Potent antitubulin tumor cell cytotoxins based on 3-aroyl indazoles

Article abstract of DOI:10.1021/jm061348t

A series of 3-aroyl indazoles was synthesized. Modification of the C-7 position resulted in a significant structure-activity relationship (SAR) with acetylene modifications conferring unusual potency in a tumor cell cytotoxicity assay. The most potent com

Full text of DOI:10.1021/jm061348t

J. Med. Chem. 2007, 50, 1001-1006
1001
Potent Antitubulin Tumor Cell Cytotoxins Based on 3-Aroyl Indazoles
Jian-Xin Duan,* Xiaohong Cai, Fanying Meng, Leslie Lan, Charles Hart, and Mark Matteucci*
Threshold Pharmaceuticals, 1300 Seaport BouleVard, Redwood City, California 94063
ReceiVed NoVember 21, 2006
A series of 3-aroyl indazoles was synthesized. Modification of the C-7 position resulted in a significant
structure-activity relationship (SAR) with acetylene modifications conferring unusual potency in a tumor
cell cytotoxicity assay. The most potent compounds exceeded the activity of combretastatin A4 (CA-4),
showing single digit nM IC50 values against all cell lines tested including those with known efflux resistance
pumps. The inhibition of in Vitro tubulin polymerization was comparable to CA-4, consistent with tubulin
being the target for these compounds. Competition binding experiments employing [3H]colchicine and purified
tubulins demonstrated that the compound specifically binds to the colchicine site.
Introduction
Tubulin-containing structures such as microtubules are im-
portant for diverse cellular functions, including chromosome
segregation during cell division, intracellular transport, cell
motility, and cell shape.1 Combretastatin A4 (CA-4a)2 is a low-
molecular weight natural product that binds to the colchicine
site of tubulin and inhibits tubulin polymerization. A water
soluble phosphate prodrug of combretastatin A4 (CA-4P)3 is
currently in clinical trials for the treatment of solid tumors. The
activity and structural simplicity of CA-4 have stimulated
enormous efforts to develop new analogues that mimic CA-4
activity and improve on its pharmacological properties. One
analogue series has utilized diphenyl ketones4 that were shown
to be tubulin inhibitors with only mild cytotoxicity. Liou5 et al.
have extended their initial observation and reported that
aroylindoles were potent tubulin inhibitors and highly cytotoxic
to tumor cells in culture.
Figure 1. Potential metal-chelated and hydrogen-bonded indazole
structures.
Scheme 1a
We report here that 3-(3,4,5-trimethoxylbenzoyl)indazoles are
highly cytotoxic agents to tumor cells in cell culture and
inhibitors of tubulin polymerization in Vitro. The interest in the
indazole series was driven by the postulate that the N-2 nitrogen
on the indazole and the oxygen on the carbonyl group could
form a chelate with a metal such as magnesium or a hydrogen-
bonded complex with water as shown below in Figure 1. Such
a preordered structure could conformationally mimic the cis
configuration of CA-4.
a (a) PdCl2(PPh3)2, Cul/TEA (89%), trimethylsilylacetylene. (b) NBu4F/
THF (82%). (c) PdCl2(PPh3)2, CuI/TEA, 4-iodo-3-nitroanisole (90%). (d)
Fe, HCl/EtOH (54%). (e) NaNO2, HCl (80%).
Scheme 2
Synthesis. The core structure was readily synthesized based
on an adoption of the literature method6,7 as shown below in
Scheme 1. 1 was coupled to trimethylsilylacetylene using
palladium catalyst followed by desilylation to give compound
2.6 Subsequent coupling with 4-iodo-3-nitroanisole gave com-
pound 3, which was readily reduced by iron powder to afford
4. The core 5 was prepared from 4 following the reported
procedure.7 Iodination of 5 was highly regioselective and gave
compound 6 in very good yield (85%) (Scheme 2). This iodo
derivative was a key synthon for further elaboration. Coupling
reaction of 6 with various terminal alkynes gave 7 in good yields
(Scheme 3). Careful hydrogenation of 10 and desilylation
resulted in compounds 12 and 13 (Scheme 4). Compound 15
was synthesized as a test of the effect of C-7 modification on
the previously published compound 14 (BPROL075) of the
indole series (Scheme 5).5
* To whom correspondence should be addressed. Phone: 650-474-8231.
Fax: 650-474-0408. E-mail: jduan@thresholdpharm.com (J.D.). Phone:
650-474-8225. Fax: 650-474-0408. E-mail: mmatteucci@thresholdpharm.com
(M. M).
a Abbreviations: CA-4, combretastatin A4; IC50, the half maximal
inhibitory concentration; MDR, multidrug drug resistance; MRP, multiple
resistant protein; GTP, guanosine triphosphate; TLC, thin layer chroma-
tography; HRMS, high resolution mass spectra; HPLC, high pressure liquid
chromatography.
Biological Evaluation. All of the reported compounds were
evaluated for cytotoxicity toward H460 cells, a human non-
small lung cancer cell line. CA-4 and 14 were included as
10.1021/jm061348t CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/08/2007

1002 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Duan et al.
Table 1. IC50 Values (nM) of 7-9, 12-14, and 16a
Scheme 3
Scheme 4a
a All assays performed in triplicate; cells were treated with compound
for 3 days.
Table 2. IC50 Values (nM) of 7a, 7b, and 8 against HT29, PC-3, and
HeLa Cell Lines
tumor cell line
tumor type
7a
7b
8
CA-4
HT29
PC3
Hela
colon
prostate
cervical
3.0
1.1
0.9
1.0
1.0
0.8
1.7
3.0
2.3
>1000
3.2
2.7
a (a) tEnDashCH2OTBMS, PdCl2(PPh3)2, Cul/TEA (90%). (b) Pd/C/
H2 (68%). (c) NBu4F (83%). (d) PtO2/H2 (74%). (e) NBu4F (85%).
allyl alcohol derivative had comparable activity to the propargyl
alcohol 7b, while the completely hydrogenated propanol 13 was
much less active (IC50 ) 8 nM for 7b vs IC50 ) 13 nM for 12
vs IC50 ) 283 nM for 13). The intriguing activity imparted by
the propyne modification in the indazole series did not transfer
to the indole series. The propyne analogue of 14 was inactive
at concentrations up to 1000 nM.
Three of most potent compounds 7a, 7b, and 8 were tested
in three other human cancer cell lines: PC-3 human prostate
cancer cells, HT29 human colorectal cancer cells, and HeLa
human cervical cancer cells. As shown in Table 2, all three
compounds exhibited similar potent cytotoxic effects against
these lines. The CA-4 control demonstrated similar low nano-
molar potency against PC-3 and HeLa cells. CA-4 was virtually
inactive against HT29 cells consistent with published data
demonstrating HT29 resistance to CA-4.8
controls. The IC50 values for the inhibition of proliferation are
shown in Table 1.
The core indazole compound 5 had an IC50 of 64 nM which
is approximately 4 times less active than the corresponding
indole analogue 14 (IC50 ) 16 nM). When acetylenes were
introduced onto the C-7 position of 5, the resulting compounds
showed dramatically improved cytotoxic activities. The activity
of the simple acetylene derivative 8 was increased by 20 times
(IC50 ) 3 nM for 8 vs 64 nM for 5), while the propyne
derivative 7a proved to be the most potent compound being 60
times more potent (IC50 ) 1 nM for 7a) than 5. Alcohols were
well tolerated with the linear substituted alcohols demonstrating
significant potency (IC50 values for 7b, 7f, and 7g are 8, 14,
and 8 nM, respectively). The tertiary alcohol was found to be
much less active (IC50 for 7e is 195 nM). Amino substitution
was less tolerated, with compound 9 being about 15 times less
active than the corresponding alcohol analogue 7b. The cis-
Tubulin Inhibitors Overcome Drug Resistance. The lead
compounds 7a, 7b, and 8 were further evaluated against cell
lines possessing the well characterized drug resistance efflux
pumps MDR-1 (P-glycoprotein, ABCB1) and MRP-1 (AB-
Scheme 5

Antitubulin Tumor Cell Cytotoxins
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1003
Figure 2. Inhibition of colchicine binding by 7b. Purified tubulin from bovine brain (A), Hela cells (B) and MCF-7 cells (C), was preincubated
with 7b for 10 min at 37 °C. [3H]Coclchicine was added into the reaction. The samples were spotted on DE81 filters, and bound and unbound
[3H]colchicine were separated by washing three times. The bound [3H]colchicine was measured using a scintillation counter.
Figure 3. 7b (A) and CA4 (B) inhibited tubulin polymerization in a concentration-dependent manner.
Table 3. IC50 Values (nM) of 7a, 7b, and 8 against Drug Resistant Cell Lines
cell line
type
origin
resistance mechanism
Messa
sensitive
gastric
none
1.6
Messa/DX5
resistant
gastric
MDR-1
1.4
HCT-15
resistant
colon
MDR-1
2.5
ACHN
resistant
renal
MDR-1
1.6
NCI-H69
sensitive
small cell lung
none
1.7
H69 AR
resistant
small cell lung
MRP-1
2.5
CA4
7a
1.6
1.9
1.9
1.6
1.6
2.5
7b
1.7
1.8
1.9
1.6
1.6
2.6
8
1.9
3.9
1.6
2.5
2.2
paclitaxel
daunorubicin
colchicine
vinblastine
1.6
20
6.3
1.6
>1000
>400
63
79.4
>400
100
25
63
39.8
25
1.9
100
2.5
>1000
8.9
79.4
1.6
1.6
CC1).9 These results along with data enabling a comparison to
some efflux-sensitive chemotherapeutics are shown in Table
3.MESSA/DX5 was derived from the parent MESSA gastric
cancer cell line and is highly resistant to daunorubicin, paclitaxel,
and colchicine due to overexpression of the MDR-1 pump.10
HCT-15 and ACHN cells also have high expression levels of
MDR-1 and display the multiple drug resistance phenotype.11
H69AR was derived from NCI-H69 and is highly resistant to
daunorubicin due to overexpression of the MRP-1 pump.12 As
shown in Table 3, all three compounds exhibited potent
antiproliferative activities in all of the drug resistant cell lines
examined, with the IC50 values ranging from 1.6 nM to 3.9 nM.
These results demonstrate that 7a, 7b, and 8 are not substrates
for the MDR-1 and MRP-1 pumps.
7b Inhibits Tubulin Polymerization in Vitro. The effects
of 7b on microtubule formation were assessed using an in Vitro
tubulin polymerization assay,13 in which microtubule formation
was monitored by the increase in fluorescent intensity of the
reaction mixture. The addition of 7b inhibited tubulin poly-
merization in a concentration-dependent manner (Figure 3) with
an IC50 of 6 µM. The control compound, CA-4, displayed a
similar inhibition effect in this assay. The concentrations
required to inhibit tubulin polymerization (IC50: 6 µM) are much
higher than those required for cytotoxicity, a phenomenon well
documented in the literature.14 These results, along with the
structural similarity to known tubulin polymerization inhibitors,
indicate that the indazole ketone’s biological activity is due to
interaction with tubulin.
A highly potent class of cytotoxic compounds has been
identified based on novel acetylene substitutions of an indazole
core. Three compounds, 7a, 7b, and 8, have emerged as lead
compounds. These compounds have been shown to be nearly
equally potent against a panel of cancer cell lines and are not
substrates of two common drug efflux pumps. On the basis of
these excellent properties, these compounds are worthy of further
in Vitro and in ViVo evaluation.
7b Specifically Binds to Colchicine Binding Site. The ability
of lead compound 7b to compete with [3H]colchicine binding
to purified tubulins, including bovine brain tubulin, HeLa cell
tubulin, and MCF-7 tubulin, was evaluated. As shown in Figure
2A, 7b inhibited 50% of the colchicine binding to bovine brain
tubulin at 500 nM. Similarly, 7b was able to compete with [3H]-
colchicine binding to purified tubulin from Hela cells and
MCF-7 cells. As shown in Figure 2B and 2C, 7b inhibited
colchicine binding to Hela tubulin and MCF-7 tubulin with IC50
values of 46 nM and 240 nM, respectively. The different
inhibitory potency to tubulins derived from various sources
might be related to differential expression of tubulin isoforms
(unpublished data).
Experimental Section
1H and 13C NMR spectra were recorded on a Varian 400 MHz
spectrometer (400 and 75 MHz, respectively) using CDCl3, CD3-
OD, or DMSO-d6 as solvents with TMS as an internal standard.

1004 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Duan et al.
High-resolution mass spectra analysis was performed on a GCT-
MS Micromass UK mass spectrometer. Column chromatography
was performed with silica gel (230-400 mesh). All chemicals and
solvents were purchased from Sigma-Aldrich and Fisher Scientific.
1-Trimethylsilyl-2-(3,4,5-trimethoxyphenyl)acetylene. A 50-
mL two-necked round-bottomed flask equipped with a septum, a
stir-bar, and a water condenser topped with a nitrogen inlet was
charged with a mixture of compound 1 (588 mg, 2.0 mmol), PdCl2-
(PPh3)2 (70 mg, 0.1 mol), CuI (19 mg, 0.1 mol), and triethylamine
(TEA, 15 mL). Trimethylsilylacetylene (0.47 mL, 3.4 mmol) was
added to it at room temperature (rt). After 30 min the solution was
heated to 50 °C under nitrogen. After complete consumption of
starting material (monitored by thin layer chromatography (TLC))
the mixture was cooled to rt and filtered, and the solid was washed
with dichloromethane (DCM, 10 mL). The filtrate was concentrated
under reduced pressure to give a crude product, which subjected
to flash chromatography purification on silica gel to give the pure
product (470 mg, 89%). 1H NMR (DMSO-d6) δ 6.73 (s, 2H), 3.78
(s, 6H), 3.66 (s, 3H), 0.22 (s, 9H).
(CDCl3 + CD3OD) δ 55.9, 57.0, 60.5, 62.1, 108.2, 109.7, 119.0,
123.4, 132.2, 142.1, 143.8, 144.4, 152.4, 157.9, 187.4. HRMS Calcd
for C18H17IN2O5: 468.0182. Found 468. 0187.
(6-Methoxy-7-(prop-1-ynyl)-1H-indazol-3-yl)(3,4,5-tri-meth-
oxyphenyl)methanone (7a). A mixture of compound 6c (46 mg,
0.1 mmol), PdCl2(PPh3)2 (7 mg, 0.1 mmol), and CuI (2 mg, 0.1
mmol) in Et3N (6 mL) was thrice degassed and exchanged with
propyne. Propyne contained in a balloon was kept in contact with
the reaction mixture. The system was stirred at 45 °C overnight
and filtered. The filtrate was concentrated under reduced pressure,
and the residue was purified with flash chromatography on silica
gel to yield compound 7a (22 mg, 58%). 1H NMR (CDCl3) δ 11.3
(br, s, NH), 8.27 (d, J ) 8.8 Hz, 1H), 7.71 (s, 2H), 7.01 (d, J )
9.2 Hz, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 3.87 (s, 6H), 2.12 (s, 3H).
13C NMR (CDCl3) δ 4.8, 56.1, 56.9, 60.8, 70.8, 93.6, 95.6, 108.4,
110.0, 118.3, 123.2, 132.4, 142.4, 143.0, 144.2, 152.6, 159.9, 187.1.
HRMS Calcd for C21H20N2O5: 380.1372. Found 380.1377.
General Procedure for Preparation of Compounds 7b-i from
Compound 6. A mixture of compound 6 (468 mg, 1 mmol), PdCl2-
(PPh3)2 (35 mg, 0.05 mmol), and CuI (10 mg, 0.05 mmol) in Et3N
(70 mL) was thrice degassed and exchanged with argon followed
by addition of alkynes at rt. The reaction mixture was stirred at
55 °C for 5-10 h (monitored by TLC) and filtered. The filtrate
was concentrated under reduced pressure, and the residue was
separated employing flash chromatography on silica gel to yield
compounds 7b-i.
3,4,5-Trimethoxyethynylbenzene (2).6 Compound 2 was syn-
thesized in 82% yield based on the literature method reported for
the compound.6 1H NMR (DMSO-d6) δ 6.78 (s, 2H), 4.13 (s, 1H),
3.77 (s, 6H), 3.66 (s, 3H).
1,2,3-Trimethoxy-5-(2-(4-methoxy-2-nitrophenyl)ethynyl)ben-
zene (3). To a solution containing compound 2 (240 mg, 1.25 mmol)
and 4-iodo-3-nitroanisole (345 mg, 1.24 mmol) were added PdCl2-
(PPh3)2 (44 mg, 5.0 mol %) and CuI (12 mg, 5.0 mol %) in TEA
(15 mL). The mixture was stirred at 55 °C for 3 h, cooled, and
filtered. The filtrate was concentrated under reduced pressure.
Chromatography of the residue on silica gel gave compound 3 (380
mg, 90%). 1H NMR (DMSO-d6) δ 8.06 (d, J ) 2.4 Hz, 1H), 7.82
(dd, J ) 2.0, 8.8 Hz, 1H), 7.42 (d, J ) 8.8 Hz), 6.89 (s, 2H), 3.97
(s, 3H), 3.81 (s, 6H), 3.7 (s, 3H).
5-Methoxy-2-(2-(3,4,5-trimethoxyphenyl)ethynyl)amine (4).
Compound 3 (140 mg, 0.41 mmol) was suspended in EtOH (95%,
15 mL) and stirred at 80 °C for 30 min. To this mixture were added
concentrated HCl (0.017 mL) and iron powder (230 mg, 8.3 mmol).
The reaction mixture was refluxed for 2 h, cooled, and filtered.
The filtrate was concentrated under reduced pressure. Chromatog-
raphy of the residue on silica gel gave compound 4 (69 mg, 54%).
1H NMR (DMSO-d6) δ 7.11 (d, J ) 11.6 Hz, 1H), 6.88 (s, 2H),
6.28 (d, J ) 2.8 Hz, 1H), 6.13 (dd, J ) 2.8, 11.6 Hz, 1H), 5.52
(br, s, NH2), 3.80 (s, 6H), 3.69 (s, 3H), 3.67 (s, 3H).
(6-Methoxy-1H-indazol-3-yl)(3,4,5-trimethoxyphenyl)metha-
none (5). To a solution containing compound 4 (170 mg, 0.54
mmol) in water/acetone (V/V ) 1:2 10 mL) was added dropwise
10% HCl (3 mL). The resulting mixture was cooled down to
-10 °C. A solution of NaNO2 (56 mg, 0.81 mmol) in water (1
mL) was added to the reaction mixture and stirred for 30 min at
-10 to -5 °C. After water (100 mL) was added, the reaction
mixture was warmed to rt, stirred for 30 min, and extracted with
AcOEt (2 × 15 mL). The organic phase was washed with 10%
NaHCO3 and brine, dried over Na2SO4, and concentrated under
reduced pressure. Chromatography of the residue on silica gel
afforded compound 5 (147 mg, 80%). 1H NMR (DMSO-d6) δ 13.8
(s, NH), 8.12 (d, J ) 8.8 Hz, 1H), 7.70 (s, 2H), 7.06 (s, 1H), 6.98
(d, J ) 8.4 Hz 1H), 3.86 (s, 9H), 3.78 (s, 3H). 13C NMR
(CDCl3 + CD3OD) δ 55.6, 56.3, 61.0, 91.3, 108.5, 115.7, 118.5,
123.4, 133.1, 142.4, 142.5, 143.2, 152.9, 160.1, 188.2. HRMS Calcd
for C18H18N2O5: 342.1216. Found 342.1214.
(7-Iodo-6-methoxy-1H-indazol-3-yl)(3,4,5-trimethoxyphenyl)-
methanone (6). To a solution of N-iodosuccinimide (45 mg, 0.2
mmol, 1.0 equiv) in AcOH (1 mL) was added a solution of 5 (68
mg, 0.2 mmol, 1.0 equiv) in AcOH (2 mL) at rt. The mixture was
stirred at rt for 2 h, diluted with 8 mL water and extracted with
EtOAc (10 mL × 3). Combined organic layers were neutralized to
pH 7 with 10% NaHCO3, dried over Na2SO4, and concentrated
under reduced pressure. The residue was crystallized from MeOH
to yield compound 6 (80 mg, 85%). 1H NMR (CDCl3) δ 10.2 (br,
s, NH), 8.36 (d, J ) 8.8 Hz, 1H), 7.75 (s, 2H), 7.03 (d, J ) 8.8
Hz, 1H), 4.04 (s, 3H), 3.97 (s, 3H), 3.96 (s, 6H). 13C NMR
(7-(3-Hydroxyprop-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (7b). Compound 7 was obtained as
1
white solid in 39% yield. H NMR (DMSO-d6) δ 14.03 (s, NH),
8.21 (d, J ) 9.2 Hz, 1H), 7.69 (s, 2H), 7.24 (d, J ) 9.2 Hz, 1H),
5.35 (br, s, OH), 4.43 (s, 2H), 3.95 (s, 3H), 3.86 (s, 6H), 3.79 (s,
3H). 13C NMR (DMSO-d6) δ 49.9, 56.0, 56.6, 60.1, 75.5, 92.5,
98.9, 108.1, 110.2, 118.0, 123.2, 132.2, 141.8, 142.6, 142.9, 152.4,
159.5, 186.1. HRMS Calcd for C21H20N2O6: 396.1321. Found
396.1319.
R-(7-(3-Hydroxybut-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (7c). Compound 7c was obtained
as a light yellow solid in 52% yield. 1H NMR (DMSO-d6) δ 13.97
(s, NH), 8.20 (d, J ) 9.2 Hz, 1H), 7.71 (s, 2H), 7.23 (d, J ) 9.2
Hz, 1H), 5.43 (d, J ) 5.2 Hz, OH), 4.72 (m, 1H), 3.94 (s, 3H),
3.87 (s, 6H), 3.79 (s, 3H), 1.48 (d, J ) 6.8 Hz, 3H). 13C NMR
(DMSO-d6) 25.2, 56.7, 57.4, 57.9, 60.9, 74.8, 93.3, 103.1, 108.9,
111.1, 118.8, 123.8, 133.0, 142.6, 143.2, 143.7, 153.1, 160.0, 186.9.
HRMS Calcd for C22H22N2O6: 410.1478. Found 420.1481.
S-(7-(3-Hydroxybut-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (7d). Compound 7d was obtained
1
as a light yellow solid in 55% yield. H NMR (DMSO-d6) δ 14.0
(br, s, NH), 8.20 (d, J ) 8.8 Hz, 1H), 7.71 (s, 2H), 7.24 (d, J )
9.2 Hz, 1H), 5.44 (br, s, OH), 4.71 (m, 1H), 3.94 (s, 3H), 3.87 (s,
6H), 3.79 (s, 3H), 1.48 (d, J ) 6.8 Hz, 3H). 13C NMR (CDCl3 +
CD3OD) 23.8, 55.9, 56.7, 58.4, 60.6, 75.1, 92.6, 100.1, 108.3, 109.8,
118.4, 123.6, 132.4, 142.1, 142.9, 143.5, 152.5, 159.5, 187.5. HRMS
Calcd for C22H22N2O6: 410.1478. Found 420.1480.
(7-(3-Hydroxy-3-methylbut-1-ynyl)-6-methoxy-1H-indazol-3-
yl)(3,4,5-trimethoxyphenyl)methanone (7e). Compound 7e was
obtained as a light yellow solid in 61% yield. 1H NMR (CDCl3) δ
12.3 (s, NH), 8.24 (d, J ) 9.2 Hz, 1H), 7.75 (s, 2H), 6.87 (d, J )
9.2 Hz, 1H), 5.02 (br, s, OH), 3.90 (s, 3H), 3.89 (s, 3H), 3.84 (s,
6H), 1.79 (s, 6H). 13C NMR (CDCl3 + CD3OD) 30.9, 55.9, 56.8,
60.6, 65.5, 73.5, 92.6, 102.8, 108.3, 110.0, 118.4, 123.6, 132.4,
142.1, 142.8, 143.6, 152.5, 159.4, 187.3. HRMS Calcd for
C23H24N2O6: 424.1634. Found 424.1636.
(7-(4-Hydroxybut-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (7f). Compound 7f was obtained
1
as a light yellow solid in 85% yield. H NMR (DMSO-d6) δ 14.0
(s, NH), 8.17 (d, J ) 8.8 Hz, 1H), 7.70 (s, 2H), 7.22 (d, J ) 9.2
Hz, 1H), 4.94 (t, J ) 5.6 Hz, HO), 3.93 (s, 3H), 3.87 (s, 6H), 3.79
(s, 3H), 3.69 (m, 2H), 2.69 (t, J ) 6.8 Hz, 1H). 13C NMR (CDCl3
+ CD3OD) δ 23.7, 55.7, 56.6, 60.2, 60.4, 72.7, 93.4, 96.6, 108.2,
109.7, 118.3, 122.9, 132.4, 141.9, 142.9, 143.3, 152.3, 159.6, 187.4.
HRMS Calcd for C22H22N2O6: 410.1478. Found 420.1482.

Antitubulin Tumor Cell Cytotoxins
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1005
(7-(5-Hydroxypent-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (7g). Compound 7g was obtained
as a light yellow solid in 71% yield. H NMR (DMSO-d6) δ 14.0
(s, NH), 8.16 (d, J ) 8.8 Hz, 1H), 7.70 (s, 2H), 7.21 (d, J ) 8.8
Hz, 1H), 3.93 (s, 3H), 3.87 (s, 6H), 3.79 (s, 3H), 3.57 (t, J ) 6.4
Hz, 2H), 2.59 (t, J ) 7.2 Hz, 2H), 1.79 (m, 2H). 13C NMR
(CDCl3+CD3OD) δ 16.6, 30.8, 56.1, 56.9, 60.8, 61.0, 72.4, 93.7,
99.1, 108.4, 110.0, 118.4, 123.1, 132.6, 142.3, 143.3, 143.7, 152.6,
159.6, 187.5. HRMS Calcd for C23H24N2O6: 424.1634. Found
424.1636.
procedures were carried out as for compound 8. Compound 11 (128
mg 1 equiv) and tetrabutylammonium fluoride solution in THF (1
M, 0.75 mL) were used as starting material to yield compound 12
(83, mg 83%). 1H NMR (CDCl3) δ 12.75 (s, NH), 8.24 (d, J ) 8.8
Hz, 1H), 7.61 (s, 2H), 7.04 (d, J ) 8.8 Hz, 1H), 8.67 (d, J ) 11.2
Hz, 1H), 6.10 (m, 1H), 4.06 (d, J ) 7.6 Hz, 2H), 3.90 (s, 3H),
3.88 (s, 9H), 3.36 (br, s, HO). 13C NMR (CDCl3) δ 56.4, 56.8,
59.1, 60.9, 106.1, 108.4, 109.9, 118.9, 123.6, 126.1, 129.2, 132.8,
140.6, 142.5, 143.8, 152.8, 157.0, 187.5. HRMS Calcd for
C21H22N2O6: 398.1478. Found 398.1482.
1
(6-Methoxy-7-trimethylsilylethynyl-1H-indazol-3-yl)(3,4,5-tri-
methoxyphenyl)methanone (7h). Crude 7h (0.43 g, 98%) was
obtained and used for the preparation of compound 8 without further
purification.
2-(3-(6-Methoxy-3-(3,4,5-trimethoxybenzoyl)-1H-indazole-7-
yl)prop-2-ynyl)isoindoline-1,3-dione (7i). Compound 6 (117 mg
1 equiv) and N-2-propynylphthalimide (93 mg 2 equiv) were reacted
to yield compound 7i (70 mg, 59%). 1H NMR (DMSO-d6) δ 14.06
(s, NH), 8.21 (d, J ) 9.2 Hz, 1H), 7.96 (m, 2H), 7.89 (m, 2H),
7.67 (s, 2H), 7.23 (d, J ) 8.8 Hz, 1H), 4.76 (s, 2H), 3.94 (s, 3H),
3.84 (s, 6H), 3.77 (s, 3H).
(7-Ethynyl-6-methoxy-1H-indazol-3-yl)(3,4,5-trimethoxyphe-
nyl)methanone (8). To a solution containing crude compound 7h
(430 mg), water (0.5 mL), and THF (9.5 mL) was added
tetrabutylammonium fluoride solution in THF (1 M, 3.0 mL) at 0
°C. The mixture was stirred from 0 °C to rt 4 h. After the solvent
was removed under reduced pressure, DCM (10 mL) was added.
The organic phase was washed with water, dried over Na2SO4, and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel to give compound 8 (260 mg, 72%). 1H NMR
(DMSO-d6) δ 14.07 (s, NH), 8.23 (d, J ) 8.8 Hz, 1H), 7.67 (s,
2H), 7.25 (d, J ) 8.8 Hz, 1H), 4.72 (s, 1H), 3.96 (s, 3H), 3.86 (s,
6H), 3.79 (s, 3H). 13C NMR (CDCl3) δ 56.3, 57.0, 60.9, 75.3, 86.6,
91.5, 108.5, 109.9, 118.4, 125.0, 132.3, 142.6, 143.0, 144.6, 152.8,
161.2, 187.0. HRMS Calcd for C20H18N2O5: 366.1216. Found
366.1219.
(7-(3-Hydroxypropyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-tri-
methoxyphenyl)methanone (13). A 7 mg amount of PtO2 was
added to a solution of 69 mg of compound 11 in AcOEt (12 mL),
purged with hydrogen thrice, stirred under hydrogen at rt overnight,
and filtered. The filtrate was concentrated under reduced pressure,
and the residue was then treated with a solution of THF (95%, 4
mL) and tetrabutylammonium fluoride solution (1 M in THF, 0.3
mL) at 0 °C for 4 h. After the solvent was removed under reduced
pressure, DCM (7 mL) was added. The organic phase was washed
with water, dried over Na2SO4, and concentrated under reduced
pressure. The residue was chromatographed on silica gel to give
compound 13 (34 mg, 85%). 1H NMR (CDCl3) δ 11.77 (br, s, NH),
8.25 (d, J ) 8.8 Hz, 1H), 7.70 (s, 2H), 7.09 (d, J ) 8.8 Hz, 1H),
3.96 (s, 3H), 3.94 (s, 3H), 3.93 (s, 6H), 3.63 (m, 2H), 3.11 (t, J )
6.4 Hz, 2H), 1.94 (m, 2H). 13C NMR (CDCl3) δ 20.0, 31.1, 56.3,
56.8, 60.87, 60.97, 108.4, 109.6, 100.6, 118.6, 121.4, 132.9, 142.4,
142.9, 144.0, 152.8, 156.3, 197.5. HRMS Calcd for C21H24N2O6:
400.1634. Found 400.1631.
(7-Iodo-6-Methoxy-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-
methanone (15). Starting material 14 was synthesized based on
the literature method,5 and 15 was prepared in a similar way as
compound 6, except the starting material was 14 and was obtained
as light yellow solid at a 71% yield. 1H NMR (CDCl3) δ 8.79 (br,
s, NH), 8.26 (d, J ) 8.4 Hz, 1H), 7.67 (d, J ) 2.8 Hz, 1H), 7.10
(s, 2H), 6.93 (d, J ) 8.4 Hz, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.88
(s, 6H).
(7-(3-Aminoprop-1-ynyl)-6-methoxy-1H-indazol-3-yl)(3,4,5-
trimethoxyphenyl)methanone (9). N2H4·H2O (0.2 mL) was added
to a solution of compound 7i (100 mg) in a mixed solvent (MeOH
(10 mL)/THF (3 mL)) at rt. The mixture was refluxed for 3 h. After
removal of solvent under vacuum, the residue was purified by flash
chromatography on silica gel to yield compound 9 (67 mg, 81%).
1H NMR (CDCl3 + CD3OD) δ 8.23 (d, J ) 9.2 Hz, 1H), 7.67 (s,
2H), 6.98 (d, J ) 9.2 Hz, 1H), 3.95 (s, 3H), 3.88 (s, 6H), 3.87 (s,
3H), 3.77 (s, 2H), 3.4 (br, s, NH2). 13C NMR (CDCl3+CD3OD) δ
31.8, 56.0, 56.8, 60.7, 75.1, 92.7, 97.4, 108.4, 109.6, 118.5, 123.8,
132.6, 142.2, 143.3, 143.7, 152.6, 159.5, 187.5. HRMS Calcd for
C21H21N3O5: 395.1481. Found 395.1479.
(6-Methoxy-7-(prop-1-ynyl)-1H-indol-3-yl)(3,4,5-trimethox-
yphenyl)methanone (16). The same reaction procedures were
carried out as for compound 7a, and compound 16 was obtained
as light yellow sold in 65% yield. 1H NMR (CDCl3) δ 8.89 (br, s,
NH), 8.25 (d, J ) 8.8 Hz, 1H), 7.67 (d, J ) 2.4 Hz, 1H), 7.11 (s,
2H), 6.98 (d, J ) 8.8 Hz, 1H), 3.99 (s, 3H), 3.94 (s, 3H), 3.90 (s,
6H), 2.22 (s, 3H).
In Vitro Proliferation Assay. Exponentially growing cells were
seeded at a density ranging from 4000 to 6000 cells per well in
96-well plates and incubated at 37 °C in 5% CO2, 95% air, and
100% relative humidity for 24 h prior to addition of compounds.
The next day, the cell population for each cell line at the time of
drug addition (T0) was measured using the AlamarBlue assay.
Compounds were solubilized in 100% DMSO at 200 times the
desired final test concentration. At the time of drug addition,
compounds were further diluted to 4 times the desired final
concentration with complete medium. Following drug addition, the
plates were incubated for 72 h at 37 °C, 5% CO2, 95% air, and
100% relative humidity. At the end of incubation, the viable cells
were quantified using AlamarBlue. Growth inhibition of 50% (IC50)
was calculated using Prism software at each of the drug concentra-
tions.
Tubulin Binding. 7b at different concentrations was incubated
with purified tubulin (bovine brain, MCF-7 cells, or Hela cells)
for 10 min at 37 °C. [3H]Colchicine was then added to the samples
and incubated for an additional 30 min at 37 °C. To separate the
bound from unbound [3H]colchicine, the samples were spotted onto
DE81 filter and washed three times to remove unbound [3H]-
colchicine. The bound colchicine on the filter was measured using
a scintillation counter.
In Vitro Tubulin Polymerization Assay. In Vitro tubulin
polymerization assays were conducted with reagents as described
by the manufacturer (Cytoskeleton Inc.). In brief, 7b was incubated
with purified bovine tubulin and buffer containing 10% glycerol
(7-(3-tert-Butyldimethylsilanyloxy-prop-1-ynyl)-6-methoxy-
1H-indazol-3-yl)(3,4,5-trimethoxyphenyl)methanone (10). The
same reaction procedures were carried out as for the compounds
7b-i. Compound 6 (468 mg 1 equiv) and tert-butyldimethyl(prop-
2-ynyloxy)silane (425 mg 2.5 equiv) were used as starting materials
1
to yield compound 10 (460 mg, 90%). H NMR (CDCl3) δ 10.6
(br, s, NH), 8.35 (d, J ) 9.2 Hz, 1H), 7.72 (s, 2H), 7.07 (d, J )
9.2 Hz, 1H), 4.71 (s, 2H), 4.02 (s, 3H), 3.96 (s, 6H), 3.95 (s, 3H),
0.97 (s, 9H), 0.22 (s, 6H).
(Z)-(7-(3-tert-Butyldimethylsilanyloxy-prop-1-enyl)-6-methoxy-
1H-indazol-3-yl)(3,4,5-trimethoxyphenyl)methanone (11). A 20
mg amount of 10% Pd/C was added to a solution of 180 mg of
compound 10 in AcOEt (25 mL) under a nitrogen atmosphere, and
the mixture was then purged with hydrogen thrice, stirred under
hydrogen at rt overnight, and filtered. The filtrate was concentrated
under reduced pressure, and the residue was purified with flash
chromatography on silica gel to yield compound 11 (123 mg, 68%).
1H NMR (CDCl3) δ 12.5 (s, NH), 8.43 (d, J ) 8.8 Hz, 1H), 7.93
(s, 2H), 7.09 (d, J ) 8.8 Hz, 1H), 6.83 (d, J ) 11.2 Hz, 1H), 6.09
(m, 1H), 4.23 (d, J ) 8.8 Hz, 2H), 3.98 (s, 3H), 3.97 (s, 6H), 3.96
(s, 3H), 1.03 (s, 9H), 0.20 (s, 6H).
(Z)-(7-(3-Hydroxyprop-1-enyl)-6-methoxy-1H-indazol-3-yl)-
(3,4,5-trimethoxyphenyl)methanone (12). The same reaction

1006 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Duan et al.
A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes
of potent antitubulin agents. J. Med. Chem. 2004, 47, 4247-4257.
(6) Provot, O.; Giraud, A.; Peyrat, J.; Alami, M.; Brion, J. Synthetic
approach to enyne and enediyne analogues of anticancer agents.
Tetrahedron Lett. 2005, 49, 8547-8550.
(7) Idiya, G. F.; Nataliya, A. Z. On the cyclization of ortho-alkynylben-
zene diazonium salts. Tetrahedron Lett. 2005, 29, 5453-5455.
(8) Beauregard, D. A.; Pedley, R. B.; Hill, S. A.; Brindle, K. M.
Differential sensitivity of two adenocarcinoma xenografts to the anti-
vascular drugs combretastatin A4 phosphate and 5,6-dimethylxan-
thenone-4-acetic acid, assessed using MRI and MRS. NMR Biomed.
2002, 15, 99-105.
(9) Atalay, C.; Deliloglu, G. I.; Irkkan, C.; Gunduz, U. Multidrug
resistance in locally advanced breast cancer. Tumor Biol. 2006, 27
(6), 309-318.
(10) Harker, W. G.; Sikic, B. I. Multidrug (pleiotropic) resistance in
doxorubicin-selected variants of the human sarcoma cell line MES-
SA. Cancer Res. 1985, 45, 4091-4096.
(11) Kochevar, J. Blockage of autonomous growth of ACHN cells by
anti-renal cell carcinoma monoclonal antibody 5F4. Cancer Res.
1990, 50, 2968-2972.
(12) Hipfner, D. R.; Gauldie, S. D.; Deeley, R. G.; Cole, S. P. Detection
of the M(r) 190,000 multidrug resistance protein, MRP, with
monoclonal antibodies. Cancer Res. 1994, 54, 5788-5792.
(13) Hamel, E. Evaluation of antimitotic agents by quantitative compari-
sons of their effects on the polymerization of purified tubulin. Cell
Biochem. Biophys. 2003, 38, 1-21.
(14) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca., S.; Genazzani,
A. A. Medicinal chemistry of combrestatin A4: present and future
directions. J. Med. Chem. 2006, 49 (11), 3033-3044.
and 1 mM GTP at 37 °C, and the effect of 7b on tubulin
polymerization was monitored kinetically using a fluorescent plate
reader.
Supporting Information Available: Characterization (1H NMR,
13C NMR, and HRMS data) of all the new compounds and the
purities of all the new compounds from normal-phase HPLC and
reverse-phase HPLC analysis. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Bacher, G.; Beckers, T.; Emig, P.; Klenner, T.; Kutscher, B.; Nickel,
B. New small-molecule tubulin inhibitors. Pure Appl. Chem. 2001,
73 (9), 1459-1464.
(2) Nam, N.-H. Combretastatin A-4 analogues as antimitotic antitumor
agents. Curr. Med. Chem. 2003, 10, 1697-7222.
(3) (a) Pettit, G. R.; Temple, C., Jr.; Narayanan.; L.; Varma, R.; Simpson,
M. J.; Boyd, M. R.; Rener, G. A.; Bansal, N. Antineoplastic agents
322. Synthesis of combretastatin A-4 prodrugs. Anticancer Drug Des.
1995, 10, 299-309. (b) Pettit, G. R.; Rhodes, M. R. Antineoplastic
agents 389. New syntheses of the combretastatin A-4 prodrug.
Anticancer Drug Des. 1998, 13 (3), 183-191.
(4) Liou, J.-P.; Chang. C.-W.; Song, S.; Yang, Y.-N.; Yeh, C.-F.; Tseng,
H.-T.; Lo, Y.-K.; Chang, Y.-L.; Chang, C.-M.; Hsieh, H.-P. Synthesis
and structure-activity relationship of 2-aminobenzophenone deriva-
tives as antimitotic agents. J. Med. Chem. 2002, 45, 2556-2567,
and references cited there.
(5) Liou, J.-P.; Chang, Y.-L.; Kuo, F.-M.; Chang, C.-W.; Tseng, H.-H.;
Wang, C.-C.; Yang, Y.-N.; Chang, J.-Y.; Lee, S.-J.; Hsieh, H.-P.
Concise synthesis and structure-activity relationships of cobrestatin
JM061348T