Microwave assisted synthesis and in vitro cytotoxicities of substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-ones against human tumor cell lines

Article abstract of DOI:10.1016/j.bmcl.2009.11.083

The synthesis of several novel substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-ones structurally related to aplysinopsin have been carried out under microwave irradiation and conventional heating methods. The analogs 3a, 3b, 3d-3g, 3k and 3l were evaluated for their in vitro cytotoxic activity against an NCI 60 human tumor cell line panel. Compound 3f exhibited good growth inhibitory properties against all but four of the human cancer cell lines examined, and afforded LC₅₀ values <10 μM for 30% of the cell lines in the panel. Compound 3e was an effective inhibitor of leukemia, CNS, melanoma, and breast cancer cell growth, but generally less effective as a cytotoxic agent. Thus, the aplysinopsin analog 3f was regarded as a useful lead compound for further structural optimization.

Full text of DOI:10.1016/j.bmcl.2009.11.083

Bioorganic & Medicinal Chemistry Letters 20 (2010) 591–593
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave assisted synthesis and in vitro cytotoxicities of substituted
(Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-
imidazol-4(5H)-ones against human tumor cell lines
*
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:
The synthesis of several novel substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-
1H-imidazol-4(5H)-ones structurally related to aplysinopsin have been carried out under microwave
irradiation and conventional heating methods. The analogs 3a, 3b, 3d–3g, 3k and 3l were evaluated
for their in vitro cytotoxic activity against an NCI 60 human tumor cell line panel. Compound 3f exhibited
good growth inhibitory properties against all but four of the human cancer cell lines examined, and affor-
Received 19 October 2009
Revised 16 November 2009
Accepted 17 November 2009
Available online 22 November 2009
ded LC50 values <10 lM for 30% of the cell lines in the panel. Compound 3e was an effective inhibitor of
Keywords:
N-Benzyl indole-3-carboxaldehydes
Creatinine
leukemia, CNS, melanoma, and breast cancer cell growth, but generally less effective as a cytotoxic agent.
Thus, the aplysinopsin analog 3f was regarded as a useful lead compound for further structural
optimization.
In vitro cytotoxicity
Ó 2009 Elsevier Ltd. All rights reserved.
In the past several decades researchers have been challenged by
the task of identifying effective clinical agents to treat cancer,
which is the second leading cause of death in the United States.1
The World Cancer Congress (WCC) has released a report stating
that 8 million people died from cancer in 2008, and 12 million peo-
ple were suffering from cancer during the same time period. Anti-
cancer drugs such as cisplatin, 5-fluorouracil, paclitaxel, and
docetaxel, are some of the major chemotherapeutic agents cur-
rently being used to treat cancer.2 However, research is still needed
to discover newer, more effective anticancer agents. Indole-derived
aplysinopsin analogs (Fig. 1. structure A) have been reported to be
cytotoxic agents against cancer cells.3,4 Li et al.5 have synthesized
and studied a series of structurally related N-heterocyclic indo-
lylglyoxylamides (Fig. 1. structure B) and found that such com-
pounds possess interesting activity against several cancer cell
lines, including multidrug resistance (MDR) cell lines. James
et al.6 also reported structure–activity relationship (SAR) studies
clidin-3-ol as potent thermal-sensitizers capable of lowering the
threshold for Hsf1 activation and thermal sensitivity. These com-
pounds were considered as potential thermal radiosensitization
agents.7
In continuation of our work on the design and synthesis of
substituted (Z)-5-(N-benzyl-1H-indol-3-yl)methylene derivatives
we focused on a series of novel (Z)-2-amino-5-(1-benzyl-1H-in-
dol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)ones structurally
related to aplysinopsin that incorporated electron donating and
electron withdrawing substituents in both the indolic ring and
the phenyl ring of the N-benzyl moiety.
The aromatic substituted N-benzylindole-3-carboxaldehydes
were synthesized in 85–90% yield by treating the appropriately
substituted indole-3-carboxaldehyde with various substituted
benzyl halides under phase-transfer catalytic (PTC) conditions uti-
lizing triethylbenzyl ammonium chloride (TEBA) and a mixture of
dichloromethane in 50% w/v aqueous NaOH solution (Scheme 1).
Aldol condensation of the appropriate N-benzylindole-3-carboxal-
dehyde with creatinine, in the presence of CH3COOH/sodium ace-
tate utilizing either conventional heating or microwave
irradiation methodologies (Scheme 1), afforded a series of novel
substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)-methylene-
1-methyl-1H-imidazol-4(5H)-one analogs. Microwave irradiation
using an open vessel in a domestic microwave oven (1100 W; Ken-
more) at atmospheric pressure was found to be more advanta-
geous than conventional heating, and afforded product yields in
the range of 85–91% compared to 70–83% for the latter method
(Table 1). In addition, reaction times were very short with
on
a series of N-benzylindole and indolizine glyoxylamides
(Fig. 1. structure C) that exhibit substantial in vitro anti-prolifera-
tive activity against various cancer cell lines, including hemato-
logic and solid tumor cell lines (i.e., leukemia, breast, colon,
and uterine). As the part of a drug discovery program to discover
and develop small molecules as potential anticancer agents, we
identified (Z)-2-(N-benzylindol-3-ylmethylidene)quinuclidin-3-ol
and (Z)-( )-2-[N-(4-chlorobenzyl)indole-3-yl-methylidene]quinu-
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.083

592
N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 20 (2010) 591–593
R3
NHR1
NHR1
O
R1
N
O
O
N
O
N
R2
O
N
N
R2
N
Het
H
B
C
A
Figure 1. Cytotoxic indole-derived aplysinopsin analogs (A–C).
Scheme 1. Synthesis of (Z)-2-amino-5-(1-benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-one analogs. Reagents and conditions: (a) appropriate benzyl
halide, aqueous NaOH solution, triethylbenzyl ammonium chloride, DCM, rt; (b) creatinine (1.1 mol equiv), NaOAc (1.2 mol equiv), AcOH, MWI, 30–60 s; (c) creatinine
(1.1 mol equiv), NaOAc (1.2 mol equiv), AcOH, reflux, 7–10 h.
microwave irradiation (30–60 s) compared to conventional heating
(7–10 h). All the synthesized compounds were fully characterized
by 1H NMR, 13C NMR and mass spectral analysis.9
The in vitro screening studies involved a two-stage process with
preliminary evaluation of compounds 3a, 3b, 3d–3g, 3k and 3l
against a 60 human tumor cell line panel at a single dose of
small cell lung, colon, CNS, melanoma, ovarian, renal, prostate,
and breast cancer cell lines. Only compounds which showed more
than 60% growth inhibition in at least eight of the 60 tumor cell
lines were selected for further dose–response studies; the remain-
ing compounds were not further investigated.
The two most active compounds (3e and 3f) from the prelimin-
ary 60 cell screen were subsequently evaluated in five dose–re-
sponse studies for their in vitro cytotoxic effects on growth
parameters against each of the 60 human tumor cell lines. Dose–
response curves were created by plotting cytotoxic effect against
the log10 of the drug concentration for each cell line. Cytotoxic ef-
fects of each compound were determined as GI50 and LC50 values,
which represent the molar 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. Except
for EVVX (non-small cell lung), UACC-257 (melanoma), A498 (re-
nal) and CAKI-1 (renal) cell lines, compound 3f afforded GI50 values
10 lM, according to the procedures described by Rubinstein
et al.8 The human tumor cell line panel included leukemia, non-
Table 1
Reaction times and yields of novel substituted (Z)-2-amino-5-(1-benzyl-1H-indol-3-
yl)methylene-1-methyl-1H imidazol-4(5H)-ones
Compds
Method A
Time (s)
(Microwave condition)
Method B
Time (h)
(Conventional heating)
Yield (%)
Yield (%)
3a
3b
3c
3d
3e
3f
3g
3h
3i
90
40
60
60
30
40
60
30
40
60
50
60
50
60
79
7
8
10
9
7
10
7
9
10
8
10
9
10
in the range 1.46–9.46
with 85% of these GI50 values falling in the range 1.46–2.93
Of particular interest was the effect of 3f on cell lines HCC-2998
(colon; GI50 = 2.99 M; LC50 = 4.71 M), SF-539, and SNB-75
(CNS; GI50 = 1.54 M and 1.46 M, respectively; LC50 = 5.56
and 5.47 M, respectively), SK-MEL-28 and SK-MEL-5 (melanoma;
GI50 = 1.78 and 1.69 M, respectively; LC50 = 6.14 and
5.75 M, respectively), 786-0 and ACHN (renal; GI50 = 1.75
and 1.76 M, respectively; LC50 = 5.85 M and 6.14 M, respec-
tively), and T-47D and MDA-MB-468 (breast; GI50 = 1.50 M and
1.63 M, respectively; LC50 = 5.74 M and 6.78 M, respectively).
lM against all the other cell lines utilized,
85
88
86
91
87
86
89
87
88
86
87
89
71
83
73
82
75
70
77
81
78
80
74
72
lM.
l
l
l
l
lM
l
lM
l
lM
3j
3k
3l
l
lM
l
l
l
l
3m
l
l
l

N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 20 (2010) 591–593
593
Table 2
Compound 3f exhibited generally poor LC50 values against leuke-
mia and non-small cell lung cancer cell lines. With the exception
of the NCI/ADR-RES ovarian cancer cell line, compound 3e exhib-
ited growth inhibitory effects against all the cell lines tested, with
Antitumor growth inhibitory activity (GI50/l /l
M)a and cytotoxicity (LC50 M)b data for
compounds 3e and 3f in five dose studies against an NCI 60-cancer cell line panel
Panel/cell line
Compound 3e
Compound 3f
GI50 values ranging from 1.19 to 82.1
affording GI50 values falling in the range 1.19–4.57
growth inhibitory activity was observed against leukemia
l
M, and with 77% of the cells
GI50
LC50
GI50
LC50
lM. Good
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
2.54
1.85
2.45
4.57
1.19
1.96
36.2
21.0
77.4
>100
65.9
72.7
2.91
2.67
2.23
2.42
3.25
2.48
>100
>100
>100
>100
>100
>100
(GI50 = 1.19–4.57
(GI50 = 1.68–4.07
l
M), CNS (GI50 = 1.58–5.38
lM), and breast
l
M) cell line sub-panels. Generally, 3e exhibited
poorer LC50 values compared to those obtained for 3f. Most notable
were the effects of 3e against HT29 (colon; GI50 = 2.18 M;
LC50 = 6.94 M), SF-539 (CNS; GI50 = 1.73 M; LC50 = 6.94 M), OV-
CAR-3 (ovarian; GI50 = 1.95 M; LC50 = 7.35 M), and MDA-MB-
468 (breast; GI50 = 1.69 M; LC50 = 6.90 M) cell lines. The results
l
l
l
l
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
28.7
58.1
8.78
2.98
1.37
8.07
11.0
3.95
5.38
>100
>100
51.2
44.5
69.9
54.8
>100
56.8
>100
5.05
>100
1.99
1.51
2.26
3.23
2.56
2.55
1.93
>100
>100
33.0
7.75
>100
58.8
>100
>100
>100
l
l
l
l
indicate that introduction of a 2-bromo group (3e) or a 4-carboxy-
methyl moiety (3f) into the N-benzyl moiety enhanced the activity
of N-benzyl-indolecreatinine (3a), compared to the introduction of
4-cyano (3b), 4-chloro (3d), and 4-fluoro (3g) groups.
In summary, a series of novel substituted (Z)-2-amino-5-(1-
benzyl-1H-indol-3-yl)methylene-1-methyl-1H-imidazol-4(5H)-one
analogs have been synthesized and evaluated for anticancer activ-
ity against a panel of 60 human cancer cell lines. Compounds 3e
and 3f were identified as molecules of interest from a single dose
assay, and were then evaluated for dose-dependent growth inhibi-
tion and cytotoxicity in all 60 human cancer cell lines. Compound
3f exhibited good growth inhibitory properties against all but four
of the human cancer cell lines examined, and afforded LC50 values
Colon
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
2.21
6.95
2.42
19.8
2.18
5.71
3.68
12.3
47.2
30.9
>100
6.94
50.8
43.3
1.91
2.99
1.79
2.27
2.17
3.18
2.20
nd
4.71
7.70
>100
nd
>100
>100
KM12
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
5.38
4.96
1.73
4.50
1.58
3.01
59.8
>100
6.94
43.8
18.6
66.2
2.56
2.36
1.54
2.39
1.46
1.81
>100
>100
5.56
31.0
5.47
7.72
<10 lM for 30% of the cell lines in the panel. Compound 3e was an
effective inhibitor of leukemia, CNS, melanoma, and breast cancer
cell growth, but was generally less effective as a cytotoxic agent.
Thus, the aplysinopsin analog 3f was regarded as a useful lead
compound for further structural optimization in the search for
anticancer agents with clinical potential.
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
1.97
3.30
4.46
4.97
5.73
4.14
3.38
12.9
9.37
73.8
47.8
68.6
>100
38.4
42.3
66.9
1.89
1.70
1.90
1.93
2.70
1.78
1.69
17.9
8.66
8.33
7.25
nd
>100
6.14
5.75
>100
Acknowledgments
We are grateful to the NCI/NIH for their financial support under
Grant Number PO1 CA104457 and to the NCI Developmental Ther-
apeutic Program (DTP) for screening data.
Ovarian
References and notes
IGR-OV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
20.0
1.95
2.41
2.12
10.1
>100
11.9
>100
7.35
25.2
29.0
>100
>100
49.9
5.91
2.29
2.18
1.90
3.45
9.46
1.98
>100
7.98
>100
18.5
>100
>100
8.68
1. De, M.; Jessica, K.; Boger, D. L. Drugs Future 2008, 33, 969.
2. Curran, W. J. Oncology 2002, 63, 29.
3. Hollenbeak, K. H.; Schmitz, F. J. Lloydia 1977, 40, 479.
4. Dobroslawa, B.; Jordan, K. Z. Mar. Drugs 2009, 7, 166.
5. 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.
6. James, D. A.; Koya, K.; Li, H.; Liang, G.; Xia, Z.; Ying, W.; Wu, Y.; Sun, L. Bioorg.
Med. Chem. Lett. 2008, 18, 1784.
Renal
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
6.15
13.9
11.8
68.1
2.77
6.07
6.84
7.95
46.2
53.1
59.0
>100
53.7
76.3
>100
>100
1.75
14.1
1.76
35.1
1.95
1.74
3.16
3.20
5.85
53.2
6.14
>100
8.71
8.59
>100
>100
7. Sonar, V. N.; Thirupathi Reddy, Y.; Sekhar, K. R.; Sasi, S.; Freeman, M. L.; Crooks,
P. A. Bioorg. Med. Chem. Lett. 2007, 17, 6821.
8. 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 lnst. 1990, 82, 1113.
9. Analytical data for compound 3e: 1H NMR (DMSO-d6): d 3.28 (s, 3H, N–CH3), 5.53
(s, 2H, CH2), 6.53 (s, 1H, CH), 6.76–6.79 (d, 1H, J = 6.9 Hz C4–H), 7.17–7.27 (m,
4H, Ar–H), 7.45–7.47 (t, 1H, J = 7.5 Hz C5–H), 7.68–7.70 (t, 1H, J = 6 Hz, C6–H),
7.71 (br s, 2H, NH2), 7.95–7.98 (d, 1H, J = 8.1 Hz, C7–H), 9.12 (s, 1H, C2–H) ppm
13C NMR (DMSO-d6): d 27.81, 49.59, 103.49, 109.08, 110.16, 118.30, 119.80,
121.97, 122.10, 127.93, 128.11, 128.61, 129.49, 131.19, 131.42, 132.62, 135.49,
136.17, 164.97, 175.11 ppm ES-API LC–MS m/z: 409.8 and 410.8 (MH+).
Analytical data for compound 3f: 1H NMR (DMSO-d6): d 3.49 (s, 3H, N–CH3), 3.81
(s, 3H, OCH3), 5.70 (s, 2H, CH2), 7.20 (s, 1H, CH), 7.23–7.26 (m, 2H, C4–H and
C7–H), 7.30–7.32 (d, 2H, J = 8.4 Hz, Ar–H), 7.52–7.55 (t, 1H, J = 9.0 Hz, C5–H),
7.90–7.93 (d, 2H, J = 8.1 Hz, Ar–H), 8.09–8.12 (t, 1H, J = 9.3 Hz, C6–H), 9.03 (s, 1H,
C2–H). 9.31 (br s, 2H, NH2) ppm 13C NMR (DMSO-d6): d 28.74, 49.36, 52.16,
108.23, 110.93, 113.56, 118.75, 120.96, 123.67, 127.09, 128.25, 128.76, 129.48,
133.59, 135.64, 142.55, 151.73, 161.55, 165.65 ppm ES-API LC–MS m/z: 388.90
(MH+).
Prostate
PC-3
DU-145
10.4
82.1
48.1
68.8
6.27
2.25
>100
14.8
Breast
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
2.25
4.07
3.97
2.05
1.68
1.69
48.1
46.7
>100
>100
13.1
6.90
2.45
2.01
2.17
1.73
1.50
1.63
77.8
14.3
>100
>100
5.74
6.78
T-47D
MDA-MB-468
nd: Not determined.
a
GI50: 50% growth inhibition, concentration of drug resulting in a 50% reduction
in net protein increase compared with control cells.
b
LC50: lethal concentration, concentration of drug lethal to 50% of cells.