Design, synthesis and anticancer activities of hybrids of indole and barbituric acids-Identification of highly promising leads

Article abstract of DOI:10.1016/j.bmcl.2009.04.014

By combining the structural features of indole and barbituric acid, new hybrid molecules were designed and synthesized. Evaluations of these molecules over 60 cell line panel of human cancer cells have identified two molecules with significant anticancer activities. Dockings of two active molecules in the active sites of COX-2, thymidylate synthase and ribonucleotide reductase indicate their strong interactions with these enzymes.

Full text of DOI:10.1016/j.bmcl.2009.04.014

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3054–3058
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and anticancer activities of hybrids of indole
and barbituric acids—Identification of highly promising leads
*
Palwinder Singh , Matinder Kaur, Pooja Verma
Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
By combining the structural features of indole and barbituric acid, new hybrid molecules were designed
and synthesized. Evaluations of these molecules over 60 cell line panel of human cancer cells have iden-
tified two molecules with significant anticancer activities. Dockings of two active molecules in the active
sites of COX-2, thymidylate synthase and ribonucleotide reductase indicate their strong interactions with
these enzymes.
Received 27 February 2009
Revised 1 April 2009
Accepted 3 April 2009
Available online 9 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Indole
Barbituric acid
Hybrid molecules
Anticancer activities
Cancer is continuing to be a major health problem in both
developed and developing countries. Surpassing heart disease, it
is taking the position of number one killer due to various world-
wide factors. Several anticancer agents including taxol, vinblastine,
vincristine, etoposide, camptothecin and its derivatives (topotecan
and irinotecan),1 mitoxantrone,2 5-fluorouracil,3 indomethacin4
etc. are in clinical use all over the world. However, these drugs suf-
fer from various side effects like low blood pressure, bone marrow
suppression, gastrointestinal toxicity, constipation and hair loss.
Therefore, the lack of effective chemotherapy of cancer is continu-
ously inciting the scientific community to explore new chemical
entities for the effective and safe cure of cancer.
The pivotal role of small chemical entities (like amino acids,
glucose, nucleobases) and the acceptability of their synthetic ana-
logues in the biological systems may be the reason for the design
and development of about 80% of the drugs based on small organic
molecules.5 Moreover, a suitable combination (keeping in mind
Lipinski’s rule of 5)6 of the structural features of two small organic,
biologically active molecules may result in the creation of new
molecules (hybrid or conjugates)5,7,8 which are proving to be more
efficacious, economical and safe for their use as drugs. Hybrid mol-
ecules or conjugates, as defined by Mehta and Singh,7 ‘are the con-
structs of different molecular entities, natural or unnatural, to
generate functional molecules in which the characteristics of vari-
ous components are modulated, amplified or give rise to entirely
new properties’.
Due to the biological significance of indoles, barbituric acids as
anti-convulsant,9 anti-hypnotic,10 anticancer,11 anti-inflamma-
tory,12 agents and also their use as agrochemicals,13 we have cho-
sen these molecular entities (1 and 2, Fig. 1) to suitably combine
them through carbon–carbon bond formation for creating new hy-
brid molecules (3, Fig. 1). Investigations of new molecules (ana-
logues of a cytotoxic marine isolate aplysinopsin14 and carrying
the components of indomethacin and 5-fluorouracil) for their tu-
mour growth inhibitory activities over 60 cell line panel of human
cancer cell lines have identified two promising candidates as leads
for anticancer drugs.
The preparation of the target compounds was achieved by irra-
diating a finely ground mixture of two reactants in microwave
O
R1
N
O
N
R1
O
R1
N
O
N
R
O
2
1
N
R1
OH
O
O
H3CO
O
N
R
N
F
CH3
HN
3
O
N
H
O
Cl
Indomethacin
5-Fluorouracil
* Corresponding author. Tel.: +91 183 2258802 09x3495; fax: +91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
Figure 1. Design of new molecules.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.014

P. Singh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3054–3058
3055
Table 1
Table 1 (continued)
Reaction time, percentage yields and melting points for compounds 3–18
Entry
R
R1
Product
Yield
(%)
Mp (°C)
O
O
O
R1
R1
H
N
R1
N
MWI
O
O
+
O
H
N
R
1-5 min
N
N
N
R1
O
N
R1
2
O
O
N
R
O
O
1
3-18
R1
6
H,
CH3
75,
80
>240,
>250
N
H
Entry
R
R1
Product
Yield
(%)
Mp (°C)
O
R1
O
N
N
13, 14
O
R1
O
H
1
H
H,
CH3
99,
93
>260,
>260
R1
N
N
O
N
O
O
H
O
R1
3, 4
N
7
H,
CH3
75,
88
>240,
>250
H
O
Cl
R1
O
Cl
H
N
O
15, 16
N
R1
R1
O
2
3
4
H,
CH3
93,
81
>250,
190
O
N
H
H
N
N
O
O
R1
O
5, 6
N
8
H,
CH3
75,
85
>240,
>240
Cl
Cl
Cl
O
R1
O
O
Cl
H
N
O
17, 18
N
R1
H,
CH3
91,
86
>250,
235
N
oven.15 Indole-3-carboxaldehyde (1) (1 mmol) and barbituric acid
(2) (1.2 mmol) on irradiating in a microwave oven for 5 min, after
washing with diethyl ether gave a solid compound (99%, mp
>260 °C, MS: (FAB) m/z 225), which from its NMR, IR and mass
spectral data has been identified as compound 3 (R1 = H). Follow-
ing the same reaction procedure, other N-substituted indole-3-
carboxaldehydes react with barbituric acids to give compounds
4–18 (Table 1) in quantitative yields.16
H
7, 8
R1
O
H
N
O
Therefore, the target compounds are synthesized in quantita-
tive yields through Knoevenagal condensation using a very conve-
nient synthetic methodology.
N
R1
O
H,
CH3
90,
80
>250,
192
N
H
In vitro tumour growth inhibitory activities of these compounds
were investigated at NCI, NIH, Bethesda on 60 cell line panel of hu-
man cancer cells using standard procedure. Compounds were first
evaluated at 10À5 M concentration for the growth inhibitory activ-
ities at entire 60 cell line panel. Compounds with appreciable activ-
ities at 10À5 M concentration (as per the standard of NCI) were
subjected to detailed tumour growth inhibitory studies at five con-
centrations viz. 10À4 M, 10À5 M, 10À6 M, 10À7 M and 10À8 M.
At 10À5 M concentration, along with the inhibition of tumour
growth of other cell lines, remarkably, most of the compounds un-
der present investigation show activity at various cell lines of renal
cancer. Percent growth inhibition of RXF 393, TK-10 and UO-31 cell
lines of renal cancer by compounds 3, 4, 9–16 is given in Table 2.
Here, compounds 9 and 10 show 71% and 95% tumour growth inhi-
bition at TK-10 cell line.
9, 10
R1
R1
O
H
N
N
O
O
S
O
O
N
S
H
O
O
5
H,
CH3
85,
82
>240,
246
CH3
On the basis of preliminary investigations, compounds 9 and
10 were selected for detailed studies at five concentrations
where they show highly encouraging results. The GI50 (50%
growth inhibitory concentration) of compound 9 (average over
CH3
11, 12

3056
P. Singh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3054–3058
Table 2
(indomethacin and 5-fluorouracil) justify the design of these
molecules.
Percent growth inhibition of renal cancer cells by compounds 3, 4, 9–16
Compound
Percent growth inhibition for renal cancer
Although the cellular target is not defined in the experimental
anticancer investigations of these molecules, to look into the pos-
sible interactions of these compounds at the enzymatic level,17
we performed the dockings of compounds 9 and 10 in the active
sites of COX-2 (probable mode of action of indomethacin) and thy-
midylate synthase (cellular target of 5-fluorouracil). Docking pro-
gramme18 was validated by docking Sc-558 in the crystal
structure of COX-2 (containing Sc-558, pdb ID 6COX). A close over-
lapping between the two ligands (docked Sc-558 and native Sc-
558) was observed (Fig. 2).
Docking of compound 9 in the active site of COX-2 (Fig. 3)
shows H-bond interactions between NH, CO of pyrimidine moiety
and S353, Q192, H90 amino acid residues of the active site. The
phenyl ring present at indole is oriented towards W387 and
Y385 residues exhibiting hydrophobic interactions. Similar inter-
actions of compound 10 with the active site amino acid residues
of COX-2 were observed. Therefore, the bindings of compounds 9
and 10 in the active site of COX-2 indicate their indomethacin like
mode of action.
Dockings of compounds 9 and 10 in the active site of thymidyl-
ate synthase (TS) shows a number of H-bond interactions between
the three carbonyl groups of pyrimidine moiety of compounds 9
and 10 and amino acid residues of the enzyme (Fig. 4).
Additionally, compounds 9 and 10 were also docked in the ac-
tive site of ribonucleotide reductase (RNR) (another enzyme in-
volved in the propagation of cancer; synthesis of raw material for
DNA replication). Here also compounds 9 and 10 interact with
the active site amino acids through a number of H-bonds (Fig. 5).
Therefore, the dockings of these two compounds in the ac-
tive sites of COX-2, TS and RNR indicate the probable mode
of action of these compounds for anticancer activities.
RXF 393
TK-10
UO-31
3
4
9
10
11
12
13
14
15
16
—
—
—
43
—
52
55
55
—
—
—
71
95
—
—
—
—
—
—
39
33
29
36
32
41
36
36
22
—
33
Table 3
GI50
(lM), total growth inhibitory concentration (TGI) and LC50
(l
M) of compounds 9
and 10 at various cell lines
Cell lines
GI50
(l
M)
TGI (lM)
LC50
(l
M)
10
9
10
9
10
9
K-562
3.1
1.5
5.4
0.3
1.4
3.3
2.9
1.6
0.48
2.0
0.3
0.03
4.6
2.0
4.5
0.1
4.3
8.3
3.4
4.1
0.7
1.1
3.0
1.0
0.06
6.1
0.1
0.2
0.5
3.1
5.0
0.02
23
15
28
16.5
10.9
70.7
23.4
16.5
10.4
44
>100
50
27
>100
9.1
>100
45.7
12.5
0.26
>100
0.67
0.64
6.4
>100
58.8
>100
63
41.6
>100
>100
>100
63
>100
39.8
1.09
93
>100
95
>100
>100
>100
>100
>100
>100
>100
>100
0.85
>100
>100
2.29
>100
>100
>100
0.69
HOP-92
NCI-H226
NCI-H460
COLO 205
SF-295
SK-MEL-2
UACC-62
IGROV1
OVCAR-4
OVCAR-5
A498
3.8
0.26
25.7
8.3
22.3
0.38
CAKI-1
TK-10
T-47D
MDA-MB-468
57.5
>100
0.06
>100
all the 60 cell lines) is 7.5
as 13.8 M. Interestingly, the GI50 values of both these com-
pounds are better than indomethacin (GI50 64.3 M) and 5-fluo-
rouracil (GI50 17.7 M).
lM while compound 10 exhibit GI50
l
l
l
Compounds 9 and 10 exhibit significant tumour growth inhibi-
tory activities at various cell lines of non-small cell lung cancer
(NSCLC), colon cancer, CNS cancer, ovarian cancer, renal cancer
and breast cancer (Table 3).
It is apparent from the data given in Table 3 that compound 9
exhibits tumour growth inhibitory activities at micro/sub-micro-
molar concentrations. It exhibits GI50 0.3
0.48 M for IGROV1, 0.3 M for OVCAR-5, 0.03
0.1
l
M
l
for NCI-H460,
M for A498 and
l
l
l
M for MDA-MB-468 cell lines of NSCLC, ovarian cancer, renal
cancer and breast cancer, respectively. Remarkably, the total
growth inhibitory concentrations (TGI) of compound 9 for A498
and MDA-MB-468 cell lines are 0.26
Compound 10 shows more promising results with GI50 0.06
0.1 M, 0.2 M and 0.02 M for IGROV1, OVCAR-5, A498 and
MDA-MB-468 cell lines, respectively. Respective TGI concentra-
tions of compound 10 at these cell lines are 0.26 M, 0.67 M,
0.64 M and 0.06 M. LC50 values (Table 3) of compounds 9 and
lM and 0.38
l
M, respectively.
lM,
l
l
l
l
l
l
l
10 are quite high in comparison to their GI50 values indicating their
toxicity at high concentrations.
Therefore, the screening of these ten rationally designed com-
pounds for anticancer activities have identified two candidates
which could be further refined to improve their anticancer activi-
ties and drug like properties. Moreover, the improved anticancer
activities of compounds 9 and 10 over their component molecules
Figure 2. Sc-558 docked in the active site pocket of COX-2. A close overlapping
between the docked molecule (pink) and one present as crystal (green) validates
the docking programme.

P. Singh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3054–3058
3057
Figure 3. Compound 9 docked in the active site of COX-2. Hs’ are omitted for
clarity. H-bonds are visible between NH, CO groups of compound 9 and S353, Q192,
H90 of COX-2. Carbon atoms of compound 9 are given different colour for clarity.
Figure 5. (A) Compound 9 docked in RNR. H-bonds between compound 9 and
different amino acid residues are visible. (B) Compound 10 docked in RNR. H-bonds
between compound 10 and different amino acid residues are visible. Carbon atoms
of 9 and 10 are given different colour.
Figure 4. Compound 10 docked in the active site of TS. Carbon atoms of compound
10 are given different colour for clarity.
Table 4
Lipinski values for compounds 9 and 10
Compound
Log P
TPSA (Å2)
nON
nOHNH
Remarkably, such correlations between the experimental data
and the docking studies are useful for refining the structure
of the molecules and improving their efficacies. Lipinski values
of compounds 9 and 10 (Table 4) are also in favour of their
drug like properties.
9
10
2.10
2.24
87.2
66.01
6
6
2
0
Acknowledgements
In conclusion, we have designed hybrid molecules on the basis
of the biological significance of indole and barbituric acid and eval-
uated for their anticancer activities. Out of a set of 10 molecules,
two compounds exhibit significant anticancer activities and could
be used as leads for further investigations.
Authors are grateful to CSIR, DST and UGC New Delhi for re-
search grants. M.K. thanks UGC and P.V. thanks CSIR for fellow-
ships. Dr. V. L. Narayanan and his team at NCI, NIH, Bethesda,

3058
P. Singh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3054–3058
12. (a) Badawey, E.; El-Ashmawey, I. M. Eur. J. Med. Chem. 1998, 33, 349; (b)
Radwan, M. A. A.; Ragab, E. A.; Sabry, N. M.; El-Shenawy, S. M. Bioorg. Med.
Chem. 2007, 15, 3832. and references therein.
13. (a) Lee, D.; Carter, C. (Stauffer Chemical Co.), U.S. Patent 4, 797, 147, 1989;
Chem. Abstr. 1989, 111, 148889c.; (b) Kratt, G.; Salbeck, G.; Bonin, W.; Duewel,
D. Ger. Offen. DE 3, 903, 404, 1990; Chem. Abstr. 1991, 114, 23984k.; (c)
Sundberg, R. J. Indoles; Academic Press: San Diego, 1996.
USA is gratefully acknowledged for screening anticancer activities.
RSIC, CDRI Lucknow is acknowledged for recording mass spectra
and CHN analysis.
References and notes
14. (a) Kazlauskas, R.; Murphy, P. T.; Quinn, R. J.; Wells, R. J. Tetrahedron Lett. 1977,
61–64; (b) Hollenbeak, K. H.; Schmitz, F. J. Lloydia 1977, 40, 479.
1. Shoeb, M. Bangladesh J. Pharmacol. 2006, 1, 35. and references cited therein.
2. (a) Zee-Cheng, R. K. Y.; Cheng, C. C. J. Med. Chem. 1978, 21, 291; (b) Cheng, K. Y.;
Zee-Cheng, R. K. Y.; Podrebarac, E. G.; Menon, C. S.; Cheng, C. C. J. Med. Chem.
1979, 22, 501.
3. NIH database, NSC 19893.
4. NIH database, NSC 77541.
5. Meunier, B. Acc. Chem. Res. 2008, 41, 69. and references therein.
6. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
7. Mehta, G.; Singh, V. Chem. Soc. Rev. 2002, 31, 324.
8. Morphy, R.; Rankovic, Z. J. Med. Chem. 2005, 48, 6523.
9. Goodman, L. S.; Gilman, A. In The Pharmacological Basis of Therapeutics; Mc
Graw-Hill: New Delhi, 1991; pp 358–360.
10. (a) Andrews, G. In Medical Pharmacology; The CV Mosby Company: Saint louis,
1976; p 243; (b) Foye, W. O. Principles of Medicinal Chemistry; Lea & Febiger:
London, 1989. 159.
15. Reddy, C. S.; Nagaraj, A.; Jalapathi, P. Indian J. Chem, Sect. B 2007, 46, 660.
16. Experimental data for selected compounds. Compound 9: 5-((1-Benzyl-1H-
indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione. Yield 90%; Yellow
solid; mp >260 °C; IR (KBr, cmÀ1): 1375, 1650 (C@O), 3505 (NH); 1H NMR
(300 MHz, CDCl3 + DMSO):d 5.39 (s, 2H, CH2), 7.18–7.39 (m, 9H, ArH), 7.39 (s, 1H,
CH), 10.00 (s, 1H, indole2-H); Anal. Calcd for C20H15N3O3: C, 69.56; H, 4.38; N,
12.17. Found: C, 69.39; H, 4.49; N, 12.24; MS (FAB) m/z 345 (M+).
Compound 10: 5-(1-Benzyl-1H-indol-3-ylmethylene)-1, 3-dimethyl-pyrimidine-
2,4,6-trione. Yield 80%; Light Lime solid; mp 192 °C; IR (KBr, cmÀ1): 1388, 1660,
1662 (C@O); 1HNMR(300MHz, CDCl3) d 3.41(s, 3H, CH3), 3.42(s, 3H, CH3), 5.47(s,
2H, CH2),7.18–7.39(m, 8H, ArH),8.00(d,J = 7.8 Hz, 1H, ArH,),9.03(s, 1H, @H),9.69
(s, 1H, indole2-H);13CNMR(75 MHz, CDCl3):d28.02, 28.81, 51.75,108.57, 110.28,
111.34, 112.3, 118.77, 122.14, 123.06, 123.33, 124.20, 126.82, 127.20, 128.35,
129.08, 130.77,134.96, 136.71,142.60, 146.33, 151.78,162.22, 163.71;Anal. Calcd
for C22H19N3O3: C, 70.76; H, 5.13; N, 11.25. Found: C, 70.85; H, 5.09; N, 11.39; MS
(FAB) m/z 374 (M+1).
11. Guerin, D. J.; Mazeas, D.; Musale, M. S.; Naguib, N. F. M.; Al Safarjalani, O. N.; el
Kouni, M. H.; Panzica, R. P. Bioorg. Med. Chem. Lett. 1999, 9, 1477. and
references cited therein.
17. Singh, P.; Bhardwaj, A. Mini-Rev. Med. Chem 2008, 8, 388.
18. Docking studies were performed as per our previous protocol: Singh, P.;
Bhardwaj, A.; Kaur, S.; Kumar, S. Eur. J. Med. Chem. 2009, 44, 1278.