Synthesis and in vitro screening of novel N-benzyl aplysinopsin analogs as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2011.01.020

A series of novel substituted (Z)-5-((1-benzyl-1H-indol-3-yl)methylene) imidazolidin-2,4-diones (3a-f) and (Z)-5-((1-benzyl-1H-indol-3-yl)methylene)-2- iminothiazolidin-4-ones (3g-o) have been synthesized utilizing microwave irradiation. These analogs wer

Full text of DOI:10.1016/j.bmcl.2011.01.020

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1411–1413
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro screening of novel N-benzyl aplysinopsin
analogs as potential anticancer agents
⇑
Narsimha Reddy Penthala, Thirupathi Reddy Yerramreddy, Peter A. Crooks
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel substituted (Z)-5-((1-benzyl-1H-indol-3-yl)methylene)imidazolidin-2,4-diones (3a–f)
and (Z)-5-((1-benzyl-1H-indol-3-yl)methylene)-2-iminothiazolidin-4-ones (3g–o) have been synthe-
sized utilizing microwave irradiation. These analogs were evaluated for in vitro cytotoxicity against a
panel of 60 human tumor cell lines. Compound 3i exhibits potent growth inhibition against melanoma
UACC-257 (GI50 = 13.3 nM) and OVCAR-8 ovarian (GI50 = 19.5 nM) cancer cells while possessing signifi-
cant cytotoxicity (LC50 = 308 nM and LC50 = 851 nM, respectively) against the same cell lines within this
series of compounds. A second analog, 3a, had GI50 values of 307 and 557 nM against SK-MEL-2 mela-
Received 8 December 2010
Revised 5 January 2011
Accepted 6 January 2011
Available online 11 January 2011
Keywords:
N-Benzyl indole-3-carboxaldehydes
Hydantoin
noma and A498 renal cancer cell lines, and exhibited GI50 values ranging from 0.30 to 6 lM against
98% of all cancer cell lines in the 60-cell panel. Thus, (Z)-5-((5-chloro-1-(4-fluorobenzyl)-1H-indol-3-
yl)methylene)-2-iminothiazolidin-4-one (3i) and (Z)-methyl 1-(4-cyanobenzyl)-3-((2,5-dioxoimidazoli-
din-4-ylidene)methyl)-1H-indole-6-carboxylate (3a) can be regarded as useful lead compounds for fur-
ther structural optimization as antitumor agents.
2-Imino thiazolidin-4-one
In vitro cytotoxicity
Ó 2011 Elsevier Ltd. All rights reserved.
Considerable effort has been focused on the development of
anti-cancer agents that contain an indole moiety as their main
structural motif.1–5 We have recently reported on the antitumor
activity of a number of novel indole barbituric acid analogs and re-
lated compounds that act as both radiosensitizing agents and anti-
cancer agents (e.g., Fig. 1. Structure A).6,7 Recently, our laboratory
reported on the cytotoxicity of a series of (Z)-2-amino-5-(1-ben-
zyl-1H-indol-3-yl)-methylene-1-methyl-1H-imidazol-4(5H)-ones
against human tumor cell lines (e.g., Fig. 1. Structure B).8 In this re-
spect, Li et al. have synthesized and studied a series of structurally
related N-heterocyclic indolyl glyoxylamides (Fig. 1. Structure C),
and found that such compounds possess marked activity against
several cancer cell lines, including multidrug resistance (MDR) cell
lines.9 N-Benzylindole and indolizine glyoxyl amides (Fig. 1. Struc-
ture D) have also been shown to exhibit substantial in vitro anti-
proliferative activity against various cancer cell lines, including
hematologic and solid tumor cell lines, that is, leukemia, breast, co-
lon, and uterine.10
In the synthesis of analogs 3a–o, a series of substituted N-ben-
zylindole-3-carboxaldehydes were initially synthesized in 85–90%
yield by reacting the appropriate indole-3-carboxaldehyde with
various substituted benzyl halides under phase-transfer catalytic
(PTC) conditions using triethylbenzyl ammonium chloride (TEBA)
and 50% w/v aqueous NaOH solution in dichloromethane
(Scheme 1). Aldol condensation of the appropriate N-benzylin-
dole-3-carboxaldehyde with hydantoin or 2-iminothiazolidin-4-
one, in the presence of CH3COOH and sodium acetate under micro-
wave irradiation conditions (1100 W; Kenmore, 30–60 s)
(Scheme 1), afforded
a series of (Z)-5-((1-benzyl-1H-indol-3-
yl)methylene)imidazolidin-2,4-diones (3a–f) and (Z)-5-((1-ben-
zyl-1H-indol-3-yl)methylene)-2-iminothiazolidin-4-ones (3g–o)
(Table 1), respectively, in yields ranging from 85–93%. All the syn-
thesized compounds were fully characterized by 1H NMR and 13C
NMR.12
The in vitro screening studies involved a two-stage process in
the evaluation of compounds (3a–o) against a 60 human tumor cell
line panel. In the first stage of the screen, compounds which
showed more than 60% growth inhibition in at least eight of the
In our continuing efforts to develop small molecules that func-
tion as novel anticancer agents we have now synthesized a series
of (Z)-5-((1-benzyl-1H-indol-3-yl)methylene)imidazolidin-2,4-
diones (3a–f) and a series of (Z)-5-((1-benzyl-1H-indol-3-yl)meth-
ylene)-2-iminothiazolidin-4-ones (3g–o) as second generation
analogs of the aplysinopsin analog B (Fig. 1).
60 tumor cell lines in single dose (10 lM) studies, were selected
for the second stage five dose–response studies, according to the
procedures described by Rubinstein et al.11
The human tumor cell line panel included leukemia, non-small
cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and
breast cancer cell lines. From the single dose–response studies
the imidazolidine-2,4-dione analog (3a) showed >60% growth inhi-
bition in 50 of the 60 cancer cell lines utilized. The remainder of the
⇑
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.020

1412
N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1411–1413
Figure 1. Cytotoxic indole-derived aplysinopsin analogs (A–D).
compounds 3i, 3j, and 3k with growth inhibitory activity against
colon HCT-116 cells. Analog 3k was also active against melanoma
LOX IMVI cells. Analog 3i exhibited the best growth inhibitory
properties in this series.
The two most active compounds (3a and 3i) from the prelimin-
ary 60 cell screen were evaluated in five dose–response studies for
their in vitro cytotoxic effects on growth parameters against each
of the 60 human tumor cell lines. Dose-response curves were cre-
ated by plotting cytotoxic effect against the log10 of the drug con-
centration for each cell line. Cytotoxic effects of each compound
were determined as GI50, and LC50 values, which represent the mo-
lar drug concentration required to cause 50% growth inhibition,
and the concentration that kills 50% of the cells, respectively. The
results are presented in Table 2.
Scheme 1. Reagents and conditions: (a) Appropriate benzyl halides, 50% NaOH,
CH2Cl2, TEBA, 2 h; (b) Hydantoin or 2-iminothiazolidin-4-one, sodium acetate,
acetic acid, microwave irradiation, 30–60 s, 85–93% yield.
Table 1
Table 2
Antitumor activity (GI50
five dose studies for the NCI 60-cell lines screen
N-Benzyl aplysinopsin analogs (3a–o)
/
l
M)a and toxicity (LC50
/
l
M)b data of compounds selected for
S.No.
R1
R2
R3
X
Y
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
COOCH3
H
H
H
Cl
OCH3
H
H
H
p-CN
p-CN
p-CN
H
H
H
o-F
o-F
p-F
o-F
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
S
S
S
S
Panel/cell line
Compound 3a
Compound 3i
COOCH3
H
H
H
H
H
Cl
Cl
Br
Br
H
H
COOCH3
COOCH3
GI50
LC50
GI50
LC50
Leukemia cancer
CCRF-CEM
HL-60(TB)
K-562
3.06
2.54
0.97
1.38
3.61
>100
>100
>100
>100
>100
>100
>100
77.6
>100
>100
>100
>100
>100
>100
>100
MOLT-4
H
H
H
RPMI-8226
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
p-F
4.44
4.22
3.58
na
>100
>100
73.0
na
0.77
>100
>100
>100
80.7
60.1
71.0
>100
11.0
>100
>100
>100
COOCH3
COOCH3
H
H
p-CN
p-COOCH3
p-CN
p-COOCH3
S
S
S
S
NCI-H226
NCI-H23
5.94
4.07
0.72
2.91
na
>100
>100
>100
>100
na
>100
>100
>100
>100
>100
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
analogs in this series did not qualify for five dose–response screen-
ing. In the 2-iminothiazolidin-4-one series, analog (3i) showed
more than 60% growth inhibition in 14 of the 60 cancer cell lines.
The other compounds in the same series (3g–h and 3j–o) did not
qualify for five dose testing.
3.16
2.43
1.21
na
>100
85.0
>100
na
>100
>100
5.41
>100
20.8
7.49
38.3
>100
>100
>100
>100
>100
>100
>100
Some structure–activity data could be generated from the initial
single dose studies. In the para-cyano-N-benzylindoleimidazoli-
dine-2,4-dione series (3a–c), the 6-carbomethoxyindole analog
3a exhibited the best growth inhibition, and moving this substitu-
ent to the 5-indolic carbon (analog 3b) resulted in a complete loss
of growth inhibition, indicating a significant regiospecific require-
ment for this substituent. However, in the N-benzyl substituted in-
dole-2-iminothiazolidin-4-one series (3g–o), both the 5- and 6-
carbomethoxyindole analogs 3o and 3m exhibited growth inhibi-
tion against colon KM12 and CNS U251 cells, although these ana-
logs contained a para-carbomethoxy-N-benzyl moiety rather that
a para-cyano-N-benzyl moiety. In addition, unlike 3m, analog 3o
was active against colon HCT-116 cells and lung NCI-H460 cells.
Interestingly, the presence of a 5-indolic halogeno substituent (Cl
or Br) and an indolic N-para-fluorobenzyl or N-ortho-fluorobenzyl
moiety in the indole-2-iminothiazolidin-4-one series afforded
5.99
3.73
2.27
>100
>100
>100
4.22
3.09
2.39
3.77
4.60
2.03
>100
>100
>100
>100
>100
59.1
32.0
>100
4.40
>100
2.47
2.94
>100
>100
>100
>100
>100
>100
SNB-75
U-251
Melanoma cancer
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
1.43
2.01
1.43
2.76
0.30
3.79
>100
>100
>100
>100
30.6
2.32
23.5
57.2
32.4
>100
>100
>100
>100
>100
>100
>100
>100
>100

N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1411–1413
1413
tively), UACC-62, LOX IMVI and M14 melanoma (GI50 = 1.27, 1.43
Table 2 (continued)
and 1.43
lines.
lM, respectively), and MOLT-4 leukemia (GI50 = 1.38) cell
Panel/cell line
Compound 3a
Compound 3i
GI50
LC50
GI50
LC50
Of particular significance was the observation that the introduc-
tion of an indolic 6-carboxymethyl group and an N-para-cyanoben-
zyl moiety afforded an analog (3a) which exhibited effective
growth inhibition in all but one of the cancer cell lines tested. In
addition, in the 2-iminothiazolidin-4-one series of compounds,
the 5-chloro-(p-fluoro-N-benzyl)indole analog (3i) exhibited very
potent growth inhibition of UACC-257 melanoma and OVCAR-8
ovarian cancer cell lines, affording GI50 values of 13.3 and
19.5 nM, respectively.
In conclusion, a series of novel substituted (Z)-5-((1-benzyl-1H-
indol-3-yl)methylene)imidazolidin-2,4-diones (3a–f) (Z)-5-((1-
benzyl-1H-indol-3-yl)methylene)-2-iminothiazolidin-4-ones
(3g–o) have been synthesized and evaluated for their anticancer
activity against a panel of 60 human tumor cell lines. Two analogs,
(Z)-methyl 1-(4-cyanobenzyl)-3-((2,5-dioxoimidazolidin-4-yli-
dene)methyl)-1H-indole-6-carboxylate (3a) and (Z)-5-((5-chloro-
1-(4-fluorobenzyl)-1H-indol-3-yl)methylene)-2-iminothiazolidin-
4-one (3i) emerged as useful lead compounds for further structural
optimization as novel anticancer agents.
SK-MEL-5
UACC-257
UACC62
3.25
4.20
1.27
>100
>100
51.0
>100
0.013
>100
>100
0.30
>100
Ovarian cancer
IGR-OV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
0.66
2.05
4.13
5.51
3.75
>100
0.86
>100
>100
>100
>100
>100
>100
79.8
3.02
>100
2.49
>100
0.019
>100
>100
>100
30.1
>100
>100
>100
>100
>100
2.36
0.55
2.08
2.74
0.58
2.80
3.87
2.70
>100
>100
>100
>100
>100
>100
>100
>100
37.4
31.5
>100
>100
1.62
>100
5.64
>100
>100
>100
>100
>100
>100
>100
>100
>100
TK-10
UO-31
Prostate cancer
PC-3
DU-145
2.44
na
>100
na
23.6
5.24
>100
>100
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
Acknowledgments
3.55
3.32
0.97
1.24
1.50
4.14
>100
>100
>100
>100
>100
>100
>100
6.45
3.67
98.8
>100
>100
>100
>100
>100
>100
>100
>100
We are grateful to the NCI/NIH for their financial support under
grant number CA 140409, and to the NCI Developmental Therapeu-
tic Program (DTP) for screening data.
T-47D
MDA-MB-468
NA: Not analyzed.
References and notes
a
GI50: 50% Growth inhibition, concentration of drug resulting in a 50% reduction
in net protein increase compared with control cells.
1. Narisawa, T.; Sato, M.; Sano, M.; Niwa, M.; Takahashi, S.; Ito, T.; Tanida, N.;
Shimoyama, T. Cancer 1985, 56, 1719.
b
LC50: Lethal concentration, concentration of drug lethal to 50% of cells.
2. Rosenbaum, C.; Rohrs, S.; Muller, O.; Waldmann, H. J. Med. Chem. 2005, 48,
1179.
3. Hollenbeak, K. H.; Schmitz, F. J. Lloydia 1977, 40, 479.
4. Bialonska, D.; Zjawiony, J. K. Mar. Drugs 2009, 7, 166.
Compound 3i exhibited growth inhibitory properties against
52% of all cancer cell lines in the panel, with GI50 values in the
range of 0.19–98.8 lM. (Table 2). Good growth inhibitory activity
was observed against UACC-257 melanoma (GI50 = 13.3 nM), OV-
CAR-8 ovarian cancer (GI50 = 19.5 nM), and A549/ATCC non-small
cell lung cancer (GI50 = 774 nM) cell lines, and moderate growth
inhibition was observed against RXF 393 renal cancer
5. Weng, J. R.; Tsai, C. H.; Kulp, S. K.; Chen, C. S. Cancer Lett. 2008, 262, 153.
6. Reddy, Y. T.; Sekhar, K. R.; Sasi, N.; Reddy, P. N.; Freeman, M. L.; Crooks, P. A.
Bioorg. Med. Chem Lett. 2010, 20, 600.
7. Vijayakumar, N. S.; Reddy, Y. T.; Sekhar, K. R.; Soumya, S.; Freeman, M. L.;
Crooks, P. A. Bioorg. Med. Chem. Lett. 2007, 17, 6821.
8. Reddy, P. N.; Reddy, Y. T.; Crooks, P. A. Bioorg. Med. Chem Lett. 2010, 20, 591.
9. Li, W.-T.; Hwang, D.-R.; Chen, C.-P.; Shen, C.-W.; Huang, C.-L.; Chen, T.-W.; Lin,
C.-H.; Chang, Y.-L.; Lo, Y.-K.; Tseng, H.-Y.; Lin, C.-C.; Song, J.-S.; Chen, H.-C.;
Chen, S.-J.; Wu, S.-H.; Chen, C.-T. J. Med. Chem. 2003, 46, 1706.
10. James, D. A.; Koya, K.; Li, H.; Liang, G.; Xia, Z.; Ying, W.; Wu, Y.; Sun, L. Bioorg.
Med. Chem. Lett. 2008, 18, 1784.
(GI50 = 1.62
and U251 CNS cancer (GI50 = 2.47 and 2.94
OVCAR-4 ovarian cancer (GI50 = 2.49 M) cell lines.
l
M), LOX IMVI melanoma (GI50 = 2.32
lM), SNB-75
l
M, respectively), and
l
11. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simo, R. M.; Tosini, S.; Skehan,
P.; Scudiero, P. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
Compound 3a exhibited growth inhibitory properties against
98% of all cancer cell lines in the panel, with GI50 values in the
12. Analytical data for compound 3a. MF:
C
22H16N4O4, mp: >300 °C, 1H NMR
(300 MHz, DMSO-d6): d 3.82 (s, 3H, OCH3), 5.68 (s, 2H, CH2), 6.71 (s, 1H, CH),
7.38–7.40 (d, J = 8.1 Hz, 2H, Ar-H), 7.72–7.93 (m, 4H, Ar-H), 8.13 (s, 1H, Ar-H),
8.45 (s, 1H, Ar-H), 10.27 (br s, 1H, NH), 11.15 (br s, 1H, NH) ppm; 13C NMR
(75 MHz, DMSO-d6): d 50.00, 52.78, 100.55, 109.65, 111.12, 112.99, 119.26,
121.84, 124.58, 125.76, 128.45 (2C), 131.55, 133.34 (2C), 133.87, 135.66,
143.56, 155.93, 161.86, 165.79, 167.29 ppm.
range of 0.30–6
inhibitory activity against SK-MEL-2 melanoma cell lines
(GI50 = 307 nM; LC50 = 30.6 M), A498 (GI50 = 557 nM; LC50
>100 M) and RXF 393 renal cancer (GI50 = 585 nM; LC50 = >100
M), IGROV1 and SK-OV-3 ovarian cancer (GI50 = 667 and
866 nM, respectively), NCI-H322M non-small cell lung cancer
(GI50 = 721 nm, LC50 = >100), HS 578T breast cancer
lM (Table 2). Compound 3a exhibited good growth
l
=
l
l
Analytical data for compound 3i. MF: C19H13ClFN3OS, mp: >300 °C, 1H NMR
(300 MHz, DMSO-d6): d 5.55 (s, 2H, CH2), 7.14–7.33 (m, 5H, Ar-H & CH), 7.57–
7.58 (d, 1H, J = 5.4 Hz, Ar-H), 7.81 (s, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.95 (s, 1H,
C2-H), 9.00 (br s, 1H, NH), 9.24 (br s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-
d6): d 49.66, 111.29, 113.74, 114.45, 116.06, 116.33, 120.81, 121.98, 125.34,
126.24, 129.74, 130.02, 131.53, 133.84, 135.41, 160.54, 163.77, 174.96, 181.07
ppm.
(GI50 = 971 nM; LC50 = >100
(GI50 = 972 nM; LC50 = >100
l
l
M), and K-562 leukemia cell lines
M), and showed moderate growth
inhibitory activity against HCT-116 colon cancer (GI50 = 1.21
lM),
BT-549 and T-47D breast cancer (GI50 = 1.24 and 1.50 M, respec-
l