New Substituted 1-Aryl-4,4,6-Trimethyl-3,4-Dihydropyrimidine-2-(1H)Thiones; A Metal-Free and Solvent-Free Synthesis, Characterization, and Lymphoid Tyrosine Phosphatase Inhibition Studies

Article abstract of DOI:10.1002/jccs.201600805

A series of fourteen 3,4-dihydropyrimidine-2-thiones (3a–n) were synthesized by a green protocol, and their structures were characterized by spectroanalytical data. The compounds were obtained in high yields by efficient annulation of mesityl oxide (4-methylpent-3-en-2-one) with anilines in the presence of potassium thiocyanate. The reaction is essentially metal-catalyst- and solvent-free, as mesityl oxide itself is the solvent as well as the reactant. The compounds were tested for their ability to inhibit the lymphoid tyrosine phosphatase PTPN22, and 5 of the 14 compounds exhibited IC₅₀ values in the mid-micromolar range, with the most potent hit being the compound 3d, having a methoxy substituent at the 2-position of the phenyl ring with an IC₅₀ = 18 ± 1 μM, and second most potent compound (3c) with an IC₅₀ value of 45 ± 3 μM, having methyl substituents at both 2- and 4-position of the phenyl ring.

Full text of DOI:10.1002/jccs.201600805

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
New Substituted 1-Aryl-4,4,6-Trimethyl-3,4-Dihydropyrimidine-2-(1H)Thiones;
A Metal-Free and Solvent-Free Synthesis, Characterization, and Lymphoid Tyrosine
Phosphatase Inhibition Studies
*
Asma Khurshid and Aamer Saeed
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
(Received: November 4, 2016; Accepted: December 13, 2016; DOI: 10.1002/jccs.201600805)
A series of fourteen 3,4-dihydropyrimidine-2-thiones (3a–n) were synthesized by a green protocol,
and their structures were characterized by spectroanalytical data. The compounds were obtained
in high yields by efficient annulation of mesityl oxide (4-methylpent-3-en-2-one) with anilines in
the presence of potassium thiocyanate. The reaction is essentially metal-catalyst- and solvent-free,
as mesityl oxide itself is the solvent as well as the reactant. The compounds were tested for their
ability to inhibit the lymphoid tyrosine phosphatase PTPN22, and 5 of the 14 compounds exhib-
ited IC50 values in the mid-micromolar range, with the most potent hit being the compound 3d,
having a methoxy substituent at the 2-position of the phenyl ring with an IC50 = 18 Æ 1 μM, and
second most potent compound (3c) with an IC50 value of 45 Æ 3 μM, having methyl substituents
at both 2- and 4-position of the phenyl ring.
Keywords: Green; Metal-free; Solvent-free; 1-Aryl-3,4-dihydropyrimidine-2-thiones; PTPN22
inhibitors.
INTRODUCTION
The pyrimidine thiones (mercapto or thioxopyri-
some drugs and derivatives containing a pyrimidine
thione nucleus.
midines) are an important class of heterocyclic com-
pounds with two ring nitrogens and a thioamide
functionality. The pyrimidine bases thyamine, cytosine,
and uracil are vital building blocks of DNA and
RNA.1 The pyrimidine thiones can exist in two regioi-
someric forms: pyrimidine-2-thiones and pyrimidine-4-
thiones. Most of the dihydropyrimidin-2-thiones men-
tioned in the literature are those obtained by variations
of the well-known three-component reaction reported
by Pietro Biginelli, in 1891. A survey of the literature
indicates that pyrimidine-2-thiones are used in a num-
ber of biological applications, such as antitubercular,
antibacterial,2 antimicrobial, insecticidal, miticidal,
hypoglygamic, and antifungal functions.3 Among the
dihydropyrimidin-2-thiones drugs are barbituric acid
derivatives such as thiopental, a heterocyclic sedative/
hypnotic drug, thiobarbital, and thymol. High-
throughput screening has identified monastrol as a new
antimitotic compound as a kinesin-5 inhibitor.4–7 Mon-
astrol analogs LaSOM 63 and LaSOM 65 have effect
on cell proliferation.8,9 Figure 1 shows the structures of
1-Aryl-4,4,6-trimethyl-3,4-dihydropyrimidine-2-(1H)-
thiones represent a unique subclass of 3,4-dihydropyri-
midine-2-thiones with only a few examples in the litera-
ture. The first report includes the unexpected formation
of
4,4,6-trimethyl-1-phenyl-3,4-dihydropyrimidine-2
(1H)-thione as a side product during the reaction of cin-
namoyl isothiocyanate and aniline to afford the corre-
sponding acyl thiourea derivative in 2005.10,11 We were
the first to demonstrate that dihydropyrimidine-2(1H)-
thiones are in fact produced by the reaction of aniline,
KSCN, and 4-methylpent-3-en-2-one, an aldol conden-
sation product of acetone formed in situ from the ace-
tone used as solvent. Accordingly, the reaction of an
acid chloride with KSCN to afford corresponding acyl
isothiocayante does not occur; instead, 3,4-dihydropyri-
midine-2-thiones are formed by the reaction of acetone
dimer with KSCN and aniline without the involvement
of the acid chloride used. We have already reported, in
2010, the synthesis and crystal structures of some
dihydropyrimidine-2(1H)-thione derivatives.12–14 We
had been involved in a systematic study on the synthesis
*Corresponding author. Email: aamersaeed@yahoo.com
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Article
Khurshid and Saeed
N(CH)3
reversible phosphorylation of tyrosine residues in pro-
teins plays a critical role in regulating numerous aspects
of cellular signaling.23,24 The enzymes responsible for
tyrosine phosphorylation, namely the protein tyrosine
kinases or PTKs, have been clinically validated as ther-
apeutic targets for a number of disease states.25 How-
ever, the protein tyrosine phosphatases or PTPs have
not yet been successfully targeted, despite overwhelming
evidence that they could be good therapeutic targets.26
The lymphoid tyrosine phosphatase PTPN22 is consid-
ered a particularly intriguing therapeutic target for the
treatment of human autoimmune disorders.27
S
HO
O2N
HO
CH3 CH3
HN
NH
O
O
O
O
CH3
EtO2C
H3C
EtO2C
H3C
EtO2C
H3C
HN
NH
NH
NH
NH
H2C
S
N
S
N
S
N
S
CH3
H
H
H
Thiobarbital
Thimylal
LaSOM 63
Monastral
LaSOM 65
Fig. 1. Some pyrimidine-2-thione containing drugs
and their derivatives.
of 3,4-dihydropyrimidine-2-thiones for detailed structural
studies and biological evaluation. During the optimiza-
tion of the reaction conditions, it was noted that the reac-
tions proceed smoothly in the presence of a Brønsted acid
catalyst. On the other hand, during the preparation of this
manuscript, another group using our designed procedure
reported the synthesis of these compounds using expen-
sive, toxic, and eco-unfriendly transition-metal catalysts
with no citation of our work.15 This prompted us to
report our original work involving a one-pot, two compo-
nent, essentially metal-catalyst- or solvent-free synthesis
of a series of new pyrimidine2-thiones. In fact, there is no
need in any way to involve any transition-metal catalyst,
and the reaction proceeds smoothly with good yields,
with a simple purification by recrystallization only.
The lymphoid tyrosine phosphatase is a protein
tyrosine phosphatase (PTP), encoded in humans by the
PTPN22 gene16,17 with a critical negative regulatory
role on signaling through the T-cell receptor (TCR).
Engagement of TCR leads to a spatially and tem-
porally regulated chain of PTK/PTP activation/deacti-
vation events, which in turn result in tyrosine
phosphorylation and dephosphorylation of numerous
cytosolic and transmembrane proteins.18,19
Considering the primary association between the
C1858T polymorphism of PTPN22 (also called LYP)
and disease, and the increased enzymatic activity of the
resulting LYP-W620, it has been predicted that a spe-
cific small-molecule inhibitor of LYP will be useful to
revert the effects of LYP-W620 at the central and/or
peripheral level, and prevent the emergence, or reap-
pearance, of autoreactive T cells in carriers of LYP-
W620 affected by autoimmunity.20 Furthermore, recent
evidence indicates that inhibition of LYP could also be
beneficial in anaphylaxis21 and chronic lymphocytic leu-
kemia.22 The newly synthesized series of compounds
was screened for their ability to inhibit the activity of
the lymphoid tyrosine phosphatase PTPN22. The
In this study, we have screened our series of
pyrimidine thione compounds and identified two novel
inhibitors of PTPN22 activity with activity in the mid-
micromolar range
EXPERIMENTAL
Melting points were determined using a Gallenkamp
melting point apparatus (MP-D) and are uncorrected.
Infrared spectra were recorded using a Shimadzu IR
460 spectrometer using KBr pellets. 1H-NMR spectra were
obtained using a Bruker 300 NMR MHz spectrometer in
deuterated solvents using TMS as internal reference. 13C-
NMR spectra were obtained by (75 MHz) the NMR spec-
trometer in deuterated solvents. Thin-layer chromatogra-
phy (TLC) was performed on precoated silica gel
aluminum plates (layer thickness 0.2 mm, HF 254, Reidal-
de-Haen from Merck). The chromatograms were detected
by using ultraviolet light (254 and 260 nm). Liquid
chromatography-mass spectrometry (LC-MS) was per-
formed using an EI source of (70 eV) on an Agilent Tech-
nologies 6890 N gas chromatograph and an inert mass-
selective detector 5973 mass spectrometer. Elemental ana-
lyses were conducted using
932 instrument.
a
LECO CHNS
General procedure for the synthesis of 4,4,6-trimethyl-1-
aryl-3,4-dihydropyrimidine-2(1H)-thiones
A total of 14 derivatives (3a–n) were synthesized
which are as follows:
Suitably substituted anilines (1a-n) (1.0 mmol)
were added portionwise to a stirred suspension of potas-
sium thiocyanate (1.0 mmol) in 4-methylpent-3-en-2-
one (2) (1.20 mmol) containing 1–2 drops of HCl, at
room temperature. The reaction mixture was heated at
50–60ꢀC for 3–5 h, and the progress was followed by
2
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Green Synthesis of Dihydropyrimidine-2-Thiones
TLC. On completion, the reaction mixture was cooled
to room temperature and poured into ice-water. The
precipitated compounds were recrystallized from etha-
nol to afford the purified dihydropyrimidine-2-thiones
(3a–n) in good to excellent yields.
C15H20N2S: calc.: C 69.14, H 7.79, N 10.72, S 12.35;
found: C 69.17, H 7.75, N 10.71, S 12.37.
1-(2-Methoxyphenyl)-4,4,6-trimethyl-3,4-dihydro-
pyrimidine-2(1H)-thione (3d). Brown amorphous solid;
Yield; 86%, Rf: 0.43; m.p: 141–142ꢀC; IR (neat): 3360
(NH), 3070 (=C–H stretch), 1699 (CS–NH), 1575
(C = C), 1299 (C–N): 1H-NMR (CDCl3, 300 MHz):
δ 8.03 (s, 1H, N–H), 7.07–6.35 (m, 5H, Ar–H), 4.45
(s, 1H, Csp2–H), 3.52 (s, 3H, Ar–OCH3), 1.54 (s, 3H,
Csp2–CH3) 1.19 (s, 3H, CH3), 1.10 (s, 3H, CH3); 13C-
NMR (CDCL3, 75 MHz): δ 185.7 (CSNH), 136.0
(−C=C–), 135.1, 130.0, 127.4, 125.3, 121.7, 121.2, 108.2
(−H–C=C–), 58.9 ((CH3)2–), 55.9 (Ar–OCH3), 35.0
((CH3)2–C), 32.0 (−C–CH3). Elemental analysis
C14H18N2OS: calc.: C 64.03, H 6.97, N 10.62, O 6.18,
S 12.20; found: C 64.07, H 6.93, N 10.64, O 6.14, S 12.22.
1-(4-Methoxyphenyl)-4,4,6-trimethyl-3,4-dihydro-
pyrimidine-2(1H)-thione (3e). Red crystalline solid;
Yield; 87%, Rf: 0.47; m.p: 163–164ꢀC; IR (neat): 3355
(NH), 3045 (=C–H stretch), 1700 (CS–NH), 1585
4,4,6-Trimethyl-1-o-tolyl-3,4-dihydroprymidine-2(1H)-
thione (3a). Brown amorphous solid; Yield; 90%, Rf:
0.33; m.p: 100–101ꢀC; IR (neat): 3251 (NH), 3050 (=C–
H stretch), 1691 (CS-NH), 1523 (C=C), 1285 (C–N):
1H-NMR ((CD3)2SO, 300 MHz): δ 8.93 (s, 1H, N–H),
7.16–7.42 (m, 5H, Ar–H), 4.95 (s, 1H, Csp2–H), 2.50 (s,
3H, Ar–CH3), 1.44 (s, 3H, Csp2–CH3) 1.29 (s, 3H,
CH3), 1.25 (s, 3H, CH3); 13C-NMR ((CD3)2SO,
75 MHz): δ 176.66 (CSNH), 143.0 (–C=C–), 132.9,
131.4, 130.9, 130.3, 129.7, 128.7, 110.2 (–H–C=C–),
52.0 ((CH3)2–C), 31.0 ((CH3)2–), 27.5 (–C–CH3), 20.61
(Ar–CH3). LC-MS (m/z)%: [M + K]+ 284.5 (100%).
Elemental analysis C14H18N2S: calc. C 68.27, H 7.35,
N 11.34, S 13.03; found: C14H18N2S: C 68.27, H 7.35,
N 11.34, S 13.03.
4,4,6-Trimethyl-1-p-tolyl-3,4-dihydroprymidine-2(1H)-
thione (3b). Yellow crystalline solid; Yield; 88%, Rf:
0.36; m.p: 139–140ꢀC; IR (neat): 3345 (NH), 3075
(=C–H stretch), 1695 (CS–NH), 1527 (C = C), 1287
(C–N): 1H-NMR ((CD3)2CO, 300 MHz): δ 7.71 (s,
1H, N–H), 7.24–7.01 (m, 5H, Ar–H), 4.97 (s, 1H,
Csp2–H), 2.35 (s, 3H, Ar–CH3), 1.50 (s, 3H, Csp2–
CH3) 1.39 (s, 6H, 2 × CH3); 13C-NMR ((CD3)2CO,
75 MHz): δ 179.16 (CSNH), 142.9 (C=C), 138.4,
133.4, 129.1, 128.3, 111.2 (–H–C=C–), 53.9 ((CH3)2–),
32.6 ((CH3)2–C), 27.9 (–C–CH3), 20.5 (Ar–CH3). Ele-
mental analysis C14H18N2S: calc.: C 68.25, H 7.37, N
11.33, S 13.04; found: C 68.28, H 7.35, N 11.34,
S 13.02.
1
(C = C), 1301 (C–N): H-NMR (CDCl3, 300 MHz):
δ 7.81 (s, 1H, N–H), 7.65–6.17 (m, 5H, Ar–H), 4.61
(s, 1H, Csp2–H), 3.71 (s, 3H, Ar–OCH3), 1.64 (s, 3H,
Csp2–CH3) 1.24 (s, 3H, CH3), 1.21 (s, 3H, CH3);
13C-NMR (CDCl3, 75 MHz): δ 186.5 (CSNH), 137.9
(−C=C–), 134.7, 126.3, 121.9, 120.2, 111.2 (−H–
C=C–), 57.9 ((CH3)2–C–), 54.6 (Ar–OCH3), 33.8
((CH3)2–), 32.4 (−C–CH3). LC–MS (m/z) %:
[M + H]+ 263.6 (100%). Elemental analysis
C14H18N2OS: calc.: C 64.05, H 6.96, N 10.64, S
12.21; found: C 64.03, H 6.93, N 10.66, S 12.20.
1-(2-Fluorophenyl)-4,4,6-trimethyl-3,4-dihydropyri-
midine-2(1H)-thione (3f ). Brown crystalline solid; Yield;
81%, Rf: 0.41; m.p: 118–119ꢀC; IR (neat): 3321 (NH),
3092 (=C–H stretch), 1689 (CS–NH), 1583 (C = C),
1-(2,4-Dimethylphenyl)-4,4,6-trimethyl-3,4-dihydro-
pyrimidine-2(1H)-thione (3c). White crystalline solid;
Yield; 85%, Rf: 0.32; m.p: 150–151ꢀC; IR (neat): 3320
(NH), 3082 (=C–H stretch), 1697 (CS–NH), 1535
1
1347 (C–N): H-NMR (CDCl3, 300 MHz): δ 8.02 (s,
1H, N–H), 7.15–6.27 (m, 5H, Ar–H), 4.39 (s, 1H,
Csp2–H), 1.59 (s, 3H, Csp2–CH3) 1.21 (s, 3H, CH3),
1.19 (s, 3H, CH3); 13C-NMR (CDCL3, 75 MHz):
δ 183.5 (CSNH), 136.9 (−C=C–), 134.1, 132.4, 126.8,
126.0, 121.4, 120.9, 110.2 (−H–C=C–), 56.0 ((CH3)2–
C–), 33.3 ((CH3)2–), 32.7 (−C–CH3). Elemental analysis
C13H15FN2S: calc.: C 62.33, H 6.08, N 7.69, S 12.85;
found: C 62.31, H 6.10, N 7.66, S 12.84.
(C = C),
1290
(C–N):
1H-NMR
((CD3)2CO,
300 MHz): δ 7.72 (s, 1H, N–H), 7.08–6.89 (m, 3H,
Ar–H), 4.97 (q, 1H, J = 1.2 Hz, Csp2–H), 2.31 (s, 3H,
Ar–CH3), 2.17 (s, 3H, Ar–CH3), 1.44 (d, 3H,
J = 1.2 Hz, Csp2–CH3), 1.39 (6H, 2 × CH3); 13C-
NMR ((CD3)2CO, 75 MHz): δ 177.2 (CSNH), 138.1
(−C=C–), 137.5, 136.5, 131.8, 130.8, 129.9, 128.8, 109.2
(−H–C=C–), 51.8 ((CH3)2–), 30.8 ((CH3)2–C), 20.2
(–C–CH3), 19.6 (2 × Ar–CH3): GC–MS: 260 (M+),
245 (100%), 179, 163, 79. Elemental analysis
1-(4-Bromo-2-fluorophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (3g). Off-white amor-
phous solid; Yield; 80%, Rf: 0.39; m.p: 169–170ꢀC; IR
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Article
Khurshid and Saeed
(neat): 3348 (NH), 3063 (=C–H stretch), 1679 (CS–
NH), 1615 (C = C), 1349 (C–N): H-NMR (CDCL3,
C–), 32.7 ((CH3)2–), 31.9 (−C–CH3). LC–MS (m/z) %:
1
[M + H]+
267.4
(100%).
Elemental
analysis
300 MHz): δ 8.22 (s, 1H, N–H), 7.27–6.23 (m, 5H, Ar–
H), 4.33 (s, 1H, Csp2–H), 1.52 (s, 3H, Csp2–CH3) 1.27
(s, 3H, CH3), 1.24 (s, 3H, CH3); 13C-NMR (CDCL3,
75 MHz): δ 184.6 (CSNH), 136.4 (−C=C–), 136.0,
135.9, 133.8, 129.9, 128.7, 127.3, 111.7 (−H–C=C–),
57.9 ((CH3)2–C–), 34.8 ((CH3)2–), 33.8 (−C–CH3). Ele-
C13H15ClN2S: calc.: C 58.65, H 5.55, N 10.63, S 12.05;
found: C 58.67, H 5.54, N 10.64, S 12.04.
1-(2,3-Dichlorophenyl)-4,4,6-trimethyl-3,4-dihydro-
pyrimidine-2(1H)-thione (3k). Brown crystalline solid;
Yield; 81%, Rf: 0.33; m.p: 130–131ꢀC; IR (neat): 3401
(NH), 3087 (=C–H stretch), 1694 (CS–NH), 1598
mental analysis C13H14BrFN2S: calc.:
C
47.63,
(C = C),
1307
(C–N):
1H-NMR
((CD3)2CO,
H 4.09, N 8.93, S 9.32; found: C 47.66, H 4.06,
N 8.95, S 9.30.
300 MHz): δ 7.99 (s, 1H, N–H), 7.64–7.29 (m, 5H, Ar–
H), 5.04 (q, 1H, J = 1.2 Hz, Csp2–H), 1.52 (d, 3H,
J = 1.2 Hz, Csp2–CH3) 1.44–1.41 (s, 6H, 2 × CH3);
13C-NMR ((CD3)2CO, 75 MHz): δ 183.1 (CSNH),
137.5 (−C=C–), 137.1, 135.9, 132.8, 128.5, 128.1, 127.6,
107.9 (−H–C=C–), 57.1 ((CH3)2–C–), 33.6 (CH3)2–),
32.7 (−C–CH3). GC–MS: 301 (M+), 285, 265 (100%),
249, 226, 185. Elemental analysis C13H14Cl2N2S: calc.:
C 51.95, H 4.56, N 9.33, S 10.65; found: C 51.98,
H 4.53, N 9.31, S 10.63.
1-(2-Chlorophenyl)-4,4,6-trimethyl-3,4-dihydropyri-
midine-2(1H)-thione (3h). White crystalline solid; Yield;
92%, Rf: 0.31; m.p: 123–124ꢀC; IR (neat): 3390 (NH),
3029 (=C–H stretch), 1645 (CS–NH), 1576 (C = C),
1
1297 (C–N): H-NMR (CDCl3, 300 MHz): δ 7.88 (s,
1H, N–H), 7.22–6.45 (m, 5H, Ar–H), 4.25 (s, 1H,
Csp2–H), 1.41 (s, 3H, Csp2–CH3) 1.28 (s, 3H, CH3),
1.24 (s, 3H, CH3); 13C-NMR (CDCL3, 75 MHz): δ
187.5 (CSNH), 138.1 (−C=C–), 137.3, 135.2, 134.7,
131.7, 130.4, 129.8, 110.6 (−H–C=C–), 57.4 ((CH3)2–
C–), 34.2 ((CH3)2–), 33.4 (−C–CH3). Elemental analysis
C13H15ClN2S: calc.: C 58.97, H 5.23, N 10.54, S 12.01;
found: C 58.94, H 5.27, N 10.53, S 12.00.
1-(3-Chloro-4-fluorophenyl)-4,4,6-trimethyl-3,4-
dihydropyrimidine-2(1H)-thione (3l). Yellow amorphous
solid; Yield; 80%, Rf: 0.35; m.p: 102–103ꢀC; IR (neat):
3333 (NH), 3020 (=C–H stretch), 1683 (CS–NH), 1617
(C = C), 1339 (C–N): 1H-NMR (CDCl3, 300 MHz):
δ 7.92 (s, 1H, N–H), 7.25–6.47 (m, 5H, Ar–H), 4.37 (s,
1H, Csp2–H), 1.56 (s, 3H, Csp2–CH3) 1.23 (s, 3H,
CH3), 1.20 (s, 3H, CH3); 13C-NMR (CDCL3,
75 MHz): δ 186.9 (CSNH), 135.9 (−C=C–), 135.5,
134.4, 130.8, 128.9, 127.4, 126.9, 109.2 (−H–C=C–),
57.1 ((CH3)2–C–), 33.8 ((CH3)2–), 32.0 (−C–CH3). LC–
MS (m/z) %: M+ 284.5 (100%). Elemental analysis
C13H14ClFN2S: calc.: C 54.96, H 4.83, N 10.86,
S 10.24; found: C 54.99, H 4.81, N 10.88, S 10.21.
4,4,6-Trimethyl-1-(4-nitrophenyl)-3,4-dihydropry-
midine-2-(1H)-thione (3m). Black amorphous solid;
Yield; 78%, Rf: 0.47; m.p: 158–159ꢀC; IR (neat): 3376
(NH), 3052 (=C–H stretch), 1664 (CS–NH), 1610
(C = C), 1343 (C–N): 1H-NMR (CDCL3, 300 MHz):
δ 8.23 (s, 1H, N–H), 7.94–6.40 (m, 3H, Ar–H), 4.01
(s, 1H, Csp2–H), 1.62 (s, 3H, Csp2–CH3) 1.29 (s, 3H,
CH3), 1.25 (s, 3H, CH3); 13C-NMR (CDCL3,
75 MHz): δ 185.2 (CSNH), 137.1 (−C=C–), 135.5,
133.5, 132.6, 1310.8, 112.7 (−H–C=C–), 107.2
((CH3)2–C–), 53.7 ((CH3)2–C), 32.8 (−C–CH3). Ele-
mental analysis C13H15N3O2S: calc.: C 55.30, H 6.53,
N 15.07, S 10.32; found: C 55.32, H 6.52, N 15.09,
S 10.31.
1-(3-Chlorophenyl)-4,4,6-trimethyl-3,4-dihydropyri-
midine-2(1H)-thione (3i). Yellow crystalline solid; Yield;
84%, Rf: 0.44; m.p: 113–114ꢀC; IR (neat): 3393 (NH),
3011 (=C–H stretch), 1676 (CS–NH), 1579 (C = C),
1
1283 (C–N): H-NMR ((CD3)2CO, 300 MHz): δ 8.06
(s, 1H, N–H), 7.02–6.30 (m, 5H, Ar–H), 4.36 (s, 1H,
Csp2–H), 1.47 (s, 3H, Csp2–CH3) 1.21 (s, 3H, CH3),
1.19 (s, 3H, CH3); 13C-NMR ((CD3)2CO, 75 MHz): δ
188.2 (CSNH), 135.1 (−C=C–), 134.0, 132.9, 131.3,
129.7, 129.4, 128.8, 108.7 (−H–C=C–), 56.2 ((CH3)2–
C–), 34.7 ((CH3)2–), 32.5 (−C–CH3). Elemental analysis
C13H15ClN2S: calc: C 58.67, H 5.53, Cl 13.07, N 10.74,
S 12.00; found: C 58.69, H 5.54, N 10.75, S 12.02.
1-(4-Chlorophenyl)-4,4,6-trimethyl-3,4-dihydropyri-
midine-2(1H)-thione (3j). Brown amorphous solid;
Yield; 90%, Rf:0.42; m.p: 148–149ꢀC; IR (neat): 3397
(NH), 3093 (=C–H stretch), 1685 (CS–NH), 1595
(C = C), 1300 (C–N): 1H-NMR (CDCl3, 300 MHz):
δ 8.11 (s, 1H, N–H), 7.02–6.41 (m, 5H, Ar–H), 4.46 (s,
1H, Csp2–H), 1.55 (s, 3H, Csp2–CH3) 1.25 (s, 3H,
CH3), 1.23 (s, 3H, CH3); 13C-NMR (CDCL3,
75 MHz): δ 185.1 (CSNH), 136.4 (−C=C–), 132.0,
131.7, 129.3, 127.7, 106.7 (−H–C=C–), 53.2 ((CH3)2–
4
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Green Synthesis of Dihydropyrimidine-2-Thiones
H
O
4,4,6-Trimethyl-1-(4-napthalen-2-yl)-3,4-dihydro-
prymidine-2-(1H)-thione (3n). White amorphous solid;
Yield; 75%, Rf: 0.49; m.p: 173–174ꢀC; IR (neat): 3258
(NH), 3029 (=C–H stretch), 1653 (CS–NH), 1601
O
N
C
S
O
N
+
C
H
N
S C
S
(2)
OH
HN
S
H
N
C
O
NH2
N
S
N
+
R
C
N
S
1
(C = C), 1327 (C–N): H-NMR ((CD3)2SO, 300 MHz):
H
OH
δ 9.00 (s, 1H, N–H), 7.99–7.70 (m, 3H, Ar–H),
7.70–7.38 (dd, 4H, J = 9 Hz, Ar–H), 5.02 (s, 1H, Csp2–
H), 1.43–1.28 (3H, Csp2–CH3), 1.43–1.28 (6H,
2 × CH3); 13C-NMR ((CD3)2SO, 75 MHz): δ 184.9
(CSNH), 138.7 (−C=C–), 136.3, 135.2, 131.1, 128.8,
127.6, 125.7, 124.9, 123.8, 122.4, 121.5, 108.7 (−C=C–),
109.5 ((CH3)2–C–), 54.7 ((CH3)2–C), 33.6 (−C–CH3).
Elemental analysis C17H18N2S: calc.: 71.30, H 7.40,
N 9.74, S 11.55; found: C 71.32, H 7.41, N 9.73, S 11.53.
S
H
H
N
S
N
N
-H2O
N
(3a-n)
Scheme 2. Plausible mechanistic pathway to
pyrmidine-2-thiones (3a–n).
every 60 s over 30 min (λex = 350 nm, λem = 455 nm).
Percent inhibition was calculated by averaging the initial
enzyme activities for each inhibitor and normalizing them
against a set of DMSO control wells. The IC50 value of
each inhibitor was determined using plots of the initial
enzyme activity against inhibitor concentration created
using Kaleidagraph. Initial screening was run in dupli-
cate, while IC50 testing was run in triplicate.
LYP inhibition assay
LYP activity and inhibition assays were performed
in black 96-well microplates in a total volume of 100 μL
using a Molecular Devices Spectramax M5 plate reader.
Unless otherwise noted, all solutions were prepared using
a Bis–Tris (50 mM, pH 6.5) buffer containing 100 mM
NaCl and 0.01% Brij35. The pyrimidine thiones and
TCEP (tris(2-carboxyethyl) phsphine) stock solutions
were prepared using dimethylsulfoxide (DMSO). Each
well in the 96-well plate formatted assay contained
3.58 nM LYP, 100 μM TCEP, 5 μM 6,8-difluoro-4-
methylumbelliferyl phosphate (DiFMUP),17 and either
100 μM of inhibitor (initial screen), 5–1000 μM inhibitor
(IC50 determination), or DMSO, resulting in a final
DMSO concentration of 6.1% (v/v). Prior to testing, LYP
was activated with 1 mM TCEP and allowed to incubate
on ice for 30 min. Inhibitor compounds were added and
allowed to incubate for 30 min at room temperature.
Reactions were initiated by the addition of DiFMUP,
and the resulting increase in fluorescence was measured
RESULTS AND DISCUSSION
Equimolar quantities of suitably substituted ani-
lines (1a–n) and potassium thiocyanate were reacted
Initial Screen of Select Compounds
100
80
60
40
20
CH3
CH3
NH2
H3
C
C
H3
C
H
R
N
S-K+
N
R
H3
HN
O
S
CH3
(3a-n)
(1a-n)
(2)
0
R=
3a 2-Me,
3b 4-Me,
3c 2,4-DiMe,
3d 2-MeO,
3e 4-MeO,
3f 4-F,
3h 2-Cl,
3i 3-Cl,
3j 4-Cl,
3k 2,3-DiCl,
3l 3-Cl-4-F,
3m 4-NO2,
3n Napthyl
3a 3c 3d 3e 3g 3h 3i 3j 3k 3l 3m 3n
3g 4-Br-2-F,
Fig. 2. Initial screening of selected compounds in
their efficacy in inhibiting PTPN22. Concen-
tration was held constant at 100 μM for all
compounds.
Scheme 1. Catalyst and solvent-free to 1-aryl-4,4,6-
trimethyl-3,4-dihydropyrimidine-2-(1H)-
thiones (3a–n).
J. Chin. Chem. Soc. 2017
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5

Article
Khurshid and Saeed
1617 cm−1. In the 1H-NMR spectra, the singlet of
thioamide proton lies in the range of 7.71–9.00 ppm. In
some cases, i.e., 3c and 3k, the allylic coupling, giving a
multiplicity pattern of the quartet and doublet with a
coupling constant of 3.0 Hz, was observed. The cou-
pling giving a multiplicity pattern of quartet and dou-
blet with coupling constant of 1.2 Hz was observed
besides other signals for 3c (2,4-dimethyl substituted)
and 3k (2,3-dichloro substituted). However, in some
cases, e.g., 3n (naphthyl derivative), overlapping of sig-
nals was observed for the germinal dimethyl protons
and the protons of methyl at the allylic position.
13C-NMR spectra showed the diagnostic signal
for thiocarbonyl in the range 177.2–188.2 ppm. In LC–
MS, the molecular ion peak appeared in the protonated
mode [M + H+], as well as an adduct of potassium
ions [M + K+].
A plausible mechanistic pathway is depicted in
Scheme 2. Accordingly, the conjugate addition of thio-
cyanic acid formed by the reaction of potassium thiocy-
anate with traces of Brønsted acid to mesityl oxide
afforded the isothiocyanate species. The latter, on
attack by substituted anilines through 1,2-addition fol-
lowed by intramolecular dehydrative cyclization, leads
to the target compounds (3a–n). The thiocarbonyl–thiol
tautomerism exhibited by these compounds offer the
possibility of further substitution on the sulfur, leading
to a variety of heterocycles.
Fig. 3. IC50 curves for compound (a) 3d (IC50 = 18
Æ 1 μM), (b) 3l (IC50 = 69 Æ 3 μM), (c) 3c
(IC50 = 45 Æ 3 μM), (d) 3g (IC50 = 119 Æ 6
μM), and (e) 3n (IC50 = 219 Æ 50 μM).
LYP inhibitor screening and SAR
The increase in fluorescence from the enzymatic turn-
over of DiFMUP is an established method of measuring
PTP activity. Several of the synthesized compounds were
initially screened at a concentration of 100 μM to test their
efficacy in inhibiting the PTP LYP (Figure 2). Compounds
3a and 3c differ only in the position of one methyl group;
yet they show a significant difference in their ability to
inhibit PTPN22. This suggests that the substituent at the 4-
position contributes to inhibitor activity, which is supported
by the fact that four of the top five compounds identified
all contain a group in the 4-position. Interestingly, the top
hit lacks a group at the 4-position but contains only a meth-
oxy group at the 2-position.
with a slight excess of 4-methylpent-3-en-2-one (2) in
the presence of a catalytic amount of concentrated HCl
at room temperature, as depicted in Scheme 1. The
reaction mixture was heated at 50–60ꢀC for 3–5 h, and
the crude products were recrystallized from ethanol to
afford the dihydropyrimidine-2-thiones (3a–n) in high
purity and good to excellent yields (Scheme 1).
The structures of the products 3a–n were substan-
tiated through spectral data. Fourier transform infrared
(FT-IR), 1H-NMR, 13C-NMR, LC-MS, and CHNS
elemental analyses gave satisfactory analytical and
spectroscopic data. FT-IR spectra displayed a charac-
teristic stretching vibration for the thioamide function-
ality in a range 1575–1700 cm−1 besides the stretching
frequency of olefinic bond between 1523 and
Compounds 3c, 3d, 3g, 3l, and 3n all showed inhi-
bition of >50% at a concentration of 100 μM, and were
further screened to identify their IC50 values. Results
are
shown
in
Figure
3.
Compound
3c
6
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Green Synthesis of Dihydropyrimidine-2-Thiones
7. T. U. Mayer, T. M. Kapoor, S. J. Haggarty, R. W. King,
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(IC50 = 45 Æ 3 μM) was 1.5 times more potent than
compound 3l (IC50 = 69 Æ 3 μM). Compound 3d, the
best hit from the initial screening, had an IC50 value of
18 Æ 1, which is 2.5 times more potent than the second
most potent compound (3c). Compounds 3g and 3n had
IC50 values of 119 Æ 6 and 219 Æ 50 μM, respectively.
These experimentally determined IC50 values are in rea-
sonable agreement with the initial screening data.
CONCLUSION
A series of 14 substituted dihydropyrimidine-2-
thione derivatives were synthesized under metal-cata-
lyst- and solvent-free conditions in good to excellent
yields. Several of these compounds inhibit PTPN22
activity in the mid-micromolar range, with the best hit
being compound 3d (IC50 = 18 Æ 1 μM). Based on
these results, preliminary structure–activity relation-
ships (SARs) were studied, which indicated that these
compounds could be promising leads for the future
development of more potent inhibitors of PTPN22.
17. S. Cohen, H. Dadi, E. Shaoul, N. Sharfe,
C. M. Roifman, Blood 1995, 93, 2013.
ACKNOWLEDGMENTS
18. A. Alonso, J. Sasin, N. Bottini, I. Friedberg, I. Friedberg,
A. Osterman, A. Godzik, T. Hunter, J. Dixon,
T. Mustelin, Cell 2004, 117, 699.
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5, 43.
21. E. Fiorillo, V. Orrú, S. M. Stanford, Y. Liu, M. Salek,
N. Rapini, A. D. Schenone, P. Saccucci, L. G. Delogu,
F. Angelini, M. L. M. Bitti, C. Schmedt, A. C. Chan,
O. Acuto, N. Bottini, J. Biol. Chem. 2010, 285, 26506.
22. A. Veillette, I. Rhee, C. M. Souza, D. Davidson, Immu-
nol. Rev. 2009, 228, 312.
We gratefully acknowledge Dr. Amy Barrios and
Brandon McCullough, University of Utah College of Phar-
macy, USA, for assistance with the PTPN22 screening
assay.
Supporting information
Additional supporting information (Appendix S1)
is available in the online version of this article.
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