Synthesis and structure-activity relationship of novel diarylpyrimidines with hydromethyl linker (CH(OH)-DAPYs) as HIV-1 NNRTIs

Article abstract of DOI:10.1016/j.bmc.2011.07.023

A series of 26 diarylpyrimidines, characterized by the hydroxymethyl linker between the left wing benzene ring and the central pyrimidine, were synthesized and evaluated for in vitro anti-HIV activity. Most of the compounds exhibited moderate to excellent

Full text of DOI:10.1016/j.bmc.2011.07.023

Bioorganic & Medicinal Chemistry 19 (2011) 5117–5124
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationship of novel diarylpyrimidines
with hydromethyl linker (CH(OH)-DAPYs) as HIV-1 NNRTIs
Shuang-Xi Gu a, Qiu-Qin He a, Shi-Qiong Yang a, Xiao-Dong Ma a, Fen-Er Chen a,b, , Erik De Clercq c,
⇑
Jan Balzarini c, Christophe Pannecouque c
a Department of Chemistry, Fudan University, Shanghai 200433, People’s Republic of China
b Institute of Biomedical Science, Fudan University, Shanghai 200433, People’s Republic of China
c 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 26 diarylpyrimidines, characterized by the hydroxymethyl linker between the left wing ben-
zene ring and the central pyrimidine, were synthesized and evaluated for in vitro anti-HIV activity. Most
of the compounds exhibited moderate to excellent activities against wild-type HIV-1. Among them, com-
pound 10i, bearing a chlorine atom at the C-2 position of left benzene ring, was the best congener and
Received 17 June 2011
Revised 12 July 2011
Accepted 13 July 2011
Available online 22 July 2011
showed potent activity against wild-type HIV-1 with an EC50 value of 0.009
activities against the double RT mutant (K103N + Y181C) HIV-1(IIIB) and HIV-2(ROD) with an EC50 value
of 6.2 and 6.0 M, respectively. The preliminary structure–activity relationship (SAR) of this new series of
compounds was also investigated.
lM, along with moderate
Keywords:
HIV-1 reverse transcriptase
NNRTIs
Diarylpyrimidines,
CH(OH)-DAPYs
l
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
confirmed as the indispensible pharmacophore, the further
modifications were mainly focused on the structural diversity of
the linker between the left benzene ring and the central pyrimidine
ring.2b In terms of different linkers, DAPYs contain a variety of
molecules (Fig. 1) as follows: CH2-DAPYs (3),10a O-DAPYs (4),10a,b,f,i
S-DAPYs (5),10a NH-DAPYs (6),10a,13 C(@NOH)-DAPYs (7),10e
CH(CN)-DAPYs (8)10g and CH(Me)-DAPYs (9).10j Based on these
different structures, we conceived that the introduction of a hydro-
philic hydroxyl group to the CH2 linker of 3 could offer a structurally
novel scaffold for further development of a new series of NNRTIs (10,
CH(OH)-DAPYs). The hydroxymethyl linker would make the mole-
cules retain good molecular flexibility, which might help the
compounds to adopt multiple conformations within the RT nonnu-
cleoside binding site.10j,14 In the present work, the synthesis and
the structure–activity relationship (SAR) of the series of CH(OH)-D-
APYs are described.
Since 1996, highly active antiretroviral therapy (HAART) has
been adopted as an efficient way to treat AIDS patients by reducing
viral loads and restoring the immune system.1 Nonnucleoside
reverse transcriptase inhibitors (NNRTIs), known as one of the
indispensable components of HAART for specifically inhibiting
HIV-1 reverse transcriptase (RT), have been paid wide attention
due to their unique antiviral potency, high specificity and low
cytotoxicity.2
With the aim to develop novel anti-HIV drugs, various structur-
ally different classes of NNRTIs, such as benzophenons,3 diaryl
ethers,4
1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymines
(HEPTs),5,6 dihydro-alkoxybenzyl-oxopyrimidines (DABOs),7 diaryl-
triazines (DATAs),8 dihydro-aryl/alkylsulfanyl-cyclohexyl-meth-
yloxo-pyrimidines (S-DACOs)9 and diaryl-pyrimidines (DAPYs),10
have been discovered. Among these series, DAPYs have been recog-
nized as one of the most successful family of NNRTIs developed so far
due to their excellent potency against HIV-1 wild-type (WT) and
mutant strains.11 In the DAPY series, etravirine (TMC125, 1,
Fig. 1)10a,12 has been approved by the US Food and Drug Administra-
tion (FDA) in 2008, and rilpivirine (TMC278, 2, Fig. 1)13 has been
regarded as another promising anti-HIV drug that has recently also
been approved. Since the right wing of the DAPY structure was
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds 10a–z were shown in
Scheme 1. The key intermediates 4-((4-chloropyrimidin-2-
yl)amino)benzonitriles (14a–c) were prepared from 11a–c through
the modified methods of our previously reported 3-step synthetic
strategy.10f,g Treatment of 14a–c with the corresponding phenyl-
acetonitriles in the presence of 60% NaH in anhydrous DMF at room
⇑
Corresponding author.
E-mail address: rfchen@fudan.edu.cn (F.-E. Chen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.023

5118
S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
CN
CN
NH
CN
CN
O
CN
NH
R3
R3
CN
X
N
NH
N
Me
Me
N
R
N
N
Me
Me
N
R2
R2
HN
NH
R1
R1
N
Br
3
:CH2-DAPYs, X= CH2
N
8
:CH(CN)-DAPYs, R= CN
NH2
4:O-DAPYs, X= O
9:CH(Me)-DAPYs, R= Me
10:CH(OH)-DAPYs, R= OH
Rilpivirine
(TMC278)
2
Etravirine
(TMC125)
1
5:S-DAPYs, X= S
6:NH-DAPYs, X= NH
7:C(=NOH)-DAPYs, X= C(=NOH)
Figure 1. Structures of DAPYs.
R1
R1
N
R1
R1
N
NC
R2
O
NC
R2
Cl
R2
R2
O
N
HN
b
a
c
HN
HN
N
H
N
N
S
N
H
11a-c
O
MeS
H
14a-c
12a-c
13a-c
CN
CN
CN
NH
R3
R3
R3
f
e
d
N
NH
N
NH
N
HO
NC
O
N
N
N
R2
R2
R2
R1
10a-z
R1
R1
16a-z
15a-z
15, 16, 10
R1
R2
H
H
H
H
H
H
H
H
H
H
H
H
H
R3
15, 16, 10
R1
Me
Me
Me
Me
H
R2
R3
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
H
H
H
H
2-F
3-F
n
o
p
q
r
H
2-F
2-Cl
2-Br
4-F
H
11, 12,
13, 14
R1
R2
4-F
H
2-Me
3-Me
4-Me
H
A
B
C
H
H
H
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
s
H
2-Cl
4-Cl
3-F
Me
g
h
i
4-OMe
4-t-Bu
t
H
u
v
w
x
y
z
H
2-Cl
H
4-F
j
2-Br
H
2-Br
4-Me
3-Me
2-Me
k
l
2, 5-diF
2, 5-diMe
3, 4-diCl
H
H
m
H
Scheme 1. Synthetic route to CH(OH)-DAPYs (10a–z). Reagents and conditions: (a) MeI, NaOH, H2O, rt, 24 h; (b) 4-cyanoaniline, 180–190 °C, 10 h; (c) POCl3, reflux, 0.5 h; (d)
R3-phenylactonitrile, 60% NaH, Ar, DMF, ꢀ20 °C to rt, 48–72 h; (e) NaH, air, DMF, rt, 36–72 h; (f) NaBH4, MeOH, 0 °C to rt, 2 h.
temperature for 48–72 h under argon atmosphere gave
4-(4-(cyanoarylmethyl)pyrimidin-2-ylamino)benzonitriles (15a–
z), which were subsequently subjected to natural oxidation by
exposing the reaction mixture to air at room temperature under
stirring conditions for 36–72 h to afford the 4-(4-benzoylpyrimi-
din-2-ylamino)-benzonitriles (16a–z).10e Reductions of 16a–z with
potassium borohydride in methanol provided the title compounds
4-((4-(hydroxy(phenyl)methyl)pyrimidin-2-yl)amino)benzonitr-
iles (10a–z) in yields of 64.8–90.9%.
(10a–z) along with five FDA-approved drugs: nevirapine, zidovu-
dine, zalcitabine, delavirdine, and efavirenz as reference com-
pounds. These compounds were assayed for their cytotoxicity
and anti-HIV activities in MT-4 cells infected with wild-type
HIV-1 (LAI strain, IIIB), double RT mutant (K103N + Y181C)
HIV-1, with Lys 103 replaced by Asn and Tyr181 by Cys), and
HIV-2 strain ROD. The results, expressed as CC50 (50% cytotoxic
concentration), EC50 (50% HIV-1 replication inhibitory concentra-
tion) and SI (selectivity index given by the CC50/EC50 ratio) values,
are summarized in Table 1.
2.2. Biological activity
As listed in Table 1, most CH(OH)-DAPYs showed moderate to
potent activities against wild-type (WT) HIV-1 with EC50 values
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-
in the range of 9.870–0.008
displayed excellent anti-HIV-1 activity against WT HIV-1 with an
lM. In particular, the compound 10i
mide (MTT) method15 was used to evaluate 26 new CH(OH)-DAPYs

S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
5119
Table 1
Anti-HIV activities and cytotoxicity of compounds 10a–z in MT-4 cellsa
Compd
EC50
(l
M)b
CC50
(
l
M)c
SId
WT (IIIB)
K103N + Y181C
HIV-2
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
10w
10x
10y
0.031 0.013
0.562 0.343
5.931 3.996
0.047 0.016
0.348 0.253
0.070 0.025
0.220 0.145
0.335 0.084
0.009 0.003
0.009 0.006
0.047 0.027
0.010 0.010
0.008 0.001
0.123 0.111
0.029 0.011
0.149 0.243
9.870 3.440
7.302 7.049
0.051 0.014
7.925 1.767
1.140 0.428
>37.177
5.516 0.835
2.815 1.544
1.029 0.575
0.333 0.121
0.180 0.083
0.007 0.001
1.468 0.237
0.005 0.001
0.160 0.112
0.003 0.001
8.1 1.8
>34.7
>252.6
>38.6
>38.4
>41.8
>151.0
>35.3
6.2 0.3
10.2 0.3
>7.6
>36.6
>1.8
>21.4
>31.6
>27.0
>34.6
>80.5
>21.1
>40.4
>36.4
–
>30.3
>32.9
>378.4
>41.4
>9.50
0.006 0.002
–
0.443 0.063
>4.380
0.026 0.010
8.1 0.9
>34.7
>252.6
>38.6
>38.4
>41.8
>151.0
>35.3
6.0 0.2
P11.4
>7.6
38.6 1.2
34.7 5.7
252.6 43.5
38.6 3.3
38.4 2.9
41.8 1.7
151.0 64.4
35.5 3.2
35.8 5.0
34.2 3.4
7.6 0.9
36.6 5.5
1.8 0.1
21.4 3.3
31.6 2.9
27.0 1.9
34.6 3.7
80.5 56.0
21.1 6.2
40.4 15.2
36.4 2.3
37.2 2.2
30.3 2.7
32.9 1.3
>378.4
1271
61
43
822
115
597
687
110
4115
3924
164
3600
223
175
1075
180
4
>36.6
>1.8
>21.4
>31.6
>27.0
>34.6
>80.5
>21.1
>40.4
>36.4
>37.2
>30.3
>32.9
>378.4
>41.4
–
11
408
5
32
<1
5
12
>364
130
>83
>13799
>65
>1146
>27
>1537
10z
41.4 33.1
>15.02
>93.55
>94.69
>6.33
Nevirapine
Zidovudine
Zalcitabine
Efavirenz
Delavirdine
Etravirine
0.006 0.001
1.56 0.14
–
–
–
>4.38
>4.6
a
b
c
Data represent the mean of at least three separate experiments.
Compound concentration required to protect MT-4 cells against viral cytopathogenicity by 50%.
Compound concentration that decreases the uninfected MT-4 cell viability by 50%.
Selectivity index: CC50/EC50 ratio (WT).
d
EC50 value of 0.009
along with moderate activities against the double RT mutant virus
(K103N + Y181C, EC50 = 6.2 M) and HIV-2 (EC50 = 6.0 M).
Apparently, it is more potent than nevirapine, zalcitabine, and del-
avirdine in inhibiting WT HIV-1. In addition, the other three com-
pounds (10j, 10l,m) also displayed the anti-HIV-1 WT activities
l
M and the greatest selectivity (SI = 4115),
resulting compounds also showed potent activities against WT
HIV-1. However, the di-halogenated analogs showed higher
toxicity and low inhibitory selectivity. Typically, the compound
10m displayed the most potent activity against WT HIV-1
l
l
(EC50 = 0.008 lM) but at a low selectivity index (SI = 223).
Among the analogs 10n–q with a methyl group at the C-6 po-
sition of the pyrimidine, the chlorine in the ortho-position (10o)
also proved to be the most favorable for the activity
ranging from 0.010 to 0.008 lM, which are also proximal to that
of the best compound 10i.
To explore the structure–activity relationship (SAR) of the new
CH(OH)-DAPYs, the modifications are focused on the left wing as
well as the central pyrimidine ring. In general, the methyl group
at the C-5 or -6 position on the pyrimidine ring affords great harm
to the anti-HIV-1 (WT) activity of CH(OH)-DAPYs, and the former
one seems to be more unfavorable. When there is no substituent
on the pyrimidine ring, most of the compounds (10a–m) displayed
potent activities with EC50 values lower than 0.1 lM, especially,
four of them (10i,j and 10l,m) are the most potent compounds
among these analogs.
(EC50 = 0.029 lM) and selectivity (SI = 1075), and the fluorine in
the para-position on the benzene ring(10q) is still intolerable in
comparison with the ortho-postion fluorine (10n). Moreover, the
compounds 10r–z, with a methyl at the C-5 position of the
pyrimidine, showed moderate to poor activity except the rela-
tively potent ortho-chlorine analog (10s). When compared with
the compound with no subsituent on the left benzene ring
(10r), the ortho- and meta-halogen could enhance the activities
and the para-halogen would result in loss of activity. The intro-
duction of para-fluorine (10v) led to the complete loss of activi-
For the compounds with an unsubstituted pyrimidine and a
mono-substituent at the C-2 position of the left wing, the elec-
tron-withdrawing halogens seem to be more advantageous than
the electron-donating alkyl and alkoxy groups. The activity of the
compounds with halogens at the benzene ring displayed following
order of activity: ortho-F (10a) > meta-F (10b) > para-F (10c), ortho-
ties against WT HIV-1 (EC50 >37 lM, SI <1). The introduction of
a methyl group is all favorable for the anti-HIV-1 WT activity
with an obvious priority of ortho- > meta- > para-.
To investigate the potency against drug-resistant virus, these
compounds were evaluated for their activity against the double
RT mutant (K103N + Y181C) HIV-1 strain which was cross-resis-
tant to most of currently available NNRTIs.3b,16 The compounds
with an ortho-halogen on the left wing and no substituent on the
dipyrimidine displayed moderateactivities against the double mu-
F
(10a) < ortho-Cl (10i) = ortho-Br (10j). For electro-donating
groups, the activity sequence is ortho-Me (10d) > para-Me
(10f) > meta-Me (10e), and the bulky group appeared to be more
disadvantageous: 4-t-Bu (10h) < 4-OMe (10g) < 4-Me (10f). When
the disubstituted halogens are installed on the benzene ring, the
tant strain at a similar level: (10a, EC50 = 8.1
6.2 M; 10j, EC50 = 10.2 M).
lM; 10i, EC50 =
l
l

5120
S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
All the CH(OH)-DAPYs were also assayed for their activities
against HIV-2 (ROD) in MT-4 cells. As depicted in Table 1, some
compounds also showed activities against HIV-2 at a micromolar
H2O, and dried to give 2-(methylthio)pyrimidin-4(1H)-ones 12a–
c to be used without further purification.
level such as compounds 10a (EC50 = 8.1
l
M) and 10i
4.1.2. General procedure for the synthesis of 13a–c
The mixture of 2-(methylthio)-pyrimidin-4(1H)-ones 12a–c
(100 mmol) and 4-cyanoaniline (35.4 g, 300 mmol) was slowly
heated to 180–190 °C and maintained at this temperature for 8 h.
After cooling, the hard mixture was crushed by ultrasound treat-
ment in CH3CN (150 mL). Then the solid was filtered off and
washed with CH3CN until there was no residue 4-cyanoaniline de-
tected by TLC.
(EC50 = 6.0 M). Although HIV-1 and HIV-2 reverse transcriptases
l
are similar in structure and functionality,17 most of previously dis-
covered NNRTIs are only active against HIV-1 and lack of anti-HIV-
2 activity.4–9 Some of the synthesized CH(OH)-DAPYs displayed
moderate anti-HIV-2 activities, whereas it is still difficult to devel-
op NNRTIs that exhibit both anti-HIV-1 and -2 activities.
Compared with the most promising C(@NOH)-DAPY,10e the most
potent CH(OH)-DAPY compound 10i has better activities against
HIV-1 WT, the double RT mutant (K103N + Y181C) and HIV-2 ROD,
which may be attributed to that the single bond CH–OH of 10i deter-
mines its better molecular flexibility. Although the activities of some
CH(CN)-DAPYs (8)10g are more potent than CH(OH)-DAPYs, the activ-
ities of the former against the double RT mutant (K103N + Y181C)
and HIV-2 ROD are strikingly weak than the latter.
4.1.3. General procedure for the synthesis of 14a–c
A mixture of POCl3 (30 mL) and intermediate 4-(4-oxo-1,4-
dihydropyrimidin-2-ylamino) benzonitriles 13a–c (80 mmol) was
stirred at reflux for 30 min. Then the mixture was poured into
ice-water (250 mL) and stirred for 30 min at room temperature.
The resulting precipitate was filtered, washed with H2O to pH
6–7, and dried to give 4-(4-chloropyrimidin-2-ylamino)-benzo-
nitriles 14a–c to be used without further purification.
Taking together the activities against HIV-1 WT, the double RT
mutant (K103N + Y181C) HIV-1 strain and HIV-2 ROD, the com-
pound 10i is undoubtedly the most potent of all the synthesized
CH(OH)-DAPYs.
4.1.4. General procedure for the synthesis of 16a–z
A solution of 14a–c (2 mmol), appropriate aryl acetonitrile
(3 mmol) and anhydrous DMF (30 mL) was stirred for 0.5 h at
ꢀ20 °C. Then NaH (0.14 g, 3.5 mmol; 60% dispersion in mineral
oil) was added portionwise at ꢀ20 °C under Ar. The mixture was
stirred at RT for 48–72 h under Ar, and then stirred under air for
another 36–72 h at RT. The resulting mixture was poured into
water and extracted with EtOAc. The combined organic layers were
dried by Na2SO4, filtered and concentrated in vacuo. Purification by
flash chromatography (silica gel; EtOAc/petroleum ether, 1:6 to
1:4) gave 16a–z.
3. Conclusion
In summary, we designed and synthesized a novel series of
CH(OH)-DAPYs. Biological evaluation indicated that most of the
designed compounds showed moderate to excellent anti-HIV activ-
ity. Among these compounds, the most active compound 10i, which
possess a chlorine atom at the C-2 position of the left benzene ring,
showed potent activity against wild-type HIV-1 with an EC50 value
of 0.009
mutant (K103N + Y181C) HIV-1 strain and HIV-2 strain ROD with
EC50 values of 6.2 and 6.0 M, respectively. These results serve to
lM, along with moderate activities against the double RT
4.1.5. General procedure for the synthesis of target compounds
10a–z
l
support further modification of diarylpyrimidines in an attempt to
search for more effective anti-HIV-1 candidates. As our continuing
work, wewouldpreparetheopticalisomersofthembychemicalres-
olution or asymmetric synthesis and assess their anti-HIV activities.
KBH4 (19 mg, 0.35 mol) was added to the mixture of 16a–z
(0.5 mmol) and methanol (15 mL) at 0–5 °C. The mixture was stir-
red for 2 h, then poured into water and extracted with EtOAc. The
combined organic layers were dried by Na2SO4, filtered and con-
centrated in vacuo. Purification by flash chromatography (silica
gel; EtOAc/petroleum ether, 1:5 to 1:3) gave 10a–z.
4. Experimental
4.1. Chemistry
4.1.5.1. 4-((4-((2-fluorophenyl)(hydroxy)methyl)pyrimidin-2-
yl)amino)-benzonitrile (10a).
Yield 81.7%; light yellow solid;
mp 154.1–155.8 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 5.87 (d,
J = 4.8 Hz, 1H, CH), 6.40 (d, J = 4.8 Hz, 1H, deuterium-exchanged,
OH), 7.19–7.85 (m, 5H, pyrimidine H5 + Ar0H), 7.62 (d, J = 8.8 Hz,
2H, ArH2,6), 7.84 (d, J = 8.8 Hz, 2H, ArH3,5), 8.59 (d, J = 5.2 Hz, 1H,
pyrimidine H6), 10.12 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
Melting points were measured on a SGW X-1 microscopic melt-
ing-point apparatus. 1H NMR and 13C NMR spectra on a Bruker AV
400 MHz spectrometer were recorded in DMSO-d6. Chemical shifts
are reported in d (ppm) units relative to the internal standard tet-
ramethylsilane (TMS). Mass spectra were obtained on a Waters
Quattro Micromass instrument using electrospray ionization (ESI)
techniques. Elemental analyses were performed on a Carlo Erba
1106 instrument. All chemicals and solvents used were of reagent
grade and were purified and dried by standard methods before use.
All air-sensitive reactions were run under a nitrogen atmosphere.
All the reactions were monitored by thin layer chromatography
(TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp
using ethyl acetate/petroleum ether as eluent. Flash chromatogra-
phy separations were obtained on silica gel (300–400 mesh).
d
(ppm): 68.78 (d, JCF = 2.1 Hz), 102.27, 109.96, 115.30 (d,
JCF = 21.5 Hz), 118.18, 119.60, 124.49 (d, JCF = 3.4 Hz), 129.02 (d,
JCF = 4.4 Hz), 129.53 (d, JCF = 7.9 Hz), 130.28 (d, JCF = 14.1 Hz),
132.82, 144.93, 158.83, 158.93, 159.70 (d, JCF = 243.6 Hz), 172.54;
MS (ESI+) m/z 321 (M+H)+; Anal. Calcd for C18H13FN4O: C, 67.49;
H, 4.09; F, 5.93; N, 17.49. Found: C, 67.42; H, 4.12; F, 5.96; N, 17.45.
4.1.5.2. 4-((4-((3-fluorophenyl)(hydroxy)methyl)pyrimidin-2-
yl)amino)-benzonitrile (10b).
Yield 83.9%; light pink solid;
mp 149.5–150.8 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 5.63
(d, J = 4.0 Hz, 1H, CH), 6.41 (d, J = 4.0 Hz, 1H, deuterium-exchanged,
OH), 7.07–7.43 (m, 5H, pyrimidine H5 + Ar0H), 7.67 (d, J = 8.4 Hz,
2H, ArH2,6), 7.92 (d, J = 8.4 Hz, 2H, ArH3,5), 8.56 (d, J = 5.2 Hz, 1H,
pyrimidine H6), 10.16 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 74.19, 102.32, 109.58, 113.34 (d, JCF=22.0 Hz), 114.18 (d,
JCF = 21.5 Hz), 118.28, 119.59, 122.85 (d, JCF = 2.4 Hz), 130.19 (d,
JCF = 8.1 Hz), 132.88, 144.91, 145.99 (d, JCF = 6.9 Hz), 158.77,
4.1.1. General procedure for the synthesis of 12a–c
NaOH (6.18 g, 155 mmol) was added portion-wise to a suspen-
sion of thiouracils 11a–c (150 mmol) in H2O at room temperature.
After the reaction mixture was stirred for 30 min and cooled to
10 °C, iodomethane (25.55 g, 180 mmol) was added. The mixture
was stirred for another 24 h at room temperature, followed by
cooling to 5–10 °C. The precipitate was filtered off, washed with

S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
5121
158.94, 162.13 (d, JCF = 241.8 Hz), 172.88; MS (ESI+) m/z 321
(M+H)+; Anal. Calcd for C18H13FN4O: C, 67.49; H, 4.09; F, 5.93; N,
17.49. Found: C, 67.52; H, 4.06; F, 5.89; N, 17.53.
4.1.5.8.
4-((4-((4-(tert-butyl)phenyl)(hydroxy)methyl)pyrimi-
Yield 87.8%; light yel-
din-2-yl)amino)-benzonitrile (10h).
low solid; mp 86.4–88.1 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 1.22 (s, 9H, t-Bu), 5.58 (d, J = 4.0 Hz, 1H, CH), 6.19 (d,
J = 4.0 Hz, 1H, OH), 7.21 (d, J = 5.2 Hz, 1H, pyrimidine H5),
7.34–7.40 (m, 4H, Ar0H), 7.68 (d, J = 8.8 Hz, 2H, ArH2,6), 7.96 (d,
J = 8.8 Hz, 2H, ArH3,5), 8.55 (d, J = 4.8 Hz, 1H, pyrimidine H6),
10.15 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 31.11,
34.17, 74.88, 102.27, 109.51, 118.28, 119.64, 124.98, 126.56,
132.90, 140.18, 145.03, 149.84, 158.65, 158.79, 173.75; MS (ESI+)
m/z 359 (M+H)+; Anal. Calcd for C22H22N4O: C, 73.72; H, 6.19; N,
15.63. Found: C, 73.81; H, 6.23; N, 15.60.
4.1.5.3. 4-((4-((4-fluorophenyl)(hydroxy)methyl)pyrimidin-2-
yl)amino)-benzonitrile (10c).
Yield 77.8%; light yellow solid;
mp 153.5–155.2 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 5.61
(d, J = 4.0 Hz, 1H, CH), 6.32 (d, J = 4.0 Hz, 1H, OH), 7.16–7.50 (m,
5H, pyrimidine H5 + Ar0H), 7.68 (d, J = 8.4 Hz, 2H, ArH2,6), 7.91 (d,
J = 8.8 Hz, 2H, ArH3,5), 8.56 (d, J = 5.2 Hz, 1H, pyrimidine H6),
10.15 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 74.19,
102.28, 109.48, 114.99 (d, JCF=21.2 Hz), 118.26, 119.62, 128.76 (d,
JCF = 7.8 Hz), 132.93, 139.36 (d, JCF = 3.4 Hz), 144.94, 158.77,
158.90, 161.52 (d, JCF = 241.7 Hz), 173.33; MS (ESI+) m/z 321
(M+H)+; Anal. Calcd for C18H13FN4O: C, 67.49; H, 4.09; F, 5.93; N,
17.49. Found: C, 67.38; H, 4.13; F, 5.97; N, 17.46.
4.1.5.9. 4-((4-((2-chlorophenyl)(hydroxy)methyl)pyrimidin-2-
yl)amino)-benzonitrile (10i).
Yield 82.9%; light yellow solid;
mp 168.3–170.2 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 5.98
(d, J = 4.8 Hz, 1H, CH), 6.44 (d, J = 4.8 Hz, 1H, OH), 7.19 (d,
J = 4.8 Hz, 1H, pyrimidine H5), 7.34–7.53 (m, 4H, Ar0H), 7.60 (d,
J = 8.4 Hz, 2H, ArH2,6), 7.82 (d, J = 8.8 Hz, 2H, ArH3,5), 8.59 (d,
J = 4.8 Hz, 1H, pyrimidine H6), 10.11 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 71.48, 102.22, 110.46, 118.18,
119.57, 127.31, 129.15, 129.21, 132.18, 132.75, 140.55, 144.87,
158.84, 158.96, 171.98; MS (ESI+) m/z 337 (M+H)+; Anal. Calcd
for C18H13ClN4O: C, 64.19; H, 3.89; Cl; 10.53; N, 16.64. Found: C,
64.24; H, 3.86; Cl, 10.49; N, 16.63.
4.1.5.4. 4-((4-(hydroxy(o-tolyl)methyl)pyrimidin-2-yl)amino)-
benzonitrile (10d).
Yield 84.6%; light yellow solid; mp
177.5–179.1 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.38 (s,
3H, CH3), 5.79 (d, J = 4.0 Hz, 1H, CH), 6.14 (d, J = 4.4 Hz, 1H, OH),
7.18–7.34 (m, 5H, pyrimidine H5 + Ar0H), 7.62 (d, J = 8.8 Hz, 2H,
ArH2,6), 7.82 (d, J = 8.8 Hz, 2H, ArH3,5), 8.56 (d, J = 5.2 Hz, 1H, pyrim-
idine H6), 10.06 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 19.29, 72.02, 102.17, 110.09, 118.17, 119.55, 125.80,
127.25, 127.28, 130.15, 132.77, 135.50, 141.46, 144.91, 158.66,
158.69, 173.48; MS (ESI+) m/z 317 (M+H)+; Anal. Calcd for
4.1.5.10.
2-yl)amino)-benzonitrile (10j).
4-((4-((2-bromophenyl)(hydroxy)methyl)pyrimidin-
Yield 79.6%; light yellow so-
C19H16N4O: C, 72.13; H, 5.10; N, 17.71. Found: C, 72.08; H, 5.13;
lid; mp 173.2–175.1 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm):
5.96 (d, J = 4.8 Hz, 1H, CH), 6.48 (d, J = 4.8 Hz, 1H, OH), 7.19 (d,
J = 5.2 Hz, 1H, pyrimidine H5), 7.24–7.66 (m, 4H, Ar0H), 7.60 (d,
J = 8.8 Hz, 2H, ArH2,6), 7.83 (d, J = 8.8 Hz, 2H, ArH3,5), 8.59 (d,
J = 5.2 Hz, 1H, pyrimidine H6), 10.12 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 73.70, 102.19, 110.48, 118.20,
119.56, 122.82, 127.87, 129.46, 129.49, 132.37, 132.75, 142.14,
144.84, 158.82, 159.01, 171.92; MS (ESI+) m/z 381 (M+H)+; Anal.
Calcd for C18H13BrN4O: C, 56.71; H, 3.44; Br, 20.96; N, 14.70.
Found: C, 56.78; H, 3.46; Br, 20.88; N, 14.65.
N, 17.66.
4.1.5.5. 4-((4-(hydroxy(m-tolyl)methyl)pyrimidin-2-yl)amino)-
benzonitrile (10e).
Yield 80.5%; light yellow solid; mp
129.4–130.7 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.29 (s,
3H, CH3), 5.53 (d, J = 4.4 Hz, 1H, CH), 6.19 (d, J = 4.0 Hz, 1H, OH),
7.07–7.26 (m, 5H, pyrimidine H5 + Ar0H), 7.67 (d, J = 8.8 Hz, 2H,
ArH2,6), 7.82 (d, J = 8.8 Hz, 2H, ArH3,5), 8.56 (d, J = 5.2 Hz, 1H, pyrim-
idine H6), 10.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 21.04, 74.96, 102.19, 109.49, 118.20, 119.56, 123.92,
127.24, 127.99, 128.08, 132.86, 137.25, 143.01, 144.94, 158.66,
158.69, 173.60; MS (ESI+) m/z 317 (M+H)+; Anal. Calcd for
4.1.5.11.
din-2-yl)amino)-benzonitrile (10k).
solid; mp 158.5–159.3 °C; 1H NMR (400 MHz, DMSO-d6)
4-((4-((2,5-difluorophenyl)(hydroxy)methyl)pyrimi-
Yield 86.0%; light pink
C19H16N4O: C, 72.13; H, 5.10; N, 17.71. Found: C, 72.18; H, 5.06;
d
N, 17.73.
(ppm): 5.85 (d, J = 4.8 Hz, 1H, CH), 6.53 (d, J = 5.2 Hz, 1H, OH),
7.21–7.30 (m, 4H, pyrimidine H5 + Ar0H), 7.62 (d, J = 8.8 Hz, 2H,
ArH2,6), 7.83 (d, J = 8.8 Hz, 2H, ArH3,5), 8.60 (d, J = 5.2 Hz, 1H, pyrim-
idine H6), 10.14 (s, 1H, NH); MS (ESI+) m/z 339 (M+H)+; Anal. Calcd
for C18H12F2N4O: C, 63.90; H, 3.58; F, 11.23; N, 16.56. Found: C,
63.87; H, 3.66; F, 11.14; N, 16.45.
4.1.5.6. 4-((4-(hydroxy(p-tolyl)methyl)pyrimidin-2-yl)amino)-
benzonitrile (10f).
Yield 82.1%; light yellow solid; mp
154.8–156.9 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.26 (s,
3H, CH3), 5.54 (d, 1H, J = 4.0 Hz, CH), 6.18 (d, 1H, J = 4.0 Hz, OH),
7.14–7.33 (m, 5H, pyrimidine H5 + Ar0H), 7.68 (d, J = 8.8 Hz, 2H,
ArH2,6), 7.93 (d, J = 8.8 Hz, 2H, ArH3,5), 8.54 (d, J = 4.8 Hz, 1H, pyrim-
idine H6), 10.13 (s, 1H, NH); MS (ESI+) m/z 317 (M+H)+; Anal. Calcd
for C19H16N4O: C, 72.13; H, 5.10; N, 17.71. Found: C, 72.20; H, 5.12;
N, 17.65.
4.1.5.12. 4-((4-((2,5-dimethylphenyl)(hydroxy)methyl)pyrimi-
din-2-yl)amino)-benzonitrile (10l).
Yield 85.2%; light yellow
solid; mp 158.7–159.9 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm):
2.24 (s, 3H, CH3), 2.33 (s, 3H, CH3), 5.75 (d, J = 4.0 Hz, 1H, CH), 6.10
(d, J = 4.0 Hz, 1H, OH), 6.97–7.15 (m, 3H, Ar0H), 7.20 (d, J = 5.2 Hz,
1H, pyrimidine H5), 7.62 (d, J = 8.8 Hz, 2H, ArH2,6), 7.84 (d,
J = 8.8 Hz, 2H, ArH3,5), 8.56 (d, J = 5.2 Hz, 1H, pyrimidine H6),
10.06 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 18.87,
20.70, 71.98, 102.16, 110.02, 118.16, 119.54, 127.74, 127.80,
130.08, 132.30, 132.74, 134.55, 141.22, 144.92, 158.63, 158.68,
173.54; MS (ESI+) m/z 331 (M+H)+; Anal. Calcd for C20H18N4O: C,
72.71; H, 5.49; N, 16.96. Found: C, 72.66; H, 5.57; N, 16.91.
4.1.5.7. 4-((4-(hydroxy(4-methoxyphenyl)methyl)pyrimidin-2-
yl)amino)-benzonitrile (10g).
Yield 64.8%; light yellow solid;
mp 167.5–168.8 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 3.72 (s,
3H, OCH3), 5.54 (d, J = 4.4 Hz, 1H, CH), 6.15 (d, J = 4.0 Hz, 1H, OH),
6.90 (d, J = 8.4 Hz, 2H, Ar0H3,5), 7.18 (d, J = 5.2 Hz, 1H, pyrimidine
H5), 7.35 (d, J = 8.8 Hz, 2H, Ar0H2,6), 7.68 (d, J = 8.8 Hz, 2H, ArH2,6),
7.93 (d, J = 8.8 Hz, 2H, ArH3,5), 8.54 (d, J = 5.2 Hz, 1H, pyrimidine
H6), 10.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
55.07, 74.56, 102.21, 109.43, 113.62, 118.23, 119.62, 127.98,
132.92, 135.22, 145.00, 158.64, 158.67, 158.74, 173.87; MS (ESI+)
m/z 333 (M+H)+; Anal. Calcd for C19H16N4O2: C, 68.66; H, 4.85; N,
16.86. Found: C, 68.74; H, 4.87; N, 16.79.
4.1.5.13.
4-((4-((3,4-dichlorophenyl)(hydroxy)methyl)pyrimi-
Yield 86.0%; light yel-
din-2-yl)amino)-benzonitrile (10m).
low solid; mp 161.7–162.9 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 5.65 (d, J = 4.4 Hz, 1H, CH), 6.51 (d, J = 4.4 Hz, 1H, OH),

5122
S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
7.20 (d, J = 4.8 Hz, 1H, pyrimidine H5), 7.42–7.71 (m, 3H, Ar0H), 7.67
(d, J = 8.8 Hz, 2H, ArH2,6), 7.90 (d, J = 8.8 Hz, 2H, ArH3,5), 8.57 (d,
J = 5.2 Hz, 1H, pyrimidine H6), 10.17 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 73.47, 102.38, 109.58, 118.32,
119.58, 127.09, 128.70, 129.99, 130.48, 130.90, 132.87, 144.22,
144.86, 158.79, 159.13, 172.38; MS (ESI+) m/z 371 (M+H)+; Anal.
Calcd for C18H12Cl2N4O: C, 58.24; H, 3.26; Cl, 19.10; N, 15.09.
Found: C, 58.29; H, 3.36; Cl, 19.01; N, 15.04.
1H, OH), 7.28–7.43 (m, 5H, Ar0H), 7.63 (d, J = 8.4 Hz, 2H, ArH2,6),
7.89 (d, J = 8.8 Hz, 2H, ArH3,5), 8.32 (s, 1H, pyrimidine H6), 10.09
(s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 14.04,
73.24, 101.70, 117.84, 119.67, 119.94, 126.63, 127.16, 128.08,
132.86, 141.86, 145.20, 157.52, 159.42, 168.88; MS (ESI+) m/z
317 (M+H)+; Anal. Calcd for C19H16N4O: C, 72.13; H, 5.10; N,
17.71. Found: C, 72.08; H, 5.17; N, 17.67.
4.1.5.19.
4-((4-((2-chlorophenyl)(hydroxy)methyl)-5-methyl-
Yield 88.4%; light
4.1.5.14.
4-((4-((2-fluorophenyl)(hydroxy)methyl)-6-methyl-
Yield 80.5%; light
pyrimidin-2-yl)amino)benzonitrile (10s).
pyrimidin-2-yl)amino)benzonitrile (10n).
yellow solid; mp 202.6–204.3 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.36 (s, 3H, CH3), 6.10 (d, J = 6.0 Hz, 1H, CH), 6.33 (d,
J = 6.0 Hz, 1H, OH), 7.41–7.85 (m, 8H, ArH + Ar0H), 8.41 (s, 1H, pyrim-
idine H6); 13C NMR (100 MHz, DMSO-d6) d (ppm): 13.88, 69.36,
101.56, 117.63, 119.68, 120.72, 126.94, 128.73, 128.85, 129.14,
131.14, 132.60, 140.34, 145.09, 157.56, 159.61, 167.15; MS (ESI+)
m/z 351 (M+H)+; Anal. Calcd for C19H15ClN4O: C, 65.05; H, 4.31; Cl,
10.11; N, 15.97. Found: C, 65.02; H, 4.39; Cl, 10.07; N, 15.94.
yellow solid; mp 153.3–154.8 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.43 (s, 3H, CH3), 5.85 (d, J = 4.4 Hz, 1H, CH), 6.33 (d,
J = 4.8 Hz, 1H, OH), 7.13 (s, 1H, pyrimidine H5), 7.19–7.48 (m, 4H,
Ar0H), 7.60 (d, J = 8.8 Hz, 2H, ArH2,6), 7.84 (d, J = 8.8 Hz, 2H,
ArH3,5), 10.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 23.86, 68.61 (d, JCF = 2.0 Hz), 102.01, 109.21, 115.22 (d,
JCF = 22.1 Hz), 118.06, 119.63, 124.41 (d, JCF = 2.9 Hz), 128.93 (d,
JCF = 4.6 Hz), 129.41 (d, JCF = 8.0 Hz), 130.43 (d, JCF = 14.0 Hz),
132.75, 145.11, 158.69, 159.66 (d, JCF = 2.9 Hz), 168.60, 172.11;
MS (ESI+) m/z 335 (M+H)+; Anal. Calcd for C19H15FN4O: C, 68.25;
H, 4.52; F, 5.68; N, 16.76. Found: C, 68.33; H, 4.57; F, 5.57; N, 16.69.
4.1.5.20.
4-((4-((4-chlorophenyl)(hydroxy)methyl)-5-methyl-
Yield 84.8%; light
pyrimidin-2-yl)amino)benzonitrile (10t).
yellow solid; mp 187.7–188.6 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.19 (s, 3H, CH3), 5.86 (s, 1H, CH), 6.19 (d, J = 2 Hz, 1H,
OH, deuterium exchange), 7.44 (s, 4H, Ar0H), 7.61 (d, J = 8.8 Hz,
2H, ArH2,6), 7.83 (d, J = 8.4 Hz, 2H, ArH3,5), 8.34 (s, 1H, pyrimidine
H6), 10.08 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
14.02, 73.50, 101.74, 117.84, 119.67, 120.13, 128.05, 128.54,
131.70, 132.80, 141.10, 145.17, 157.52, 159.61, 168.41; MS (ESI+)
m/z 351 (M+H)+; Anal. Calcd for C19H15ClN4O: C, 65.05; H, 4.31;
Cl, 10.11; N, 15.97. Found: C, 64.98; H, 4.39; Cl, 10.07; N, 15.92.
4.1.5.15.
4-((4-((2-chlorophenyl)(hydroxy)methyl)-6-methyl-
Yield 90.9%; light
pyrimidin-2-yl)amino)benzonitrile (10o).
yellow solid; mp 93.2–94.7 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.42 (s, 3H, CH3), 5.97 (d, J = 4.4 Hz, 1H, CH), 6.42 (d,
J = 4.4 Hz, 1H, OH), 7.10 (s, 1H, pyrimidine H5),7.32–7.53 (m, 4H,
Ar0H), 7.58 (d, J = 8.8 Hz, 2H, ArH2,6), 7.82 (d, J = 8.8 Hz, 2H,
ArH3,5), 10.07 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 23.82, 71.37, 101.98, 109.72, 118.08, 119.63, 127.29,
129.10, 129.18, 132.17, 132.72, 140.73, 145.07, 158.71, 168.63,
171.63; MS (ESI+) m/z 351 (M+H)+; Anal. Calcd for C19H15ClN4O:
C, 65.05; H, 4.31; Cl, 10.11; N, 15.97. Found: C, 64.98; H, 4.38; Cl,
10.09; N, 15.94.
4.1.5.21.
4-((4-((3-fluorophenyl)(hydroxy)methyl)-5-methyl-
Yield 83.9%; light
pyrimidin-2-yl)amino)benzonitrile (10u).
yellow solid; mp 169.4–170.8 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.20 (s, 3H, CH3), 5.88 (d, J = 5.2 Hz, 1H, CH), 6.26 (d,
J = 5.2 Hz, 1H, OH), 7.11–7.43 (m, 4H, Ar0H), 7.61 (d, J = 8.8 Hz,
2H, ArH2,6), 7.84 (d, J = 8.4 Hz, 2H, ArH3,5), 8.35 (s, 1H, pyrimidine
H6), 10.10 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
4.1.5.16.
4-((4-((2-bromophenyl)(hydroxy)methyl)-6-methyl-
Yield 83.8%; light
pyrimidin-2-yl)amino)benzonitrile (10p).
yellow solid; mp 82.8–84.2 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.42 (s, 3H, CH3), 5.94 (d, J = 4.8 Hz, 1H, CH), 6.42 (d,
J = 4.8 Hz, 1H, OH), 7.10 (s, 1H, pyrimidine H5), 7.25–7.66 (m, 4H,
Ar0H), 7.60 (d, J = 8.8 Hz, 2H, ArH2,6), 7.84 (d, J = 8.8 Hz, 2H,
ArH3,5), 10.08 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 23.82, 73.62, 101.96, 109.74, 118.11, 119.64, 122.84,
127.84, 129.42, 129.44, 132.32, 132.72, 142.34, 145.04, 158.69,
168.65, 171.60; MS (ESI+) m/z 397 (M+H)+; Anal. Calcd for
14.06, 72.55, 101.77, 113.37 (d,
JCF=21.5 Hz), 113.92 (d,
JCF=20.8 Hz), 117.84, 119.71, 120.19, 122.70 (d, JCF=2.9 Hz),
130.07 (d, JCF=8.1 Hz), 132.84, 145.16 (d, JCF=7.3 Hz), 157.58,
159.68, 162.20 (d, JCF=241.9 Hz), 168.32; MS (ESI+) m/z 335
(M+H)+; Anal. Calcd for C19H15FN4O: C, 68.25; H, 4.52; F, 5.68; N,
16.76. Found: C, 68.21; H, 4.60; F, 5.63; N, 16.72.
4.1.5.22.
4-((4-((4-fluorophenyl)(hydroxy)methyl)-5-methyl-
Yield 79.5%; light
C19H15BrN4O: C, 57.74; H, 3.83; Br, 20.22; N, 14.17. Found: C,
pyrimidin-2-yl)amino)benzonitrile (10v).
57.67; H, 3.86; Br, 20.17; N, 14.20.
yellow solid; mp 181.1–181.8 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.17 (s, 3H, pyrimidine-CH3), 5.86 (s, 1H, CH), 6.15 (s, 1H,
OH), 7.17–7.47 (m, 4H, Ar0H), 7.63 (d, J = 8.8 Hz, 2H, ArH2,6), 7.88
(d, J = 8.4 Hz, 2H, ArH3,5), 8.32 (s, 1H, pyrimidine H6), 10.12 (s,
1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 14.08, 72.51,
101.78, 114.89 (JCF = 21.0 Hz), 117.86, 119.75, 119.99, 128.70
(JCF = 8.1 Hz), 132.90, 138.16 (JCF = 3.1 Hz), 145.24, 157.61, 159.57,
161.44 (JCF = 241.3 Hz), 168.67; MS (ESI+) m/z 335 (M+H)+; Anal.
Calcd for C19H15FN4O: C, 68.25; H, 4.52; F, 5.68; N, 16.76. Found:
C, 68.19; H, 4.63; F, 5.61; N, 16.74.
4.1.5.17.
4-((4-((4-fluorophenyl)(hydroxy)methyl)-6-methyl-
pyrimidin-2-yl)amino)benzonitrile (10q).
Yield 74.3%; light
pink solid; mp 182.3–184.4 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.41 (s, 3H, CH3), 5.58 (s, 1H, CH), 6.27 (d, J = 3.6 Hz, 1H,
OH), 7.09 (s, 1H, pyrimidine H5), 7.16–7.50 (m, 4H, Ar0H), 7.67 (d,
J = 8.8 Hz, 2H, ArH2,6), 7.92 (d, J = 8.8 Hz, 2H, ArH3,5), 10.09 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 23.89, 74.16,
102.05, 108.71, 114.95 (d, JCF = 21.5 Hz), 118.16, 119.70, 128.73
(d, JCF = 8.1 Hz), 132.92, 139.55 (d, JCF = 2.6 Hz), 145.15, 158.66,
161.50 (d, JCF = 241.2 Hz), 168.61, 172.95; MS (ESI+) m/z 335
(M+H)+; Anal. Calcd for C19H15FN4O: C, 68.25; H, 4.52; F, 5.68; N,
16.76. Found: C, 68.36; H, 4.57; F, 5.74; N, 16.68.
4.1.5.23.
4-((4-((2-bromophenyl)(hydroxy)methyl)-5-methyl-
Yield 80.3%; light
pyrimidin-2-yl)amino)benzonitrile (10w).
yellow solid; mp 174.1–175.5 °C; 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.21 (s, 3H, CH3), 5.85 (s, 1H, CH), 6.22 (s, 1H, OH), 7.33–
7.64 (m, 6H, ArH2,6 + Ar0H), 7.82 (d, J = 8.8 Hz, 2H, ArH3,5), 8.34 (s,
1H, pyrimidine H6), 10.08 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 14.01, 72.55, 101.73, 117.82, 119.65, 120.15,
120.22, 128.89, 130.94, 132.78, 141.54, 145.15, 157.49, 159.59,
4.1.5.18.
2-yl)amino)benzonitrile (10r).
4-((4-(hydroxy(phenyl)methyl)-5-methylpyrimidin-
Yield 82.6%; light yellow so-
lid; mp 205.4–207.1 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm):
2.16 (s, 3H, CH3), 5.84 (d, J = 4.4 Hz, 1H, CH), 6.06 (d, J = 4.8 Hz,

S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
5123
168.34; MS (ESI+) m/z 397 (M++1), Calcd for C19H15BrN4O: C, 57.74;
References and notes
H, 3.83, Br 20.22; N, 14.17. Found: C, 57.67; H, 3.86, Br 20.20; N,
14.13.
1. Hirschel, B.; Francioli, P. N. Engl. J. Med. 1998, 338, 906.
2. (a) De Clercq, E. Curr. Med. Chem. 2001, 8, 1543; (b) Chen, X.; Zhan, P.; Li, D.; De
Clercq, E.; Liu, X. Curr. Med. Chem. 2011, 18, 359.
4.1.5.24. 4-((4-(hydroxy(p-tolyl)methyl)-5-methylpyrimidin-2-
3. (a) Wyatt, P. G.; Bethell, R. C.; Cammack, N.; Charon, D.; Dodic, N.; Dumaitre, B.;
Evans, D. N.; Green, D. V. S.; Hopewell, P. L. J. Med. Chem. 1995, 38, 1657; (b) Chan,
J. H.; Freeman, G. A.; Tidwell, J. H.; Romines, K. R.; Schaller, L. T.; Cowan, J. R.;
Gonzales, S. S.; Lowell, G. S.; Andrews, C., III; Reynolds, D. J. J. Med. Chem. 2004, 47,
1175; (c) Romines, K. R.; Freeman, G. A.; Schaller, L. T.; Cowan, J. R.; Gonzales, S.
S.; Tidwell, J. H.; Andrews, C. W., III; Stammers, D. K.; Hazen, R. J.; Ferris, R. G. J.
Med. Chem. 2006, 49, 727; (d) Ren, J.; Chamberlain, P. P.; Stamp, A.; Short, S. A.;
Weaver, K. L.; Romines, K. R.; Hazen, R.; Freeman, A.; Ferris, R. G.; Andrews, C. W.;
Boone, L.; Chan, J. H.; Stammers, D. K. J. Med. Chem. 2008, 51, 5000.
yl)amino)benzonitrile (10x).
Yield 79.5%; light yellow solid;
mp 182.7–184.3 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.13 (s,
3H, Ar0-CH3), 2.28 (s, 3H, pyrimidine-CH3), 5.80 (s, 1H, CH), 5.99 (s,
1H, OH), 7.15 (d, J = 8.0 Hz, 2H, Ar0H3,5), 7.29 (d, J = 8.0 Hz, 2H,
Ar0H2,6), 7.64 (d, J = 8.8 Hz, 2H, ArH2,6), 7.93 (d, J = 8.8 Hz, 2H,
ArH3,5), 8.30 (s, 1H, pyrimidine H6), 10.10 (s, 1H, NH); 13C NMR
4. (a) Sweeney, Z. K.; Kennedy-Smith, J. J.; Wu, J.; Arora, N.; Billedeau, J. R.;
Davidson, J. P.; Fretland, J.; Hang, J. Q.; Heilek, G. M.; Harris, S. F.; Hirschfeld, D.;
Inbar, P.; Javanbakht, H.; Jernelius, J. A.; Jin, Q.; Li, Y.; Liang, W.; Roetz, R.;
Sarma, K.; Smith, M.; Stefanidis, D.; Su, G.; Suh, J. M.; Villaseñor, A. G.; Welch,
M.; Zhang, F.-J.; Klumpp, K. ChemMedChem 2009, 4, 88; (b) Su, D.-S.; Lim, J. J.;
Tinney, E.; Wan, B.-L.; Young, M. B.; Anderson, K. D.; Rudd, D.; Munshi, V.;
Bahnck, C.; Felock, P. J.; Lu, M.; Lai, M.-T.; Touch, S.; Moyer, G.; DiStefano, D. J.;
Flynn, J. A.; Liang, Y.; Sanchez, R.; Perlow-Poehnelt, R.; Miller, M.; Vacca, J. P.;
Williams, T. M.; Anthony, N. J. J. Med. Chem. 2009, 52, 7163; (c) Tucker, T. J.;
Saggar, S.; Sisko, J. T.; Tynebor, R. M.; Williams, T. M.; Felock, P. J.; Flynn, J. A.;
Lai, M.-T.; Liang, Y.; McGaughey, G. Bioorg. Med. Chem. Lett. 2008, 18, 2959; (d)
Su, D.-S.; Lim, J. J.; Tinney, E.; Tucker, T. J.; Saggar, S.; Sisko, J. T.; Wan, B.-L.;
Young, M. B.; Anderson, K. D.; Rudd, D. Bioorg. Med. Chem. Lett. 2010, 20, 4328.
5. Meng, G.; Chen, F. E.; De Clercq, E.; Balzarini, J.; Pannecouque, C. Chem. Pharm.
Bull. 2003, 51, 779.
(100 MHz, DMSO-d6)
d (ppm): 14.03, 20.66, 73.12, 101.73,
117.87, 119.68, 119.79, 126.57, 128.65, 132.86, 136.26, 138.81,
145.23, 157.52, 159.30, 169.02; MS (ESI+) m/z 331 (M+H)+; Anal.
Calcd for C20H18N4O: C, 72.71; H, 5.49; N, 16.96. Found: C, 72.59;
H, 5.56; N, 16.93.
4.1.5.25. 4-((4-(hydroxy(m-tolyl)methyl)-5-methylpyrimidin-2-
yl)amino)benzonitrile (10y).
Yield 84.1%; light yellow solid;
mp 198.6–199.8 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.15
(s, 3H, Ar0-CH3), 2.28 (s, 3H, pyrimidine-CH3), 5.79 (d, J = 5.2 Hz,
1H, CH), 6.00 (d, J = 4.8 Hz, 1H, OH), 7.08–7.26 (m, 4H, Ar0H), 7.64
(d, J = 8.8 Hz, 2H, ArH2,6), 7.91 (d, J = 8.8 Hz, 2H, ArH3,5), 8.31 (s,
1H, pyrimidine H6), 10.09 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 14.07, 21.09, 73.24, 101.72, 117.85, 119.67,
119.89, 123.76, 127.13, 127.78, 127.98, 132.85, 137.12, 141.78,
145.22, 157.51, 159.36, 168.91; MS (ESI+) m/z 331 (M+H)+; Anal.
Calcd for C20H18N4O: C, 72.71; H, 5.49; N, 16.96. Found: C, 72.67;
H, 5.54; N, 16.90.
6. (a) Dolle, V.; Fan, E.; Nguyen, C. H.; Aubertin, A. M.; Kirn, A.; Andreola, M. L.;
Jamieson, G.; Tarrago-Litvak, L.; Bisagni, E. J. Med. Chem. 1995, 38, 4679; (b) Ji,
L.; Chen, F. E.; Feng, X. Q.; De Clercq, E.; Balzarini, J.; Pannecouque, C. Chem.
Pharm. Bull. 2006, 54, 1248.
7. (a) Mai, A.; Artico, M.; Sbardella, G.; Massa, S.; Novellino, E.; Greco, G.; Loi, A.
G.; Tramontano, E.; Marongiu, M. E.; La Colla, P. J. Med. Chem. 1999, 42, 619; (b)
Ji, L.; Chen, F.-E.; De Clercq, E.; Balzarini, J.; Pannecouque, C. J. Med. Chem. 2007,
50, 1778; (c) Cancio, R.; Mai, A.; Rotili, D.; Artico, M.; Sbardella, G.; Clotet-
Codina, I.; Esté, J. A.; Crespan, E.; Zanoli, S.; Hübscher, U.; Spadari, S.; Maga, G.
ChemMedChem 2007, 2, 445; (d) Radi, M.; Falciani, C.; Contemori, L.; Petricci, E.;
Maga, G.; Samuele, A.; Zanoli, S.; Terrazas, M.; Castria, M.; Togninelli, A.; Esté, J.
A.; Clotet-Codina, I.; Armand-Ugón, M.; Botta, M. ChemMedChem 2008, 3, 573;
(e) Wang, Y.-P.; Chen, F.-E.; Balzarini, J.; De Clercq, E.; Pannecouque, C. Chem.
Biodivers. 2008, 5, 168.
8. (a) Ludovici, D. W.; Kavash, R. W.; Kukla, M. J.; Ho, C. Y.; Ye, H.; De Corte, B. L.;
Andries, K.; de Béthune, M.-P.; Azijn, H.; Pauwels, R.; Moereels, H. E. L.; Heeres,
J.; Koymans, L. M. H.; de Jonge, M. R.; Van Aken, K. J. A.; Daeyaert, F. F. D.; Lewi,
P. J.; Das, K.; Arnold, E.; Janssen, P. A. J. Bioorg. Med. Chem. Lett. 2001, 11, 2229;
(b) Xiong, Y.-Z.; Chen, F.-E.; Balzarini, J.; De Clercq, E.; Pannecouque, C. Eur. J.
Med. Chem. 2008, 43, 1230.
4.1.5.26. 4-((4-(hydroxy(o-tolyl)methyl)-5-methylpyrimidin-2-
yl)amino)benzonitrile (10z).
Yield 85.7%; light yellow solid;
mp 222.8–224.3 °C; 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.13
(s, 3H, Ar0-CH3), 2.23 (s, 3H, pyrimidine-CH3), 5.97 (s, 2H, OH + CH,
1H was deuterium-exchanged), 7.14–7.51 (m, 6H, ArH2,6 + Ar0H),
7.67 (d, J = 8.8 Hz, 2H, ArH3,5), 8.36 (s, 1H, pyrimidine H6), 10.02
(s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 14.98,
18.89, 70.00, 101.56, 117.78, 119.73, 120.48, 125.56, 126.98,
127.05, 129.90, 132.71, 134.95, 140.44, 145.20, 157.55, 159.40,
168.13; MS (ESI+) m/z 331 (M+H)+; Anal. Calcd for C20H18N4O: C,
72.71; H, 5.49; N, 16.96. Found: C, 72.69; H, 5.56; N, 16.92.
9. He, Y.-P.; Long, J.; Zhang, S.-S.; Li, C.; Lai, C. C.; Zhang, C.-S.; Li, D.-X.; Zhang, D.-
H.; Wang, H.; Cai, Q.-Q. Bioorg. Med. Chem. Lett. 2011, 21, 694.
10. (a) 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, A.; 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;
(b) Liang, Y. H.; Feng, X. Q.; Zeng, Z. S.; Chen, F. E.; Balzarini, J.; Pannecouque, C.;
De Clercq, E. ChemMedChem 2009, 4, 1537; (c) Liang, Y.-H.; Chen, F.-E. Eur. J.
Med. Chem. 2009, 44, 625; (d) Liang, Y.-H.; He, Q.-Q.; Zeng, Z.-S.; Liu, Z.-Q.; Feng,
X.-Q.; Chen, F.-E.; Balzarini, J.; Pannecouque, C.; De Clercq, E. Bioorg. Med. Chem.
2010, 18, 4601; (e) Feng, X.-Q.; Zeng, Z.-S.; Liang, Y.-H.; Chen, F.-E.;
Pannecouque, C.; Balzarini, J.; De Clercq, E. Biorg. Med. Chem. 2010, 18, 2370;
(f) Feng, X.-Q.; Liang, Y.-H.; Zeng, Z.-S.; Chen, F.-E.; Balzarini, J.; Pannecouque,
C.; De Clercq, E. ChemMedChem 2009, 4, 219; (g) Zeng, Z.-S.; Liang, Y.-H.; Feng,
X.-Q.; Chen, F.-E.; Pannecouque, C.; Balzarini, J.; De Clercq, E. ChemMedChem
2010, 5, 837; (h) Zeng, Z.-S.; He, Q.-Q.; Liang, Y.-H.; Feng, X.-Q.; Chen, F.-E.; De
Clercq, E.; Balzarini, J.; Pannecouque, C. Bioorg. Med. Chem. 2010, 18, 5039; (i)
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; (j) Rotili, D.; Tarantino, D.; Artico, M.;
Nawrozkij, M. B.; Gonzalez-Ortega, E.; Clotet, B.; Samuele, A.; Este´, J. A.; Maga,
G.; Mai, A. J. Med. Chem. 2011, 54, 3091.
11. (a) Mehellou, Y.; De Clercq, E. J. Med. Chem. 2010, 53, 521; (b) Moreno, S.; López
Aldeguer, J.; Arribas, J. R.; Domingo, P.; Iribarren, J. A.; Ribera, E.; Rivero, A.;
Pulido, F. J. Antimicrob. Chemother. 2010, 65, 827.
12. (a) Udier-Blagovic, M.; Tirado-Rives, J.; Jorgensen, W. L. J. Am. Chem. Soc. 2003,
125, 6016; (b) Nadler, J. P.; Berger, D.; Blick, G.; Cimoch, P.; Cohen, C.;
Greenberg, R.; Hicks, C.; Hoetelmans, R.; Iveson, K.; Jayaweera, D. AIDS 2007,
21, F1; (c) Andries, K.; Azijn, H.; Thielemans, T.; Ludovici, D.; Kukla, M.; Heeres,
J.; Janssen, P.; De Corte, B.; Vingerhoets, J.; Pauwels, R. Antimicrob. Agents
Chemother. 2004, 48, 4680; (d) De Corte, B. L. J. Med. Chem. 2005, 48, 1689; (e)
Heeres, J.; Lewi, P. J. Adv. Antiviral Drug Des. 2007, 5, 213.
4.2. Anti-HIV activity assay
The anti-HIV activity and cytotoxicity of the compounds were
evaluated against wild-type HIV-1 strain IIIB, double RT mutant
(K103N + Y181C) HIV-1 and HIV-2 strain ROD in MT-4 cell cultures
using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) method.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
1 ꢁ 105 cells/mL and infected with HIV at a multiplicity of infection
of 0.02. Immediately after viral infection, 100 lL of the cell suspen-
sion was placed in each well of a flat-bottomed microtiter tray con-
taining various concentrations of the test compounds. The tested
compounds were dissolved in DMSO at 50 mM. After 4 days of
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.
Acknowledgments
13. (a) Janssen, P. A. J.; Lewi, P. J.; Arnold, E.; Daeyaert, F.; de Jonge, M.; Heeres, J.;
Koymans, L.; Vinkers, M.; Guillemont, J.; Pasquier, E. J. Med. Chem. 2005, 48,
1901; (b) Mordant, C.; Schmitt, B.; Pasquier, E.; Demestre, C.; Queguiner, L.;
Masungi, C.; Peeters, A.; Smeulders, L.; Bettens, E.; Hertogs, K. Eur. J. Med. Chem.
2007, 42, 567; (c) van Roey, J.; von Schoen-Angerer, T.; Ford, N.; Calmy, A. Drug
Discovery Today 2008, 13, 601.
We are grateful to the National Natural Science Foundation of
China (No. 30672536) and the K.U. Leuven (GOA No. 10/14) for
the financial support of this research. We thank Mrs. K. Erven
and Mr. K. Uyttersprot for excellent technical assistance.

5124
S.-X. Gu et al. / Bioorg. Med. Chem. 19 (2011) 5117–5124
14. Das, K.; Clark, A. D.; Lewi, P. J.; Heeres, J.; de Jonge, M. R.; Koymans, L. M. H.;
Vinkers, H. M.; Daeyaert, F.; Ludovici, D. W.; Kukla, M. J.; De Corte, B.; 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.
15. (a) Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Virol. Methods 1988, 20, 309; (b) Pannecouque, C.;
Daelemans, D.; De Clercq, E. Nat. Protocols 2008, 3, 427.
16. Miller, V.; de Béthune, M.-P.; Kober, A.; Sturmer, M.; Hertogs, K.; Pauwels, R.;
Stoffels, P.; Staszewski, S. Antimicrob. Agents Chemother. 1998, 42, 3123.
17. (a) Witvrouw, M.; Pannecouque, C.; Van Laethem, K.; Desmyter, J.; De Clercq,
E.; Vandamme, A. M. AIDS 1999, 13, 1477; (b) Witvrouw, M.; Pannecouque, C.;
Switzer, W. M.; Folks, T. M.; De Clercq, E.; Heneine, W. Antiviral Ther. 2004, 9,
57; (c) Dang, Z.; Lai, W.; Qian, K.; Ho, P.; Lee, K.-H.; Chen, C.-H.; Huang, L. J. Med.
Chem. 2009, 52, 7887.