Structural modifications of CH(OH)-DAPYs as new HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.02.030

A series of CR₂(OH)-diarylpyrimidine derivatives (CR ₂(OH)-DAPYs) featuring a hydrophobic group at CH(OH) linker between wing I and the central pyrimidine were synthesized and evaluated for their anti-HIV activity in MT-4 cell cultur

Full text of DOI:10.1016/j.bmc.2014.02.030

Bioorganic & Medicinal Chemistry 22 (2014) 2535–2541
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural modifications of CH(OH)-DAPYs as new HIV-1
non-nucleoside reverse transcriptase inhibitors
a,b,
Zi-Hong Yan a, Xia-Yun Huang a, Hai-Qiu Wu a, Wen-Xue Chen a, Qiu-Qin He a, , Fen-Er Chen
⇑
⇑
,
Erik De Clercq 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 CR2(OH)-diarylpyrimidine derivatives (CR2(OH)-DAPYs) featuring a hydrophobic group at
CH(OH) linker between wing I and the central pyrimidine were synthesized and evaluated for their
anti-HIV activity in MT-4 cell cultures. All the target compounds except for compound 3k displayed
inhibitory activity against HIV-1 wild-type with EC50 values ranging from 7.21 1.99 to
Received 25 December 2013
Revised 18 February 2014
Accepted 22 February 2014
Available online 4 March 2014
0.067 0.006
(EC50 = 0.067 0.006
l
M. Among them, compound 3d showed the most potent anti-HIV-1 activity
M, SI > 592), which was approximately 2-fold more potent than the reference
l
Keywords:
HIV-1
NNRTIs
Diarylpyrimidine
SAR
drugs nevirapine (NVP) and delaviridine (DLV) in the same assay. In addition, the binding modes with
HIV-1 RT and the preliminary SAR studies of these new derivatives were also investigated.
Ó 2014 Elsevier Ltd. All rights reserved.
Antiviral activity
1. Introduction
These CH(OH)-DAPYs had inhibitory potencies in the nanomolar
range against wild-type HIV-1 without significant cytotoxicity at
Diarylpyrimidine derivatives (DAPYs, 1, Fig. 1), represented by
etravirine (1a, Fig. 1) and rilpivirine (1b, Fig. 1), have been regarded
as one of the most successful family of non-nucleoside reverse
transcriptase inhibitors (NNRTIs) developed so far due to their high
potency against both wild-type and drug-resistant HIV-1.1–4 The
crystal structures of HIV-1 RT in complex with DAPYs inhibitors
and SAR studies have revealed that (i) the RT-bound conformations
of DAPYs resemble a ‘U’ or horseshoe shape in the non-nucleoside
inhibitor binding pocket (NNIBP), (ii) the NH linker and the
nitrogen atom on the pyrimidine ring form two hydrogen bonds
with K101 residue of RT, (iii) the para-cyanoaniline substitution
at the position C-2 of the pyrimidine ring is crucial for significant
inhibitory activity.5,6
Over the past few years, considerable structural modifications
of DAPYs focused on wing I, pyrimidine ring, and X linker have
been introduced in an effort to obtain more potent and selective
RT inhibitors.7–20 Our recent program on DAPYs disclosed that
the introduction of a CH(OH) linker between wing I and the central
pyrimidine led to a new promising class of NNRTIs (2, Fig. 1).18
high concentration. As part of our further optimization of these
CH(OH)-DAPYs, we introduced a hydro-phobic group into CH(OH)
linker to investigate whether the added hydrophobic moiety would
occupy more space in hydrophobic cavity formed by the aromatic
residues Tyr181, Tyr188, Phe227, Trp229 of NNIBP and thus reflect
improved antiviral activity. Herein, we report the synthesis and
antiviral activities of this series of CR2(OH)-DAPYs (3, Fig. 1).
2. Chemistry
The synthetic route to the target compounds 3a–r is depicted in
Scheme 1. The key intermediates 4-(4-benzoylpyrimidin-2-ylami-
no)-benzonitriles 9a–o were prepared from 4a–c according to our
previously reported protocol in 5 steps.7 Grignard reaction of 9a–o
with appropriate alkyl magnesium chloride in the present of anhy-
drous THF afforded the target compounds 3a–r in 37–72% yield.
3. Results and discussion
3.1. Biological activity
⇑
Corresponding authors. Tel.: +86 21 65643809; fax: +86 21 65643811.
E-mail addresses: qqhe@fudan.edu.cn (Q.-Q. He), rfchen@fudan.edu.cn
The newly synthesized compounds 3a–r were evaluated
for their anti-HIV activity in comparison with nevirapine (NVP),
(F.-E. Chen).
http://dx.doi.org/10.1016/j.bmc.2014.02.030
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2536
Z.-H. Yan et al. / Bioorg. Med. Chem. 22 (2014) 2535–2541
CN
CN
NH
modificaiton
allowed for
improving
potency
crucial for
anti-HIV
potency
Trp229
Phe227
Tyr181
R1
R1
I
II
NH
HO
R2
R3
X
N
N
Tyr188
N
N
R3
H-bonds
with Lys101
changeable for
potency &
physicochemical
properties
R4
R4
CR2(OH)-DAPYs (3)
DAPYs (1)
CN
CN
O
CN
CN
CN
R1
N
NH
N
NH
HO
R3
HN
N
NH
N
N
Br
N
NH2
R4
etravirine (1a)
rilpivirine (1b)
CH(OH)-DAPYs (2)
Figure 1. The structures of DAPYs, CH(OH)-DAPYs and newly designed CR2(OH)-DAPYs.
H
N
H
N
H
S
N
O
S
O
N
Cl
N
N
O
a
b
c
HN
R4
HN
N
HN
R4
NC
R4
NC
R4
R3
R3
R3
R3
5a-c
7a-c
4a-c
6a-c
O
R2
OH
R1
CN
R1
R4
H
N
H
N
H
N
N
N
N
d
e
f
R1
N
N
N
R4
NC
R4
NC
NC
R3
R3
R3
9a-o
8a-o
R2
3a-r
R3
1
R1
4-OMe-Ph
4-t-butyl-Ph
Ph
4-Me-Ph
4-F-Ph
4-Cl-Ph
4-Br-Ph
3-F-Ph
3-Cl-Ph
R3
R4
1
R1
R2
R4
H
Me
H
H
H
H
3-Br-Ph
4-F-Ph
4-F-Ph
1-Naphthyl
4-Br-Ph
4-Br-Ph
4-Br-Ph
4-t-butyl-Ph
4-t-butyl-Ph
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
vinyl
Et
cyclopropanyl
Me
Me
H
Me
H
Me
Me
Scheme 1. Synthetic route to compounds 3a–r. Reagents and conditions: (a) MeI, NaOH, H2O, rt, 24 h; (b) 4-cyanoaniline, 180–190 °C, 8 h; (c) POCl3, reflux, 0.5 h; (d)
substituted phenylactonitrile, 60% NaH, Ar, DMF, À20 °C to rt, 48–72 h; (e) air, rt, 48–72 h; (f) R2MgCl, N2, anhydrous THF, À20 °C to rt, 2 h.
zidovudine (AZT), efavirenz (EFV), delaviridine (DLV) and
etravirine (ETV) as reference drugs. The results are summarized
in Table 1, expressed as EC50 (anti-HIV activity), CC50 (cytotoxicity)
and SI (selectivity index).
As shown in Table 1, all the target compounds 3a–r exhibited
moderate to good antiviral activity against wild-type HIV-1 (LAI
strain IIIB) with EC50 values ranging from 7.21 1.99 to
0.067 0.006 M, except for compound 3k. Most of them were
as potent as DLV against wild-type HIV-1 in submicromolar level,
but they lacked inhibitory activity against the double mutant virus
(K103N+Y181C) and HIV-2 strain ROD. Compound 3d displayed
the highest potency against HIV-1 with an EC50 value of
l

Z.-H. Yan et al. / Bioorg. Med. Chem. 22 (2014) 2535–2541
2537
Table 1
Anti-HIV-1 activities and cytotoxicity of compounds 3a–r in MT-4 cellsa
b
c
Compounds
EC50
[lM]
CC50
[
lM]
SId
WT (IIIB)
K103N+Y181C
HIV-2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
NVP
AZT
EFV
DLV
ETVf
0.346 0.144
>35.9
>31.8
>45.4
>39.9
>43.1
>39.0
>28.2
>53.2
>37.5
>33.7
NDe
>38.2
>30.7
>31.7
>31.3
>33.2
>31.1
>30.4
0.901
0.008
0.507
>43.80
0.034 0.00045
>35.9
>31.8
>45.4
>39.9
>43.1
>39.0
>28.2
>49.6
>37.5
>33.7
>61.5
>38.2
>30.7
>31.7
>31.3
>33.2
>31.1
>30.4
NDe
35.9 3.6
31.8 1.2
45.4 2.1
39.3 3.7
43.1 2.7
39.0 2.4
28.2 2.5
53.2 10.7
37.5 3.2
33.7 1.4
61.5 5.9
38.2 2.0
30.7 3.0
31.7 1.9
31.3 1.5
33.2 1.8
31.1 0.3
30.4 1.2
>15.0
104
56
158
592
17
123
92
264
146
176
<1
11
79
89
15
11
4
24
>148
0.564
0
0.288 0.057
0.067 0.006
2.503 1.226
0.317 0.114
0.305 0.025
0.201 0.074
0.257 0.051
0.191 0.005
>61.5
3.549 2.153
0.388 0.081
0.355 0.025
2.073 0.147
3.109 0.190
7.206 1.992
1.246 0.051
0.101 0.015
0.007 0.001
0.022
0.006 0.0
>93.6
>6.3
>43.8
27.79 11.54
>14124
>280
>54
NDe
0.810
0.002 0.0004
NDe
>27.79
12884
a
b
c
d
e
f
Data represent the mean of at least three separate experiments.
Compound concentration required to inhibit MT-4 cells against viral cytopathogenicity by 50%.
Compound concentration that decreases the uninfected MT-4 cell viability by 50%.
Selectivity index:CC50/IC50 ratio.
Not detected.
The data were evaluated in the same biological laboratory with the same essay.
0.067 0.006
l
M and a selectivity index value of 592. It proved
as the template and primer, NVP, EFV and ETV as references. All
the tested compounds showed moderate inhibitory activity against
HIV-1 RT. As consistent with SAR information obtained in antiviral
evaluation in MT-4 cells, compound 3d still exhibited the most
more potent by approximately 2-fold than NVP and DLV.
In terms of SARs, it seemed that the introduction of a bulky
hydrophobic substituent into CH(OH) linker between wing I and
the central pyrimidine is not tolerable. The inhibitory activity
generally decreased in the order: Me (3g) > Vinyl (3n) > Et
(3o) > cycloPr (3p), with a bromide atom at the para position of
the wing I.
potent activity against HIV-1 RT with an IC50 of 0.16 lM, which
was higher than that of NVP but lower than those of EFV
and ETV. The results suggested that these newly synthesized
CR2(OH)-DAPYs target HIV-1 RT.
The substitution at pyrimidine ring could significantly influence
the antiviral potency. Compound 3b (R1 = 4-t-Butyl, R2 = Me,
R3 = R4 = H) was 2-fold more active than compound 3r (R1 = 4-t-
Butyl, R2 = Me, R3 = Me, R4 = H), and 13-fold more active than 3q
(R1 = 4-t-Butyl, R2 = Me, R3 = H, R4 = Me). Similarly, compound 3e
(R1 = 4-F, R2 = Me, R3 = R4 = H) showed higher activity than that of
3l (R1 = 4-F, R2 = Me, R3 = Me, R4 = H), whereas compound 3k
(R1 = 4-F, R2 = Me, R3 = H, R4 = Me) completely lost potency against
HIV-1. These results suggested that no substituent at pyrimidine
ring may be favorable for antiviral potency.
Following these observations, structural modifications at wing I
were investigated. An appropriate electron-donating group intro-
duced at the C-4 position of Wing I of 3c is favorable to anti-HIV
activities. For example, 3d featuring a methyl group at C-4 position
of Wing I displayed improved anti-HIV-1 activity. However, the
anti-HIV activities of 3a with a methoxy and 3b with a tert-butyl
group decreased to a certain degree. Interestingly, the introduction
of a halogen group at the C-4 position of 3a led to compounds
3e–g, whose inhibitory activity increased in the following order:
F (3e) < Cl (3f) < Br (3g). Among them, compound 3g showed an
3.2. Molecular modeling analysis
To understand the binding modes of the newly synthesized
CR2(OH)-DAPYs with HIV-1 RT, molecular modeling was conducted
using Sybyl Surflex-Dock program and the crystal structure of
wild-type RT in complex with ETV (PDB ID: 3MEC). The represen-
tative compounds 3d and 3p were chosen for docking analysis,
considering their structural diversity and potency. Compound 3d
was the most active one among the newly synthesized compounds
with a methyl group at CH(OH) linker, while 3p showed less active
with a cyclopropyl group at CH(OH) linker.
As shown in Figure 2, both representative compounds and ETV
were superposed into NNIBP in a similar horseshoe conformational
shape. The docking simulation clearly showed that 3d and 3p
formed two typical hydrogen bonds, which were crucial for
Table 2
Activity of selected compounds against wt HIV-1 RT polymerization
a
Compound
IC50
[
l
M]
EC50 value with 0.305 0.025 lM, which was comparable to that
3c
2.00 0.11
of compound 3c. Additionally, the inhibitory activity also appeared
to be influenced by the position of the introduced halogen group.
Compounds 3e–g were less active than the corresponding com-
pounds 3h–j with a halogen substituent at the C-3 position of 3c.
To further confirm the binding target, compounds 3c, 3d, 3h
and 3j were taken as the representative compounds to carry out
HIV-1 RT inhibitory assays (Table 2), using poly(rA)/oligo(dt)16
3d
3h
3j
NVP
EFV
ETV
0.16 0.00
0.63 0.084
0.39 0.030
1.78 0.19
0.038 0.019
0.016 0.0069
a
Compound concentration required to inhibit HIV-1 RT wt activity by 50%.

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Z.-H. Yan et al. / Bioorg. Med. Chem. 22 (2014) 2535–2541
Figure 2. Predicted binding mode of the representative compounds in NNIBP of wild type HIV-1 RT (PDB ID: 3MEC): (a) compound 3d (red) and 3p (blue), ETV (green) was
superposed into the same pocket for comparison, (b) compound 3d (red) and 3p (blue). Possible hydrogen bonds between the inhibitors and the residues are indicated with
dashed lines (yellow). The side chains of key amino acids on the binding site surface are illustrated with solid lines (gray).
anti-HIV activity of CR2(OH)-DAPYs. One hydrogen bond was be-
tween the nitrogen atom of pyrimidine ring and the residue
Lys101, and the other involved the linker of NH and Lys103. Nota-
bly, the 4-substitued-benzyl moiety was almost parallel with the
tetramethylsilane (TMS). Mass spectra were obtained on a Waters
Quattro Micromass instrument using electrospray ionization (ESI)
techniques. Infrared spectra (IR) were recorded with a nexus FT/
IR-4200 spectro-meter. All chemicals and solvents used were of re-
agent grade and were purified and dried by standard methods be-
fore use. All air-sensitive reactions were run under a nitrogen
atmosphere. All the reactions were monitored by thin layer chro-
matography (TLC) on pre-coated silica gel G plates at 254 nm un-
der a UV lamp using ethyl acetate/petroleum ether as eluent.
Flash chromatography separations were obtained on silica gel
(300–400 mesh). All yields are unoptimized and only represent
one experiment’s result.
aromatic amino acid residues Tyr181, which resulted in strong p-
stacking interaction. However, the orientation of the horseshoe
shape for 3d and 3p moved downward when comparing with
ETV and no interaction with the residues Phe227 and Trp229 could
be obviously observed, which may determine the loss of the inhib-
itory activity against mutant virus. As observed in Figure 2b, the
cyclopropyl group of 3p was protruded out of NNIBP when com-
pared to 3d bound with HIV-1 RT structure, indicating that the
large moiety is not favorable for inhibitory activity.
5.2. General procedure for preparation of target compounds
4. Conclusion
To a solution of 9a–o (1 mmol) in THF (3.5 ml) was added
appropriate alkyl magnesium chloride (2 M, in THF, 3 mmol) drop-
wise at À20 °C under N2. After addition, the resulting mixture was
stirred at room temperature for 2 h, then quenched by saturated
NH4Cl solution and extracted with ethyl acetate (20 ml  3). The
combined organic phases was washed with brine, dried over anhy-
drous Na2SO4, filtered and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (ethyl acetate/
hexane, 1:3 to 1:1) to give the desired compounds.
In conclusion, we designed and synthesized a new series of
CR2(OH)-DAPYs featuring a hydrophobic group at CH(OH) linker
between wing I and the central pyrimidine. Most of these target
compounds displayed anti-HIV-1 activity with micromolar to
nanomolar EC50 values in infected MT-4 cells. Compound 3d
showed the most potency against wild-type HIV-1 with an EC50 va-
lue of 0.067 0.006 lM and a selectivity index value of 592, which
was 2-fold more active than the reference drugs NVP and DLV.
However, these compounds were inactive against the double mu-
tant type virus K103N+Y181C and HIV-2 strain ROD. The HIV-1
RT assay confirmed that the biological target of these newly syn-
thesized CR2(OH)-DAPYs is HIV-1 RT. The binding modes with
HIV-1 RT and the preliminary SAR studies revealed that the intro-
duction of a small hydrophobic group into CH(OH) linker could oc-
cupy more space in hydrophobic cavity lined with Tyr181, Tyr188,
Phe227, Trp229 of NNIBP and retained low cellular inhibitory
potencies, but large moiety is not favorable for inhibitory activity.
5.2.1. 4-((4-(1-Hydroxy-1-(4-methoxyphenyl)ethyl)pyrimidin-
2-yl)amino)benzonitrile (3a)
Yield 57%; light yellow solid; mp 184.1–184.9 °C; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 1.87 (s, 3H, Me), 3.69 (s, 3H, OMe),
5.99 (s, 1H, OH), 6.87 (d, J = 8.8 Hz, 2H, ArH2,6), 7.23 (d, J = 5.2 Hz,
1H, pyrimidine H5), 7.44 (d, J = 8.8 Hz, 2H, ArH3,5), 7.69 (d,
J = 8.8 Hz, 2H, ArH2,6), 7.93 (d, J = 8.8 Hz, 2H, ArH3,5), 8.49 (d,
J = 5.2 Hz, 1H, pyrimidine H6), 10.08 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm) 28.71, 55.08, 75.06, 102.27, 109.21,
113.29, 118.26, 126.90, 133.00, 139.15, 145.06, 158.11, 158.63,
158.66, 176.71; IR (KBr, cmÀ1) 2215 (CN); MS (ESI) m/z 347
[M+H]+.
5. Material and methods
5.1. Chemistry
5.2.2. 4-((4-(1-(4-(tert-Butyl)phenyl)-1-ydroxyethyl)pyrimidin-
2-yl)amino)benzonitrile (3b)
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
Yield 61%; white solid; mp 141.7–142.2 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.20 (s, 9H, t-butyl), 1.87 (s, 3H, Me), 6.01 (s, 1H,

Z.-H. Yan et al. / Bioorg. Med. Chem. 22 (2014) 2535–2541
2539
OH), 7.23 (d, J = 5.2 Hz, 1H, pyrimidine H5), 7.31 (d, J = 8.4 Hz,
2H, ArH2,6), 7.44 (d, J = 8.4 Hz, 2H, ArH3,5), 7.68 (d, J = 8.8 Hz, 2H,
ArH2,6), 7.92 (d, J = 8.8 Hz, 2H, ArH3,5), 8.48 (d, J = 5.2 Hz, 1H,
pyrimidine H6), 10.07 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
75.23, 102.42, 109.29, 118.35, 120.20, 128.17, 130.90, 133.04,
145.01, 146.51, 158.73, 158.96, 175.93; IR (KBr,cmÀ1) 2224 (CN);
MS (ESI) m/z 393 [M–H]À.
d
(ppm) 28.68, 31,20, 34,14, 75.25, 102.30, 109.36, 118.31,
5.2.8. 4-((4-(1-(3-Fluorophenyl)-1-hydroxyethyl)pyrimidin-2-
yl)amino)benzonitrile (3h)
124.75, 125.44, 133.02, 144.07, 145.09, 149.12, 158.69, 176.59; IR
(KBr, cmÀ1) 2217 (CN); MS (ESI) m/z 373 [M+H]+.
Yield 60%; white solid; mp 150.1–150.8 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.88 (s, 3H, Me), 6.23 (s, 1H, deuterium-ex-
changed, OH), 7.02–7.08 (m, 1H, ArH5), 7.23 (d, J = 5.2 Hz, 1H,
pyrimidine H5), 7.32–7.38 (m, 3H, ArH2,4,6), 7.69 (d, J = 8.8 Hz, 2H,
ArH2,6), 7.91 (d, J = 8.8 Hz, 2H, ArH3,5), 8.52 (d, J = 5.2 Hz, 1H,
pyrimidine H6), 10.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm) 28.65, 75.08, 102.29, 109.21, 112.51 (d, JCF = 22.3 Hz),
113.61, 118.22, 119.57, 121.82, 122.28 (d, JCF = 2.5 Hz), 129.87 (d,
JCF = 8.1Hz), 132.93, 144.91, 158.73 (d, JCF = 22.3 Hz), 176.16; IR
(KBr, cmÀ1) 2220 (CN); MS (ESI) m/z 335 [M+H]+.
5.2.3. 4-((4-(1-Hydroxy-1-phenylethyl)pyrimidin-2-yl)amino)
benzonitrile (3c)
Yield 37%; yellow solid; mp 149.0–150.2 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.89 (s, 3H, Me), 6.07 (s, 1H, OH), 7.18–7.22
(m, 1H, ArH3), 7.24 (d, J = 4.8 Hz, 1H, pyrimidine H5), 7.29–7.33
(d, J = 7.6 Hz, 2H, ArH3,5), 7.54 (d, J = 7.6 Hz, 2H, ArH2,6), 7.69
(d, J = 8.8 Hz, 2H, ArH2,6), 7.92 (d, J = 8.8 Hz, 2H, ArH3,5), 8.50 (d,
J = 4.8 Hz, 1H, pyrimidine H6), 10.10 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6)
d (ppm) 29.65, 75.33, 102.25, 109.32,
118.22, 125.65, 126.72, 127.91, 132.93, 144.99, 146.98, 158.61,
158.63, 176.39; IR (KBr,cmÀ1): 2217 (CN); MS (ESI) m/z 317
[M+H]+.
5.2.9. 4-((4-(1-(3-Chlorophenyl)-1-hydroxyethyl)pyrimidin-2-
yl)amino)benzonitrile (3i)
Yield 56%; yellow solid; mp 103.7–104.1 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.87 (s, 3H, Me), 6.25 (s, 1H, OH), 7.23 (d,
J = 5.2 Hz, 1H, pyrimidine H5), 7.30 (d, J = 8.0 Hz, 1H, ArH6), 7.34–
7.38 (m, 1H, ArH5), 7.48 (d, J = 7.6 Hz, 1H, ArH4), 7.57 (s, 1H,
ArH2), 7.70 (d, J = 8.8 Hz, 2H, ArH2,6), 7.89 (d, J = 8.8 Hz, 2H,
ArH3,5), 8.53 (d, J = 5.2 Hz, 1H, pyrimidine H6), 10.13 (s, 1H, NH);
5.2.4. 4-((4-(1-Hydroxy-1-(p-tolyl)ethyl)pyrimidin-2-yl)amino)
benzonitrile (3d).
Yield 40%; light yellow solid; mp 144.1–144.7 °C; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 1.87 (s, 3H, Me), 2.23 (s, 3H, Me),
5.99 (s, 1H, OH), 7.11 (d, J = 7.6 Hz, 2H, ArH3,5), 7.22(d, J = 4.8 Hz,
1H, pyrimidine H5), 7.41 (d, J = 7.6 Hz, 2H, ArH2,6), 7.70
(d, J = 8.8 Hz, 2H, ArH2,6), 7.94 (d, J = 8.0 Hz, 2H, ArH3,5), 8.49 (d,
J = 4.8 Hz, 1H, pyrimidine H6), 10.10 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm) 20.56, 28.62, 75.22, 102.25, 109.29,
118.24, 125.58, 128.48, 132.96, 135.81, 144.11, 145.04, 158.58,
158.63, 176.57; IR (KBr, cmÀ1) 2214 (CN); MS (ESI) m/z 329
[MÀH]À.
13C NMR (100 MHz, MSO-d6)
d (ppm) 29.60, 75.04, 102.28,
109.12, 118.21, 124.51, 125.52, 126.72, 129.87, 132.73, 132.91,
144.87, 149.47, 158.61, 158.91, 175.59; IR (KBr, cmÀ1) 2218 (CN);
MS (ESI) m/z 351 [M+H]+.
5.2.10. 4-((4-(1-(3-Bromophenyl)-1-hydroxyethyl)pyrimidin-2-
yl)amino)benzonitrile (3j)
Yield 69%; light yelllow solid; mp 132.1–132.7 °C; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 1.88 (s, 3H, Me), 6.25 (s, 1H, OH),
7.24 (d, J = 5.2 Hz, 1H, pyrimidine H5), 7.29 (d, J = 8.0 Hz, 1H,
ArH5), 7.40 (d, J = 8.0 Hz, 1H, ArH6), 7.53 (d, J = 8.0 Hz, 1H, ArH4),
7.69 (d, J = 8.8 Hz, 2H, ArH2,6), 7.74 (s, 1H, ArH2), 7.91 (d,
J = 8.8 Hz, 2H, ArH3,5), 8.52 (d, J = 5.2 Hz, 1H, pyrimidine H6),
10.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm) 28.65,
75.06, 102.36, 109.12, 118.27, 121.48, 124.94, 128.47, 129.63,
130.15, 132.91, 144.91, 149.74, 158.67, 158.90, 175.63; IR (KBr,
cmÀ1) 2224 (CN); MS (ESI) m/z 395 [M+H]+.
5.2.5. 4-((4-(1-(4-Fluorophenyl)-1-hydroxyethyl)pyrimidin-2-
yl)amino)benzonitrile (3e)
Yield 72%; yellow solid; mp 132.7–133.6 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.88 (s, 3H, Me), 6.15 (s, 1H, OH), 7.10–7.15(m,
2H, Ar H3,5), 7.23(d, J = 5.2 Hz, 1H, pyrimidine H5), 7.54–7.58 (m,
2H, ArH2,6), 7.69 (d, J = 8.8 Hz, 2H, ArH2,6), 7.92 (d, J = 8.8 Hz, 2H,
ArH3,5), 8.50 (d, J = 4.8 Hz, 1H, pyrimidine H6), 10.11 (s, 1H, NH);
13C NMR (100 MHz, DMSO-d6) d (ppm) 28.82, 75.08, 102.35,
109.18, 114.58 (d, JCF = 21 Hz), 118.27, 119.65, 127.77 (d, JCF
=
)
8.0 Hz), 132.96, 144.99, 158.69, 158.79, 176.19; IR (KBr, cmÀ1
5.2.11. 4-((4-(1-(4-Fluorophenyl)-1-hydroxyethyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (3k)
2219(CN); MS (ESI) m/z 335 [M+H]+.
Yield 65%; white solid; mp 205.4–206.2 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.84 (s, 3H, Me), 1.92 (s, 3H, Me), 6.11 (s, 1H,
OH), 7.09–7.13 (m, 2H, ArH3,5), 7.33–7.36 (m, 2H, ArH2,6), 7.71 (d,
J = 8.8 Hz, 2H, ArH2,6), 7.98 (d, J = 8.8 Hz, 2H, ArH3,5), 8.50 (s, 1H,
pyrimidine H6), 10.06 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
5.2.6. 4-((4-(1-(4-Chlorophenyl)-1-hydroxyethyl)pyrimidin-2-
yl)amino)benzonitrile (3f)
Yield 65%; light yellow solid; mp 162.9–163.7 °C; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 1.87 (s, 3H, Me), 6.18 (s, 1H, OH),
7.22 (d, J = 5.2 Hz, 1H, pyrimidine H5), 7.36 (d, J = 8.4 Hz, 2H, Ar
d
(ppm) 15.62, 31.38, 76.95, 101.80, 114.64 (d, JCF = 22 Hz),
H
3,5), 7.54 (d, J = 8.4 Hz, 2H, ArH2,6), 7.68 (d, J = 8.8 Hz, 2H,
117.85, 119.67, 120.83, 126.82 (d, JCF = 8.1Hz), 133.00, 145.28,
156.77, 160.43, 170. 91; IR (KBr, cmÀ1): 2209 (CN); MS (ESI) m/z
349 [M+H]+.
ArH2,6), 7.90 (d, J = 8.8 Hz, 2H, ArH3,5), 8.50 (d, J = 4.8 Hz, 1H, pyrim-
idine H6), 10.11 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm)
28.62, 75.07, 102.33, 109.19, 118.24, 127.67, 127.86, 131.55,
132.93, 144.94, 146.00, 158.66, 158.81, 175.90; IR (KBr, cmÀ1
)
5.2.12. 4-((4-(1-(4-Fluorophenyl)-1-hydroxyethyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (3l)
2220 (CN); MS (ESI) m/z 349 [MÀH]À.
Yield 52%; white solid; mp 175.2–176.6 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.84 (s, 3H, Me), 1.92 (s, 3H, Me), 6.11
(s, 1H, OH), 7.11–7.15 (m, 3H, pyrimidine H6+ArH3,5), 7.53–7.57
(m, 2H, ArH2,6), 7.68 (d, J = 8.8 Hz, 2H, ArH2,6), 7.91 (d, J = 8.8 Hz,
2H, ArH3,5), 10.06 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm) 23.85, 28.87, 74.94, 102.04, 108.34, 114.49 (d, JCF = 21Hz),
118.09, 119.65, 127.70 (d, JCF = 8.0 Hz), 132.92, 145.14, 158.53,
168.43, 175. 81; IR (KBr, cmÀ1) 2223 (CN); MS (ESI) m/z 349
[M+H]+.
5.2.7. 4-((4-(1-(4-Bromophenyl)-1-hydroxyethyl)pyrimidin-2-
yl)amino)benzonitrile (3g)
Yield 70%; yellow solid; mp 169.4–170.6 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.86 (s, 3H, Me), 6.21 (s, 1H, OH), 7.21
(d, J = 4.8 Hz, 1H, pyrimidine H5), 7.47 (d, J = 9.6 Hz, 2H, ArH3,5),
7.50 (d, J = 9.2 Hz, 2H, ArH2,6), 7.68 (d, J = 8.0 Hz, 2H, ArH2,6), 7.88
(d, J = 8.0 Hz, 2H, ArH3,5), 8.50 (d, J = 4.4 Hz, 1H, pyrimidine H6),
10.09 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm) 28.63,

2540
Z.-H. Yan et al. / Bioorg. Med. Chem. 22 (2014) 2535–2541
5.2.13. 4-((4-(1-Hydroxy-1-(naphthalen-1-yl)ethyl)pyrimidin-2-
yl)amino)benzonitrile (3m)
ArH2,6), 7.94 (d, J = 8.8 Hz, 2H, ArH3,5), 10.04 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6) d (ppm) 24.32, 29.17, 31,59, 75. 52,
102.41, 108.88, 118.53, 120.12, 125.06, 125.77, 133.38, 144.62,
145.67, 149.37, 158.97, 168.66, 176.64; IR (KBr, cmÀ1) 2218 (CN);
MS (EI) m/z 385 [MÀH]À.
Yield 60%; yellow solid; mp 188.4–189.0 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.99 (s, 3H, Me), 6.35 (s, 1H, OH), 7.25–8.16
(m, 12H, pyrimidine H5+ArH4+napH7), 8.57 (d, J = 5.2 Hz, 1H,
pyrimidine H6), 9.99 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
(ppm) 30.51, 76.62, 102.01, 109.33, 118.00, 124.48, 124.88,
124.99, 125.06, 126.86, 128.34, 128.50, 130.81, 132.57, 134.17,
141.84, 144.82, 158.50, 158.78, 177.40; IR (KBr, cmÀ1) 2217 (CN);
MS (ESI) m/z 367 [M+H]+.
5.3. Anti-HIV activity assay
The anti-HIV activity and cytotoxicity of the compounds 3a–r
were evaluated against wild type HIV-1 strain IIIB, a double RT
mutant (K103N+Y181C) HIV-1 strain and HIV-2 strain ROD in
MT-4 cell cultures using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) method.21,22 Briefly, stock
solutions (10Â final concentration) of test compounds were added
5.2.14. 4-((4-(1-(4-Bromophenyl)-1-hydroxyallyl)pyrimidin-2-
yl)amino)benzonitrile (3n)
Yield 57%; light yellow solid; mp 107.7–108.5 °C; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 1.86 (s, 3H, Me), 6.21 (s, 1H, OH),
7.21 (d, J = 4.8 Hz, 1H, pyrimidine H5), 7.47 (d, J = 9.6 Hz,
2H, ArH3,5), 7.50 (d, J = 9.2 Hz, 2H, ArH2,6), 7.68 (d, J = 8.0 Hz, 2H,
ArH2,6), 7.88 (d, J = 8.0 Hz, 2H, ArH3,5), 8.50 (d, J = 4.4 Hz, 1H,
pyrimidine H6), 10.09 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
d (ppm) 77.98, 102.35, 109.70, 114.37, 118.25, 120.41, 128.95,
130.85, 132.91, 142.05, 144.70, 1446.84, 158.52, 159.05, 174.26;
IR (KBr, cmÀ1) 2218 (CN); MS (ESI) m/z 405 [MÀH]À.
in 25 ll volumes to two series of triplicate wells so as to allow
simultaneous evaluation of their effects on mock-and HIV-infected
cells at the beginning of each experiment. Serial 5-fold dilutions of
test compounds were made directly in flat-bottomed 96-well
microtiter trays using a Biomek 3000 robot (Beckman instru-
ments). Untreated control HIV-1 and mock-infected cell samples
were included for each sample. Virus stock (50 ll) at 100–300
CCID50 (50% cell culture infectious dose) or culture medium was
added to either the virus-infected or mock-infected cells of the
microtiter tray. Mock-infected cells were used to evaluate the
effect of test compounds on uninfected cells in order to assess
the cytotoxicity of the test compounds. Exponentially growing
MT-4 cells were centrifuged for 5 min at 220 g and the supernatant
was discarded. The MT-4 cells were resuspended at 6 Â 10 cells/ml
5.2.15. 4-((4-(1-(4-Bromophenyl)-1-hydroxypropyl)pyrimidin-
2-yl)amino)benzonitrile (3o)
Yield 66%; yellow solid; mp 114.6–115.8 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 0.74–0.78 (m, 3H, Me), 2.21–2.40 (m, 2H, CH2),
5.93 (s, 1H, OH), 7.21 (d, J = 5.2 Hz, 1H, pyrimidine H5), 7.46–7.52
(m, 4H, ArH2,6+ArH3,5), 7.68 (d, J = 8.8 Hz, 2H, ArH2,6), 7.90
(d, J = 8.8 Hz, 2H, ArH3,5), 8.48 (d, J = 5.2 Hz, 1H, pyrimidine H6),
10.08 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d (ppm) 8.05,
32.79, 77.76, 102.50, 110.04, 118.38, 119.76, 120.20, 128.44,
and 50 lL volumes were transferred to the microtiter tray wells.
Five days after infection, the viability of mock-and HIV-infected
cells was examined spectrophoto-metrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored MTT
(Acros Organics) by mitochondrial dehydrogenase activity of meta-
bolically active cells to a blue-purple formazan that can be measured
spectrophotometrically. The absorbances were read in an eight-
channel computer-controlled photometer (Infinite M1000, Tecan)
at two wavelengths (540 and 690 nm). All datas were calculated
using the median OD (optical density) values of tree wells. The
50% cytotoxic concentration (CC50) was defined as the concentration
of the test compounds that reduced the absorbance (OD540) of the
mock-infected control sample by 50%. The concentration achieving
50% protection from the cytopathic effect of the virus in infected
cells was defined as the 50% effective concentration (EC50).
130.88, 133.10, 145.04, 145.38, 158.80, 175.19; IR (KBr, cmÀ1
)
2216 (CN); MS (ESI) m/z 407 [MÀH]À.
5.2.16. 4-((4-((4-Bromophenyl)(cyclopropyl)(hydroxy)methyl)
pyrimidin-2-yl)amino)benzonitrile (3p)
Yield 67%; light yellow solid; mp 102.6–103.1 °C; 1H NMR
(400 MHz, DMSO-d6) d (ppm) 0.33–0.55 (m, 4H, cyclopropyl
H2–5), 1.87–1.92 (m, 1H, cyclopropyl H1), 5.64 (s, 1H, OH), 7.23
(d, J = 4.8 Hz, 1H, pyrimidine H5), 7.49–7.56 (m, 4H, ArH3,5+ArH2,6),
7.64 (d, J = 8.4 Hz, 2H, ArH2,6), 7.83 (d, J = 8.8 Hz, 2H, ArH3,5), 8.55
(d, J = 5.2 Hz, 1H, pyrimidine H6), 10.09 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) d (ppm) 14.09, 19.69, 20.77, 75.51, 102.24,
109.57, 118.18, 120.21, 129.08, 130.71, 132.86, 144.91, 146.03,
158.56, 158.79, 175.52; IR (KBr, cmÀ1) 2224 (CN); MS (ESI) m/z
419 [MÀH]À.
5.4. HIV-1 RT assay
Recombinant wild type p66/p51 HIV-1 RT was expressed and
purified as described by Auwerx et al.23 The RT assay is performed
with the EnzCheck Reverse Transcriptase Assay kit (Molecular
Probes, Invitrogen), as described by the manufacturer. The assay
is based on the dsDNA quantitation reagent PicoGreen. This
reagent shows a pronounced increase in fluorescence signal upon
binding to dsDNA or RNA-DNA heteroduplexes. Single-stranded
nucleic acids generate only minor fluorescence signal enhance-
ment when a sufficiently high dye:base pair ratio is applied.24 This
condition is met in the assay.
5.2.17. 4-((4-(1-(4-(tert-Butyl)phenyl)-1-hydroxyethyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (3q)
Yield 58%; white solid; mp 119.2–120.3 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.23 (s, 9H, t-butyl), 1.84 (s, 3H, Me), 1.93 (s, 3H,
Me), 5.96 (s, 1H, OH), 7.25 (d, J = 8.4 Hz, 2H, ArH2,6), 7.31
(d, J = 8.4 Hz, 2H, ArH3,5), 7.71 (d, J = 8.8 Hz, 2H, ArH2,6), 8.00 (d,
J = 8.8 Hz, 2H, ArH3,5), 8.21 (s, 1H, pyrimidine H6), 10.05 (s, 1H,
NH); 13C NMR(100 MHz, DMSO-d6) d (ppm) 16.26, 31,63, 31.82,
77.55, 102.20, 118.29, 120.15, 121.37, 125.01, 125.10, 133.47,
A poly(rA) template of approximately 350 bases long, and an
143.91, 145.81, 149.14, 157.22, 160.69, 171.81; IR (KBr, cmÀ1
)
oligo(dT)16 primer, are annealed in
(60 min at room temperature). Fifty-two ng of the RNA/DNA is
brought into each well of a 96-well plate in a volume of 20
polymerization buffer (60 mM Tris–HCl, 60 mM KCl, 8 mM MgCl2,
13 mM DTT, 100 M dTTP, pH 8.1). 5 l of RT enzyme solution,
a molar ratio of 1:1.2
2219 (CN); MS (ESI) m/z 387 [M+H]+.
ll
5.2.18. 4-((4-(1-(4-(tert-Butyl)phenyl)-1-hydroxyethyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (3r)
l
l
Yield 70%; white solid; mp 198.9–199.3 °C; 1H NMR (400 MHz,
DMSO-d6) d (ppm) 1.22 (s, 9H, t-butyl), 1.86 (s, 3H, Me), 2.38 (s, 3H,
Me), 5.93 (s, 1H, OH), 7.15 (s, 1H, pyrimidine H5), 7.32 (d, J = 8.4 Hz,
2H, ArH2,6), 7.45 (d, J = 8.4 Hz, 2H, ArH3,5), 7.68 (d, J = 8.8 Hz, 2H,
diluted to a suitable concentration in enzyme dilution buffer
(50 mM Tris–HCl, 20% glycerol, 2 mM DTT, pH 7.6), is added. The
reactions are incubated at 25 °C for 40 min and then stopped by
the addition of EDTA (15 mM fc). Heteroduplexes are then detected

Z.-H. Yan et al. / Bioorg. Med. Chem. 22 (2014) 2535–2541
2541
by addition of PicoGreen. Signals are read using an excitation
Acknowledgments
wavelength of 490 nm and emission detection at 523 nm using a
spectrofluorometer (Safire2, Tecan). To test the activity of com-
pounds against RT, 1 lL of compound in DMSO is added to each
well before the addition of RT enzyme solution. Control wells with-
out compound contain the same amount of DMSO. Results are ex-
pressed as relative fluorescence i.e. the fluorescence signal of the
reaction mix with compound divided by the signal of the same
reaction mix without compound.
This research was financially supported by grants from
Chinese National Science and Technology Major Project (No.
2012ZX09103101-068), National Natural Science Foundation of
China (No. 81172918), Shanghai Municipal Natural Science
Foundation (No.13ZR1402200) and KU Leuven (GOA 10/014).
References and notes
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5.5. Molecular simulation
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 then optimized using the Tripos force field for 1000
generations two times or more, with an energy gradient of
0.001 kcal/(mol Å) and Gasteiger–Hückel charges loaded, until
the minimized conformers of the ligand were the same. The
X-ray co-crystal structure of wild type RT in complex with ETV in
the Brookhaven Protein Data Bank (PDB entry code: 3MEC) was
used for docking. Prior to docking, the protein receptor was prepro-
cessed by removing the initial ligand. Then the free protein was
prepared by removing water molecules and other unnecessary
small molecules, and adding hydrogen atoms. The atomic charges
were recalculated using the Kollman all-atom approach for the
protein and the Gasteiger–Hückel approach for the ligand. The
geometry of the non-nucleoside binding site (NNIBP) was defined
by selecting all the residues within of the initial inhibitor. Sur-
flex-Dock default settings were used for other parameters, such
as the number of starting conformations per molecule (set to 0),
the size to expand search grid (set to 8 Å), the maximum number
of rotatable bonds per molecule (set to 100), and the maximum
number of poses per ligand (set to 20). During the docking proce-
dure, all of the single bonds in residue side chains inside the de-
fined RT binding pocket were regarded as rotatable or flexible,
and the ligand was allowed to rotate on all single bonds and move
flexibly within the tentative binding pocket. The binding interac-
tion energy was calculated to include van der Waals, electrostatic
and torsional energy terms defined in the Tripos force field. The
structure optimization was performed for 20,000 generations
using a genetic algorithm, and the 20 best scoring ligand–protein
complexes were kept for further analyses. The Àlog(Kd)2 values
of the 20 best scoring complexes, which represented the binding
affinities of ligand with RT, ranged over a wide scope of functional
classes. Therefore, only the highest scoring 3D structural model of
the ligand-bound RT was chosen to define the binding interaction.
18. Gu, S. X.; He, Q. Q.; Yang, S. Q.; Ma, X. D.; Chen, F. E.; De Clercq, E.; Balzarini, J.;
Pannecouque, C. Bioorg. Med. Chem. 2011, 19, 5117.
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20. Zhang, L. G.; Zhan, P.; Chen, X. W.; Li, Z. Y.; Xie, Z. M.; Zhao, T.; Liu, H. Q.;
Pannecouque, C.; Balzarini, J.; De Clercq, E.; Liu, X. Y. Bioorg. Med. Chem. 2014,
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