A novel synthesis and preliminary in vitro cytotoxic evaluation of dihydropyrimidine-2,4(1H, 3 H)-dione derivatives

Article abstract of DOI:10.1007/s12039-017-1223-4

We report the synthesis and characterization of four new compounds: 6-(2-(1H-inden-2-yl) phenyl)-3-(2-morpholinoethyl)dihydropyrimidine-2,4(1H,3H)-dione (13a), 6-(2-(1H-inden-2-yl)phenyl)-3-(2- (pyrrolidin-1-yl)ethyl)dihydropyrimidine-2,4(1H,3H)-dione (13

Full text of DOI:10.1007/s12039-017-1223-4

c
J. Chem. Sci. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-017-1223-4
REGULAR ARTICLE
A novel synthesis and preliminary in vitro cytotoxic evaluation
of dihydropyrimidine-2,4(1H,3H)-dione derivatives
V UDAYAKUMARa,∗, J GOWSIKAb and A PANDURANGANa
aDepartment of Chemistry, Anna University, Guindy, Chennai 600 025, India
bDepartment of Chemistry, Pachaiyappa’s College, University of Madras, Chennai 600 030, India
Email: udaya_taurus@yahoo.co.in
MS received 8 September 2016; revised 1 October 2016; accepted 25 December 2016
Abstract. We report the synthesis and characterization of four new compounds: 6-(2-(1H-inden-2-yl)
phenyl)-3-(2-morpholinoethyl)dihydropyrimidine-2,4(1H,3H)-dione (13a), 6-(2-(1H-inden-2-yl)phenyl)-3-(2-
(pyrrolidin-1-yl)ethyl)dihydropyrimidine-2,4(1H,3H)-dione (13b), 6-(2-(1H-inden-2-yl)phenyl)-3-phenethyl-
dihydropyrimidine-2,4(1H,3H)-dione (13c) and 3-(3-(1H-imidazol-1-yl)propyl)-6-(2-(1H-inden-2-yl)phenyl)
dihydropyrimidine-2,4(1H,3H)-dione (13d). A series of dihydropyrimidine-2,4(1H, 3H)-dione moieties was
derived from methyl 3-amino-3-(2-chlorophenyl)propanoate precursor in a multi-step synthesis. Acid-amine
coupling of 3-(2-(1H-inden-2-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoic acid with a series of amine
derivatives under mild reaction conditions led to form the corresponding dihydropyrimidine-2,4(1H, 3H)-
dione derivatives via de-Boc and cyclization reaction in modest yield. Spectroscopic (1H, 13C NMR, and Mass)
and analytical techniques have been used to identify and confirm the structure of the products.
Keywords. Triflic anhydride; Boc anhydride; Negishi coupling; acid-amine coupling; cyclization reaction;
cytotoxicity; MCF-7.
pyridin-pyrimidin-2-amines4 and 2-Anilino-4-(benzi-
midazol-2-yl)-pyrimidines derivatives4 have shown
anticancer activity against various cancer cell lines
(NCI 60, HCT-116, MCF-7, Aurora B, PLK1, FAK and
VEGF-R2). Since the diverse applications in pyrimi-
dine contain heterocycles, we proceeded to synthesize
novel 6-(2-(1H-inden-2-yl)phenyl)-3-(2-morpholino-
ethyl)dihydropyrimidine-2,4(1H, 3H)-dione and sub-
stituted derivatives in a stepwise way. The synthesized
products shown in Figure 1 have been tested in few
cancer cell lines.
1. Introduction
Synthesis of organic scaffolds from a mere precursor
through a multi-step reaction is a challenge in synthetic
organic chemistry. Heterocyclic Scaffolds play a vital
role in biological and pharmacologic activities in medi-
cinal chemistry.1 Pyrimidine-fused heterocycles has
found a wide range of biological applications as anti-
cancer, anti-inflammatory, antibacterial, antifungal,
anthelmintic, antitopoisomerase, antitumour, antiviral
and antioxidant agents.2 Nucleoside and non-nucleo-
side3 reverse transcriptase inhibitors have gained a
significant role in HIV infection. In particular, com-
poundslike1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)
thymine (HEPT), 6-benzyl-1-(ethoxymethyl)-5-isopro-
pyl pyrimidine-2,4-dione (MKC-442) and 6-benzyl-
1-(benzyloxymethyl)-5-isopropylpyrimidine-2,4-dione
(TNK-651) have high activity against HIV-1. N-hete-
rocyclic compounds have gained interest over the
past few years in pharmaceutical industries. Recently,
pyrimidine derivatives like 4-(2-chlorophenyl)-6-(2,
4-dichlorophenyl)pyrimidin-2-amine,4 4-indazolyl-N-
phenylpyrimidin-2-amines, N-phenyl-4-pyrazolo[3,4-b]
2. Experimental
2.1 Materials and methods
1H and 13C NMR spectra were recorded in deuterated sol-
vents on Bruker 500, 400 MHz and 125, 100 MHz spectrom-
eter, respectively. LC-MS were recorded on Agilent Technol-
ogy LCMS-1200 mass spectrometer. Reaction progress was
monitored by using thin-layer chromatography (TLC) on 60
F254 silica gel plates and visualized by UV light (254 nm) or
KMnO4. Column chromatography was performed using 60–
120 μm and 230–400 μm silica gel. All the reactions were
carried out under argon atmosphere and distilled solvents
were used.
∗For correspondence

V Udayakumar et al.
Figure 1. Biological active dihydropyrimidine-2,4(1H, 3H)-dione derivatives 13a–d.
anhydride (11.22 g, 51.4 mmol, 1.1 equiv.). The mixture was
2.2 1H-inden-2-yl trifluoromethanesulfonate (2)
heated at 60◦C for 3 h. After checking TLC, the resulting
solution was then washed with water and aqueous layer was
extracted with ethyl acetate, dried (Na2SO4) and concen-
trated under reduced pressure. The crude was then purified
by column chromatography over silica gel (60–120 mesh)
using 10–15% EtOAC in hexane as eluent to give the target
compound 4 (12 g, 82% yield) as brown solid (Rf-0.38,
3:7 ethylacetate/hexane, visualized by UV light (254 nm));
Stirred solution of 1H-inden-2(3H)-one (1; 10 g,
75.6 mmol) in DCM (50 mL) was added to N, N-Diiso-
propylethylamine (11.73 g, 90.7 mmol, 1.2 equiv.). Triflic
anhydride (25.6 g, 90.7 mmol, 1.2 equiv.) was then treated
dropwise at 40◦C for 1 h, until the consumpion of (1) as
shown by TLC and the residual mass was washed with
water and extracted with DCM. The organic layer was dried
(Na2SO4) and concentrated under reduced pressure. The
crude was then purified by column chromatography over
silica gel using 7–10% EtOAc in hexane as eluent to give the
target compound 2 (13 g, 65% yield) as yellow oil (Rf-0.57,
3:7 ethylacetate/hexane, visualized by UV light (254 nm));
1H NMR (500 MHz, DMSO-d6) δ 7.37 (d, 1H, ArH), 7.26
(d, 1H, ArH), 7.24 (m, 2H, ArH), 5.81 (s, 1H, = CH), 3.21
(s, 2H, CH2); 13C NMR (125 MHz, DMSO-d6) δ 174.1,
144.1, 142.9, 127.6, 126.1, 125.8, 125.4, 118.6, 98.5; LCMS
265.21 (M+H)+. Anal. Calcd. (%) for C10H7F3O3S: C,
45.46; H, 2.67; S, 12.14. Found (%): C, 45.44; H, 2.65; S,
12.13.
1
M.p. 179–181◦C; H NMR (500 MHz, DMSO-d6) δ 8.91
(s, 1H, NH), 7.69 (d, 1H, ArH), 7.21–7.28 (m, 3H, ArH),
5.58 (m, 1H, CH), 3.70 (s, 3H, CH3), 2.62–2.83 (d, 2H,
CH2), 1.42 (s, 9H, CH3); 13C NMR (125 MHz, DMSO-d6) δ
171.2, 155.4, 143.7, 132.5, 128.4, 126.1, 79.6, 51.3, 49.6,
37.1, 28.4; LCMS 314.75 (M+H)+. Anal. Calcd. (%) for
C15H20ClNO4: C, 57.42; H, 6.42; N, 4.46. Found (%) C,
57.45; H, 6.39; N, 4.43.
2.4 Methyl 3-(2-(1H-inden-2-yl)phenyl)-3-
((tert-butoxycarbonyl)amino) propanoate (5)
Zinc dust (5.17 g, 79.6 mmol, 2.5 equiv.) is activated by
heating with a hot air gun under vacuum over 10 min,
cooled to room temperature, then dried DMF (20 mL) and
iodine (0.485 g, 1.9 mmol, 0.06 equiv.) were added which
2.3 Methyl 3-((tert-butoxycarbonyl)amino)-3-
(2-chlorophenyl) propanoate (4)
To
a stirred mixture of methyl 3-amino-3-(2-chlo- decolorized within few minutes. N-Boc protected amine (4;
rophenyl) propanoate (3; 10 g, 46.8 mmol), DMAP (5.17 g, 10 g, 31.8 mmol) was added, followed by a pinch of iodine
46.8 mmol, 1 equiv.) in THF (50 mL) was added Boc (0.485 g, 1.9 mmol, 0.06 equiv.). The resulting mixture

Synthesis of dihydropyrimidine-2,4(1H, 3H)-dione derivatives
was stirred at room temperature for 2 h. After checking DCM (20 mL), the mixture was further treated with 1-[Bis
TLC, starting material was consumed (4) and zinc inser- (dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi-
tion was formed (Rf-0.17, 3:7 ethylacetate/hexane, visual- nium 3-oxid hexafluorophosphate, N-[(Dimethylamino)-1H-
ized by UV light (254 nm)). Into this mixture Pd2(dba)3 1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmetha-
(0.583 g, 0.6 mmol, 0.02 equiv.), SPhos (0.523 g, 1.2 mmol, naminium hexafluorophosphate N-oxide (1.5 g, 3.9 mmol, 1.2
0.04 equiv.) were charged and inden-O-triflate (2; 10.1 g, equiv.), 2-morpholinoethanamine (7; 0.85 g, 6.5 mmol) and
38.2 mmol, 1.2 equiv.) in dry DMF (20 mL) was added drop- N-ethylmorpholine (1.1 g, 9.8 mmol, 3 equiv.), and stirred
wise through a cannula. After complete addition, the reac- at room temperature for 12 h. The resulting mixture was
tion mixture was allowed to stir at room temperature for diluted with water and extracted with DCM, and the organic
12 h. After checking TLC, intermediate was consumed and layers were washed with brine, dried over Na2SO4 and sol-
the reaction mixture was then filtered through celite bed. vent was removed by rotary evaporation. The crude was then
The filtrate was then washed with water and extracted with purified by column chromatography over silica gel (60–120
dichloromethane, the organic layers were dried over Na2SO4 mesh) using 2–10% MeOH in chloroform as eluent to give
and concentrated under reduced pressure. The crude was the target compound 11a (1.2 g, 74% yield) as a pale white
then purified by column chromatography over silica gel (60– solid (Rf-0.66, 1:9 MeOH/chloroform, visualized by UV
1
120 mesh) using 7–12% EtOAC in hexane as eluent to give light (254 nm)). H NMR (400 MHz, DMSO-d6) δ 8.94 (s,
the target compound 5 (8.2 g, 66% yield) as off-white solid 2H, NH), 7.21–7.47 (m, 8H, ArH), 6.82 (s, 1H, =CH), 5.42
(Rf-0.43, 3:7 ethylacetate/hexane, visualized by UV light (t, 1H, CH), 3.68 (t, 4H, CH2), 3.12–3.22 (m, 4H, CH2),
1
(254 nm)). H NMR (500 MHz, DMSO-d6) δ 8.95 (s, 1H, 2.38–2.62 (m, 8H, CH2), 1.42 (s, 9H, CH3); 13C NMR (100
NH), 7.21–7.47 (m, 8H, ArH), 6.81 (s, 1H, =CH), 5.44 MHz, DMSO-d6) δ 173.6, 156.1, 146.5, 141.1, 137.2, 130.2,
(t, 1H, CH), 3.71 (s, 3H, CH3), 3.24 (s, 1H, CH), 2.65– 129.3, 128.6, 126.3, 125.2, 122.3, 80.1, 67.1, 58.4, 56.2,
2.89 (d, 2H, CH2), 1.42 (s, 9H, CH3); 13C NMR (125 MHz, 53.4, 41.1, 40.1, 28.6. LCMS 492.72 (M+H)+. Anal. Calcd.
DMSO-d6) δ 171.9, 156.1, 146.5, 144.2, 137.2, 130.1, 129.2, (%) for C29H37N3O4 C, 70.85; H, 7.59; N, 8.55. Found (%)
128.6, 126.3, 125.2, 122.3, 79.6, 52.6, 40.1, 39.6, 38.4, 28.5. C, 70.74; H, 7.61; N, 8.52.
LCMS 394.51 (M+H)+. Anal. Calcd. (%) for C24H27NO4
C, 73.26; H, 6.92; N, 3.56. Found (%) C, 73.22; H, 6.89; N,
2.7 Tert-butyl (1-(2-(1H-inden-2-yl)phenyl)-3-oxo-3-
((2-pyrrolidin-1-yl)ethyl)amino)propyl) carbamate (11b)
3.54.
2.5 3-(2-(1H-inden-2-yl)phenyl)-3-((tert-butoxycar-
Adopting the procedure described for the synthesis of 11a,
bonyl)amino) propanoicacid (6)
6 (1.25 g, 3.2 mmol) was coupled with 8 (750 mg, 6.5 mmol,
2 equiv.). The crude was then purified by column chro-
Starting material (5; 8 g, 20.3 mmol), H2O/dioxane (240 mL)
matography over silica gel (60–120 mesh) using 2–10%
in 1:1 ratio and then LiOH.H2O (1.7 g, 40.6 mmol, 2 equiv.)
MeOH in chloroform as eluent to give the target compound
was added. The reaction mixture was stirred at room temper-
11b (1.2 g, 76% yield) as a pale white solid (Rf-0.69, 1:9
ature for 3 h. After checking TLC, the reaction mixture was
1
MeOH/chloroform, visualized by UV light (254 nm)). H
concentrated, and water (100 mL) and ethyl acetate (100 mL)
NMR (400 MHz, DMSO-d6) δ 8.93 (s, 2H, NH), 7.22–7.54
was added. The aqueous layer was then acidified with 1 M
HCl under ice and then extracted with ethyl acetate (100 mL).
(m, 8H, ArH), 6.82 (s, 1H, =CH), 5.42 (t, 1H, CH), 3.28
(s, 2H, CH2), 3.14 (m, 2H, CH2), 2.43–2.84 (m, 8H, CH2),
The organic layers were combined, washed with brine, then
1.64 (m, 4H, CH2), 1.41 (s, 9H, CH3); 13C NMR (100 MHz,
dried with Na2SO4 and concentrated under reduced pres-
sure. The crude was co-evaporated with hexane to afford
acid 6 (6.5 g, 84% yield) as a pale white solid (Rf-0.12, 1:9
DMSO-d6) δ 173.6, 156.2, 146.2, 141.3, 137.4, 130.4, 129.3,
128.6, 126.3, 125.2, 121.8, 80.4, 58.4, 56.4, 53.4, 41.1, 40.1,
28.6, 23.4. LCMS 476.53 (M+H)+. Anal. Calcd. (%) for
methanol/chloroform, visualized by UV light (254 nm) and
1
C29H37N3O3C, 73.23; H, 7.84; N, 8.83. Found (%)C, 73.18;
H, 7.84; N, 8.79.
KMnO4 activity on heating). H NMR (500 MHz, DMSO-
d6) δ 10.5 (s, 1H, OH), 8.94 (s, 1H, NH), 7.21–7.49 (m,
8H, ArH), 6.81 (s, 1H, =CH), 5.44 (t, 1H, CH), 3.24 (s,
1H, CH), 2.65–2.89 (d, 2H, CH2), 1.42 (s, 9H, CH3); 13C
NMR (125 MHz, DMSO-d6) δ 173.1, 156.1, 146.5, 145.8,
137.2, 130.1, 129.2, 128.6, 126.3, 125.2, 122.3, 79.6, 53.3,
39.6, 28.5. LCMS 380.32 (M+H)+. Anal. Calcd. (%) for
C23H25NO4 C, 72.80; H, 6.64; N, 3.69. Found (%) C, 72.69;
H, 6.61; N, 3.65.
2.8 Tert-butyl (1-(2-(1H-inden-2-yl)phenyl)-3-oxo-3-
(phenethylamino)propyl) carbamate (11c)
Adoptng the procedure described for the synthesis of
11a, 6 (1.25 g, 3.2 mmol) was coupled with 9 (790 mg,
6.5 mmol, 2 equiv.). The crude was then purified by col-
umn chromatography over silica gel (60–120 mesh) using
2.6 Tert-butyl (1-(2-(1H-inden-2-yl)phenyl)-3-((2-mor- 2–10% MeOH in chloroform as eluent to give the tar-
get compound 11c (1.25 g, 79% yield) as a pale white
pholinoethyl)amino)-3-oxopropyl) carbamate (11a)
solid (Rf-0.71, 1:9 MeOH/chloroform, visualized by UV
3-(2-(1H-inden-2-yl)phenyl)-3-((tert-butoxycarbonyl)amino) light (254 nm)). 1H NMR (400 MHz, DMSO-d6) δ 8.92
propanoic acid (6; 1.25 g, 3.2 mmol) was dissolved in (s, 2H, NH), 7.22–7.49 (m, 13H, ArH), 6.79 (s, 1H,

V Udayakumar et al.
=CH), 5.41 (t, 1H, CH), 3.37 (m, 2H, CH2), 3.25 (s, 2H, NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H, NH), 7.22–7.54
CH2), 2.61–2.87 (m, 4H, CH2), 1.41 (s, 9H, CH3); 13C NMR (m, 8H, ArH), 6.82 (s, 1H, =CH), 5.25 (s, 2H, NH2), 4.35
(100 MHz, DMSO-d6) δ 173.6, 156.2, 146.2, 144.3, 139.4, (t, 1H, CH), 3.28 (s, 2H, CH2), 3.14 (m, 2H, CH2), 2.53–
136.4, 129.2, 128.8, 127.4, 125.2, 124.2, 121.8, 80.4, 53.4, 2.84 (m, 8H, CH2), 1.64 (m, 4H, CH2); 13C NMR (100 MHz,
41.1, 40.1, 35.7, 28.6. LCMS 483.32 (M+H)+. Anal. Calcd. DMSO-d6) δ 173.6, 146.2, 141.3, 137.4, 130.4, 129.3, 128.6,
(%) for C31H34N2O3C, 77.15; H, 7.10; N, 5.80. Found (%)C, 126.3, 125.2, 121.8, 58.4, 56.4, 48.2, 42.1, 40.1, 23.4. LCMS
77.27; H, 7.08; N, 5.78.
376.42 (M+H)+. Anal. Calcd. (%) for C24H29N3O C, 76.76;
H, 7.78; N, 11.19. Found (%) C, 76.75; H, 7.75; N, 11.20.
2.9 Tert-butyl (3-((3-(1H-imidazol-1-yl)propyl)amino)
-1-(2-(1H-inden-2-yl)phenyl)-3-oxopropyl) carbamate
(11d)
2.12 3-(2-(1H-inden-2-yl)phenyl)-3-amino-N-phene-
thylpropanamide hydrochloride (12c)
Following the procedure for the synthesis of 12a, using
11c (0.9 g, 1.86 mmol) and dichloromethane (18 mL) yielded
12c (0.51 g, 71% yield) as a white solid (Rf-0.53, 2:8
Adopting the procedure described for the synthesis of 11a, 6
(1.25 g, 3.2 mmol) was coupled with 10 (820 mg, 6.5 mmol,
2 equiv.). The crude was then purified by column chro-
matography over silica gel (60–120 mesh) using 2–15%
MeOH in chloroform as eluent to give the target compound
11d (0.93 g, 72% yield) as a pale white solid (Rf-0.57, 1:9
1
MeOH/chloroform, visualized by UV light (254 nm)). H
NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H, NH), 7.22–7.44
(m, 13H, ArH), 6.79 (s, 1H, =CH), 5.24 (s, 2H, NH2), 4.31
(t, 1H, CH), 3.37 (m, 2H, CH2), 3.25 (s, 2H, CH2), 2.61–2.87
(m, 4H, CH2); 13C NMR (100 MHz, DMSO-d6) δ 173.6,
146.2, 144.3, 139.4, 136.4, 129.7, 128.1, 127.5, 125.4, 124.2,
121.1, 48.4, 44.8, 40.1, 35.7. LCMS 383.52 (M+H)+. Anal.
Calcd. (%) for C26H26N2O C, 81.64; H, 6.85; N, 7.32. Found
(%) C, 81.61; H, 6.82; N, 7.31.
1
MeOH/chloroform, visualized by UV light (254 nm)). H
NMR (400 MHz, DMSO-d6) δ 8.93 (s, 2H, NH), 8.02 (s,
1H, CH), 7.22–7.55 (m, 8H, ArH), 6.85 (s, 2H, =CH), 5.41
(t, 1H, CH), 4.53 (t, 2H, CH2), 3.62 (t, 2H, CH2), 3.28 (s,
2H, CH2), 2.53–2.84 (m, 4H, CH2), 1.41 (s, 9H, CH3); 13C
NMR (100 MHz, DMSO-d6) δ 173.6, 156.2, 146.2, 141.3,
138.4, 136.2, 130.4, 129.3, 128.6, 126.3, 125.2, 121.5, 120.7,
80.4, 53.4, 47.3, 41.1, 40.1, 38.4, 32.8, 28.6. LCMS 487.71
(M+H)+. Anal. Calcd. (%) for C29H34N4O3C, 71.58; H,
7.04; N, 11.51. Found (%)C, 71.57; H, 7.06; N, 11.48.
2.13 N-(3-(1H-imidazol-1-yl)propyl)-3-(2-(1H-inden-
2-yl)phenyl)-3-aminopropanamide hydrochloride (12d)
Following the procedure for the synthesis of 12a, using
11d (0.7 g, 1.43 mmol) and dichloromethane (18 mL) yielded
12d (0.39 g, 70% yield) as a white solid (Rf-0.43, 2:8
2.10 3-(2-(1H-inden-2-yl)phenyl)-3-amino-N-(2-mor-
pholinoethyl)propanamide hydrochloride (12a)
1
MeOH/chloroform, visualized by UV light (254 nm)). H
NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H, NH), 8.02 (s, 1H,
CH), 7.22–7.63 (m, 8H, ArH), 6.85 (s, 2H, =CH), 5.26 (s,
2H, NH2), 4.34 (m, 1H, CH), 4.19 (m, 2H, CH2), 3.62 (t, 2H,
CH2), 3.28 (s, 2H, CH2), 2.53–2.84 (m, 4H, CH2); 13C NMR
(100 MHz, DMSO-d6) δ 173.6, 146.2, 144.3, 138.4, 136.2,
130.4, 129.3, 128.6, 126.3, 125.2, 122.3, 121.5, 48.8, 47.1,
45.3, 40.1, 38.4, 32.8. LCMS 387.49 (M+H)+. Anal. Calcd.
(%) for C24H26N4O C, 74.58; H, 6.78; N, 14.50. Found (%)
C, 74.63; H, 6.75; N, 14.29.
Removal of Boc group was done by bubbling HCl gas into a
stirred solution of 11a (0.9 g, 1.83 mmol) in dichloromethane
(18 mL) at room temperature for 3 h, until completion of the
reaction as shown by TLC. The solvent was vaporized under
reduced pressure and the crude was then purified by column
chromatography over neutral alumina (70–290 mesh) using
5–25% MeOH in chloroform as eluent to give 12a (0.5 g,
70% yield) as a white solid (Rf-0.48, 2:8 MeOH/chloroform,
visualized by UV light (254 nm)). 1H NMR (400 MHz,
DMSO-d6) δ 8.94 (s, 1H, NH), 7.21–7.47 (m, 8H, ArH),
6.82 (s, 1H, =CH), 5.24 (s, 2H, NH2), 4.38 (t, 1H, CH),
3.68 (t, 4H, CH2), 3.12–3.22 (m, 4H, CH2), 2.91–2.62 (m,
4H, CH2), 2.35 (m, 4H, CH2); 13C NMR (100 MHz, DMSO-
d6) δ 173.6, 146.5, 141.1, 137.2, 130.2, 129.3, 128.6, 126.3,
125.2, 122.3, 67.1, 58.4, 56.2, 48.6, 43.2, 40.1. LCMS 392.62
(M+H)+. Anal. Calcd. (%) for C24H29N3O2C, 73.63; H,
7.47; N, 10.73. Found (%) C, 73.59; H, 7.46; N, 10.71.
2.14 6-(2-(1H-inden-2-yl)phenyl)-3-(2-morpholino-
ethyl)dihydropyrimidine-2,4(1H,3H)-dione (13a)
12a (300 mg, 0.76 mmol) was added to ethyl chlorofor-
mate (4.5 mL), heated and stirred at reflux for 2 h. The
intermediate was confirmed by TLC analysis (Rf-0.60, 2:8
MeOH/chloroform, visualized by UV light (254 nm)). The
reaction mixture was then cooled to room temperature and
2.11 3-(2-(1H-inden-2-yl)phenyl)-3-amino-N-(2-(pyr- solid was collected by filtration, washed with ethanol and
the residue was charged with ethanol (6 mL), anhydrous
K2CO3 for 3 h at reflux. The volatiles were removed under
rolidin-1-yl)ethyl)propanamide hydrochloride (12b)
Following the procedure for the synthesis of 12a, using reduced pressure, and the crude was dissolved in water and
11b (0.9 g, 1.89 mmol) and dichloromethane (18 mL) gave adjusted the pH (6-7) using acetic acid. The white pre-
12b (0.51 g, 70% yield) as a white solid (Rf-0.51, 2:8 cipitate was collected by filtration, washed with ethanol to
1
MeOH/chloroform, visualized by UV light (254 nm)). H afford the cyclic compound 13a (281 mg, 90% yield) as a

Synthesis of dihydropyrimidine-2,4(1H, 3H)-dione derivatives
white solid (Rf-0.53, 2:8 MeOH/chloroform, visualized by adjusts the pH (6–7) using acetic acid. The white precipi-
UV light (254 nm)). 1H NMR (400 MHz, DMSO-d6) δ 9.12 tate was collected by filtration, washed with ethanol to afford
(s, 1H, NH), 7.21–7.49 (m, 8H, ArH), 6.82 (s, 1H, =CH), cyclic compound 13b (290 mg, 90% yield) as a white solid
5.15 (t, 1H, CH), 3.68 (t, 4H, CH2), 3.12–3.22 (m, 4H, CH2), (Rf-0.57, 2:8 MeOH/chloroform, visualized by UV light
1
2.87–2.62 (m, 4H, CH2), 2.35 (m, 4H, CH2); 13C NMR (254 nm)). H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H,
(100 MHz, DMSO-d6) δ 172.9, 153.3, 146.5, 144.1, 137.2, NH), 7.22–7.50 (m, 8H, ArH), 6.82 (s, 1H, =CH), 5.15
130.2, 129.3, 128.6, 126.3, 125.2, 122.3, 67.1, 56.2, 47.3, (t, 1H, CH), 3.22–3.28 (m, 4H, CH2), 2.53–2.84 (m, 8H,
43.2, 40.1, 38.2. LCMS 418.63 (M+H)+. Anal. Calcd. (%) CH2), 1.64 (m, 4H, CH2); 13C NMR (100 MHz, DMSO-
for C25H27N3O3C, 71.92; H, 6.52; N, 10.06. Found (%) C, d6) δ 172.1, 153.2, 146.5, 144.7, 137.4, 130.4, 129.3, 128.6,
71.86; H, 6.50; N, 10.09.
126.3, 125.2, 121.8, 56.7, 52.3, 46.9, 40.1, 39.3, 23.4. LCMS
402.45 (M+H)+. Anal. Calcd. (%) for C25H27N3O2C, 74.79;
H, 6.78; N, 10.47. Found (%) C, 74.77; H, 6.56; N, 10.44.
2.15 6-(2-(1H-inden-2-yl)phenyl)-3-(2-(pyrrolidin-1-
yl)ethyl)dihydropyrimidine-2,4(1H,3H)-dione (13b)
12b (300 mg, 0.79 mmol) was added to ethyl chlorofor- 2.16 6-(2-(1H-inden-2-yl)phenyl)-3-phenethyldihydro-
mate (4.5 mL), heated and stirred at reflux for 2 h. The
intermediate was confirmed by TLC analysis (Rf-0.66, 2:8
pyrimidine-2,4(1H,3H)-dione (13c)
MeOH/chloroform, visualized by UV light (254 nm)). The 12c (300 mg, 0.78 mmol) was added to ethyl chlorofor-
reaction mixture was then cooled to room temperature and mate (4.5 mL), heated and stirred at reflux for 2 h. The
solid was collected by filtration, washed with ethanol and intermediate was confirmed by TLC analysis (Rf-0.70, 2:8
the residue was charged with ethanol (6 mL) and anhydrous MeOH/chloroform, visualized by UV light (254 nm)). The
K2CO3 for 3 h at reflux. The volatiles were removed under reaction mixture was then cooled to room temperature and
reduced pressure, and the crude was dissolved in water and the solid was collected by filtration, washed with ethanol
Scheme 1. Synthesis of 1H-inden-2-yl trifluoromethanesulfonate (2).
Scheme 2. Protecting Boc group in methyl 3-amino-3-(2-chlorophenyl)propanoate.
Scheme 3. Coupling of 4 with 2 and synthesis of methyl 3-(2-(1H-inden-2-
yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate.

V Udayakumar et al.
Scheme 4. Synthesis of 3-(2-(1H-inden-2-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoic acid.
Scheme 5. Synthesis of amide derivatives by coupling of acid 6 with series of amines 7–10.

Synthesis of dihydropyrimidine-2,4(1H, 3H)-dione derivatives
Scheme 6. Removal of Boc group to make primary amines.
and the residue was charged with ethanol (6 mL), anhy- DMSO-d6) δ 172.4, 153.2, 147.1, 144.3, 139.4, 136.4, 128.8,
drous K2CO3 for 3 h at reflux. The volatiles were removed 124.2, 121.8, 47.5, 44.8, 40.1, 39.2. LCMS 409.51 (M+H)+.
under reduced pressure, and the crude was dissolved in Anal. Calcd. (%) for C27H24N2O2C, 79.39; H, 5.92; N, 6.86.
water and The volatiles were removed the pH (6–7) The Found (%) C, 79.35; H, 5.94; N, 6.82.
volatiles were removed using acetic acid. The white pre-
cipitate was collected by filtration, washed with ethanol
to afford cyclic compound 13c (301 mg, 94% yield) as a
white solid (Rf-0.58, 2:8 MeOH/chloroform, visualized by
UV light (254 nm)). 1H NMR (400 MHz, DMSO-d6) δ
9.12 (s, 1H, NH), 7.22–7.52 (m, 13H, ArH), 6.79 (s, 1H,
=CH), 5.15 (t, 1H, CH), 3.37 (m, 2H, CH2), 3.25 (s,
2H, CH2), 2.61–2.87 (m, 4H, CH2); 13C NMR (100 MHz,
2.17 3-(3-(1H-imidazol-1-yl)propyl)-6-(2-(1H-inden-
2-yl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione (13d)
12d (200 mg, 0.51 mmol) was added to ethyl chlorofor-
mate (3 mL), heated and stirred at reflux for 2 h. The
intermediate was confirmed by TLC analysis (Rf-0.58, 2:8

V Udayakumar et al.
3.28 (s, 2H, CH2), 2.53–2.84 (m, 4H, CH2); 13C NMR (100
MHz, DMSO-d6) δ 172.6, 153.2, 146.2, 144.3, 138.4, 136.2,
130.4, 129.3, 128.6, 126.3, 125.2, 121.5, 47.3, 46.2, 41.7,
40.1, 39.8, 28.3. LCMS 413.64 (M+H)+. Anal. Calcd. (%)
for C25H24N4O2C, 72.80; H, 5.86; N, 13.58. Found (%) C,
72.74; H, 5.83; N, 13.56.
MeOH/chloroform, visualized by UV light (254 nm)). The
reaction mixture was then cooled to room temperature and
the solid was collected by filtration, washed with ethanol.
and the residue was charge with ethanol (4 mL), anhydrous
K2CO3 for 3 h at reflux. The volatiles were removed under
reduced pressure, and the crude was dissolved in water and
adjusted the pH (6–7) using acetic acid. The white precip-
itate was collected by filtration and washed with ethanol
to afford cyclic compound 13d (191 mg, 89% yield) as a
white solid (Rf-0.48, 2:8 MeOH/chloroform, visualized by
UV light (254 nm)). 1H NMR (400 MHz, DMSO-d6) δ 9.13
(s, 1H, NH), 8.02 (s, 1H, CH), 7.22–7.73 (m, 8H, ArH),
3. Results and Discussion
Treatment of 2-indanone (1) with potassium bis(trime-
6.85 (d, 2H, =CH), 5.15 (t, 1H, CH), 4.11 (m, 4H, CH2), thylsilyl)amide in dichloromethane followed by enolate
Scheme 7. Synthesis of dihydropyrimidine-2,4(1H, 3H)-dione.

Synthesis of dihydropyrimidine-2,4(1H, 3H)-dione derivatives
5
some of the intermediates (12a–d) where the IC50 value
was found to be above 2 μM. These results suggested
that the synthesized compounds (13a–d) will work as
good scaffolds for cancer therapy research.
with (tBu)2Si(OTf)2 gave poor yield, but the desired
product 1H-inden-2-yl trifluoromethanesulfonate (2;
Scheme 1) was obtained in moderate yield (65%) using
N, N-Diisopropylethylamine and trifluoromethanesul-
fonic anhydride.
Methyl 3-amino-3-(2-chlorophenyl)propanoate(3)was
added with Boc anhydride and 4-dimethylaminopy-
ridine (DMAP) to give N-Boc protected amine (4;
Scheme 2) in 82% yield.6
4. Conclusions
In summary, we have developed a new method for
the synthesis of dihydropyrimidine-2,4(1H, 3H)-dione
derivatives through a multi-step reaction with modest
to high yields. NMR spectroscopy confirmed the struc-
tures of the synthesized compounds in each step. The
biological evaluation of the synthesized compounds
against A431 cell line exhibited an active cytotoxic
effect compared with other cell lines. When compared
among the series of compounds, we found that com-
pound 13d has highest cytotoxic effect with the IC50
value of 1.07 μM. We anticipate that this route can
be useful towards the synthesis of dihydropyrimidine-
2,4(1H, 3H)-dione scaffolds.
Negishi coupling reaction was carried out with me-
thyl 3-((tert-butoxycarbonyl)amino)-3-(2-chlorophen-
yl)propanoate (4) and activated zinc dust (Scheme 3).7
N-Boc protected amine (4) was converted into corres-
ponding PhZnCl intermediate which was coupled
with 1H-inden-2-yl trifluoromethanesulfonate (2) using
Pd2(dba)3 catalyst, to give methyl 3-(2-(1H-inden-
2-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate
(5) in a yield of 66%. 3-N-Boc-propanoic acid (6,
Scheme 4) was prepared from 3-N-Boc-methylpro-
panoate (5) by reacting with LiOH.H2O.8
Amide derivatives (11 a–d, Scheme 5)8 were synthe-
sized by the coupling reaction of acid (6) on treatment
with different amines (7–10) in the presence of HATU
Supporting Information (SI)
and N-ethylmorpholine which gave a good yield (75– 1H, 13C NMR spectra of all compounds are available at www.
80%) when compared with the reaction proceeded with ias.ac.in/chemsci.
HATU and DIEA. Elimination of Boc group in amide
derivatives (11 a–d) with HCl in dichloromethane
gave the corresponding amine hydrochloride (12 a–d,
Scheme 6).8
Acknowledgements
Theauthors would like to thank the Department of Chem-
istry, Pachaiyappa’s College, University of Madras, Chennai
for the instrumental facilities provided under the DST-FIST
programme. We also like to thank Mr. R. Balamurali, Senior
Research Associate for his contribution to the LC-MS analysis.
Cyclic compounds (13 a–d, Scheme 7)9 gave 90%
yield by cyclization of corresponding unprotected
amines (12 a–d) on heating with ethyl chloroformate
and ethanol.
The anticancer efficacy of the synthesized dihydro-
pyrimidine-2,4(1H,3H)-dione derivatives (13a–d) were
tested against a few cancer cell lines including
MCF-7 (breast), U87 (glioma), HeLa (cervix), A549
(lung), A431 (vulvar) using MTT assay. The obtained
inhibitory activity (IC50) of the synthesized com-
pounds (12a–d and 13a–d) using 5-fluorouracil as
standard are tabulated in Table S1 (in Supplementray
Information).
The incubation of organic molecules on cancer cell
lines was taken in 24 well plates for 24 h. The via-
bility of the cells were calculated by seeding the
organic molecules in different concentrations; normal
concentration has 100% of cell spreading. After incu-
bation, all the synthesized molecules (13a–d) exhib-
ited moderate cytotoxic activity against cancer cell
lines at the concentration of 62.5 μg/L. Cytotoxicity
results exhibited that compound 13d in A431 cell line
showed IC50 value of 1.07 μM, indicating better activ-
ity. Besides, we also investigated the bioactivity of
References
1. (a) Clercq E D and Bernaerts R 1987 Specific phospho-
rylation of 5-ethyl-2ꢁ-deoxyuridine by herpes simplex
virus-infected cells and incorporation into viral DNA
J. Biol. Chem. 262 14905; (b) Gready J E, McKinlay
C and Gebauer M G 2003 Synthesis of quaternised 2-
aminopyrimido[4,5-d]pyrimidin-4(3H)-ones and their
biological activity with dihydrofolate reductase Eur. J.
Med. Chem. 38 719; (c) Gangjee A, Adair O O and
Queener S F 1999 Pneumocystis carinii and Toxo-
plasma gondii Dihydrofolate Reductase Inhibitors and
Antitumor Agents: Synthesis and Biological Activities
of 2,4-Diamino-5-methyl-6-[(monosubstituted anilino)
methyl]- pyrido[2,3-d]pyrimidines J. Med. Chem. 42
2447; (d) Gangjee A, Adair O O and Queener S F
2008 N9-Substituted 2,4-Diaminoquinazolines: Synthe-
sis and Biological Evaluation of Lipophilic Inhibitors
of Pneumocystis carinii and Toxoplasma gondii Dihy-
drofolate Reductase J. Med. Chem. 51 6195; (e) Chan
D C M, Fu H, Forsch R A, Queener S F and Rosowsky
A 2005 Design, Synthesis, and Antifolate Activity of

V Udayakumar et al.
New Analogues of Piritrexim and Other Diaminopy-
as new anticancer agents Eur. J. Med. Chem. 83 630;
(b) Lukasik P M, Elabar S, Lam F, Shao H, Liu X,
Abbas A Y and Wang S 2012 Synthesis and biological
evaluation of imidazo[4,5-b]pyridine and 4-heteroaryl-
pyrimidine derivatives as anti-cancer agents Eur. J. Med.
Chem. 57 311; (c) Determann R, Dreher J, Baumann K,
Preu L, Jones P G, Totzke F, Schachtele C, Kubbutat
M H and Kunick C 2012 2-Anilino-4-(benzimidazol-2-
yl)pyrimidines – A multikinase inhibitor scaffold with
antiproliferative activity toward cancer cell lines Eur. J.
Med. Chem. 53 254
rimidine Dihydrofolate Reductase Inhibitors with ω-
Carboxyalkoxy or ω-Carboxy-1-alkynyl Substitution in
the Side Chain J. Med. Chem. 48 4420; (f) Radi M,
Schenone S and Botta M 2009 Recent highlights in
the synthesis of highly functionalized pyrimidines Org.
Biomol. Chem. 7 2841
2. (a) Gibson C L, Huggan J K, Kennedy A, Kiefer L, Lee
J H, Suckling C J, Clements C, Harvey A L, Hunter
W N and Tulloch L B 2009 Diversity oriented syntheses
of fused pyrimidines designed as potential antifolates
Org. Biomol. Chem. 7 1829; (b) Sakata K I, Someya
M, Matsumoto Y, Tauchi H, Kai M, Toyota M, Takagi
M, Hareyama M and Fukushima M 2011 Gimeracil, an
inhibitor of dihydropyrimidine dehydrogenase, inhibits
the early step in homologous recombination Cancer Sci.
102 1712; (c) Ramesh B and Bhalgat C M 2011 Novel
dihydropyrimidines and its pyrazole derivatives: Synthe-
sis and pharmacological screening Eur. J. Med. Chem.
46 1882; (d) Zhu L, Cheng P, Lei N, Yao J, Sheng C,
Zhuang C, Guo W, Liu W, Zhang Y, Dong G, Wang S,
Miao Z and Zhang W 2011 Synthesis and Biological
Evaluation of Novel Homocamptothecins Conjugating
with Dihydropyrimidine Derivatives as Potent Topoiso-
merase I Inhibitors Arch. Pharm. Chem. Life Sci. 344
726; (e) Gangjee A, Zhu Y and Queener S F 1998 6-
Substituted 2,4-Diaminopyrido[3,2-d]pyrimidine Ana-
logues of Piritrexim as Inhibitors of Dihydrofolate
Reductase from Rat Liver, Pneumocystis carinii, and
Toxoplasma gondii and as Antitumor Agents J. Med.
Chem. 41 4533
3. (a) Wang X, Lou Q, Guo Y, Xu Y, Zhang Z
and Liu J 2006 The design and synthesis of 9-
phenylcyclohepta[d]pyrimidine-2,4-dione derivatives as
potent non-nucleoside inhibitorsof HIV reverse tran-
scriptase Org. Biomol. Chem. 4 3252; (b) Yachoan R,
Mitsuya H, Thomas R V, Pioda J M, Hartman N R,
Perno C F, Marczky K S, Allain J P, Johns D G and
Broder S 1989 In Vivo Activity Against HIV and Favor-
able Toxicity Profile of 2ꢁ,3ꢁ-Dideoxyinosine Science
245 412; (c) Mellors J W, Im G J, Tramontano E, Winkler
S R, Medina D J, Dutschman G E, Bazmi H Z, Piras G,
Gonzalez C J and Cheng Y C 1993 A single conserva-
tive amino acid substitution in the reverse transcriptase
of human immunodeficiency virus-1 confers resistance
to (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-
butenyl)imidazo[4,5, 1-jk][1,4]benzodiazepin-2(1H)-thione
(TIBO R82150) Mol. Pharmacol. 43 11
5. (a) Van den Berg R J B H N, Korevaar C G N,
Van der Marel G A, Overkleeft H S and Van Boom
J H 2002 A simple and low cost synthesis of d-erythro-
sphingosine and d-erythro-azidosphingosine from d-
ribo-phytosphingosine: Glycosphingolipid precursors
Tetrahedron Lett. 43 8409; (b) Mangfei Y, Qian Z, Jia
W, Peng H, Pengfei Y, Rui Z and Dewen D 2016 Triflic
Anhydride-Mediated Beckmann Rearrangement Reac-
tion of β-Oximyl Amides: Access to 5-Iminooxazolines
J. Chem. Sci. 128 951
6. (a) Darnbrough S, Mervic M, Condon S M and Burns
C J 2001 An improved synthesis of N-Boc protected
aryl amines Synth. Commun. 31 3273; (b) Helgen C
and Bochet C G 2003 Photochemical Protection of
Amines with Cbz and Fmoc Groups J. Org. Chem. 68
2483
7. (a) Knappke C E I and Von Wangelin A J 2011 35 years
of palladium-catalyzed cross-coupling with Grignard
reagents: How far have we come? Chem. Soc. Rev. 40
4948; (b) Suzuki A 1999 Preparation of Cyclobutarenes
via the Steam Pyrolysis of Aromatic Derivatives J. Org.
Chem. 576 147; (c) Olsen J A, Severinsen R, Ras-
mussen T B, Hentzer M, Givskov M and Nielsen J
2002 Synthesis of new 3- and 4-substituted analogues of
acyl homoserine lactone quorum sensing autoinducers
Bioorg. Med. Chem. Lett. 12 325
8. (a) Kaul R, Surprenant S and Lubell W D 2005 Sys-
tematic Study of the Synthesis of Macrocyclic Dipeptide
β-Turn Mimics Possessing 8-, 9-, and 10-Membered
Rings by Ring-Closing Metathesis J. Org. Chem. 70
3838; (b) Atmuri N D P and Lubell W D 2015 Insight
into Transannular Cyclization Reactions To Synthesize
Azabicyclo[X.Y.Z]alkanone Amino Acid Derivatives
from 8-, 9-, and 10-Membered Macrocyclic Dipeptide
Lactams J. Org. Chem. 80 4904
9. El-Sherief H A H, El-Naggar G M, Hozien Z A
and El-Sawaisi S M 2008 Studies on the reaction
of N-(3-carbethoxy-4,5,6,7-tetrahydrobenzo[b]thien-2-
yl)-Nꢁ-phenylthiourea with hydrazine hydrate (Part 1) J.
Heterocycl. Chem. 45 467
4. (a) Rashid M, Husain A, Shaharyar M, Mishra R,
Hussain A and Afzal O 2014 Design and synthesis of
pyrimidine molecules endowed with thiazolidin-4-one