Synthesis and anti-proliferative activity of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H) -trione analogs against human tumor cell lines

Article abstract of DOI:10.1016/j.bmcl.2013.12.013

Based on previous SAR studies on N-benzylindole and barbituric acid hybrid molecules, we have synthesized a series of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H) -trione analogs (3a-i) and evaluated th

Full text of DOI:10.1016/j.bmcl.2013.12.013

Bioorganic & Medicinal Chemistry Letters 24 (2014) 601–603
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-proliferative activity of aromatic substituted
5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,
4,6(1H,3H,5H)-trione analogs against human tumor cell lines
⇑
Nikhil Reddy Madadi, Narsimha Reddy Penthala, Venumadhav Janganati, Peter A. Crooks
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on previous SAR studies on N-benzylindole and barbituric acid hybrid molecules, we have
synthesized a series of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrim-
idine-2,4,6(1H,3H,5H)-trione analogs (3a–i) and evaluated them for their in vitro growth inhibition and
cytotoxicity against a panel of 60 human tumor cell lines. Compounds 3c, 3d, 3f and 3g were identified as
highly potent anti-proliferative compounds against ovarian, renal and breast cancer cell lines with GI50
values in low the nanomolar range. The 4-methoxy-N-benzyl analog (3d) was the most active compound
with GI50 values of 20 nM and 40 nM against OVCAR-5 ovarian cancer cells and MDA-MB-468 breast
cancer cells, respectively. Two other analogs, 3c (the 4-methyl-N-benzyl analog) and 3g (the 4-fluoro-
N-benzyl analog) exhibited equimolar potency against MDA-MB-468 cells GI50 = 30 nM). Analog 3f
(the 4-chloro-N-benzyl analog) exhibited a GI50 value of 40 nM against renal cancer cell line A498. These
results suggest that aromatic substituted N-benzylindole dimethylbarbituric acid hybrids may have
potential for development as clinical candidates to treat a variety of solid tumors.
Received 10 October 2013
Revised 25 November 2013
Accepted 2 December 2013
Available online 9 December 2013
Keywords:
N-Benzyl indole
Dimethylbarbituric acid
Percentage growth inhibition
Growth inhibitory activity (GI50
Lethal concentration (LC50
)
)
Ó 2013 Elsevier Ltd. All rights reserved.
Cancer is the second most life threatening disease after cardio-
vascular disease, affecting more than six million people per year
worldwide.1 Drastic changes in life style during the end of the
19th century has increased the risk of humans developing different
types of cancers. Also, considerable effort has been put into identi-
fying molecules with anti-cancer properties from both natural and
synthetic sources.
inhibitors of DNA repair and replication stress response
polymerases.12
In our continuing studies on improving the potencies of newly
identified anti-cancer leads, we now report on the synthesis
and antiproliferative properties of some aromatic substituted
5-(indolin-3-ylmethylene)-1,3-dimethylpyrimidine-2,4,6-triones as
second generation indole barbituric acid hybrids.
Indole and barbituric acids derivatives are known to have a
wide range of beneficial biological activities such as anti-cancer,2
anti-inflammatory,3 anti-convulsant,4 anti-psychotic,5 anti-hyper-
tensive,6 and anti-bacterial properties.7 Singh et al. have synthe-
sized and evaluated some novel N-benzyl indole-barbituric acid
hybrid molecules against a panel of 60 human tumor cell lines.
They identified compound 1 (Fig. 1) as a promising lead compound
with significant tumor growth inhibitory activity (GI50) against a
variety of human cancer cell lines; the molecule also had good
maximum tolerable dose (MTD) characteristics.8 Our laboratory
has also reported on several novel indole barbiturates as
anti-cancer and radio-sensitization agents9,10 (e.g., compound 2,
Fig. 1). Recently,11 we have reported that N-aroyl indole thiobarbi-
turic acids (e.g., compound 3, Fig. 1) possess both anticancer
and anti-inflammatory properties. Several of these analogs are also
A series of N-benzylindole-3-carboxaldehydes (2a–i) were syn-
thesized by reacting an appropriate indole carboxaldehyde (1a–c)
with various aromatic substituted benzyl halides utilizing the
phase transfer catalyst triethylbenzyl ammonium chloride (TEBAC)
in a mixture of 50% w/v aq NaOH solution and dichloromethane.
The resulting N-benzyl products were obtained in 80–85% yield.5
The N-benzylindole-3-carboxaldehydes (2a–i) (1 mmol) were each
then reacted with N,N-dimethylbarbituric acid (1.2 mmol) in
methanol at room temperature to afford a series of 5-((1-benzyl-
1H-indol-3-yl)methylene)-1,3-dimethyl-pyrimidine-2,4,6-(1H,3H,
5H)-trione analogs (3a–i) (Scheme 1 and Table 1) in 75–90% yield.
The synthesized compounds were fully characterized by 1H NMR
and 13C NMR spectrometric analysis.13
In vitro screening of the above compounds was carried out
against a panel of 60 human tumor cell lines utilizing the proce-
dure described by Rubinstein et al.14 Compounds 3a–i were ini-
tially screened at 10À5 M to determine growth inhibition and
cytotoxic properties (Table 2). Compounds 3c, 3d, 3f and 3g
showed more than 60% growth inhibition in at least eight cell lines
⇑
Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.013

602
N. R. Madadi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 601–603
Table 3
Growth inhibition (GI50
/l /l
M)a and cytotoxicity (LC50 M)b data for compounds 3c, 3d,
3f and 3g against human cancer cellsc
Panel/cell line
3c
3d
3f
3g
GI50
LC50
GI50 LC50
GI50
LC50
GI50
LC50
Leukemia
K-562
SR
2.23
1.89
>100 2.18 >100 NA
>100 5.43 >100 70.3
>100 NA
>100 >100 >100
>100
Non-small cell lung cancer
HOP-62
7.44
1.95
8.19
0.91
>100 6.22 >100 10.7
>100 7.70
>100 2.56
>100 51.5
>100 0.37
>100
>100
>100
>100
Figure 1. Lead compounds from previous studies (1–3).8–10
NCI-H226
NCI-H23
NCI-H460
>100 1.44 53.9
1.26
>100 4.67 >100 10.2
>100 0.81 82.8
>100 0.63 45.3
0.40
Colon cancer
COLO 205
HCT-116
1.89
5.45
NA
2.78
29.5
1.44
>100
>100
>100 5.01 >100 4.47
>100 6.89 >100 >100 >100 >100 >100
>100 6.26
SW-620
CNS cancer
SF-295
SNB-19
2.02
26.8
>100 1.54 >100 1.21
>100 15.8 >100 18.3
>100 1.97
>100 26.7
>100
>100
Melanoma
LOX IMVI
MDA-MB-435
SK-MEL-2
SK-MEL-5
UACC-257
UACC-62
27.8
NA
3.36
9.17
2.02
2.42
>100 5.45 >100 17.3
>100 3.29 >100 >100 >100 >100 >100
>100 >100 >100
Scheme 1. Reagents and conditions: (a) appropriate benzyl halide, 50% NaOH,
CH2Cl2, TEBAC, 2 h; (b) dimethylbarbituric acid in methanol, room temp, 75–90%
yield.
>100 2.78 6.25
>100 5.08 64.4
>100 1.54 38.2
>100 0.90 68
2.84
75.8
6.35
>100
>100 >100 >100 >100
2.01
2.06
>100 2.52
>100 2.89
>100
>100
Table 1
Ovarian cancer
IGROV1
OVCAR-5
5-((1-Benzyl-1H-indol-3-yl)methylene)-1,3-dimethyl-pyrimi- dine-2,4,6(1H,3H,5H)-
trione analogs (3a–3i)
2.95
0.07
>100 1.93 >100 2.88
>100 0.02 >100 0.16
>100 4.33
>100 0.11
>100
>100
OVCAR-8
NCI/ADR-RES
SK-OV-3
>100 >100 13.4 88.6
21.3
>100 >100 >100
>100 >100 >100
Compound
R1
R2
6.23
23.7
>100 3.59 >100 16.3
>100 5.03 >100 6.08
3a
3b
3c
3d
3e
3f
3g
3h
3i
COOCH3
H
H
H
H
H
H
Cl
p-SO2Ph
o-Br
p-CH3
p-OCH3
p-CN
p-Cl
p-F
p-F
p-COOCH3
>100 15.3
>100
Renal cancer
786-0
A498
RXF 393
SN12C
TK-10
22.1
0.12
15.2
>100 11.2 >100 6.90
>100 22.7
>100
0.67
>100
0.69
0.06 5.27
0.04
0.64
0.07
>100 14.8 >100 12.5
>100 14.9
>100 >100 12.5 >100 92.1
>100 >100 >100
50.4 0.59 >100
0.28
>100 0.10 72.5
0.18
Breast cancer
MCF7
MDA-MB-231/
ATCC
COOCH3
6.76
13.6
>100 5.26 >100 >100 >100 >100 >100
>100 5.86 >100 12.2
>100 23.6
>100
BT-549
T-47D
MDA-MB-468
13.5
5.00
0.03
>100 4.94 >100 12.5
>100 3.53 >100 3.53
>100 24.1
>100 5.38
>100
>100
0.50
Table 2
Percentage growth inhibition of five human cancer cell lines by compounds (3a–3i)a
at 10
0.62
0.04 0.76
0.06
0.70
0.03
l
M
NA: not analyzed.
Cell line
Percentage growth inhibition
a
GI50: 50% growth inhibition, concentration of drug resulting in a 50% reduction
in net protein increase compared with control cells.
3a
98
108
83
100
88
3b
3c
3d
3e
3f
3g
3h
3i
88
98
105
113
99
b
LC50: lethal concentration, concentration of drug lethal to 50% of cells.
GI50 and LC50 values below 1 lM are bolded.
NCI-H226
OVCAR-5
A498
TK-10
MDA-MB-468
–32
75
74
105
56
–63
–23
–91
–4
–55
–38
–92
–12
–65
–72
6
68
62
–71
–82
–23
–94
–26
–71
–78
–33
–85
–15
–71
–79
–19
–95
19
c
PG = 100 Â (ODtest À ODtzero)/(ODctrl À ODtzero) and
percentage growth is shown as positive.
–62
–70
a
If (ODtest À ODtzero) > 0. Then PG is shown as positive. If (ODtest À ODtzero) <0, PG
If (ODtest À ODtzero) < 0, then
is shown as negative, implying cell death.
PG = 100 Â (ODtest À ODtzero)/ODtzero and
percentage growth is shown as negative, which implies cell
death.15
from the panel of sixty cell lines, and were selected for a complete
dose response study at five different concentrations, viz. 10À4 M,
10À5 M, 10À6 M, 10À7 M and 10À8 M.
The four compounds selected for full dose response studies
were effective against lung cancer cell line NCI-H226, renal cancer
cell line A498 and breast cancer cell line MDA-MB-468 in the single
dose screen (Table 2). Activities of all four compounds against
tumor cell line 498 were good, with ꢀÀ90 percentage growth at
The growth inhibitory or cytotoxicity effect of the test com-
pounds in the above cellular assay is measured by determining
percentage cell growth (PG) inhibition. Optical density (OD) mea-
surements of SRB-derived color just before exposing the cells to
the test compound (ODtzero) and after 48hrs exposure to the test
compound (ODtest) or the control vehicle (ODctrl) are recorded.15
Growth percentage is calculated utilizing one of the two formu-
las below. A negative growth percentage implies cytotoxicity
(Table 2).
10 lM. Although compounds 3h and 3e were not selected for com-
plete dose–response studies, compound 3h was effective against
the A498 cell line (À95 percentage growth), while compound 3e
was active against the MDA-MB-468 cell line (À71 percentage
growth) (Table 2).
Further evaluation of lead compounds 3c, 3d, 3f and 3g in the
five dose screen showed that these compounds were very effective
If (OD test - ODtzero) P 0, then

N. R. Madadi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 601–603
603
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against five particular cancer cell lines: NCI-H460, OVCAR-5, A498,
TK-10 and MDA-MB-468, with GI50 values in the nanomolar range.
Breast cancer cell line MDA-MB-468 appeared to be the most sen-
sitive to the growth inhibition effects of these compounds; 3c, 3d,
3f and 3g exhibited GI50 values of 30 nM, 40 nM, 60 nM, and
30 nM, respectively, with LC50 values of 620 nM, 760 nM, 700 nM,
and 500 nM, respectively, against this cell type. Compounds 3c,
3d, 3f and 3g also exhibited good growth inhibition against renal
cancer cell line A498, with GI50 values of 120 nM, 60 nM, 40 nM,
and 70 nM, respectively, and LC50 values of 690 nM, 527 nM,
640 nM, and 670 nM, respectively. All four compounds were active
against renal cancer cell line TK-10 with GI50 values of 280 nM,
100 nM, 180 nM, and 590 nM, respectively, and also exhibited
growth inhibitory effects against ovarian cancer cell line OVCAR-
5 (GI50 = 70 nM, 20 nM, 160 nM, and 110 nM, respectively) and
non-small cell lung cancer cell line NCI-H460 (GI50 = 910 nM,
810 nM, 400 nM, and 370 nM, respectively). Compound 3d also
inhibited the growth of colon cancer cell line COLO 205
(GI50 = 630 nM) and melanoma cell line UACC-62 (GI50 = 900 nM)
(see Table 3).
In conclusion, a series of novel aromatic substituted 5-((1-ben-
zyl-1H-indol-3-yl)-methyl-ene)-1,3-dimethylpyrimidine-2,4,6-
(1H,3H,5H)-trione analogs have been synthesized and evaluated
for growth inhibition properties against a panel of 60 human can-
cer cell lines, and their GI50 and LC50 values have been determined.
Four lead compounds (3c, 3d, 3f and 3g) have been identified with
GI50’s in the nanomolar range against 5 different cell lines. All four
compounds exhibited GI50 values in the range 30–60 nM and 40–
120 nM against breast cancer MDA-MB-468 and renal cancer A49
cell lines, respectively; compounds 3c and 3d afforded GI50 values
of 70 nM and 20 nM, respectively, against the ovarian cancer cell
line OVCAR-5. The above four compounds generally have superior
GI50 values compared to the previous lead compound 1 against
most of the cell lines in the 60 tumor cell line panel. The biggest
difference was the GI50 value of compound 1 against renal cancer
cell line A49 (GI50 = 300 nM) compared to GI50 values over the
range 40–120 nM for compounds 3c, 3d, 3f, and 3g. These novel
aromatic substituted 5-((1-benzyl-1H-indol-3-yl)-methyl-ene)-1,
3-dimethylpyrim-idine-2,4,6-(1H,3H,5H)-triones represent prom-
ising new analogs that may have clinical potential in treating a
variety of solid tumors.
12. Coggins, G. E.; Maddukuri, L.; Penthala, N. R.; Hartman, J. R.; Eddy, S.; Ketkar,
A.; Crooks, P. A.; Eoff, R. L. Chem. Biol. 2013, 8, 1722.
13. General experimental procedure: In a 50 ml round bottom flask the appropriate
indole carboxaldehyde (1 mmol), benzyl halide (1.1 mmol), triethylbenzyl
ammonium chloride (0.01 mmol) and 50% w/v aq NaOH were added to 5
volumes of DCM. The reaction mixture was stirred at room temperature and
monitored by TLC. When the reaction was completed, water was added and the
mixture extracted into DCM. The organic layer was concentrated under
reduced pressure at 40 °C and the residue was purified by flash
chromatography using methanol/DCM as mobile phase to afford the
corresponding N-benzylindole-3-carboxaldehydes in 80–85% yield. The N-
benzylindole-3-carboxaldehyde (1 mmol) and N,N-dimethylbarbituric acid
(1.2 mmol) were added to 10 volumes of methanol and the resulting mixture
stirred at room temperature. The final product, the appropriate 5-((1-benzyl-
1H-indol-3-yl)methylene)-1,3-dimethyl-pyrimidine-2,4,6(1H, 3H,5H)-trione
crashed out of the solution once the reaction was complete (1–2 h). The final
product was filtered and recrystallized from methanol to afford the 5-((1-
benzyl-1H-indol-3-yl)methylene)-1,3-dimethyl-pyrimidine-2,4,6-(1H,3H 5H)-
trione in 75–90% yield. Analytical data for compound 3d: Yellow solid; yield,
90%; mp >300 °C, 1H NMR (400 MHz, DMSO-d6): d 3.36 (s, 6H, N-CH3), 3.70 (s,
3H, –OCH3), 5.45 (s, 2H, –CH2), 6.89–6.91 (d, J = 8.8 Hz, 2H, ArH), 7.23–7.31 (m,
J = 30 Hz, 4H, ArH), 7.61–7.63 (d, J = 7.6 Hz, 1H, ArH), 8.09–8.12 (d, J = 7.6 Hz,
1H, ArH), 8.45 (s, 1H, ArH), 9.93 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): d
49.69, 55.44, 111.85, 111.96, 114.52, 117.74, 121.46, 121.53, 122.97, 124.01,
125.23, 128.99, 129.45, 137.31, 141.21, 159.31, 185.09. HRMS (ESI): m/z calcd
for C23H22N3O4 [MÀH] 404.1610; found 404.1606. Compound 3g: Yellow solid;
yield 85%; mp >300 °C, 1H NMR (400 MHz, DMSO-d6): d 3.24 (s, 6H, –N–CH3),
5.70 (s, 2H, –CH2), 7.17–7.22 (t, J = 17.6 Hz, 2H, ArH), 7.34–7.40 (m, J = 25.6 Hz,
4H, ArH), 7.67-7.69 (d, J = 8Hz, 1H, ArH), 7.87-7.89 (d, J = 8.4Hz, 1H, ArH), 8.75
(s, 1H, ArH), 9.66 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): 28.22, 28.85,
50.02, 109.33, 111.43, 112.61, 118.34, 118.43, 123.63, 124.47, 129.26, 129.65,
130.42, 133.00, 135.87, 136.77, 142.42, 144.15, 151.66, 162.03, 163.38. HRMS
(ESI): m/z calcd for C22H19N3O3F [MÀH] 392.1410; found 392.1389. Compound
3f: Yellow solid; yield, 90%; mp >300 °C, 1H NMR (400 MHz, DMSO-d6): d 3.24–
3.25 (d, J = 3.6 Hz, 6H, N-CH3), 5.71 (s, 2H, –CH2), 7.31–7.35 (m, J = 16.8 Hz, 4H,
ArH), 7.41–7.43 (d, J = 8 Hz, 2H, ArH), 7.63–7.65 (d, J = 8 Hz, 1H, ArH), 7.87–
7.89 (d, J = 6.4 Hz, 1H, ArH), 8.75 (s, 1H, ArH), 9.66 (s, 1H, ArH). 13C NMR
(100 MHz, DMSO-d6):
112.61, 118.35, 118.44, 123.63, 124.47, 129.26, 129.66, 130.43, 133.00, 135.88,
d 28.22, 28.27, 28.85, 28.90, 50.02, 109.34, 111.43,
Acknowledgements
136.77, 142.42, 144.16, 151.66, 162.03, 163.39. HRMS (ESI): m/z calcd for
We thank Dr. Howard Hendrickson for providing the HRMS
data. This research was supported by NIH/National Cancer Institute
grant CA140409, and by an Arkansas Research Alliance grant. We
are grateful to the National Cancer Institute for anticancer screen-
ing data.
C
22H19N3O3Cl [MÀH] 408.1115; found 408.1122. Compound 3c: Yellow solid;
yield, 90%; mp >300 °C, 1H NMR (400 MHz, DMSO-d6): d 2.24 (s, 3H, –CH3),
3.23–3.24 (d, J = 3.2 Hz, 6H, N–CH3), 5.62 (s, 2H, –CH2), 7.14–7.16 (d, J = 7.6 Hz,
2H, ArH), 7.20–7.22 (d, J = 7.6 Hz, 2H, ArH), 7.32–7.33 (t, J = 5.6 Hz, 2H, ArH),
7.63-7.65 (d, J = 7.6 Hz, 1H, ArH), 7.85–7.86 (d, J = 6.8 Hz, 1H, ArH), 8.73 (s, 1H,
ArH), 9.64 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): d 28.21, 28.84, 109.16,
111.34, 118.47, 123.46, 123.75, 127.68, 127.92, 129.72, 129.82, 129.90, 130.47,
133.78, 136.90, 137.67, 142.38, 142.56, 144.32, 151.72, 162.09, 163.47. HRMS
(ESI): m/z calcd for C23H22N3O3 [MÀH] 388.1661; found 388.1636.
References and notes
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15. NCI screening services. www.dtp.nci.nih.gov.
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