. 2004, 14, 3173.
12. Wang, Y. P.; Chen, F. E.; De Clercq, E.; Balzarini, J.; Pannecouque, C. Bioorg. Med.
Chem. 2008, 16, 3887.
5.2. Anti-HIV activity assays
13. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Virol. Methods 1988, 20, 309.
14. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protocol. 2008, 3, 427.
The anti-HIV activity and cytotoxicity were evaluated against
wild-type HIV-1 strain IIIB in MT-4 cells using the 3-(4,5-dimethyl-