Design and synthesis of a new series of cyclopropylamino-linking diarylpyrimidines as HIV non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejps.2014.06.003

A new series of 29 diarylpyrimidine analogues featuring a cyclopropylamino group between the pyrimidine scaffold and the aryl wing have been synthesized. All of the new compounds have been characterized by spectra analysis. The target molecules were evalu

Full text of DOI:10.1016/j.ejps.2014.06.003

European Journal of Pharmaceutical Sciences 62 (2014) 334–341
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Design and synthesis of a new series of cyclopropylamino-linking
diarylpyrimidines as HIV non-nucleoside reverse transcriptase inhibitors
b
c
c
d
Yang Liu a, Ge Meng a, , Aqun Zheng , Fener Chen , Wenxue Chen , Erik De Clercq ,
⇑
Christophe Pannecouque d, Jan Balzarini d
a School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, PR China
b School of Science, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, PR China
c Department of Chemistry, Fudan University Research, Shanghai 200433, PR China
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, 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 new series of 29 diarylpyrimidine analogues featuring a cyclopropylamino group between the
pyrimidine scaffold and the aryl wing have been synthesized. All of the new compounds have been char-
acterized by spectra analysis. The target molecules were evaluated for their in vitro anti-HIV activity with
FDA-approved drugs as references. Some of the compounds exhibited moderate to potent activities
against wild-type HIV-1. The compound 4-((4-((cyclopropylamino)(2,5-difluorophenyl)methyl)pyrimi-
din-2-yl)amino)benzonitrile (1e) displayed potent anti-HIV-1 activity against WT HIV-1 with an IC50 of
Received 19 February 2014
Received in revised form 3 June 2014
Accepted 3 June 2014
Available online 20 June 2014
PubChem:
Cyclopropylamine (PubChem CID:
24847670)
0.099
lM and a selectivity index of 2302. The preliminary structure–activity relationship (SAR) of this
new series of compounds was also investigated.
Ó 2014 Published by Elsevier B.V.
Keywords:
Non-nucleoside RT inhibitors
Diarylpyrimidine
Cyclopropylamine
Anti-HIV activity
SAR
1. Introduction
Although the docking results showed that these groups could fill
the Val179-including active binding pocket of HIV-1 RT (Das
et al., 2008) and generate suitable electrostatic interactions with
the amino acid residues at the wall of the active site, this
preliminary CADD result still needed more experimental support-
ing. Therefore, we aimed at more conclusive evidence that the
substitution position would be effectively beneficial for the
binding between the inhibitor and the NNBP of RT.
On the other hand, while considering the fact that the chemical
structures of two FDA approved NNRTIs used in HIV therapy shared
the same cyclopropyl group (4, 5, Fig. 1), we envisaged that the intro-
duction of a cyclopropylamino (Cpa) group might be better accom-
modated in the appropriated position of the NNBPs in HIV-1 RT
(Hassam et al., 2012). This improvement in the steric interactions
between the ligand and the active binding pocket might be an effi-
cient strategy to design more suitable drug candidates according
to the already known hydrophobic nature of the HIV-1 RT binding
pocket (Antunes et al., 2011; Das et al., 2008; Hassam et al., 2012).
We describe the design and synthesis of a new series of 4-(cyclopro-
pylamino)aryl methyl DAPYs derivatives (1, Fig. 2) in this paper. The
biological activity of all the compounds was also evaluated to
delineate the structure activity relationship (SAR).
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI)
are the key drugs in efficient treatment of AIDS (de Béthune,
2010; Zhan et al., 2009). Diarylpyrimidine (DAPY) derivatives are
one of the most successful families of NNRTIs developed so far
(Chen et al., 2011), which have been confirmed by the approval
of Etravirine (2, Fig. 1) and Rilpivirine (3, Fig. 1) for clinical use
(Mordant et al., 2007).
To identify more potent DAPYs as possible NNRTIs (Meng et al.,
2003; Meng et al., 2005), we have made considerable efforts and
structural modifications on the linker of CH2-DAPYs, which
resulted in a variety of groups between the left benzene ring and
the central pyrimidine ring (Feng et al., 2010; Zeng et al., 2010).
The introductions of these groups, especially hydrazine group in
candidate 6 or hydroxyl group in compound 7 (Gu et al., 2011,
2012) (Fig. 2) (Ma et al., 2011), were proved to be very beneficial
for the improvement in antiviral activity (Ma et al., 2011).
⇑
Corresponding author. Tel./fax: +86 29 82656327 801.
E-mail address: mengge@mail.xjtu.edu.cn (G. Meng).
http://dx.doi.org/10.1016/j.ejps.2014.06.003
0928-0987/Ó 2014 Published by Elsevier B.V.

Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
335
Fig. 1. Structures of some currently FDA-approved HIV-1 NNRTIs.
Fig. 2. Molecular design of CH(Cpa)-DAPYs.
CN
NH
CN
CN
NH
R3
R3
b
a
e
Cl
N
N
NH
N
NC
O
N
N
N
R2
R2
R2
R1
8
R1
R1
10 a-10ac
9
d
3
10j
10p
R
= 4-Br
3 = 4-C2H2CN
R
3 = 4-CN
CN
NH
CN
NH
c
10q
R
R3
R3
f
N
N
N
N
H
N
N
R2
R2
R1
R1
11a-11ac
1a-1ac
10, 11, 1
R1
R2
R3
10, 11, 1
R1
R2
R3
10, 11, 1
R1
R2
R3
a
b
c
H
H
H
H
H
H
4-H
4-Me
4-F
k
l
H
H
H
H
H
H
3-Br
4-tBu
u
v
Me
Me
Me
H
H
H
4-Br
4-tBu
m
4-OMe
w
4-OMe
d
e
H
H
H
H
3-F
2,5-diF
n
o
H
H
H
H
2,3-CH=CH-CH=CH-
3,4-CH=CH-CH=CH-
x
y
H
H
Me
Me
4-H
4-F
f
g
h
i
H
H
H
H
H
H
H
H
H
H
4-Cl
3-Cl
p
q
r
s
H
H
H
H
H
H
4-CN
4-C2H4CN
4-H
z
H
H
H
H
Me
Me
Me
Me
4-Cl
4-Br
H
aa
ab
ac
2-Cl
Me
Me
Me
4-tBu
4-OMe
2,4-diCl
4-Br
4-F
j
t
4-Cl
Scheme 1. Synthetic route to CH(Cpa)-DAPYs (1a–1ac). Reagents and conditions: (a) R3-Phenylacetonitrile, 60% NaH, Ar, DMF, -20 °C to r. t., 48–72 h; (b) NaH, air, DMF, r. t.,
36–72 h; (c) CuCN, DMF, 150 °C, 10 h; (d) acrylonitrile, Pd(OAc)2, P(o-Tol)3, NaOAc, DMA, 150 °C, overnight; (e) Cyclopropylamine, EtOH, reflux, 4–8 h; and (f) NaBH3CN, EtOH,
60 °C, 4 h.

336
Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
as half maximal (50%) inhibitory concentration (IC50). Moreover,
2. Results and discussion
the cytotoxicities (CC50) of the compounds were also determined.
The selective index (SI = CC50/IC50), which indicates the specificity
of the antiviral effect, was also given for the biological data within
the wild-type HIV strain.
The biological testing results of these target DAPY analogues
together with the relative data of the five reference compounds
are listed in Table 1. Some of the target compounds showed mod-
erate to potent activities against wild-type (WT) HIV-1 with IC50
2.1. Chemistry
The synthesis of the target compounds 1a–1ac is shown in
Scheme 1. The key intermediates, oxo-CH2-DAPYs (10a–10ac) were
conveniently synthesized from 4-(4-chloro pyrimidin-2-ylami-
no)benzonitriles (8) through two steps of reactions, via the modi-
fied procedures of our previously reported methods (Hassam
et al., 2012). Cyanidation of 10j with CuCN at 150 °C for 10 h in
anhydrous DMF gave the corresponding 4-cyano intermediate
10p. The acrylonitrile derivative 10q was prepared according to
the coupling reaction which was conducted on 10j with classical
Heck conditions using palladium (II) diacetate as the catalyst in
the presence of sodium acetate in DMA. Imino compounds
(11a–11ac) were synthesized by refluxing 10a–10ac with
cyclopropylamine in ethanol with the assistance of acetic acid
and anhydrous sodium sulfate. Reductions of 11a–11ac with
sodium cyanoborohydride in ethanol solution provided the final
compounds 1a–1ac.
values in the range of 9.70–0.099 lM. In this series, the compounds
containing the 2,5-difluoro, 2-chloro and 4-cyanovinyl on the left
phenyl ring (1e, 1h and 1q) displayed good anti-HIV-1 activity
against WT HIV-1 and showed excellent selectivity. Unfortunately,
none of them exhibited activity against the double RT mutant virus
(K103N + Y181C) and HIV-2 strain ROD.
All of the unsubstituted parent compounds (1a, 1r and 1x) dem-
onstrated good biological activity against HIV-1. With the intro-
duction of a methyl group in the para-position of the phenyl ring,
the biological activity of these compounds were kept almost
unchanged. The replacement of the methyl group in these com-
pounds by an electron-withdrawing group such as a halogen atom
(1c, 1f and 1j) or a cyano group (1p) might result in a reduced bio-
logical potency. Replacement of the cyano substituent by an acry-
lonitrile group (1q) could lead to a very potent compound highly
active against single mutant strains and with a elevated selectivity.
This result provide a hint for the future research that a favorable
para-substituent on the A-ring might be flexible in conformation
and more linear in shape in order to insert into the narrow tunnel
of the HIV-1 NNRTIs binding site. Introduction of a methoxy or a
2.2. Biological activity
The anti-HIV activity of 4-(cyclopropylamino)arylmethyl DAPYs
was measured using the MTT method and compared to five FDA-
approved drugs: Nevirapine, Zidovudine, Efavirenz, Delavirdine
and Etravirine. The cells were infected with HIV-1 wild-type virus
(LAI) or double RT mutant (K103N/Y181C) strain derived from
wild-type LAI, and HIV-2 strain (ROD). The results are reported
Table 1
Anti-HIV activities and cytotoxicities of compounds 1a–1ac MT-4 cells.
Compounds
IC50
IIIBa
(l
M)
CC50
(
lM)
SI
RES056b
ROD
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
1x
1y
1z
1aa
1ab
1.83 0.58
3.68 1.50
>33.18
0.91 0.34
0.099 0.038
>33.52
1.14 0.27
0.12 0.01
1.08 0.20
>28.73
1.102 0.15
>28.68
>40.54
>96.95
>33.18
>105.79
>228.941
ND
>101.74
>37.89
>31.39
ND
>35.55
ND
ND
>182.19
ND
>34.71
>56.108
>315.10
ND
ND
>267.08
ND
ND
>351.68
ND
ND
ND
>28.96
ND
0.90
>40.54
>96.95
>33.18
>105.79
>228.941
>33.52
>101.74
>37.89
>31.39
>28.73
>35.55
>28.68
>38.84
>182.19
>29.06
>34.71
>56.108
>315.10
>37.96
>261.79
>267.08
>29.22
>36.81
>351.68
>5.49
40.54 4.52
96.95 68.50
33.18 1.99
105.79 67.89
228.941 58.061
33.52 1.32
101.74 31.31
37.89 1.98
31.39 0.95
28.73 1.05
35.55 1.11
28.68 1.03
38.84 2.78
182.19 10.59
29.06 2.50
34.71 1.08
56.108 9.788
P315.10
37.96 0.83
261.79 70.00
P267.08
29.22 0.50
36.81 0.68
>351.68
5.49 0.69
1.19 0.11
2.13 1.20
28.96 1.14
2.80 1.64
>15.021
22
26
<1
116
2302
<1
89
317
29
<1
32
<1
<1
39
<1
<1
287
P33
<1
<1
<1
<1
<1
>172
<1
<1
<1
3
<1
>148
>22
>14,124
>280
>54
>38.84
4.66 0.89
>29.06
>34.71
0.196 0.03
9.69 2.67
>37.96
>261.79
>267.08
>29.22
>36.81
2.05 0.32
>5.49
>1.19
>2.13
9.70 2.66
>2.80
0.101 0.015
3.89 2.89
0.0066 0.0006
0.0226
>1.19
>2.13
>28.96
>2.80
1ac
Nevirapine
Zidovudine
Efavirenz
Delavirdine
Etravirine
15.53 11.17
0.0017
>87.24
>93.5489
6.3356
43.84
0.0077
0.16
0.81
a
IIIB means the wild type of HIV-1 virus strain.
ES056 means the double mutant (K103N/Y181C) HIV-1 virus strain.
b

Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
337
Fig. 3. Comparison of the binding modes between the representative potential compound 1e and TMC278 in NNRTBP of HIV-1 RT (PDB: 2ZD1(Das et al., 2008)). (a) TMC278/
RTWT, (b) 1e/RTWT. The figures are generated by the software of SYBYL-X 2.0. Possible hydrogen bonds are indicated with dashed lines in red. The amino acid residues are
shown in liner frame and the docked conformations of the small molecules are shown in ball and stick both for 1e and TMC278, respectively. (For interpretation of the
references to color in these figures legend, the reader is referred to the web version of this article.)
tert-butyl group at para-position could not exert any effects on the
activities, which could be explained by the presence of the steric
hindrance effect generated by the bulky substituents.
It could be deduced from the obtained biological data that the
meta and ortho substitutions might lead the corresponding
compounds to exhibit good potency against wild-type virus, espe-
cially for the ortho position. The introduction of chlorine or fluorine
atoms onto the meta and ortho position resulted in compounds 1e,
substituted HEPT analogues as the potential NNRTIs (Feng et al.,
2009; Zeng et al., 2010).
The introduction of the methyl substituent in C-5 or C-6 posi-
tion of the pyrimidine ring clearly resulted in unfavorable activity
against most of virus strains ever tested. In these series, none of the
substituted phenyl compounds exhibited activity except that the
non-substituted parent compounds 1r and 1x displayed weak
potency. Moreover, the compounds 1y–1ac with a methyl group
at the C-6 position of the pyrimidine also exhibited very high
toxicity.
Although the introduction of the cyclopropylamino group to the
methylene linker did not bring the excellent result which we had
expected at first, the relating research result still might provide
some deep insight of the relative SAR analysis. Compared with
the hydrazine (Das et al., 2008) or hydroxyl (Ma et al., 2011) groups
that we used in our previous research work, cyclopropylamino
group brought more steric hindrance to the neighboring groups.
This bulky group maybe liable to destroy the proper binding orien-
tation position between the compounds and the NNRTI binding
site, which have been called as the horseshoe conformational
shape (Gu et al., 2011). Superposition of all the compounds using
the conformations after docking (Jain, 2009) in the NNRTI pocket
showed that an unfavorable connection appeared in some com-
pounds with lower activities, in which the cyclopropyl group was
protruding into the binding pocket instead of the phenyl ring A
(Fig. 3) (Spitzer and Jain, 2012). Because the previous relative
1h with excellent activity of 0.099 lM and 0.12 lM, respectively.
Additionally, 11e was also the best one with highest selectivity
(SI = 2302) and lowest cytotoxicity (CC50 = 228.9) out of all the
active compounds. This was partially due to the presence of the
groups at the ortho position, limiting the rotational freedom of
the phenyl ring. Removal of these substituents might increase
the conformational flexibility and therefore decrease the
p–p
interaction with the HIV-1 RT NNBP. In addition, the halogen atoms
were also involved in hydrophobic interactions with the side
chains of amino residues exposed on the surface of this allosteric
site.
With the aim to enhance the p–p stacking interactions with the
NNBP, 1-naphthyl- or 2-naphthyl-substituted parent compounds
(1n and 1p) were synthesized and also assayed. The results indi-
cated that only 1-naphthyl substituted compound 1n was deter-
mined to have IC50 value of 4.66 lM, while the 2-naphthyl
substituted 1p exhibited no activity against HIV-1. The decrease
in biological activity might be explained by both the protruding
effect and the stereospecific blockade formed between the large
2-naphthyl and the large cyclopropyl group, which therefore might
SAR studies have showed that p–p main interactions were present
between the phenyl ring A of the ligands and residues Tyr181 and
cause further weakening of the p–p interactions. This similar result
Tyr188 in the allosteric binding pocket (Tian et al., 2010), a cyclo-
about the different effects between the 1-naphthyl- and the
2-naphthyl-substitutions on the biological activities against the
HIV were also agreed with our previous discoveries in the naphthyl
propyl might therefore weaken the p–p interaction (Thakur et al.,
2008). With this point of view, it is not surprising that all the new
compounds exhibit relatively lower activities against HIV-1 virus
Table 2
Comparison of the torsion angles of the binding conformations of TMC278 and compound 1e.
Compound-RT complex
Torsion angles (°)
s
1
s
2
s
3
s4
TMC278/RTWT
85.0
ꢀ35.1
16.1
28.0
ꢀ11.5
ꢀ48.9
ꢀ12.0
1e/RTWT
0.6

338
Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
compared with other FDA-approved drugs. The comparisons
between the torsion angles of the active binding conformations
of both 1e with RTWT and TMC278-RT cocrystal structure (Das
et al., 2008) could also explain the biological difference of com-
pound 1e from that of TMC278 (Table 2).
4.1.3. General procedures for the synthesis of target compounds
1a–1ac
To
a mixture of 10a–1ac (1.00 mmol), Na2SO4 (213 mg,
1.50 mmol) and ethanol (20.0 mL) was added cyclopropyl-amine
(285 mg, 5.00 mmol) and 5 drops of acetic acid. The mixture was
heated to reflux for 4–8 h until the reaction completed which
was monitored by TLC, then cooled. Sodium cyanoborohydride
was added at room temperature and subsequently the mixture
was heated at 60 °C for 4 h, then poured into saturated sodium
bicarbonate and extracted with CH2Cl2 (20.0 mL ꢁ 3). The organic
layers were combined, dried over anhydrous MgSO4, and was con-
centrated under reduced pressure. Next, the residue was purified
by column chromatography (silica gel; EtOAc/petroleum ether,
1:5–1:3).
4-((4-((Cyclopropylamino)(phenyl)methyl)pyrimidin-2-yl)
amino)benzonitrile (1a) Yield: 58%; mp 149.8–151.8 °C; 1H NMR
(400 MHz, DMSO) d 10.15 (s, 1H, NH), 8.49 (d, J = 5.1 Hz, 1H, pyrim-
idine H6), 7.94 (d, J = 8.7 Hz, 2H, ArH3,5), 7.69 (d, J = 8.7 Hz, 2H,
ArH2,6), 7.42 (d, J = 7.4 Hz, 2H, Ar0H2,6), 7.32 (t, J = 7.5 Hz, 2H,
Ar0H3,5), 7.24 (d, J = 7.2 Hz, 1H, Ar0H4), 7.15 (d, J = 5.1 Hz, 1H, pyrim-
idine H5), 4.81 (s, 1H, CH), 2.04–1.91 (m, 1H, CH), 0.39–0.29 (m, 4H,
C2H4); 13C NMR (100 MHz, DMSO) d 173.14, 159.51, 158.80,
145.42, 142.57, 133.43 (2C), 128.80 (2C), 127.96 (2C), 127.62,
120.07, 118.71 (2C), 111.61, 102.68, 67.87, 29.37, 6.81 (2C); MS
(ESI+) m/z 342 (M + H)+; HR-MS: m/z Calcd for C21H19N5:
341.1640. Found: 341.1649.
3. Conclusions
A
series of 4-(cyclopropylamino)aryl methyl DAPYs were
successfully synthesized via several steps as part of our anti-
HIV program. Biological evaluation indicated that the newly syn-
thesized derivatives showed moderate to potent activities against
wild-type (WT) HIV-1. The exploratory study clearly enriched the
SAR of CH2-DAPYs as anti-HIV agents. Compounds 1e displayed
good anti-HIV-1 activity against WT HIV-1 with an IC50 value of
0.099 lM and a selectivity index of 2302. None of the compounds
exhibited activity against the mutant virus. Therefore, this study
clearly highlighted the necessary to keep a proper volume of
the substituent group to the CH2-linker and a satisfactory flexibil-
ity of the whole molecule to achieve a higher level of inhibition
on NNRTI-resistant viruses. Furthermore, the result has enriched
the SAR of DAPY analogues as potent anti-HIV-1 compounds.
These conclusions could provide valuable information for the
rational design of effective inhibitors with better therapeutic
profiles against AIDS.
4-((4-((Cyclopropylamino)(p-tolyl)methyl)pyrimidin-2-yl)
amino)benzonitrile (1b) Yield: 62%; mp 177.8–179.4 °C; 1H NMR
(400 MHz, DMSO) d 10.14 (s, 1H, NH), 8.48 (d, J = 5.1 Hz, 1H, pyrim-
idine H6), 7.95 (d, J = 8.7 Hz, 2H, ArH3,5), 7.69 (d, J = 8.7 Hz, 2H,
ArH2,6), 7.30 (d, J = 7.9 Hz, 2H, Ar0H2,6), 7.21–7.05 (m, 3H, Ar0H3,5 + -
pyrimidine H5), 4.77 (s, 1H, CH), 2.24 (s, 3H, CH3), 2.02–1.91 (m,
1H, CH), 0.41–0.23 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d
173.31, 159.49, 158.71, 145.44, 139.59, 136.73, 133.43 (2C),
129.35 (2C), 127.82 (2C), 120.08, 118.70 (2C), 111.54, 102.66,
67.60, 29.35, 21.10, 6.78 (2C); MS (ESI+) m/z 356 (M + H)+; HR-
MS: m/z Calcd for C22H21N5: 355.1797. Found: 355.1806.
4-((4-((Cyclopropylamino)(4-fluorophenyl)methyl)pyrimidin-
2-yl)amino)benzonitrile (1c) Yield: 48%; mp 157.9–159.1 °C; 1H
NMR (400 MHz, DMSO) d 10.16 (s, 1H, NH), 8.50 (d, J = 5.0 Hz,
1H, pyrimidine H6), 7.93 (d, J = 8.5 Hz, 2H, ArH3,5), 7.69 (d,
J = 8.5 Hz, 2H, ArH2,6), 7.59–7.39 (m, 2H, 2H, Ar0H2,6), 7.32–7.05
(m, 3H, Ar0H3,5 + pyrimidine H5), 4.83 (s, 1H, CH), 2.03–1.92 (m,
1H, CH), 0.41–0.26 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d
172.87, 161.77 (d, JCF = 243.2 Hz), 159.53, 158.93, 145.38, 138.77,
133.43 (2C), 129.89 (2C, d, JCF = 8.0 Hz), 120.07, 118.72 (2C),
115.50 (2C, d, JCF = 21.2 Hz), 111.56, 102.72, 67.05, 29.32, 6.82,
6.77; MS (ESI+) m/z 360 (M + H)+; HR-MS: m/z Calcd for
4. Experimental section
4.1. Chemistry
Chemical reagents and solvents, purchased from commercial
sources, were of analytical grade and were used without further
purification. Melting points were measured on a SGW X-1 micro-
scopic melting point apparatus. TLC analyses were run on
pre-coated silica gel G plates at 254 nm under a UV lamp using a
variety of solvent systems. Column chromatography separations
were performed with silica gel (300–400 mesh). 1H NMR and 13C
NMR spectra were recorded on a Bruker AV400 MHz spectrometer
in DMSO-d6. Chemical shifts are reported in d (ppm) units relative
to the internal standard tetramethylsilane (TMS). Mass spectra
were obtained on a Waters Quattro Micromass instrument using
electron spray ionization (ESI) techniques.
4.1.1. Procedure for the synthesis of 10p
A
mixture of 10j (756 mg, 2.00 mmol), CuCN (178 mg,
2.00 mmol) and DMF (2.00 mL) was heated at 150 °C for 10 h with
stirring, then cooled. The suspension was a solution of iron (III)
chloride (1.00 g), concentrated HCl (0.20 mL), and water
(20.0 mL). The mixture was stirred at 70 °C for 20 min and
extracted with CH2Cl2 (20.0 mL ꢁ 3). The combined organic layers
were dried over anhydrous MgSO4, filtered and concentrated in
vacuo. Purification by column chromatography (silica gel; CH2Cl2)
gave 10p.
C21H18FN5: 359.1546. Found: 359.1555.
4-((4-((Cyclopropylamino)(3-fluorophenyl)methyl)pyrimidin-
2-yl)amino)benzonitrile (1d) Yield: 46%; mp 160.4–161.9 °C; 1H
NMR (400 MHz, DMSO) d 10.16 (s, 1H, NH), 8.50 (d, J = 5.1 Hz,
1H, pyrimidine H6), 7.92 (d, J = 8.8 Hz, 2H, ArH3,5), 7.67 (d,
J = 8.8 Hz, 2H, ArH2,6), 7.36 (dd, J = 14.1, 7.8 Hz, 1H, Ar0H), 7.27 (t,
J = 10.9 Hz, 2H, Ar0H), 7.14 (d, J = 5.1 Hz, 1H, pyrimidine H5),
7.10–7.01 (m, 1H, Ar0H), 4.85 (s, 1H, CH), 3.52 (s, 1H, NH), 2.03–
1.91 (m, 1H, CH), 0.39–0.26 (m, 4H, C2H4); 13C NMR (100 MHz,
4.1.2. Procedures for the synthesis of 10q
A mixture of 10j (756 mg, 2.00 mmol), acrylonitrile (1.06 g,
20.0 mmol), Pd(OAc)2 (89.9 mg, 0.40 mmol), tri-o-tolyl phosphine
(608 mg, 2.00 mmol) and sodium acetate (656 mg, 8.00 mmol) in
anhydrous DMA (30.0 mL) was stirred in a sealed tube at 150 °C
overnight. The reaction mixture was then hydrolyzed with water
and extracted with CH2Cl2 (20.0 mL ꢁ 3). The organic layer was
dried over anhydrous MgSO4 and concentrated under reduced
pressure. The crude product 10q was recrystallized from acetoni-
trile to afford the pure product, which could be used directly in
the next step.
DMSO)
d 172.46, 162.70 (d, JCF = 243.5 Hz), 159.52, 159.01,
145.57 (d, JCF = 6.8 Hz,) 133.40 (2C), 130.68 (d, JCF = 8.2 Hz),
124.20, 120.06, 118.74 (2C), 114.68, 114.48 (d, JCF = 2.3 Hz),
114.29, 111.60, 102.74, 67.24, 29.26, 6.86, 6.79; MS (ESI+) m/z
360 (M + H)+; HR-MS: m/z Calcd for C21H18FN5: 359.1546. Found:
359.1559.
4-((4-((Cyclopropylamino)(2,5-difluorophenyl)methyl)pyrim-
idin-2-yl)amino)benzonitrile (1e) Yield: 44%; mp 178.1–179.2 °C;
1H NMR (400 MHz, DMSO) d 10.12 (s, 1H, NH), 8.52 (d, J = 5.0 Hz,

Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
339
1H, pyrimidine H6), 7.83 (d, J = 8.7 Hz, 2H, ArH3,5), 7.62
(d, J = 8.7 Hz, 2H, ArH2,6), 7.46–7.38 (m, 1H, Ar0H), 7.29–7.20
(m, J = 9.1, 4.5 Hz, 1H, Ar0H), 7.20–7.13 (m, 1H, Ar0H), 7.11
(d, J = 5.0 Hz, 1H, pyrimidine H5), 5.10 (d, J = 8.1 Hz, 1H, CH), 3.57
(s, 1H, NH), 2.06–1.94 (m, 1H, CH), 0.45–0.27 (m, 4H, C2H4); 13C
(m, CH), 0.39–0.27 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d
172.55, 159.52, 158.99, 145.37, 142.08, 133.42 (2C), 131.65 (2C),
130.28 (2C), 120.72, 120.06, 118.72 (2C), 111.58, 102.73, 67.14,
29.31, 6.90, 6.77; MS (ESI+) m/z 420 (M + H)+; HR-MS: m/z Calcd
for C21H18BrN5: 419.0746. Found: 419.0751.
NMR (100 MHz, DMSO)
d
171.21, 159.49, 159.24, 158.85(d,
4-((4-((3-Bromophenyl)(cyclopropylamino)methyl)pyrimi-
din-2-yl)amino)benzonitrile (1k) Yield: 52%; mp 170.1–171.9 °C;
1H NMR (400 MHz, DMSO) d 10.16 (s, 1H, NH), 8.51 (d, J = 5.0 Hz,
1H, pyrimidine H6), 7.91 (d, J = 8.7 Hz, 2H, ArH3,5), 7.68
(d, J = 8.5 Hz, 3H, ArH2,6 + Ar0H2), 7.43 (t, J = 7.7 Hz, 2H, Ar0H4,6),
7.29 (t, J = 7.8 Hz, 1H, Ar0H5), 7.14 (d, J = 5.1 Hz, 1H, pyrimidine
H5), 4.83 (s, 1H, CH), 3.51 (d, J = 29.4 Hz, 1H, NH), 2.01–1.89
(m, 1H, CH), 0.40–0.28 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO)
d 172.34, 159.52, 159.10, 145.43, 145.34, 133.40 (2C), 130.98,
130.65, 130.48, 127.22, 122.18, 120.06, 118.76 (2C), 111.60,
110.01, 102.74, 67.15, 29.26, 6.90, 6.77; MS (ESI+) m/z 420
(M + H)+; HR-MS: m/z Calcd for C21H18BrN5: 419.0746. Found:
419.0758.
4-((4-((4-(tert-Butyl)phenyl)(cyclopropylamino)methyl)pyr-
imidin-2-yl)amino)benzonitrile (1l) Yield: 55%; mp 139.3–
141.3 °C; 1H NMR (400 MHz, DMSO) d 10.15 (s, 1H, NH), 8.48
(d, J = 5.1 Hz, 1H, pyrimidine H6), 7.95 (d, J = 8.7 Hz, 2H, ArH3,5),
7.70 (d, J = 8.6 Hz, 2H, ArH2,6), 7.33 (s, 4H, Ar0H), 7.15
(d, J = 5.1 Hz, 1H, pyrimidine H5), 4.77 (s, 1H, CH), 2.01–1.91
(m, 1H, CH), 1.23 (s, 9H, t-Bu), 0.37–0.29 (m, 4H, C2H4); 13C NMR
J = 242.0 Hz), 156.73 (d, J = 242.0 Hz), 145.30, 133.27 (2C), 131.62,
120.02, 118.71 (2C), 117.26 (2C, dd, J = 25.0, 8.8 Hz), 116.18–
115.55 (2C, m), 111.68, 102.78, 60.57, 29.21, 7.02, 6.68; MS
(ESI+) m/z 378 (M + H)+; HR-MS: m/z Calcd for C21H17F2N5:
377.1452. Found: 377.1461.
4-((4-((4-Chlorophenyl)(cyclopropylamino)methyl)pyrimidin-
2-yl)amino)benzonitrile (1f) Yield: 42%; mp 159.6–161.4 °C; 1H
NMR (400 MHz, DMSO) d 10.17 (s, 1H, NH), 8.51 (d, J = 5.1 Hz,
1H, pyrimidine H6), 7.92 (d, J = 8.6 Hz, 2H, ArH3,5), 7.69
(d, J = 8.6 Hz, 2H, ArH2,6), 7.42 (dd, J = 22.1, 8.4 Hz, 4H, Ar0H), 7.14
(d, J = 5.1 Hz, 1H, pyrimidine H5), 4.84 (d, J = 6.0 Hz, 1H, CH), 3.49
(s, 1H, NH), 2.02–1.92 (m, 1H, CH), 0.38–0.29 (m, 4H, C2H4); 13C
NMR (100 MHz, DMSO) d 172.62, 159.53, 158.99, 145.37, 141.65,
133.42 (2C), 132.18, 129.90 (2C), 128.73 (2C), 120.06, 118.72
(2C), 111.58, 102.73, 67.08, 29.31, 6.77 (2C); MS (ESI+) m/z 376
(M + H)+; HR-MS: m/z Calcd for C21H18ClN5: 375.1251. Found:
375.1259.
4-((4-((3-Chlorophenyl)(cyclopropylamino)methyl)pyrimidin-
2-yl)amino)benzonitrile (1g) Yield: 38%; mp 171.2–172.1 °C; 1H
NMR (400 MHz, DMSO) d 10.17 (s, 1H, NH), 8.52 (d, J = 5.1 Hz,
1H, pyrimidine H6), 7.92 (d, J = 8.8 Hz, 2H, ArH3,5), 7.68
(d, J = 8.7 Hz, 2H, ArH2,6), 7.54 (s, 1H, Ar0H2), 7.43–7.26 (m, 3H,
Ar0H4,5,6), 7.16 (d, J = 5.1 Hz, 1H, pyrimidine H5), 4.84 (s, 1H, CH),
3.57 (s, 1H, NH), 2.01–1.91 (m, 1H, CH), 0.38–0.28 (m, 4H, C2H4);
13C NMR (100 MHz, DMSO) d 172.39, 159.53, 159.09, 145.36,
145.20, 133.51, 133.40 (2C), 130.66, 127.78, 127.58, 126.86,
120.06, 118.75 (2C), 111.59, 102.74, 67.20, 29.26, 6.90, 6.79; MS
(ESI+) m/z 376 (M + H)+; HR-MS: m/z Calcd for C21H18ClN5:
375.1251. Found: 375.1261.
4-((4-((2-Chlorophenyl)(cyclopropylamino)methyl)pyrimidin-
2-yl)amino)benzonitrile (1h) Yield: 21%; mp 169.6–171.4 °C; 1H
NMR (400 MHz, DMSO) d 10.13 (s, 1H, NH), 8.53 (d, J = 5.0 Hz,
1H, pyrimidine H6), 7.84 (d, J = 8.7 Hz, 2H, ArH3,5), 7.62
(d, J = 8.7 Hz, 2H, ArH2,6), 7.57 (d, J = 7.1 Hz, 1H, Ar0H3), 7.51–7.24
(m, 3H, Ar0H4,5,6), 7.11 (d, J = 5.1 Hz, 1H, pyrimidine H5), 5.28 (d,
J = 8.6 Hz, 1H, CH), 3.54 (d, J = 8.8 Hz, 1H, NH), 2.06–1.97 (m, 1H,
CH), 0.56–0.20 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d
171.54, 159.51, 159.07, 145.33, 139.89, 133.53, 133.29 (2C),
129.90, 129.74, 129.29, 127.80, 120.05, 118.67 (2C), 112.03,
102.66, 63.92, 29.35, 7.04, 6.86; MS (ESI+) m/z 376 (M + H)+; HR-
MS: m/z Calcd for C21H18ClN5: 375.1251. Found: 375.1265.
4-((4-((Cyclopropylamino)(2,4-dichlorophenyl)methyl)pyrim-
idin-2-yl)amino)benzonitrile (1i) Yield: 33%; mp 139.7–141.1 °C;
1H NMR (400 MHz, DMSO) d 10.15 (s, 1H, NH), 8.54 (d, J = 5.0 Hz,
1H, pyrimidine H6), 7.81 (d, J = 8.6 Hz, 2H, ArH3,5), 7.74–7.56
(m, 4H, ArH2,6 + Ar0H3,5), 7.50 (d, J = 6.8 Hz, 1H, Ar0H6), 7.11
(d, J = 5.0 Hz, 1H, pyrimidine H5), 5.25 (d, J = 9.0 Hz, 1H, CH), 3.61
(d, J = 8.6 Hz, 1H, NH), 2.07–1.93 (m, 1H, CH), 0.56–0.19 (m, 4H,
C2H4); 13C NMR (100 MHz, DMSO) d 171.01, 159.49, 159.21,
145.29, 139.20, 134.48, 133.27 (2C), 132.89, 131.37, 129.11,
127.99, 120.04, 118.68 (2C), 112.05, 102.71, 63.58, 29.32, 7.12,
6.79; MS (ESI+) m/z 410 (M + H)+; HR-MS: m/z Calcd for C21H17Cl2
N5: 409.0861. Found: 409.0869.
(100 MHz, DMSO)
d 173.34, 159.52, 158.74, 149.96, 145.44,
139.55, 133.44 (2C), 127.54 (2C), 125.56 (2C), 120.08, 118.70
(2C), 111.56, 102.66, 67.60, 34.62, 31.58 (3C), 29.40, 6.89, 6.68;
MS (ESI+) m/z 398 (M + H)+; HR-MS: m/z Calcd for C25H27N5:
397.2266. Found: 397.2275.
4-((4-((Cyclopropylamino)(4-methoxyphenyl)methyl)pyrim-
idin-2-yl)amino)benzonitrile (1m) Yield: 39%; mp 163.3–
163.7 °C; 1H NMR (400 MHz, DMSO) d 10.15 (s, 1H, NH), 8.48
(d, J = 5.1 Hz, 1H, pyrimidine H6), 7.96 (d, J = 8.7 Hz, 2H, ArH3,5),
7.70 (d, J = 8.7 Hz, 2H, ArH2,6), 7.33 (d, J = 8.5 Hz, 2H, Ar0H2,6), 7.11
(d, J = 5.1 Hz, 1H, pyrimidine H5), 6.88 (d, J = 8.5 Hz, 2H, Ar0H3,5),
4.76 (s, 1H, CH), 3.70 (s, 3H, OCH3), 2.04–1.92 (m, 1H, CH), 0.42–
0.24 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d 173.45, 159.51,
158.85, 158.70, 145.46, 134.59, 133.43 (2C), 129.02 (2C), 120.09,
118.70 (2C), 114.18 (2C), 111.48, 102.66, 67.28, 55.51, 29.36,
6.80, 6.76; MS (ESI+) m/z 372 (M + H)+; HR-MS: m/z Calcd for
C22H21N5O: 371.1746. Found: 371.1755.
4-((4-((Cyclopropylamino)(naphthalen-1-yl)methyl)pyrimi-
din-2-yl)amino)benzonitrile (1n) Yield: 45%; mp 173.7–175.2 °C;
1H NMR (400 MHz, DMSO) d 10.14 (s, 1H, NH), 8.49 (d, J = 5.1 Hz,
1H, pyrimidine H6), 8.37 (d, J = 8.4 Hz, 1H, Ar0H8), 8.04–7.41
(m, 10H, ArH2,3,5,6+ Ar0H2,3,4,5,6,7), 7.18 (d, J = 5.1 Hz, 1H, pyrimidine
H5), 5.63 (d, J = 5.3 Hz, 1H, CH), 3.56 (s, 1H, NH), 2.15–2.03 (m, 1H,
CH), 0.46–0.23 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d 173.02,
159.42, 158.85, 145.33, 138.41, 133.92, 133.32 (2C), 131.52,
129.11, 128.10 (2C), 126.61, 126.07, 125.99, 125.54, 124.26,
120.07, 118.64, 111.93, 102.62, 63.79, 29.74, 6.93, 6.84; MS
(ESI+) m/z 392 (M + H)+; HR-MS: m/z Calcd for C25H21N5:
391.1797. Found: 391.1805.
4-((4-((Cyclopropylamino)(naphthalen-2-yl)methyl)pyrimi-
din-2-yl)amino)benzonitrile (1o) Yield: 40%; mp 143.1–144.3 °C;
1H NMR (400 MHz, DMSO) d 10.17 (s, 1H, NH), 8.51 (d, J = 5.1 Hz,
1H, pyrimidine H6), 7.99–7.42 (m, 11H, ArH + Ar0H), 7.21
(d, J = 5.1 Hz, 1H, pyrimidine H5), 5.00 (d, J = 7.6 Hz, 1H, CH), 3.57
(d, J = 6.7 Hz, 1H, NH), 2.08–1.97 (m, 1H, CH), 0.43–0.29 (m, 4H,
C2H4); 13C NMR (100 MHz, DMSO) d 172.94, 159.50, 158.89,
145.40, 140.20, 133.36 (2C), 133.33, 132.76, 128.37, 128.19,
127.97, 126.67, 126.63, 126.33, 126.23, 120.07, 118.68 (2C),
111.71, 111.71, 102.64, 67.97, 29.45, 6.92, 6.79; MS (ESI+) m/z
392 (M + H)+; HR-MS: m/z Calcd for C25H21N5: 391.1797. Found:
391.1809.
4-((4-((4-Bromophenyl)(cyclopropylamino)methyl)pyrimidin-
2-yl)amino)benzonitrile (1j) Yield: 42%; mp 179.4–180.1 °C; 1H
NMR (400 MHz, DMSO) d 10.17 (s, 1H, NH), 8.51 (d, J = 5.1 Hz,
1H, pyrimidine H6), 7.92 (d, J = 8.8 Hz, 2H, ArH3,5), 7.69
(d, J = 8.8 Hz, 2H, ArH2,6), 7.53 (d, J = 8.4 Hz, 2H, Ar0H2,6), 7.39
(d, J = 8.4 Hz, 2H, Ar0H3,5), 7.13 (d, J = 5.1 Hz, 1H, pyrimidine H5),
4.82 (d, J = 8.3 Hz, 1H, CH), 3.48 (d, J = 7.0 Hz, 1H, NH), 2.01–1.92

340
Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
4-((4-((4-Cyanophenyl)(cyclopropylamino)methyl)pyrimidin-
C2H4); 13C NMR (100 MHz, DMSO) d 172.10, 168.68, 159.39,
145.56, 142.24, 133.40 (2C), 131.60 (2C), 130.28 (2C), 120.64,
120.12, 118.62 (2C), 110.84, 102.49, 67.02, 29.32, 24.26, 6.93,
6.73; MS (ESI+) m/z 434 (M + H)+; HR-MS: m/z Calcd for C22H20
BrN5: 433.0902. Found: 433.0911.
4-((4-((4-(tert-Butyl)phenyl)(cyclopropylamino) methyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (1v) Yield: 40%; mp
163.1–164.0 °C; 1H NMR (400 MHz, DMSO) d 10.09 (s, 1H, NH),
7.98 (d, J = 8.7 Hz, 2H, ArH3,5), 7.69 (d, J = 8.7 Hz, 2H, ArH2,6),
7.43–7.19 (m, 4H, Ar0H), 7.05 (s, 1H, pyrimidine H5), 4.74
(d, J = 8.4 Hz, 1H, CH), 3.28 (d, J = 6.6 Hz, 1H, NH), 2.38 (s, 3H,
CH3), 2.03–1.90 (m, 1H, CH), 1.23 (s, 9H, t-Bu), 0.39–0.27 (m, 4H,
C2H4); 13C NMR (100 MHz, DMSO) d 172.91, 168.41, 159.41,
149.85, 145.66, 139.72, 133.42 (2C), 127.52 (2C), 125.51 (2C),
120.14, 118.59 (2C), 110.75, 102.43, 67.48, 34.62, 31.60 (3C),
29.39, 24.27, 6.87, 6.73; MS (ESI+) m/z 412 (M + H)+; HR-MS: m/z
Calcd for C26H29N5: 411.2423. Found: 4112432.
4-((4-((Cyclopropylamino)(4-methoxyphenyl)methyl)-6-
methylpyrimidin-2-yl)amino)benzonitrile (1w) Yield: 29%; mp
166.5–167.1 °C; 1H NMR (400 MHz, DMSO) d 10.08 (s, 1H, NH),
7.97 (d, J = 8.8 Hz, 2H, ArH3,5), 7.68 (d, J = 8.7 Hz, 2H, ArH2,6), 7.33
(d, J = 8.6 Hz, 2H, Ar0H2,6), 7.00 (s, 1H, pyrimidine H5), 6.87
(d, J = 8.6 Hz, 2H, Ar0H3,5), 4.72 (d, J = 8.2 Hz, 1H, CH), 3.70 (s, 3H,
OCH3), 3.26 (d, J = 6.4 Hz, 1H, NH), 2.37 (s, 3H, CH3), 2.02–1.91
(m 1H, CH), 0.42–0.23 (m, C2H4); 13C NMR (100 MHz, DMSO) d
172.99, 168.37, 159.40, 158.80, 145.66, 134.75, 133.41 (2C),
129.00 (2C), 120.14, 118.59 (2C), 114.13 (2C), 110.70, 102.43,
67.13, 55.50, 29.35, 24.25, 6.77 (2C); MS (ESI+) m/z 386 (M + H)+;
HR-MS: m/z Calcd for C23H23N5O: 385.1903. Found: 385.1912.
4-((4-((Cyclopropylamino)(phenyl)methyl)-5-methylpyrimi-
din-2-yl)amino)benzonitrile (1x) Yield: 37%; mp 225.1–227.2 °C;
1H NMR (400 MHz, DMSO) d 10.09 (s, 1H, NH), 8.31 (s, 1H, pyrim-
idine H6), 7.91 (d, J = 8.7 Hz, 2H, ArH3,5), 7.68 (d, J = 8.6 Hz, 2H,
ArH2,6), 7.32 (ddd, J = 36.2, 16.1, 7.1 Hz, 5H, Ar0H), 5.04
(d, J = 8.0 Hz, 1H, CH), 2.17 (s, 3H, CH3), 1.97–1.89 (m, 1H, CH),
0.41–0.27 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d 169.19,
159.21, 158.33, 145.66, 141.44, 133.47 (2C), 128.80 (2C), 128.28
(2C), 127.57, 120.51, 120.17, 118.33 (2C), 102.22, 63.06, 28.89,
14.53, 6.96, 6.81; MS (ESI+) m/z 356 (M + H)+; HR-MS: m/z Calcd
for C22H21N5: 355.1797. Found: 355.1790.
2-yl)amino)benzonitrile (1p) Yield: 38%; 1H NMR (400 MHz,
DMSO) d 10.16 (s, 1H, NH), 8.51 (d, J = 5.1 Hz, 1H, pyrimidine H6),
7.88 (d, J = 8.7 Hz, 2H, ArH3,5), 7.81 (d, J = 8.2 Hz, 2H, ArH2,6), 7.65
(dd, J = 15.9, 8.4 Hz, 4H, Ar0H), 7.14 (d, J = 5.1 Hz, 1H, pyrimidine
H5), 4.93 (s, 1H, CH), 2.01–1.90 (m, 1H, CH), 0.39–0.26 (m, 4H,
C2H4); 13C NMR (100 MHz, DMSO) d 171.90, 159.54, 159.19,
148.28, 145.28, 133.41 (2C), 132.75 (2C), 129.13 (2C), 120.04,
119.29, 118.75 (2C), 111.71, 110.42, 102.81, 67.36, 29.28, 6.95,
6.75; MS (ESI+) m/z 367 (M + H)+; HR-MS: m/z Calcd for
C
22H18N6: 366.1593. Found: 366.1602.
4-((4-((4-(2-Cyanovinyl)phenyl)(cyclopropylamino)methyl)
pyrimidin-2-yl)amino)benzonitrile (1q) Yield: 40%; 1H NMR
(400 MHz, DMSO) d 10.14 (s, 1H, NH), 8.49 (d, J = 5.0 Hz, 1H,
pyrimidine H6), 7.90 (d, J = 8.8 Hz, 2H, ArH3,5), 7.67 (d, J = 8.8 Hz,
2H, ArH2,6), 7.64–7.56 (m, 3H, Ar0H + alkenyl H), 7.49
(d, J = 8.2 Hz, 2H, Ar0H), 7.13 (d, J = 5.1 Hz, 1H, pyrimidine H5),
6.40 (d, J = 16.8 Hz, 1H, alkenyl H), 4.85 (s, 1H, CH), 2.02–1.91
(m, 1H, CH), 0.39–0.27 (m, 4H, C2H4); 13C NMR (100 MHz, DMSO)
d 172.49, 159.51, 159.00, 150.74, 145.79, 145.35, 133.41 (2C),
133.24, 128.62 (2C), 128.24 (2C), 120.06, 119.31, 118.74 (2C),
111.62, 102.74, 96.96, 67.51, 29.32, 6.90, 6.75; MS (ESI+) m/z 393
(M + H)+; HR-MS: m/z Calcd for C24H20N6: 392.1749. Found:
392.1758.
4-((4-((Cyclopropylamino)(phenyl)methyl)-6-methylpyrimi-
din-2-yl)amino)benzonitrile (1r) Yield: 59%; mp 166.4–167.6 °C;
1H NMR (400 MHz, DMSO) d 10.07 (s, 1H, NH), 7.94 (d, J = 8.8 Hz,
2H, ArH3,5), 7.67 (d, J = 8.8 Hz, 2H, ArH2,6), 7.42 (d, J = 7.4 Hz, 2H,
Ar0H2,6), 7.32 (t, J = 7.5 Hz, 2H, Ar0H3,5), 7.22 (t, J = 7.3 Hz, 1H, Ar0H4),
7.03 (s, 1H, pyrimidine H5), 4.77 (s, 1H, CH), 2.37 (s, 3H, CH3), 2.02–
1.91 (m, 1H, CH), 0.40–0.27 (m, 4H, C2H4); 13C NMR (100 MHz,
DMSO) d 172.66, 168.51, 159.39, 145.61, 142.71, 133.41 (2C),
128.75 (2C), 127.95 (2C), 127.54, 120.13, 118.61 (2C), 110.84,
102.45, 67.72, 29.36, 24.25, 6.82, 6.78; MS (ESI+) m/z 356
(M + H)+; HR-MS: m/z Calcd for C22H21N5: 355.1797. Found:
355.1790.
4-((4-((Cyclopropylamino)(4-fluorophenyl)methyl)-6-meth-
ylpyrimidin-2-yl)amino)benzonitrile (1s) Yield: 37%; mp 167.0–
167.9 °C; 1H NMR (400 MHz, DMSO) d 10.06 (s, 1H, NH), 7.93
(d, J = 8.7 Hz, 2H, ArH3,5), 7.67 (d, J = 8.7 Hz, 2H, ArH2,6), 7.45
(dd, J = 8.3, 5.8 Hz, 2H, Ar0H2,6), 7.14 (t, J = 8.8 Hz, 2H, Ar0H3,5),
7.01 (s, 1H, pyrimidine H5), 4.79 (s, 1H, CH), 2.37 (s, 3H, CH3),
2.00–1.91 (m, 4.4 Hz, 1H, CH), 0.39–0.26 (m, 4H, C2H4); 13C NMR
(100 MHz, DMSO) d 172.37, 168.67, 161.74 (d, JCF = 242.6 Hz),
159.41, 145.57, 138.93, 133.40 (2C), 129.88 (2C, d, JCF = 8.1 Hz),
120.12, 118.63 (2C), 115.44 (2C, d, JCF = 21.2 Hz), 110.82, 102.49,
66.90, 29.32, 24.24, 6.85, 6.72; MS (ESI+) m/z 374 (M + H)+;
HR-MS: m/z Calcd for C22H20FN5: 373.1703. Found: 373.1712.
4-((4-((4-Chlorophenyl)(cyclopropylamino)methyl)-6-meth-
ylpyrimidin-2-yl)amino)benzonitrile (1t) Yield: 32%; mp 188.1–
190.4 °C; 1H NMR (400 MHz, DMSO) d 10.06 (s, 1H, NH), 7.91
(d, J = 8.8 Hz, 2H, ArH3,5), 7.66 (d, J = 8.7 Hz, 2H, ArH2,6), 7.41
(dd, J = 25.3, 8.4 Hz, 4H, Ar0H), 7.01 (s, 1H, pyrimidine H5), 4.79
(s, 1H, CH), 2.37 (s, 3H, CH3), 2.01–1.90 (m, 1H, CH), 0.38–0.27
(m, 4H, C2H4); 13C NMR (100 MHz, DMSO) d 172.09, 168.74,
159.39, 145.54, 141.76, 133.39 (2C), 132.12, 129.90 (2C), 128.68
(2C), 120.12, 118.64 (2C), 110.87, 102.50, 66.93, 29.31, 24.24,
6.90, 6.69; MS (ESI+) m/z 390 (M + H)+; HR-MS: m/z Calcd for
4-((4-((Cyclopropylamino)(4-fluorophenyl)methyl)-5-meth-
ylpyrimidin-2-yl)amino)benzonitrile (1y) Yield: 33%; mp 209.8–
212.0 °C; 1H NMR (400 MHz, DMSO) d 10.09 (s, 1H, NH), 8.32
(s, 1H, pyrimidine H6), 7.90 (d, J = 8.8 Hz, 2H, ArH3,5), 7.68
(d, J = 8.7 Hz, 2H, ArH2,6), 7.44 (dd, J = 8.4, 5.7 Hz, 2H, Ar0H2,6),
7.17 (t, J = 8.8 Hz, 2H, Ar0H3,5), 5.06 (d, J = 8.9 Hz, 1H, CH), 2.18
(s, 3H, CH3), 1.96–1.87 (m, 1H, CH), 0.39–0.30 (m, 4H, C2H4); 13C
NMR (100 MHz, DMSO)
d 168.96, 161.74 (d, JCF = 243.1 Hz),
159.33, 158.34, 145.63, 137.69, 133.44 (2C), 130.24 (2C, d,
JCF = 8.0 Hz), 120.53, 120.16, 118.35 (2C), 115.50 (2C, d,
J = 21.3 Hz), 102.25, 62.34, 28.90, 14.50, 6.99, 6.78; MS (ESI+) m/z
374 (M + H)+; HR-MS: m/z Calcd for C22H20FN5: 373.1703. Found:
373.1718.
4-((4-((4-Chlorophenyl)(cyclopropylamino)methyl)-5-meth-
ylpyrimidin-2-yl)amino)benzonitrile (1z) Yield: 33%; mp 215.6–
216.4 °C; 1H NMR (400 MHz, DMSO) d 10.08 (s, 1H, NH), 8.32
(s, 1H, pyrimidine H6), 7.86 (d, J = 8.6 Hz, 2H, ArH3,5), 7.67
(d, J = 8.6 Hz, 2H, ArH2,6), 7.42 (s, 4H, Ar0H), 5.06 (d, J = 10.2 Hz,
1H, CH), 2.19 (s, 3H, CH3), 2.01–1.85 (m 1H, CH), 0.40–0.26 (m,
4H, C2H4); 13C NMR (100 MHz, DMSO) d 168.71, 159.40, 158.30,
145.60, 140.60, 133.42 (2C), 132.11, 130.24 (2C), 128.72 (2C),
120.63, 120.17, 118.34 (2C), 102.24, 62.48, 28.94, 14.51, 7.06,
6.76; 390.59; MS (ESI+) m/z 390 (M + H)+; HR-MS: m/z Calcd for
C
22H20ClN5: 389.1407. Found: 389.1421.
4-((4-((4-Bromophenyl)(cyclopropylamino)methyl)-6-meth-
ylpyrimidin-2-yl)amino)benzonitrile (1u) Yield: 35%; mp 193.1–
194.5 °C; 11H NMR (400 MHz, DMSO) d 10.10 (s, 1H, NH), 7.92
(d, J = 8.6 Hz, 2H, ArH3,5), 7.67 (d, J = 8.6 Hz, 2H, ArH2,6), 7.53
(d, J = 8.2 Hz, 2H, Ar0H2,6), 7.39 (d, J = 8.2 Hz, 2H, Ar0H3,5), 7.02
(s, 1H, pyrimidine H5), 4.78 (d, J = 7.4 Hz, 1H, CH), 3.43 (s, 1H,
NH), 2.38 (s, 3H, CH3), 0.02–1.89 (m, CH), 0.40–0.26 (m, 4H,
C22H20ClN5: 389.1407. Found: 389.1415.
4-((4-((4-Bromophenyl)(cyclopropylamino)methyl)-5-meth-
ylpyrimidin-2-yl)amino)benzonitrile (1aa) Yield: 40%; mp

Y. Liu et al. / European Journal of Pharmaceutical Sciences 62 (2014) 334–341
341
235.7–237.2 °C; 1H NMR (400 MHz, DMSO) d 10.07 (s, 1H, NH),
8.32 (s, 1H, pyrimidine H6), 7.85 (d, J = 8.6 Hz, 2H, ArH3,5), 7.66
(d, J = 8.5 Hz, 2H, ArH2,6), 7.55 (d, J = 8.1 Hz, 2H, Ar0H2,6), 7.37
(d, J = 8.1 Hz, 2H, Ar0H3,5), 5.05 (d, J = 9.1 Hz, 1H, CH), 2.19 (s, 3H,
CH3), 1.99–1.88 (m, 1H, CH), 0.40–0.28 (m, 4H, C2H4); 13C NMR
the National Natural Science Foundation of China (Grant No.
81172918).
Appendix A. Supplementary data
(100 MHz, DMSO)
d 168.65, 159.40, 158.29, 145.59, 141.04,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2014.06.003.
133.41 (2C), 131.63 (2C), 130.62 (2C), 120.66, 120.16, 118.34
(2C), 110.01, 102.24, 62.57, 28.95, 14.51, 7.07, 6.75; MS (ESI+) m/
z 434 (M + H)+; HR-MS: m/z Calcd for C22H20BrN5: 433.0902.
Found: 433.0916.
References
Antunes, A.M., Novais, D.A., da Silva, J.F., Santos, P.P., Oliveira, M.C., Beland, F.A.,
4-((4-((4-(tert-Butyl)phenyl)(cyclopropylamino)methyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (1ab) Yield: 23%; mp
204.4–205.4 °C; 1H NMR (400 MHz, DMSO) d 10.10 (s, 1H, NH),
8.30 (s, 1H, pyrimidine H6), 7.95 (d, J = 8.8 Hz, 2H, ArH3,5), 7.70
(d, J = 8.7 Hz, 2H, ArH2,6), 7.33 (q, J = 8.4 Hz, 4H, Ar0H), 4.99
(d, J = 10.4 Hz, 1H, CH), 2.17 (s, 3H, CH3), 1.95–1.87 (m 1H, CH),
1.24 (s, 9H, t-Bu), 0.40–0.28 (m 4H, C2H4); 13C NMR (100 MHz,
DMSO) d 169.33, 159.14, 158.37, 149.88, 145.69, 138.39, 133.50
(2C), 127.88 (2C), 125.58 (2C), 120.38, 120.17, 118.34 (2C),
102.21, 62.61, 34.65, 31.60, 28.85, 14.55, 6.88; MS (ESI+) m/z 412
(M + H)+; HR-MS: m/z Calcd for C26H29N5: 411.2423. Found:
411.2431.
Marques, M.M., 2011. Synthesis and oxidation of 2-hydroxynevirapine,
a
metabolite of the HIV reverse transcriptase inhibitor nevirapine. Org. Biomol.
Chem. 9, 7822–7835.
Chen, X., Zhan, P., Li, D., De Clercq, E., Liu, X., 2011. Recent advances in DAPYs and
related analogues as HIV-1 NNRTIs. Curr. Med. Chem. 18, 359–376.
Das, K., Bauman, J.D., Clark, A.D., Frenkel, Y.V., Lewi, P.J., Shatkin, A.J., Hughes, S.H.,
Arnold, E., 2008. High-resolution structures of HIV-1 reverse transcriptase/
TMC278 complexes: strategic flexibility explains potency against resistance
mutations. Proc. Natl. Acad. Sci. 105, 1466–1471.
de Béthune, M.-P., 2010. Non-nucleoside reverse transcriptase inhibitors (NNRTIs),
their discovery, development, and use in the treatment of HIV-1 infection: a
review of the last 20 years (1989–2009). Antiviral Res. 85, 75–90.
Feng, X.-Q., Liang, Y.-H., Zeng, Z.-S., Chen, F.-E., Balzarini, J., Pannecouque, C., De
Clercq, E., 2009. Structural modifications of DAPY analogues with potent anti-
HIV-1 activity. ChemMedChem 4, 219–224.
4-((4-((Cyclopropylamino)(4-methoxyphenyl)methyl)-5-
methylpyrimidin-2-yl)amino)benzonitrile (1ac) Yield: 21%; mp
210.9–212.7 °C; 1H NMR (400 MHz, DMSO) d 10.09 (s, 1H, NH),
8.29 (s, 1H, pyrimidine H6), 7.94 (d, J = 8.8 Hz, 2H, ArH3,5), 7.70
(d, J = 8.7 Hz, 2H, ArH2,6), 7.29 (d, J = 8.6 Hz, 2H, Ar0H2,6), 6.89
(d, J = 8.6 Hz, 2H, Ar0H3,5), 4.97 (s, 1H, CH), 3.71 (s, 3H, OCH3),
2.14 (s, 3H, CH3), 1.95–1.87 (m, 1H, CH), 0.38–0.27 (m 4H, C2H4);
13C NMR (100 MHz, DMSO) d 169.41, 159.08, 158.79, 158.31,
145.69, 145.08, 133.49 (2C), 133.33, 129.39 (2C), 120.35, 118.33
(2C), 114.20 (2C), 102.20, 62.37, 55.50, 28.77, 14.49, 6.91; MS
(ESI+) m/z 386 (M + H)+; HR-MS: m/z Calcd for C23H23N5O:
385.1903. Found: 385.1913.
Feng, X.-Q., Zeng, Z.-S., Liang, Y.-H., Chen, F.-E., Pannecouque, C., Balzarini, J., De
Clercq, E., 2010. Synthesis and biological evaluation of 4-(hydroxyimino)
arylmethyl diarylpyrimidine analogues as potential non-nucleoside reverse
transcriptase inhibitors against HIV. Bioorg. Med. Chem. 18, 2370–2374.
Gu, S.-X., He, Q.-Q., Yang, S.-Q., Ma, X.-D., Chen, F.-E., De Clercq, E., Balzarini, J.,
Pannecouque, C., 2011. Synthesis and structure–activity relationship of novel
diarylpyrimidines with hydromethyl linker (CH(OH)-DAPYs) as HIV-1 NNRTIs.
Bioorg. Med. Chem. 19, 5117–5124.
Gu, S.-X., Li, Z.-M., Ma, X.-D., Yang, S.-Q., He, Q.-Q., Chen, F.-E., De Clercq, E.,
Balzarini, J., Pannecouque, C., 2012. Chiral resolution, absolute configuration
assignment and biological activity of racemic diarylpyrimidine CH(OH)-DAPY
as potent nonnucleoside HIV-1 reverse transcriptase inhibitors. Eur. J. Med.
Chem. 53, 229–234.
Hassam, M., Basson, A.E., Liotta, D.C., Morris, L., van Otterlo, W.A., Pelly, S.C., 2012.
Novel cyclopropyl-indole derivatives as HIV non-nucleoside reverse
transcriptase inhibitors. ACS Med. Chem. Lett. 3, 470–475.
Jain, A.N., 2009. Effects of protein conformation in docking: improved pose
prediction through protein pocket adaptation. J. Comput. Aided Mol. Des. 23,
355–374.
4.2. Anti-HIV activity assay
Ma, X.-D., Yang, S.-Q., Gu, S.-X., He, Q.-Q., Chen, F.-E., De Clercq, E., Balzarini, J.,
Pannecouque, C., 2011. Synthesis and anti-HIV activity of aryl-2-[(4-
cyanophenyl) amino]-4-pyrimidinone hydrazones as potent non-nucleoside
reverse transcriptase inhibitors. ChemMedChem 6, 2225–2232.
Meng, G., Chen, F., De Clercq, E., Balzarini, J., Pannecouque, C., 2003. Nonnucleoside
HIV-1 reverse transcriptase inhibitors: Part I. Synthesis and structure–activity
relationship of 1-alkoxymethyl-5-alkyl-6-naphthylmethyl uracils as HEPT
analogues. Chem. Pharm. Bull. (Tokyo) 51, 779–789.
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-diphenyl tetrazolium
bromide (MTT) method. 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
Meng, G., Kuang, Y., Ji, L., Chen, F.e., 2005. Synthesis of 1-[(2-
hydroxyethoxy)methyl]-6-(5,6,7,8-tetrahydronaphthylmethyl-1)thymine
as
novel inhibitor against drug-resistant HIV mutants. Synth. Commun. 35,
1095–1102.
of 0.02. Immediately after viral infection, 100 lL of the cell suspen-
Mordant, C., Schmitt, B., Pasquier, E., Demestre, C., Queguiner, L., Masungi, C.,
Peeters, A., Smeulders, L., Bettens, E., Hertogs, K., 2007. Synthesis of novel
diarylpyrimidine analogues of TMC278 and their antiviral activity against HIV-1
wild-type and mutant strains. Eur. J. Med. Chem. 42, 567–579.
Spitzer, R., Jain, A.N., 2012. Surflex-Dock: docking benchmarks and real-world
application. J. Comput. Aided Mol. Des. 26, 687–699.
Thakur, A., Thakur, M., Bharadwaj, A., Thakur, S., 2008. SAR and QSAR studies:
modelling of new DAPY derivatives. Eur. J. Med. Chem. 43, 471–477.
Tian, X., Qin, B., Wu, Z., Wang, X., Lu, H., Morris-Natschke, S.L., Chen, C.H., Jiang, S.,
Lee, K.-H., Xie, L., 2010. Design, synthesis, and evaluation of diarylpyridines and
diarylanilines as potent non-nucleoside HIV-1 reverse transcriptase inhibitors.
J. Med. Chem. 53, 8287–8297.
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
Zeng, Z.-S., Liang, Y.-H., Feng, X.-Q., Chen, F.-E., Pannecouque, C., Balzarini, J., De
Clercq, E., 2010. Lead optimization of diarylpyrimidines as non-nucleoside
Inhibitors of HIV-1 reverse transcriptase. ChemMedChem 5, 837–840.
Zhan, P., Liu, X., Li, Z., 2009. Recent advances in the discovery and development of
novel HIV-1 NNRTI platforms: 2006–2008 update. Curr. Med. Chem. 16, 2876–
2889.
The authors are grateful to the Scientific Research Foundation
for the Returned Overseas Chinese Scholars, the Ministry of
National Education, PR China (JWSL 20101561), the Shaanxi
Province Science and Technology Research and Development
Program of China, International Cooperation (2013KW31-04) and