Design, Synthesis, and Cytotoxicity of Novel 3-Arylidenones Derived from Alicyclic Ketones

Article abstract of DOI:10.1111/j.1747-0285.2011.01176.x

Forty-four novel chalcone-inspired analogs having a 3-aryl-2-propenoyl moiety derived from alicyclic ketones were designed, synthesized, and investigated for cytotoxicity against murine B16 and L1210 cancer cell lines. The analogs belong to four structurally divergent series, three of which (series g, h, and i) contain differently substituted cyclopentanone units and the fourth (series j) contains a 3,3-dimethyl-4-piperidinone moiety. Of these, the analogs in series j showed potential cytotoxic activity against murine B16 (melanoma) and L1210 (lymphoma) cells. The most active compounds 5j, 11j, 15j, and 12h produced IC₅₀ values from 4.4 to 15μm against both cell lines. A single-crystal X-ray structure analysis and molecular modeling studies confirmed that these chalcones have an E-geometry about the alkene bond and possess a slightly 'twisted' conformation similar to that of combretastatin A-4. At a concentration of 30μm, compounds 5j, 11j, and 15j did not cause microtubule depolymerization in cells, suggesting that they have a different mechanism of action. Forty-four novel chalcone-inspired analogs were designed and synthesized. Several compounds gave IC₅₀ values 4.4-15μm against the growth of L1210 and B16 murine cancer cells.

Full text of DOI:10.1111/j.1747-0285.2011.01176.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01176.x
Chem Biol Drug Des 2011; 78: 700–708
Research Letter
Design, Synthesis, and Cytotoxicity of Novel
3-Arylidenones Derived from Alicyclic Ketones
Vijay Satam1, Ravi K. Bandi2, Ajaya K.
Combretastatin A-4 (CA-4 or 1 as shown in Figure 1) is a natural
product, which consists of a core cis-stilbene moiety. It originates
from the African willow tree, Combretum caffrum, and is known to
inhibit tubulin polymerization via interaction with the colchicine
binding site of tubulin (1–4). Like combretastatins, chalcones (2,
Figure 1) also derive their antitumor properties through the inhibi-
Behera2, Bijay K. Mishra2, Samuel Tzou1,
Olivia Brockway1, Balaji Babu1, Matthias
Zeller3, Cara Westbrook4, Susan L.
Mooberry4,5, Moses Lee1,* and Hari Pati2
1Division of Natural and Applied Sciences and Department of
tion of tubulin polymerization (5–8). Chalcones differ from combre-
tastatin by having a trans-enone moiety between the aromatic rings
(Figure 1). This 1,3-diarylprop-1-enone system is essential for chal-
cones to elicit their cytotoxic properties. The ease of synthesis of
chalcones, from substituted benzaldehydes and acetophenones,
makes them an attractive drug scaffold. Many of the chalcone ana-
logs exhibit powerful anticancer activities, causing renewed interest
in this class of molecules (9–15).
Chemistry, Hope College, Holland, MI 49423, USA
2Department of Chemistry, Sambalpur University, Jyoti Vihar-768019,
Sambalpur, Orissa, India
3Department of Chemistry, Youngstown State University,
Youngstown, OH 44555, USA
4Department of Pharmacology, University of Texas Health Science
Center at San Antonio, San Antonio, TX 78229, USA
5Department of Medicine, University of Texas Health Science Center
at San Antonio, San Antonio, TX 78229, USA
Structurally, chalcones are open-chained molecules bearing two aro-
matic rings linked by a three-carbon enone pharmacophore. We
envisaged that the attachment of this 3-aryl-2-propenoyl pharmaco-
phore to alicyclic scaffolds would lead to additional series of com-
pounds, which may exhibit cytotoxic activities against malignant
cells. This rationale was inspired by several reports that described
superior cytotoxic effects of the cyclic chalcone analogs (3,
Figure 2) (16–21). Some of these analogs were found to display sig-
nificant CYP1A inhibitor activity (22,23).
*Corresponding author: Moses Lee, lee@hope.edu
Forty-four novel chalcone-inspired analogs having
a 3-aryl-2-propenoyl moiety derived from alicyclic
ketones were designed, synthesized, and investi-
gated for cytotoxicity against murine B16 and
L1210 cancer cell lines. The analogs belong to
four structurally divergent series, three of which
(series g, h, and i) contain differently substituted
cyclopentanone units and the fourth (series j) con-
This study was aimed at preparing analogs that contained a 3-aryl-
2-propenoyl core structure 4 (Figure 2) and at investigating their
ability to inhibit the proliferation of cancer cells in culture. A
diverse group of compounds were included in this study because
earlier structure–activity studies on cyclic chalcone analogs have
revealed that the cytotoxicity of these compounds was influenced
by the shape of the molecules (22,23). Accordingly, four structurally
divergent series of compounds, each differing significantly in the
substitution pattern of cyclic ketone moiety, were synthesized and
evaluated for their cytotoxic activity against murine B16 and L1210
cancer cell lines. Selected molecules were also assayed in compari-
son with a known tubulin inhibitor, CA-4, for their ability to cause
microtubule depolymerization in A-10 cells.
tains
a 3,3-dimethyl-4-piperidinone moiety. Of
these, the analogs in series j showed potential
cytotoxic activity against murine B16 (melanoma)
and L1210 (lymphoma) cells. The most active com-
pounds 5j, 11j, 15j, and 12h produced IC50 values
from 4.4 to 15 lM against both cell lines. A single-
crystal X-ray structure analysis and molecular
modeling studies confirmed that these chalcones
have an E-geometry about the alkene bond and
possess a slightly ’twisted’ conformation similar
to that of combretastatin A-4. At a concentration
of 30 lM, compounds 5j, 11j, and 15j did not cause
microtubule depolymerization in cells, suggesting
that they have a different mechanism of action.
Key words: 3-aryl-2-propenoyl, chalcones, combretastatin A-4,
cyclopentanone, cytotoxicity, tubulin
Material and Methods
Solvents and organic reagents were purchased from Aldrich and
used without further purification. Melting points (mp) were
determined using a Bꢀchi B-545 melting point apparatus, and the
results were uncorrected. Infrared (IR) spectra were recorded on a
Midac M1700 FT-IR instrument as films on KBr disks, unless stated
otherwise. Proton NMR spectra were recorded on a Varian INOVA
Despite recent advances in the understanding of the biological pro-
cesses leading to the development of cancer, there is still a need
for new and effective agents to help bring this disease under con-
trol. Among the recently identified antitumor agents, combretasta-
tins and chalcones represent important classes of molecules.
700

Design and Synthesis of Novel Cytotoxic 3-Arylidenones
terization of compounds that were selected for detailed cytotoxicity
studies is given below.
R1
R4
O
R10
R2
R3
R9
H3CO
H3CO
OH
R5 R6
R8
OCH3
OCH3
R7
3-(3-Methoxybenzylidene)bicyclo[2.2.1]heptan-
2-one (6g)
1
2
Yield: 77%; white solid; mp: 93–95 ꢀC; FTIR (KBr, ⁄ cm) 1727, 1643,
1596, 1577, 1434, 1266, 1207, 1092, 942, 916, 810, 739; H NMR
Figure 1: Structures of combretastatin A-4 and chalcone.
1
(CDCl3, 400 MHz) d 7.32 (t, 1H, J = 8.0 Hz), 7.12 (s, 1H), 7.08 (dt,
1H, J = 1.0, 8.0 Hz), 7.01 (dd, 1H, J = 1.0, 2.6 Hz), 6.91 (ddd, 1H,
J = 1.0, 2.6, 8.0 Hz), 3.83 (s, 3H), 3.64 (m, 1H), 2.77 (m, 1H), 2.09–
1.99 (m, 2H), 1.78–1.63 (m, 4H); MS (ESI, m ⁄ z) 229 (M+H+).
R1
O
R9
O
R9
R6
R2
R3
R8
R7
R8
R7
R5
R5
R4
R6
3-(4-Chlorobenzylidene)bicyclo[2.2.1]heptan-2-
one (9g)
4
3
Yield: 65%; white solid; mp: 88–90 ꢀC; FTIR (KBr, ⁄ cm) 1724, 1640,
1490, 1087, 914, 830, 788, 719; H NMR (CDCl3, 400 MHz) d 7.41
Figure 2: Structures of cyclic chalcone analogs.
1
(d, 2H, J = 8.0 Hz), 7.37 (d, 2H, J = 8.0 Hz), 7.09 (s, 1H), 3.58 (m,
1H), 2.80 (m, 1H), 2.08–1.97 (m, 2H), 1.79–1.62 (m, 4H); MS (EI,
m ⁄ z) 232 (M+); HRMS m ⁄ z calc. for C14H13ClO 232.0655, found
232.0657.
400 MHz Fourier transform spectrometer. Chemical shifts are quoted
in parts per million downfield from tetramethylsilane (TMS). Low-
and high-resolution mass spectra were provided by the Mass Spec-
trometry Laboratory, University of South Carolina, Columbia, SC.
Reactions were monitored by thin-layer chromatography (TLC) using
commercially available precoated plates (Merck Kieselgel 60 F254
silica), and developed plates were examined under UV light
(254 nm) or using iodine vapor staining. Column chromatography
was performed using 200-mesh silica gel. Crystallographic data for
compound 10j were collected using a Bruker AXS SMART APEX
CCD diffractometer with monochromatic Mo Ka radiation (Wave-
length k = 0.71073 ꢁ) with the X scan technique. The crystal was
mounted on a Mitegen micromesh mount using a trace of mineral
oil and cooled in situ to 100(2) K for data collection. Cell refine-
ment, data reduction, and absorption corrections were carried out
using APEX2. The structure was solved by direct methods using
SHELXTL 6.14 and was refined by full-matrix least-squares calcula-
tions on F2 with SHELXTL. Non-hydrogen atoms were refined with
anisotropic displacement parameters. Carbon-bound hydrogen atoms
were placed in geometrically idealized positions with Uiso(H) = 1.2–
1.5 Ueq(C). The position of the amine H atom was refined; Uiso(H)
was set to 1.2 Ueq(N).
3-(3,4,5-Trimethoxybenzylidene)bicyclo[2.2.1]
heptan-2-one (11g)
Yield: 55%; light yellow solid; mp: 57–60 ꢀC; FTIR (KBr, ⁄ cm) 1725,
1639, 1593, 1472, 1255, 1111, 943, 776, 732; 1H NMR (CDCl3,
400 MHz) d 7.28 (s, 1H), 6.73 (s, 2H), 3.89 (s, 9H), 3.64 (m, 1H),
2.79 (m, 1H), 2.06–1.90 (m, 2H), 1.80–1.68 (m, 4H); MS (ESI, m ⁄ z)
289 (M+H+).
3-(3-Bromobenzylidene)bicyclo[2.2.1]heptan-2-
one (14g)
Yield: 64%; white solid, mp: 102–105 ꢀC; FTIR (KBr, ⁄ cm) 1732,
1650, 1558, 1447, 1241, 1098, 954, 767, 719; 1H NMR (CDCl3,
400 MHz) d 7.60 (t, 1H, J = 1.6 Hz), 7.47 (dd, 1H, J = 1.6, 8.0 Hz),
7.39 (dd, 1H, J = 1.6, 8.0 Hz); 7.27 (t, 1H, J = 8.0 Hz), 7.06 (s, 1H),
3.59 (m, 1H), 2.78 (m, 1H), 2.09–1.95 (m, 2H), 1.79–1.64 (m, 4H);
MS (ESI, m ⁄ z) 277 (M+H+).
General procedure for the synthesis of
chalcone analogs in the series g and h
3-(5-Bromo-2-methylbenzylidene)bicyclo[2.2.1]
heptan-2-one (15g)
Sodium hydroxide solution (3% in ethanol, 2.0 mL) was added
dropwise to the solution of bicyclo[2.2.1]heptan-2-one (for series g)
or 2-methylcyclopentanone (for series h) (1.0 mmol) and the appro-
priate aryl aldehyde (1.0 mmol) in ethanol (4.0 mL) at room temper-
ature. The reaction mixture was stirred at room temperature for
12 h. The reaction was monitored by TLC using 20% ethyl acetate
in hexane as the eluent system. Upon completion of the reaction,
water (10.0 mL) was added to the reaction mixture and stirred for
10 min. The reaction mixture was then extracted with ethyl acetate
(2 · 15.0 mL). The ethyl acetate layer was washed with water
(2 · 10.0 mL), dried over anhydrous sodium sulfate, and concen-
trated on a rotary evaporator to obtain the crude product, which
was purified by column chromatography using silica gel and 10%
ethyl acetate in hexane as the eluent system. The spectral charac-
Yield: 64%; white solid; mp: 86–89 ꢀC; FTIR (KBr, ⁄ cm) 1719, 1599,
1432, 1067, 979, 749, 722; 1H NMR (CDCl3, 400 MHz) d 7.46 (d,
1H, J = 2.0 Hz), 7.36 (dd, 1H, J = 2.0, 8.0 Hz), 7.21 (s, 1H), 7.08 (d,
1H, J = 8.0 Hz), 3.45 (m, 1H), 2.81 (m, 1H), 2.31 (s, 3H), 2.07–1.94
(m, 2H), 1.78–1.64 (m, 4H); MS (ESI, m ⁄ z) 291 (M+H+).
2-(4-Fluorobenzylidene)-5-methylcyclopentanone
(12h)
Yield: 58%; yellow solid; mp: 60–64 ꢀC; FTIR (KBr, ⁄ cm) 1706, 1631,
1
1600, 1507, 1415, 1229, 1101, 835, 912; H NMR (CDCl3, 400 MHz)
d 7.60 (dd, 2H, JH–H = 8.7 Hz, JH–F = 5.4 Hz), 7.39 (s, 1H); 7.11 (dd,
2H JH–H = 8.7 Hz, JH–F = 8.6 Hz), 2.96–2.88 (m, 1H), 2.84–2.64 (m,
Chem Biol Drug Des 2011; 78: 700–708
701

Satam et al.
1H), 2.42–2.23 (m, 2H), 1.58–1.52 (m, 1H), 1.18 (d, 3H, J = 6.4 Hz);
MS (EI, m ⁄ z) 204 (M+, 100); HRMS m ⁄ z calc. for C13H13FO
204.0948, found 204.0950.
in saturated sodium carbonate solution (10.0 mL) for 30 min and
again filtered, washed with water, and dried. The crude compound
was purified by column chromatography using silica gel and 40%
ethyl acetate in hexane as the eluent system. The spectral character-
ization of compounds selected for cytotoxicity studies is given below.
General procedure for the synthesis of
chalcone analogs in the series i
To a solution of the appropriate 2-arylidene-5-methylcyclopentanone
5h–15h (1 mmol) in dry tetrahydrofuran (5.0 mL) was added potas-
sium tert-butoxide (2 mmol) and stirred for 30 min at room temper-
ature. To the reaction mixture was then added methyl iodide
(1.2 mmol) and stirred at room temperature for 12 h. The reaction
was monitored and worked up in the same procedure as described
for series g and h, and the characterization data for 'active' com-
pounds according to the cytotoxicity screen are given below.
5-Benzylidene-3,3-dimethylpiperidin-4-one (5j)
Yield: 57%; pale yellow solid; mp: 89–93 ꢀC; FTIR (KBr, ⁄ cm) 1693,
1
1594, 1421, 1265, 895, 739, 704; H NMR (CDCl3, 400 MHz) d 7.80
(s, 1H), 7.46–7.42 (m, 3H), 7.33–7.30 (m, 2H), 4.41 (s, 2H), 3.31 (s,
2H), 1.37 (s, 6H); MS (EI, m ⁄ z) 215 (M+); HRMS m ⁄ z calc. for
C14H17NO 215.1310, found 215.1314.
5-(3-Methoxybenzylidene)-3,3-dimethyl-
piperidin-4-one (6j)
2-(2-Chlorobenzylidene)-2,2-dimethylcyclo-
pentanone (8i)
Yield: 67%; pale yellow solid; mp: 98–101 ꢀC; FTIR (KBr, ⁄ cm) 1705,
1682, 1597, 1489, 1317, 1160, 1048, 993, 781; H NMR (DMSO-d6,
1
Yield: 68%; white solid; mp: 82–85 ꢀC; FTIR (KBr, ⁄ cm) 1717, 1626,
1476, 1436, 1183, 1095, 989, 766, 745; H NMR (CDCl3, 400 MHz)
d 7.75 (t, 1H, J = 8.0 Hz), 7.56–7.53 (m, 1H), 7.45–7.43 (m, 1H),
7.33–7.28 (m, 2H), 2.82 (dt, 2H, J = 4.0, 8.0 Hz), 1.84 (t, 2H,
J = 8.0 Hz), 1.15 (s, 6H); MS (ESI, m ⁄ z) 235 (M+H+).
400 MHz) d 7.39–7.33 (m, 1H), 7.24 (s, 1H), 7.03–6.95 (m, 3H), 3.94
(s, 2H), 3.77 (s, 3H), 2.81 (s, 2H), 1.53 (s br, 1H), 1.07 (s, 6H); MS
(ESI, m ⁄ z) 246 (M+H+).
1
5-(2-Chlorobenzylidene)-3,3-dimethylpiperidin-4-
one (8j)
5-(4-Chlorobenzylidene)-2,2-dimethylcyclo-
pentanone (9i)
Yield: 71%; off-white solid; mp: 89–92 ꢀC; FTIR (KBr, ⁄ cm) 1687,
1605, 1469, 1436, 1317, 1150, 1054, 970, 762, 737; 1H NMR
(DMSO-d6, 400 MHz) d 7.46–7.45 (m, 1H), 7.44–7.41 (m, 1H), 7.28–
7.25 (m, 2H), 7.16–7.13 (m, 1H), 3.88 (s, 2H), 2.96 (s, 2H), 1.73 (s br,
1H), 1.20 (s, 6H); MS (ESI, m ⁄ z) 250 (M+H+).
Yield: 71%; off-white solid; mp: 110–114 ꢀC; FTIR (KBr, ⁄ cm) 1706,
1
1623, 1492, 1265, 1183, 1089, 1012, 988, 735, 705; H NMR (CDCl3,
400 MHz) d 7.48 (dt, 2H, J = 2.6, 8.0 Hz), 7.38 (d, 2H, J = 8.0 Hz),
7.36 (t, 1H, J = 2.6 Hz), 2.86 (dt, 2H, J = 4.0, 8.0 Hz), 1.86 (t, 2H,
J = 8.0 Hz), 1.14 (s, 6H); MS (EI, m ⁄ z) 234 (M+); HRMS m ⁄ z calc.
for C14H15ClO 234.0811, found 234.0807.
5-(4-Chlorobenzylidene)-3,3-dimethylpiperidin-4-
one (9j)
Yield: 55%; off-white solid; mp: 142–144 ꢀC; FTIR (KBr, ⁄ cm) 1711,
1684, 156, 1283, 1095 1013, 811, 745; H NMR (CDCl3, 400 MHz) d
1
5-(2,6-Dimethoxybenzylidene)-2,2-
dimethylcyclo-
pentanone (10i)
7.39 (s, 1H), 7.36 (d, 2H, J = 8.0 Hz), 7.23 (d, 2H, J = 8.0 Hz), 4.02
(s, 2H), 2.97 (s, 2H), 1.21 (s, 6H); MS (ESI, m ⁄ z) 250 (M+H+).
Yield: 60%; white solid; mp: 78–81 ꢀC; FTIR (KBr, ⁄ cm) 1711, 1628,
1
1593, 1472, 1254, 1119, 1106, 990, 887, 776, 751; H NMR (CDCl3,
400 MHz) d 7.51 (t, 1H, J = 2.7 Hz), 7.28 (t, 1H, J = 8.0 Hz), 6.57
(d, 2H, J = 8.0 Hz), 3.84 (s, 6H), 2.48 (dt, 2H, J = 4.0, 8.0 Hz), 1.72
(t, 2H, J = 8.0 Hz), 1.13 (s, 6H); MS (ESI, m ⁄ z) 261 (M+H+).
5-(2,6-Dimethoxybenzylidene)-3,3-
dimethylpiperidin-4-one (10j)
Yield: 57%; off-white solid; mp: 108–111 ꢀC; FTIR (KBr, ⁄ cm) 1681,
1
1602, 1582, 1471, 1254, 1180, 778, 747; H NMR (CDCl3, 400 MHz)
d 7.30 (s, 1H), 7.27 (t, 1H, J = 8.0 Hz), 6.55 (d, 2H, J = 8.0 Hz),
3.80 (s, 6H), 3.60 (s, 2H), 2.97 (s, 2H), 1.19 (s, 6H); MS (EI, m ⁄ z)
275 (M+); HRMS m ⁄ z calc. for C16H21NO3 275.1521, found
275.1520.
General procedure for the synthesis of
chalcone analogs in the series j
To the solution of tert-butyl 3,3-dimethyl-4-piperidone-1-carboxylate j
(1 mmol) and the appropriate aryl aldehyde (1 mmol) in ethanol
(5.0 mL) at room temperature was added sodium hydroxide solution
(10%, 2.0 mL), and the reaction mixture was stirred at room temper-
ature for 12 h. The reaction was monitored by TLC using 20% ethyl
acetate in hexane as the eluent system. On completion of the reac-
tion, the precipitated solid was filtered off and washed with cold
ethanol (3.0 mL). The solid was dried and then stirred with 20% HCl
in dioxane (5.0 mL) for 3 h at room temperature. The precipitate was
filtered and washed with cold methanol (3.0 mL). It was then stirred
5-(3,4,5-Trimethoxybenzylidene)-3,3-
dimethylpiperidin-4-one (11j)
Yield: 67%; light yellow solid; mp: 99–103 ꢀC; FTIR (KBr, ⁄ cm)
1
1700, 1654, 1265, 1103, 1083,738, 704; H NMR (CDCl3, 400 MHz)
d 7.41 (m, 1H), 6.54 (s, 2H), 4.14 (s, 2H), 3.88 (s, 3H), 3.86 (s, 6H),
2.99 (s, 2H), 1.16 (s, 6H); MS (EI, m ⁄ z) 305 (M+); HRMS m ⁄ z calc.
for C17H23NO4: 305.1627, found 305.1624.
702
Chem Biol Drug Des 2011; 78: 700–708

Design and Synthesis of Novel Cytotoxic 3-Arylidenones
5-(4-Fluorobenzylidene)-3,3-dimethylpiperidin-4-
one (12j)
synthesized in good-to-excellent yields (50–85%) by aldol cross-con-
densation of the appropriate ketone with appropriately substituted
benzaldehydes. The series h compounds were stirred with potas-
sium tert-butoxide in THF for 30 min at room temperature, and the
resulting solution was treated with methyl iodide and stirred for
12 h to obtain series i compounds in good yields (60–70%). To syn-
thesize the analogs of series j, commercially available Boc-pro-
tected 3,3-dimethyl-4-piperidone was treated with an appropriate
aldehyde in ethanol and aqueous sodium hydroxide at ambient
conditions for 12 h. The Boc protection was removed by stirring the
resulting compounds in 20% HCl in dioxane to afford the desired
analogs (5j–15j) in moderate-to-good yields (35–50%). All com-
pounds were purified by column chromatography using silica gel.
Yield: 74%; light brown solid; mp: 92–95 ꢀC; FTIR (KBr, ⁄ cm) 1724,
1
1599, 1508, 1225, 1158, 1085, 836, 668; H NMR (CDCl3, 400 MHz)
d 7.35 (s, 1H), 7.28 (dd, 2H, JH–H = 8.7 Hz, JH–F = 5.4 Hz), 6.90 (dd,
2H JH–H = 8.7 Hz, JH–F = 8.6 Hz), 4.05 (s, 2H), 3.06 (s, 2H), 1.32 (s,
6H); MS (ESI, m ⁄ z) 234 (M+H+).
5-(5-Bromo-2-methylbenzylidene)-3,3-
dimethylpiperidin-4-one (15j)
Yield: 65%; off-white solid; mp: 111–114 ꢀC; FTIR (KBr, ⁄ cm) 1695,
1604, 1475, 1319, 1138, 1098, 813; 1H NMR (CDCl3, 400 MHz) d
7.61 (s, 1H), 7.33 (d, 1H, J = 8.0 Hz), 7.17 (s, 1H), 7.06 (d, 1H,
J = 8.0 Hz), 4.14 (s, 2H), 3.24 (s, 2H), 2.21 (s, 3H), 1.23 (s, 6H); MS
(EI, m ⁄ z) 307 (M+); HRMS m ⁄ z calc. for C15H18BrNO 307.0572, found
307.0558.
Cytotoxicity of the chalcone-inspired analogs
With the target molecules 5–15 (series g, h, i, and j) at hand,
their ability to inhibit the growth of murine L1210 lymphoma cells
in culture was screened at a concentration of 10 lM. For compari-
son, the previously reported cytotoxic agent seco-hydroxy-CBI-TMI
16 (24–26) (Figure 3) was used as a positive control. Screening of
the compounds was conducted after the cells were incubated con-
tinuously for 72 h, and growth inhibition was measured using an
MTT assay (27). For each compound, the average growth inhibition
from four wells was determined by calculating the inverse of cell
viability. Viable, living cells readily convert MTT to formazan, which
absorbs strongly at 570 nm. The inverse of cell viability is therefore
an inverse of the absorbance at 570 nm after adjustment of the
Results and Discussion
Synthesis of novel chalcone-inspired analogs
Four series of cyclic chalcone-inspired analogs (g, h, i, and j) were
synthesized as outlined in Scheme 1. Three (g, h, and i) of the four
series were derived from cyclopentanones substituted differently at
the a-position, while the fourth series (j) was derived from a nitro-
gen-containing, a-substituted, six-membered cyclic ketone (3,3-
dimethyl-4-piperidinone). The compounds in series g and h were
O
O
O
aq. NaOH (10%)
ethanol, rt, 12 h
R
H
R
Series g (5g-15g)
g
O
O
O
aq. NaOH (10%)
ethanol, rt, 12 h
H3C
H3C
R
H
R
Series h (5h-15h)
h
O
O
pot. tert-butoxide
CH3I, THF
H3C
H3C
R
R
H3C
rt, 12 h
Series i (5i-15i)
5h-15h
O
O
H3C
O
H3C
H3C
O
H
H3C
H3C
HCl in dioxane
20%
aq. NaOH (10%)
ethanol, rt, 12 h
H3C
R
R
R
N
CH3
CH3
CH3
N
CH3
N
H
CH3
O
O
O
O
CH3
j
Series j (5j-15j)
Cl
-R =
OCH3
OCH3
OCH3
Cl
5
6
9
7
8
H3CO
OCH3
H3CO
H3C
Br
-R =
Scheme 1: Synthetic pathway
of the compounds in series g, h,
i, and j.
Br
F
CH3
OCH3
12
14
15
10
11
13
Chem Biol Drug Des 2011; 78: 700–708
703

Satam et al.
OCH3
OCH3
Cl
OCH3
NH
N
O
OH
seco-Hydroxy-CBI-TMI (16)
series-g
series-h
Figure 3: Structure of the
cytotoxic agent seco-hydroxy-CBI-
TMI 16 and charts showing the
results from an MTT-based in vitro
screening of the compounds at
10 lM concentration for activity
against murine L1210 (lymphoma)
cells. Difference in cell viability
(x-axis) is defined as A ) B, in
which A = 1 ⁄ (absorbance at
16
15g
14g
13g
12g
11g
10g
9g
8g
7g
6g
5g
16
15h
14h
13h
12h
11h
10h
9h
8h
7h
6h
5h
O
O
R1
R1
R2
H3
C
R2
R3
R5
R3
R5
R4
R4
–0.005
0
0.005
0.01
0.015
0.02
–0.005
0
0.005
0.01
0.015
0.02
Difference in cell viability
Difference in cell viability
570 nm for an individual com-
pound ) absorbance at 570 nm for
the negative untreated control).
B = average of A for all com-
pounds within each series. Bars to
the right of the average indicate
compounds that are more active
than the average for each series
of molecules. The activity of com-
pound 16 was not included in the
calculations.
series-i
series-j
16
16
15j
14j
13j
12j
11j
10j
9j
8j
7j
6j
5j
15i
14i
13i
12i
11i
10i
9i
8i
7i
6i
5i
O
O
R1
R1
H3
3C
C
H3
H3
C
R2
R3
R2
R3
H
C
N
H
R5
R5
R4
R4
–0.005
0
0.005
0.01
0.015
0.02
–0.005
0
0.005
0.01
0.015
0.02
Difference in cell viability
Difference in cell viability
controls (see legend of Figure 3). The screening results are depicted
in Figure 3. Compounds that are more cytotoxic than the average
for each series are indicated by bars pointing to the right from the
average. Accordingly, the following compounds were selected for
detailed cytotoxicity studies: five compounds in series g (6g, 9g,
11g, 14g, and 15g), one compound in series h (12h), three com-
pounds in series i (8i, 9i, and 10i), and eight compounds in series
j (5j, 6j, 8j, 9j, 10j, 11j, 12j, and 15j). These compounds were
tested against the growth of murine B16 (melanoma) and L1210 cell
lines using a 72-h continuous exposure MTT assay technique (27).
The concentration at which 50% cell growth was inhibited (IC50,
lM) was determined for each compound in triplicate experiments;
the values were averaged and are presented in Table 1.
oxy-substituted compound 11j emerged as the most cytotoxic
compounds in the series with IC50 values of 9.2 and 8.4 lM,
respectively, for B16. These compounds gave even lower IC50 values
of 5.1 and 4.4 lM, respectively, for L1210 cell lines. The monohalo-
genated compounds 8j, 9j, and 12j were only moderately active
against the B16 cell line (IC50 = 30, 59, and 41 lM, respectively).
Compounds 8j and 12j showed remarkable cytotoxicity against the
L1210 cell line with lower IC50 values of 4.4 and 4.9 lM, respec-
tively. However, the para-chloro-substituted compound 9j did not
show cytotoxicity against L1210 cells, indicating that the position of
substitution on the phenyl ring has an impact on activity. It was
interesting to note that compound 15j, which has a 2-methyl-5-
bromo substitution, was equally active against B16 and L1210 can-
cer cell lines (IC50 = 14 and 15 lM, respectively). It is noteworthy
that any substitution on the aromatic ring decreased cytotoxicity
except for the trimethoxy analog. Interestingly, the 3,4,5-trimethoxy
substitution pattern in compound 11j is consistent with the A-ring
of CA-4.
The results given in Table 1 showed that none of the compounds in
series g, h, and i was active against murine L1210 and B16 cancer
cells with the sole exception of the compound 12h, derived from
2-methylcyclopentanone. Compound 12h, which contains a fluorine
atom in the para position of the phenyl moiety, showed remarkable
cytotoxicity against both B16 and L1210 cell lines. The recorded
IC50 values are 12 and 6.8 lM, respectively. The series j compounds
furnished more promising results. Five compounds, 5j, 8j, 11j, 12j,
and 15j, were cytotoxic against both B16 and L1210 cell lines (IC50
values between 4.4 and 41 lM). On the other hand, three com-
pounds 6j, 9j, and 10j showed moderate cytotoxicity against B16
melanoma cell lines with IC50 values of 60, 59, and 50 lM, respec-
tively. The simple non-substituted compound 5j and 3,4,5-trimeth-
Conformation
The configuration about the double bond between the alicyclic
moiety and the aromatic ring was determined by aromatic
solvent–induced shift (ASIS) data by recording the 1H NMR spec-
trum in CDCl3 and in an aromatic solvent (C7D8, deuterated toluene-
d8). For example, in CDCl3, the peak for the b-alkene proton on
compound 10j appeared as a singlet at 7.30 ppm, which shifted
704
Chem Biol Drug Des 2011; 78: 700–708

Design and Synthesis of Novel Cytotoxic 3-Arylidenones
Table 1: IC50 values of the selected compounds in the series g, h, i, and j
IC50 (lM)
IC50 (lM)
IC50 (lM)
IC50 (lM)
Compounds
Structure
B16
L1210
Compounds
Structure
B16
L1210
O
6g
>100
>100
>100
>100
>100
>86
5j
9.2
5.1
>100
4.4
O
H3C
H3C
OCH3
N
H
O
9g
6j
8j
60
O
H3C
H3C
OCH3
Cl
N
H
O
11g
30
O
Cl
H3C
H3C
OCH3
OCH3
N
H
H3CO
O
14g
15g
>100
>100
>100
>100
9j
59
50
>100
>100
O
H3C
H3C
Br
N
H
Cl
O
10j
CH3
H3CO
O
H3C
H3C
N
H
H3CO
Br
O
12h
12
6.8
11j
8.4
4.4
O
H3C
H3C
H3C
OCH3
OCH3
F
N
H
OCH3
Cl
O
8i
9i
>100
>100
>100
>100
12j
15j
41
14
4.9
O
H3C
H3C
H3C
H3C
N
H
F
O
15
O
CH3
H3C
H3C
H3C
H3C
Cl
N
H
Br
OCH3
10i
O
>100
>100
H3C
H3C
H3CO
downfield (7.74 ppm) when the solvent was changed from CDCl3 to
deuterated toluene, while all other peaks shifted upfield. This
downfield shift of 0.44 ppm of the b-alkene proton in an aromatic
solvent suggests that the proton is syn to a keto group (Figure 4)
(28,29). Unambiguous evidence for the E-configuration of compound
10j was gained from a single-crystal X-ray crystallographic analysis
Chem Biol Drug Des 2011; 78: 700–708
705

Satam et al.
Microtubule depolymerization activity
A
B
Proton syn to keto group
The effects of compounds 5j, 11j, and 15j on interphase cellular
microtubules were evaluated with A-10 smooth muscle cells (32).
Exposure of the cells to the three compounds did not show any signif-
icant microtubule depolymerization up to 30 lM concentration. In
contrast, CA-4 strongly causes microtubule depolymerization giving
an EC50 value of 0.007 lM (33). This indicates that the novel
compounds described herein do not act via a similar mechanism of
inhibiting cell growth as CA-4. This is interesting because related bis-
arylidene analogs of the target compounds, which gave equal cyto-
toxicity, also did not display microtubule disruption properties (34).
OCH3
O
H
H3C
H3C
N
H
H3CO
Figure 4: Chemical structure (A) and single-crystal X-ray crystal-
lographic structure (B) of the compound 10j. The crystals of 10j
were grown from a solution in methanol that was kept in the
refrigerator. Complete X-ray structural data for 10j were deposited
with the Cambridge Structural Database.a
Conclusions
Forty-four alicyclic chalcone-inspired analogs belonging to four
unique series were designed and synthesized for the investigation
of their cytotoxicity. Generally, the chalcone analogs derived from
substituted cyclopentanones (series g, h, and i) were inactive at
inhibiting the growth of cancer cells in the culture. However, sev-
eral chalcones containing a 3,3-dimethyl-4-piperidinone unit (series
j) showed significant cytotoxicity against both murine L1210 and
B16 cancer cell lines. These chalcone analogs possess an E-con-
figuration and a twisted conformation similar to that of combre-
tastatin A-4 as revealed by single-crystal X-ray structure analysis
and molecular modeling studies. However, their mechanism of
action was found to be different from that of combretastatin A-4
as they failed to cause microtubule depolymerization. Further stud-
ies on the j series of compounds are planned which include
experiments to determine their mechanism of action and in vivo
studies.
(Figure 4). The X-ray structure of 10j confirmed the E-geometry
about the alkene and showed that the two rings are almost perpen-
dicular to each other.a
The conformation of the compounds 5j and 11j was also exam-
ined by molecular modeling studies using the suite of programs in
MacSpartan, version '04. Upon optimization of the structure using
molecular mechanics (MMFF) and molecular dynamics (equilibrium
conformer search option and molecular mechanics), the structure
was energy-optimized using Hartree–Fock (3-21G), followed by den-
sity function theory (B3LYP and 6-31G*) calculations. For compari-
son, the conformation of CA-4 1, determined using the same
protocol and as previously reported, was used in the analysis (30).
The resulting conformations of compounds 5j and 11j are depicted
in Figure 5, and the conformations are remarkably similar to the
X-ray diffraction-derived conformation of 10j, indicating the molec-
ular modeling can quite reasonably predict the conformation of the
compounds described herein. It is evident from these results that
in general the steric hindrance between the methylene-hydrogen
atoms of the piperidino unit and the phenyl moiety on the alkene
causes the molecule to adopt a slightly 'twisted' conformation
between 5 and 10ꢀ. Interestingly, this conformation is similar to
that observed in an X-ray structure of CA-4 (31) and prompted us
to investigate whether some of the active compounds may
enhance microtubule depolymerization in cells in a similar way as
CA-4.
Acknowledgments
Support from the Department of Chemistry, Sambalpur University
for providing facilities for research is acknowledged. The X-ray dif-
fractometer at Youngstown State University was funded by NSF
Grant 0087210, Ohio Board of Regents Grant CAP-491, and
Youngstown State University. Support from the President's Council
Research Excellence Award and from the CTRC Cancer Center
Support Grant, CCSG (CA054174) is gratefully acknowledged
(SLM).
A
B
Figure 5: Molecular models of
the compounds 5j (A) and 11j (B)
determined using Hartree–Fock and
density functional calculations.
706
Chem Biol Drug Des 2011; 78: 700–708

Design and Synthesis of Novel Cytotoxic 3-Arylidenones
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Note
aX-ray structural information for 10j in cif format, CCDC 807546.
These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_
request/cif.
708
Chem Biol Drug Des 2011; 78: 700–708