Synthesis, biological evaluation, and molecular modeling studies of novel heterocyclic compounds as anti-proliferative agents

Article abstract of DOI:10.1007/s00044-013-0556-x

Two novel series of heterocyclic compounds have been synthesized. In first series, isatin was allowed to react with substituted aromatic/cyclic carbonyl compounds to get desired mannich bases (2a-e). In second series, 4,5-disubstituted oxazoles (6a-p) wer

Full text of DOI:10.1007/s00044-013-0556-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0556-x
ORIGINAL RESEARCH
Synthesis, biological evaluation, and molecular modeling studies
of novel heterocyclic compounds as anti-proliferative agents
•
Anurag Chaudhary P. P. Sharma Gautam Bhardwaj
•
•
•
•
•
Vaibhav Jain P. V. Bharatam Birendra Shrivastav
R. K. Roy
Received: 30 November 2012 / Accepted: 18 February 2013
Ó Springer Science+Business Media New York 2013
Abstract Two novel series of heterocyclic compounds
have been synthesized. In first series, isatin was allowed to
react with substituted aromatic/cyclic carbonyl compounds to
get desired mannich bases (2a–e). In second series, 4,5-
disubstituted oxazoles (6a–p) were synthesized. Eight com-
pounds (2c, 6a, 6e, 6f, 6i, 6j, 6m, and 6n) were screened for
anticancer activity in 60 cell lines. Compound 2c, 1-[(4,7,7-
trimethyl-3-oxobicyclo[2.2.1]heptan-2-yl)methyl]indoline-2,
3-dione, showed maximum activity and thus, selected for
further evaluation at five dose level screening. Furthermore,
molecular docking studies of compounds 2c into the col-
chicine-binding site of tubulin, revealed possible mode of
inhibition by the compound.
Keywords DAMA-colchicine ꢀ Combretastatin A-4 ꢀ
Anticancer ꢀ Molecular docking
Introduction
Malignant tumor is one of the most serious threats against
human health in the world, and the clinical prognosis remains
relatively poor. Therefore, it needs to develop the new
therapeutic strategies for the improvement of drugs that are
currently in use. There has been more and more interest in the
search for antitumor compounds with high efficacy and low
toxicity in recent years. There are three major approaches in
the drug discovery: empiric screening, rational design of new
compounds, and chemical modification of known com-
pounds with established pharmacological actions. During
the course of identifying compounds active for the preven-
tion and treatment of cancer, stilbene derivatives, such as
plant micro-components resveratrol and combretastatin A-4
(CA-4, Fig. 1), have emerged to hold promise for their potent
in vitro and in vivo anticancer bioactivities (Murias et al.,
2005; Pettit et al., 2005; Latruffe et al., 2002; Bhat and
Pezzuto 2001; Ciolino and Yeh 2001; Pettit et al., 1998; Jang
et al., 1997; Pettit et al., 1989; Lin et al., 1988). The anti-
cancer potential of CA-4 is attributed to the finding that, this
compound binds to the colchicine-binding site of tubulin,
which in turn inhibits the microtubule assembly (Lin et al.,
1989). CA-4 is active in cis form. The reason for this is that in
cis conformation the two phenyl rings are noncoplanar and
within an appropriate distance (Gaukroger et al., 2001; Pettit
et al., 1995; Lin et al., 1988). In fact, the anticancer potential
of resveratrol, CA-4 and their derivatives has been widely
studied, but it is still important to contribute for an ample
knowledge on the biological effects of related compounds
(Kang et al., 2003).
A. Chaudhary (&)
Kharvel Subharti College of Pharmacy, Swami Vivekanand
Subharti University, NH-58,
Meerut Bypass Road, Meerut 250005, India
e-mail: anurag15182@rediffmail.com
P. P. Sharma
Department of Pharmaceutical Science, Gurukul Kangri
University, Haridwar 249404, India
G. Bhardwaj
Department of Pharmacy, IILM College, Gr. Noida, India
V. Jain ꢀ P. V. Bharatam
Department of Pharmacoinformatics, National Institute
of Pharmaceutical Education and Research (NIPER),
Sector-67, S. A. S. Nagar, Mohali 160062, Punjab, India
B. Shrivastav
School of Pharmaceutical Sciences, Jaipur National University,
Jaipur 302025, India
R. K. Roy
Dr. K.N. Modi Institute of Pharmaceutical Education
and Research, Modinagar 201201, India
123

Med Chem Res
HS
O
NH
O
OH
B
CH3
C
B
O
HO
A
B
O
OH
A
A
H3C
O
O
O
O
CH3
O
CH3
CH3
O
CH3
CH3
CH3
OH
DAMA-Colchicine (COL)
Combretastatin A-4 (CA-4)
Resveratrol
Fig. 1 Chemical structures of antitubulin agent COL and cytotoxic stilbenes (CA-4 and resveratrol)
Different authors worldwide have reported anticancer
activity of N-substituted isatin derivatives (Vine et al.,
2009; Solomon et al., 2009). Many CA-4 related hetero-
cyclic compounds, containing fused ring system, have
shown potent anticancer activity (Dupeyre et al., 2006;
Liou et al., 2004; Bailly et al., 2003; Tahir et al., 2003;
Flynn et al., 2002; Xia et al., 1998). It has been revealed
that potent activity of some of these heterocyclic com-
pounds is based on the fact that two phenyl rings, present in
their structure, are noncoplanar and at an appropriate dis-
tance, as in CA-4 (Bailly et al., 2003). Therefore, we can
presume that mannich bases of isatin, formed by using
cyclic ketones, have some structure resemblance with
CA-4 and hence, their mechanism of action in terms of
cytotoxic potential may also be similar. Thus, this rational
gave us immense confidence to synthesize some novel
mannich bases of isatin by using cyclic ketones, which
might be having potential anticancer activity.
systems, compounds with drug-like properties utilizing the
concept of privileged scaffolds or structures based on com-
puter-aided design are particularly encouraged for submis-
sion. Highly flexible acyclic analogs with accompanying
entropic liabilities or without prior activity data are generally
not accepted. Structures containing problematic linkages or
functional groups for successful drug development (e.g.,
nitro, nitroso, –N–N–, –N=N–, imine, semicarbazone) are
also not accepted. From an analog series, representative
compounds which will most efficiently provide the greatest
information are selected. As per the protocol of NCI, only
eight representative compounds, of the series, were selected
and granted NSC codes viz; NSC 742530, NSC 753088,
NSC 753089, NSC 753090, NSC 753091, NSC 753092,
NSC 753093, NSC 753094 and screened at NCI, USA for
anti-proliferative activity at a single high dose (10-5 M) in
full NCI cell panel. Compound 2c (NSC 742530) exhibited
the best result at single dose and was selected for further
evaluation at five dose level screening. This article presents
the in vitro anticancer profile of all the selected compounds
including the most potent compound of the series (2c),
1-[(4,7,7-trimethyl-3-oxobicyclo[2.2.1]heptan-2-yl)methyl]
indoline-2,3-dione (NSC 742530), against NCI’s human
cancer cell lines. In addition, molecular docking study was
also taken into account to explore the possible binding mode
of these compounds.
CA-4 does not show in vivo efficacy due to isomeriza-
tion of cis-double bond to the more thermally stable trans-
isomer, which is inactive (Pettit et al., 1998). Isomerization
of cis to trans form can be prevented by forming locked
cis-type bridge between the two phenyl rings. Various
researchers have reported structurally rigid analogs having
locked cis-type bridge, and many of them have shown
potent activity (Peifer et al., 2006; Kaffy et al., 2006;
Simoni et al., 2005; Wang et al., 2002; Ohsumi et al.,
1998). Therefore, to further explore in this area, we plan-
ned to synthesize some novel analogs, in which cis-type
bridge is locked by forming an oxazole ring, to find novel
anticancer agents.
Experimental
Chemistry
The data, of these synthesized compounds, was submitted
to National Cancer Institute (NCI) for anti-proliferative
activity. Selection of structures, for anticancer screening at
NCI, is based on their ability to add diversity to the NCI small
molecule compound collection. Novel heterocyclic ring
All the chemicals used were laboratory grade and procured
from Fisher Scientific, S.D. Fine Chemicals and CDH.
Melting points were determined by open tube capillary
method and are uncorrected. Thin layer chromatography
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Med Chem Res
1-((1-Methyl-4-oxopiperidin-3-yl)methyl)indoline-2,3-
dione (2a)
(TLC) plates (silica gel G) were used to confirm the purity
of commercial reagents used, compounds synthesized
and to monitor the reactions as well. Two different solvent
systems; petroleum ether:toluene:acetic acid in ratio
5:4:1 (for compounds of Scheme 1) and n-hexane:ethyl
acetate:methanol in ratio 10:10:1 (for compounds of
Scheme 2) were used to run the TLC. The spots were
located under iodine vapors/UV light. IR spectra were
obtained on a Shimadzu 8400S FT-IR spectrometer (KBr
Pellets). 1H NMR spectra were recorded on a 300 MHz dpx
300, spectrometer-using TMS as internal standard in
dimethyl sulfoxide (DMSO). 13C NMR spectra were
recorded on an AC 300 BRUKER spectrometer. Mass
spectra’s were recorded on an API 3000 LC/MS/MS Q3
(SHIMADZU) spectrometer.
FTIR (KBr) cm-1 3034 and 753 (Ar–H), 2996, 2947, 2859,
1454, 1422 and 1326 (C–H), 1732, 1722, 1681 (C=O), 1602
1
(C=C of Ar), 1175 (NR3). H NMR (DMSO) d 7.73–7.41
(3H, m), 7.19 (1H, d), 4.15 (1H, dd), 3.90 (1H, dd), 3.29 (1H,
quintet), 2.69–2.43 (6H, m), 2.21 (3H, s). 13C NMR
(75 MHz, DMSO-d6) d 38.8, 45.3, 48.7, 49.8, 55.6, 59.4,
117.5, 121.9, 124.6, 130.0, 134.7, 148.3, 160.4, 184.6, 211.7.
MS (TISI) 272.3 (M?). Anal. Calcd: C, 66.16; H, 5.92; N,
10.29. Found: C, 66.18; H, 5.84; N, 10.33.
1-((2-Oxotetrahydro-2H-pyran-3-yl)methyl)indoline-2,3-
dione (2b)
FTIR (KBr) cm-1 3033 and 751 (Ar–H), 2948, 2849, 1467
and 1327 (C–H), 1726, 1719, 1685 (C=O), 1604 (C=C of
General procedure for synthesis of N-substituted
isatin derivatives (2a–e)
1
Ar), 1108 (C–O). H NMR (DMSO) d 7.71–7.38 (3H, m),
7.21 (1H, d), 4.35–4.23 (3H, m), 4.01 (1H, dd), 3.08 (1H,
quintet), 2.05–1.84 (4H, m). 13C NMR (75 MHz, DMSO-
d6) d 19.7, 28.1, 43.3, 49.6, 71.9, 117.7, 122.0, 124.4,
130.2, 134.3, 148.6, 160.7, 172.5, 184.5. MS (TISI) 259.2
(M?). Anal. Calcd: C, 64.86; H, 5.05; N, 5.40. Found: C,
66.83; H, 5.04; N, 5.43.
A mixture of isatin (0.326 M), paraformaldehyde (0.33
mol), and ketones (1a–e, 0.25 M) was taken in 250 ml
round bottom flask attached to a reflux condenser. To it
40 ml of 95 % ethanol, containing 0.5 ml of concentrated
hydrochloric acid, was added, and mixture was refluxed on
a water bath for 2 h. Resulting hot solution was filtered
through a hot water funnel, and to the filtrate 200 ml of
acetone was added. Solution was cooled to room temper-
ature and left overnight in refrigerator. Crystals of the
crude product were filtered by using vacuum pump and
washed with 10 ml of acetone, and dried. Product was
purified by re-crystallization from ethanol–acetone (1:5).
1-((4,7,7-Trimethyl-3-oxobicyclo[2.2.1]heptan-2-
yl)methyl)indoline-2,3-dione (2c).
FTIR (KBr) cm-1 3039 and 759 (Ar–H), 2957, 2926, 2869,
1457, 1423, 1343 and 1323 (C–H), 1742, 1727, 1689
Scheme 1 Synthesis route of
N-substituted isatin derivatives
O
Cyclic/Aromatic Carbonyl Compounds
HCHO
O
+
+
N
H
1a-e
1a=1-Methylpiperidine-4-one
1b=Tetrahydropyranone
1c=Camphor
1d=Tropinone
1e=3,4,5-Trimethoxyphenylacetic acid
Ethanol
HCl
O
O
N
R
2a-e
123

Med Chem Res
Scheme 2 Synthesis route
of 4,5-disubstituted oxazole
derivatives
O
O
NH
p-toluenesulfinic acid, HCONH2
10-camphorsulfonic acid
R
O
S
O
3a-d
R
3a R=3,4,5-tri-OCH3
3b R=p-N(CH3)2
3c R=p-Cl
4a-d
CH3
3d R=m-Br
POCl3
DME
O
C-
R
N+
R1
R1
O
S
N
O
O
K2CO3, C2H5OH/DME (6:4)
R
6a-p
R1=3,4,5-tri-OCH3/p-N(CH3)2/p-Cl/m-Br
5a-d
CH3
1
(C=O), 1599 (C=C of Ar). H NMR (DMSO) d 7.75–7.39
(3H, m), 7.19 (1H, d), 4.11 (1H, dd), 3.87 (1H, dd), 2.76
(1H, q), 2.04–1.39 (5H, m), 1.27 (3H, s), 0.97 (6H, s). 13C
NMR (75 MHz, DMSO-d6) d 15.5, 22.0, 31.3, 36.5, 37.3,
46.5, 51.6, 59.8, 117.4, 122.2, 124.7, 130.3, 134.3, 148.4,
160.6, 184.9, 222.9, 258.1. MS (TISI) 311.3 (M?). Anal.
Calcd: C, 66.16; H, 5.92; N, 10.29. Found: C, 66.18; H,
5.84; N, 10.33.
(C=C of Ar), 1578 and 1418 (COO-), 1241 and 1003
1
(C–O). H NMR (DMSO) d 12.04 (1H, s), 7.68–7.39 (3H,
m), 7.18 (1H, d), 6.54 (2H, s), 4.19 (1H, t), 3.93 (1H, dd),
3.81 (3H, s), 3.78 (6H, s), 3.63 (1H, dd). 13C NMR
(75 MHz, DMSO-d6) d 43.6, 51.3, 56.3, 56.9, 106.8, 107.4,
117.6, 122.2, 124.7, 129.1, 130.4, 134.8, 138.3, 148.6,
151.2, 160.5, 177.1, 184.3. MS (TISI) 385.3 (M?). Anal.
Calcd: C, 62.33; H, 4.97; N, 3.63. Found: C, 62.38; H,
4.99; N, 3.61.
1-((8-Methyl-3-oxo-8-azabicyclo[3.2.1]octan-2-
yl)methyl)indoline-2,3-dione (2d)
General procedure for synthesis of N-{[(4-
methylphenyl)sulfonyl](phenyl)methyl}formamide
derivatives (4a–d)
FTIR (KBr) cm-1 3043 and 755 (Ar–H), 2999, 2936, 2857,
1459, 1420 and 1318 (C–H), 1739, 1725, 1683 (C=O), 1595
(C=CofAr), 1184(NR3). 1HNMR(DMSO) d7.79–7.45(3H,
m), 7.23 (1H, d), 4.03 (1H, dd), 3.81 (1H, dd), 3.19 (1H, q),
2.69–2.40 (4H, m), 2.23 (3H, s), 1.71 (2H, m), 1.41 (2H, m).
13C NMR (75 MHz, DMSO-d6) d 28.6, 31.2, 38.4, 43.5, 44.3,
52.1, 58.7, 64.7, 117.5, 122.1, 124.4, 130.2, 134.6, 148.4,
160.5, 185.1, 211.5. MS (TISI) 298.3 (M?). Anal. Calcd: C,
68.44; H, 6.08; N, 9.39. Found: C, 68.48; H, 6.09; N, 9.33.
A solution of toluenesulfinic acid (22.3 g, 0.15 mol), 3a or
3b or 3c or 3d (0.18 mol), and camphorsulfonic acid
(3.48 g, 1.5 mmol) in formamide (40 ml) was stirred vig-
orously at 65 °C for 16 h, cooled, and filtered. The
resulting solid was washed with methanol and dried to
provide the desired compound.
N-{[(4-methylphenyl)sulfonyl](3,4,5-
trimethoxyphenyl)methyl}formamide (4a)
3-(2,3-Dioxoindolin-1-yl)-2-(3,4,5-
trimethoxyphenyl)propanoic acid (2e)
FTIR (KBr) cm-1 3436 and 1515 (N–H), 3027, 840 and
748 (Ar–H), 2935, 1460 and 1371 (CH3), 2893 and 1336
(C–H), 1672 (C=O), 1598 (C=C of Ar), 1326 and 1144
FTIR (KBr) cm-1 3037, 827 and 749 (Ar–H), 2941, 2843,
1459 and 1321 (C–H), 1725, 1707, 1679 (C=O), 1601
123

Med Chem Res
(S=O), 1233 and 999 (C–O). 1H NMR (DMSO) d 8.17 (1H,
s), 7.85 (2H, d), 7.30 (2H, d), 6.41 (1H, s), 6.07 (2H, s),
3.89 (6H, s), 3.78 (3H, s), 2.29 (3H, s), 1.97 (1H, s). 13C
NMR (75 MHz, DMSO-d6) d 24.2, 56.4, 56.8, 74.7, 104.0,
128.5, 129.8, 135.8, 136.9, 137.2, 143.5, 150.8, 163.2.
Anal. Calcd: C, 56.98; H, 5.58; N, 3.69. Found: C, 57.04;
H, 5.54; N, 3.66.
this solution was added phosphorus oxychloride (10.3 ml,
110 mmol) followed by dropwise addition of triethylamine
(25.3 ml, 181.5 mmol) in DME (20 ml). The reaction
mixture was stirred at -5 °C for 3 h, then poured into ice
cold water (500 ml), and extracted with ethylacetate
(3 9 120 ml). The combined extracts were washed with
10 % sodium bicarbonate and brine, dried with magnesium
sulfate, concentrated in vacuo to 20 % of the original
volume, and filtered to afford the desired compound.
N-{[4-(dimethylamino)phenyl]
[(4-methylphenyl)sulfonyl]methyl}formamide (4b)
5-{Isocyano[(4-methylphenyl)sulfonyl]methyl}-1,2,3-
trimethoxybenzene (5a)
FTIR (KBr) cm-1 3434 and 1515 (N–H), 3028, 844 and 749
(Ar–H), 2937, 1456 and 1375 (CH3), 2897 and 1336 (C–H),
1673 (C=O), 1600 (C=C of Ar), 1327 and 1144 (S=O), 1173
(C-NR2). 1H NMR (DMSO) d 8.20 (1H, s), 7.84 (2H, d), 7.28
(2H, d), 6.84 (2H, d), 6.49 (2H, d), 6.38 (1H, s), 2.93 (6H, s),
2.26 (3H, s), 2.01 (1H, s). 13C NMR (75 MHz, DMSO-d6) d
24.7, 40.6, 74.5, 114.0, 127.6, 128.2, 128.6, 130.4, 131.5,
135.7, 143.5, 147.3, 163.3. Anal. Calcd: C, 61.42; H, 6.06; N,
8.43. Found: C, 61.39; H, 6.10; N, 8.41.
FTIR (KBr) cm-1 3024, 837 and 747 (Ar–H), 2930, 1456
and 1372 (CH3), 2898 and 1332 (C–H), 2171 (NC), 1596
(C=C of Ar), 1329 and 1145 (S=O), 1236 and 1002 (C–O).
1H NMR (DMSO) d 7.87 (2H, d), 7.33 (2H, d), 6.23 (1H,
s), 6.09 (2H, s), 3.84 (6H, s), 3.73 (3H, s), 2.28 (3H, s). 13
C
NMR (75 MHz, DMSO-d6) d 24.3, 56.3, 56.7, 80.6, 106.4,
128.0, 128.5, 130.4, 132.2, 135.7, 136.3, 143.6, 150.5,
157.2. Anal. Calcd: C, 59.82; H, 5.30; N, 3.88. Found: C,
59.88; H, 5.28; N, 3.84.
N-{(4-chlorophenyl)
[(4-methylphenyl)sulfonyl]methyl}formamide (4c)
4-{Isocyano[(4-methylphenyl)sulfonyl]methyl}-N,N-
dimethylaniline (5b)
FTIR (KBr) cm-1 3431 and 1517 (N–H), 3033, 847 and
750 (Ar–H), 2938, 1454 and 1373 (CH3), 2895 and 1334
(C–H), 1675 (C=O), 1602 (C=C of Ar), 1326 and 1143
(S=O), 719 (C–Cl). 1H NMR (DMSO) d 8.14 (1H, s), 7.80
(2H, d), 7.32 (2H, d), 7.17 (2H, d), 6.97 (2H, d), 6.45 (1H,
s), 2.37 (3H, s), 1.94 (1H, s). 13C NMR (75 MHz, DMSO-
d6) d 24.3, 74.3, 128.1, 128.5, 128.9, 130.5, 132.4, 135.6,
140.7, 143.5, 163.1. Anal. Calcd: C, 55.64; H, 4.36; N,
4.33. Found: C, 55.59; H, 4.33; N, 4.31.
FTIR (KBr) cm-1 3027, 836 and 749 (Ar–H), 2933, 1457
and 1372 (CH3), 2896 and 1331 (C–H), 2173 (NC), 1597
1
(C=C of Ar), 1330 and 1146 (S=O), 1168 (C-NR2). H
NMR (DMSO) d 7.89 (2H, d), 7.36 (2H, d), 6.83 (2H, d),
6.49 (2H, d), 6.24 (1H, s), 2.97 (6H, s), 2.22 (3H, s). 13C
NMR (75 MHz, DMSO-d6) d 24.4, 40.4, 79.6, 114.4,
127.5, 128.5, 130.3, 130.6, 135.6, 143.8, 146.7, 157.1.
Anal. Calcd: C, 64.94; H, 5.77; N, 8.91. Found: C, 64.91;
H, 5.76; N, 8.94.
N-{(3-bromophenyl)
[(4-methylphenyl)sulfonyl]methyl}formamide (4d)
(4-Chlorophenyl)(isocyano)methyl 4-methylphenyl
sulfone (5c)
FTIR (KBr) cm-1 3433 and 1514 (N–H), 3034, 846 and
748 (Ar–H), 2936, 1458 and 1370 (CH3), 2897 and 1333
(C–H), 1673 (C=O), 1601 (C=C of Ar), 1327 and 1143
(S=O), 595 (C–Br). 1H NMR (DMSO) d 8.19 (1H, s), 7.86
(2H, d), 7.30–7.24 (4H, m), 7.05–6.98 (2H, m), 6.40 (1H,
s), 2.34 (3H, s), 2.03 (1H, s). 13C NMR (75 MHz, DMSO-
d6) d 24.4, 74.5, 122.6, 126.0, 128.7, 129.4, 130.4, 130.6,
131.6, 135.3, 143.8, 144.9, 163.4. Anal. Calcd: C, 48.92; H,
3.83; N, 3.80. Found: C, 48.95; H, 3.83; N, 3.78.
FTIR (KBr) cm-1 3031, 839 and 750 (Ar–H), 2933, 1456
and 1376 (CH3), 2899 and 1335 (C–H), 2177 (NC), 1600
1
(C=C of Ar), 1327 and 1146 (S=O), 723 (C–Cl). H NMR
(DMSO) d 7.86 (2H, d), 7.30 (2H, d), 7.13 (2H, d), 6.96
(2H, d), 6.27 (1H, s), 2.28 (3H, s). 13C NMR (75 MHz,
DMSO-d6) d 24.6, 79.7, 128.4, 128.5, 130.1, 130.3, 131.1,
135.6, 135.7, 143.7, 157.0. Anal. Calcd: C, 58.92; H, 3.96;
N, 4.58. Found: C, 58.97; H, 3.93; N, 4.57.
General procedure for synthesis of 1-
{[isocyano(phenyl)methyl]sulfonyl}-4-methylbenzene
derivatives (5a–d)
(3-Bromophenyl)(isocyano)methyl 4-methylphenyl
sulfone (5d)
A solution of 4a or 4b or 4c or 4d (36.3 mmol) in 200 ml
of 1,2-dimethoxyethane (DME) was cooled to -10 °C. To
FTIR (KBr) cm-1 3034, 840 and 751 (Ar–H), 2936, 1459
and 1376 (CH3), 2896 and 1332 (C–H), 2175 (NC), 1603
123

Med Chem Res
1
(C=C of Ar), 1326 and 1144 (S=O), 589 (C–Br). H NMR
145.6, 151.2, 151.9. MS (TISI) 345.0 (M?). Anal. Calcd:
C, 62.52; H, 4.66; N, 4.05. Found: C, 62.54; H, 4.67; N,
4.07.
(DMSO) d 7.83 (2H, d), 7.32–7.23 (4H, m), 7.07–7.01 (2H,
m), 6.26 (1H, s), 2.29 (3H, s). 13C NMR (75 MHz, DMSO-
d6) d 24.5, 79.4, 123.4, 128.1, 128.3, 128.5, 130.4, 130.7,
133.7, 135.8, 140.0, 144.1, 157.2. Anal. Calcd: C, 51.44; H,
3.45; N, 4.00. Found: C, 51.42; H, 3.47; N, 4.02.
5-(3-Bromophenyl)-4-(3,4,5-trimethoxyphenyl)-
1,3-oxazole (6d)
General procedure for synthesis of 4,5-disubstituted
FTIR (KBr) cm-1 3026, 833 and 750 (Ar–H), 2941 and
1455 (CH3), 1675 (C=N–C), 1601 (C=C of Ar), 1235 and
998 (C–O), 593 (C–Br). 1H NMR (DMSO) d 8.12 (1H, d),
7.97 (1H, s), 7.54–7.40 (3H, m), 6.85 (2H, s), 3.82 (6H, s),
3.72 (3H, s). 13C NMR (75 MHz, DMSO-d6) d 56.6, 56.7,
104.4, 123.8, 124.1, 126.6, 131.5, 131.9, 132.5, 133.4,
136.5, 139.3, 145.4, 150.7, 151.5. MS (TISI) 389.0 (M?).
Anal. Calcd: C, 55.40; H, 4.13; N, 3.59. Found: C, 55.41;
H, 4.14; N, 3.56.
oxazoles (6a–p)
A mixture of 5a or 5b or 5c or 5d (1.2 mmol), substituted
benzaldehyde (1 mmol), and potassium carbonate
(2.4 mmol) in 10 ml of methanol and 3 ml of DME was
refluxed for 2 h. After it was cooled to room temperature,
the solution was concentrated in vacuo and diluted with
ethyl acetate. The solution was washed with water and
brine, dried with magnesium sulfate, filtered and concen-
trated in vacuo. The residue was purified with column
chromatography by eluting with methanol to give the
product.
N,N-dimethyl-4-[5-(3,4,5-trimethoxyphenyl)-
1,3-oxazol-4-yl]aniline (6e)
FTIR (KBr) cm-1 3030, 835 and 750 (Ar–H), 2939, 2901,
1454 and 1375 (CH3), 1676 (C=N–C), 1601 (C=C of Ar),
4,5-Bis(3,4,5-trimethoxyphenyl)oxazole (6a)
1
1240 and 997 (C–O), 1171 (C-NR2). H NMR (DMSO) d
FTIR (KBr) cm-1 3033, 837 and 751 (Ar–H), 2936 and
1456 (CH3), 1658 (C=N–C), 1593 (C=C of Ar), 1238 and
1002 (C–O). 1H NMR (DMSO) d 7.98 (1H, s), 6.85 (4H, s),
3.86 (12H, s), 3.76 (6H, s). 13C NMR (75 MHz, DMSO-d6)
d 56.4, 56.7, 104.7, 124.2, 127.6, 136.5, 139.6, 145.1,
151.0, 151.8. MS (TISI) 401.1 (M?). Anal. Calcd: C,
62.83; H, 5.78; N, 3.49. Found: C, 62.81; H, 5.79; N, 3.45.
7.95 (1H, s), 7.64 (2H, d), 6.85–6.69 (4H, m), 3.85 (6H, s),
3.75 (3H, s), 3.15 (6H, s). 13C NMR (75 MHz, DMSO-d6)
d 40.5, 56.3, 56.6, 104.3, 114.1, 122.6, 124.4, 128.7, 136.9,
145.6, 149.8, 151.0, 151.5. MS (TISI) 354.1 (M?). Anal.
Calcd: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.79; H,
6.23; N, 7.91.
4,40-(1,3-Oxazole-4,5-diyl)bis(N,N-dimethylaniline) (6f)
N,N-dimethyl-4-[4-(3,4,5-trimethoxyphenyl)oxazol-5-
yl]aniline (6b)
FTIR (KBr) cm-1 3027, 812 and 727 (Ar–H), 2901 and
1371 (CH3), 1661 (C=N–C), 1598 (C=C of Ar), 1231
FTIR (KBr) cm-1 3025, 835 and 752 (Ar–H), 2938, 2903,
1454 and 1378 (CH3), 1673 (C=N–C), 1602 (C=C of Ar),
(C–O), 1165 (C-NR2). H NMR (DMSO) d 7.96 (1H, s),
1
7.63 (4H, d), 6.84 (4H, d), 3.13 (12H, s). 13C NMR
(75 MHz, DMSO-d6) d 40.6, 114.4, 119.7, 122.0, 128.3,
136.5, 145.7, 149.2, 149.4, 151.8. MS (TISI) 307.1 (M?).
Anal. Calcd: C, 74.24; H, 6.89; N, 13.67. Found: C, 74.25;
H, 6.90; N, 13.68.
1
1240 and 995 (C–O), 1169 (C-NR2). H NMR (DMSO) d
7.98 (1H, s), 7.63 (2H, d), 6.84–6.71 (4H, m), 3.83 (6H, s),
3.72 (3H, s), 3.09 (6H, s). 13C NMR (75 MHz, DMSO-d6)
d 40.9, 56.3, 56.6, 104.5, 115.1, 119.7, 127.6, 128.1, 136.8,
139.1, 145.2, 149.8, 151.0, 151.7. MS (TISI) 354.1 (M?).
Anal. Calcd: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.79;
H, 6.27; N, 7.91.
4-[5-(4-Chlorophenyl)-1,3-oxazol-4-yl]-N,N-
dimethylaniline (6g)
5-(4-Chlorophenyl)-4-(3,4,5-trimethoxyphenyl)-1,3-
oxazole (6c)
FTIR (KBr) cm-1 3034, 819 and 749 (Ar–H), 2903 and
1377 (CH3), 1661 (C=N–C), 1605 (C=C of Ar), 1234
1
(C–O), 1173 (C-NR2), 717 (C–Cl). H NMR (DMSO) d
FTIR (KBr) cm-1 2943, 1458 and 1392 (CH3), 1687
(C=N–C), 1589 (C=C of Ar), 1233 and 992 (C–O), 845 and
757 (Ar–H), 729 (C–Cl). 1H NMR (DMSO) d 7.95 (1H, s),
7.77 (2H, d), 7.51 (2H, d), 6.83 (2H, s), 3.85 (6H, s), 3.75
(3H, s). 13C NMR (75 MHz, DMSO-d6) d 56.4, 56.9,
104.9, 124.5, 128.7, 129.3, 129.9, 134.6, 136.2, 139.1,
7.95 (1H, s), 7.82 (2H, d), 7.63 (2H, d), 7.51 (2H, d), 6.83
(2H, d), 3.15 (6H, s). 13C NMR (75 MHz, DMSO-d6) d
40.7, 114.6, 122.8, 128.0, 128.1, 128.9, 129.9, 134.4,
136.2, 145.5, 149.3, 151.6. MS (TISI) 298.1 (M?). Anal.
Calcd: C, 68.34; H, 5.06; N, 9.38. Found: C, 68.35; H,
5.07; N, 9.36.
123

Med Chem Res
1
4-[5-(3-Bromophenyl)-1,3-oxazol-4-yl]-N,N-
dimethylaniline (6h)
(C–Br). H NMR (DMSO) d 8.15 (1H, d), 8.08 (2H, d),
7.95 (1H, s), 7.72–7.30 (5H, m). 13C NMR (75 MHz,
DMSO-d6) d 123.8, 126.8, 128.4, 128.9, 129.6, 131.2,
131.4, 132.4, 133.5, 134.2, 136.7, 145.2, 152.5. MS (TISI)
334.9 (M?). Anal. Calcd: C, 53.84; H, 2.71; N, 4.19.
Found: C, 53.85; H, 2.72; N, 4.16.
FTIR (KBr) cm-1 3029, 822 and 747 (Ar–H), 2907 and
1371 (CH3), 1655 (C=N–C), 1598 (C=C of Ar), 1231
1
(C–O), 1165 (C-NR2), 595 (C–Br). H NMR (DMSO) d
8.11 (1H, d), 7.96 (1H, s), 7.76–7.32 (5H, m), 6.83 (2H, d),
3.15 (6H, s). 13C NMR (75 MHz, DMSO-d6) d 40.6, 114.3,
122.8, 123.7, 126.2, 128.7, 131.3, 131.5, 132.2, 133.4,
136.4, 145.0, 149.9, 151.5. MS (TISI) 342.0 (M?). Anal.
Calcd: C, 59.49; H, 4.41; N, 8.16. Found: C, 59.47; H,
4.39; N, 8.15.
4-(3-Bromophenyl)-5-(3,4,5-trimethoxyphenyl)-1,3-
oxazole (6m)
FTIR (KBr) cm-1 3033, 834 and 750 (Ar–H), 2945 and
1456 (CH3), 1677 (C=N–C), 1598 (C=C of Ar), 1235 and
1007 (C–O), 595 (C–Br). 1H NMR (DMSO) d 7.96 (1H, s),
7.79 (1H, d), 7.59–7.35 (3H, m), 6.83 (2H, s), 3.83 (6H, s).
3.72 (3H, s). 13C NMR (75 MHz, DMSO-d6) d 56.0, 56.3,
104.5, 123.4, 124.8, 126.8, 131.3, 131.5, 133.3, 135.6,
136.6, 139.0, 145.1, 151.7, 151.9. MS (TISI) 389.0 (M?).
Anal. Calcd: C, 55.40; H, 4.13; N, 3.59. Found: C, 55.41;
H, 4.14; N, 3.60.
4-(4-Chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-1,3-
oxazole (6i)
FTIR (KBr) cm-1 3032, 841 and 754 (Ar–H), 2947 and
1453 (CH3), 1684 (C=N–C), 1589 (C=C of Ar), 1236 and
993 (C–O), 721 (C–Cl). 1H NMR (DMSO) d 8.08 (2H, d),
7.97 (1H, s), 7.53 (2H, d), 6.82 (2H, s), 3.84 (6H, s), 3.73
(3H, s). 13C NMR (75 MHz, DMSO-d6) d 56.3, 56.6,
104.8, 124.4, 128.2, 129.1, 131.5, 134.6, 136.5, 139.8,
145.7, 151.1, 151.3. MS (TISI) 345.0 (M?). Anal. Calcd:
C, 62.52; H, 4.66; N, 4.05. Found: C, 62.54; H, 4.67; N,
4.087.
4-[4-(3-Bromophenyl)-1,3-oxazol-5-yl]-N,N-
dimethylaniline (6n)
FTIR (KBr) cm-1 3024, 823 and 749 (Ar–H), 2907 and
1373 (CH3), 1657 (C=N–C), 1593 (C=C of Ar), 1231
1
(C–O), 1167 (C-NR2), 595 (C–Br). H NMR (DMSO) d
4-[4-(4-Chlorophenyl)-1,3-oxazol-5-yl]-N,N-
dimethylaniline (6j)
7.95 (1H, s), 7.78 (1H, d), 7.69–7.33 (5H, m), 6.84 (2H, d),
3.14 (6H, s). 13C NMR (75 MHz, DMSO-d6) d 40.7, 114.7,
120.2, 123.1, 126.2, 128.9, 131.4, 131.6, 133.4, 135.7,
136.4, 145.0, 149.3, 151.1. MS (TISI) 342.0 (M?). Anal.
Calcd: C, 59.49; H, 4.41; N, 8.16. Found: C, 59.49; H,
4.42; N, 8.14.
FTIR (KBr) cm-1 3026, 821 and 749 (Ar–H), 2905 and
1375 (CH3), 1664 (C=N–C), 1601 (C=C of Ar), 1236
1
(C–O), 1173 (C-NR2), 719 (C–Cl). H NMR (DMSO) d
8.10 (2H, d), 7.95 (1H, s), 7.64 (2H, d), 7.51 (2H, d), 6.83
(2H, d), 3.13 (6H, s). 13C NMR (75 MHz, DMSO-d6) d
40.5, 115.1, 119.7, 128.2, 128.9, 129.2, 131.3, 134.4,
136.9, 145.0, 149.4, 151.6. MS (TISI) 298.1 (M?). Anal.
Calcd: C, 68.34; H, 5.06; N, 9.38. Found: C, 68.35; H,
5.07; N, 9.39.
4-(3-Bromophenyl)-5-(4-chlorophenyl)-1,3-oxazole (6o)
FTIR (KBr) cm-1 3025, 823 and 751 (Ar–H), 1670 (C=N–
C), 1603 (C=C of Ar), 1239 (C–O), 719 (C–Cl), 598
1
(C–Br). H NMR (DMSO) d 7.98 (1H, s), 7.85 (1H, d),
7.77 (2H, d), 7.68–7.31 (5H, m). 13C NMR (75 MHz,
DMSO-d6) d 123.8, 126.7, 128.7, 129.1, 131.3, 131.5,
132.9, 134.6, 135.9, 136.6, 145.5, 151.7. MS (TISI) 334.9
(M?). Anal. Calcd: C, 53.84; H, 2.71; N, 4.19. Found: C,
53.86; H, 2.72; N, 4.17.
4,5-Bis(4-chlorophenyl)-1,3-oxazole (6k)
FTIR (KBr) cm-1 3030, 823 and 750 (Ar–H), 1668
1
(C=N–C), 1599 (C=C of Ar), 1240 (C–O), 721 (C–Cl). H
NMR (DMSO) d 8.08 (2H, d), 7.96 (1H, s), 7.79 (2H, d),
7.62 (2H, d), 7.52 (2H, d). 13C NMR (75 MHz, DMSO-d6)
d 128.3, 128.7, 129.8, 131.6, 134.1, 136.4, 145.5, 152.2.
MS (TISI) 289.0 (M?). Anal. Calcd: C, 62.09; H, 3.13; N,
4.83. Found: C, 62.07; H, 3.14; N, 4.85.
4,5-Bis(3-bromophenyl)-1,3-oxazole (6p)
FTIR (KBr) cm-1 3029, 827 and 752 (Ar–H), 1665 (C=N–
1
C), 1605 (C=C of Ar), 1239 (C–O), 596 (C–Br). H NMR
(DMSO) d 8.09 (1H, d), 7.97 (1H, s), 7.78 (1H, d),
7.69–7.31 (6H, m). 13C NMR (75 MHz, DMSO-d6) d
123.5, 126.3, 131.2, 131.4, 132.1, 133.3, 135.5, 136.9,
145.8, 151.6. MS (TISI) 378.9 (M?). Anal. Calcd: C,
47.53; H, 2.39; N, 3.70. Found: C, 47.54; H, 2.40; N, 3.71.
5-(3-Bromophenyl)-4-(4-chlorophenyl)-1,3-oxazole (6l)
FTIR (KBr) cm-1 3027, 818 and 753 (Ar–H), 1673
(C=N–C), 1603 (C=C of Ar), 1241 (C–O), 717 (C–Cl), 596
123

Med Chem Res
Pharmacological studies
½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ ꢃ 100ðconcentrations for which Ti ꢄ TzÞ
½ðTi ꢁ TzÞ=Tzꢂ ꢃ 100ðconcentrations for which Ti \ TzÞ
In vitro cancer screen
Three dose–response parameters were calculated for each
experimental agent. GI50 was calculated from [(Ti - Tz)/
(C - Tz)] 9 100 = 50, which is the drug concentration
resulting in a 50 % reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug
incubation. The drug concentration resulting in total growth
inhibition (TGI) was calculated from Ti = Tz. The
concentration of drug resulting in a 50 % reduction in the
measured protein at the end of the drug treatment as
compared to that at the beginning (LC50) indicating a net loss
of cells following treatment was calculated from [(Ti - Tz)/
Tz] 9 100 = -50. Values were calculated for each of these
three parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded,
the value for that parameter was expressed as greater or less
than the maximum or minimum concentration tested (Boyd
and Paull 1995; DeVita et al., 1989; Alley et al., 1988).
The human tumor cell lines of the cancer screening panel
were grown in RPMI-1640 medium containing 5 % fetal
bovine serum and 2 mM L-glutamine. For a typical
screening experiment, cells were inoculated into 96-well
microtiter plates in 100 ll at plating densities ranging from
5,000 to 40,000 cells/well depending on the doubling time
of individual cell lines. After cell inoculation, the micro-
titer plates were incubated at 37 °C, 5 % CO2, 95 % air,
and 100 % relative humidity for 24 h prior to addition of
experimental drugs. After 24 h, two plates of each cell line
were fixed in situ with trichloroacetic acid (TCA), to rep-
resent a measurement of the cell population for each cell
line at the time of drug addition (Tz). Experimental drugs
were solubilized in DMSO at 400-fold the desired final
maximum test concentration and stored frozen prior to use.
At the time of drug addition, an aliquot of frozen con-
centrate was thawed and diluted to twice the desired final
maximum test concentration with complete medium con-
taining 50 lg/ml gentamicin. Additional four, tenfold or ‘
log serial dilutions were made to provide a total of five
drug concentrations plus control. Aliquots of 100 ll of
these different drug dilutions were added to the appropriate
microtiter wells already containing 100 ll of medium,
resulting in the required final drug concentrations.
Molecular modeling
The 3D structure of the most active compound of the series
(2c) as well as combretastatin (CA-4), were built using
SYBYL 7.1 molecular modeling package (SYBYL 7.1 2005)
installed on a silicon graphics fuel work station running IRIX
6.5. The full optimizations of these built structures were
carried out by AM1 method implemented in MOPAC inter-
face of SYBYL. The following parameters were set while
performing the minimization. Spin state of wave functionwas
set to singlet and normal SCF convergence was used for the
optimization, rest all other parameters were set as default.
For the purpose of molecular docking, crystal structure of
tubulin (PDB ID: 3SA0) (Ravelli et al., 2004) solved at
Following drug addition, the plates were incubated for
an additional 48 h at 37 °C, 5 % CO2, 95 % air, and 100 %
relative humidity. For adherent cells, the assay was ter-
minated by the addition of cold TCA. Cells were fixed
in situ by the gentle addition of 50 ll of cold 50 % (w/v)
TCA (final concentration, 10 % TCA) and incubated for
60 min at 4 °C. The supernatant was discarded, and the
plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 ll) at 0.4 % (w/v)
in 1 % acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. After staining,
unbound dye was removed by washing five times with 1 %
acetic acid and the plates were air dried. Bound stain was
subsequently solubilized with 10 mM trizma base, and the
absorbance was read on an automated plate reader at a
wavelength of 515 nm. For suspension cells, the method-
ology was the same except that the assay was terminated by
fixing settled cells at the bottom of the wells by gently
adding 50 ll of 80 % TCA (final concentration, 16 %
TCA). Using the seven absorbance measurements [time
zero, (Tz), control growth, (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the
percentage growth was calculated at each of the drug
concentrations levels. Percentage growth inhibition was
calculated as:
˚
3.58 A resolution was utilized. Ligand docking was accom-
plished by the Grid-based Ligand Docking with Energetics
¨
(GLIDE) (GLIDE 2009) program incorporated in Schro-
dinger package. GLIDE algorithm is based on a systematic
search of positions, orientations, and conformations of the
ligand in the receptor binding site using funnel type approach.
Protein and ligand preparation
The X-ray crystallographic structures of a,b-tubulin (PDB
˚
ID: 1SA0; 3.58 A resolution) in complex with DAMA-
colchicine and with the stathmin-like domain (SLD) of
RB3 was downloaded from Protein Data Bank (PDB).
Tubulin dimer 1 (chains A and B) was extracted along with
the associated GTP, GDP, DAMA-colchicine (COL), and
Mg. While other two chains (C and D) and SLD of RB3
was removed.
123

Med Chem Res
Further, the tubulin dimer structure was prepared for the
docking study using the protein preparation wizard tool
implemented in Maestro. The right bond orders were
assigned and the hydrogen atoms were added to the protein.
The orientation of hydroxyl groups, amide groups of Asn and
Gln, and charge state of His residues were optimized using
the utility protassign. The final step was a restrained mini-
mization by the impref utility until the average root-mean-
square-deviation (RMSD) of the hydrogen atoms reached
respectively, described in this study are outlined in
Schemes 1 and 2. The chemical structure and physical data
of these compounds is presented in Table 1. N-substituted
isatin derivatives (2a–e) were synthesized by mannich
reaction of isatin with aromatic/cyclic carbonyl compounds
in the presence of HCl and ethanol. 4,5-Disubstituted
oxazole derivatives (6a–p) were synthesized by reaction of
substituted benzaldehydes with p-toluenesulfinic acid and
formamide in the presence of 10-camphorsulfonic acid,
followed by dehydration and subsequent reaction with
substituted benzaldehydes. N-substituted isatin derivatives
were recrystallized with ethanol, while 4,5-disubstituted
oxazole derivatives were recrystallized with methanol.
The purity of the compounds was checked by TLC.
˚
0.3 A, leaving heavy atoms in place. Ligands were prepared
using LigPrep by generating all possible low energy ioni-
zation, tautomeric, and stereoisomeric states within the
range of pH 7.0 2.0 using OPLS_2005 force field.
1
Grid generation and protocol setting
Spectral data H and 13C NMR, and IR of all the synthe-
sized compounds, and mass of final compounds, were
recorded and found in full agreement with the proposed
structures. Structural formulae of the compounds were
further confirmed by elemental analysis.
An interaction grid was generated for protein structure by
using the receptor grid generation wizard of GLIDE 5.5, by
taking bound inhibitor DAMA-colchicine as the reference
structure for defining active site. The grid box was centered
on the bound ligand in the protein structure by selecting the
ligand from the workspace, and used its centroid (XYZ
coordinates: 117.1827, 89.9909, 6.2823) for grid genera-
Pharmacological evaluation
Primary single high dose (10-5 M) full NCI 60 cell
panel in vitro assay
˚
tion. The grid box was extended 10 A from the center, with
˚
the outer box extending an additional 10 A, completely
covering the active site cavity.
All the eight selected compounds, submitted to NCI for
in vitro anticancer assay, were evaluated for their anti-
proliferative activity. Primary in vitro one dose anticancer
assay was performed in full NCI 60 cell panel representing
leukemia, melanoma and cancers of lung, colon, brain,
breast, ovary, kidney and prostate in accordance with the
protocol of the NCI, USA. The compounds were added at a
single concentration (10-5 M) and the culture was incu-
bated for 48 h. End point determinations were made with a
protein binding dye, SRB. Results for each compound were
reported as a mean graph of the percent growth of the
treated cells when compared to the untreated control cells.
There after obtaining the results for one dose assay, anal-
ysis of historical Developmental Therapeutics Program
(DTP) was performed and compound 2c (NSC 742530),
which satisfied pre-determined threshold inhibition criteria
was selected for NCI full panel 5 dose assay. Data of 5
dose assay for compound 2c is shown in Table 2.
Docking and scoring of compounds using GLIDE
To set a docking protocol, the bound ligand (DAMA-
colchicine) was extracted from the prepared protein struc-
ture. The atom and bond type of the extracted ligand were
corrected, and was subjected to re-docking by imple-
menting abovementioned grid parameters, and finally
RMSD was determined.
After the validation of docking protocol, the set of
prepared ligands were docked into the colchicine-binding
site of a,b-tubulin by using GLIDE 5.5 at SP (Standard
Precision) mode. The docking and scoring algorithms of
GLIDE have been fully described elsewhere (Friesner
et al., 2004; Halgren et al., 2004). All docking calculations
were performed using the OPLS-2005, an implementation
of the optimized potential for liquid simulation - all atom
¨
force field (Schrodinger, Portland, OR). All the other
parameters were kept at default for the docking.
In vitro 5 dose full NCI 60 cell panel assay
All the cell lines (about 60), representing nine tumor sub-
panels, were incubated at five different concentrations
(0.01, 0.1, 1, 10, and 100 mM). The outcomes were used to
create log concentration vs. % growth inhibition curves,
and three response parameters (GI50, TGI, and LC50) were
calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the concentration of the
Results and discussion
Chemistry
The synthesis of N-substituted isatin derivatives and 4,5-
disubstituted oxazole derivatives, (2a–e) and (6a–p),
123

Med Chem Res
Table 1 Physical properties of the synthesized compounds
O
R
R1
O
N
N
O
R
(2a-e)
(6a-p)
Compound
code
NSC
number
R
R1
Molecular
formula
Molecular
weight
Melting
point
2a
2b
2c
–
1-Methyl-4-oxopiperidin-3-yl
3-Oxotetrahydro-2H-pyran-2-yl
–
–
–
C15H16N2O3
272.30
259.26
311.37
[250
[250
[250
–
C
C
14H13NO4
19H21NO3
742530
4,7,7-Trimethyl-3-
oxobicyclo[2.2.1]heptan-1-yl
2d
–
8-Methyl-3-oxo-8-azabicyclo[3.2.1]octan-
2-yl
–
C
17H18N2O3
298.34
[250
2e
6a
–
(3,4,5-Trimethoxyphenyl) carboxymethyl
3,4,5-Trimethoxy
–
C
C
20H19NO7
21H23NO7
385.37
401.40
[250
[250
753088
3,4,5-
Trimethoxy
6b
–
3,4,5-Trimethoxy
p-
C
20H22N2O4
354.39
108–110
Dimethylamino
p-Chloro
6c
6d
6e
–
3,4,5-Trimethoxy
3,4,5-Trimethoxy
p-Dimethylamino
C
C
C
18H16ClNO4
18H16BrNO4
20H22N2O4
345.77
390.22
354.39
163–165
155–157
149–151
–
m-Bromo
753089
3,4,5-
Trimethoxy
6f
753092
p-Dimethylamino
p-
C
19H21N3O
307.38
145–147
Dimethylamino
6g
6h
6i
–
p-Dimethylamino
p-Dimethylamino
p-Chloro
p-Chloro
m-Bromo
C
C
C
17H15ClN2O
17H15BrN2O
18H16ClNO4
298.76
343.21
345.77
152–154
167–169
179–181
–
753090
3,4,5-
Trimethoxy
6j
753093
p-Chloro
p-
C
17H15ClN2O
298.76
190–192
Dimethylamino
6k
6l
–
p-Chloro
p-Chloro
m-Bromo
p-Chloro
m-Bromo
C
C
C
15H9Cl2NO
15H9BrClNO
18H16BrNO4
290.14
334.59
390.22
140–142
182–184
171–173
–
6m
753091
3,4,5-
Trimethoxy
6n
753094
m-Bromo
p-
C
17H15BrN2O
343.21
138–140
Dimethylamino
6o
6p
–
–
m-Bromo
m-Bromo
p-Chloro
m-Bromo
C
C
15H9BrClNO
15H9Br2NO
334.59
379.04
131–133
202–204
compound causing 50 % decrease in net cell growth, the
TGI value (cytostatic activity) is the concentration of the
compound resulting in TGI and LC50 value (cytotoxic
activity) is the concentration of the compound causing net
50 % loss of initial cells at the end of the incubation period
of 48 h. Compound 2c (NSC 742530) under investigation
exhibited remarkable anticancer activity against most of
the tested cell lines representing nine different subpanels
with GI50 values between ‘‘1.53 and 26.9 lM’’ (Table 2).
With regard to the sensitivity against individual cell line
(Table 2) the compound showed highest activity against
leukemia SR cell line with GI50 1.53 lM and least for
melanoma MALME-3M cell line with GI50 26.9 lM.
Obtained data revealed that compound sensitivity is more
in leukemic (GI50 1.53–7.46 lM) and colon cancer (GI50
1.95–13.5 lM except HCT-15) cell lines, and least in non-
small cell lung cancer (GI50 13.5–23.4 lM) and melanoma
(GI50 10.4–26.9 lM) cell lines. The criterion for selectivity
of a compound depends upon the ratio obtained by dividing
the full panel MID (the average sensitivity of all cell lines
123

Med Chem Res
Table 2 NCI developmental therapeutics program, in vitro testing results of compound NSC 742530 (compound 2c) in lM
Panel
Cell line
GIc50
TGId
LCe50
Concentration per cell line
Subpanel MIDb
3.51
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
7.46
4.40
2.46
3.51
3.22
1.53
16.9
19.6
15.6
17.9
21.8
14.5
23.4
16.9
13.5
12.6
13.5
1.95
17.1
5.45
4.12
3.21
15.8
11.13
12.8
13.8
17.1
15.4
10.4
15.9
26.9
15.6
19.0
19.7
17.3
13.9
16.2
15.6
18.8
14.8
2.86
12.7
14.8
28.6
15.9
8.20
10.6
17.2
4.02
32.9
37.9
30.1
38.3
61.0
29.6
38.3
36.5
29.4
29.2
27.5
3.76
34.7
20.4
12.5
9.25
36.0
24.3
25.8
26.6
33.1
29.3
24.5
33.5
44.8
32.9
41.7
39.5
33.0
31.1
30.6
33.5
44.1
28.4
6.97
35.3
28.5
[100
59.8
96.9
91.1
MOLT-4
RPMI-8226
SR
[100
13.4
64.1
73.3
58.1
81.9
Non-small cell
Lung cancer
A549/ATCC
EKVX
17.78
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
[100
60.4
62.7
78.4
64.1
67.4
56.2
Colon cancer
8.27
7.25
70.4
65.5
45.8
35.5
81.9
52.5
51.8
51.6
64.2
56.0
58.0
70.9
74.5
69.2
91.3
78.9
63.0
69.3
57.7
72.2
HT29
KM12
SW-620
CNS cancer
SF-268
14.33
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
M-14
17.21
MALME-3M
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Ovarian cancer
13.26
[100
54.6
38.7
98.0
54.8
123

Med Chem Res
Table 2 continued
Panel
Cell line
GIc50
TGId
LCe50
Concentration per cell line
Subpanel MIDb
13.67
Renal cancer
786-0
16.8
16.2
16.3
5.46
15.3
14.8
13.2
11.9
14.9
5.80
15.1
13.8
12.3
20.6
13.21
30.8
29.7
31.3
16.9
31.6
28.5
26.2
25.9
28.1
24.6
30.0
32.6
24.8
46.7
56.4
54.5
60.1
44.0
65.0
54.8
51.8
56.4
53.0
90.0
59.8
81.2
49.8
[100
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
Breast cancer
PC-3
13.4
DU-145
MCF7
13.38
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MIDa
a
Average sensitivity of all cell lines in lM
b
c
d
e
Average sensitivity of all cell lines of a particular sub panel in lM
Concentration of the compound causing 50 % decrease in net cell growth
Concentration of the compound resulting in total growth inhibition
Concentration of the compound causing net 50 % loss of initial cells at the end of the incubation period of 48 h
toward the test agent) by their individual subpanel MID
(the average sensitivity of all cell lines of a particular
subpanel toward the test agent). Ratio between 3 and 6
refer to moderate selectivity; ratios [6 indicate high
selectivity toward the corresponding cell line, while com-
pounds not meeting either of these criteria were rated non-
selective (Rostom 2006). As per this criterion, compound
in the study was found to be mild selective toward leuke-
mia subpanel only. Whereas, on overall basis it was found
to be non-selective.
interaction of RB3-SLD with two tubulin heterodimers
(a,b-subunits), adopting a hook-like shape and extending
from head to tail of a complex in a curved manner. This
type of arrangement suggested that the stathmin plays an
important role in the regulation of microtubule assembly,
by not allowing the tubulin heterodimers to be incorporated
in the microtubule.
Molecular modeling studies were performed to investi-
gate the binding ability of the N-substituted isatin deriva-
tive (2c) to the colchicine-binding site of a,b-tubulin.
Before performing any molecular docking study of ligands
of interest, the first most imperative step is to set up a
docking protocol and its validation.
Molecular modeling studies
The colchicine-binding site
Thus, in order to check whether the selected docking
process was adequate enough for finding the correct
binding mode of ligands binding to the colchicine-binding
site, DAMA-colchicine (COL) was extracted from the
original X-ray crystallographic tubulin structure (PDB ID:
1SA0), and re-docked using GLIDE 5.5 (GLIDE 2009).
The set docking protocol was successfully able to dock the
extracted bound ligand inside the protein with an almost
identical binding mode (GLIDE score -8.39), when
compared with the co-crystallized X-ray structure. The
superimposition of the best scored docked conformer and
X-ray structure bound DAMA-colchicine showed the
The X-ray crystal structure of tubulin in complex with
N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-col-
chicine, Fig. 1) and with the SLD of RB3 (PDB ID: 1SA0),
˚
at 3.5 A resolution was reported by Ravelli et al., 2004.
˚
The same group also determined the 4.2 A structure of a
ternary tubulin-podophyllotoxin: RB3-SLD complex (PDB
ID: 1SA1), and found that podophyllotoxin also binds to
tubulin at the same site as colchicine. Thus, it can compete
with the colchicine to occupy the binding pocket in tubulin
(Wilson et al., 1974). The X-ray structure 1SA0 shows the
123

Med Chem Res
Fig. 2 Superimposition of co-crystallized X-ray bound (PDB ID:
˚
1SA0; cyan) and docked (magenta) COL (RMSD 1.33 A)
Fig. 3 Comparison of CA-4 (magenta) and COL (green) binding
models inside the a,b-tubulin binding pocket (PDB ID: 1SA0)
˚
RMSD of 1.33 A and is depicted in Fig. 2. It indicates that
the docking protocol is acceptable and can be further
extended to dock other ligands.
conformer of CA-4 showed GLIDE score of -8.30 which
is almost comparable to that of COL (-8.39). The com-
parison of the binding mode of CA-4 and COL is pre-
sented in Fig. 3. This figure clearly indicates that CA-4
binds to the colchicine-binding site in a similar fashion and
orientation like COL (rings A and B of CA-4 and rings A
and C of COL almost superimposes).
The detailed analyses of the docked COL revealed that it
binds at the a/b interface, but mostly buried in the interme-
diate domain of b-subunit, which is surrounded by the
strands S8 and S9, loop T7 and helices H7 and H8. However,
in addition to the interactions of COL with b-subunit, it was
also found to interact with loop T5 (Ser178, Thr179, Ala180,
Val181) of the neighboring a-subunit; consistent with the
fact that colchicine stabilizes the tubulin a,b-heterodimer
(Shearwin and Timasheff 1994). Thus, binding model of
COL correlates well with biochemical data showing that
mutants of tubulin formed by substitution at Valb318 have a
reduced sensitivity to colchicine (Burns 1992), and that the
oxygen atom of methoxy groups in ring A can form strong
hydrogen bonding (cross-linked) with the side chain of
Cysb241 (Bai et al., 2000).
Moreover, we also observed that the interaction pattern
of docked CA-4 is parallel to the COL. The methoxy
groups at ring A of CA-4 forms strong hydrogen bonding
with the side chain of Cysb241. The distance of these
˚
methoxy groups from –SH of Cysb241 are 5.43 A
˚
˚
(3-OꢀꢀꢀꢀS), 2.95 A (4-OꢀꢀꢀꢀS), and 3.10 A (5-OꢀꢀꢀꢀꢀS),
respectively, while the three methoxy groups at ring A of
˚
˚
˚
COL are 3.19 A (2-OꢀꢀꢀꢀꢀS), 2.93 A (3-OꢀꢀꢀꢀꢀS), and 5.49 A
(4-OꢀꢀꢀꢀꢀS), respectively, away from the same residue. The
binding pose of docked CA-4 showed that the 3-hydroxyl
group of the ring B of CA-4 forms hydrogen bonding
Docking of CA-4
˚
(3.09 A) with the main chain carbonyl group of Thr179 of
CA-4, a natural cis-stilbene product is the most promising
compound for the treatment of solid tumors (Pettit et al.,
1989). Its water-soluble phosphate prodrug (CA4P) is
currently in phase II and III clinical trials (Patterson and
Rustin 2007; Lippert 2007; Hinnen and Eskens 2007; Cai
2007; Horsman and Siemann 2006; West and Price 2004).
The enormous amount of literature for the CA-4 (Fig. 1)
and its analogs suggested that, these compounds also
inhibit tubulin polymerization by occupying the same
binding pocket similar to that of COL (Simoni et al., 2006;
Tron et al., 2006; Woods et al., 1995).
a-tubulin subunit. The same hydroxyl moiety may also be
˚
involved in a hydrogen bonding (3.55 A) with the side
chain of Lys352 of b-tubulin. In addition, the 4-methoxy
group of the ring B of CA-4 might also form long range
˚
hydrogen bond (4.07 A) with the main chain amide nitro-
gen of Lysb352. Thus, our proposed binding model is
consistent with the fact that CA-4 induces GTPase activity
(Pandit et al., 2006). Beside this, some weaker interactions
were also observed. The Ala180 of a-tubulin subunit
interacts by the weak CHꢀꢀꢀꢀꢀO hydrogen bonds, Leub255
by CHꢀꢀꢀꢀꢀp interaction with ring A. The side chains of the
residues Alab316 and Vala181 may be involved in
hydrophobic, van der Waals interactions.
In order to compare the docking score and binding mode
of CA-4 with the COL and synthesized potent anticancer
compound 2c, CA-4 was docked into the colchicine-binding
site using our adopted docking methodology.
Docking of synthesized, active compound 2c
The combination of GLIDE scores and visual inspection
of the docked conformation was taken into account to
select the most plausible binding mode. The best docked
Molecular docking studies showed that the synthe-
sized, bioactive isatin derivative (2c) occupied the
123

Med Chem Res
Fig. 4 Binding model of
compound 2c and its hydrogen
bonding interactions (blue
dashed lines) with tubulin (PDB
ID: 1SA0) binding pocket (blue
color residues). A and B in the
amino acid residues label
signifies a- and b-tubulin,
respectively
colchicine-binding site of a,b-tubulin (mostly buried in the
b-subunit). The binding model of compounds 2c and its
interactions with the tubulin heterodimer are presented in
Fig. 4. The best docked conformer of compound 2c showed
GLIDE score of 6.51. In consistent with the most promi-
nent interaction of tubulin inhibitors (COL, CA-4 and
its analogs), the O3 atom of indoline ring in compound 2c
4,5-disubstituted oxazole derivatives (6a–p) synthesized by
reaction of substituted benzaldehydes with p-toluenesulfinic
acid and formamide in the presence of 10-camphorsulfonic
acid, followed by dehydration and subsequent reaction with
substituted benzaldehydes. Data of the synthesized com-
pounds was submitted to NCI, and only eight compounds
(2c, 6a, 6e, 6f, 6i, 6j, 6m, and 6n) were subsequently selected
for their in vitro anticancer activity. On the basis of results
obtained, it was found that compound 2c, 1-[(4,7,7-tri-
methyl-3-oxobicyclo[2.2.1]heptan-2-yl)methyl]indoline-2,3-
dione (NSC 742530) was most active compound of the series
and also satisfied pre-determined threshold inhibition criteria.
Hence, it was subjected to NCI full panel 5 dose assays for
further evaluation. The result of these assays indicated that
compound 2c exhibited significant anticancer activity against
most of the tested cell lines representing nine different
subpanels with GI50 values between ‘‘1.53 and 26.9 lM.’’
Molecular docking studies, of compound 2c, revealed possi-
ble mode of action, plausibly by blocking the colchicine-
binding site of a,b-tubulin heterodimer. The binding model of
the compound 2c, suggest that, this compounds form key
interactions with the tubulin a,b-subunits in the similar fash-
ion to that of DAMA-colchicine (COL) and CA-4. Finally, it
is conceivable that further derivatization of such compounds
will be of interest with the hope to get more selective anti-
cancer agents.
˚
also formed hydrogen bonding interaction (2.72 A) with
the side chain of Cys241 of b-tubulin subunit. Weaker
C–Hꢀꢀꢀꢀꢀp interactions were observed between the benzene
of indoline ring and Leub248 and Leub255 hydrophobic
residues. The same benzene ring also involved in hydro-
phobic interaction with Valb318. The oxobicycloheptane
ring in compound 2c showed multiple interactions with the
residues of a,b-tubulin subunit: (1) the carbonyl group
present in the ring showed two hydrogen bonding interac-
˚
tions (2.54 and 3.10 A) with the side chain of Asnb258; (2)
hydrophobic interaction with the Leub248 and; (3) weak
C–HꢀꢀꢀꢀꢀO type of interaction with side and main chain of
˚
Sera178 and Thra179, respectively (distance B 3.0 A)
(Fig. 4).
Overall, the docking simulation of compound 2c shows
similar interactions with the tubulin heterodimers as
revealed by previous molecular modeling studies of tubulin
inhibitors (La Regina et al., 2007; Do Yoon et al., 2006; De
Martino et al., 2006; De Martino et al., 2004). Thus, this
compound is showing anticancer activity most plausibly
via blocking the colchicine-binding site of a,b-tubulin
subunit and making interactions with its key residues.
Acknowledgments The author’s are thankful to the staff members
of National Cancer Institute (NCI), USA, for in vitro anticancer
screening of the newly synthesized compounds.
Conclusion
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