Synthesis of a 2,4,6-trisubstituted 5-cyano-pyrimidine library and evaluation of its immunosuppressive activity in a Mixed Lymphocyte Reaction assay

Article abstract of DOI:10.1016/j.bmc.2012.12.032

A series of novel pyrimidine analogues were synthesized and evaluated for immunosuppressive activity in the Mixed Lymphocyte Reaction assay, which is well-known as the in vitro model for in vivo rejection after organ transplantation. Systematic variation of the substituents at positions 2, 4 and 6 of the pyrimidine scaffold led to the discovery of 2-benzylthio-5-cyano-6-(4- methoxyphenyl)-4-morpholinopyrimidine with an IC₅₀ value of 1.6 μM in the MLR assay.

Full text of DOI:10.1016/j.bmc.2012.12.032

Bioorganic & Medicinal Chemistry 21 (2013) 1209–1218
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a 2,4,6-trisubstituted 5-cyano-pyrimidine library
and evaluation of its immunosuppressive activity in a Mixed
Lymphocyte Reaction assay
Alessandro Stella a,b, Kristien Van Belle a,c, Steven De Jonghe a,b, Thierry Louat a,c, Jean Herman a,d
,
Jef Rozenski b, Mark Waer a,c, Piet Herdewijn a,b,
⇑
a KU Leuven, Interface Valorisation Platform, Kapucijnenvoer 33, 3000 Leuven, Belgium
b KU Leuven, Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Minderbroedersstraat 10, 3000 Leuven, Belgium
c KU Leuven, Laboratory of Experimental Transplantation, Campus Gasthuisberg O&N I box 811, Herestraat 49, B-3000 Leuven, Belgium
d University Hospitals Leuven, Department of Pediatric Transplantation, Herestraat 49, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pyrimidine analogues were synthesized and evaluated for immunosuppressive activity
in the Mixed Lymphocyte Reaction assay, which is well-known as the in vitro model for in vivo rejection
after organ transplantation. Systematic variation of the substituents at positions 2, 4 and 6 of the pyrim-
idine scaffold led to the discovery of 2-benzylthio-5-cyano-6-(4-methoxyphenyl)-4-morpholinopyrimi-
Received 11 June 2012
Revised 20 December 2012
Accepted 21 December 2012
Available online 4 January 2013
dine with an IC50 value of 1.6 lM in the MLR assay.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Pyrimidines
Mixed Lymphocyte Reaction
SAR
Immunosuppressive drugs
1. Introduction
auto-immune diseases have been described. The most prevalent
conditions are rheumatoid arthritis, psoriasis, multiple sclerosis,
Crohn’s disease and systemic lupus erythematosus. Because of
the high prevalence of these diseases, the pharmaceutical sector
is heavily interested in pursuing new therapies for these diseases.
Pyrimidines were extensively explored in medicinal chemistry
and they were shown to be associated with a wide range of biolog-
ical activities. Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs)
were potent HIV-1 non-nucleoside reverse transcriptase inhibitors
(NNRTIs).3 Pyrimidine-based compounds were prepared as dual re-
verse transcriptase and integrase inhibitors of HIV.4 Pyrimidines
were often used as central core structure for the design and syn-
thesis of inhibitors of a wide range of kinases, such as for example
Axl kinase,5 phosphoinositide-3-kinase,6 fibroblast growth factor
receptor,7 cyclin-dependent kinase 4/6,8 Tie kinase9 and Janus
tyrosine kinase 3.10
Although the biological activity of a wide variety of pyrimidines
was described, their immunosuppressive activity has, to our
knowledge, not been reported in literature. As part of our ongoing
interest in the synthesis of small-molecule based heterocyclic
structures as potential novel immunosuppressive agents, we re-
port the discovery of a series of pyrimidines with immunosuppres-
sive activity in a Mixed Lymphocyte Reaction (MLR) assay.
The central issue in organ transplantation remains suppression
of allograft rejection. In recent years, the patient and graft survival
dramatically increased, mainly due to improvements in immuno-
suppressive therapy.1
Current immunosuppressive regimens are mostly based on a
combination of a calcineurin inhibitor (cyclosporine or tacrolimus),
a glucocorticoid (prednisone) and an antiproliferative agent (aza-
thioprine or mycophenolate). In recent years, two new drugs (sirol-
imus and everolimus) were made available. Both compounds
inhibit a kinase, called the mammalian-target of rapamaycin
(mTOR). This current armamentarium of immunosuppressive
drugs is efficient for the inhibition of T-lymphocyte dependent
rejection, but is quite ineffective in preventing or treating chronic
rejection. In addition, most of these drugs show toxicities that im-
pair patient and graft survival. So, there is a continuous need for
novel immunosuppressive agents. Besides their use for the preven-
tion of graft rejection, immunosuppressive agents are also often
used for the treatment of auto-immune diseases.2 More than 70
⇑
Corresponding author. Tel.: +32 (0)16/33 73 87; fax: +32 (0)16/33 73 40.
E-mail address: piet.herdewijn@rega.kuleuven.be (P. Herdewijn).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.032

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A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
tion of
a 2-anilino-pyrimidine skeleton. By the selection of
2. Chemistry
different substituents on the benzaldehyde and phenylguanidine
counterpart, a small library of 2-anilino-5-cyano-pyrimidin-4-
(3H)-one analogues 5a–i was prepared. A summary of the synthe-
sized compounds is shown in Table 2. For further derivatisation of
the scaffold, a series of nitrogen-containing nucleophiles was
introduced at C-4 of the pyrimidine ring by a one-step amination
reaction, using benzotriazol-1-yloxytris(dimethylamino)-phos-
phonium hexafluorophosphate (BOP) as coupling partner,14 yield-
ing compounds 7a–h. Moreover, alkylation of the lactam moiety
of the pyrimidine scaffold was carried out with different alkyl
halides (methyl iodide, ethyl iodide and benzyl bromide), using
potassium carbonate as base in DMF as solvent. These reaction
conditions led selectively to O-alkylated derivatives 6a–e
(Scheme 2). The presence of an O-alkyl group was easily derived
from the 13C NMR spectra. The signals of the C-atom linked to
oxygen were observed between 50–60 ppm. On the contrary, if
the C-atom would be directly linked to the nitrogen, the 13C signals
should be visible between 35 and 40 ppm.
In order to expand the structural variety, thiouracil derivatives
1a, 1b and 1d were alkylated with different alkyl and benzyl ha-
lides, affording compounds 8a–k (Scheme 3 and Table 3 of the Sup-
plementary data). These 4-oxo-derivatives were used for further
derivatisation by introducing nitrogen containing nucleophiles
using the BOP-mediated nucleophilic arylic substitution reaction
(compounds 9c–x, Tables 3 and 4). Alternatively, compound 8a
was treated with POCl3, furnishing a 4-chloro-pyrimidine analogue
10. The latter was used as starting point for direct nucleophilic aro-
matic substitution reaction with morpholine and isopropylamine,
yielding compounds 9a and 9b, respectively.
The generation of a pyrimidine library started with the synthe-
sis of 2-thiouracil derivatives 1a–e as key intermediates
(Scheme 1), which are versatile building block as the thio-urea
moiety, as well as the lactam group, can be further derivatised.
The construction of the pyrimidine core was achieved via a Biginel-
li–type multicomponent reaction11 between a substituted benzal-
dehyde, ethylcyanoacetate, thiourea, and piperidine as a base,
yielding the 2-thiouracil congeners 1a–e. Treatment of compound
1a (X = Cl) with iodomethane in the presence of potassium carbon-
ate yielded the thiomethyl derivative 2. It was anticipated that this
compound would be an ideal key intermediate for structural vari-
ation at position 2 by nucleophilic aromatic substitutions. In order
to introduce an aniline substituent at position 2, a wide range of
different conditions (solvents, temperature, reaction time) have
been explored, but the desired compounds were never been iso-
lated in acceptable yields. The best result was obtained performing
the reaction with p-chloroaniline, under microwave irradiation at
160 °C, using DMF as solvent. Under these reaction circumstances,
the desired 2-(4-chloroanilino)pyrimidine derivative 5a was iso-
lated in only 24% yield, after complex and tedious chromatographic
separations. Attempts to oxidize the thiomethyl group of com-
pound 2 to the corresponding sulfone or sulfoxide with m-chloro-
peroxybenzoic (m-CPBA) prior to nucleophilic displacement were
unsuccessful, as only decomposition of the starting material was
observed.
Therefore, alternative strategies for the introduction of an anili-
no moiety at C-2 were explored. The availability of a substituted
phenylguanidine moiety such as 4a–e in a Biginelli-type multicom-
ponent reaction would allow the direct synthesis of a 2-anilino-
pyrimidine library. Two different methods were explored for the
synthesis of these phenylguanidine counterparts 4a–e (Scheme 2).
A first methodology was based on the N-iodosuccinimide (NIS)-
promoted guanylation of thiourea.12 As this reaction only works
with electron-withdrawing groups on both nitrogens, commer-
cially available di-Boc-thiourea was selected as starting material.
Reaction with p-chloroaniline, in the presence of NIS and triethyl-
amine afforded the protected guanidine 3a in good yields. Acidic
deprotection furnished the desired phenylguanidines 4a. Although
this method worked well, the protection/deprotection sequence
makes the synthetic route long and not suitable for parallel chem-
istry. A straightforward and shorter approach was based on the
reaction between a substituted aniline and cyanamide (in a mix-
ture of ethanol/water, under acidic conditions), affording directly
the desired substituted phenylguanidines 4b–e in good yield.
Guanidines 4a–e were used in the multicomponent reaction with
substituted benzaldehydes and ethylcyanoacetate,13 with piperi-
dine as base and ethanol as solvent, permitting the direct construc-
The 2-thiobenzyl derivatives 8a, 8b and 8d were alkylated with
alkyl iodides or benzylbromide using K2CO3 as base and DMF as
solvent. These conditions were identical to the procedure used
for the alkylation of 2-anilino-pyrimidin-4-(3H)-one derivatives
5a–k. However, for the 2-thiobenzyl congeners the reaction was
less selective affording a mixture of O- and N-alkylated products
(Table 1). In most cases, the two isomers were separated by flash
chromatography and the ratio between O- versus N-alkylated
derivatives was determined by mass balance after chromato-
graphic separation. In some cases, only one of the regioisomers
was isolated in pure form (entries 6 and 7).
3. Biological evaluation
To screen for compounds that have immunosuppressive proper-
ties, the MLR assay was used, which is considered to be a model as-
say for T-cell responses against allogenic antigens. This test was
developed 50 years ago15 and was used extensively to discover no-
vel immunosuppressive compounds,16 to detect immune activity
O
O
CN
CHO
CN
S
NC
b
HN
a
HN
+
+
X
H2N
NH2
EtOOC
S
N
2
S
N
H
1a-e
X
ForX=Cl
Cl
Cl
O
N
Cl
CN
HN
N
H
5a
Scheme 1. Reagents and conditions: (a) piperidine, EtOH, reflux, overnight; (b) MeI, K2CO3, CH3CN.

A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
1211
X
Cl
NH2
N
S
N
c
b
a
NH2CN
+
H2N
NH2
BocHN
NHBoc
BocHN
NHBoc
4a-e
(Table 2,
X
3a
Supporting Information)
d
Nu
O
OR
N
CN
CN
CN
f
N
HN
e
N
X
X
X
N
H
N
N
H
N
N
H
Y
Y
Y
7a-h
5a-i
6a-e
Scheme 2. Reagents and conditions: (a) p-chloroaniline, NIS, Et3N, CH2Cl2, 24 h, rt; (b) TFA, CH2Cl2, rt; (c) HNO3, EtOH/H2O; (d) substituted benzaldehydes, ethylcyanoacetate,
piperidine, EtOH, reflux; (e) alkyliodides, K2CO3, DMF, rt; (f) NuH, DBU, BOP, MeCN, 12 h, rt.
Table 1
O/N alkylation of 2-thiobenzylpyrimidine derivatives
O
OR
N
O
N
R
CN
CN
a
CN
HN
BnS
N
N
N
BnS
BnS
X
X
X
11a-e
12a-g
R=Me, Et, Pr, Bn
8a,b,d
Entry
Substrate
R
Product
Ratio O-alkyl/N-alkyl
1
2
3
4
5
6
7
8a (X = 4-Cl)
8a (X = 4-Cl)
8a (X = 4-Cl)
8b (X = 3-Cl)
8d (X = 4-OMe)
8b (X = 3-Cl)
8d (X = 4-OMe)
Me
Et
Bn
Me
Et
11a/12a
11b/12b
11c/12c
11d/12d
11e/12e
12f
(11a) 2:1 (12a)
(11b) 3:1 (12b)
(11c) 1:2 (12c)
(11d) 3:1 (12d)
(11e) 2:1 (12e)
Only N-Pr derivative was isolated in pure form (12f)
Only N-Me derivative was isolated in pure form (12g)
Pr
Me
12g
Table 2
SAR of 2-anilino-4-substituted-5-cyano-6-aryl pyrimidine analogues
R
X
CN
N
N
H
N
Y
Ma
MLR IC50
(l
M)
a
Compd
X
Y
R
% Inhibition at 10
l
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
7f
7g
7h
CsAb
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH3
CH3
Cl
Cl
Cl
Cl
H
Benzyloxy
Methoxy
Ethoxy
Ethoxy
Propoxy
Morpholine
Isopropylamino
Piperidino
Cyclohexylamino
Isopropylamino
Piperidino
26
0
0
38
44
45
0
15
1
72
0
88
70
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
5.7
H
Cl
Cl
Cl
Cl
H
Cl
H
H
NDc
4.3
Piperidino
Piperidino
6.2
0.054
a
b
c
Values are means of two independent experiments.
Cyclosporin A.
ND = not determined.

1212
A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
Nu
O
O
CN
CN
CN
N
HN
HN
b)
a)
RS
N
RS
N
S
N
H
X
X
X
9c-x
Table 4 and 5
8a-k
(Table 3
1a X=4-Cl
1b X=3-Cl
Supporting Information)
1d X=4-OMe
(For R=Bn
c)
and X=4-Cl)
Cl
N
Nu
CN
CN
N
d
N
BnS
BnS
N
10
Cl
Cl
9a Nu=morpholine
9b Nu=isopropylamino
(Table 4)
Scheme 3. Reagents and conditions: (a) RBr or RI, K2CO3, CH3CN, rt; (b) NuH, BOP, DBU, CH3CN, rt; (c) POCl3, dioxane, reflux; (d) NuH, dioxane, rt.
Table 3
SAR of 2-thiobenzyl-4-substituted-5-cyano-6-aryl pyrimidines
R
N
CN
N
S
Y
Ma
MLR IC50 (lM)
a
Compd
Y
R
% Inhibition at 10
l
11a
11b
11c
11d
11e
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
4-Cl
4-Cl
4-Cl
3-Cl
4-OMe
4-Cl
4-Cl
4-Cl
4-Cl
Methoxy
Ethoxy
Benzyloxy
Ethoxy
Ethoxy
Morpholine
Isopropylamine
Cyclohexylamine
Isonipecotinamide
Nipecotinamide
Benzylpiperidine
Benzoylpiperidine
2-Thienylmethylamine
Benzylamine
Phenethylamine
Morpholine
Thiomorpholine
4-Me-piperidine
4-Benzylpiperidine
Isonipecotinamide
4-Benzoylpiperidine
4-Phenylpiperidine
Morpholine
10
13
22
10
46
63
48
56
57
64
17
55
65
73
11
90
13
35
31
73
63
31
80
NDc
NDc
NDc
NDc
NDc
8.3
NDc
6.4
6.4
4-Cl
4-Cl
4-Cl
NDc
NDc
9.8
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
3-Cl
5.0
2.5
NDc
1.6
9k
9l
NDc
NDc
NDc
4.9
9m
9n
9o
9p
9q
9r
2.1
NDc
5.5
CsAb
0.054
a
b
c
Values are means of two independent experiments.
Cyclosporin A.
ND = not determined.
of samples from AIDS patients17 or to predict immune rejection of
kidney allograft.18 It is a clinically relevant test for histocompatibil-
ity between donor and recipient where lymphocytes from one
individual (effector) are incubated with the lymphocytes of an-
other individual (stimulator), which have been previously ren-
dered incapable of blast transformation by treatment with
mitomycin. The degree of incompatibility is reflected in the
amount of cell division as measured by the uptake of tritium-
labeled thymidine, whereby incompatibility increases its uptake.
In a first round of screening, compounds were tested at a con-
tion were subjected to dose-response curves in order to determine
IC50 values. Cyclosporine A, a marketed immunosuppressive agent,
was included in the MLR assay to compare their in vitro immuno-
suppressive activity.
The first series of compounds screened for immunosuppressive
activity were the 2-anilino-5-cyano-pyrimidin-4-(3H)-one ana-
logues 5a–i, bearing structural variety on both phenyl rings. These
compounds lacked immunosuppressive activity, all displaying IC50
values greater than 10
the nitrogen of the lactam moiety with a number of alkylhalides
afforded series of N(3)-substituted pyrimidin-4-(3H)-one
lM (see Supplementary data). Alkylation of
centration of 10
lM. Compounds displaying more than 50% inhibi-
a

A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
1213
Table 4
Primary, secondary, aliphatic, as well as aromatic amines were
incorporated to maximize structural diversity. In a first series of
analogues, a 3- or 4-chlorophenyl moiety was fixed at position 6
of the pyrimidine scaffold. A small, aliphatic substituent at position
4 such as isopropylamine (compound 9b) did not impart any
immunosuppressive activity to the pyrimidine scaffold. On the
other hand, bulkier amines displayed immunosuppressive activity.
The 4-N-cyclohexylamino derivative (compound 9c) displayed an
SAR of 2-thioalkyl-4-morpholino-5-cyano-6-phenyl-pyrimidines
O
N
CN
N
R
S
N
IC50 value of 6.4 lM. The presence of a morpholine moiety in com-
O
bination with a 3-Cl-phenyl (compound 9r) or 4-Cl-phenyl moiety
(compound 9a) furnished pyrimidine analogues with immunosup-
pressive activity (MLR IC50 values of 5.5 and 8.3 lM, respectively).
Compd
R
% Inhibition at 10
l
Ma
MLR IC50 (lM)
a
A number of larger substituents was also introduced. The insertion
of a carboxamide group on the piperidine moiey at the right posi-
tion (i.e., an isonipecotinamide) yielded derivative 9d, which dis-
9s
9t
9u
9v
CH2Bn
Me
4-Br-Bn
n-Butyl
n-Hexyl
Allyl
62
80
36
67
69
82
3.9
4.9
NDc
2.3
played an MLR IC50 value of 6.4 lM. The insertion of the
9w
9x
5.3
3.4
0.054
corresponding isomeric nipecotinamide led to compound 9e,
which was devoid of immunosuppressive activity. The addition
of a benzyl group on the piperidine moiety led to an inactive com-
CsAb
a
b
c
Values are means of two independent experiments.
Cyclosporin A.
ND = not determined.
pound 9f (MLR IC50 >10
logue 9g showed weak immunosuppressive activity (MLR
IC50 = 9.8 M).
lM), whereas the benzoylpiperidine ana-
l
In order to probe the influence of the substituents on the 6-phe-
nyl ring, a number of analogues was made carrying a 4-methoxy-
phenyl moiety at position 6 instead of the Cl-phenyl group.
Among these congeners, aromatic amines, such as 2-thienylm-
ethylamine (compound 9h) and benzylamine (compound 9i) dis-
played strong immunosuppressive activity with IC50 values of 5.0
Table 5
Toxicity data on Jurkat T cell line
Compd
% Inhibition at 10 l
Ma
7h
7g
9a
9c
9d
9g
9h
9i
9k
9o
9p
9r
9s
9t
9v
9w
9x
9
10
5
0
19
6
13
5
10
12
2
15
9
6
and 2.5 lM, respectively. However, insertion of an additional
methylene linker between the amino group and the phenyl ring
(phenethylamine at position 4; compound 9j) led to a loss of activ-
ity. Within the 6-(4-OCH3-phenyl) series, a morpholine at position
4 seems to be optimal for immunosuppressive activity with an IC50
value of 1.6 lM (compounds 9k). Remarkably, changing the mor-
pholine substituent for a thiomorpholine moiety (compound 9l)
resulted in complete loss of immunosuppressive activity. Analo-
gously to the Cl-phenyl series, a number of substituted piperidine
substituents were also incorporated. The insertion of a methyl
group (compound 9m) or a benzyl group (compound 9n) led to
pyrimidine analogues lacking immunosuppressive activity,
whereas a nipecotinamide (compound 9o) or a benzoylpiperidine
(compound 9p) gave potent immunosuppressive activity (MLR
13
13
5
a
Values are means of two independent experiments.
IC50 values of 4.9 and 2.1 lM, respectively).
To examine the optimal substitution pattern at position 2, dif-
ferent thio-alkyl groups were introduced. This was combined with
a 4-methoxyphenyl moiety at C-(6) and a morpholino at C-(4) as
this was the optimal substitution pattern for immunosuppressive
activity in compound 9k.
analogues 12a–g. Similarly, none of these N-substituted deriva-
tives showed any immunosuppressive activity at 10
Supplementary data).
lM (see
In an attempt to increase the biological activity, a novel series of
pyrimidine analogues was prepared with structural modifications
at position 4 of the pyrimidine scaffold (Table 2). The presence of
an alkoxy substituent (compounds 6a–e) never imparted immuno-
suppressive activity to these compounds. On the other hand, the
presence of a piperidino or isopropyl amino moiety, combined with
an appropriate substitution pattern at positions 2 and 6, yielded
compounds 7e, 7g and 7h, with immunosuppressive activity, dis-
From the data in Table 4, it is clear that structural variety is tol-
erated at position 2. Replacement of the bulky benzyl group by
smaller alkyl groups furnished analogues (compounds 9t, 9v, 9w,
9x) that showed noteworthy inhibitory activity in the MLR assay
with IC50 values ranging from 2.3 to 5.3 lM. Elongation of the lin-
ker between the sulfur atom and the phenyl group from a methy-
lene group in compound 9k to an ethylene spacer in derivative 9s
led to a modest decrease of activity in the MLR assay. Surprisingly,
a 4-bromothiobenzyl group is not tolerated, as compound 9u
playing IC50 values of 5.7, 4.3 and 6.2 lM, respectively.
Driven by the fact that simultaneous variation of substituents at
positions 2, 4 and 6 of the pyrimidine scaffold can lead to immo-
suppressive compounds, a focused library of 5-cyano-pyrimidines
was prepared (Table 3). For the ease of synthesis, a thiobenzyl
group was selected at position 2. The installation of alkoxy substit-
uents at position 4 (compounds 11a–e) did not afford any immu-
nosuppressive agents. This is in complete agreement with the
MLR data of the 4-alkoxy-2-anilino pyrimidine analogues 6a–e,
which were also completely inactive.
shows a IC50 higher than 10 lM.
In order to exclude that the immunosuppressive activity is due
to a general cytotoxic effect, the cellular toxicity of the most potent
immunosuppressive compounds was evaluated by a WST assay
using Jurkat cells. From the data in Table 5, it can be deduced that
all the compounds lack cytotoxic activity, displaying CC50 values
higher than 10 lM.
Using a cell-based MLR assay, the molecular target of these
immunosuppressive compounds remains elusive. Substituted

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A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
pyrimidines are a well-known template for kinase inhibitors and
therefore, the most potent analogue 9k was assayed for its binding
affinity towards 451 kinases, using the KINOMEscan™ screening
Precoated aluminum sheets (Fluka Silica gel/TLC-cards, 254 nm)
were used for TLC. Column chromatography (CC) was performed
on ICN silica gel 63-200, 60 Å.
platform. At a concentration of 10 lM, pyrimidine 9k did bind very
weakly to the following kinases: BRAF (34% of control), BRAF
(V600E, 28% of control), RIPK4 (43% of control), RSK4 (kin dom
1-N-terminal; 35% of control), LOK (45% of control), JAK1 (JH2-do-
5.2. General procedure A for the synthesis of 5-cyano-6-phenyl-
2-thiouracils (1a–e)
main-pseudokinase; 39% of control); IKK-
a
(44% of control), IKK-b
To a solution of an appropriate benzaldehyde (6.0 mmol) in
EtOH (20 mL) were added ethylcyanoacetate (6.3 mmol), thiourea
(6.3 mmol) and piperidine (12 mmol). The resulting reaction mix-
ture was refluxed overnight. The solution was cooled down at
0 °C, the precipitate was filtered off and washed several times with
cold EtOH. The crude products were used in the next step without
any further purification.
(48% of control). As compound 9k did not bind strongly to any of
the 451 kinases, it is unlikely that the immunosuppressive activity
of compound 9k is due to inhibition of one of these kinases. How-
ever, the most potent compounds do possess structural features
(i.e., the morpholine moiety) that are typical for ATP-competitive
mTOR inhibitors and, in addition, some of the evolving SAR is typ-
ical for mTOR inhibitors (e.g., the loss of immunosuppressive activ-
ity by changing a morpholine into a thiomorpholino).19 To rule out
mTOR inhibition as target responsible for the immunosuppression,
compound 9k was also evaluated in a functional mTOR assay, using
the KinaseProfiler™ service from Millipore.20 At a compound con-
5.2.1. 5-Cyano-6-(4-toluil)-2-thiouracil (1c)
This compound was prepared according to general procedure A
using p-tolualdehyde in a yield of 61%. 1H NMR (300 MHz, DMSO-
d6): 7.59 (d, J = 7.95 Hz, 2H), 7.40 (d, J = 7.95 Hz, 2H), 2.40 (s, 3H).
13C NMR (75 MHz, DMSO-d6): 168.2, 153.2, 135.3, 121.2, 120.1,
118.4, 105.9, 81.7, 12.2. ESI-MS (pos): 244.0 ([M+H]+).
centration of 10 lM and an ATP concentration of 70 lM, only 10%
inhibition was observed, which further supports the findings of the
binding screening assay that mTOR is not the target.
5.2.2. 5-Cyano-6-(4-methoxyphenyl)-2-thiouracil (1d)
This compound was prepared according to general procedure A
using 4-anisaldehyde in a yield of 47%. 1H NMR (300 MHz, DMSO-
d6): 13.1 (br s, 1H), 7.65 (d, J = 8.55 Hz, 2H), 7.11 (d, J = 8.55 Hz, 2H),
3.86 (s, 3H). 13C NMR (75 MHz, DMSO-d6) 176.3, 162.5, 160.6,
158.8, 131.0, 121.2, 115.2, 114.0, 89.9, 55.7. ESI-MS (pos): 260.1
([M+H]+).
4. Conclusion
A library of novel 2,4,6-trisubstituted-5-cyano-pyrimidine ana-
logues has been synthesized and evaluated for its immunosuppres-
sive activity in the MLR assay. The SAR study revealed that a
hydroxyl group or an alkoxy group at position 4 is not tolerated
for immunosuppressive activity, whereas the presence of larger
amines (in particular morpholine) yielded compounds that showed
a better activity in the MLR assay. Position 2 of the pyrimidine scaf-
fold tolerates structural variety with respect to immunosuppres-
sive activity, as the presence of aniline groups, as well as
different thioalkyl moieties, furnished compounds with IC50 values
5.2.3. 5-Cyano-6-phenyl-2-thiouracil (1e)
This compound was prepared according to general procedure A
using benzaldehyde in a yield of 41%. 1H NMR (300 MHz, DMSO-
d6): 12.86 (br s, 1H), 8.5 (br s, 1H), 7.68 (d, J = 7.56 Hz, 2H),
7.61 m, 1H), 7.56 (d, J = 7.56 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6): 176.0, 160.2, 158.2, 132.0, 128.6, 128.2, 113.8, 90.2. ESI-MS
(pos): 230.1 ([M+Na]+).
of less than 5 lM in the MLR assay. At position 6, only phenyl
groups were investigated as substituent, whereby the 4-OCH3-phe-
nyl moiety seems to be optimal for immunosuppressive activity,
5.2.4. Synthesis of N,N0-Bis(tert-butoxycarbonyl)-N00-p-
chlorophenylguanidine (3a)
yielding compound 9k with a IC50 of 1.6
Counterscreening revealed that pyrimidine derivative 9k was de-
void of cellular toxicity (CC50 >10 M) and is therefore an interest-
lM in the MLR assay.
To a solution of p-chloroaniline (0.3 mmol), di-Boc-thiourea
(0.25 mmol) and Et3N (0.50 mmol) in CH2Cl2, was added N-iodo-
succinimide (0.25 mmol) in one portion and the reaction was stir-
red at room temperature for 12 h. The reaction was quenched by
adding 3 mL of a Na2S2O3 solution. The organic phase was sepa-
rated and washed twice with brine. The crude product was purified
by silica gel chromatography (heptane/AcOEt 5:1). 1H NMR
(300 MHz, DMSO-d6): 11.6 (s, 1H), 10.4 (s, 1H), 7.58 (d,
J = 8.79 Hz, 2H), 7.29 (d, J = 8.79 Hz, 2H), 1.55 (s, 9H), 1.53 5s,
9H). ESI-MS (pos): 370.2 ([M+H]+).
l
ing starting point for further optimization towards more potent
immunosuppressive drugs.
5. Experimental section
5.1. General
For all reactions, analytical grade solvents were used. All mois-
ture-sensitive reactions were carried out in oven-dried glass-ware
(135 °C). 1H and 13C NMR spectra: Bruker Advance 300 (1H NMR:
300 MHz, 13C NMR: 75 MHz), using tetramethylsilane as internal
standard for 1H NMR spectra and DMSO-d6 (39.5 ppm) or CDCl3
(77.2 ppm) for 13C NMR spectra. Abbreviations used are: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, br. s = broad sin-
glet. Coupling constants are expressed in Hz. Mass spectra are ob-
tained with a Finnigan LCQ Advantage Max (ion trap) mass
spectrophotometer from Thermo Finnigan, San Jose, CA, USA. High
resolution mass spectrometry: spectra were acquired on a quadru-
pole orthogonal acceleration time-of-flight mass spectrometer
(Synapt G2 HDMS, Waters, Milford, MA). Samples were infused
5.2.5. Synthesis of p-Chlorophenylguanidine (4a)
A solution of 3a in a mixture of CH2Cl2/TFA (1:1; 2 mL) was stir-
red at room temperature for 12 h. The mixture was basified by
slowly adding a saturated NaHCO3 solution till pH = 8. The crude
product was used in the next step without any further purification.
1H NMR (300 MHz, DMSO-d6): 7.50–7.25 (m, 4H), 7.05 (br s, 4H).
ESI-MS (pos): 170.0 ([M+H]+).
5.3. General procedure B for the synthesis of substituted
arylguanidines (4b–e)
at 3 lL/min and spectra were obtained in positive (or negative)
ionization mode with a resolution of 15,000 (FWHM) using leucine
enkephalin as lock mass.
To a solution of the appropriate aniline (7 mmol) in a mixture of
EtOH/H2O (1:1; 20 mL) were added cyanoamide (10 mmol) and a
concentrated nitric acid solution (7 mmol). The resulting reaction
mixture was refluxed overnight. The solvent was evaporated under

A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
1215
reduced pressure and the crude product was purified by flash chro-
matography, using the mobile phase as mentioned below for each
derivative.
6:1), affording the title compound in 79% yield. 1H NMR (300 MHz,
CDCl3): 10.1 (br s, 1H), 7.93 (d, J = 8.40 Hz, 2H), 7.74 (br s, 2H), 7.64
(d, J = 8.40 Hz, 2H), 7.47 (br d, 3H), 7.43 (d, J = 8.52 Hz, 2H), 7.38 (d,
J = 8.52 Hz, 2H), 5.57 (s, 2H). 13C NMR (75 MHz, DMSO-d6): 169.0,
159.1, 137.8, 136.3, 135.7, 134.6, 130.5, 128.8, 128.7, 128.4,
127.2, 122.1, 115.6, 69.1. HRMS: ESI+ calcd for (C24H17Cl2N4O)+
447.0779, found: 447.0782.
5.3.1. 3-Fluorophenylguanidine (4b)
This compound was prepared using 3-fluoro-aniline, following
the general procedure B. The crude residue was purified by silica
gel chromatography (CH2Cl2/MeOH 99:1 ? 95:5), affording the ti-
tle compound in 39% yield. 1H NMR (300 MHz, DMSO-d6): 9.84 (br
s, 1H), 7.50 (br m, 4H), 7.65 (d, J = 7.89 Hz, 1H) 7.16–7.06 (m, 4H).
ESI-MS (pos): 154.2 ([M+H]+).
5.5.2. 2-(4-Chlorophenylamino)-4-methoxy-5-cyano-6-(4-
chlorophenyl)pyrimidine (6b)
This compound was prepared according to the general proce-
dure D, starting from compound 5a and methyliodide. The crude
residue was purified by silica gel chromatography (heptane/AcOEt
8:1), affording the title compound in 61% yield. 1H NMR (300 MHz,
CDCl3): 7.97 (d, J = 7.89 Hz, 2H), 7.60 (d, J = 7.89 Hz, 2H), 7.52 (d,
J = 8.55 Hz, 2H), 7.37 (d, J = 8.55 Hz, 2H), 4.13 (s, 3H). 13C NMR
(75 MHz, CDCl3): 168.8, 158.6, 137.6, 136.1, 129.8, 129.0, 128.8,
128.7, 121.2, 114.9, 93.4, 55.0. HRMS: ESI+ calcd for
(C18H13Cl2N4O)+ 371.0466, found: 371.0450.
5.4. General procedure C for the synthesis of 2-anilino-4-oxo-5-
cyano-6-phenylpyrimidines (5a–j)
To a solution of the substituted arylguanidines (3 mmol) in
EtOH (10 mL), were added the appropriate substituted benzalde-
hyde (3.15 mmol), ethylcyanoacetate (3.15 mmol) and piperidine
(6 mmol) and the mixture was refluxed overnight. The solution
was cooled down at 0 °C. The reaction was then quenched with
brine and the organic phase was extracted with AcOEt. The crude
product was purified by silica gel chromatography, using a mobile
a phase as mentioned below for each derivative.
5.5.3. 2-Benzylthio-4-methoxy-5-cyano-6-(4-
chlorophenyl)pyrimidine (11a) and 2-benzylthio-N(3)-methyl-
5-cyano-6-(4-chlorophenyl)pyrimidine (12a)
These compounds were prepared according to the general pro-
cedure D, starting from compound 8a and methyliodide. The mix-
ture of isomers was purified by silica gel chromatography
(heptane/AcOEt 5:1), after which the ratio O-alkyl/N-alkyl was
determined to be as 2:1, and the overall yield was 71%. Compound
11a: 1H NMR (300 MHz, CDCl3): 8.0 (d, J = 8.61 Hz, 2H), 7.97 (d,
J = 8.61 Hz, 2H), 7.49 (d, J = 6.66 Hz, 2H), 7.35–7.28 (m, 3H), 4.48
(s, 2H), 4.14 (s, 3H). 13C NMR (75 MHz, CDCl3): 174.6, 169.6,
166.9, 137.9, 136.3, 133.1, 130.0, 128.7, 128.5, 128.3, 127.2,
114.2, 87.6, 55.2, 35.5. HRMS: ESI+ calcd for (C19H15ClN3OS)+
368.0624, found: 368.0625. Compound 12a: 1H NMR (300 MHz,
CDCl3): 7.99 (d, J = 8.28 Hz, 2H), 7.51 (d, J = 8.28 Hz, 2H), 7.38–
7.28 (m, 5H), 4.57 (s, 2H), 3.60 (s, 3H). 13C NMR (75 MHz, CDCl3):
165.9, 164.0, 159.7, 138.0, 134.1, 133.0, 130.0, 128.8, 128.7,
128.6, 127.9, 114.8, 92.7, 37.0, 30.6. HRMS: ESI+ calcd for
(C19H15ClN3OS)+ 368.0624, found: 368.0622.
5.4.1. 2-(4-Chlorophenylamino)-4-oxo-5-cyano-6-(4-
chlorophenyl)-3,4-dihydropyrimidine (5a)
This compound was prepared according to general procedure C,
using p-chlorophenylguanidine and p-chlorobenzaldehyde. The
crude product was purified by silica gel chromatography, using a
mixture of CH2Cl2/MeOH (in a ratio of 99:1) as mobile phase,
affording the title compound in
a
yield of 49%. 1H NMR
(300 MHz, DMSO-d6): 10.19 (s, 1H); 7.88 (d, J = 8.52 Hz, 1H), 7.65
(dd, J = 4.38, 8.79 Hz, 4H), 7.62 (d, J = 8.52 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6): 169.2, 136.4, 136.2, 135.1, 130.4, 128.9,
128.8, 128.3, 123.2, 116.6, 88.5. HRMS: ESI+ calcd for
(C17H11Cl2N4O)+ 357.0310, found: 357.0321
5.4.2. 2-(4-Chlorophenylamino)-4-oxo-5-cyano-6-(3-
chlorophenyl)-3,4-dihydropyrimidine (5b)
This compound was prepared according to general procedure C,
using a mixture of p-chlorophenylguanidine and m-chlorobenzalde-
hyde. The crude product was purified by silica gel chromatography,
using a mixture of CH2Cl2/MeOH (in a ratio of 99:1) as mobile phase,
affording the title compound in 39% yield. 1H NMR (300 MHz, DMSO-
5.5.4. 2-Benzylthio-4-ethoxy-5-cyano-6-(4-
chlorophenyl)pyrimidine (11b) and 2-benzylthio-N(3)-ethyl-5-
cyano-6-(4-chlorophenyl)pyrimidine (12b)
These compounds were prepared according to the general pro-
cedure D, starting from compound 8a and ethyliodide. The mixture
of isomers was purified by silica gel chromatography (heptane/
AcOEt 5:1), after which the ratio O-alkyl/N-alkyl was determined
to be 3:1 and the overall yield was 63%. Compound 11b: 1H NMR
(300 MHz, CDCl3): 8.00 (d, J = 8.58 Hz, 2H), 7.52 (d, J = 8.58 Hz,
2H), 7.44 (br d, 2H), 7.34–7.28 (m, 3H), 4.63 (q, J = 7.08 Hz, 2H),
4.47 (s, 2H), 1.48 (t, J = 7.08 Hz, 3H). 13C NMR (75 MHz, DMSO-
d6): 174.5, 169.2, 167.0, 137.8, 136.3, 133.2, 130.0, 128.7, 128.4,
128.3, 127.2, 114.3, 87.7, 64.5, 35.5, 13.9. HRMS: ESI+ calcd for
(C20H17ClN3OS)+ 382,0781, found: 382,0796. Compound 12b: 1H
NMR (300 MHz, CDCl3): 7.99 (d, J = 8.67 Hz, 2H), 7.51 (d,
J = 8.67 Hz, 2H), 7.40–7.35 (m, 5H), 4.56 (s, 2H), 4.20 (q,
J = 7.14 Hz, 2H), 1.40 (t, J = 7.14 Hz, 3H). 13C NMR (75 MHz, CDCl3):
165.1, 164.0, 159.4, 138.0, 134.1, 133.1, 130.0, 128.8, 128.7, 128.6,
127.9, 114.4, 93.1, 40.5, 36.9, 11.9. HRMS: ESI+ calcd for
(C20H17ClN3OS)+ 382.0781, found: 382.0794.
d6): 7.83 (m, 4H), 7.57 (d, J = 8.41 Hz, 2H), 7.33 (d, J = 8.58 Hz, 2H). 13
C
NMR (75 MHz, DMSO-d6): 172.2, 168.0, 139.6, 138.8, 133.1, 130.4,
130.2, 128.5, 128.1, 127.1, 125.9, 121.5, 118.7, 86.4. HRMS: ESI+ calcd
for (C17H11Cl2N4O)+ 357.0310, found: 357.0320
5.5. General procedure D for the synthesis of compounds 6a–e,
11a–e, 12a–g
To a solution of a pyrimidine derivative (0.3 mmol) in DMF
(7 mL) were added an appropriate alkylating agent (1.05 equiv)
and K2CO3 (1.5 equiv). The solution was stirred at room tempera-
ture for 12 h. The reaction was quenched with brine and the organ-
ic phase was extracted twice with AcOEt, dried over MgSO4,
filtered and evaporated under reduced pressure. The crude residue
was purified by silica gel chromatography, using the mobile phase
as specified below for each derivative.
5.5.1. 2-(4-Chlorophenylamino)-4-benzyloxy-5-cyano-6-(4-
chlorophenyl)pyrimidine (6a)
5.6. General procedure E for the synthesis of compounds 2 and
8a–k
This compound was prerared according to the general proce-
dure D, starting from compound 5a and benzylbromide. The crude
residue was purified by silica gel chromatography (heptane/AcOEt
To a solution of a 2-thiouracil analogue (0.61 mmol) in CH3CN
(8 mL), were added K2CO3 (0.64 mmol) and an alkyl or benzyl

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A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
halide (0.64 mmol). The mixture was stirred overnight at room
temperature. The reaction was quenched with brine and diluted
with AcOEt. The organic phase was extracted, dried over MgSO4
and evaporated under reduced pressure. The crude product was
purified by silica gel chromatography using the mobile phase as
mentioned below for each derivative.
DMSO-d6): 10.2 (br s, 1H), 7.88 (d, J = 8.19 Hz, 2H), 7.71 (d,
J = 8.64 Hz, 2H), 7.62 (d, J = 8.64 Hz, 2H), 7.37 (d, J = 8.19 Hz, 2H),
3.88 (br s, 4H), 3.76 (br s, 4H). 13C NMR (75 MHz, DMSO-d6)
170.6, 162.4, 158.3, 138.3, 135.6, 130.8, 128.4, 126.3, 124.1,
121.6, 118.6, 65.8, 47.3. HRMS: ESI+ calcd for (C21H18Cl2N5O)+
426.0888, found: 426.0885.
5.6.1. 2-Methylthio-5-cyano-6-(4-chlorophenyl)uracil (2)
This compound was prepared according to general procedure E,
starting from 1a and methyliodide. The crude residue was purified
by silica gel chromatography (heptane/AcOEt1:1), affording the ti-
tle compound in 88% yield. 1H NMR (300 MHz, DMSO-d6): 7.91 (d,
J = 8.07 Hz, 2H), 7.79 (d, J = 8.07 Hz, 2H), 2.60 (s, 3H). ESI-MS (pos):
278.0 ([M+H]+).
5.7.2. 2-(4-Chlorophenylamino)-4-isopropylamino-5-cyano-6-
(4-chlorophenyl)pyrimidine (7b)
This compound was prepared according to general procedure F
starting from compound 5a and isopropylamine. The crude residue
was purified by silica gel chromatography (heptane/AcOEt 2:1),
affording the title compound in 62% yield. 1H NMR (300 MHz,
DMSO-d6): 10.05 (br s, 1H), 7.85 (d, J = 8.43 Hz, 2H), 7.81 (d,
J = 8.88 Hz, 2H), 7.614 (d, J = 8.43 Hz, 2H), 7.37 (d, J = 8.88 Hz,
2H), 4.41 (br m, 1H), 1.27 (d, J = 6.42 Hz, 6H). 13C NMR (75 MHz,
DMSO-d6): 168.1, 162.0, 159.2, 138.9, 135.8, 135.4, 130.3, 128.6,
128.5, 126.1, 12 1.4, 117.2, 43.0, 21.9. HRMS: ESI+ calcd for
(C20H18Cl2N5)+ 398.0939, found: 398.0951
5.6.2. 2-Benzylthio-5-cyano-6-(4-chlorophenyl)uracil (8a)
This compound was prepared according to general procedure E,
starting from 1a and benzylbromide. The crude residue was puri-
fied by silica gel chromatography (heptane/AcOEt 5:1 ? 1:1),
affording the title compound in 61% yield. 1H NMR (300 MHz,
DMSO-d6): 7.90 (d, J = 8.55 Hz, 2H), 7.61 (d, J = 8.55 Hz, 2H), 7.40
(d, J = 6.75 Hz, 2H), 7.33–7.24 (m, 3H), 4.45 (s, 2H). HRMS: ESI+
calcd for (C18H13ClN3OS)+ 354.0468, found: 354.0486.
5.7.3. 2-(4-Chlorophenylamino)-4-piperidino-5-cyano-6-(4-
chlorophenyl)pyrimidine (7c)
This compound was prepared according to general procedure F
starting from compound 5a and piperidine. The crude residue was
purified by silica gel chromatography (heptane/AcOEt 2:1), yield-
ing the title compound in 71% yield. 1H NMR (300 MHz, DMSO-
d6): 10.11 (br s, 1H), 7.87 (d, J = 8.46 Hz, 2H), 7.76 (d, J = 8.82 Hz,
2H), 7.63 (d, J = 8.46 Hz, 2H), 8.76 (d, J = 8.82 Hz, 2H), 3.84 (br s,
4H), 1.67 (br s, 6H). HRMS: ESI+ calcd for (C22H20Cl2N5)+
424.1096, found: 424.1085.
5.6.3. 2-Benzylthio-5-cyano-6-(3-chlorophenyl)uracil (8b)
This compound was prepared according to general procedure E,
starting from 1b and benzylbromide. The crude residue was puri-
fied by silica gel chromatography (CH2Cl2/MeOH 100:0 ? 97:3),
yielding the title compound in a yield of 57%. 1H NMR (300 MHz,
DMSO-d6): 7.88 (d, J = 7.94 Hz, 2H), 7.68 (d, J = 8.07 Hz, 1H), 7.62
(d, J = 8.12 Hz, 1H), 7.35 (d, J = 7.45 Hz, 2H), 7.33–7.27 (m, 3H),
4.52 (s, 2H). 13C NMR (75 MHz, DMSO-d6): 166.3, 165.9, 161.1,
137.4, 136.6, 133.4, 131.9, 131.6, 130.7, 129.1, 128.7, 128.5,
127.6, 127.5, 93.9, 34.4. HRMS: ESI+ calcd for (C18H13ClN3OS)+
354.0468, found: 354.0456.
5.7.4. 2-Benzylthio-5-cyano-6-(4-chlorophenyl)-4-
cyclohexylaminopyrimidine (9c)
This compound was prepared according to general procedure F
starting from compound 8a and cyclohexylamine. The crude prod-
uct was purified by silica gel chomarography (heptane/AcOEt 5:1)
affording the title compound in 58% yield. 1H NMR (300 MHz,
DMSO-d6): 7.83 (d, J = 8.55 Hz, 2H), 7.60 (d, J = 8.55 Hz, 2H), 7.41
(d, J = 6.87 Hz, 2H), 7.33-7.24 (m, 3H), 4.42 (d, 2H), 4.05 (br m,
1H), 1.75 (t, J = 12.18 Hz, 3H), 1.60 (d, J = 11.52 Hz, 1H), 1.51–1.40
(m, 2H), 1.29–1.22 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 174.2,
165.4, 160.6, 137.2, 137.0, 034.1, 029.7, 128.6, 128.4, 128.2,
126.9, 83.0, 50.0, 35.1, 32.5, 25.1, 24.5. HRMS: ESI+ calcd for
(C24H24ClN4S)+ 435.1110, found: 435.1122.
5.6.4. 2-Benzylthio-5-cyano-6-(4-methylphenyl)uracil (8c)
This compound was prepared according to general procedure E,
starting from 1c and benzylbromide. The crude residue was puri-
fied by silica gel chromatography (CH2Cl2/MeOH 100:0 ? 95:5),
furnishing the title compound in
a
yield of 61%. 1H NMR
(300 MHz, DMSO-d6): 7.87 (d, J = 7.89 Hz, 2H), 7.43–7.37 (m, 4H),
7.35–7.27 (m, 3H), 4.45 (s, 2H), 2.40 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): 167.2, 165.5, 161.2, 142.6, 142.3, 136.6, 132.5, 129.3,
129.1, 128.9, 128.7, 127.6, 116.1, 92.7, 34.3, 21.22. HRMS: ESI+
calcd for (C19H16N3OS)+ 334.1014, found: 334.1031.
5.7.5. 2-Benzylthio-4-(4-carboxyamidepiperidine)-5-cyano-6-
(4-chlorophenyl)pyrimidine (9d)
5.7. General procedure F for the BOP mediated nucleophilic
arylic substitution
This compound was prepared according to general procedure F,
starting from compound 8a and 4-carboxyamide-piperidine. The
crude residue was purified by silica gel chromatography (hep-
tane/AcOEt 8:1), affording the title compound in 55% yield. 1H
NMR (300 MHz, DMSO-d6): 7.88 (d, J = 8.58 Hz, 2H), 7.63 (d,
J = 8.58 Hz, 2H), 7.44 (br d, J = 7.08 Hz, 2H), 7.34-7.25 (m, 3H),
6.85 (br s, 1H), 4.60 (d, J = 13.38 Hz, 2H), 3.28 (m, 3H), 1.91-1.85
(m, 2H), 1.69-1.55 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 175.8,
172.2, 169.0, 161.3, 137.7, 136.2, 135.1, 131.2, 128.9, 128.7,
128.6, 128.4, 127.3, 118.0, 83.9, 46.7, 41.0, 34.7, 28.4. HRMS: ESI+
calcd for (C24H23ClN5OS)+ 464.1312, found: 464.1315.
To a solution of a 4-oxopyrimidine derivative (0.280 mmol) in
MeCN (5 mL), were added BOP (0.363 mmol), the appropriate
amine (0.7 mmol) and DBU (0.42 mmol). The reaction mixture
was stirred at room temperature for 12 h. The solution was diluted
with AcOEt and brine, the organic phase was separated and
washed twice with brine. The combined organic phases were dried
over MgSO4 and evaporated under reduced pressure. The crude
product was purified by silica gel chromatography using the mo-
bile phase as mentioned below for each derivative.
5.7.6. 2-Benzylthio-4-(3-carboxyamidepiperidine)-5-cyano-6-
(4-chlorophenyl)pyrimidine (9e)
5.7.1. 2-(4-Chlorophenylamino)-4-morpholino-5-cyano-6-(4-
chlorophenyl)pyrimidine (7a)
This compound was prepared according to general procedure F
starting from compound 5a and morpholine. The crude residue
was purified by silica gel chromatography (heptane/AcOEt 4:1),
furnishing the title compound in 71% yield. 1H NMR (300 MHz,
This compound was prepared according to general procedure F,
starting from compound 8a and 3-carboxyamide piperidine. The
crude residue was purified by silica gel chromatography (CH2Cl2/
MeOH 99:1), affording the title compound in 71% yield. 1H NMR
(300 MHz, DMSO-d6) 7.87 (d, J = 8.58 Hz, 2H), 7.61 (d, J = 8.58 Hz,

A. Stella et al. / Bioorg. Med. Chem. 21 (2013) 1209–1218
1217
2H), 7.42 (d, J = 6.96 Hz, 2H), 7.35-7.25 (m, 3H), 6.85 (br s, 1H), 4.58
(d, J = 13.44 Hz, 2H), 4.43 (s, 2H), 3.27 (t, J = 11.52 Hz, 2H), 2.54 (s,
1H), 1.91–1.88 (br m, 2H), 1.68–1.61 (br m, 2H). 13C NMR (75 MHz,
DMSO-d6): 175.8, 172.2, 169.0, 161.4, 137.7, 136.2, 135.1, 131.3,
128.9, 128.7, 128.6, 127.3, 118.0, 83.9, 46.7, 41.0, 36.6, 34.7, 28.4.
HRMS: ESI+ calcd for (C24H23ClN5OS)+ 464.1312, found: 464.1328.
37 °C in a humidified atmosphere of 5% CO2 and 95% air. DNA syn-
thesis was assayed by the addition of 1
Ci (methyl-3H) thymidine
per well during the last 18 h of culture. Thereafter, the cells were
harvested on glass filter paper and the counts per minute deter-
mined in a liquid scintillation counter.
l
7. Cytotoxicity assay
5.7.7. 2-Benzylthio-4-chloro-5-cyano-6-(4-
chlorophenyl)pyrimidine (10)
To a solution of 8a (100 mg, 0.282 mmol) in dioxane (5 mL) was
added POCl3 (155 lL, 1.69 mmol) and the reaction was stirred at
80 °C for 2 h. The solvent was then evaporated and the crude prod-
uct was purified by silica gel chromatography using a mixture of
heptane and AcOEt (in a ratio of 4:1) as mobile phase, furnishing
the title compound in a yield of 85%. 1H NMR (300 MHz, DMSO-
d6): 8.00 (d, J = 8.43 Hz, 2H), 7.73 (d, J = 8.43 Hz, 2H), 7.47 (d,
J = 6.84 Hz, 2H), 7.36 (m, 3H), 4.53 (s, 2H).
Cytotoxicity of compounds was measured using a colorimetric
cell viability assay, the WST-1 assay, on Jurkat T-cell line. Cells
were seeded in 96-well plates (5.104 cells per well) and exposed
to serial dilutions of compounds for 48 h at 37 °C and 5% CO2. After
removal of 100
lL of medium, cell viability was checked by adding
10 L of a solution of tetrazolium salt, 4-[3-[4-iodophenyl]-2-4-(4-
l
nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate (WST-1,
Roche Diagnostics, Switzerland). After incubation at 37 °C and 5%
CO2, absorbance at 450 nm was measured with a spectrophotome-
ter (2103 Envision Multilabel Reader, Perkin Elmer Waltham, MA).
Results were expressed as dilution as percentage of cytotoxicity at
each dilution and concentration at which 50% of the cells had died
(CC50) was calculated.
5.8. General Procedure F for the synthesis of compounds 9a–b
To a solution of 10 (0.25 mmol) in dioxane (5 mL) was added
the appropriate amine (0.5 mmol) and the mixture was stirred at
room temperature for 2 h. The solution was diluted with brine
and AcOEt. The organic phase was extracted, dried over MgSO4
and evaporated under reduced pressure. The crude mixture was
purified by silica gel chromatography using the mobile phase as
mentioned below for each derivative.
Acknowledgments
Alessandro Stella is deeply indebted to the IWT (Agentschap
voor Innovatie door Wetenschap en Technologie) for providing a
PhD-scholarship. Authors thank FWO (FWO research project
G.0614.09 N) for financial support. Mass spectrometry was made
possible by the support of the Hercules Foundation of the Flemish
Government (Grant 20100225–7).
5.8.1. 2-Benzylthio-4-morpholino-5-cyano-6-(4-
chlorophenyl)pyrimidine (9a)
This compound was prepared according to general procedure F
using morpholine. The crude residue was purified by silica gel
chromatography (heptane/AcOEt 6:1) yielding the title compound
in a yield of 55%. 1H NMR (300 MHz, DMSO-d6): 7.81 (d, J = 8.61 Hz,
2H), 7.46 (m, 4H), 7.33 (m, 3H), 4.41 (s, 2H), 3.97 (s, 4H), 3.80 (s,
4H). 13C NMR (75 MHz, DMSO-d6): 173.2, 169.4, 162.1, 137.3,
136.6, 134.2, 130.4, 128.5, 128.4, 128.3, 127.0, 117.7, 83.4, 77.2,
76.7, 76.3, 69.3, 47.2, 35.3. HRMS: ESI+ calcd for (C22H20ClN4OS)+
423.1046, found: 423.1050.
Supplementary data
Supplementary data (characterization data for the compounds
1a–b, 4c–e, 5c–i, 6c–e, 11c–e, 12c–g, 8d–k, 7d–h, 9f–x, as well as a
number of SAR tables) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.032.
References and notes
5.8.2. 2-Benzylthio-4-isopropylamino-5-cyano-6-(4-
chlorophenyl)pyrimidine (9b)
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