An investigation into the cytotoxicity and mode of action of some novel N-alkyl-substituted isatins

Article abstract of DOI:10.1021/jm0704189

A range of substituted N-alkylisatins were synthesized and their cytotoxicity evaluated against several cancer cell lines in vitro. SAR studies indicated that the introduction of an aromatic ring with a one or three carbon atom linker at N1 enhanced the activity from that of the allyl, 2′-methoxyethyl, and 3′-methylbutyl N-substituted isatins. Furthermore, electron-withdrawing groups substituted at the meta or para position of the ring were favored over the ortho orientation. Of the 24 compounds screened, nine displayed sub-micromolar IC₅₀ values and in general demonstrated greater selectivity toward leukemia and lymphoma cell lines over any of the carcinoma cell lines tested. 5,7-Dibromo-N-(p-methylbenzyl) isatin (6) was the most active compound, inhibiting the metabolic activity of both U937 and Jurkat cancer cell lines at 0.49 μM. Various N-alkylisatins were also found to dramatically alter lymphocyte morphology, destabilize microtubules, inhibit tubulin polymerization, induce G2/M cell cycle arrest, and activate the effector caspase-3 and -7.

Full text of DOI:10.1021/jm0704189

J. Med. Chem. 2007, 50, 5109-5117
5109
An Investigation into the Cytotoxicity and Mode of Action of Some Novel N-Alkyl-Substituted
Isatins
Kara L. Vine,*,†,‡ Julie M. Locke,‡ Marie Ranson,† Stephen G. Pyne,‡ and John B. Bremner‡
School of Biological Sciences, and Department of Chemistry, UniVersity of Wollongong, NSW 2522, Australia
ReceiVed April 10, 2007
A range of substituted N-alkylisatins were synthesized and their cytotoxicity evaluated against several cancer
cell lines in vitro. SAR studies indicated that the introduction of an aromatic ring with a one or three carbon
atom linker at N1 enhanced the activity from that of the allyl, 2′-methoxyethyl, and 3′-methylbutyl
N-substituted isatins. Furthermore, electron-withdrawing groups substituted at the meta or para position of
the ring were favored over the ortho orientation. Of the 24 compounds screened, nine displayed
sub-micromolar IC50 values and in general demonstrated greater selectivity toward leukemia and lymphoma
cell lines over any of the carcinoma cell lines tested. 5,7-Dibromo-N-(p-methylbenzyl)isatin (6) was the
most active compound, inhibiting the metabolic activity of both U937 and Jurkat cancer cell lines at 0.49
µM. Various N-alkylisatins were also found to dramatically alter lymphocyte morphology, destabilize
microtubules, inhibit tubulin polymerization, induce G2/M cell cycle arrest, and activate the effector caspase-3
and -7.
Introduction
the aim of the current study was to screen a range of N-alkylated
isatins including those with both aliphatic and aromatic substi-
tuted groups, as well as different bromine substitution patterns
on the isatin moiety, for cytotoxicity against a panel of cancer
cell lines and to determine the SAR. A further aim was to
investigate the biological mode of action using a range of cell-
based and cell-independent approaches. The results of this work
are reported herein.
The isatin molecule (1H-indole-2,3-dione) is a versatile
moiety that displays diverse biological activities,1 including
anticancer activity.2,3 The synthetic flexibility of isatin has led
to the synthesis of a variety of substituted derivatives; however,
the susceptibility of isatin to attack by nucleophiles at C3 has
resulted in the generation of a large number of 3-substituted
isatins in particular. This is reflected by numerous biologically
active 3-substituted indolin-2-ones that are reported in the
literature.4-6 Despite the significant amount of anticancer
research being devoted to this chemical class, it should not be
assumed that 3-substituted isatins are richer in bioactivity than
other 1H-indole-2,3-dione derivatives. N-Alkylated indoles have
also been reported to exhibit anticancer activity. For example,
the indolyl amide D-24851 (1) has been found to block cell
cycle progression in a variety of malignant cell lines including
those derived from the prostate, brain, breast, pancreas, and
colon.7 In vivo, compound 1 proved efficacious in a rat Yoshida
AH13 sarcoma model and induced complete tumor regression
and curative treatment of the animals, with no toxic side effects.7
Structurally related compounds have also been reported to
activate caspases in a cytochrome c-dependent manner and
therefore induce apoptosis in cancer cell lines but not normal
cells.8 Furthermore, Liu and colleagues identified a class of isatin
O-acyl oximes that selectivity inhibited neuronal ubiquitin
C-terminal hydrolase (UCH-L1) in a H1299 lung cancer cell
line, which is proposed to be linked to tumor progression upon
upregulation.9
Results and Discussion
Chemistry. The structures of the N-alkylated isatins synthe-
sized are shown in Table 1. Several methods have been
employed to produce N-alkylated isatins. Generally, the isatin
is exposed to an alkylating agent in the presence of a base. For
our purposes, the alkylation of the isatins was carried out using
the general method shown in Scheme 1, with Cs2CO3 or
K2CO3 employed as a base. This procedure was based on a
method by Torres,11 which was reported to give good yields
with 7-substituted isatins. Formation of the dark violet isatin
anion was more facile when Cs2CO3 was used; however, the
yields of final product were comparable using either base. The
time required for the alkylation reaction varied depending upon
Although very few studies have described SARsa attributable
to modifications to the benzene ring of isatin,2,9,10 we have
previously shown that the introduction of electron-withdrawing
groups to the benzene ring is associated with increased biological
activity for a range of indole-based compounds.3 Given this,
* To whom correspondence should be addressed. Telephone: +61 (0)2
42214356. Fax: +61 (0)2 42214135. E-mail: klv04@uow.edu.au.
† School of Biological Sciences.
‡ Department of Chemistry.
a Abbreviations: SAR, structure activity relationship; PBL, peripheral
blood lymphocyte; MDR, multi-drug resistance; EDTA, ethylenediamine-
tetraacetic acid.
10.1021/jm0704189 CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/21/2007

5110 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Vine et al.
Table 1. Cytotoxicity of Isatin Derivatives 2-25 on U937,a Jurkat,b and MCF-7c Cells As Calculated from Dose Response Curvesd
a U937: human monocyte-like, histiocytic lymphoma cell line. b Jurkat: human leukemic T-cell line. c MCF-7: human mammary gland adenocarcinoma
(non-metastatic) cell line. d Sigmoidal dose response curves (variable slope) were generated using GraphPad Prism V. 4.02 (GraphPad Software Inc.). e Values
are the mean of triplicates of at least two independent experiments. f NT ) not tested.
Scheme 1. General Method for N-Alkylation of Isatin
Derivatives To Give 2-20 and 22-25a
(U937, Jurkat, and MCF-7, Table 1). The general trend observed
among the three cell lines indicated that:
(1) Introduction of an aromatic ring with a one carbon atom
linker at N1 (e.g., compound 5) enhanced biological activity
by at least a factor of 2 against U937 and Jurkat cell lines as
compared to the allyl, 2′-methoxyethyl, and 3′-methylbutyl
N-substituted isatins (2-4), in the 5,7-dibrominated series.
However, no significant change in activity was observed for
compounds tested against the MCF-7 cell line.
(2) Introduction of electron-withdrawing groups such as
the nitro group were favored in the para (14) position over the
ortho (15) orientation, but no significant difference in activity
was observed for para (12)- or meta (13)-substituted methoxy
groups.
(3) Introduction of two bromine atoms at C5 and C7 for the
N-(p-methoxybenzyl)isatin series of compounds (12) substan-
tially increased biological activity in comparison to the non-
(7) or mono-brominated (8-11) N-alkylisatins (Figure 1).
(4) Increasing the carbon chain length from 1 (5) to 3 (24)
resulted in no significant difference in activity against all three
cancer cell lines tested.
Focusing on the most active compounds in the 5,7-dibromo-
N-alkylisatin series, nine were found to exhibit IC50 values in
the sub-micromolar range (Table 1). 5,7-Dibromo-N-(p-meth-
ylbenzyl)isatin (6) was the most active compound, inhibiting
50% of the metabolic activity and hence cellular proliferation
in both U937 and Jurkat cells at a concentration of 0.49 µM
(Table 1). This was 22 times more potent than the unsubstituted
5,7-dibromoisatin,3 thus emphasizing the importance of N1
substitution for cytotoxic activity. In addition, compound 6 was
a (a) K2CO3 or Cs2CO3 (1.2 equiv), DMF (1 mL per 0.1 mmol of isatin
derivative), 1 h, 4 °C, RT; (b) R1X (1.1 equiv, X ) Cl or Br), KI (cat., 0.2
equiv), 80 °C, 5-24 h.
the reactivity of the alkyl halide and so the reaction with allyl
bromide to produce 2 required only 4 h for completion, whereas
the reaction with the less reactive 1-bromo-3-methylbutane to
yield 4 required a much longer reaction time (24 h). The
synthesis of the acid 21 was accomplished via hydrolysis of
the methyl ester 22 under acidic conditions. The characterization
1
of all compounds was accomplished using H and 13C NMR
spectroscopy as well as low-resolution EI MS for known
compounds and high-resolution EI MS for novel compounds.
The purity of all compounds was greater than 95% as determined
by RP HPLC (see Supporting Information).
Biological Activity. Cytotoxicity and SAR Analysis. As part
of an ongoing effort to identify potent inhibitors of cancer cell
proliferation, the prepared N-alkylated isatins 2-25 were
investigated for cytotoxicity against a range of cancer cell types
(Table 1). A structure-activity-based selection process was used
to identify several crucial structural requirements needed to
enhance cytotoxicity (based on a cell proliferation assay), which
were determined initially against three human cancer cell lines

Cytotoxicity of N-Alkyl-Substituted Isatins
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5111
Table 2. Cytotoxicity of Selected N-Alkylisatins against Various Cancer
Cell Lines As Determined by Dose Response Curvesa
IC50 (µM)b
compound
PC-3c
MDA-MB-231d
HCT-116e
A375f
6
12
14
16
17
18
19
23
25
4.57
3.50
NTg
2.17
3.44
10.9
1.17
3.06
3.42
1.91
5.29
2.75
2.77
1.90
2.92
0.54
0.98
2.06
1.22
2.59
2.70
1.84
0.53
3.36
0.84
1.66
0.83
2.00
4.66
2.50
1.68
1.22
2.40
1.25
0.89
1.19
Figure 1. Viability of U937 cells after treatment with different
concentrations of (9) N-(p-methoxybenzyl)isatin (7), (2) 4-bromo-N-
(p-methoxybenzyl)isatin (8), (1) 5-bromo-N-(p-methoxybenzyl)isatin
(9), ([) 6-bromo-N-(p-methoxybenzyl)isatin (10), (b) 7-bromo-N-(p-
methoxybenzyl)isatin (11), and (0) 5,7-dibromo-N-(p-methoxybenzyl)-
isatin (12). Briefly, cells were incubated for 24 h at 37 °C with
increasing concentrations of test compound, then analyzed for a change
in metabolic activity and expressed as percent viability in reference to
the DMSO control. Each data point is a mean of triplicates ( SE.
a Sigmoidal dose response curves (variable slope) were generated using
GraphPad Prism V. 4.02 (GraphPad Software Inc.). b Values are the mean
of triplicates of at least two independent experiments. c PC-3: human
prostate adenocarcinoma cell line. d MDA-MB-231: human mammary gland
carcinoma (metastatic) cell line. e HCT-116: human colorectal carcinoma
cell line. f A375: human malignant melanoma cell line. g NT ) not tested.
>22 times more selective for leukemic (Jurkat) and lymphoma
(U937) cancer cell lines than for the breast adenocarcinoma
(MCF-7) cell line (Table 1). Five other N-alkylisatins (14, 16,
17, 19, and 25) also demonstrated a similar pattern of specificity,
but none were as potent as 6. This effect may be due in part to
the expression of the anti-apoptotic Bcl-2 group of proteins that
have been identified in MCF-7 cells, for which Bcl-2 and Bcl-
XS expression specifically has been correlated with resistance
and poor response to chemotherapy.12-15
Interestingly, activity was compromised in all three cancer
cells lines when a carboxylic acid was substituted at the para
position of the aromatic ring (21, Table 1). Such a decrease in
activity was not correlated with electronic (σ) or steric effects
(ES), hydrophobicity (clog P), or localized hydrophobicity at
the p-substituent (π) (see Supporting Information). Considering
that the pKa of benzoic acid is 4.2,16 it is hypothesized that the
carboxylic acid of 21 is deprotonated upon addition to the cell
culture media (pH ca. 7.4) and forms the carboxylate anion;
thus the lipophilicity of the molecule is significantly reduced
(see Supporting Information). This implies that the p-substituent
may play an important role in the cytotoxicity of the N-
alkylisatins, but the large variety of atoms that can be substituted
at this position suggests that there is no highly specific binding
interaction occurring in this region. Alternatively, carboxylate
anion formation may reduce cell uptake and hence cytotoxicity.
The most potent compounds (i.e., IC50 < 1 µM) determined
from the initial cell line screen (Table 1) were subsequently
tested against a panel of four additional adherent cancer cell
lines (PC-3, MDA-MB-231, HCT-116, and A-375, Table 2) and
one normal, non-transformed human cell line suspension (PBL,
Figure 2). Of the nine compounds tested, four exhibited IC50
values in the sub-micromolar range against at least one cell line
(17, 19, 23, and 25, Table 2). The colorectal carcinoma cell
line (HCT-116) was the most susceptible to treatment, with
compound 17 reducing the metabolic activity by 50% at a
concentration of 0.53 µM. The MDA-MB-231 and A375 cell
lines were moderately sensitive, while the prostate carcinoma
cell line PC-3 was the least susceptible to treatment, as none of
the compounds tested exhibited IC50 values less than 1 µM.
This differential effect is unlikely to be due to proliferative
disparities, as all four cell lines have similar population doubling
times. Furthermore, cancer cell line selectivity was observed
for compound 19, which exhibited >5 times more activity
toward the lymphoma and leukemic cell lines than freshly
Figure 2. Cancer cell line selectivity. Different concentrations of 5,7-
dibromo-N-(p-trifluoro-methylbenzyl)isatin (19) were incubated with
either (9) freshly isolated, non-transformed, human peripheral blood
lymphocytes (PBL), (2) human monocyte-like, histiocytic lymphoma
cells (U937), or (1) a human leukemic T-cell line (Jurkat) for 24 h at
37 °C. Cells were then analyzed for a change in metabolic activity and
expressed as percent viability in reference to the DMSO control. Each
data point is a mean of triplicates ( SE.
isolated, human PBLs (Figure 2). This is promising considering
the lack of effective and nontoxic drugs available for the
treatment of MDR lymphoid malignancies.17-19
Mode of Action Investigations. Apoptosis and Cell Cycle
Arrest. As it is known that several N-alkylindole derivatives
trigger apoptosis8 and induce G2/M arrest,4,5,7 the effect of the
most potent N-alkylisatins on the activation of the effector
caspases-3 and -7 and cell cycle progression was investigated.
Compound 19 was found to activate caspase-3 and -7 in a dose-
(Figure 3A) and time-dependent manner (see Supporting
Information) in Jurkat cells. At 3.3 µM, the activity of compound
19 was comparable to that of the staurosporine positive control.
Unlike staurosporine, however, 19 exhibited a caspase activation
trend in U937 cells similar to that seen in the Jurkat cell line,
while only minimal caspase activity was observed in PBLs
(Figure 3B). This compares closely with cytotoxicity data
(Figure 2), whereby a decrease in the viability of cells correlates
to an increase in the activation of effector caspases-3 and -7
and hence apoptosis. Such an effect was further supported by
the appearance of fragmented nuclei in morphologically altered
U937 cells after treatment with compound 18 (see Supporting
Information). Furthermore, U937 cells treated with compounds
6 and 19 exhibited an increase in the accumulation of cells in
G2/M, in comparison to vehicle control treated cells (Figure 4,
panels G and H). This effect was both dose and time dependent,
whereby mitotic arrest was most apparent at 0.5 µM after 24 h.
At higher concentrations (i.e., 2 and 1 µM), cells treated with

5112 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Vine et al.
Figure 3. Activation of the effector caspases-3 and -7 in Jurkat, U937, and PBL cells after treatment with various N-alkylisatins. (A) Jurkat cells
were exposed to increasing concentrations of 5,7-dibromo-N-(p-trifluoromethylbenzyl)isatin (19) for 5 h at 37 °C. Values were normalized to a
staurosporine (positive control). (B) Jurkat, U937, and PBL cells were exposed to three concentrations of compound 19 for 5 h at 37 °C. In all
cases, cells were then incubated with the caspase-3/7 reagent for 1 h at room temperature, and fluorescence was measured at an excitation wavelength
of 485 and 520 nm emission. Data are means ( SE of one representative experiment performed in triplicate.
Figure 4. The effect of N-alkylisatins on the cell cycle. U937 cells were treated with either (A) and (E) DMSO vehicle control, (B) and (F) 0.5
µM vinblastine sulfate, (C) and (G) 0.5 µM 5,7-dibromo-N-(p-methylbenzyl)isatin (6), or (D) and (H) 0.5 µM 5,7-dibromo-N-(p-trifluoromethylbenzyl)-
isatin (19) for 4 h (upper panels) or 24 h (lower panels). Cells were then ethanol fixed, stained with propidium iodide, and analyzed for DNA
distribution by FACS. Cell cycle arrest was additionally determined after treatment with high and low concentrations of compounds 6 (panels I and
J) and 19 (panels K and L).
compounds 6 and 19 (respectively) showed an increase in S
phase and sub-G1 peak distribution (Figure 4, panels I and K).
Although not observed in vinblastine treated cells, this latter
effect was consistent with the induction of apoptosis (Figure
3).
Effect on Lymphocyte Morphology. In addition to their
potent cytotoxicity, various N-alkylisatins were also found to
dramatically alter lymphocyte morphology (Figure 5). Within
5 h of incubation with less than 1 µM of various N-alkylisatins,
a sub-population of U937 cells developed an elongated forma-
tion as well as irregular membrane protrusions, of which the
latter appeared to be consistent with an apoptotic response20
(see Supporting Information). After 24 h, however, the elongated
morphology was more pronounced (Figure 5). An analogous
effect has also been reported for the benzylamide sulindac de-
rivatives (1Z)-N-benzyl-5-fluoro-2-methyl-1-[(4-pyridyl)methyl
ene]-1H-indene-3-acetamide hydrochloride (CP461) and (1Z)-
N-benzyl-5-fluoro-2-methyl-1-{[4-(3,4,5-trimethoxyphenyl)-

Cytotoxicity of N-Alkyl-Substituted Isatins
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5113
Figure 6. The effect of various N-alkylisatins and commercial
anticancer agents on tubulin polymerization as determined in the in
vitro microtubule polymerization assay. Purified bovine neuronal tubulin
was used to assay for microtubule formation in the presence of either:
black circle, vehicle control; red circle, paclitaxel 10 µM; dark blue
circle, vinblastine sulfate salt 10 µM; green circle, 5,7-dibromo-N-(p-
methylbenzyl)isatin (6) 10 µM; teal circle, 5,7-dibromo-N-(p-trifluo-
romethylbenzyl) isatin (19) 10 µM; dark brown circle, 5,7-dibromo-
N-(cinnamyl)isatin (24) 10 µM; or orange circle, 5,7-dibromo-N-(p-
phenylbenzyl)isatin (25) 10 µM at 37 °C. A shift of the curve to the
left or right of the control is indicative of either an increase or a
decrease, respectively, in the rate of tubulin polymerization. Changes
in fluorescence were measured at an excitation wavelength of 360 (
10 nm, and the fluorescence was collected at 440 ( 10 nm. Data points
are the means of duplicate experiments.
activity and morphological change in this study, compounds 6,
19, 24, and 25 were chosen as representative molecules to further
investigate their ability to alter tubulin polymerization in vitro.
As expected, the potent microtubule stabilizing agent paclitaxel
resulted in a dramatic increase in the rate of tubulin polymer-
ization at 10 µM, in comparison to the vehicle control, while
vinblastine strongly inhibited microtubule formation at the same
concentration (Figure 6). This was greater than the effect seen
at 3 µM (see Supporting Information), indicating that the rate
of microtubule stabilization or destabilization is dose-dependent.
The N-alkylisatins 6, 19, 24, and 25 also strongly inhibited the
rate of microtubule polymerization at 10 µM, albeit not as
potently as vinblastine (Figure 6). This effect appeared to be
independent of the nature of the p-substituent and size of the
benzene carbon linker, because compounds 6, 24, and 25 all
inhibited microtubule polymerization at a similar rate. By
comparison, compound 19 was slightly less active, but consider-
ing 19 shows broad cell-line cytotoxicity (Tables 1 and 2), this
suggests that cell death induced by direct microtubule destabi-
lization may not be its primary mechanism of action.
The role 19 played in the alteration of microtubule organiza-
tion in comparison to DMSO and vinblastine treated lymphoma
cells was further investigated by fluorescence microscopy, using
fluorescently labeled paclitaxel. Compound 19 induced the
accumulation of cells with fragmented microtubules, abnormal
mitotic spindles, and reduced cell-associated fluorescence
(Figure 7, panels B-D). Conversely, cells treated with vinblas-
tine revealed highly fragmented microtubules at both high
(Figure 7, panel E) and low (Figure 7, panel F) concentrations.
Compound 19 and N-alkylisatins in general are a collection of
low molecular weight compounds that show no structural
similarities to the Vinca alkaloids, and while binding at the same
site as these alkaloids may still be a possibility, interaction at
another tubulin site is also feasible. Recently, a methylindole-
2-carboxylate derivative was reported to adopt an orientation
similar to that of an analogue, N-deacetyl-N-(2-mercaptoacetyl)-
colchicine (DAMA-colchicine) of colchicine (26), when docked
into the colchicine binding site of tubulin.28 Similarly, A-432411
Figure 5. Morphological effects of various N-alkylisatins and com-
mercial anticancer agents on U937 cells. Cells (1.0 × 104) were
incubated with either (A) DMSO vehicle control, (B) 5,7-dibromo-N-
(cinnamyl)isatin (24), (C) vinblastine sulfate salt, (D) 5,7-dibromo-N-
(isopentyl)isatin (4), (E) paclitaxel, (F) 5,7-dibromo-N-(p-iodobenzyl)-
isatin (18), (G) 5-fluorouracil, or (H) 5,7-dibromo-N-(p-tert-
butylbenzyl)isatin (23) all at 0.39 µg/mL for 24 h. Images were obtained
by brightfield microscopy on an inverted light microscope using a Leica
DC500 12-megapixel high-performance FireWire camera system.
Images (panels A-D and F-H) were viewed at 1000× magnification,
and image E was viewed at 400× magnification.
methylene]}-1H-indene-3-acetamide (CP2-48), whereby exami-
nation of WSU-CLL cells after an 18 h treatment revealed a
marked affect on cell shape.21 Interestingly, the structurally
similar parent compounds, sulindac sulfide and sulindac sulfone,
currently being studied as chemo-preventatives in patients with
familial adenomatous polyposis (FAP),22 did not.
A change in leukemic T-cell morphology was also noted after
treatment with compound 18 (see Supporting Information) but
was not as obvious as that observed in the monocytic cell line.
Morphological alterations were also seen in vinblastine (Figure
5, panel C) and paclitaxel (Figure 5, panel E) treated cells, but
not in cells treated with 5-fluorouracil (Figure 5, panel G) or
staurosporine (see Supporting Information), suggesting that
N-alkylisatins may either disrupt or stabilize microtubules in a
fashion similar to the Vinca alkaloids23 or taxanes.24
Effects on Tubulin Polymerization and Microtubule
Formation. The indolic heterocyclic nucleus is central to a large
number of tubulin polymerization inhibitors.7,25-27 Isatins are
oxidized derivatives of an indolic moiety, and considering the
broad range of N-alkylisatins that showed potent cell-based

5114 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Vine et al.
Figure 7. The effect of 5,7-dibromo-N-(p-trifluoromethylbenzyl) isatin (19) on the stability of microtubules in U937 cells. Treatment of cells with
(A) DMSO was used as the control. Cells were exposed to either (B)-(D) 0.78 µg/mL (1.7 µM) of compound 19 or (E) 0.78 µg/mL (0.9 µM) or
(F) 0.20 µg/mL (0.2 µM) of the commercial anticancer agent vinblastine sulfate salt for 24 h. Microtubules were then visualized by direct fluorescence
microscopy after incubation for 30 min with fluorescently labeled paclitaxel.
(27) was found to compete strongly with [3H]colchicine (IC50
0.13 µM) in a competition-binding scintillation proximity assay.6
Conversely, the indole derivative 1 did not compete for the
binding of either radiolabeled vincristine or colchicine to
tubulin.7 This suggests that 1 may bind to a novel binding site
on tubulin and induce inhibition of polymerization. Because it
is known that there are three major, well-characterized binding
sites on tubulin,23,29,30 it would be of interest to conduct future
competitive binding studies, as well as molecular modeling and
docking studies, to verify the exact binding interactions between
the various N-alkylisatins (from this study) and tubulin and
correlate this with SAR data.
Alternatively, it is also possible that N-alkylisatin-induced
microtubule disassembly is a secondary phenomenon, despite
some characteristics resembling those induced by the destabiliz-
ing agent vinblastine. For example, treatment of cells with the
microtubule-dissociating agent nocodazole (28) has been re-
ported to result in Raf1 activation by Pak1, a kinase downstream
of the small GTPases Rac/Cdc42.31 Activated Rac and Cdc42
induce cell shape changes at the plasma membrane by formation
of multimolecular focal complexes as well as lamellipodia and
filopodia.32 It may therefore be of interest in future studies to
also determine whether various N-alkylisatins activate the Rac/
Cdc42/Pak1/Raf1 signaling pathway.
as in a cell-free system. The potent cytotoxicity displayed by
the various N-alkylisatins as compared to the commercial
anticancer agents, vinblastine and taxol, together with the
possibility of binding to a novel site on tubulin, suggest that
these compounds may be beneficial in the treatment of anti-
mitotic drug-resistant tumors in the future.
Experimental Section
General Synthesis and Experimental Methods. The desired
isatin starting materials were either purchased or synthesized using
previously reported methods.3 4-Bromoisatin, 6-bromoisatin, and
7-bromoisatin were obtained from Butt Park Ltd. (U.K.). All other
chemicals were purchased from the major vendors. Reactions were
carried out using dried glassware and under an atmosphere of
nitrogen. All solvents were AR grade except dichloromethane
(DCM), which was LR grade and distilled before use. Reactions
were monitored using thin layer chromatography (TLC) on
aluminum backed precoated silica gel 60 F254 plates (E. Merck).
The N-alkylisatins were highly colored and would usually be clearly
seen on a TLC plate; colorless compounds were detected using
UV light and/or iodine vapor. Flash chromatography33 was carried
out using silica gel 60 (230-400 mesh, E. Merck) with the solvent
system indicated in the individual procedures. All solvent ratios
are quoted as vol/vol. 1H and 13C NMR spectra of compounds 2-25
were acquired at 300 and 75 MHz, respectively, on a Varian Unity-
300 spectrometer and 500 and 126 MHz, respectively, on a Varian
Inova-500 spectrometer with a probe temperature of 298 K. The
NMR spectra are referenced to the residual solvent peak in the
solvent stated in the individual procedures. Hydrogen and carbon
assignments were made using standard NOE, APT, DEPT, gCOSY,
gHSQC, and gHMBC spectroscopic techniques. Low-resolution EI
mass spectra (LREI-MS) were determined on a Shimadzu QP5050
spectrometer. High-resolution EI mass spectra (HREI-MS) were
determined on VG Autospec spectrometer operating at 70 eV with
a source temperature of 250 °C and were referenced with PFK.
Melting points were determined on a Reichert melting point
apparatus and are uncorrected.
Conclusions
N-Alkyl- and arylalkylisatins, in particular compounds 6, 12,
14, 16, 17, 18, 19, 23, and 25, are novel synthetic anticancer
agents with potent cytotoxicity in vitro, some of which show
>22 times selectivity for leukemic cells over the MCF-7,
epithelial breast adenocarcinoma. Compound 19 also exerted
significant anticancer activity on a broad range of cancer cell
lines including leukemic, lymphoma, colorectal, breast, prostate,
and melanoma cells and was found, together with the most active
compound, 6, to destabilize microtubules in tumor cells as well

Cytotoxicity of N-Alkyl-Substituted Isatins
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5115
General Method for the Alkylation of Isatins. The appropriate
isatin (∼200 mg, 1 equiv) was taken up in anhydrous DMF (1 mL
per 0.1 mmol of isatin) and cooled on ice with stirring. Solid
K2CO3 (1.2 equiv) or Cs2CO3 (1.2 equiv) was added in one portion,
and the dark colored suspension was brought to room temperature
and stirred for a further 1 h. The appropriate alkyl chloride (1.1
equiv for compounds 7-12 and 16) or bromide (1.1 equiv for all
other relevant compounds) and KI (0.2 equiv) were added, and the
reaction mixture was stirred at 80 °C for 5-24 h, until the isatin
starting material had been consumed (TLC). The reaction mixture
was poured into HCl (0.5 M, 50 mL) and extracted with ethyl
acetate (1 × 50 mL). The ethyl acetate layer was washed with brine
and dried over MgSO4. The solvent was removed, and the crude
product was purified via flash column chromatography using
isocratic elution with CH2Cl2 unless otherwise stated.
Leica DC500 12-megapixel high-performance FireWire camera
system (Leica Microsystems, AG, Germany) and the Leica IM50
image manager software (Leica Microsystems AG, Heerbrugg,
Germany).
Cell Lines and Culture Conditions. The human lymphoma
(U937), leukemic (Jurkat), breast (MDA-MB-231 and MCF-7),
prostate (PC-3), and colorectal (HCT-116) carcinoma cell lines as
well as the melanoma (A375) cell line were all obtained from
American Type Culture Collection (ATCC, VA) distributed by
Cryosite, NSW, Australia. Cell lines were routinely cultured at
37 °C in a Heracell incubator (Kendro Laboratory Products,
Langenselbold, Germany) in 95% humidified atmosphere, contain-
ing 5% CO2. Cells were grown in culture media (10.4 g/L RPMI-
1640 with 2 mM L-glutamine and 2 g/L NaHCO3) supplemented
with 5% (v/v) fetal calf serum (FCS), and routinely passaged at
confluency for up to 20 passages. Adherent cell lines were detached
using sterile trypsin-EDTA, washed with culture media, and
centrifuged at 1500 rpm (514g) for 5 min at room temperature (RT)
before reseeding. All cell experiments were performed using cells
in exponential growth, cultured 48 h before without change of
media. Adherent cells were detached prior to experiments with
sterile phosphate-buffered saline (PBS) containing 5 mM EDTA
(pH 7.4), and cells were then centrifuged at 1500 rpm (514g) for
5 min at RT. Human peripheral blood lymphocytes (PBL) were
prepared fresh, on the day of use, by density centrifugation. Briefly,
Lymphoprep (7 mL) was carefully layered beneath fresh, human,
peripheral blood (10 mL) that had been diluted 1:2 in PBS (pH
7.4). The preparation was then centrifuged (Heraeus megafuge) at
1800 rpm (800g) for 20 min at RT. After centrifugation, the top,
diluted, plasma layer was carefully removed and discarded. The
opaque layer, at the interface, containing peripheral blood lym-
phocytes (PBL) was collected and placed into a sterile tube.
Collected cells were then washed twice with PBS by centrifugation
at 1800 rpm (800g) for 30 min at RT, and the supernatant was
discarded. Pelleted cells were finally resuspended in 2 mL of
complete media RPMI-1640 supplemented with 5% FCS. Cell
viability and cell number were assessed by the Trypan blue
exclusion method, and viable cells were counted with the aid of a
hemocytometer.
Cell Viability (MTS) Assay. Cytotoxicity of the isatin deriva-
tives on various cancer and non-transformed cell lines was
determined using the CellTiter 96 AQueous One Solution Cell
Proliferation Assay (MTS), in 96-well microplates, as described
previously.35 Test compounds were incubated with different cell
lines at increasing concentrations ranging from 0 to 100 µg/mL
for 24 h (unless stated otherwise) prior to the addition of MTS
reagent. Cytotoxic activity was then determined spectrophotometri-
cally at 490 nm. Results for each compound are reported as the
concentration (µM) required to inhibit the metabolic activity of 50%
of the cell population (IC50) in comparison to vehicle-treated
(DMSO) control cells. These values were calculated from loga-
rithmic sigmoidal dose response curves using the variable slope
parameter, generated from GraphPad Prism V. 4.02 software
(GraphPad Software Inc.).
N-Allyl-5,7-dibromoisatin (2). The product was a bright red
solid (102 mg, 45%), mp 103-105 °C (lit.34 98-100 °C), Rf 0.45
(CH2Cl2, silica). 1H NMR (CDCl3, 500 MHz) δ 4.79 (d, J ) 5 Hz,
2H, CH2), 5.23 (d, J ) 17 Hz, 1H, CHcisHtrans), 5.26 (d, J ) 10 Hz,
1H, CHcisHtrans), 5.96 (ddt, J ) 5, 10, 17 Hz, 1H, CH), 7.70 (d, J
) 2 Hz, 1H, H4), 7.86 (d, J ) 2 Hz, 1H, H6). 13C NMR (CDCl3,
126 MHz) δ 43.1, 104.9, 116.8, 117.6, 121.1, 127.3, 131.4, 145.1,
1
146.6, 157.7, 181.2. The quoted H and 13C NMR spectral data
were in agreement with published values.34 LREI-MS m/z 343/
345/347 ([M]+/([M + 2]+/[M + 4]+).
5,7-Dibromo-N-(p-methylbenzyl)isatin (6) (CAS No. 620932-
72-1). The product was a bright red solid (252 mg, 93%), mp 121-
1
123 °C, Rf 0.61 (CH2Cl2, silica). H NMR (CDCl3, 500 MHz) δ
2.30 (s, 3H, CH3), 5.34 (s, 2H, H1′), 7.11 (d, J ) 8 Hz, 2H, phenyl
ArH), 7.13 (d, J ) 8 Hz, 2H, phenyl ArH), 7.66 (d, J ) 2 Hz, 1H,
isatin ArH), 7.77 (d, J ) 2 Hz, 1H, isatin ArH). 13C NMR (CDCl3,
126 MHz) δ 21.0, 44.3, 105.1, 116.9, 121.3, 126.3, 127.3, 129.3,
132.5, 137.3, 145.1, 146.6, 158.2, 181.2. LREI-MS m/z 407/409/
411 ([M]+/[M + 2]+/[M + 4]+). HREI-MS m/z calcd for [M]+
C16H1179Br2NO2, 406.9157; found, 406.9175.
4-[(5,7-Dibromo-2,3-dihydro-2,3-dioxo-1H-indol-1-yl)methyl]-
benzoic Acid (21). The methyl ester 22 (70 mg, 0.15 mmol) was
dissolved in a mixture of concentrated HCl (8 mL), glacial acetic
acid (8 mL), and water (2 mL) and stirred at reflux for 1.5 h. The
reaction mixture was poured onto ice (100 mL) and extracted with
ethyl acetate (2 × 100 mL), and the combined ethyl acetate layers
were washed with brine and dried over MgSO4. The solvent was
removed to yield a bright orange solid. The solid was suspended
in CH2Cl2 (20 mL) and then filtered, and the resulting solid was
washed with CH2Cl2 to remove any trace of starting material. The
product 21 was a bright red-orange solid (59 mg, 90%), mp
1
>250 °C, Rf 0.13 (9:1 CH2Cl2:MeOH, silica). H NMR (DMSO-
d6, 500 MHz) δ 5.30 (s, 2H, CH2), 7.50 (d, J ) 8 Hz, 2H, ArH),
7.79 (d, J ) 2 Hz, 1H, isatin ArH), 7.88 (d, J ) 8 Hz, 2H, ArH),
7.98 (d, J ) 2 Hz, 1H, isatin ArH), 12.80 (br s, 1H, OH). 13C
NMR (DMSO-d6, 126 MHz) δ 44.9, 104.8, 116.4, 123.5, 127.0,
130.1, 130.3, 143.0, 143.9, 146.8, 159.8, 167.8, 181.2. LREI-MS
m/z 437/439/451 ([M]+/[M + 2]+/([M + 4]+). HREI-MS m/z calcd
for [M + 2]+ C16H979Br81BrNO4, 438.8878; found, 438.8880.
Materials and Reagents for Cell Culture and Bioassays. The
culture medium RPMI-1640 was purchased from Gibco (Invitrogen
Life Technologies, NSW, Australia). Fetal calf serum (FCS) was
obtained from MultiSer (ThermoTrace, Vic., Australia). Sodium
bicarbonate was purchased from Univar Analytical Reagents (Ajax
Chemicals, Australia). Trypsin-EDTA solution was purchased from
Sigma-Aldrich (MO). Lymphoprep was purchased from Pharmacia
Biotech, Australia. The CellTiter 96 AQueous One Solution Cell
Proliferation Assay and Apo-ONE Homogeneous Caspase-3/7
Assay were purchased from Promega Co. (Madison, WI). Tubulin
Tracker Green Reagent (fluorescently labeled paclitaxel) was
obtained from Molecular Probes (Invitrogen Aust. Pty Ltd.,
Australia), and the Tubulin Polymerization Assay was from
Cytoskeleton Inc. (Jomar Diagnostics, Australia). The Diff-Quik
staining kit was purchased from Lab Aids (Sydney, NSW Australia),
and propidium iodide was from Sigma-Aldrich (St. Louis, MO).
Light and fluorescence microscopy images were obtained using a
Caspase-3/7 Assay. The activation of effector caspases-3 and
-7 was determined in Jurkat, U937, and PBL cells after incubation
with test compound or 2 µM staurosporine (positive control) using
the Apo-ONE Homogeneous Caspase-3/7 Assay kit. The assay was
carried out according to the method described previously.3
Cell Cycle Analysis by Flow Cytometry. Flow cytometry
analysis of cellular DNA content was performed using a modifica-
tion to the method described previously.6 Briefly, cells (2.0 × 104)
were harvested by centrifugation at 1500 rpm (514g) for 5 min
and fixed with ice-cold ethanol (70%) for 30 min to overnight at
-20 °C. The ethanol was then removed by centrifugation, and cells
were stained with PI master mix (100 µg/mL RNase A, 40 µg/mL
PI, and PBS pH 7.4) for 30 min at 37 °C. DNA content was then
measured using a Becton Dickinson BD LSR II FACSort flow
cytometer (BD Biosciences, USA), and the proportion of cells in
G0/G1, S, and G2/M phases of cell cycle was calculated on the

5116 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21
Vine et al.
(5) Andreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.;
Rambaldi, M.; Garaliene, V.; Welsh, W.; Arora, S.; Farruggia, G.;
Masotti, L. Antitumor activity of new substituted 3-(5-imidazo[2,1-
b]thiazolylmethylene)-2-indolinones and study of their effect on the
cell cycle. J. Med. Chem. 2005, 48, 5604-5607.
(6) Chen, Z.; Merta, P. J.; Lin, N. H.; Tahir, S. K.; Kovar, P.; Sham, H.
L.; Zhang, H. A-432411, a novel indolinone compound that disrupts
spindle pole formation and inhibits human cancer cell growth. Mol.
Cancer Ther. 2005, 4, 562-568.
(7) Bacher, G.; Nickel, B.; Emig, P.; Vanhoefer, U.; Seeber, S.; Shandra,
A.; Klenner, T.; Beckers, T. D-24851, a novel synthetic microtubule
inhibitor, exerts curative antitumoral activity in vivo, shows efficacy
toward multidrug-resistant tumor cells, and lacks neurotoxicity.
Cancer Res. 2001, 61, 392-399.
(8) Nguyen, J. T.; Wells, J. A. Direct activation of the apoptosis
machinery as a mechanism to target cancer cells. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 7533-7538.
(9) Liu, Y.; Lashuel, H. A.; Choi, S.; Xing, X.; Case, A.; Ni, J.; Yeh, L.
A.; Cuny, G. D.; Stein, R. L.; Lansbury, P. T., Jr. Discovery of
inhibitors that elucidate the role of UCH-L1 activity in the H1299
lung cancer cell line. Chem. Biol. 2003, 10, 837-846.
(10) Lee, J. W.; Moon, M. J.; Min, H. Y.; Chung, H. J.; Park, E. J.; Park,
H. J.; Hong, J. Y.; Kim, Y. C.; Lee, S. K. Induction of apoptosis by
a novel indirubin-5-nitro-3′-monoxime, a CDK inhibitor, in human
lung cancer cells. Bioorg. Med. Chem. Lett. 2005, 15, 3948-3952.
(11) Torres, J. C.; Pinto, A. C.; Garden, S. J. Application of a catalytic
palladium biaryl synthesis reaction via C-H functionalization, to the
total synthesis of Amaryllidaceae alkaloids. Tetrahedron 2004, 60,
9889-9900.
(12) Piche, A.; Grim, J.; Rancourt, C.; Gomez-Navarro, J.; Reed, J. C.;
Curiel, D. T. Modulation of Bcl-2 protein levels by an intracellular
anti-Bcl-2 single-chain antibody increases drug-induced cytotoxicity
in the breast cancer cell line MCF-7. Cancer Res. 1998, 58, 2134-
2140.
(13) Sakakura, C.; Sweeney, E. A.; Shirahama, T.; Hakomori, S.; Igarashi,
Y. Suppression of bcl-2 gene expression by sphingosine in the
apoptosis of human leukemic HL-60 cells during phorbol ester-
induced terminal differentiation. FEBS Lett. 1996, 379, 177-180.
(14) Zhang, B.; Zhang, D.; Ren, H. Resistance of Bcl-2 adenovirus vector
to HepG(2) cell apoptosis induced by ethanol. Zhonghua Gan Zang
Bing Za Zhi 2000, 8, 215-217.
basis of DNA distribution histograms using FlowJo software (V7.1,
Tree Star Inc., USA).
Effect on Tubulin Polymerization. (a) Live Cell Staining.
U937 cells (2.2 × 105 cells/mL, 90 µL) were seeded into the wells
of a 96-well microtiter plate and incubated for 24 h (37 °C, 95%
humidity, 5% CO2) prior to the addition of test compound.
Compound 19 (10 µL) was then added to the cells at a concentration
that induced the greatest amount of morphological change (as
determined from light microscopy) and incubated for a further 24
h. Control samples were also prepared by incubating cells with
either 2 µM vinblastine sulfate salt (positive control) or 2.5%
DMSO (negative control) for 24 h. Cells were then stained with
freshly prepared TubulinTracker Green reagent at a final concentra-
tion of 250 nM for 0.5 h (37 °C, 95% humidity, 5% CO2) as per
the manufacturer’s instructions. Stained cells were washed once
with Dulbecco’s PBS, and fluorescence was visualized by confocal
microscopy. Confocal laser scanning microscopy images were
acquired using a Leica TCS SP system and the UV 63 × 1.32 N.A.
oil PALPO immersion objective lens (Leica, Heidelberg, Germany).
Cells were excited using the 488 nm spectral line of the Argon ion
laser, and fluorescence was measured at 500-540 nm.
(b) Tubulin Polymerization Assay. Compounds were tested for
their anti-mitotic ability in reference to vinblastine sulfate and
paclitaxel positive controls in a fluorescence-based Tubulin Po-
lymerization Assay.36 Briefly, 50 µL of tubulin reaction mix (2
mg/mL purified bovine brain tubulin in 80 mM PIPES pH 6.9, 2.0
mM MgCl2, 0.5 mM EGTA, 1.0 mM GTP, and 20% glycerol) was
added to duplicate wells of a half area 96-well black plate containing
5 µL of either vehicle control, paclitaxel, vinblastine sulfate, or
compounds 6, 19, 24, and 25 all at a final concentration of 3 or 10
µM. The rate of polymerization was followed for 1 h at 37 °C
using an excitation wavelength of 360 ( 10 nm, and the
fluorescence was collected at 440 ( 10 nm.
Acknowledgment. We are grateful to the University of
Wollongong for support through the Institute for Biomolecular
Science (IBS), a URC Small Grant, a University Cancer
Research Grant, and a UPA student scholarship for K.L.V. We
also thank Dr. K. Benkendorff (Flinders University, South
Australia) for support, and the Illawarra Cancer Carers Inc.,
Kiama, Minnamurra, and Gerringong Sunrise Rotary, The
Robert East Memorial Fund, Prof. P. Clingan, and other private
donors for funding assistance. Assoc. Prof. M. Ranson is a
recipient of a Cancer Institute NSW Fellowship award.
(15) Campos, L.; Rouault, J. P.; Sabido, O.; Oriol, P.; Roubi, N.; Vasselon,
C.; Archimbaud, E.; Magaud, J. P.; Guyotat, D. High expression of
bcl-2 protein in acute myeloid leukemia cells is associated with poor
response to chemotherapy. Blood 1993, 81, 3091-3096.
(16) The Merck Index: An Encyclopedia of Chemicals, Drugs, and
Biologicals, 14th ed.; O’Neil, M. J. (Editor), Heckelman, P. E. (Senior
Associate Editor), Koch, C. B. (Associate Editor), Roman, K. J.
(Assistant Editor); Merck and Co., Inc.: Whitehouse Station, NJ,
2006; p 2564.
(17) Drenou, B.; Lamy, T.; Amiot, L.; Fardel, O.; Caulet-Maugendre, S.;
Sasportes, M.; Diebold, J.; Le Prise, P. Y.; Fauchet, R. CD3- CD56+
non-Hodgkin’s lymphomas with an aggressive behavior related to
multidrug resistance. Blood 1997, 89, 2966-2974.
(18) Newcomb, E. W. P53 gene mutations in lymphoid diseases and their
possible relevance to drug resistance. Leuk. Lymphoma 1995, 17,
211-221.
(19) Nooter, K.; Sonneveld, P. Multidrug resistance (MDR) genes in
haematological malignancies. Cytotechnology 1993, 12, 213-230.
(20) Kroemer, G.; Dallaporta, B.; Resche-Rigon, M. The mitochondrial
death/life regulator in apoptosis and necrosis. Annu. ReV. Physiol.
1998, 60, 619-642.
(21) Moon, E. Y.; Lerner, A. Benzylamide sulindac analogues induce
changes in cell shape, loss of microtubules and G(2)-M arrest in a
chronic lymphocytic leukemia (CLL) cell line and apoptosis in
primary CLL cells. Cancer Res. 2002, 62, 5711-5719.
Supporting Information Available: Spectroscopic (1H NMR,
13C NMR, and MS) data for compounds 3-5, 7-20, and 22-25,
HPLC purity data for compounds 2-25, physiochemical properties
of selected N-alkylisatins, activation of the effector caspases-3 and
-7 in Jurkat cells, morphological evaluation of nuclei stained with
Diff Quik, morphological effects of compounds 24, 18, and
staurosporine on U937 or Jurkat cells, and the effect of compound
19 on tubulin polymerization. This material is available free of
charge via the Internet at http://pubs.acs.org.
(22) van Stolk, R.; Stoner, G.; Hayton, W. L.; Chan, K.; DeYoung, B.;
Kresty, L.; Kemmenoe, B. H.; Elson, P.; Rybicki, L.; Church, J.;
Provencher, K.; McLain, D.; Hawk, E.; Fryer, B.; Kelloff, G.;
Ganapathi, R.; Budd, G. T. Phase I trial of exisulind (sulindac sulfone,
FGN-1) as a chemopreventive agent in patients with familial
adenomatous polyposis. Clin. Cancer Res. 2000, 6, 78-89.
(23) Rai, S. S.; Wolff, J. Localization of the vinblastine-binding site on
beta-tubulin. J. Biol. Chem. 1996, 271, 14707-14711.
(24) Schiff, P. B.; Fant, J.; Horwitz, S. B. Promotion of microtubule
assembly in vitro by taxol. Nature 1979, 277, 665-667.
(25) Gastpar, R.; Goldbrunner, M.; Marko, D.; von Angerer, E. Methoxy-
substituted 3-formyl-2-phenylindoles inhibit tubulin polymerization.
J. Med. Chem. 1998, 41, 4965-4972.
References
(1) Pandeya, S. N.; Smitha, S.; Jyoti, M.; Sridhar, S. K. Biological
activities of isatin and its derivatives. Acta Pharm. 2005, 55, 27-
46.
(2) Cane, A.; Tournaire, M. C.; Barritault, D.; Crumeyrolle-Arias, M.
The endogenous oxindoles 5-hydroxyoxindole and isatin are anti-
proliferative and proapoptotic. Biochem. Biophys. Res. Commun.
2000, 276, 379-384.
(3) Vine, K. L.; Locke, J. M.; Ranson, M.; Benkendorff, K.; Pyne, S.
G.; Bremner, J. B. In vitro cytotoxicity evaluation of some substituted
isatin derivatives. Bioorg. Med. Chem. 2007, 15, 931-938.
(4) Bramson, H. N.; Corona, J.; Davis, S. T.; Dickerson, S. H.; Edelstein,
M. Oxindole-based inhibitors of cyclin-dependent kinase
(CDK2): design, synthesis, enzymatic activities, and X-ray crystal-
lographic analysis. J. Med. Chem. 2001, 44, 4339-4358.
2
(26) Beckers, T.; Mahboobi, S. Natural, semisynthetic and synthetic
microtubule inhibitors for cancer therapy. Drugs Future 2003, 28,
767-785.

Cytotoxicity of N-Alkyl-Substituted Isatins
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 21 5117
(27) Brancale, A.; Silvestri, R. Indole, a core nucleus for potent inhibitors
of tubulin polymerization. Med. Res. ReV. 2007, 27, 209-238.
(28) De Martino, G.; Edler, M. C.; La Regina, G.; Coluccia, A.; Barbera,
M. C.; Barrow, D.; Nicholson, R. I.; Chiosis, G.; Brancale, A.; Hamel,
E.; Artico, M.; Silvestri, R. New arylthioindoles: potent inhibitors
of tubulin polymerization. 2. Structure-activity relationships and
molecular modeling studies. J. Med. Chem. 2006, 49, 947-954.
(29) Uppuluri, S.; Knipling, L.; Sackett, D. L.; Wolff, J. Localization of
the colchicine-binding site of tubulin. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 11598-11602.
(30) Rao, S.; Orr, G. A.; Chaudhary, A. G.; Kingston, D. G.; Horwitz, S.
B. Characterization of the taxol binding site on the microtubule. 2-(m-
Azidobenzoyl)taxol photolabels a peptide (amino acids 217-231)
of beta-tubulin. J. Biol. Chem. 1995, 270, 20235-20238.
(32) Nobes, C. D.; Hall, A. Rho, rac and cdc42 GTPases: regulators of
actin structures, cell adhesion and motility. Biochem. Soc. Trans.
1995, 23, 456-459.
(33) Still, W. C.; Kahn, M.; Mitra, A. Rapid chromatographic technique
for preparative separations with moderate resolution. J. Org. Chem.
1978, 43, 2923-2925.
(34) Garden, S. J.; Torres, J. C.; da Silva, L. E.; Pinto, A. C. A convenient
methodology for the N-alkylation of isatin compounds. Synth.
Commun. 1998, 28, 1679-1689.
(35) Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Use of an
aqueous soluble tetrazolium/formazan assay for cell growth assays
in culture. Cancer Commun. 1991, 3, 207-212.
(36) Bonne, D.; Heusele, C.; Simon, C.; Pantaloni, D. 4′,6-Diamidino-
2-phenylindole, a fluorescent probe for tubulin and microtubules. J.
Biol. Chem. 1985, 260, 2819-2825.
(31) Zang, M.; Waelde, C. A.; Xiang, X.; Rana, A.; Wen, R.; Luo, Z.
Microtubule integrity regulates Pak leading to Ras-independent
activation of Raf-1. insights into mechanisms of Raf-1 activation. J.
Biol. Chem. 2001, 276, 25157-25165.
JM0704189