Synthesis and evaluation of thiouracil derivatives as dipeptidyl peptidase iv inhibitors

Article abstract of DOI:10.1111/cbdd.12070

A series of thiouracil derivatives were designed, synthesized and screened for in vitro inhibition of dipeptidyl peptidase IV. The SAR study indicated the influence of substituted chemical modifications on thiouracil scaffold. Compounds 8 (IC₅₀

Full text of DOI:10.1111/cbdd.12070

Chem Biol Drug Des 2013; 81: 257–264
Research Article
Synthesis and Evaluation of Thiouracil Derivatives as
Dipeptidyl Peptidase IV Inhibitors
as novel class of antidiabetic drugs, which prevent proteo-
lytic degradation of incretin hormones and stimulate glu-
cose-dependent insulin secretion (8).
Mani Sharma*, Divya Singh and Monica Gupta*
Department of Pharmaceutical Chemistry, Delhi Institute of
Pharmaceutical Sciences and Research, Pushp Vihar,
Sector-3, M B Road, New Delhi 110017, India
*Corresponding author: Mani Sharma and Monica Gupta,
mani120484@gmail.com
Pyrimidine and related fused heterocyclic derivatives, such
as purines (9), aminomethylpyrimidines (10,11), pyridyl
acetamide (12), pyridopyrimidines (13), phenylethylamines
(14), quinoxaline diones (15), quinazolinone (16), pyrimidi-
none, and pyrimidinedione (17), are well-known pharmaco-
phores in drug discovery and are also responsible for
providing series of potent DPP IV inhibitors (Figure 1). The
earlier studies show that conserving heterocyclic integrity
of the pharmacophore may establish a magnetic strategy
for development of DPP IV inhibitors with desirable bioac-
tivity. The extended investigation on heterobicyclic systems
led us to examine thiouracil derivatives against DPP IV
inhibitory activity. Thus, in an attempt to establish new
therapeutic agents, we designed a series of substituted
thiouracil derivatives as structurally modified and targeted
DPP IV inhibitors. Our interest in the synthesis of heterocy-
clic core as thiouracil made us to study the stabilized inter-
actions that may happen to occur with conserved features
of the core. We envisioned that targeted substitution on
the pharmacophore can prove to be an attractive strategy
for synthesis of DPP IV inhibitors with significant bioactivity.
In an effort to find more targeted compounds, we focused
on affinity of substituted thiouracil derivatives, which may
be responsible for DPP IV inhibition. However, the
designed heterocycle core revealed the close resemblance
with various available DPP IV inhibitors (Figure 1) (18,19).
A series of thiouracil derivatives were designed, syn-
thesized and screened for in vitro inhibition of dipept-
idyl peptidase IV. The SAR study indicated the
influence of substituted chemical modifications on
thiouracil scaffold. Compounds 8 (IC50 = 0.32 lM), 9
(IC50 = 0.29 lM), and 12 (IC50 = 0.25 lM) showed excel-
lent dipeptidyl peptidase IV inhibition having heterocy-
clic substituted piperazine with acetamide linker
resulted as most potent dipeptidyl peptidase IV inhibi-
tors among all the compounds screened. Single dose
(10 mg ⁄ kg) of the compounds 8, 9, and 12 significantly
reduced glucose excursion during oral glucose toler-
ance test in streptozotocin-induced diabetic rat model.
The present study on substituted thiouracil derivatives
shows good-to-moderate inhibitory potential of dipept-
idyl peptidase IV enzyme.
Key words: diabetes, DPP IV, microwave synthesis, thiouracil
Received 12 July 2012, revised 31 August 2012 and
accepted for publication 2 October 2012
Incretin hormones represent a promising approach for
treatment of type 2 diabetes. Glucagon-like peptide 1
(GLP-1) and glucose-dependent insulinotropic polypeptide
(GIP) are incretin hormones responsible for 50–70% of
postprandial insulin release from pancreatic beta cells (1).
Chronic infusion of GLP-1 has significantly improved both
blood glucose and hemoglobin A1c (HbA1c) level in
patients with T2D (2). Additionally, GLP-1 also plays bene-
ficial role in treatment of diabetes by regulating various
metabolic changes as improving insulin biosynthesis, inhib-
iting glucagon secretion, reducing food intake, slow gastric
emptying time, and feeling of satiety (3). However, its ther-
apeutic efficacy is limited due to short half-life and rapid
degradation in plasma by serine protease dipeptidyl pepti-
dase IV (DPP IV) (4,5). Inhibition of DPP IV will result in
regulating level of endogenous intact incretin hormones by
extending its half-life, consequently potentiating insulin
secretion (6,7). Therefore, DPP IV inhibitors have emerged
Herein, we report proposed methodology with the result in
respect to the synthesis, SAR of synthesized thiouracil
derivatives across the pharmacophore. The synthetic com-
pounds have been investigated by in vitro DPP IV inhibition
and in vivo antihyperglycemic activity.
Methods and Materials
Experimental
All commercial chemicals and solvents were purchased
from Sigma-Aldrich Chemical Company, St. Louis, USA and
Spectrochem Pvt. Ltd (USA), India. Chemicals and solvents
were used as such without further purification. 1H spectra
were recorded on a Bruker NMR spectrometer operating at
300 MHz in CDCl3 or DMSO-d6 with tetramethylsilane as an
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12070
257

Sharma et al.
Figure 1: Dipeptidyl Peptidase IV
inhibitors I11, II10, III12b, IV.
internal standard. The Chemical shifts are reported as parts
per million (ppm, d), and signals are reported as s (singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). All the Fou-
rier transform infrared (FT-IR) spectra were recorded on Jas-
co-410 spectrophotometer. Elemental analysis was carried
out using Vario EL III C, H, N elemental analyzer, and the
values were found within acceptable limits. Melting points of
compounds were determined in open capillary tubes using
a Hicon melting point instrument and are uncorrected.
Microwave-assisted synthetic reactions were performed
using a CEM Discover Benchmate microwave reactor (CEM
Microwave technology ltd., Buckingham). All reactions were
carried out under dried condition and performed using
oven-dried glassware.
(3.33 mol) followed immediately by stirring under reflux
for 2 h then at room temperature for 30 min. The pro-
gress of reaction was continuously monitored by thin-
layer chromatography (TLC) (Developing solvent: chloro-
from ⁄ methanol = 9.5:0.5). The reaction solution was con-
centrated under reduced pressure, and the resulting
residue was neutralized with saturated bicarbonate solu-
tion. The resulting residue was diluted with water and
extracted with chloroform. The chloroform layer was
washed with water and brine and dried over anhydrous
sodium sulfate. The organic layers were evaporated to
dryness under reduced pressure using rotary evaporator.
The resultant intermediates (2–7) were obtained in excel-
lent yields.
Synthetic procedures
2-chloro-1-[4-(pyrimidin-2-yl)piperazin-1-
yl]ethanone (2)
Step I: synthesis of 4-hydroxy-6-phenyl-2-
sulfanylpyrimidine-5-carbonitrile (1)
Yellow oil; yield: 87%; Rf: 0.71; 1H NMR (300 MHz,
CDCl3) d ppm: 3.70–3.64 (m, 4H), 3.91–3.73 (m, 4H),
4.28 (s, 2H), 6.66 (t, J = 9, 1H), 8.38 (d, J = 9, 2H);
The compound 1 is prepared by the previously reported
procedure (20) obtained as white solid after neutralization
with 1 M hydrochloric acid. Resultant compound identity
was confirmed by further characterization. White solid;
yield: 93%; mp: 145–149 ꢀC; Rf: 0.65; 1H NMR (300MHz,
CDCl3) d ppm: 7.43 (t, J = 15, 1H), 7.55 (d, J = 9, 2H),
7.94 (d, J = 9, 2H), 8.18 (s, 1H); FT-IR (mmax; per cm,
KBr): 2954 (C-Hstretch), 1628 (C=N), 1549 (C=C ringstretch),
1453 (C-N), 1247 (C-Sstretch), 1153 (C-O), 765 (Phenyl);
Anal. Calcd. For C11H7N3OS: C, 57.63; H, 3.08; N, 18.33;
found: C, 57.65; H, 3.05; N, 18.39.
FT-IR (mmax
(C=O), 1493 (C=N), 640 (C-Clstretch); Anal. Calcd. For
10H13ClN4O: C, 49.90; H, 5.44; N, 23.28; found: C,
; per cm, neat): 2931 (C-Hstretch), 1629
C
49.94; H, 5.39; N, 23.24.
2-chloro-1-[4-(pyridin-2-yl)piperazin-
1-yl]ethanone (3)
1
Brown oil; yield: 87%; Rf: 0.70; H NMR (300 MHz, CDCl3)
d ppm: 3.71–3.66 (m, 4H), 3.91–3.73 (m, 4H), 4.43 (s,
2H), 6.73 (t, J = 12 Hz, 1H), 6.99 (d, J = 9, 1H), 7.71 (t,
J = 15.3, 1H), 8.17 (s, 1H); FT-IR (mmax; per cm, neat):
3004 (C-Hstretch), 2920 (C-Hstretch), 1647 (C=O), 1541
(C=N), 775 (C-Clstretch); Anal. Calcd. For C11H14ClN3O: C,
55.12; H, 5.89; N, 17.53; found: C, 55.17; H, 5.85; N,
17.58.
Step II: General procedure for synthesis of N-
substituted amides (2–7)
To a solution of secondary amine (2.9 mol) in 1, 4-
dioxan (10 mL) was added chloroacetyl chloride
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Thiouracil Derivatives as Potent DPP IV Inhibitors
2-chloro-1-[4-(4-chlorophenyl)piperazin-1-
yl]ethanone (4)
Yellow solid; yield: 84%; mp: 129–133 ꢀC; Rf: 0.69; 1H
NMR (300 MHz, CDCl3) d ppm: 3.23–3.15 (m, 4H),
3.72–3.68 (m, 4H), 4.12 (s, 2H), 6.88 (d, J = 6 Hz, 2H),
7.28 (d, J = 6 Hz, 2H); FT-IR (mmax; per cm, KBr): 2917 (C-
were further concentrated to the dryness using rotary
evaporator. Compounds obtained as solid were recrystal-
lized using appropriate solvent, and the oily compounds
were purified by column chromatography (5% MeOH in
DCM). The final desired synthetic compounds were
obtained in good yields.
H
stretch), 1594 (C=O), 1497 (C=C ringstretch), 667 (Ar-
Clstretch), 624 (C-Clstretch); Anal. Calcd. For C12H14Cl2N2O:
C, 52.76; H, 5.17; N, 10.26; found: C, 52.79; H, 5.19; N,
10.29.
2-{2-[4-(4-Chloro-phenyl)-piperazin-1-yl]-2-oxo-
ethylsulfanyl}-4-hydroxy-6-phenyl-pyrimidine-5-
carbonitrile (8)
Yellow solid; yield: 89%; mp: 155–158 ꢀC; Rf: 0.62; 1H
NMR (300 MHz, CDCl3) d ppm: 3.1–3.15 (m, 4H), 3.62–
3.69 (m, 4H), 4.13 (s, 2H), 6.79–6.82 (d, J = 9, 2H), 7.17–
7.20 (d, J = 9, 2H), 7.39–7.42 (t, J = 9, 2H), 8.28–8.30 (d,
J = 6, 1H), 8.69–8.71 (d, J = 6, 2H); FT-IR (mmax; per cm,
KBr): 2922 (C-Hstretch), 1727 (C=O), 1632 (C=N), 1496
(C=C ringstretch), 1454 (C-N), 1232 (C-Sstretch), 640 (Ar-
Clstretch), 764 (Phenyl); Anal. Calcd. For C21H18ClN7O2S:
C, 53.90; H, 3.88; N, 20.95; found: C, 53.87; H, 3.91; N,
20.93
1-(1,4’-bipiperidin-1’-yl)-2-chloroethanone (5)
Yellow oil; yield: 64%; Rf: 0.65; 1H NMR (300 MHz,
CDCl3) d ppm : 1.15–1.20 (t, J = 15, 2H), 1.5–1.7 (m,
4H), 2.15–2.26 (m, 4H), 2.78–2.86 (m, 4H), 3.06–3.11
(m, 4H), 5.02 (s, 2H), 5.19–5.30 (m, 1H);); FT-IR (mmax
;
per cm, neat): 2940 (C-Hstretch), 1645 (C=O), 1150 (C-
Nstretch), 659 (C-Clstretch); Anal. Calcd. For C12H21ClN2O:
C, 58.89; H, 8.65; N, 11.45; found: C, 58.92; H, 8.63;
N, 11.47.
4-hydroxy-2-({2-oxo-2-[4-(pyridin-2-yl)piperazin-
1-yl]ethyl}sulfanyl)-6-phenylpyrimidine-5-
carbonitrile (9)
1-(4-aminopiperidin-1-yl)-2-chloroethanone (6)
Yellow oil; yield: 75%; Rf: 0.69; 1H NMR (300MHz, CDCl3)
d ppm: 2.00 (q, J = 12, 4H), 3.2 (t, J = 9, 4H), 4.46 (d,
J = 12, 2H), 4.8 (s, 2H); FT-IR (mmax; per cm, neat): 2936
(C-Hstretch), 1649 (C=O), 1169 (C-Nstretch), 654 (C-Clstretch);
Anal. Calcd. For C7H13ClN2O: C, 47.60; H, 7.42; N,
15.86; found: C, 47.62; H, 7.45; N, 15.89.
Light brown solid; yield: 75%; mp: 151–153 ꢀC; Rf: 0.62;
1H NMR (300 MHz, CDCl3) d ppm: 3.42–3.57 (m, 8H),
5.47 (s, 2H), 6.65–6.69 (t, J = 12, 2H), 6.83–6.86 (d,
J = 9, 2H), 7.32–7.35 (d, J = 9, 2H), 7.54–7.58 (t, J = 12,
2H), 8.12–8.13 (d, J = 3, 1H); FT-IR (mmax; per cm, KBr):
2919 (C-Hstretch), 1725 (C=N), 1638 (C=O), 1561 (C=C
ringstretch), 1478 (C-N), 1232 (C-Sstretch), 772 (Phenyl);
Anal. Calcd. For C22H20N6O2S: C, 61.10; H, 4.66; N,
19.43; found: C, 61.07; H, 4.69; N, 19.40.
1-(azepan-1-yl)-2-chloroethanone (7)
Light brown oil; yield: 78%; Rf: 0.69; 1H NMR (300 MHz,
CDCl3) d ppm: 1.79–1.73 (m, 4H), 1.87–1.82 (m, 4H),
3.58–3.42 (m, 4H), 4.11 (s, 2H); FT-IR (mmax; per cm, neat):
1647 (C=O), 1099 (C-Nstretch), 651 (C-Clstretch); Anal.
Calcd. For C8H14ClNO: C, 54.70; H, 8.03; N, 7.97; found:
C, 54.74; H, 8.05; N, 7.99.
4-hydroxy-2-({2-oxo-2-[4-(pyrimidin-2-yl)piperazin-
1-yl]ethyl}sulfanyl)-6-phenylpyrimidine-5-
carbonitrile (10)
Yellow solid; yield: 79%; mp: 152–154 ꢀC; Rf: 0.67; 1H
NMR (300 MHz, CDCl3) d ppm: 3.4–3.6 (m, 8H), 5.47 (s,
2H), 6.67–6.65 (t, J = 6, 1H), 6.80–6.86 (t, J = 18, 2H),
7.32–7.35 (d, J = 9, 2H), 7.44 (t, J = 6, 1H), 8.12–8.13 (d,
J = 3, 2H); FT-IR (mmax; per cm, KBr): 2923 (C-Hstretch),
1631 (C=N), 1585 (C=O), 1549 (C=C ringstretch), 1442 (C-
N), 1230 (C-Sstretch), 765 (Phenyl); Anal. Calcd. For
C21H19N7O2S: C, 58.19; H, 4.42; N, 22.62; found: C,
58.21; H, 4.45; N, 22.59.
General procedures for synthesis of 8–14
A solution of 4-hydroxy-6-phenyl-2-sulfanylpyrimidine-5-
carbonitrile 1 (0.43 mol), diisopropylethylamine (0.86 mol)
in dry tetrahydrofuran was placed in a 10-mL microwave
vial. A solution of N-substituted acetamide ⁄ alkyl chloride
(0.43 mol) in dry tetrahydrofuran (3 mL) was then added to
the reaction mixture and subjected to microwave irradia-
tion for 10 min at 120 ꢀC. The completion of reaction was
optimized and confirmed through TLC (Developing solvent:
ethyl acetate: hexane = 2:8) visualized under iodine
vapors. The reaction solution was concentrated under
reduced pressure. The resulting mixture was diluted with
cold water and extracted with chloroform. The organic
layer was washed with water and brine. The extraction
was repeated twice, and combined organic layers were
dried over anhydrous sodium sulfate. The organic layers
2-({2-[1-(4’-bipiperidinyl)-1’-yl]-2-oxoethyl}sulfanyl)-
4-hydroxy-6-phenylpyrimidine-5-carbonitrile (11)
Yellow solid; yield: 65%; mp: 147–149 ꢀC; Rf: 0.62; 1H
NMR (300 MHz, CDCl3) d ppm: 1.15–1.20 (t, J = 15, 2H),
1.5–1.7 (m, 4H), 2.15–2.26 (m, 4H), 2.78–2.86 (m, 4H),
3.06–3.11 (m, 4H), 5.02 (s, 2H), 5.19–5.30 (m, 1H) 7.21–
7.33 (m, 3H), 7.69–7.71 (d, J = 6, 2H); FT-IR (mmax; per
Chem Biol Drug Des 2013; 81: 257–264
259

Sharma et al.
cm, KBr): 2939 (C-Hstretch), 1627 (C=N), 1542 (C=C ring-
stretch), 1449 (C-N), 1241 (C-Sstretch), 764 (Phenyl); Anal.
Calcd. For C23H27N5O2S: C, 63.13; H, 6.22; N, 16.01;
found: C, 63.11; H, 6.25; N, 16.04.
4-hydroxy-2-[(oxiran-2-ylmethyl)sulfanyl]-6-
phenylpyrimidine-5-carbonitrile (14)
Yellow solid; yield: 68%; mp: 149–151 ꢀC; Rf: 0.65; 1H
NMR (300 MHz, CDCl3) d ppm: 2.11–2.18 (q, J = 21, 2H),
3.68–3.79 (m, 1H), 4.99–5.02 (d, J = 9, 2H), 5.23–5.30
(m, 2H), 7.26–7.33 (m, 3H), 7.86–7.88 (t, J = 6, 2H); FT-IR
(mmax; per cm, KBr): 2930 (C-Hstretch), 1725 (C=O), 1547
(C=C ringstretch), 1453 (C-N), 1240 (C-Sstretch), 764 (Phe-
nyl); Anal. Calcd. For C14H11N3O2S: C, 58.93; H, 3.89; N,
14.73; found: C, 58.90; H, 3.91; N, 14.75.
2-{[2-(4-aminopiperidin-1-yl)-2-oxoethyl]sulfanyl}-
4-hydroxy-6-phenylpyrimidine-5-carbonitrile (12)
Yellow solid; yield: 79%; mp: 145–148 ꢀC; Rf: 0.59; 1H
NMR (300 MHz, CDCl3) d ppm: 1.62 (m, 2H), 2.02 (m,
2H), 2.35 (m, 1H), 3.16 (d, J = 3, 2H), 3.30–3.31 (t, J = 3,
2H), 3.78 (s, 2H), 6.83–6.85 (t, J = 6, 3H), 7.49 (m, 2H);
FT-IR (mmax; per cm, KBr): 3430 (N-Hstretch), 2932 (C-
Pharmacological activity
H
stretch), 1631 (C=O), 1543 (C=C ringstretch), 1459 (C-N),
1248 (C-Sstretch), 712 (Phenyl); Anal. Calcd. For
18H19N5O2S: C, 58.52; H, 5.18; N, 18.96; found: C,
The Institutional Animal Ethical Committee (IAEC) approved
the experiments for oral glucose tolerance test (OGTT).
Healthy Wistar rats used for the study were obtained from
Animal House of Delhi Institute of Pharmaceutical Sciences
and Research. Rats were housed in colony cages (three
female and one male rats per cage for breeding), at an
ambient temperature of 25 ꢀC with 12-h light ⁄ 12-h dark
cycle. Animals were kept in polypropylene cages and were
fed with rat food as pellets and water ad libitum. All experi-
mental procedures were performed according to IAEC (Pro-
tocol No.1 ⁄ DIPSAR ⁄ IAEC ⁄ 2010) and CPCSEA guidelines.
C
58.49; H, 5.20; N, 18.91.
2-{[2-(azepan-1-yl)-2-oxoethyl]sulfanyl}-4-hydroxy-
6-phenylpyrimidine-5-carbonitrile (13)
Off white solid; yield: 70%; mp: 152–155 ꢀC; Rf: 0.62; 1H
NMR (300 MHz, CDCl3) d ppm: 1.25 (m, 4H), 2.36 (m,
4H), 3.36–3.41 (t, J = 15, 4H), 3.76 (s, 2H), 6.85 (m, 3H),
7.49 (m, 2H); FT-IR (mmax; per cm, KBr): 2930 (C-Hstretch),
1717 (C=O), 1623 (C=N), 1566 (C=C ringstretch), 1447 (C-
N), 1242 (C-Sstretch), 765 (Phenyl); Anal. Calcd. For
Induction of experimental diabetes
C
19H20N4O2S: C, 61.94; H, 5.47; N, 15.21; found: C,
The experimental diabetes was induced using freshly pre-
pared solution of streptozotocin (STZ; Sigma-Aldrich) at the
61.96; H, 5.45; N, 15.19.
SH
O
H
S
OEt
N
N
NC
a
NH2
H2N
+
+
O
OH
CN
1
O
O
b
R2
H
Cl
Cl
R2
+
c
Cl
- HCl
(a-f)
d
2-7
R2
R1
S
S
O
N
N
N
N
OH
OH
CN
CN
8-13
14
, R2 = 4 - aminopiperidinyl
2
2
12
(4-chlorophenyl)piperazin - 1 -yl
(pyridin-2-yl)piperazin -1-yl
, R =
(pyrimidyl-2-yl)piperazin-1-yl
1,4'-bipiperidynyl
8
9
, R =
10
, R2 =
2
, R2 =
azepanyl
, R =
13
14
11
1
(oxirane-2-yl)methyl
, R =
Scheme 1: Reagents and Conditions: (A) potassium carbonate, ethanol, reflux, 6 h, (B) H-R2 (A–F) secondary amines, diisopropylethyl-
amine, 1,4-dioxan, reflux, 2 h, (C) 2–7, dry tetrahydrofuran, MW, 10 min, 120 ꢀC, (d) RCl, dry tetrahydrofuran, MW, 10 min, 120 ꢀC.
260
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Thiouracil Derivatives as Potent DPP IV Inhibitors
Table 1: Dipeptidyl peptidase IV inhibitory effect of thiouracil
derivatives 1, 8–14
dose level of 90 mg ⁄ kg (I.P.) in 0.1 M freshly prepared cit-
rate buffer pH 4.5 to 2-day-old neonatal pups (21,22). A
total of 30 diabetic and six non-diabetic male rats were
used in the study. After 6 weeks of injection, animals were
screened for fasting blood glucose level. Diabetic rats were
further divided into four groups with six rats per group.
Animals showing fasting glucose levels >150 mg ⁄ dL were
considered diabetic and selected for screening of com-
pounds.
R
S
N
N
OH
CN
Compound
R
IC50 (lM)a
1
29.19
Antihyperglycemic activity
H
Male Wistar rats weighing 150–200 g having blood glu-
cose level >150 mg ⁄ dL were selected for OGTT. Animals
of diabetic control group received oral administration of
vehicle (0.5% w ⁄ v methylcellulose), and standard group
received vildagliptin (10 mg ⁄ kg). Animals of experimental
group were administered suspension of desired synthetic
compound orally (made in 0.5% w ⁄ v methylcellulose) at a
single dose of 10 mg ⁄ kg body weight (9,23). A glucose
load (2 g ⁄ kg) was given orally to overnight fasting rats after
30 min prior to administration of the test compound ⁄ vehi-
cle. Blood samples were taken from the tail vein at regular
intervals of 0, 30, 60, 90, 120 min after glucose load. The
blood glucose level was monitored using glucometer
(ACCU-CHEK, Active; Roche Diagonistics, Mannheim,
Germany).
Cl
N
8
0.32
N
O
N
N
N
N
9
0.29
1.03
N
N
N
O
O
O
N
N
10
11
10.38
Statistical evaluation
Statistical evaluation was performed by one-way ANOVA fol-
lowed by Dunnett’s post-test and expressed as mean
-
H2N
SEM. IC50 values were determined using nonlinear
regression analysis and expressed as results obtained
from three independent experiments. Statistical studies
and data analyses were performed using GRAPHPAD PRISM
Version 5.0 (San Diego, CA, USA).
12
0.25
4.33
33.79
2.5
N
O
N
13
O
In vitro DPP IV enzyme inhibition assay
O
All the synthesized compounds were evaluated for in vi-
tro DPP IV enzyme inhibition. The enzyme assay was
performed using a DPP IV drug discovery kit (BML-AK
499; Enzo Life Sciences, Pennsylvania, USA). The activ-
ity of test compounds was assayed using human
recombinant DPP IV enzyme, chromogenic substrate (H-
Gly-Pro-AMC, Km 114 lM), DPP IV inhibitor (P32 ⁄ 98),
assay buffer, and calibration standard as provided in the
kit. The assay was performed using 96-well flat-bot-
tomed microtiter plate followed by addition of assay buf-
fer, DPP IV enzyme, and chromogenic substrate (HGly-
Pro-pNA). The assay principle and procedure were fol-
lowed as per a manufacturer’s guidelines. Solutions of
the test compounds were made in dimethyl sulfoxide
(DMSO) at different concentrations of 25, 50, 100,
200 lg ⁄ mL, and 20 lL were added to each well after
further dilutions. The plate was incubated at 37 ꢀC for
10 min to allow enzyme-inhibitor reaction and read con-
14
O
H3C
P 32 ⁄ 98
H3C
N
S
NH2
OH
O
NH
Vildagliptin
<0.05
N
NC
aIC50 represents inhibitory concentration determined by nonlinear
regression analysis using GRAPHPAD PRISM software. Values are
expressed as mean of three independent experiments.
tinuously at A-405 nm using Bio-Rad Elisa Plate Reader
Philadelphia, PA, USA.
Chem Biol Drug Des 2013; 81: 257–264
261

Sharma et al.
ment for the series are reported as their micromolar inhibi-
tory concentration, IC50 (lM). With respect to in vitro DPP
IV inhibition as shown in Table 1, almost all the com-
pounds showed good-to-moderate response against DPP
IV inhibition. Among the synthesized analogs, parent com-
pound 1 (IC50 = 29.2 lM) showed weak inhibitory activity.
The in vitro study indicated that different substitutions over
SH group of thiouracil ring by N-substituted acetamide
derivatives exerted variable DPP IV inhibitory activity. The
N-substituted acetamide derivatives (2–7) were synthe-
sized using secondary amines and chloroacetyl chloride
and have been explored due to their substrate specificity
for DPP IV inhibition (24). Significant inhibition was
Results and Discussion
Chemistry
A convergent synthesis was designed for synthesis of
targeted compounds as outlined in Scheme 1. 4-hydroxy-
6-phenyl-2-sulfanylpyrimidine-5-carbonitrile 1 was synthe-
sized by cyclo-condensation of benzaldehyde with thiourea
and ethylcyanoacetate according to the reported procedure
(20). The second part of the scheme included synthesis of
N-substituted acetamide derivatives 2–7 as versatile inter-
mediate. The synthesis of N-substituted acetamide deriva-
tives was accomplished by reaction of various secondary
amines with chloroacetyl chloride by nucleophilic substitu-
tion of chlorine by amines. Condensation of both parts was
carried out via S-alkylation on heterocyclic core under
microwave irradiation to obtain the targeted compounds 8–
14 (Scheme 1). The structures of the titled analogs were
confirmed by spectral analysis.
recorded with 4-chlorophenyl piperazine derivative
8
(IC50 = 0.32 lM). It was noted that the presence of elec-
tron withdrawing chloro group at 4-position of phenyl ring
may be responsible for enhancement of activity. This indi-
cation prompted us to prepare pyridyl piperazine derivative
9 (IC50 = 0.29 lM) having an electron-deficient aromatic
pyridyl moiety, leading the most potent compound of ser-
ies. The replacement by the pyridyl piperazine moiety
resulted in a more potent inhibitor than the replacement by
the pyrimidyl piperazine moiety 10 (IC50 = 1.03 lM). No
considerable difference in activity was seen with 4-piperidi-
no piperidine derivative 11 (IC50 = 10.38 lM) with the
Biological activity
In vitro DPP IV inhibitory activity
All the synthesized compounds 8–14 were investigated for
DPP IV inhibition, and the results of initial SAR develop-
A
Figure 2. Effect of compounds 8,
9 and 12 on blood glucose level
in an oral glucose tolerance test
(OGTT) using streptozotocin-
induced diabetic rats. The test
compounds were given orally
(10 mg ⁄ kg), vildagliptin (standard,
10 mg ⁄ kg) and 0.5% CMC (dia-
betic control) followed by oral glu-
cose challenge (2 g ⁄ kg) after
30 min: (A) time–course of
B
changes in blood glucose level
and (B) area under the blood glu-
cose concentration–time curve
(AUC) for the period of 0–2 h dur-
ing OGTT. Data are expressed as
mean SEM for six animals in
each group. ***p < 0.0001 versus
diabetic group.
262
Chem Biol Drug Des 2013; 81: 257–264

Thiouracil Derivatives as Potent DPP IV Inhibitors
exception of incorporation of substituted 4-aminopiperidine
ring 12 (IC50 = 0.25 lM). The slight retention in activity was
encountered with seven membered ring analog, azepenyl
derivative 13 (IC50 = 4.33 lM) may be tolerated as substi-
tution. The epichlorohydrin derivative 14 (IC50 = 33.79 lM)
displays marked decrease in activity, which suggested that
removal of the carbonyl group was unfavorable for DPP IV
inhibition. Analysis of structural activity relationship of syn-
thesized derivatives revealed that replacement of SH by N-
substituted acetamide derivatives effectively influences the
DPP IV inhibitory activity. These results indicated that
effect of different cyclic analogs did not produce much
impact with regards to DPP IV inhibitory effect. However,
presence of the carbonyl oxygen of an acetamide linker is
necessary for DPP IV inhibition as evidenced by synthetic
compounds 8, 9, and 12.
Acknowledgment
The funding of this study was supported by Govt. of NCT
Delhi, India. The authors acknowledge Novartis Switzer-
land for providing vildagliptin.
Conflict of Interest
None.
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