Discovery and SAR of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based HTS assay

Article abstract of DOI:10.1016/j.bmc.2004.04.017

A series of indole-2-carboxylic acid benzylidene-hydrazides has been identified as a new class of potent apoptosis inducers through a novel cell-based caspase HTS assay. The screening hit, 5-chloro-3-methyl-indole-2- carboxylic acid (4-nitrobenzylidene)-hydrazide (3a), was found to arrest T47D cells in G₂/M and to induce apoptosis as measured by the flow cytometric analysis assay. A SAR study was carried out by modification of the substitutions on the indole and benzene rings. Substitution at the 3-position of the indole ring was found to be important for apoptotic activity. A 20-fold increase of apoptotic activity was achieved from screening hit 3a to 5-methyl-3-phenyl-indole-2-carboxylic acid (4-methylbenzylidene)-hydrazide (9a) and 5-chloro-3-phenyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (9b), with EC₅₀ value of 0.1μM in the caspase activation assay in T47D breast cancer cells. Compound 9b also was found to be highly active in a standard growth inhibition assay with a GI₅₀ value of 0.9μM in T47D cells. Compound 3a and its analogs were found to inhibit tubulin polymerization, which is the most probable primary mechanism of action of these compounds.

Full text of DOI:10.1016/j.bmc.2004.04.017

Bioorganic & Medicinal Chemistry 12 (2004) 3649–3655
Discovery and SAR of indole-2-carboxylic acid benzylidene-
hydrazides as a new series of potent apoptosis inducers using a cell-
based HTS assay
Han-Zhong Zhang, John Drewe, Ben Tseng, Shailaja Kasibhatla and Sui Xiong Cai*
Maxim Pharmaceuticals, Inc., 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
Received 5 February 2004; revised 7 April 2004; accepted 15 April 2004
Available online 18 May 2004
Abstract—A series of indole-2-carboxylic acid benzylidene-hydrazides has been identified as a new class of potent apoptosis inducers
through a novel cell-based caspase HTS assay. The screening hit, 5-chloro-3-methyl-indole-2-carboxylic acid (4-nitrobenzylidene)-
hydrazide (3a), was found to arrest T47D cells in G2/M and to induce apoptosis as measured by the flow cytometric analysis assay.
A SAR study was carried out by modification of the substitutions on the indole and benzene rings. Substitution at the 3-position of
the indole ring was found to be important for apoptotic activity. A 20-fold increase of apoptotic activity was achieved from
screening hit 3a to 5-methyl-3-phenyl-indole-2-carboxylic acid (4-methylbenzylidene)-hydrazide (9a) and 5-chloro-3-phenyl-indole-
2-carboxylic acid (4-nitrobenzylidene)-hydrazide (9b), with EC50 value of 0.1 lM in the caspase activation assay in T47D breast
cancer cells. Compound 9b also was found to be highly active in a standard growth inhibition assay with a GI50 value of 0.9 lM in
T47D cells. Compound 3a and its analogs were found to inhibit tubulin polymerization, which is the most probable primary
mechanism of action of these compounds.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
types. Utilizing this assay, we reported recently the
discovery and SAR studies of substituted N-phenyl
nicotinamides, exemplified by 6-methyl-N-(4-ethoxy-2-
nitro-phenyl)-pyridine-3-carboxamide (1),10 and gam-
bogic acid (2)11 as potent apoptosis inducers. Herein we
report the discovery, characterization, and SAR of
substituted indole-2-carboxylic acid benzylidene-
hydrazides as a new series of potent inducers of apop-
tosis using our cell-based caspase-3 HTS assay.
Apoptosis, or programmed cell death, plays a vital role
in normal embryonic development as well as adult life,
such as elimination of dispensable or excess cells. It has
been known that defects of apoptosis pathways and the
ability to evade cell death is one of the hallmarks of
cancers, which results in uncontrollable tumor cell
growth, as well as tumor resistance to chemotherapeutic
treatment.1 Therefore the discovery and development of
apoptosis inducers as new chemotherapeutic agents is a
promising approach and has been a focus of research
recently.2–7 Caspases, especially caspase-3, are known to
be the executioners of apoptosis by cleavage of protein
substrates, leading to apoptotic cell death.8 We have
O
OH
O
NO2
H
O
O
O
O
N
N
therefore developed
a cell-based high throughput
screening assay (HTS) for apoptosis inducers, using our
proprietary fluorescent caspase-3 substrate.9 The assay
measures the phenotypic induction of apoptosis by
measuring caspase-3 activity, regardless of mechanism
of apoptosis induction and has been run with several cell
O
OEt
OH
1
2
Cl
O
N
H
N
HN
Keywords: Apoptosis inducers; Anticancer drugs.
* Corresponding author. Tel.: +1-858-202-4006; fax: +1-858-202-4000;
e-mail: scai@maxim.com
NO2
3a
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.017

3650
H.-Z. Zhang et al. / Bioorg. Med. Chem. 12 (2004) 3649–3655
of apoptosis in T47D breast cancer cells from com-
Cl
O
H2NNH2
CDI
Cl
O
mercial compound library using our cell-based caspase-3
HTS assay. Briefly, human breast cancer T47D cells, in
a 96-well microtiter plate containing 10 lM of testing
compound, were incubated for 24 h for the induction of
apoptosis. To the treated cells in the well was then added
the fluorogenic caspase-3 substrate N-(Ac-DEVD)-N0-
ethoxycarbonyl-R1109 in a caspase buffer and the sam-
ple incubated at room temperature for 3 h. Using a
fluorescent plate reader, employing excitation at 485 nm
and emission at 530 nm, the fluorescence of cleaved
ethoxycarbonyl-R110 was measured, thereby determin-
ing the level of caspase activation. Compounds that in-
duce apoptosis and activate the caspases produced a
fluorescent signal that is higher than the background
(signal/background ratio). Compounds found to give a
ratio of >3, are considered active and retested in triplet
for confirmation. Compounds confirmed to be active are
then tested at several concentrations to give a dose–
response and the caspase activation activity EC50 is
calculated. Compound 3a has an EC50 of 2.2 lM with a
maximal ratio of approximately 7 versus untreated cells.
This level of caspase activation is similar to that of 6-
methyl-N-(4-ethoxy-2-nitro-phenyl)-pyridine-3-carbox-
amide (1), which has a ratio of 6, and is less than that of
gambogic acid (2), which has a ratio of around 14.
N
H
OH
N
H
HN NH2
4
5
O
R
Cl
O
H
N
H
HN
N
EtOH
R
6
Scheme 1.
2. Results and discussion
2.1. Chemistry
5-Chloro-3-methyl-indole-2-carboxylic acid (4-nitro-
benzylidene)-hydrazide (3a) was obtained from a com-
mercial source (ChemDiv, San Diego) and its structure
was confirmed by NMR and MS. Indole-2-carboxylic
acid benzylidene-hydrazides (6a–j) in general were pre-
pared in two steps as shown in Scheme 1 from the cor-
responding indole-2-carboxylic acid. Coupling of
indole-2-carboxylic acid 4 with hydrazine produced the
hydrazide 5,12 which was condensed with aromatic
aldehydes to produce 6. 3-Phenyl-indole-2-carboxylic
acid benzylidene-hydrazides (9a) were prepared as
shown in Scheme 2. Treatment of ethyl a-acetyl-b-
phenyl propionate with p-methylbenzenediazonium
chloride,13 which was prepared from p-methylaniline
and sodium nitrite, generated ethyl 5-methyl-3-phenyl-
indole-2-carboxylate (7). Refluxing of 7 with aqueous
hydrazine produced 5-methyl-3-phenyl-indole-2-car-
boxylic acid hydrazide (8), which was condensed with an
aromatic aldehyde to yield compound 9a.
2.3. Flow cytometric assay
The apoptosis-inducing activities of compound 3a were
also characterized by the flow cytometric analysis assay.
Nuclear fragmentation is one of the hallmarks of cas-
pase-mediated apoptosis.14 T47D cells were treated with
compound 3a for 24 or 48 h at 37 ꢁC, then labeled by
propidium iodide and analyzed on a flow cytometer. An
increase in G2/M DNA content (M4) in cells treated for
24 h with compound 3a was observed as shown in Figure
1B. At 48 h the sub-diploid DNA content of cells in-
creased from 5% to 20% with compound treatment,
indicating apoptotic cells, which have undergone DNA
degradation or fragmentation (Fig. 1C). The results
show that treatment of T47D cells by compound 3a for
48 h results in mitotic arrest in the G2/M phase, as well
as induction of apoptosis (M1, apoptotic sub-G1 area).
2.2. HTS assays
5-Chloro-3-methyl-indole-2-carboxylic acid (4-nitro-
benzylidene)-hydrazide (3a) was identified as an inducer
O
O
1) NaOH
2) HCl
Me
H2NNH2
O
OEt
CH2Ph
+
N
H
OEt
+
N2
7
ArCHO
reflux
Me
O
Me
O
HN NH2
N
H
HN
N
N
H
Me
8
9a
Scheme 2.

H.-Z. Zhang et al. / Bioorg. Med. Chem. 12 (2004) 3649–3655
3651
Table 1 shows that compound 3a has an EC50 of 2.2, 1.4,
and 2.0 lM in T47D, H-1299, and DLD-1 cell lines in
the caspase activation assay, respectively. For SAR
studies, we first explored substitution in the benzene
ring. The non-substituted analog 3b was about 2-times
less active than 3a, suggesting that substitution in the
para-position might be important for activity. Indeed,
the para-position of the benzene ring was found to tol-
erate a variety of groups, from electron-withdrawing
groups (NO2 and Cl) to electron-donating groups (Me,
OMe, NMe2). The methyl substituted analog 6a was 2-
fold more potent than compound 3a, suggesting that an
electron-donating group might be slightly preferred.
Interestingly, the o-Me-substituted analog 6g and m-
Me-substituted analogs 6f were both not active up to
10 lM, a reduction of potency by at least 10-fold com-
pared to p-Me 6a, suggesting that there might be a size
limited pocket around the ortho- and meta-positions.
Similarly, the m-NO2-substituted analog 6e also was not
active up to 10 lM.
Next, the SAR of substitution in the 3-position of the
indole ring was explored. Analog 6h was >2-times less
active than 3a, suggesting that a substitution at the 3-
position of indole is important for apoptotic activity.
Analog 9b, with a 3-phenyl group, was >20-fold more
potent than 3a, indicating that there is a bulky hydro-
phobic pocket in the 3-position. Analog 9c was >10-fold
more potent than 6c, confirming that a bulky group in
the 3-position is preferred.
We then explored the SAR of substitution at the 5-po-
sition of indole, by replacing the chloro group with a
nitro and methyl group, respectively. Compound 6i was
about as active as 6b, indicating that the nitro group is a
good replacement for the Cl group. Compound 6j was 4-
to 5-fold more potent than 6b and 6i in the T47D cell
line, suggesting that an electron-donating group might
be preferred in the 5-position. Compound 9a, with a 5-
Me group and a 3-phenyl group, was among the most
potent compounds in this series with an EC50 value of
0.1 lM in T47D cells.
Figure 1. (A)–(C) depict drug-induced apoptosis in T47D cells as
measured by flow cytometric analysis. The x-axis is the fluorescence
intensity and the y-axis is the number of cells with that fluorescence
intensity. (A) Control cells showing most of the cells in G1. (B) Cells
treated with 5 lM of compound 3a for 24 h showing most of the cells
have arrested in G2/M (M4). (C) Cells treated for 48 h showing a
progression from G2/M to in cells with sub-diploid DNA content.
Selective compounds were also tested by the traditional
inhibition of cell proliferation (GI50) assay to confirm
that the active compounds can inhibit the growth of
tumor cells, as well as to find whether there is a corre-
lation between the activity from the caspase activation
assay and the growth inhibition assay. Table 2 sum-
marizes the GI50 values in comparison with the caspase
activation activity (EC50).
This is supported by the finding that compounds 3a, as
well as more potent analogs, inhibit microtubule
assembly.
Table 2 shows that compounds 6c and 9b both inhibited
the growth of T47D and DLD-1 cells, while the inactive
6e also does not inhibit growth up to 10 lM. Compound
9b had a GI50 value of 0.9 and 0.4 lM in T47D and
DLD-1 cancer cells, respectively. In general, compounds
more active in the apoptosis induction assay, as mea-
sured by caspase activation, also are more potent in the
growth inhibition assay. As have been observed with
other series of apoptosis inducers,10;11 these data indi-
cate that the cell-based caspase activation HTS assay is
not only useful for the identification of inducers of
2.4. Structure–activity relationship (SAR) studies
The cell-based caspase HTS assay was also used for the
testing of analogs of compound 3a for SAR studies. The
caspase activation activity (EC50) of compound 3a and
its analogs in three cell lines, T47D breast cancer cells,
H-1299 non-small cell lung cancer cells and DLD-1
colon cancer cells are summarized in Table 1.

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H.-Z. Zhang et al. / Bioorg. Med. Chem. 12 (2004) 3649–3655
Table 1. Caspase activation activity of substituted indole-2-carboxylic acid benzylidene-hydrazides
R2
R1
O
N
H
HN
N
R3
Entry
R1
R2
R3
EC50 (lM)a
H-1299
T47D
DLD
3a
3b
6a
6b
6c
6d
6e
6f
Cl
Cl
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
p-NO2
H
2.2 0.2
4.0 0.4
1.04 0.1
2.5 0.07
2.4 0.3
1.3 0.1
>10
1.4 0.14
3.5 0.3
0.7 0.1
2.0 0.4
0.98 0.3
2.8 0.4
>10
2.0 0.3
4.3 0.5
0.7 0.4
1.5 0.07
2.7 0.09
1.6 0.2
>10
Cl
Cl
p-Me
p-Cl
Cl
Cl
p-OMe
p-NMe2
m-NO2
m-Me
o-Me
p-NO2
p-Cl
Cl
Cl
>10
>10
>10
>10
>10
>10
6g
6h
6i
Cl
Cl
5.8 0.4
2.1 0.4
0.5 0.08
0.1 0.03
0.1 0.06
0.2 0.03
4.9 0.4
2.6 0.07
1.5 0.1
0.5 0.07
0.2 0.07
0.2 0.03
>10
NO2
Me
Me
Cl
Me
Me
Ph
2.7 0.1
2.2 0.5
0.4 0.1
0.6 0.1
0.4 0.07
6j
p-Cl
9a
9b
9c
p-Me
p-NO2
p-OMe
Ph
Ph
Cl
a Data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM).
Table 2. Comparison of caspase activation activity and inhibition of cell proliferation activity of substituted indole-2-carboxylic acid benzylidene-
hydrazides
Compound
EC50 (lM)
DLD-1
GI50 (lM)
DLD-1
T47D
T47D
6c
6e
9b
2.4 0.3
>10
0.1 0.06
2.7 0.09
>10
0.6 0.1
3.1 0.7
>10
0.9 0.2
4.8 2.3
>10
0.4 0.05
Data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM).
apoptosis, but also is useful for subsequent optimization
and SAR study of the hit found from the screening.
agents,15 such as taxanes, a class of microtubule stabi-
lizer and vincas, a class of microtubule depolymerizer.
Since compound 3a was found to be a G2/M blocker
from the flow cytometric assay, we suspected that these
series of compounds might be tubulin inhibitors. We
therefore tested selective compounds in an in vitro
tubulin polymerization assay. Compound 3a, with an
EC50 value of 2 lM in the caspase activation assay,
inhibited tubulin polymerization with an IC50 value of
10 lM. Compounds 9a, 9b, and 9c, which are all with an
EC50 value of less than 0.2 lM and at least 10-fold more
potent than 3a in the caspase assay, all inhibited tubulin
polymerization with IC50s at least 10-fold more active
than compound 3a, at or below the level of sensitivity
for the assay of 1 lM. Therefore the caspase activation
potency is correlated with inhibition of tubulin poly-
merization potency for the indole-2-carboxylic acid
benzylidene-hydrazide series of apoptosis inducers,
suggesting that inhibition of microtubule assembly most
probably is the primary mechanism of action of this
class of compounds. The interference with microtubule
function represents an important mechanistic principle
in anticancer drug discovery that underlies the antitu-
mor activity of a variety of clinical validated anticancer
3. Conclusion
In conclusion, by applying a cell-based caspase HTS
assay, a series of indole-2-carboxylic acid benzylidene-
hydrazides has been identified as a new class of potent
inducers of apoptosis. SAR studies indicate that the sub-
stitution at 3-position of the indole ring was important
for apoptotic activity. A 20-fold increase of apoptotic
activity was achieved from screening hit 5-chloro-3-
methyl-indole-2-carboxylic acid (4-nitrobenzylidene)-
hydrazide (3a) to compounds 9a and 9b with an EC50 of
0.1 lM in the caspase activation assay in T47D breast
cancer cells. Compounds that are more potent in the
caspase activation also were found to be more potent in
the cell proliferation assay. Compound 3a arrested
T47D cells in the G2/M phase, and induced apoptosis as
measured by the flow cytometric analysis. Compound 3a
and its analogs were found to inhibit tubulin polymeri-
zation, and the more potent compounds 9a–c were
also more potent inhibitors of tubulin polymerization,

H.-Z. Zhang et al. / Bioorg. Med. Chem. 12 (2004) 3649–3655
3653
suggesting that inhibition of tubulin polymerization
might be the primary mechanism of action for these
compounds as inducers of apoptosis.
4.1.4. 5-Chloro-3-methyl-indole-2-carboxylic acid (3-ni-
trobenzylidene)-hydrazide (6e). (44%). 1H NMR
(DMSO-d6): 11.8 (br s, 1H), 11.6 (br s, 1H), 8.57 (s, 1H),
8.47 (br s, 1H), 8.27 (d, J ¼ 7:5, 1H), 8.17 (d, J ¼ 7:5,
1H), 7.80 (t, J ¼ 8:4, 1H), 7.72 (s, 1H), 7.46 (d, J ¼ 8:4,
1H), 7.24 (m, 1H), 2.51 (s, 3H). Anal. Calcd for
C17H13ClN4O3Æ2H2O: C, 51.98; H, 4.36; N, 14.26
Found: C, 51.92; H, 4.41; N, 14.03.
4. Experimental section
4.1. General methods and materials
4.1.5. 5-Chloro-3-methyl-indole-2-carboxylic acid (3-
methylbenzylidene)-hydrazide (6f). (20%). 1H NMR
(DMSO-d6): 11.5 (br s, 2H), 8.65 (br s, 1H), 7.84 (br s,
1H), 7.71 (br s, 1H), 7.46 (s, 1H), 7.44 (d, J ¼ 8:7Hz,
1H), 7.34–7.20 (m, 3H), 2.50 (s, 3H), 2.45 (s, 3H). Anal.
Calcd for C18H16ClN3OÆ0.2H2O: C, 65.63; H, 5.00; N,
12.76. Found: C, 65.62; H, 4.68; N, 12.40.
The 1H NMR spectra were recorded at Varian
300 MHz. Chemical shifts are reported in ppm (d) and J
coupling constants are reported in Hz. Elemental anal-
yses were performed by Numega Resonance Labs, Inc.
(San Diego, CA). Reagent grade solvents were used
without further purification unless otherwise specified.
Compounds 3a, 3b, 6c, 6i, 6j, and 9c were purchased
from ChemDiv (San Diego, CA). Compound 9b was
purchased from Chemstar (Moscow, Russia), and their
structures were confirmed by 1H NMR and MS. Human
breast cancer cell T47D, human non-small cell lung
cancer cells H-1299 and human colon cancer cells DLD-
1 were obtained from American Type Culture Collection
(Manassas, VA).
4.1.6. 5-Chloro-3-methyl-indole-2-carboxylic acid (2-
methylbenzylidene)-hydrazide (6g). (34%). 1H NMR
(DMSO-d6): 11.46 (br s, 2H), 8.65 (br s, 1H), 7.88 (br s,
1H), 7.71(s, 1H), 7.42 (d, J ¼ 8:7, 1H), 7.34–7.22 (m,
4H), 2.50 (s, 3H), 2.45 (s, 3H). Anal. Calcd for
C18H16ClN3O: C, 66.36; H, 4.95; N, 12.90. Found: C,
65.99; H, 4.82; N, 12.53.
4.1.1. 5-Chloro-3-methyl-indole-2-carboxylic acid (4-
methylbenzylidene)-hydrazide (6a). A solution of 5-
chloro-3-methyl-indole-2-carboxylic acid hydrazide
4.1.7. 5-Chloro-indole-2-carboxylic acid (4-nitrobenzylid-
1
ene)-hydrazide (6h). (60%). H NMR (DMSO-d6): 12.3
(br s, 1H), 12.1 (br s, 1H), 8.55 (s, 1H), 8.32 (d, J ¼ 8:7,
2H), 8.03 (d, J ¼ 8:7, 2H), 7.82 (s, 1H), 7.48 (d, J ¼ 8:4,
1H), 7.36 (s, 1H), 7.25 (d, J ¼ 8:4, 1H), 7.24 (m, 1H),
2.51 (s, 3H). Anal. Calcd. for C16H11ClN4O3: C, 56.07;
H, 3.23; N, 16.35. Found: C, 55.97; H, 3.51; N, 16.46.
(13 mg,
0.054 mmol),
p-tolualdehyde
(6.5 mg,
0.054 mmol) in ethanol (5 mL) was refluxed for 8 h. It
was evaporated in vacuo and the residue was purified by
column chromatography (silica gel, EtOAc/hex-
ane ¼ 4:1) to give 7.8 mg (42%) of 6a. 1H NMR
(CD3OD): 8.26 (s, 1H), 7.72 (d, J ¼ 8:1, 2H), 7.62 (s,
1H), 7.39 (d, J ¼ 8:7, 1H), 7.27 (d, J ¼ 8:1, 2H), 7.25
(m, 1H), 2.58 (s, 3H), 2.39 (s, 3H). Anal. Calcd for
C18H16ClN3O: C, 66.36; H, 4.95; N, 12.90. Found: C,
66.02; H, 4.98; N, 12.88.
4.1.8. Ethyl 5-methyl-3-phenyl-indole-2-carboxylate (7).
A solution of sodium hydroxide (6 g) in water (15 mL)
was added to a solution of ethyl a-acetyl-b-phenyl
propionate (11 g, 0.05 mmol) in ethanol (80 mL), fol-
lowed immediately by addition of a solution of p-
methylbenzenediazonium chloride (prepared from 4.65 g
of p-methylaniline and 3.45 g of sodium nitrite in 25 mL
of concentrated HCl), resulted in the precipitation of
brown oil. The mixture was diluted by water (100 mL)
and it was extracted with ethyl acetate (3 · 50 mL). The
extracts were dried and evaporated to give a crude
intermediate hydrazone. A solution of the crude
hydrazone in hydrogen chloride saturated methanol
(100 mL) was refluxed for 4 h. It was concentrated and
the residue was purified by column chromatography
(EtOAc/CH2Cl2 ¼ 4:1) to give 3.2 g (24%) of 7. 1H NMR
(CDCl3): 8.90 (br s, 1H), 7.60–7.20 (m, 8H), 4.28 (q,
2H), 2.41 (s, 3H), 1.23 (t, J ¼ 7:2, 3H).
Compounds 6b, 6d–h were prepared by a method similar
to that described for the preparation of compound 6a
from 5-chloro-3-methyl-indole-2-carboxylic acid hydr-
azide and the corresponding substituted benzaldehyde.
4.1.2. 5-Chloro-3-methyl-indole-2-carboxylic acid (4-chlo-
robenzylidene)-hydrazide (6b). (22%). 1H NMR
(CD3OD): 8.90 (s, 1H), 8.18 (br s, 1H), 7.70 (d, J ¼ 7:80,
2H), 7.50 (s, 1H), 7.32 (d, J ¼ 7:80, 2H), 7.27 (m, 1H),
7.10 (m, 1H), 2.45 (s, 3H). Anal. Calcd for
C17H13Cl2N3O: C, 58.98; H, 3.78; N, 12.14. Found: C,
58.15; H, 3.44; N, 12.27.
4.1.3. 5-Chloro-3-methyl-indole-2-carboxylic acid (4-
dimethylaminobenzylidene)-hydrazide (6d). (21%). 1H
NMR (CD3OD): 10.9 (br s, 1H), 9.07 (br s, 1H), 8.2–6.6
(m, 6H), 3.06 (s, 6H), 2.70 (s, 3H). Anal. Calcd for
C19H19ClN4O: C, 75.73; H, 6.10; N, 14.10. Found: C,
75.35; H, 6.28; N, 14.11.
4.1.9. 5-Methyl-3-phenyl-indole-2-carboxylic acid hydr-
azide (8). A suspension of 7 in H2NNH2/H2O (4:1, 15 mL)
was refluxed for 7 h. It was cooled to room temperature
and concentrated. The residue was diluted with water
(50 mL) and it was extracted with ethyl acetate
(3 · 20 mL). The extracts were dried and evaporated,

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H.-Z. Zhang et al. / Bioorg. Med. Chem. 12 (2004) 3649–3655
and the crude product was washed with hexane, and
1
plate reader (Model Spectrafluor Plus Tecan Instru-
ment). GI50 values were calculated from dose–response
curves using XLFit3 (IDBS) software. The GI50 (lM)
are summarized in Table 2 in comparison with the cas-
pase activation activity (EC50).
dried to give 1.4 g (99%) of 8. H NMR (CDCl3): 9.20
(br s, 1H), 7.60–7.05 (m, 8H), 2.39 (br s, 2H), 2.39 (s,
3H).
4.1.10. 5-Methyl-3-phenyl-indole-2-carboxylic acid (4-
methylbenzylidene)-hydrazide (9a). Compound 9a was
prepared by a method similar to that described for
compound 6a from 8 and p-tolualdehyde (44%). 1H
NMR (CDCl3): 9.42 (br s, 1H), 8.85 (s, 1H), 7.60–7.57
(m, 7H), 7.40 (d, J ¼ 8:4, 1H), 7.26–7.15 (m, 4H), 2.41(s,
3H), 2.36 (s, 3H). Anal. Calcd for C24H21N3OÆ0.5H2O:
C, 76.57; H, 6.20; N, 10.80. Found: C, 76.80; H, 5.89; N,
11.16.
4.5. Tubulin depolymerization assay (IC50)
Lyophilized tubulin (Cytoskeleton #ML113, 1 mg,
MAP-rich) was assayed for the effect of the test com-
pound on tubulin polymerization as measured by
change in fluorescence for 40,6-diamidino-2-phenyl-
indole (DAPI).16 One microliter of serial dilutions of
each experimental compound (from 100· DMSO stock)
was added in 96 well plate format and preincubated for
30 min with 94 lL of the non-GTP supplemented tubu-
lin supernatant. Five microliters of DAPI/GTP solution
was added to initiate polymerization and incubated for
30 min at 37 ꢁC. Fluorescence was read with excitation
350 nm, emission wavelength 485 nm on a Tecan Spec-
traflour Plus. Polymerized tubulin (sample treated with
DMSO and sample treated with the tubulin stabilizer
taxol) gives a higher DAPI fluorescence as compared to
non-polymerized tubulin (vinblastine and colchicine
were used to determine baseline). The IC50 for tubulin
inhibition was the concentration found to decrease the
fluorescence of DAPI by 50% as calculated with Prism
3.0.
4.2. Caspase activation assay (EC50)
The potency of indole-2-carboxylic acid benzylidene-
hydrazides analogs as inducers of apoptosis was mea-
sured by our caspase-based cell assay as previously
described.10 Briefly, human breast cancer cell lines
T47D, human non-small cell lung cancer cells H-1299,
and human colon cancer cell line DLD-1 were treated
with various concentrations of test compound and
incubated at 37 ꢁC for 24 h. The samples were then
treated with the fluorogenic substrate N-(Ac-DEVD)-
N0-ethoxycarbonyl-R110, and incubated for 3 h. The
fluorescent signal was measured using a fluorescent plate
reader (Model Spectrafluor Plus Tecan). The EC50 (lM)
was determined by a sigmoidal dose–response calcula-
tion (XLFit3, IDBS), as the concentration of compound
that produces the 50% maximum response. The caspase
activation activity (EC50) in three cancer cell lines,
T47D, H-1299 and DLD, are summarized in Table 1.
Acknowledgements
The authors acknowledge the fine technical support
given by Candace Crogan-Grundy.
4.3. Flow cytometric analysis assay
References and notes
Cell cycle analysis was performed as previously
described.10 Briefly, T47D cells were treated with 10 lM
of compound 3a for 24–48 h at 37 ꢁC. Control cells were
treated with the solvent (DMSO) for 48 h. After the
incubation, cells were treated with propidium iodide and
RNAse A, and analyzed on a flow cytometer. All flow
cytometry analyses were performed on FACScalibur
(Becton Dickinson) using Cell Quest analysis software.
On the x-axis is plotted the fluorescence intensity and on
the y-axis is plotted the number of cells with that fluo-
rescence intensity.
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