Novel (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones with restricted conformation as potent non-nucleoside reverse transcriptase inhibitors against HIV-1

Article abstract of DOI:10.1016/j.ejmech.2016.06.026

To elucidate the structure-geometry-activity relationship in diarylpyrimidine family (DAPYs) containing carbonyl linker between the central pyrimidine core and phenyl type B-arm, a series of (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones wa

Full text of DOI:10.1016/j.ejmech.2016.06.026

European Journal of Medicinal Chemistry 122 (2016) 185e195
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel (2,6-difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)
methanones with restricted conformation as potent non-nucleoside
reverse transcriptase inhibitors against HIV-1
a
a
a
b
b
ꢀ
ꢀ
ꢀ
ꢀ
Petr Simon , Ondrej Baszczynski , David Saman , George Stepan , Eric Hu ,
Eric B. Lansdon b, Petr Jansa b, Zlatko Janeba a
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, Prague-6 CZ-16610, Czech Republic
b Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA 94404, USA
,
*
a
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
To elucidate the structure-geometry-activity relationship in diarylpyrimidine family (DAPYs) containing
Received 15 April 2016
Received in revised form
1 June 2016
Accepted 15 June 2016
Available online 17 June 2016
carbonyl linker between the central pyrimidine core and phenyl type B-arm,
a series of (2,6-
difluorophenyl)(2-(phenylamino)pyrimidin-4-yl)methanones was designed, prepared and tested for
their anti-HIV-1 activity. The carbonyl linker bearing B phenyl arm was successfully attached at both C-2
and C-4 positions of the central pyrimidine ring using a new synthetic approach. Further modifications of
target compounds are present at C-5 position of the pyrimidine ring. In vitro anti-HIV-1 activity study
performed on a series of 22 compounds confirmed the crucial importance of both conformational rigidity
between phenyl B arm and the pyrimidine core linked through the carbonyl bridge, as well as presence of
fluoro substituents in ortho-positions of phenyl B moiety. The most potent derivative of the series,
compound 17, having almost perpendicular angle within the two planes made from the B aromatic arm
and the pyrimidine ring, exhibited low nanomolar anti-HIV-1 activity (EC50 ¼ 4 nM) with no significant
Keywords:
Diarylpyrimidine (DAPY)
Etravirine
Human immunodeficiency virus (HIV)
Non-nucleoside reverse transcriptase
inhibitors
toxicity (CC50 > 57.1 mM).
NNRTIs
Rilpivirine
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
with the approved NNRTIs had key implications for further drug
design [18].
Etravirine (Fig. 1) [1e3] and rilpivirine (Fig. 1) [4,5] represent
highly potent and FDA approved non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs) of human immunodeficiency virus
(HIV-1) [6e11]. NNRTIs play an important role in highly active
antiretroviral therapy (HAART) and in prevention and treatment of
HIV infections. Due to the emergence of severe side effects and due
to the development of multidrug-resistant HIV strains, intensive
and continuous efforts have led to the identification of a number of
promising NNRTIs hits, leads, and candidates [12e17]. Etravirine
and rilpivirine belong to the diarylpyrimidine (DAPY) family and
specifically bind to an allosteric hydrophobic site of reverse tran-
scriptase (RT) of HIV [6]. As RT function is utterly essential in the
HIV lifecycle, the area of DAPY analogues has been intensively
studied during the last two decades due to their high potency
[6e17]. Especially, determination of crystal structures of HIV-1 RT
A large number of SAR studies on DAPY analogues were per-
formed covering various substitutions of the aromatic rings, as well
as diverse linkers between C-4 position of the pyrimidine ring and
aryl moiety B (Fig. 2a). Among the latter modifications, carbon
based linkers have been extensively studied, namely compounds
having linkers such as: a) carbonyl group [19] or its derivatives as
Schiff bases with hydrazine [20] or hydroxylamine [21] (such an-
alogues were low nanomolar inhibitors of HIV-1 RT); b) (cyclo-
propylamino)methylenes [22] and (alkylamino)methylenes [23]
(prepared by reduction of the corresponding imino derivatives); c)
hydroxymethylene moiety (prepared by reduction of the carbonyl
linker) [24]; d) halomethylene [25] and cyanomethylene [26]
linkers (with comparable potency to analogues with the hydroxy-
methylene linkers); e) hydroxyl(alkyl)methylene linkers [27]
(formed by the reaction of the corresponding alkylmagnesium re-
agent with the carbonyl group); and f) eCH2-NH- diatomic linker
[28] (to improve conformational flexibility and positional adapt-
ability of traditional DAPY compounds).
* Corresponding author.
E-mail address: janeba@uochb.cas.cz (Z. Janeba).
http://dx.doi.org/10.1016/j.ejmech.2016.06.026
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
moiety (as B arm) at C-4 position of the central pyrimidine core
(Fig. 2c).
2. Results and discussion
2.1. Chemistry
The reported synthetic pathways leading to carbonyl modified
DAPY derivatives usually consist of two key synthetic sequences
[19]: 1) the (4-cyanophenyl)amino moiety is connected to C-2 po-
sition of the pyrimidine ring; 2) the chlorine atom in C-4 position of
pyrimidine is replaced by a second aromatic arm, using corre-
spondingly substituted phenylacetonitriles under basic conditions.
But, such a synthetic approach has not been successful to produce
the desired analogues bearing 2,6-difluorophenyl B arm connected
to the central pyrimidine core via the carbonyl linker. To overcome
this synthetic obstacle, we have designed new synthetic sequence
where B arm is attached to 2,4-dichloropyrimidine in the first step,
followed by the subsequent modification of C-2 position of the
pyrimidine ring [32]. In order to test the efficiency of such synthetic
approach and to easily characterize the two possible regioisomers
formed in the first reaction step, namely in the reaction of 2,4-
dichloropyrimidine with the corresponding arylaldehyde, several
model compounds without ortho-substituents on B phenyl ring
were synthesized first (Scheme 1).
Fig. 1. Structures of the FDA approved NNRTIs e etravirine and rilpivirine.
When comparing anti-HIV-1 activity of previously published
compounds with methylene or carbonyl linker (Fig. 2a) their ac-
tivity strongly depends on the position of substituents on B aryl
ring. Presence of substituents in both C-2 and C-6 positions of
phenyl B ring is crucial for the biological activity of NNRTIs, as is
also the case of 2,6-dimethylphenyl moiety in etravirine and rilpi-
virine (Fig. 1). The two ortho-substituents generate almost
perpendicular geometry of B aryl ring towards the pyrimidine
plane and modulation at both B ring ortho-positions resulted in a
significant increase of anti-HIV-1 activity [29]. Such structural
features were previously combined in compounds bearing B aryl
ring substituted with either chloro [30], bromo [30], and fluoro [31]
atoms in both ortho-positions. In particular, the recently reported
series based on a combination of structural features of DAPY and
DABO (dihydroxy-alkoxy-benzyl-oxopyrimidines) analogues and
bearing 2,6-difluorophenyl B ring (Fig. 2b) exhibited nanomolar
anti-HIV-1 activity against wild-type, as well as clinically relevant
mutant strains [31]. Based on the above data we have designed and
synthesized a novel series of NNRTIs having 2,6-difluorobenzoyl
Thus, commercially available 2,4-dichloropyrimidine was reac-
ted with 4-formylbenzonitrile under the N,Nꢁ-dimethylbenzimida-
zolium iodide catalysis [33] to give a mixture of regioisomers 1 and
2 (Scheme 1) in a 4:3 ratio (20% and 15% yields, respectively).
Analogous reaction of 4-methoxybenzaldehyde with 2,4-
dichloropyrimidine afforded only the desired regioisomer
3
(Scheme 1), modified at C-4 position of pyrimidine, though in low
27% yield. Intermediates 1e3 were subsequently treated with 4-
aminobenzonitrile under the Buchwald coupling conditions [34]
to yield corresponding derivatives 4e6 (Scheme 1) in satisfactory
yields (53e68%). Although the synthesis of compounds 4 [22] and 6
[19] has recently been reported, the original synthetic route con-
sisted of five steps offering the final products in significantly lower
Fig. 2. a) Numbering and general structure of DAPY analogues; b) structure of potent DAPYeDABO hybrides [31]; c) geometry and structure of target molecules featuring the
carbonyl linker and 2,6-difluorophenyl B ring.

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
187
Scheme 1. Reagents and conditions: (a) NaH, N,Nꢁ-dimethylbenzimidazolium iodide, DMF, r.t., 4 h; (b) 4-aminobenzonitrile, Pd(OAc)2, XantPhos, Cs2CO3, dioxane, 100 ꢀC, 4 h.
overall yields compared to our novel approach.
Subsequently, the intended NNRTIs series bearing 2,6-
difluorobenzoyl moiety as B arm was prepared using the above
developed reaction sequence. First, the reactions of 2,4-
dichloropyrimidine with 4-substituted 2,6-difluorobenzaldehydes
under the N,Nꢁ-dimethylbenzimidazolium iodide catalysis [33]
yielded intermediates 13e15 (Scheme 3) in variable yields
(28e73%). Bromo derivative 15 was then converted into cyano
analogue 16 (Scheme 3) in 88% yield using Negishi coupling con-
ditions. The reaction of 2-chloropyrinidine derivatives 13, 14 and 16
with 4-aminobenzonitrile under Buchwald coupling conditions
[34] afforded the desired products 17e19 (Scheme 3) in moderate
yields. When the Buchwald reaction was applied to bromo
derivative 15, both halogen atoms were replaced with 4-
aminobenzonitrile giving derivative 20 (Scheme 3) in 18% yield.
Finally, bromination of derivative 17 using NBS in DMF afforded
both desired 5-bromopyrimidine derivative 21 (12%) and bis-
brominated by-product 22 (13%, Scheme 4). To evaluate the
For correct assignment of true structures of pyrimidine
regioisomers 1 and 2 (Scheme 1), and subsequently of the whole
NNRTIs series (see details in 2.2 chapter of NMR studies), the
hydroxymethylene derivatives 7 and 8 (Scheme 2) were prepared
from compounds 4 and 5, respectively, using sodium borohydride
in MeOH [24].
In order to prepare structural analogues of etravirine (Fig. 1),
compounds 4e6 were converted into their 5-bromopyrimidine
derivatives 9e11 (Scheme 2) using N-bromosuccinimide (NBS).
The yields of the bromination highly depend on the aryl substitu-
tion of B phenyl arm as quite complex mixtures were observed in
the cases of electron withdrawing 4-cyano group. In this case,
desired 5-bromopyrimidines 9 (17%) and 11 (3%), as well as the by-
product 12 (8%, Scheme 2), were obtained in very low yields.
Electron richer derivative 10 (Scheme 2) was, on the other hand,
isolated in a 92% yield.
Scheme 2. Reagents and conditions: (a) NaBH4, MeOH, r.t., 1 h; (b) NBS, DMF, 100 ꢀC, overnight.

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
Scheme 3. Reagents and conditions: (a) NaH, N,Nꢁ-dimethylbenzimidazolium iodide, dioxane, 80 ꢀC, overnight; (b) Zn(CN)2, Pd(t-Bu3P)2, DMF, 80 ꢀC, 1 h; (c) 4-aminobenzonitrile,
Pd(OAc)2, XantPhos, Cs2CO3, dioxane, 100 ꢀC, 4 h.
Table 1
biological effect of the 4-methoxy versus 4-hydroxy group present
on B phenyl ring, compound 21 was treated with boron tribromide
in DMC to give derivative 23 (in 9% yield, Scheme 4).
Anti-HIV activity and toxicity of all tested derivatives (n ¼ 2).
Compound
EC50-MT4 (
mM)
CC50-MT4 (
m
M)
SIa
1
2
>57
>57
>57
4.5
43
0.814
3.7
31.9
16.5
39.6
>57
>57
>57
>57
>57
>57
2.45
>57
>57
40.1
>57
30.5
>57
>57
>57
51.9
38.7
>57
>57
5.88
20.6
<0.56
<0.29
<0.69
>12.7
>1.33
>70.0
>15.4
~1.00
>7.70
<0.04
>2.06
~1.00
<0.70
~1.00
<0.54
>14,250
>1676
>1118
167
2.2. NMR studies
3
4b
The pyrimidine regioisomers modified either in C-2 or in C-4
positions have very similar 1H and 13C NMR spectra. Due to lack of
protons in such derivatives the unambiguous assignment of carbon
signals and confirmation of the structures are difficult. Generally,
the 2D NMR HSQC and HMBC spectra are used. Unfortunately, it is
quite a complicated task to distinguish between positions of the
substituents in isomeric compounds 1 and 2, as well as 4 and 5
(Scheme 1) due to the absence of cross peaks Py-H5/C]O. In order
to determine the absolute structures we decided to reduce the
carbonyl function in compounds 4 and 5 to prepare the corre-
sponding hydroxymethyl analogues 7 and 8 (Scheme 2). The ability
to observe CHeOH/Py-C5 interaction allowed us to unequivocally
confirm the structure of compound 7.
5
6c
7
8
9
10
11
13
14
15
16
17
18
19
20
21
22
23
>57
7.4
>57
27.7
>57
>57
>57
>57
0.004
0.034
0.051
0.310
0.069
18.9
>57
0.002
0.001
561
>3.02
~1.00
2940
2.3. Biological activity
Etravirine
Efavirenz
20,600
The prepared carbonyl bridged DAPY analogues were tested for
their in vitro anti-HIV-1 activity (Table 1). Compounds 17e21
exhibited submicromolar activities and no or low cytotoxicity. The
common structural feature of the most potent compounds is 4-
cyanophenyl as A ring and 4-substituted 2,6-difluorobenzoyl as B
arm. The most active compound 17 (EC50 ¼ 4 nM) represents new
NNRTIs lead structure for further optimization.
Evidently, the synthetic intermediates, namely compounds 1e3
and 13e16, lacking the 4-aminobenzonitrile moiety (A arm) do not
possesses any antiviral properties (Table 1). This corroborates the
relevance of U-shaped diarylpyrimidine structural feature for
potent antiretroviral properties of DAPY analogues. Furthermore,
the absence of one or two ortho-substituents on B ring in DAPY
a
Selectivity index: CC50/EC50 ratio.
Known compound 4 [22].
Known compound 6 with reported EC50 ¼ 0.239
b
c
mM [19].
series (compounds 11 and 4e10, respectively) does not lead to
substantial perpendicular geometry of B aryl moiety towards the
central pyrimidine plane, which explains much worse (several or-
ders of magnitude) anti-HIV-1 activity of these compounds
(Table 1).
Finally, as the effectiveness of current NNRTI agents is usually
hampered by the rapid emergence of drug-resistant viruses, there
is an urgent need to develop novel NNRTIs without such a
Scheme 4. Reagents and conditions: (a) NBS, DMF, 100 ꢀC, overnight; (b) BBr3, DCM, r.t., overnight.

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
189
limitation. Thus, the most potent compound 17 was also tested
towards two major HIV mutant strains K103 N and Y181C (Table 2).
Regretably, compound 17 did not exhibit any promising activity
against the two mutant strains compared to approved antiviral
agents etravirine and efavirenz.
2.4. Crystallography results
To investigate the binding mode of the most potent compound
17, the co-crystal structure with HIV-1 RT was determined. Com-
pound 17 was found to occupy the NNRTI binding pocket akin to
previously reported DAPY analogues (Fig. 3) [18]. Near the pyrim-
idine core, residues E138 from the p51 subunit and K101 from the
p66 subunit form a salt bridge which defines part of the pocket. The
pyrimidine core is sandwiched between residues L100 and V179
and a hydrogen bond is found between the aniline nitrogen and the
carbonyl of K101 (2.7 Å). The pyrimidine nitrogen is also in close
proximity to the backbone nitrogen of K101 (3.4 Å) however the
distance suggests that it is not forming a strong hydrogen bond. The
A arm is relatively coplanar with the pyrimidine core with torsion
of 11ꢀ out of plane while the B arm is closer to perpendicular with
the pyrimidine (torsion 63ꢀ). The B arm lies across the aromatic
residues Y181 and Y183. The carbonyl linker between the pyrimi-
dine and B arm is well accommodated in the pocket and not
forming any specific interactions with the protein.
Fig. 3. Co-crystal structure of HIV-1 reverse transcriptase (RT) and compound 17.
Dashed lines show hydrogen bond contacts in the pocket between the compound and
the protein.
derivatives with required 2,6-difluorobenzoyl moiety connected to
C-4 position of the pyrimidine ring. From the series prepared,
compounds 17e19 and 21 exhibited nanomolar activities
(4e69 nM) against HIV-1 with no significant toxicity. A common
feature of these potent compounds is the presence of 2,6-
difluorophenyl B arm, that leads to almost perpendicular posi-
tioning of B ring with the pyrimidine core. This key conformational
feature has been corroborated by determination of co-crystal
structure of the most potent compound 17 with HIV-1 RT. On the
other hand, compounds 4e6 and 9e11, which do not possess this
conformational advantage and are practically planar, were shown
to be several orders of magnitude less active against HIV-1
compared to their counterparts with both ortho-substituents on B
phenyl ring. In other words, the conformational rigidity and nearly
perpendicular position of the B arm towards pyrimidine core are
utterly essential for activity of the reported analogues. Unfortu-
nately, the most potent derivative 17 did not exhibit any interesting
antiviral activity against major NNRTI-resistant single mutants
K103 N and Y181C. The resistance profile of the reported DAPY
series thus needs to be improved within the future antiretroviral
drug discovery design. The most potent NNRTIs reported herein,
nevertheless, represent a promising starting point in the develop-
ment of even more efficacious anti-HIV drugs.
2.5. Computational modeling
Molecular dockings using Glide XP (Schrodinger 2015) have
been performed with various in-house NNRTI X-ray crystal struc-
tures to guide compound design. Re-dockings of co-crystal ligands
show on the average less than 1 Å RMSD comparing to their crys-
tallographic conformations. Docking modes of compounds 17 and
21 show high overall similarities (Fig. 4). The binding mode change
in compound 21, in comparison to compound 17, is mainly due to
the extra Br group (Fig. 4a). Compound 21 binds in the NNRTI
pocket with a less ideal and bigger out-of-plane carbonyl dihedral
angel (34ꢀ) comparing to compound 17 (24ꢀ). The BreO distance is
3.1 Å which is smaller than sum of individual van der Waals radius
(Rvdw,Br ¼ 1.9 Å and Rvdw,O ¼ 1.6 Å). There is potentially more
negative charge repulsion between Br and neighbouring carbonyl
group (Fig. 4b). This could explain why compound 21 is less potent
than 17 contrary to the potency gains from the Br group in the
etravirine series where there is no carbonyl group and the oxygen
atom simply acts as the linker between the pyrimidine and phenyl
ring.
4. Experimental part
3. Conclusion
4.1. Chemistry
A novel series of NNRTIs was designed that combines structural
features of 2,6-difluorophenyl B ring [31] and NNRTI analogues
having carbonyl group as a linker between the B aryl ring and
central pyrimidine [19]. New synthetic route has been developed to
prepare the target NNRTIs, where a reaction of commercially
available 2,4-dichloropyrimidine with various arylaldehydes was
used as the first step. This approach allowed us to prepare unique
Chemical reagents and solvents were of analytical grade and
were used as purchased. 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance III NMR spectrometer at 600.1 MHz
(for 1H) equipped by 5 mm TCI cryoprobehead in DMSO-d6 (Aldrich,
99.8% D) or CD3OD (Aldrich, 99.8% D). The chemical shifts are re-
ported in d units, for standardization the residual peaks of solvents
(DMSO-d6 1H 2.5 ppm and 13C 39.5 ppm; CD3OD 1H 3.31 ppm and
13C 49.8 ppm) were used. For details concerning the experimental
techniques and methods see part 2.2. Following abbreviations were
used to describe peak patterns: Py ¼ corresponds to pyrimidine
part of molecule, An ¼ aromatic system bonded to amino (e.g. 4-
cyanophenylamino) group, Ar ¼ carbon bonded aromatic system
(e.g. 4-cyanobenzoyl). The high resolution mass spectra (HRMS)
were measured on LTQ Orbitrap XL spectrometer (Thermofisher
Table 2
Anti-HIV activity of compound 17 against wild type (w.t.) and K103 N and Y181C
mutant strains (n ¼ 2).
Compound
EC50
(
m
M) w.t.
EC50
(
mM) K103 N
EC50 (mM) Y181C
17
Etravirine
Efavirenz
0.004
0.002
0.001
0.347
0.002
0.101
>0.500
0.012
0.005

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
Fig. 4. Docked conformations for compound 17 (green carbon) and 21 (grey carbon) in (a) side view and (b) top view. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Scientific) using ESI ionisation. All the progress of the reactions was
monitored by thin-layer chromatography (TLC) on silica gel plates
at 254 nm under a UV lamp or by Waters UPLC/MS system using
water-acetonitrile gradient (0.1% formic acid as modifier) on Phe-
4.1.2. (2-Chloropyrimidin-4-yl)(4-methoxyphenyl)methanone (3)
4-Methoxybenzaldehyde (1 mL, 8.2 mmol) and sodium hydride
(60% dispersion in mineral oil, 0.5 g, 12 mmol) were added to a
solution of 2,4-dichloropyrimidine (1 g, 6.7 mmol) and N,Nꢁ-dime-
thylbenzimidazolium iodide (0.5 g, 1.8 mmol) in anhydrous DMF
(50 mL). The reaction mixture was stirred at room temperature for
5 h and poured into ice cold water. The product was extracted with
EtOAc (2 ꢁ 100 mL). Organic phase was washed with brine
(2 ꢁ 100 mL), dried over sodium sulphate and evaporated to dry-
ness. Column chromatography on silica gel using gradient from 0%
to 50% ethyl acetate in hexane afforded 3 (450 mg, 27%) off-white
nomenex Gemini-NX 3
m
C18 110 Å, 100 ꢁ 2.00 mm, flow 0.5 mL/
min. Flash chromatography separations were performed on silica
gel (300e400 mesh) using Isco Teledyne apparatus. All compounds
were standardised before biological testing by preparative HPLC
(Waters Delta 600) using water-acetonitrile gradient (0.1% tri-
fluoroacetic acid as modifier) on Phenomenex Gemini 10
mm C18,
250 ꢁ 21.2 mm, flow 10 mL/min followed by lyophilisation.
solid. 1H NMR (600.1 MHz, DMSO-d6)
d
9.05 (d, J ¼ 4.9 Hz, 1H, Py-
H6), 8.00e7.97 (m, 2H, Ar-ortho), 7.93 (d, J ¼ 4.9 Hz, 1H, Py-H5),
4.1.1. Preparation of 4-(2-chloropyrimidine-4-carbonyl)benzonitrile
(1) and 4-(4-chloropyrimidine-2-carbonyl)benzonitrile (2)
7.13e7.10 (m, 2H, Ar-meta), 3.89 (s, 3H, CH3); 13C NMR
(150.9 MHz, DMSO-d6)
d 189.02 (C]O), 164.80 (Py-C4), 164.21 (Ar-
Sodium hydride (60% dispersion in mineral oil, 0.5 g, 12 mmol)
was added to a solution of 2,4-dichloropyrimidine (1 g, 6.7 mmol),
4-formylbenzonitrile (1 g, 7.6 mmol) and N,Nꢁ-dimethylbenzimi-
dazolium iodide (1 g, 3.6 mmol) in anhydrous DMF (100 mL). The
reaction mixture was stirred at room temperature for 4 h and
poured into ice cold water. The product was extracted with EtOAc
(2 ꢁ 100 mL). Organic phase was washed with brine (2 ꢁ 100 mL),
dried over sodium sulphate and evaporated to dryness. Column
chromatography on silica gel using gradient from 0% to 50% ethyl
acetate in hexane afforded 1 (320 mg, 20%) and 2 (250 mg, 15%).
4-(2-Chloropyrimidine-4-carbonyl)benzonitrile (1). 1H NMR
para), 162.45 (Py-C6), 159.36 (Py-C2), 133.19 (Ar-ortho), 126.69 (Ar-
ipso), 119.18 (Py-C5), 114.16 (Ar-meta), 55.76 (CH3); HRMS (ESI):
Calculated for
C
12H10N2O2Cl [MþH]þ: 249.04253, Found:
249.04262; UPLC/MS (m/z) 248.75 [MþH]þ, Tr 3.96 min.
4.1.3. 4-((4-(4-Cyanobenzoyl)pyrimidin-2-yl)amino)benzonitrile
(4)
[22] A solution of 1 (200 mg, 0.82 mmol), 4-aminobenzonitrile
(100 mg, 0.85 mmol), palladium(II) acetate (20 mg, 0.09 mmol),
(600.1 MHz, DMSO-d6)
(m, 2H, Ar-ortho), 8.08e8.06 (m, 2H, Ar-meta), 8.05 (d, J ¼ 4.9 Hz,
1H, Py-H5); 13C NMR (150.9 MHz, DMSO-d6)
190.11 (C]O), 162.92
(Py-C6),162.52 (Py-C4),159.43 (Py-C2), 137.79 (Ar-ipso),132.44 (Ar-
meta), 131.13 (Ar-ortho), 119.46 (Py-C5), 118.06 (CN), 115.73 (Ar-
para); HRMS (ESI): Calculated for C12H7N3OCl [MþH]þ: 244.0278,
Found: 244.0271; UPLC/MS (m/z) 243.90 [MþH]þ, Tr 3.82 min.
4-(4-Chloropyrimidine-2-carbonyl)benzonitrile (2). 1H NMR
d
9.12 (d, J ¼ 4.9 Hz, 1H, Py-H6), 8.15e8.13
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(100
mg,
0.17 mmol) and cesium carbonate (1 g, 3.1 mmol) in dry dioxane
(5 mL) was heated at 90 ꢀC under argon atmosphere for 3 h. The
reaction mixture was evaporated to dryness. Column chromatog-
raphy on silica gel using gradient from 0% to 100% ethyl acetate in
hexane gave 4 (180 mg, 68%). 1H NMR (600.1 MHz, DMSO-d6)
d
d
10.47 (bs,1H, NH), 8.89 (d, J ¼ 4.9 Hz,1H, Py-H6), 8.17e8.14 (m, 2H,
Ar-ortho), 8.08e8.05 (m, 2H, Ar-meta), 7.90e7.87 (m, 2H, An-
ortho), 7.70e7.67 (m, 2H, An-meta), 7.43 (d, J ¼ 4.9 Hz, 1H, Py-
(600.1 MHz, DMSO-d6)
(m, 2H, Ar-ortho), 8.06e8.04 (m, 2H, Ar-meta), 8.01 (d, J ¼ 5.4 Hz,
1H, Py-H5); 13C NMR (150.9 MHz, DMSO-d6)
188.89 (C]O), 161.04
d
9.02 (d, J ¼ 5.4 Hz, 1H, Py-H6), 8.13e8.11
H5); 13C NMR (150.9 MHz, DMSO-d6)
d 191.91 (C]O), 160.77 (Py-
d
C6), 160.62 (Py-C4), 158.74 (Py-C2), 144.32 (An-ipso), 138.61 (Ar-
ipso), 132.98 (An-meta), 132.41 (Ar-meta), 130.90 (Ar-ortho), 119.41
(An-CN), 118.80 (Py-C5), 118.55 (An-ortho), 118.16 (Ar-CN), 115.47
(Ar-para), 103.03 (An-para); HRMS (ESI): Calculated for
(Py-C4*), 160.91 (Py-C2*), 159.55 (Py-C6), 138.14 (Ar-ipso), 132.48
(Ar-meta), 131.04 (Ar-ortho), 124.03 (Py-C5), 118.07 (CN), 115.70
(Ar-para); HRMS (ESI): Calculated for
C
12H7N3OCl [MþH]þ:
244.0278, Found: 244.0268; UPLC/MS (m/z) 243.71 [MþH]þ, Tr
C
19H11N5ONa [MþNa]þ: 348.08558, Found: 348.08570; UPLC/MS
3.63 min.
(m/z) 326.12 [MþH]þ, Tr 4.13 min.

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
191
4.1.4. 4-((2-(4-Cyanobenzoyl)pyrimidin-4-yl)amino)benzonitrile
(5)
A solution of 2 (200 mg, 0.82 mmol), 4-aminobenzonitrile
(100 mg, 0.85 mmol), palladium(II) acetate (20 mg, 0.09 mmol),
stirred at room temperature for 1 h and then evaporated to dryness.
The product was extracted with EtOAc (10 mL) and washed with
water. Organic phase was separated, dried over sodium sulphate
and evaporated to dryness to give 8 (25 mg, 76%) as yellowish
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(100
mg,
powder. 1H NMR (600.1 MHz, CD3OD)
d
8.31 (d, J ¼ 6.8 Hz, 1H, Py-
0.17 mmol) and cesium carbonate (1 g, 3.1 mmol) in dry dioxane
(5 mL) was heated at 90 ꢀC under argon atmosphere for 3 h. The
reaction mixture was evaporated to dryness. Column chromatog-
raphy on silica gel using gradient from 0% to 100% ethyl acetate in
hexane gave 5 (140 mg, 53%). 1H NMR (600.1 MHz, DMSO-d6)
H6), 7.80e7.78 (m, 2H, Ar-meta), 7.76e7.74 (m, 2H, Ar-ortho),
7.75e7.73 (m, 2H, An-ortho), 7.73e7.71 (m, 2H, An-meta), 6.94 (d,
J ¼ 6.8 Hz, 1H, Py-H5), 5.96 (s, 1H, CHO); 13C NMR (150.9 MHz,
CD3OD) d 168.75 (Py-C2), 163.14 (Py-C4), 148.59 (Py-C6), 147.68 (Ar-
ipso), 143.57 (An-ipso), 134.78 (An-meta), 134.05 (Ar-meta), 129.66
(Ar-ortho), 123.20 (An-ortho), 120.02 (An-CN), 119.91 (Ar-CN),
113.88 (Ar-para), 109.49 (An-para), 108.62 (Py-C5), 75.26 (CH);
HRMS (ESI): Calculated for C19H14N5O [MþH]þ: 328.11929, Found:
328.11945; UPLC/MS (m/z) 327.82 [MþH]þ, Tr 3.14 min.
d
10.46 (bs, 1H, NH), 8.55 (d, J ¼ 5.9 Hz, 1H, Py-H6), 8.12e8.09 (m,
2H, Ar-ortho), 8.02e7.99 (m, 2H, Ar-meta), 7.89e7.86 (m, 2H, An-
ortho), 7.72e7.69 (m, 2H, An-meta), 7.08 (d, J ¼ 5.9 Hz, 1H, Py-
H5); 13C NMR (150.9 MHz, DMSO-d6)
d 190.26 (C]O), 160.24 (Py-
C2), 159.76 (Py-C4), 155.76 (Py-C6), 143.70 (An-ipso), 138.78 (Ar-
ipso), 133.14 (An-meta), 132.37 (Ar-meta), 130.78 (Ar-ortho), 119.23
(An-ortho), 119.18 (An-CN), 118.14 (Ar-CN), 115.37 (Ar-ipso), 109.85
(Py-C5), 103.95 (An-para); HRMS (ESI): Calculated for C19H12N5O
[MþH]þ: 326.10364, Found: 326.10372; UPLC/MS (m/z) 325.87
[MþH]þ, Tr 3.76 min.
4.1.8. 4-(5-Bromo-2-((4-cyanophenyl)amino)pyrimidine-4-
carbonyl)benzonitrile (9)
A solution of 4 (410 mg, 1.26 mmol) and N-bromosuccinimide
(600 mg, 3.37 mmol) in DMF (20 mL) was heated at 100 ꢀC for 5 h
and then evaporated to dryness. Column chromatography on silica
gel using gradient from 0% to 100% ethyl acetate in hexane afforded
9 (84 mg, 17%) as yellow solid. 1H NMR (600.1 MHz, DMSO-d6)
4.1.5. 4-((4-(4-Methoxybenzoyl)pyrimidin-2-yl)amino)benzonitrile
(6) [19].
A solution of 3 (450 mg, 1.81 mmol), 4-aminobenzonitrile
(300 mg, 2.54 mmol), palladium(II) acetate (30 mg, 0.13 mmol),
d
10.69 (bs,1H, NH), 8.95 (s, 1H, Py-H6), 8.14e8.11 (m, 2H, Ar-ortho),
8.10e8.07 (m, 2H, Ar-meta), 7.87e7.84 (m, 2H, An-ortho), 7.74e7.71
(m, 2H, An-meta); 13C NMR (150.9 MHz, DMSO-d6)
190.52 (C]O),
d
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(150
mg,
161.26 (Py-C6), 161.22 (Py-C4), 157.48 (Py-C2), 143.87 (An-ipso),
136.33 (Ar-ipso), 133.30 (Ar-meta), 133.10 (An-meta), 130.55 (Ar-
ortho), 119.28 (An-CN), 118.71 (An-ortho), 117.85 (Ar-CN), 116.92
(Ar-para), 104.70 (Py-C5), 103.45 (An-para); HRMS (ESI): Calculated
for C19H10N5OBrNa [MþNa]þ: 425.99609, Found: 425.99615; UPLC/
MS (m/z) 404.18 [MþH]þ, Tr 4.41 min.
0.26 mmol) and cesium carbonate (2 g, 6.1 mmol) in dry dioxane
(10 mL) was heated at 90 ꢀC under argon atmosphere for 1 h. The
reaction mixture was evaporated to dryness. Column chromatog-
raphy on silica gel using gradient from 0% to 100% ethyl acetate in
hexane afforded 6 (330 mg, 55%). 1H NMR (600.1 MHz, DMSO-d6)
d
10.46 (bs, 1H, NH), 8.83 (d, J ¼ 4.8 Hz, 1H, Py-H6), 8.02e7.99 (m,
2H, Ar-ortho), 7.97e7.94 (m, 2H, An-ortho), 7.73e7.70 (m, 2H, An-
meta), 7.28 (d, J ¼ 4.8 Hz, 1H, Py-H5), 7.13e7.10 (m, 2H, Ar-meta),
4.1.9. 4-((5-Bromo-4-(4-methoxybenzoyl)pyrimidin-2-yl)amino)
benzonitrile (10)
3.88 (s, 3H, CH3); 13C NMR (150.9 MHz, DMSO-d6)
d
190.78 (C]
A solution of 6 (280 mg, 0.85 mmol) and N-bromosuccinimide
(400 mg, 2.25 mmol) in DMF (5 mL) was heated at 100 ꢀC overnight
and then evaporated to dryness. Column chromatography on silica
gel using gradient from 0% to 100% ethyl acetate in hexane afforded
10 (320 mg, 92%) as yellow solid. 1H NMR (600.1 MHz, DMSO-d6)
O), 163.98 (Ar-para), 163.11 (Py-C4), 160.12 (Py-C6), 158.61 (Py-C2),
144.49 (An-ipso), 133.00 (An-meta), 133.00 (Ar-ortho), 127.24 (Ar-
ipso), 119.41 (An-CN), 118.45 (An-ortho), 114.08 (Ar-meta), 111.83
(Py-C5), 102.83 (An-para), 55.71 (CH3); HRMS (ESI): Calculated for
C
19H14N4O2Na [MþNa]þ: 353.10090, Found: 353.10107; UPLC/MS
d
10.64 (bs, 1H, NH), 8.89 (s, 1H, Py-H6), 7.89e7.86 (m, 4H, Ar-ortho,
An-ortho), 7.76e7.73 (m, 2H, An-meta), 7.13e7.10 (m, 2H, Ar-meta),
3.88 (s, 3H, CH3); 13C NMR (150.9 MHz, DMSO-d6)
189.52 (C]O),
(m/z) 331.03 [MþH]þ, Tr 4.22 min.
d
4.1.6. 4-((4-((4-Cyanophenyl)(hydroxy)methyl)pyrimidin-2-yl)
amino)benzonitrile (7)
164.66 (Ar-para), 163.21 (Py-C4), 160.63 (Py-C6), 157.32 (Py-C2),
143.99 (An-ipso), 133.09 (An-meta), 132.54 (Ar-ortho), 126.13 (Ar-
ipso), 119.31 (An-CN), 118.60 (An-ortho), 114.69 (Ar-meta), 104.69
(Py-C5), 103.27 (An-para), 55.81 (OCH3); HRMS (ESI): Calculated for
Sodium borohydride (5 mg, 0.13 mmol) was added to a solution
of 4 (33 mg, 0.10 mmol) in MeOH (5 mL). The reaction mixture was
stirred at room temperature for 1 h and then evaporated to dryness.
The product was extracted with EtOAc (10 mL) and washed with
water. Organic phase was separated, dried over sodium sulphate
and evaporated to dryness to give 7 (14 mg, 43%) as a yellowish
C
19H13N4O2BrNa [MþNa]þ: 431.01141, Found: 431.01142; UPLC/MS
(m/z) 409.03 [MþH]þ, Tr 4.42 min.
4.1.10. Preparation of 4-(5-bromo-4-((4-cyanophenyl)amino)
pyrimidine-2-carbonyl)benzonitrile (11) and 4-(5-bromo-4-((2-
bromo-4-cyanophenyl)amino)pyrimidine-2-carbonyl)benzonitrile
(12)
powder. 1H NMR (600.1 MHz, CD3OD)
d
8.49 (d, J ¼ 5.1 Hz, 1H, Py-
H6), 7.84e7.81 (m, 2H, An-ortho), 7.73e7.71 (m, 2H, Ar-meta),
7.70e7.68 (m, 2H, Ar-ortho), 7.60e7.57 (m, 2H, An-meta), 7.17 (dd,
J ¼ 0.6, 5.1 Hz, 1H, Py-H5), 5.74 (s, 1H, CHO); 13C NMR (150.9 MHz,
A solution of 5 (30 mg, 0.09 mmol) and N-bromosuccinimide
(120 mg, 0.67 mmol) in DMF (3 mL) was heated at 100 ꢀC for 3 h and
then evaporated to dryness. Column chromatography on silica gel
using gradient from 0% to 100% ethyl acetate in hexane afforded 11
(1 mg, 3%) as yellow powder and 12 (3 mg, 8%) as yellow solid.
4-(5-Bromo-4-((4-cyanophenyl)amino)pyrimidine-2-carbonyl)
CD3OD) d 174.22 (Py-C4), 160.95 (Py-C2), 160.46 (Py-C6),150.07 (Ar-
ipso), 146.67 (An-ipso), 134.46 (An-meta), 133.80 (Ar-meta), 129.41
(Ar-ortho), 120.91 (An-CN), 120.20 (An-ortho), 120.16 (Ar-CN),
112.97 (Ar-para), 111.16 (Py-C5),105.18 (An-para), 76.73 (CH); HRMS
(ESI): Calculated for C19H13N5ONa [MþNa]þ: 350.10123, Found:
350.10132; UPLC/MS (m/z) 327.88 [MþH]þ, Tr 3.75 min.
benzonitrile (11). 1H NMR (600.1 MHz, DMSO-d6)
d 9.55 (bs, 1H, NH),
8.87 (s, 1H, Py-H6), 8.12e8.09 (m, 2H, Ar-ortho), 8.04e8.01 (m, 2H,
Ar-meta), 7.91e7.88 (m, 2H, An-ortho), 7.77e7.74 (m, 2H, An-meta);
4.1.7. 4-((2-((4-Cyanophenyl)(hydroxy)methyl)pyrimidin-4-yl)
amino)benzonitrile (8)
13C NMR (150.9 MHz, DMSO-d6)
d 189.79 (C]O), 158.44 (Py-C2),
Sodium borohydride (5 mg, 0.13 mmol) was added to a solution
of 5 (33 mg, 0.10 mmol) in MeOH (5 mL). The reaction mixture was
157.86 (Py-C6), 156.50 (Py-C4), 142.65 (An-ipso), 138.69 (Ar-ipso),
132.62 (An-meta), 132.36 (Ar-meta), 130.77 (Ar-ortho), 122.25 (An-

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
ortho), 118.93 (An-CN), 118.12 (Ar-CN), 115.37 (Ar-para), 107.49 (Py-
C5), 105.70 (An-para); HRMS (ESI): Calculated for C19H10N5OBrNa
[MþNa]þ: 425.99609, Found: 425.99614; UPLC/MS (m/z) 403.99
[MþH]þ, Tr 4.07 min.
163.75 (Py-C6), 161.91 (Py-C4), 160.69 (Py-C2), 160.26 (dd, J ¼ 8.0,
256.6 Hz, Ar-ortho), 126.34 (t, J ¼ 12.7 Hz, Ar-para), 117.73 (Py-C5),
116.47 (dd, J ¼ 4.4, 24.1 Hz, Ar-meta), 113.49 (t, J ¼ 19.1 Hz, Ar-ipso);
HRMS (ESI): Calculated for
C
11H4N2OBrClF2Na [MþNa]þ:
4-(5-Bromo-4-((2-bromo-4-cyanophenyl)amino)pyrimidine-2-
354.90558, Found: 354.90567; UPLC/MS (m/z) 332.99 [MþH]þ, Tr
carbonyl)benzonitrile (12). 1H NMR (600.1 MHz, DMSO-d6)
d
9.24
4.49 min.
(bs, 1H, NH), 8.89 (s, 1H, Py-H6), 8.31 (d, J ¼ 1.9 Hz, 1H, An-H3),
8.06e8.03 (m, 2H, Ar-ortho), 8.02 (d, J ¼ 8.4 Hz, 1H, An-H6),
7.99e7.97 (m, 2H, Ar-meta), 7.88 (dd, J ¼ 1.9, 8.4 Hz, 1H, An-H5);
4.1.14. 4-(2-Chloropyrimidine-4-carbonyl)-3,5-difluorobenzonitrile
(16)
13C NMR (150.9 MHz, DMSO-d6)
d
189.43 (C]O), 158.55 (Py-C2),
A solution of 15 (1300 mg, 3.9 mmol), zinc(II) cyanide (800 mg,
6.8 mmol) and bis(tri-t-butylphosphine)palladium(0) (300 g,
0.59 mmol) in DMF (15 mL) was heated under argon at 80 ꢀC for 1 h.
The reaction mixture was evaporated to dryness and column
chromatography on silica gel using gradient from 0% to 10%
methanol in chloroform afforded 16 (960 mg, 88%) as yellowish
157.52 (Py-C6), 156.71 (Py-C4), 140.94 (An-C1), 138.42 (Ar-ipso),
136.31 (An-C3), 132.29 (Ar-meta), 132.29 (An-C5), 130.74 (Ar-
ortho), 126.86 (An-C6), 119.35 (An-C4), 118.05 (Ar-CN), 117.27 (An-
CN), 115.38 (Ar-para), 109.31 (An-C2), 107.15 (Py-C5); HRMS (ESI):
Calculated for
C
19H9N5OBr2Na [MþNa]þ: 503.90661, Found:
503.90675; UPLC/MS (m/z) 482.05 [MþH]þ, Tr 4.52 min.
powder. 1H NMR (600.1 MHz, DMSO-d6)
d
9.20 (d, J ¼ 4.9 Hz,1H, Py-
H6), 8.18 (d, J ¼ 4.9 Hz, 1H, Py-H5), 8.08e8.05 (m, 2H, Ar-meta); 13
C
4.1.11. (2-Chloropyrimidin-4-yl)(2,6-difluoro-4-methoxyphenyl)
methanone (13)
NMR (150.9 MHz, DMSO-d6) d 186.09 (CO), 163.94 (Py-C6), 160.32
(Py-C4), 159.83 (Py-C2), 159.30 (dd, J ¼ 7.7, 254.3 Hz, Ar-ortho),
118.72 (t, J ¼ 20.0 Hz, Ar-ipso), 117.73 (Py-C5), 117.20 (dd, J ¼ 4.5,
24.0 Hz, Ar-meta), 116.34 (t, J ¼ 12.7 Hz, Ar-para), 116.07 (t,
J ¼ 3.3 Hz, CN); HRMS (ESI): Calculated for C12H5N3OClF2 [MþH]þ:
280.00837, Found: 280.00847; UPLC/MS (m/z) 280.06 [MþH]þ, Tr
4.00 min.
Method A: Sodium hydride (60% dispersion in mineral oil, 1.2 g,
29 mmol) was added to a solution of 2,4-dichloropyrimidine (3 g,
20 mmol), 2,6-difluoro-4-methoxybenzaldehyde (4 g, 23 mmol)
and N,Nꢁ-dimethylbenzimidazolium iodide (1 g, 3.6 mmol) in
anhydrous dioxane (50 mL). The reaction mixture was heated un-
der argon at 90 ꢀC for 14 h and poured into ice cold water. The
product was extracted with EtOAc (2 ꢁ 100 mL). Organic phase was
washed with brine (2 ꢁ 100 mL), dried over sodium sulphate and
evaporated to dryness. Column chromatography on silica gel using
gradient from 0% to 50% ethyl acetate in hexane afforded 13 (4.15 g,
4.1.15. 4-((4-(2,6-Difluoro-4-methoxybenzoyl)pyrimidin-2-yl)
amino)benzonitrile (17)
A solution of 13 (1000 mg, 3.51 mmol), 4-aminobenzonitrile
(500 mg, 4.24 mmol), palladium(II) acetate (100 mg, 0.45 mmol),
73%) as yellowish powder. 1H NMR (600.1 MHz, DMSO-d6)
d
8.43 (d,
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(500
mg,
J ¼ 4.9 Hz, 1H, Py-H6), 7.33 (d, J ¼ 4.9 Hz, 1H, Py-H5), 6.24 (dd,
0.85 mmol), and cesium carbonate (5 g, 15.5 mmol) in dry dioxane
(10 mL) was heated at 90 ꢀC under argon atmosphere for 3 h. The
reaction mixture was evaporated to dryness. Column chromatog-
raphy on silica gel using gradient from 0% to 100% ethyl acetate in
hexane gave 17 (780 mg, 60%) as yellowish solid. 1H NMR
J ¼ 1.6, 12.1 Hz, 2H, Ar-meta), 3.19 (s, 3H, CH3); 13C NMR (150.9 MHz,
DMSO-d6)
d
186.06 (CO), 164.59 (t, J ¼ 15.0 Hz, Ar-para), 163.36 (Py-
C6), 162.52 (Py-C4), 161.68 (dd, J ¼ 10.0, 252.4 Hz, Ar-ortho), 160.02
(Py-C2), 117.70 (Py-C5), 106.64 (t, J ¼ 17.9 Hz, Ar-ipso), 99.10 (dd,
J ¼ 3.5, 28.4 Hz, Ar-meta), 56.72 (OCH3); HRMS (ESI): Calculated for
(600.1 MHz, DMSO-d6)
Py-H6), 7.82e7.79 (m, 2H, An-ortho), 7.63e7.60 (m, 2H, An-meta),
7.45 (d, J ¼ 4.9 Hz, 1H, Py-H5), 6.98 (dd, J ¼ 1.3, 11.6 Hz, 2H, Ar-
d
10.48 (bs, 1H, NH), 8.90 (d, J ¼ 4.9 Hz, 1H,
C
12H8N2O2ClF2 [MþH]þ: 285.02369, Found: 285.02372; UPLC/MS
(m/z) 284.85 [MþH]þ, Tr 4.10 min.
meta), 3.91 (s, 3H, CH3); 13C NMR (150.9 MHz, DMSO-d6)
d 188.30
4.1.12. (2-Chloropyrimidin-4-yl)(2,4,6-trifluorophenyl)methanone
(14)
(CO), 163.90 (t, J ¼ 14.7 Hz, Ar-para), 161.19 (dd, J ¼ 10.5, 250.2 Hz,
Ar-ortho), 161.18 (Py-C6), 160.25 (Py-C4), 159.32 (Py-C2), 144.25
(An-ipso), 132.80 (An-meta), 119.31 (An-CN), 118.44 (An-ortho),
109.98 (Py-C5), 107.72 (t, J ¼ 19.5 Hz, Ar-ipso), 103.03 (An-para),
99.00 (dd, J ¼ 3.2, 25.1 Hz, Ar-meta), 56.71 (OCH3); HRMS (ESI):
Calculated for C19H13N4O2F2 [MþH]þ: 367.10011, Found: 367.10012;
UPLC/MS (m/z) 367.07 [MþH]þ, Tr 4.32 min.
Treatment of 2,4-dichloropyrimidine (0.75 g, 5 mmol), 2,4,6-
trifluorobenzaldehyde (1 g, 6.2 mmol), N,Nꢁ-dimethylbenzimida-
zolium iodide (0.25 g, 0.9 mmol), and sodium hydride (60%
dispersion in mineral oil, 0.3 g, 7 mmol) in anhydrous dioxane
(10 mL) by Method A afforded 14 (840 mg, 62%) as yellowish
powder. 1H NMR (600.1 MHz, DMSO-d6)
d
9.03 (d, J ¼ 4.9 Hz,1H, Py-
H6), 8.03 (d, J ¼ 4.9 Hz, 1H, Py-H5), 7.48e7.43 (m, 2H, Ar-meta); 13
C
4.1.16. 4-((4-(2,4,6-Trifluorobenzoyl)pyrimidin-2-yl)amino)
benzonitrile (18)
A solution of 14 (273 mg, 1.0 mmol), 4-aminobenzonitrile
(200 mg, 1.7 mmol), palladium(II) acetate (20 mg, 0.09 mmol),
NMR (150.9 MHz, DMSO-d6)
d
186.14 (CO), 164.57 (dt, J ¼ 16.1,
243.5 Hz, Ar-para), 163.71 (Py-C6), 161.01 (Py-C4), 160.78 (ddd,
J ¼ 9.7, 16.1, 254.0 Hz, Ar-ortho), 160.23 (Py-C2), 117.79 (Py-C5),
111.10 (dt, J ¼ 4.5, 19.0 Hz, Ar-ipso), 101.90 (dt, J ¼ 3.3, 26.9 Hz, Ar-
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(100
mg,
meta); HRMS (ESI): Calculated for
C
11H5N2OClF3 [MþH]þ:
0.17 mmol), and cesium carbonate (1 g, 3.1 mmol) in dry dioxane
(5 mL) was heated at 100 ꢀC under argon atmosphere for 1 h. The
reaction mixture was evaporated to dryness. Flash column chro-
matography on silica gel using gradient from 0% to 10% methanol in
chloroform afforded 18 (80 mg, 23%) as yellowish solid. 1H NMR
273.00370, Found: 273.00363; UPLC/MS (m/z) 272.88 [MþH]þ, Tr
4.13 min.
4.1.13. (2-Chloropyrimidin-4-yl)(4-bromo-2,6-difluorophenyl)
methanone (15)
(600.1 MHz, DMSO-d6)
d
10.47 (bs, 1H, NH), 8.94 (d, J ¼ 4.8 Hz, 1H,
Treatment of 2,4-dichloropyrimidine (3 g, 20 mmol), 4-bromo-
2,6-difluorobenzaldehyde (4 g, 18 mmol), N,Nꢁ-dimethylbenzimi-
dazolium iodide (1 g, 3.6 mmol), and sodium hydride (60%
dispersion in mineral oil, 1.2 g, 29 mmol) by Method A gave 15
Py-H6), 7.76e7.73 (m, 2H, An-ortho), 7.63e7.60 (m, 2H, An-meta),
7.52 (d, J ¼ 4.8 Hz, 1H, Py-H5), 7.49e7.46 (m, 2H, Ar-meta); 13C
NMR (150.9 MHz, DMSO-d6)
d
188.21 (CO), 163.98 (dt, J ¼ 15.9,
252.2 Hz, Ar-para), 161.53 (Py-C6), 160.13 (ddd, J ¼ 10.3, 15.8,
251.5 Hz, Ar-ortho), 159.43 (Py-C4), 158.78 (Py-C2), 144.05 (An-
ipso), 132.69 (An-meta), 119.17 (An-CN), 118.43 (An-ortho), 112.25
(dt, J ¼ 4.3, 20.5 Hz, Ar-ipso), 109.86 (Py-C5), 103.19 (An-para),
(1.9 g, 28%) as yellowish solid. 1H NMR (600.1 MHz, DMSO-d6)
d
9.18
(d, J ¼ 4.9 Hz, 1H, Py-H6), 8.12 (d, J ¼ 4.9 Hz, 1H, Py-H5), 7.77e7.75
(m, 2H, Ar-meta); 13C NMR (150.9 MHz, DMSO-d6)
186.35 (CO),
d

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P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
193
101.60 (dt, J ¼ 2.9, 26.9 Hz, Ar-para); HRMS (ESI): Calculated for
J ¼ 14.8 Hz, Ar-para), 162.52 (dd, J ¼ 10.1, 256.1 Hz, Ar-ortho), 161.64
(Py-C4), 161.64 (Py-C6), 157.55 (Py-C2), 143.84 (An-ipso), 133.05
(An-meta), 119.28 (An-CN), 118.68 (An-ortho), 106.71 (t, J ¼ 14.6 Hz,
Ar-ipso), 103.71 (Py-C5), 103.49 (An-para), 99.50 (dd, J ¼ 3.6,
27.1 Hz, Ar-meta), 56.97 (OCH3); HRMS (ESI): Calculated for
C
18H9N4OF3Na [MþNa]þ: 377.06207, Found: 377.06213; UPLC/MS
(m/z) 354.97 [MþH]þ, Tr 4.35 min.
4.1.17. 4-(2-((4-Cyanophenyl)amino)pyrimidine-4-carbonyl)-3,5-
difluorobenzonitrile (19)
A solution of 16 (200 mg, 0.72 mmol), 4-aminobenzonitrile
(150 mg, 1.27 mmol), palladium(II) acetate (25 mg, 0.11 mmol),
C
19H12N4O2BrF2 [MþH]þ: 445.01062, Found: 445.01062; UPLC/MS
(m/z) 445.19 [MþH]þ, Tr 4.52 min.
3-Bromo-4-((5-bromo-4-(2,6-difluoro-4-methoxybenzoyl)pyr-
imidin-2-yl)amino)benzonitrile (22). 1H NMR (600.1 MHz, DMSO-d6)
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(120
mg,
0.21 mmol), and cesium carbonate (1.2 g, 3.68 mmol) in dry dioxane
(5 mL) was heated at 80 ꢀC under argon atmosphere for 1 h. The
reaction mixture was diluted with EtOAc (20 mL) and washed with
brine (20 mL). Solvent was evaporated and column chromatog-
raphy on silica gel using gradient from 0% to 10% methanol in
chloroform gave 19 (80 mg, 31%) as yellowish solid. 1H NMR
d
9.66 (bs, 1H, NH), 8.83 (s, 1H, Py-H6), 8.17 (dd, J ¼ 0.4, 1.8 Hz, 1H,
An-H3), 7.82 (dd, J ¼ 0.4, 8.5 Hz, 1H, An-H6), 7.78 (dd, J ¼ 1.8, 8.5 Hz,
1H, An-H5), 6.94 (dd, J ¼ 1.5, 12.6 Hz, 2H, Ar-meta), 3.90 (s, 3H,
OCH3); 13C NMR (150.9 MHz, DMSO-d6)
d 184.82 (CO), 165.56 (t,
J ¼ 15.5 Hz, Ar-para), 162.53 (dd, J ¼ 9.5, 255.9 Hz, Ar-ortho), 161.96
(Py-C4), 161.63 (Py-C6), 158.07 (Py-C2), 141.75 (An-C1), 136.47 (An-
C3), 132.04 (An-C5), 125.71 (An-C6), 118.02 (An-C2), 117.52 (An-CN),
108.09 (An-C4), 106.59 (t, J ¼ 15.0 Hz, Ar-ipso), 103.70 (Py-C5),
99.56 (dd, J ¼ 4.2, 27.1 Hz, Ar-meta), 56.96 (OCH3); HRMS (ESI):
(600.1 MHz, DMSO-d6)
d
10.49 (bs, 1H, NH), 8.97 (d, J ¼ 4.8 Hz, 1H,
Py-H6), 8.11e8.07 (m, 2H, Ar-meta), 7.68e7.65 (m, 2H, An-ortho),
7.62e7.59 (m, 2H, An-meta), 7.57 (d, J ¼ 4.8 Hz, 1H, Py-H5); 13C
NMR (150.9 MHz, DMSO-d6)
d
188.00 (CO), 161.77 (Py-C6), 159.44
Calculated for
C
19H11N4O2Br2F2 [MþH]þ: 522.92113, Found:
(Py-C4), 158.80 (dd, J ¼ 18.4, 252.1 Hz, Ar-ortho), 157.66 (Py-C2),
143.85 (An-ipso), 132.62 (An-meta), 120.31 (t, J ¼ 21.5 Hz, Ar-ipso),
119.07 (An-CN), 118.38 (An-ortho), 116.97 (dd, J ¼ 15.5, 23.4 Hz, Ar-
meta), 116.04 (t, J ¼ 2.8 Hz, Ar-CN), 115.44 (t, J ¼ 12.5 Hz, Ar-para),
109.68 (Py-C5), 103.29 (An-para); HRMS (ESI): Calculated for
522.92096; UPLC/MS (m/z) 523.13 [MþH]þ, Tr 4.87 min.
4.1.20. 4-((5-Bromo-4-(2,6-difluoro-4-hydroxybenzoyl)pyrimidin-
2-yl)amino)benzonitrile (23)
A solution of boron tribromide (1.5 mL, 1 M in methylene
chloride, 1.5 mmol) was added to a solution of 21 (125 mg,
0.28 mmol) in methylene chloride (4 mL) at 0 ꢀC. The reaction
mixture was stirred overnight at room temperature. MeOH (3 mL)
was added dropwise and the reaction mixture was evaporated to
dryness. Column chromatography on silica gel using gradient from
0% to 10% methanol in chloroform gave 23 (11 mg, 9%) as of-white
C
19H9N5OF2Na [MþNa]þ: 384.06674, Found: 384.06685; UPLC/MS
(m/z) 362.09 [MþH]þ, Tr 4.26 min.
4.1.18. 4-((4-(4-((4-Cyanophenyl)amino)-2,6-difluorobenzoyl)
pyrimidin-2-yl)amino)benzonitrile (20)
A solution of 15 (100 mg, 0.30 mmol), 4-aminobenzonitrile
(50 mg, 0.42 mmol), palladium(II) acetate (10 mg, 0.04 mmol),
solid. 1H NMR (600.1 MHz, DMSO-d6)
d 10.62 (bs, 1H, NH), 8.91 (s,
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(50
mg,
1H, Py-H6), 7.85e7.81 (m, 2H, An-ortho), 7.73e7.69 (m, 2H, An-
0.09 mmol), and cesium carbonate (0.5 g,1.53 mmol) in dry dioxane
(3 mL) was heated at 80 ꢀC under argon atmosphere for 1 h. The
reaction mixture was evaporated to dryness and column chroma-
tography on silica gel using gradient from 0% to 100% ethyl acetate
in hexane gave 20 (25 mg, 18%) as yellowish solid. 1H NMR
meta), 6.61 (dd,
(150.9 MHz, DMSO-d6)
J
¼
1.4, 12.6 Hz 2H, Ar-meta); 13C NMR
d
183.91 (CO),165.80 (t, J ¼ 14.7 Hz, Ar-para),
163.42 (dd, J ¼ 19.7, 255.8 Hz, Ar-ortho), 162.90 (Py-C4), 161.79 (Py-
C6), 158.05 (Py-C2), 144.37 (An-ipso), 133.48 (An-meta), 119.73 (An-
CN), 119.18 (An-ortho), 105.63 (t, J ¼ 15.5 Hz, Ar-ipso), 104.13 (Py-
C5), 103.97 (An-para), 100.81 (dd, J ¼ 3.7, 24.9 Hz, Ar-meta); HRMS
(600.1 MHz, DMSO-d6)
d 10.49 (bs, 1H, NH), 9.76 (bs, 1H, NH), 8.89
(d, J ¼ 4.9 Hz, 1H, Py-H6), 7.87e7.84 (m, 2H, An-ortho), 7.79e7.76
(m, 2H, ArAn-meta), 7.65e7.62 (m, 2H, An-meta), 7.41 (d, J ¼ 4.9 Hz,
1H, Py-H5), 7.38e7.35 (m, 2H, ArAn-ortho), 6.96e6.91 (m, 2H, Ar-
(ESI): Calculated for
C
18H9N4O2BrF2Na [MþNa]þ: 452.97692,
Found: 452.97691; UPLC/MS (m/z) 431.08 [MþH]þ, Tr 4.17 min.
meta); 13C NMR (150.9 MHz, DMSO-d6)
d
187.76 (CO), 161.60 (dd,
4.2. MT-4 HIV antiviral and cytotoxicity assay
J ¼ 10.2, 250.6 Hz, Ar-ortho), 161.04 (Py-C6), 160.90 (Py-C4), 159.24
(Py-C2), 147.48 (t, J ¼ 14.2 Hz, Ar-para), 144.91 (AnAr-ipso), 144.32
(An-ipso), 133.89 (ArAn-meta), 132.83 (An-meta), 119.32 (An-CN),
119.19 (ArAn-CN), 118.44 (An-ortho), 118.30 (ArAn-ortho), 110.01
(Py-C5), 106.93 (t, J ¼ 19.0 Hz, Ar-ipso), 103.31 (ArAn-para), 102.97
(An-para), 99.69 (dd, J ¼ 3.6, 24.5 Hz, Ar-meta); HRMS (ESI):
Compounds were tested in a high-throughput 384-well assay
format for their ability to inhibit the replication of HIV-1 (IIIB) in
MT-4 cells. Compounds were serially diluted (1:3) in DMSO on 384-
well polypropylene plates and further diluted 200-fold into com-
plete RPMI media (10% FBS, 1% P/S) using the Biotek Micro Flow and
Agilent ECHO acoustic dispenser. Each plate contained up to 8 test
Calculated for
C
25H14N6OF2Na [MþNa]þ: 475.10894, Found:
475.10896; UPLC/MS (m/z) 453.32 [MþH]þ, Tr 4.38 min.
compounds, with negative (No Drug Control) and 5
mM AZT positive
controls. MT-4 cells were pre-infected with 10 L of either RPMI
m
4.1.19. Preparation of 4-((5-bromo-4-(2,6-difluoro-4-
(mock-infected) or a fresh 1:250 dilution of an HIV-1 (IIIB)
concentrated virus stock. Infected and uninfected MT-4 cells were
further diluted in complete RPMI media and added to each plate
using a Micro Flow dispenser. After 5 days incubation in a humid-
ified and temperature controlled incubator (37 ꢀC), Cell Titer Glo
(Promega) was added to the assay plates to quantify the amount of
luciferase. EC50 and CC50 values were defined as the compound
concentration that causes a 50% decrease in luminescence signal,
and were calculated using a sigmoidal dose-response model to
generate curve fits.
methoxybenzoyl)pyrimidin-2-yl)amino)benzonitrile (21) and 3-
bromo-4-((5-bromo-4-(2,6-difluoro-4-methoxybenzoyl)pyrimidin-
2-yl)amino)benzonitrile (22)
A solution of 17 (100 mg, 0.27 mmol) and N-bromosuccinimide
(100 mg, 0.56 mmol) in DMF (3 mL) was heated at 90 ꢀC for 1 h and
evaporated to dryness. Column chromatography on silica gel using
gradient from 0% to 100% ethyl acetate in hexane gave 21 (14 mg,
12%) as yellow solid and 22 (18 mg, 13%) as yellow solid.
4-((5-Bromo-4-(2,6-difluoro-4-methoxybenzoyl)pyrimidin-2-yl)
amino)benzonitrile (21). 1H NMR (600.1 MHz, DMSO-d6)
d 10.62 (bs,
1H, NH), 8.94 (s, 1H, Py-H6), 7.83e7.80 (m, 2H, An-ortho), 7.71e7.68
(m, 2H, An-meta), 6.97 (dd, J ¼ 1.6, 12.6 Hz, 2H, Ar-meta), 3.57 (s,
4.3. RT co-crystallization, data collection, and structure refinement
Co-crystals of the HIV-1 RT and 17 were grown by hanging drop
3H, OCH3); 13C NMR (150.9 MHz, DMSO-d6)
d 184.77 (CO), 165.57 (t,

ꢀ
194
Table 3
P. Simon et al. / European Journal of Medicinal Chemistry 122 (2016) 185e195
K. Iterbeke, F. Stappers, P. Stevens, L. Schueller, P. Van Remoortere, G. Kraus,
P. Wigerinck, J. Rosier, Development of a long-acting injectable formulation
with nanoparticles of rilpivirine (TMC278) for HIV treatment, Eur. J. Pharm.
Biopharm. 72 (2009) 502e508.
X-ray data collection and refinement statistics for compound 17.
Data collection
Space group
Resolution (Å)
No. unique
C2221
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their discovery, development, and use in the treatment of HIV-1 infection: a
review of the last 20 years (1989e2009), Antivir. Res. 85 (2010) 75e90.
[7] C. Reynolds, C.B. de Koning, S.C. Pelly, W.A. van Otterlo, M.L. Bode, In search of
a treatment for HIV e current therapies and the role of non-nucleoside
reverse transcriptase inhibitors (NNRTIs), Chem. Soc. Rev. 41 (2012)
4657e4670.
[8] E. De Clercq, The role of non-nucleoside reverse transcriptase inhibitors
(NNRTIs) in the therapy of HIV-1 infection, Antivir. Res. 38 (1998) 153e179.
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diversity, pharmacophore similarity, and implications for drug design, Med.
Res. Rev. 33 (2013) E1eE72.
50.00e2.40 (2.45e2.40)
53 535
I/
s
28.7 (2.4)
5.8 (70.1)
a
Rmerge (%)
Completeness (%)
Refinement statistics
Resolution (Å)
96.6 (98.2)
50.0e2.4
51 444
20,4
No. reflections (F ꢂ 0)
R-factorb
R-freeb
26,3
RMS bond lengths (Å)
RMS bond angles (ꢀ)
0,009
1,19
[10] E. De Clercq, Non-nucleoside reverse transcriptase inhibitors (NNRTIs): past,
present, and future, Chem. Biodivers. 1 (2004) 44e64.
P P
P P
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related analogues as HIV-1 NNRTIs, Curr. Med. Chem. 18 (2011) 359e376.
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novel HIV-1 NNRTI platforms: 2006-2008 update, Curr. Med. Chem. 16 (2009)
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patent review (2005-2010), Expert Opin. Ther. Pat. 21 (2011) 717e796.
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[19] F.-E. Chen, X.-Q. Feng, Y.-H. Liang, Z.-S. Zeng, CN101723903, 2009.
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C. Pannecouque, Synthesis and anti-HIV activity of aryl-2-[(4-cyanophenyl)
amino]-4-pyrimidinone hydrazones as potent non-nucleoside reverse tran-
scriptase inhibitors, ChemMedChem 6 (2011) 2225e2232.
a
Rmerge ¼ [
h
i|Ih e Ihi|/
h
iIhi] where Ih is the mean of Ihi obser-
vations of reflection h. Numbers in parenthesis represent highest resolution
shell.
P
P
b
R-factor and R-free ¼ ||Fobs|ꢃ|Fcalc||/ |Fobs| ꢁ 100 for 95% of recorded
data (R-factor) or 5% of data (R-free).
vapor diffusion as described previously [18]. Briefly, purified RT was
concentrated and combined with 17 to a final concentration of
20 mg/ml RT and 0.5 mM 17. This protein/inhibitor solution was
mixed in equal parts with mother liquor containing 0.8 M K/Na
tartrate and MES pH 6.5 at 20 ꢀC. Prior to data collection, the
crystals were transferred into a solution of 25% glycerol in addition
to the mother liquor and then cryocooled in liquid nitrogen. Data
was collected at a temperature of 100 K and processed with
HKL2000 [35].
Molecular replacement and structure refinement was per-
formed with Phenix [36] using the starting model PDB code 3MEE.
Model building was performed with the molecular graphics pro-
gram Coot [37]. The final model statistics are listed in Table 3 and
the data have been deposited into the Protein Data Bank (PDB code:
5K14).
[21] X.-Q. Feng, Z.-S. Zeng, Y.-H. Liang, F.-E. Chen, C. Pannecouque, J. Balzarini, E. De
Clercq, Synthesis and biological evaluation of 4-(hydroxyimino)arylmethyl
diarylpyrimidine analogues as potential non-nucleoside reverse transcriptase
inhibitors against HIV, Bioorg. Med. Chem. 18 (2010) 2370e2374.
[22] Y. Liu, G. Meng, A. Zheng, F. Chen, W. Chen, E. De Clercq, C. Pannecouque,
J. Balzarini, Design and synthesis of a new series of cyclopropylamino-linking
diarylpyrimidines as HIV non-nucleoside reverse transcriptase inhibitors, Eur.
J. Pharm. Sci. 62 (2014) 334e341.
Acknowledgements
This work was supported by the subvention for development of
research organization (Institute of Organic Chemistry and
Biochemistry CAS, RVO 61388963).
[23] G. Meng, Y. Liu, A. Zheng, F. Chen, W. Chen, E. De Clercq, C. Pannecouque,
J. Balzarini, Design and synthesis of
diarylpyrimidines as drug-resistant HIV non-nucleoside reverse transcrip-
tase inhibitors, Eur. J. Med. Chem. 82 (2014) 600e611.
a new series of modified CH-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.06.026.
[24] S.-X. Gu, Q.-Q. He, S.-Q. Yang, X.-D. Ma, F.-E. Chen, E. De Clercq, J. Balzarini,
C. Pannecouque, Synthesis and structureeactivity relationship of novel dia-
rylpyrimidines with hydromethyl linker (CH(OH)-DAPYs) as HIV-1 NNRTIs,
Bioorg. Med. Chem. 19 (2011) 5117e5124.
[25] Z.-H. Yan, H.-Q. Wu, W.-X. Chen, Z. Wu, H.-R. Piao, Q.-Q. He, F.-E. Chen, E. De
Clercq, C. Pannecouque, Synthesis and biological evaluation of CHX-DAPYs as
HIV-1 non-nucleoside reverse transcriptase inhibitors, Bioorg. Med. Chem. 22
(2014) 3220e3226.
[26] Z.-S. Zeng, Y.-H. Liang, X.-Q. Feng, F.-E. Chen, C. Pannecouque, J. Balzarini, E. De
Clercq, Lead optimization of diarylpyrimidines as non-nucleoside inhibitors of
HIV-1 reverse transcriptase, ChemMedChem 5 (2010) 837e840.
[27] Z.-H. Yan, X.-Y. Huang, H.-Q. Wu, W.-X. Chen, Q.-Q. He, F.-E. Chen, E. De Clercq,
C. Pannecouque, Structural modifications of CH(OH)-DAPYs as new HIV-1
non-nucleoside reverse transcriptase inhibitors, Bioorg. Med. Chem. 22
(2014) 2535e2541.
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