Design, synthesis, and biological evaluation of (E)-N-Aryl-2- arylethenesulfonamide analogues as potent and orally bioavailable microtubule-targeted anticancer agents

Article abstract of DOI:10.1021/jm400575x

A series of novel (E)-N-aryl-2-arylethenesulfonamides (6) were synthesized and evaluated for their anticancer activity. Some of the compounds in this series showed potent cytotoxicity against a wide spectrum of cancer cell-lines (IC₅₀ values ranging from 5 to 10 nM) including all drug resistant cell-lines. Nude mice xenograft assays with compound (E)-N-(3-amino-4- methoxyphenyl)-2-(2′,4′,6′-trimethoxyphenyl)ethenesulfonamide (6t) showed dramatic reduction in tumor size, indicating their in vivo potential as anticancer agents. A preliminary drug development study with compound 6t is predicted to have increased blood-brain barrier permeability relative to many clinically used antimitotic agents. Mechanistic studies indicate that 6t and some other analogues disrupted microtubule formation, formation of mitotic spindles, and arrest of cells in mitotic phase. Compound 6t inhibited purified tubulin polymerization in vitro and in vivo and circumvented drug resistance mediated by P-glycoprotein. Compound 6t specifically competed with colchicine binding to tubulin and with similar avidity as podophylltoxin, indicating its binding site on tubulin.

Full text of DOI:10.1021/jm400575x

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Article
Design, Synthesis and Biological Evaluation of (E)-N-
Aryl-2-arylethene-sulfonamide Analogues as Potent and
Orally Bioavailable Microtubule-targeted Anticancer Agents
M.V. Ramana Reddy, Muralidhar R Mallireddugari, Venkat R Pallela, Stephen C
Cosenza, Vinaykumar Billa, Balaiah Akula, D.R.C. Venkata Subbaiah, Earla Vijaya
Bharathi, Amol padgaonkar, Hua Lv, James M. Gallo, and E. Premkumar Reddy
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400575x • Publication Date (Web): 10 Jun 2013
Downloaded from http://pubs.acs.org on June 12, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Design, Synthesis and Biological Evaluation of (E)ꢀNꢀArylꢀ2ꢀaryletheneꢀ
sulfonamide Analogues as Potent and Orally Bioavailable Microtubuleꢀ
targeted Anticancer Agents
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2#
2#
M. V. Ramana Reddy *, Muralidhar R. Mallireddigari , Venkat R. Pallela , Stephen C.
1#
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2
1
1
Cosenza , Vinay K. Billa , Balaiah Akula , D. R. C. Venkata Subbaiah , E. Vijaya Bharathi ,
Amol Padgaonkar , Hua Lv , James M. Gallo and E. Premkumar Reddy *
1
3
3
1
1
Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison
Ave., New York, NY 10029ꢀ6514, United States
2
Department of Medicinal Chemistry, Onconova Therapeutics Inc., 375 Pheasant Run,
Newtown, PA 18940ꢀ3423, United States
3
Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount
Sinai, One Gustave L. Levy Place, Box 1603, New York, NY 10029ꢀ6514, United States
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ABSTRACT
A series of novel (E)ꢀNꢀarylꢀ2ꢀarylethenesulfonamides (6) were synthesized and evaluated for
their anticancer activity. Some of the compounds in this series showed potent cytotoxicity
against a wide spectrum of cancer cellꢀlines (IC values ranging from 5 to 10 nM) including all
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drug resistant cellꢀlines. Nude mice xenograft assays with compound (E)ꢀNꢀ(3ꢀAminoꢀ4ꢀ
methoxyphenyl)ꢀ2ꢀ(2',4',6'ꢀtrimethoxyphenyl)ethenesulfonamide (6t) showed dramatic reduction
in tumor size indicating their in vivo potential as anticancer agents. A preliminary drug
development study with compound 6t is predicted to have increased bloodꢀbrain barrier
permeability relative to many clinically used antiꢀmitotic agents. Mechanistic studies indicate
that 6t and some other analogs disrupted microtubule formation, formation of mitotic spindles
and arrest of cells in mitotic phase. Compound 6t inhibited purified tubulin polymerization in
vitro and in vivo and circumvented drug resistance mediated by Pꢀglycoprotein. Compound 6t
specifically competed with colchicine binding to tubulin and with similar avidity as
podophylltoxin indicating its binding site on tubulin.
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INTRODUCTION
Microtubules, formed by αꢀ and βꢀtubulin heterodimers, are essential constituents of the
cytoskeleton in eukaryotic cells and are involved in a number of important structural and
regulatory functions, including the maintenance of cell shape, intracellular transport machinery,
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as well as cell growth and mitosis. Their highly ordered structures, rigidity, and their ability to
grow and shrink via polymerization/depolymerization mechanisms are critical to their function in
several cellular processes. Perhaps the most important role of microtubules is during mitosis,
where they serve to organize and segregate chromosomes. Tubulin is the major structural
component of the microtubules and a well verified target for a variety of highly successful
2
3
anticancer drugs. Thus, vinca alkaloids 1 (vincristine and vinblastine) have been successfully
used for the therapy of hematological disorders for the past three decades and the seminal
4
discovery of paclitaxel (Taxol) by Wani and Wall in 1971 had a profound impact on the
treatment of breast and ovarian cancers. The success of these agents has also led to the
identification of several new tubulin interactive agents that have found application in cancer
5
chemotherapy. Based on the mechanism of action of alternation of microtubule dynamics, drugs
can be classified into two categories. Amongst the compounds (Chart 1) that inhibit tubulin
polymerization and destabilize microtubules are the combretastatins (2), colchicine (3),
Plinabulin (NPIꢀ2358) (4), podophyllotoxin (5), curcumin and the vinca (1) alkaloids, and those
that promote the polymerization of tubulins and stabilize the microtubules in their polymerized
state include discodermolide, eleutherobins, the epothilones, laulimalide, the sarcodictyins, and
6
the taxanes. Both microtubule stabilizers and destabilizers alter the tubulinꢀmicrotubule
7
equilibrium causing mitotic arrest at G2/M phase and ultimately apoptotic cell death. Because of
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the clinical success of microtubuleꢀaffecting compounds such as paclitaxel, the vinca alkaloids,
and epothilone derivatives in the treatment of a wide variety of cancers, it has been argued that
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microtubules represent the single most important protein target for anticancer therapy.
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OH
MeO
MeO
MeO
N
A
NHCOMe
O
N
MeO
B
OAc
HO
OMe
R1
MeO
N
H
CO Me
OMe
MeO C
N
H
2
2
MeO
1a. vincristine R1 = CHO
b. vinblastine R1 = Me
R1
2a. combretastatin R1 = OH (CA4)
b. R = OP(O)(ONa)2 (CA4P)
1
OMe
2
2
3. colchicine
c. R = NHCOCHNH CH OH (AVE-8062)
1
2
2
1
OH
O
MeO
MeO
O
O
O2
S
N
H
O
NHCOMe
O
N
NH
N
NH
HN
NH
O
MeO
MeO
O
P
OH
O
HO
MeO
OMe
OH
OMe
4
. plinabulin (NPI-2358)
6. ZD6126
7
. ABT-751
5. podophyllotoxin
OMe
OMe
F
OMe
OH
F
F
O2
S
OMe
OMe
O2
S
F
F
O2
S
H CO
3
N
N
H
H
N
H
F
H CO
3
N
OCH3
Me
10
8
. T-138067
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Chart 1. Structures of microtubule depolymerizing agents
These antimitotic drugs, however, are not without limitations. Many, including paclitaxel and the
vinca alkaloids, are large (MW>700Da) natural products that display ADMEꢀTox shortcomings
(including poor water solubility, bioavailability, and significant doseꢀlimiting toxicity). In
addition, a common problem observed with this class of compounds is that these large natural
products are substrates for efflux pumps of the ABC transporter family, such as Pꢀglycoprotein
(Pꢀgp) and multidrug resistance (MDR) proteins that can alter their pharmacokinetic
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characteristics. Furthermore, drugs such as taxanes are typically poor chemotherapeutics for the
treatment of many brain cancers, as high levels of Pꢀgp in the bloodꢀbrain barrier (BBB) and the
chemical properties of the molecules themselves prevent significant accumulation of drug in the
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brain. Because of these factors, there has been an intense search for more effective antimitotics.
1
1
A variety of synthetic small molecules have also been reported as inhibitors of polymerization,
1
2
which compete with the colchicineꢀbinding site of tubulin. While no colchicineꢀsite binders are
currently approved for cancer chemotherapy, compounds like combretastatin Aꢀ4P (CA4P)
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(
2b), AVEꢀ8062 (2c), ZD6126 (6),
ABTꢀ751(7), Tꢀ138067 (8), Nꢀ(3ꢀHydroxyꢀ4ꢀ
1
8
methoxyphenyl)ꢀ3,4,5ꢀtrimethoxybenzenesulfonamide (9) and 1ꢀMethylꢀ1Hꢀindoleꢀ5ꢀsulfonic
1
9
acid (3,4,5ꢀtrimethoxyphenyl)amide (10) are now under clinical investigation as potential new
20
chemotherapeutic agents (Chart 1). However, a report of the activity and SAR information for
these compounds, especially in vivo efficacy, is limited.
Hence herein we report the synthesis, in vitro evaluations, cell cycle progression and structure
activity relationship (SAR) of (E)ꢀNꢀarylꢀ2ꢀarylethenesulfonamides, which cause cell death
through destabilization of microtubules. In this study, we also report the caspase activation and
tubulin depolymerization study along with bloodꢀbrain barrier (BBB) permeability of the active
compound (E)ꢀNꢀ(3ꢀAminoꢀ4ꢀmethoxyphenyl)ꢀ2ꢀ(2',4',6'ꢀtrimethoxyphenyl)ethenesulfonamide
(
6t).
CHEMISTRY
The syntheses of (E)ꢀNꢀarylꢀ2ꢀarylethenesulfonamides (6) were achieved by multiple synthetic
routes as illustrated in Schemes 1, 5 & 6. The initial method involved for the synthesis of 6 by
the condensation of chlorosulfonylacetic acid ethyl ester (2) with various anilines 1 in the
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presence of triethylamine in DCM to obtain the arylsulfamoylacetic acid ethyl esters (3) in high
yields. Hydrolysis of 3 with 10% NaOH in water afforded the corresponding arylsulfamoylacetic
acids (4) in good yields. Knovenagel condensation of 4 with various aromatic aldehydes 5 either
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in benzylamine/acetic acid or piperidine/benzoic acid in toluene afforded 6 in good yields
(Scheme 1). All 3ꢀnitro and 3,5ꢀdinitro substituted arylethenesulfonamides were reduced to their
Scheme 1. Synthesis of (E)ꢀNꢀArylꢀ2ꢀarylethenesulfonamides (6) from NꢀArylsulfonylacetic
a
Acids
H
O
N
O
NH2
O2
S
S
b
a
R
O
2
R
+
Cl
O
O
2
1a-k
3a-k
O
H
H
N
R1
N
OH
+ R1
S
H
c or d
S
R
O
2
R
O
O
2
4a-k
6
5
1a R= H
1g R = 3-NO 4-OCH
3
2
,
1b R= 4-Cl
1c R = 4-F
1h R = 2,3,4,5,6-F5
1i R = 3-NO ,4-F
2
1d R = 4-OCH3
1e R = 3-F,4-OCH3
1f R = 2,4,6-(OCH3)3
1j R = 3,5-(NO ) ,4-OCH
1k R = 3-OH,4-OCH3
2 2
3
a
Reagents and conditions: (a) Et N, DCM, room temp, 3 h, 80%; (b) 10% NaOH, H O, room temp, 3 h,
3
2
8
4%; (c) C H CH NH , AcOH, reflux, 8 h, 50%; (d) piperidine, benzoic acid, toluene,reflux, 4 h, 60%.
6 5 2 2
corresponding amino analogs either with sodium hydrosulfite in acetone/water or with iron
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powder in methanol/acetic acid as shown in the scheme 2. The substituted anilines 1g and 1j
which are not available commercially were synthesized as shown in the Scheme 3. 1g was
prepared by the treatment of 4ꢀfluoroꢀ3ꢀnitroꢀaniline (7) with 6% methanolic KOH in absolute
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methanol at room temperature to afford the required 1g in moderate yields. The dinitro aniline 1j
Scheme 2. Conversion of 3ꢀNitro and 3,5ꢀDinitro NꢀArylꢀ2ꢀarylethenesulfonamides to
a
corresponding Amines (6)
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H
R1
H
R1
N
N
a or b
S
S
R
R
O
2
O
2
6
s
R = 3-NO ,4-OCH ; R = 2,4,6-(OCH )
6t R = 3-NH ,4-OCH ; R = 2,4,6-(OCH )
2 3 1 3 3
6v R = 3-NH ,4-OCH ; R = 3,4,5-(OCH )
2
3
1
3 3
6u R = 3-NO ,4-OCH ; R = 3,4,5-(OCH )
2
3
1
3 3
2 3 1 3 3
6w R = 3-NO ,4-OCH ; R = 2,6-(OCH ) ,4-O(CH ) COOH 6x R = 3-NH ,4-OCH ; R = 2,6-(OCH ) ,4-O(CH ) COOH
2
3
1
3 2
2 3
2
3
1
3 2
2 3
6z R = 3-NO ,4-F; R = 2,4,6-(OCH )
6aa R = 3-NH ,4-F; R = 2,4,6-(OCH )
2 1 3 3
6ac R = 3,5-(NH ) ,4-OCH ; R = 2,4,6-(OCH )
2 2 3 1 3 3
6ag R = 3-NH ,4-OCH ; R = 2,3,4,5,6-F
2 3 1 5
6ai R = 2,3,4,5,6-F ; R = 3-NH ,4-OCH
5 1 2 3
2
1
3 3
6ab R = 3,5-(NO ) ,4-OCH ; R = 2,4,6-(OCH )
af R = 3-NO ,4-OCH ; R = 2,3,4,5,6-F
ah R = 2,3,4,5,6-F ; R = 3-NO ,4-OCH
2
2
3
1
3 3
6
6
2
3
1
5
5
1
2
3
a
0
Reagents and conditions: (a) Sodium hydrosulfite, acetone / water (2:1), 50 C, 30 min, 40%;
0
(
b) Iron powder, MeOH / AcOH (2:1), 80 C, 2 h, 55%.
was synthesized starting from 4ꢀfluorobenzoic acid (8) by nitration with fuming nitric acid and
0% sulfuric acid to get 3,5ꢀdinitroꢀ4ꢀfluorobenzoic acid (9) which on treatment with 20% oleum
3
and ethylene dichloride in presence of sodium azide afforded 3,5ꢀdinitroꢀ4ꢀfluoro aniline (10).
Treatment of 10 with 6% methanolic KOH in absolute methanol gave the required 3,5ꢀdinitroꢀ4ꢀ
24
methoxy aniline (1j) in moderate yields (Scheme 3).
Commercially not available
chlorosulfonylacetic acid ethyl ester (2) was in turn made as shown in Scheme 4. Treatment of
Scheme 3. Synthesis of 4ꢀMethoxyꢀ3ꢀnitro Aniline (1g) and 4ꢀMethoxyꢀ3,5ꢀdinitrophenyl
a
Amine (1j)
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NH2
NH2
a
NO2
NO2
F
OCH3
7
1
g
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O
OH
O
OH
NH2
NH2
b
c
a
O N
NO2
O
2
N
NO
2
O N
NO2
2
2
F
F
F
OCH3
8
10
1j
9
a
Reagents and conditions: (a) absolute methanol, 6% methanolic KOH, room temp, 30 min, 50%; (b) fuming
0
H SO (30%), HNO (90%), 95 C, 3 h, 70%; (c) fuming H SO (20%), ClCH CH Cl, NaN , reflux, 1 h,
2
4
3
2
4
2
2
3
7
0%.
ethyl bromoacetate (11) with sodium sulfite gave sodium ethoxycarbonylmethanesulfonate (12)
0
25
which on reaction with PCl at 100 C resulted in 2 in moderate yields. The 2 was also prepared
5
directly by the esterification of chlorosulfonyl acetyl chloride (13) with absolute ethanol in
2
6
diethyl ether in 55% yield.
a
Scheme 4. Synthesis of Chlorosulfonylacetic acid ethyl ester (2)
a
O
Na
O
O
Br
S
O
2
O
O
11
12
b
O
O
O2
S
O2
S
c
Cl
Cl
Cl
O
13
2
a
0
Reagents and conditions: (a) Na SO , H O, EtOH, 50 C, 30 min, 70 %;
2
3
2
0
0
(
b) PCl , 100 C, 45 min, 85%; (c) EtOH, diethyl ether, 0 C, 3 h, 55%.
5
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Alternatively, the sulfonamides 6 were also prepared by acylation of the acids 4d, 4g or 4k by
thionyl chloride in DCM to get the corresponding acid chlorides 14aꢀc which on FriedelꢀCrafts
acylation with 1,3,5ꢀtrimethoxy benzene (15) afforded 2ꢀarylꢀ2ꢀoxoethanesulfonic acid aryl
amides (16aꢀc). Sequential reduction of 16 with sodium borohydride and subsequent dehydration
with pꢀtoluenesulfonic acid (pꢀTSA) afforded the desired product 6 in moderate yields (Scheme
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
7
5
). To explore for the simple reaction conditions, less number of steps to obtain the targeted
compounds and better overall yields, sulfonamides 6 were also prepared by the condensation of
anilines (1aꢀc) with 2ꢀarylethenesulfonyl chlorides (19aꢀc) in the presence of triethylamine in
DCM in good yields. The sulfonyl chlorides 19 were in turn made by the addition of sulfuryl
2
7c
0
chloride to styrenes (18aꢀc) in DMF at 0 C to room temperature to get the desired product in
quantitative yields (Scheme 6). The method described in Scheme 6 is superior to the other two
methods as it involves less steps, cheaper chemicals and relatively higher yields.
Scheme 5. Synthesis of (E)ꢀNꢀArylꢀ2ꢀarylethenesulfonamides (6) from Phenacyl Nꢀ
a
Arylsulfones
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
H
N
OH
N
Cl
+
S
a
S
R
b
O
R
R1
2
O
2
O
O
1
4a-c
15
R = 1,3,5-(OCH )
4d,4g,4k
14a R = 4-OCH3
4b R= 3-NO ,4-OCH3
4c R= 3-OH,4-OCH3
1
3 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
H
R1
H
N
S
R1
N
d
S
c
6
R
O
R
2
O
2
O
OH
7a-c
16a, 17a R = 4-OCH ; R = 2,4,6-(OCH )
16a-c
1
3
1
3 3
16b, 17b R= 3-NO ,4-OCH ; R = 2,4,6-(OCH )
3 3
2
3
1
1
6c, 17c R= 3-OH,4-OCH ; R = 2,4,6-(OCH )
3 1 3 3
a
Reagents and conditions: (a) SOCl , DCM, room temp, 6 h, 81% or (COCl) , DMF, anhydrous CH Cl ,
2
2
2
2
room temp, 12 h, 67%; (b) AlCl , DCM, room temp, 4 h, 80%; (c) NaBH , THF, room temp, 3 h, 88%; (d)
3
4
p-TSA, toluene, reflux, 3 h, 78%.
Scheme 6. Synthesis of (E)ꢀNꢀArylꢀ2ꢀarylethenesulfonamides (6) from (E)ꢀStyryl sulfonyl
a
chloride
O2
NH2
S
Cl
R
R1
a
R1
+
1
8a-c
19a-c
1a-c
1
1
1
8a R = H
1
8b R = 4-Br
1
8c R = 4-CH O
1
3
H
R1
N
b or c
S
R
O
2
6
a
0
Reagents and conditions: (a) SO Cl , DMF, 0 C to room temp, 3 h, 90%; (b) Et N, DCM,
2
2
3
room temp, 5 h, 85%; (c) pyridine, room temp, 6 h, 80%.
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9
To enhance the bioavailability of the active sulfonamide (E)ꢀ2ꢀ(2,4,6ꢀtrimethoxyphenyl)ꢀ2ꢀ(3ꢀ
hydroxyꢀ4ꢀmethoxyphenyl)ethanesulfonamide (6p), we explored to make its ester analog 6q.
Condensation of 3ꢀ(2,5ꢀdimethylꢀ3,6ꢀdioxocyclohexaꢀ1,4ꢀdienyl)ꢀ3ꢀmethylbutanoic acid (20)
with 6p in the presence of EDAC and DMAP in dichloromethane resulted in ester (E)ꢀ2ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
methoxyꢀ5ꢀ(2ꢀ(2,4,6ꢀtrimethoxyphenyl)vinylsulfonamido)phenyl
2ꢀ(2,5ꢀdimethylꢀ3,6ꢀdioxoꢀ
cyclohexaꢀ1,4ꢀdienyl)ꢀ2ꢀmethylpropanoate (6q) in moderate yields (Scheme 7). The
benzoquinone ester 20 in turn was synthesized from 2,5ꢀDimethyl[1,4]benzoquinone (21) in
presence of 3,3ꢀdimethylacrylic acid, aqueous sodiumhydrosulfite and methane sulfonic acid in
0
diethyl ether at 85 C to yield 6ꢀHydroxyꢀ4,4,5,8ꢀtetramethylchromanꢀ2ꢀone (22) in 59% yield.
NꢀBromosuccinimide ring opening reaction of 22 afforded the target compound 20 in moderate
2
8
yields (Scheme 8).
Scheme 7. Synthesis of (E)ꢀ2ꢀMethoxyꢀ5ꢀ(2ꢀ(2,4,6ꢀtrimethoxyphenyl)vinylsulfonamido)phenyl
2ꢀ(2,5ꢀdimethylꢀ3,6ꢀdioxocyclohexaꢀ1,4ꢀdienyl)ꢀ2ꢀmethylpropanoate (6q)
O
O
O
O
O
O
O
OH
O
O
O
O
EDAC, DMAP, CH Cl2
2
+
SO2
O
rt, 12 h, 69%.
N
H
SO2
O
HO
N
H
O
6
q
6p
20
O
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 8. Synthesis of 3ꢀ(2,5ꢀdimethylꢀ3,6ꢀdioxocyclohexaꢀ1,4ꢀdienyl)ꢀ3ꢀmethylbutanoic acid
a
(20)
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
OH
O
a
b
OH
21
22
20
a
0
Reagents and conditions: (a) diethyl ether, aq. Na S O , (CH ) C=CHCOOH, CH SO H, 85 C,
2
2
4
3 2
3
3
3 h, 81%; (b) CH CN, acetone, water, NBS, room temp, 30 min, 60%.
3
To further enhance the bioavailability and water solubility of the active sulfonamide (E)ꢀ2ꢀ
2,4,6ꢀtrimethoxyphenyl)ꢀ2ꢀ(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)ethenesulfonamide 6p, its disodium
(
phosphate prodrug was synthesized in two steps as shown in the Scheme 9. Phosphorylation of
the phenolic group in 6p employing phosphorous oxychloride under basic conditions gave 3ꢀOꢀ
phosphate 23. Treatment of the phosphate 23 with 25% aq. sodium hydroxide in ethylene glycol
2
9
dimethyl ether yielded disodium Oꢀphosphate 6r (Scheme 9).
Scheme 9. Synthesis of Sodium (E)ꢀ2ꢀMethoxyꢀ5ꢀ(2ꢀ(2',4',6'ꢀtrimethoxyphenyl)vinylsulfonꢀ
a
amido)phenyl phosphate (6r)
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9
O
O
O
O
O
O
O
O
O
b
O
a
O
O
O
O
O
P
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
SO2
Cl
SO2
HO P
SO2
HO
N
H
N
N
H
Cl
H
OH
6p
23
O
O
O
O
O
c
O
NaO P
SO2
N
H
ONa
6r
a
0
Reagents and conditions: (a) phosphorus oxychloride, THF, triethylamine, 0 C-room temp, 3 h; (b) water,
0
room temperature,18 h, 69%; (c) 25% aq.NaOH, CH OCH CH OCH , 0 C-room temp, 3 h, 85%.
3
2
2
3
To further enhance the bioavailability and water solubility of the active sulfonamide 6t, several
ꢀ amino substituted esters and acids were made by the reaction of αꢀbromo esters in the
presence of mild base sodium acetate in ethanol to give amine esters (24) which on subsequent
3
3
0
hydrolysis with sodium hydroxide in ethanol afforded the corresponding acids 25 (Scheme 10).
Scheme 10. Synthesis of amine esters and acids of (E)ꢀNꢀ(3ꢀAminoꢀ4ꢀmethoxyphenyl)ꢀ2ꢀ
(
2',4',6'ꢀtrimethoxyphenyl)ethenesulfonamide (6t)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
Br-R -COOCH ,
2
3
O
O
O
O
NaOH, EtOH
O
O
O
CH COONa, EtOH, 48 h
O
0
3
O
60 C, 1 h
SO2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SO2
HO
R2
H CO
R2
SO2
3
N
H
N
H
N
H
N
H N
N
H
2
H
O
O
6t
2
4
25
StructureꢀActivity Relationships (SAR)
The newly synthesized compounds were tested for their in vitro cytotoxicity activity against two
different human cell lines derived from human prostate (DU145) and leukemic (K562) cancers.
The study results are presented in Table 1. These studies reveal that the cytotoxicity of the Nꢀ
arylꢀ2ꢀarylethenesulfonamide (6) is totally dependent on the nature and position of the
substituents present on the two aromatic rings. For the purpose of structureꢀactivity relationship,
we have selected few compounds from a library of more than 500 arylethenesulfonamides
synthesized in our laboratory. The cytotoxicity data (Table 1) clearly shows that the molecules
Table 1. In vitro Cytotoxicity of (E)ꢀNꢀArylꢀ2ꢀarylethenesulfonamides (6)
R1
R
SO2
N
H
a
IC (ꢀM)
5
0
Compd
R
R1
DU145
K562
6
6
6
a
b
c
H
H
10
20
10
20
4ꢀCl
4ꢀF
H
4ꢀBr
10
10
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6
d
e
4ꢀF
4ꢀOCH
2ꢀOCH
4ꢀOCH
3
3
3
10
5
10
5
6
4ꢀOCH
4ꢀOCH
4ꢀOCH
4ꢀOCH
4ꢀOCH
4ꢀOCH
3
3
3
3
3
3
6f
5
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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45
46
47
48
49
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52
53
54
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56
57
58
59
60
6g
2,4ꢀ(OCH
2,6ꢀ(OCH
3
3
)
2
2
15
15
5
6
6
6
h
)
0.375
0.2
35
i
2,4,6ꢀ(OCH
3,4,5ꢀ(OCH
3
3
)
)
3
0.075
35
2.5
j
3
6k
2,4,6ꢀ(OCH
3
)
3
4ꢀOCH
2,6ꢀ(OCH ) ,4ꢀ
OH
3
7.5
3
2
6
6
6
l
4ꢀOCH
4ꢀOCH
4ꢀOCH
3
3
3
10
75
10
15
m
n
2,4,6ꢀF
3
2,3,4,5,6ꢀF
2,4,6ꢀ(OCH
2,4,6ꢀ(OCH
10
5.0
5
6o
3ꢀF,4ꢀOCH
3
3
3
)
3
3
0.2
0.03
0.075
0.015
6p
3ꢀOH,4ꢀOCH
ꢀOCOCH C(CH ) ꢀ
3
)
3
2
3 2
C6H(CH ) O ,
4ꢀOCH
3ꢀOPO(ONa) ,4ꢀ
3
2
2
6q
3
2,4,6ꢀ(OCH
3
)
3
0.009
0.008
2
6r
OCH
3
2,4,6ꢀ(OCH
2,4,6ꢀ(OCH
2,4,6ꢀ(OCH
3,4,5ꢀ(OCH
3,4,5ꢀ(OCH
3
3
3
3
3
)
3
)
3
)
3
)
3
)
3
0.04
2.5
0.0075
1.0
6
6
6
s
3ꢀNO2,4ꢀOCH
3ꢀNH2,4ꢀOCH
3ꢀNO2,4ꢀOCH
3ꢀNH2,4ꢀOCH
3
3
3
3
t
0.005
75
0.003
75
u
6v
35
15
2
,6ꢀ(OCH ) ,4ꢀ
3 2
6w
3ꢀNO2,4ꢀOCH
3
O(CH
2
)
3
COOH
100
10
100
10
2
,6ꢀ(OCH ) ,4ꢀ
3
2
6x
3ꢀNH
2
,4ꢀOCH
3
O(CH
2
)
3
COOH
2,6ꢀ(OCH ) ,4ꢀ
3
2
6
6
6
y
3ꢀOH,4ꢀOCH
3
O(CH
2
)
3
COOH
10
100
10
10
75
5
z
3ꢀNO2,4ꢀF
2,4,6ꢀ(OCH
2,4,6ꢀ(OCH
3
3
)
3
3
aa
3ꢀNH2,4ꢀF
)
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
ab
ac
3,5ꢀ(NO
3,5ꢀ(NH
2
2
)
2
2
,4ꢀOCH
3
3
2,4,6ꢀ(OCH
2,4,6ꢀ(OCH
3
3
)
3
3
10
2.5
5
10
7.5
1
6
)
,4ꢀOCH
)
6ad
6ae
3ꢀF,4ꢀOCH
3ꢀF,4ꢀOCH
3
3
4ꢀOCH
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2,3,4,5,6ꢀF
2,3,4,5,6ꢀF
2,3,4,5,6ꢀF
10
10
100
75
75
35
35
5
5
5
6
6
6
af
3ꢀNO
2
2
,4ꢀOCH
3
3
100
75
ag
ah
3ꢀNH
,4ꢀOCH
2,3,4,5,6ꢀF
2,3,4,5,6ꢀF
2,3,4,5,6ꢀF
3ꢀNO
3ꢀNH
,4ꢀOCH
,4ꢀOCH
100
35
5
5
5
2
2
3
6ai
3
6aj
2,3,4,5,6ꢀF
5
35
a
IC values are the compound concentrations (ꢁM) required to inhibit cell
5
0
proliferation by 50% of tumor cells following 96 h treatment with the tested
compound; values represent the mean SD from the dose response curves of
two independent experiments and are within 5ꢀ10%.
are less active when the aromatic rings are without any substitution or mono substituted with
halogens or with a combination of a methoxy and a halogen atom (6aꢀ6d). The improvement in
the cytotoxicity profile was observed when each aromatic ring has a methoxy group on it (6e,
6
f). Based on this observation, additional methoxy groups were added on the styryl aromatic ring
keeping a methoxy group constantly at the 4th position of the aryl sulfamyl ring to evaluate the
influence of these groups on the cytotoxicity of the molecules (6gꢀ6i). From the cytotoxicity data
(
Table 1) it is clear that 2,4 and 2,6ꢀdimethoxy substitutions (6g, 6h) moderately enhanced the
potency of the molecules while 2,4,6ꢀtrimethoxy substitution (6i) enhanced the cytotoxicity
potency to several folds higher in the molecule. Altering the positions of the methoxy groups
from 2,4,6 to 3,4,5 (6j) resulted in the loss of the cytotoxicity indicating that the optimum
activity could be achieved only when the substitution pattern is 2,4,6 on the styryl aromatic ring.
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Also to evaluate the importance of the location of 2,4,6ꢀtrimethoxy groups on styryl aromatic
ring, we switched these methoxy groups on to sulfamyl aromatic ring and tested the resulting
molecule (6k) for the cytotoxicity. The loss of cytotoxicity activity in 6k clearly shows that the
trimethoxy groups not only should be on the styryl aromatic ring but also at 2,4,6ꢀ positions.
Further to make 6i more water soluble we replaced the methoxy group with a hydroxyl group at
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
th
the 4 position of the 2,4,6ꢀtrimethoxy styryl ring and the resulted molecule (6l) lost the activity
indicating that hydrophobic group at that position is required for the activity of the molecule.
When fluorine atoms are introduced in place of 2,4,6ꢀtrimethoxy groups on 6i, the resulting
molecules (6m, 6n) lost their cytotoxic potential on cancer cells showing that the methoxy
groups at those positions are critical for the activity of the molecule. After fixing the styryl
aromatic ring with 2,4,6ꢀtrimethoxy groups to attain the best activity, we then focused our
attention on to aryl sulfamyl ring of the molecule. Addition of a fluorine atom at the third
position of the sulfamyl ring in 6i retained the activity of the molecule (6o) indicating that the
meta position in the ring is vulnerable and can be used for introducing groups that enhance water
solubility and bioavailability without affecting the potency of the molecule. Replacing the
fluorine in 6o with a hydroxyl group resulted in a potent molecule (6p) that has 5ꢀ7 folds higher
cytotoxicity than 6o. Conversion of 6p into dimethyl quinone prodrug (6q) enhanced the
cytotoxicity of the molecule compared to 6p probably due to better cell permeability compared
to 6p. But the solubility of 6q in aqueous buffers has not much improved compared to 6p. Also
introduction of the hydroxyl group on the sulfamyl ring created the possibility of producing a
highly waterꢀsoluble disodium phosphate salt (6r) having more or less same potency as that of
rd
6p. Further attempts to enhance the cytotoxicity with an electron withdrawing nitro group at 3
position of the sulfamyl ring resulted in a molecule (6s) that is several folds less active than 6p,
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
q and 6r. Surprisingly reduction of the nitro group (6s) to an electron releasing amino group
resulted in the formation of the most active molecule (6t) of this series. Any other alterations and
modifications on 6t resulted in molecules (6uꢀ6z and 6aaꢀ6aj) with reduced or total loss of
cytotoxicity indicating that the best activity in Nꢀarylꢀ2ꢀarylethenesulfonamide series could be
obtained only when a molecule bears 3ꢀamino, 4ꢀmethoxy on the arylsulfamyl ring and 2,4,6ꢀ
trimethoxy groups on the styryl ring.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
After identifying the most potent molecule (6t) in the series, we made several modifications on
the amino group of 6t to make it more waterꢀsoluble and bioavailable. Alkylation of the metaꢀ
amino group in 6t with methyl bromoacetate and 2ꢀalkyl, aryl substituted bromoacetates
produced 3ꢀglycine esters (24aꢀg) which on hydrolysis resulted in acids (25aꢀg). All the glycine
analogs (25aꢀg) showed enhanced water solubility (10ꢀ20 mg/mL) and excellent cytotoxicity
(Table 2) compared to the glycine esters (24aꢀg).
Table 2. In vitro cytotoxicity of Amine esters (24) and Acids (25)
O
O
O
O
O
O
O
O
SO2
SO2
H CO
3
R2
HO
R2
N
N
N
N
H
H
H
H
O
O
24
25
a
IC (ꢀM)
5
0
Compd R2
DU145
K562
24a
CH
2
0.35
0.35
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0.1
0.30
2
4b
4c
CH(CH
3
)
2
C(CH
CH(C
CH(C
CH(C
CH(C
3
6
6
6
6
)
2
0.2
2.5
0.30
2.5
24d
24e
H
H
H
H
5
4
4
4
)
10
11
12
13
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15
16
17
18
19
20
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22
23
24
25
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4ꢀF)
4ꢀCl)
4ꢀBr)
2.5
2.5
2
2
2
4f
5.0
2.5
4g
5a
7.5
1.0
CH
CH(CH
0.4
0.02
0.008
0.005
2
25b
25c
3
)
0.04
0.07
C(CH
CH(C
CH(C
CH(C
CH(C
)
2
3
6
6
6
6
2
2
2
5d
5e
5f
H
H
H
H
5
4
4
4
)
0.075
0.075
0.30
0.02
4ꢀF)
4ꢀCl)
4ꢀBr)
0.075
0.25
0.015
0.075
25g
a
IC50 values are the compound concentrations (ꢁM)
required to inhibit cell proliferation by 50% of tumor
cells following 96 h treatment with the tested compound;
values represent the mean SD from the dose response
curves of two independent experiments and are within 5ꢀ
10%.
BIOLOGICAL RESULTS AND DISCUSSION
In vitro Antiꢀtumor Effects of 6i, 6p, 6t, and 25c Compounds. Screening of the novel
arylethenesulfonamide compound library of molecules for antiꢀtumor activity using cell
biological assays yielded several candidate molecules that induced doseꢀdependent growth
inhibition and death of tumor cells (Tables 1 & 2). To identify the arylethenesulfonamides with
the broadest range of activity in a wider selection of cancer cell types we chose four of the most
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
potent compounds from the series shown in Chart 2 and found high potency with similar GI50
values (selected data shown in Table 3). The appreciable cell killing across multiple tumor types
suggests that these compounds are inhibiting an intrinsically important process of tumor cell
division. 6t was found to be the most active of the four compounds exhibiting IC values of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
between 0.003 ꢀ 0.01 ꢁM (Table 3), and on that basis, we further investigated the mechanism of
action of this compound.
O
O
O
O
O
O
O
O
SO2
SO2
HO
N
N
H
H
6p
6i
O
O
O
O
O
O
O
O
HO
SO2
SO2
N
N
H
H N
2
N
H
H
O
6t
25c
Chart 2. Chemical structures of lead compounds
Table 3. In vitro cytotoxicity profiles of most active molecules 6i, 6p, 6t and 25c in various
tumor cell lines.
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a
IC (ꢁM)
5
0
Cell Line
Tumor Type
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
i
6p
6t
25c
0.15
0.01
0.10
N/D
N/D
0.08
N/D
0.15
0.10
N/D
0.15
N/D
0.20
N/D
N/D
N/D
0.03
N/D
N/D
0.08
N/D
N/D
N/D
0.03
0.03
0.09
DU145
K562
LNCAP
PC3
SKꢀMELꢀ28
MCFꢀ7
SK BRꢀ3
BT474
BT20
Prostate
Leukemia
Prostate
Prostate
Melanoma
Breast
Breast
Breast
Breast
Breast
Epidermoid
ColoꢀRectal
ColoꢀRectal
Ovarian
Ovarian
NSCLC
SCLC
Gastric
Gastric
Pancreatic
Glioma
Uterine
0.075
0.075
0.100
0.100
0.050
0.050
0.050
0.250
0.250
0.100
0.030
0.100
0.100
0.100
0.100
0.100
0.050
0.025
0.050
0.100
0.100
0.050
0.100
0.100
0.100
0.229
0.050
0.015
0.050
0.025
0.025
0.025
0.025
0.025
0.250
0.025
0.600
0.010
0.025
0.025
0.100
0.010
0.010
0.010
0.010
0.010
0.025
0.025
0.025
0.025
0.025
0.026
0.008
0.003
0.008
0.008
0.008
0.004
0.004
0.010
0.008
0.008
0.005
0.004
0.008
0.008
0.004
0.004
0.004
0.004
0.004
0.004
0.004
0.004
0.008
0.008
0.008
0.009
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
T47D
A431
HCT116
HCT15
SKOV3
OVCAR3
H187
N417
RFꢀ48
RFꢀ1
MIAPaCa2
H80
MESꢀSA
NAMALWA Lymphocytic
DAUDI
MOLTꢀ4
U87
Lymphocytic
Lymphocytic
b
Glioma
a
GI (growth inhibitory concentrationꢀ50%). Cells were treated with 6i, 6p, 6t and 25c in a 96 h
dose response assay. The GI concentration (ꢁM) was determined from duplicate cell counts using
a Hemocytometer following trypsinization and trypan blue staining. Values are IC as measured
5
0
5
0
b
5
0
by SRB assay. ND = not determined
Effects of 6t on Cell Cycle Progression of Tumor Cells. The effect of 6t on cell cycle
progression in DU145 human prostate cancer cells using fluorescenceꢀactivated cell sorting
(
FACS) analysis indicated that the addition of 6t to tumor cells resulted in gradual accumulation
of cells in the G2/M phase of the cell cycle in a concentrationꢀdependent manner. Figure 1 shows
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5
5
5
6
that tumor cells accumulated in the G2/M phase of the cell cycle at 0.005 ꢁM concentration of
6t, and a majority of cells showed G2/M arrest at a 0.02 ꢁM concentration, whereas a normal
cell cycle distribution was seen in the vehicle treated cells. In addition, treatment of the tumor
0
1
2
3
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7
8
9
0
1
2
3
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1
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8
9
0
cells with 6t resulted in an accumulation of cells containing subG1 content of DNA which is an
31
indication of mitotic arrest.
0
50
100
150
200
25
0
50
100
150
200
25
0
50
100
150
200
250
0
50
100
150
200
250
Channels (FL2-A)
Channels (FL2-A)
Channels (FL2-A)
Channels (FL2-A)
Vehicle
0.005 ꢁM
0.01 ꢁM
0.02 ꢁM
Figure 1. Cell cycle arrest and apoptosis of DU145 cells treated with 6t. DU145 cells were
treated with various concentrations of 6t for 24 h and subjected to flow cytometry following
propidium iodide staining. DU145 cells were arrested in the G2/M phase of the cell cycle.
Compound 6t Treatment Induces Abnormal Mitotic Spindle Development. We tested if 6t
was able to depolymerize cellular microtubules in the same way as other depolymerizing agents.
Exposure of DU145 cells to different concentrations of 6t for 24 h led to a complete
depolymerization of the microtubule cytoskeleton. Confocal laser microscopy showed that while
DMSOꢀtreated cells (vehicle) went through various phases of mitosis without any abnormality,
6t treated cells exhibited profound abnormalities in spindleꢀformation, resulting in the
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appearance of abnormal spindles, misalignment of chromosomes and complete loss of coꢀ
ordination in mitotic spindle assembly (Figure 2). Some cells were microꢀnucleated, and others
were arrested in proꢀmetaphase with a ball or rosette of condensed DNA without a mitotic
10
11
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32
spindle (type IV spindle). The mitotic arrest caused by 6t was accompanied by net microtubule
depolymerization. These data suggested that 6t acts as a mitotic inhibitor by blocking cell cycle
progression at a time between proꢀmetaphase and metaphase by its ability to disrupt spindle
assembly that led to the caspase activation and apoptosis as evidenced by PARP [Poly(ADPꢀ
3
3
ribose) polymeraseꢀ1] cleavage (Figure 3). Treatment of 6t selectively induced PARP cleavage
in the tumor cell line while there was no PARP cleavage in the treated normal cell line (data not
shown). These results correlate quite well with those obtained in tubulin assembly experiments.
VEHICLE
6t
Figure 2. DU145 cells were plated onto glass coverslips and exposed to either 25 nM 6t or
DMSO and then fixed, stained with FITC conjugated (green) antiꢀtubulin antibodies and
propidium iodide (red) and analyzed by confocal microscope (Olympus). 6t inhibits the normal
formation of mitotic microtubules.
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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5
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5
5
5
5
5
6
1
0 nM
2
0 nM
V
24
48
72
V
24
48
72 h
0
1
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0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
PARP
Figure 3. DU145 cells were treated with the indicated concentrations of 6t and harvested at
the indicated times. Protein lysates were subjected to western blotting against PARP
antibodies. Full length PARP gets cleaved in the 6t treated cells indicating the cells are
undergoing apoptosis.
Compound 6t Destabilizes Tubulin Polymerization by Binding to the Colchicine Binding
Site on Tubulin. Since chromosomal architecture showed abnormalities in spindle formation,
we examined whether 6t effected polymerization of tubulin in vitro. For these assays, the
spontaneous polymerization of purified bovine brain tubulin was measured to determine if the
antimitotic properties of 6t were due to either stabilization or destabilization of microtubule
polymerization. The extent of tubulin polymerization was determined spectrophotometrically by
the increasing absorbance at 340 nM. DMSO vehicle is known to slightly stabilize microtubules
3
4
and produces an intermediate polymerization phenotype. As expected, the microtubule
stabilizer paclitaxel induced a strong increase in turbidity, whereas nocodazole destabilized
growing microtubules such that depolymerization at the (ꢀ)ꢀend is faster than polymerization at
the (+)ꢀend, giving rise to a net “depolymerization” phenotype and no rise in turbidity (Figure
4
A). Attempted polymerization of tubulin in the presence of 6t produced a depolymerization
phenotype, suggesting that 6t and related arylethenesulfonamides, 25a and 25c, caused
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cytotoxicity through destabilization of microtubule polymerization (see Figure 4B). The ability
of 6t compounds to compete for known binding sites on tubulin was determined using a mass
3
5
spectrophotometric (MS) competitive binding assay. Three tubulin ligands, corresponding to
the 3 binding sites on tubulin, colchicine, vinblastine, and paclitaxel were used for these
competitive binding studies. 6t (IC = 3.68 ꢁM) specifically competed with colchicine binding
10
11
12
13
14
15
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24
25
26
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5
0
to tubulin and with similar avidity as podophylltoxin, (IC = 3.71 ꢁM), but it did not compete
5
0
with either vinblastine or paclitaxel binding to tubulin (Figure 5). Known tubulin binding
ligands, podophylltoxin, vincristine and docetaxel effectively competed for the colchicineꢀ,
vinblastineꢀ and paclitaxelꢀtubulin binding sites, respectively, indicating the validity of the MS
binding assay.
Figure 4. 6t inhibit in vitro tubulin polymerization. A. Purified tubulin was incubated in the
0
presence of 10 ꢁM concentration of taxol, nocodazole, 6t and DMSO at 37 C. The extent of
tubulin polymerization was determined by the increasing absorbance at 340 nM over 40 min. 6t
and nocodazole (a known tubulin depolymerizer) were able to completely inhibit tubulin
polymerization compared to Taxol (tubulin polymerizer) and DMSO suggesting that 6t interfere
with normal tubulin polymerization kinetics. Data shown are representative of three independent
experiments. B. Purified tubulin was incubated in the presence of 10 ꢁM concentration of 6t,
0
2
5a, 25c and DMSO at 37 C. The extent of tubulin polymerization was determined by the
increasing absorbance at 340 nM over 40 min. All three compounds were able to completely
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1
1
2
2
2
2
2
2
2
2
2
2
3
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5
5
6
inhibit tubulin polymerization suggesting that these compounds interfere with normal tubulin
polymerization kinetics. Data shown are representative of three independent experiments.
B
A
1
00
Podophyllotoxin
9
7
5
0
0
0
0
1
2
3
4
5
6
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8
9
0
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2
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0
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0
1
2
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5
6
7
8
9
0
8
6
0
0
6
t
40
20
0
Vincristine
30
10
6
t
-10
0
.1
1
10
100
0.1
1
10
100
Concentration of competitor (uM)
Concentration of competitor (uM)
C
1
00
Docetaxel
6t
8
0
0
.1
1
Concentration of competitor (uM)
10
100
Figure 5. Tubulin competitive binding studies. 6t competes with colchicine (A), but not with
vinblastine (B) and paclitaxel (C) to bind to tubulin. In panel A and B, tubulin was incubated
with colchicine or vinblastine (1.2 ꢁM) in the absence of GTP with increasing concentrations of
the test compounds. In panel C, performed microtubules were incubated with paclitaxel (1.2
ꢁM) and 1 mM GTP, and increasing concentration of the test compounds. Podophyllotoxin,
vincristine and docetaxel were used as positive controls for competitive binding with colchicine,
vinblastine and paclitaxel, respectively.
In Vivo Tubulin Polymerization of 6t. To determine whether this compound also inhibits
tubulin polymerization in vivo, we treated human prostate cancer cells (DU145) with increasing
concentrations of 6t for 24 h and determined the extent of polymerized tubulin present in cell
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lysates. This assay, based on differential precipitation of depolymerized (soluble) and
polymerized (precipitated) tubulin, detects the abundance of polymerized tubulin (spindle
formation) present in normal mitotic cells. Paclitaxel and colchicine were used as controls since
paclitaxel promotes tubulin polymerization while colchicine induces deꢀpolymerization of
tubulin. Figure 6A shows that 6t caused a concentrationꢀdependent depolymerization of tubulin
in treated cells, similar to colchicine. Furthermore, 6t caused tubulin depolymerization even in
cells preꢀtreated with paclitaxel for 24 h that had high levels of polymerized tubulin; colchicine
had a similar effect (Figure 6B). These studies clearly indicate that 6t acts as a tubulinꢀ
depolymerizing agent and this property explains its ability to induce mitotic arrest of human
tumor cells which results in their apoptotic death.
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Figure 6. 6t inhibits in vivo tubulin polymerization. A. DU145 cells were treated with vehicle
(DMSO), paclitaxel (PTX), colchicine (COLC) or the indicated concentrations of 6t for 24 h and
lysed in a hypotonic buffer. The pellet and supernatants were resolved by SDSꢀPAGE. The
polymerized tubulin in the pellet (P) and the soluble tubulin in supernatant (S) were analyzed for
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presence of tubulin by western blotting. Like colchicine, a known tubulin depolymerizer, 6t
treatment completely inhibits the polymerization of tubulin. B. DU145 cells were first treated
with paclitaxel for 24 h and subsequently treated for 4 h with colchicine or 6t. As expected,
paclitaxel treatment induced a shift from soluble to polymerized tubulin (see arrow) while both
colchicine and 6t were both effective at depolymerizing paclitaxel induced polymerized tubulin
0
1
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0
(see arrows).
In vivo Antiꢀtumor Effects of 6t. 6t was next tested for its antiꢀtumor activity in soft agar and
nude mouse assays. For soft agar assays, we used a wellꢀknown pancreatic cancer cell line,
MIAꢀPaꢀCa2. Treatment of these cells line with 6t resulted in inhibition of anchorageꢀ
independent growth of tumor cells in a dose dependent manner (Figure 7A). In these assays,
paclitaxel was used as a positive control, which showed similar potency (data not shown). To
examine the anticancer efficacy, we used ERꢀnegative human breast tumor cells (BTꢀ20) grown
as xenografts in nude mice. Female athymic (NCrꢀnu/nu) mice were injected subcutaneously
7
with 0.5ꢀ1x10 tumor cells in 0.2 mL of PBS and the tumors allowed to grow for 7ꢀ10 days to a
size of 100ꢀ150 mm prior to treatment. The mice were then paired such that the pairs harbored
equal sized tumors which were used to test the therapeutic effects of 6t. Of the pairs, one mouse
received the vehicle alone while the second mouse received the compound by intraperitoneal (IP)
injection. The tumor size was measured on alternate days for a total of 14 days. Figure 7B shows
that 6t readily inhibited tumor growth in this xenograft model system. Of the 8 mice included in
each group, 100% of the control mice (placebo administered) showed a doubling of the total
tumor volume. On the other hand, most of the mice administered with 6t showed growth arrest or
a gradual reduction in their tumor volume, suggesting that this compound could be valuable antiꢀ
cancer therapeutic. Measurement of the body weights during the experimental period showed no
reduction in body weight indicated the safety profile of the drug.
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Page 29 of 96
Journal of Medicinal Chemistry
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Figure 7. 6t Inhibition of tumor growth. A. MIAꢀPaCaꢀ2 cells were plated in soft agar
containing increasing concentrations of 6t in triplicates. After three weeks of growth, the plates
were stained for 48ꢀh using 0.05% nitroblue tetrazolium solution. Representative plates were
photographed using an Olympus stereoscope mounted with a Sony digital camera system (DKC
5000, Sony Inc). 6t inhibits the semiꢀsolid growth of pancreatic cells indicating activity. B.
Human estrogen receptor negative breast cancer cells (BT20) were implanted into the hind
quarter of female nude mice. When the tumors became palpable, they were treated with either
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0 mg/kg 6t or an equal volume of vehicle every other day for a total of 6 injections. The tumor
volumes and body weights were determined and plotted (Blue diamonds: Vehicle; Red circles:
t) as the average per group along with SEM values (Vehicle: N=7, 6t: N=8). Trend lines were
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added using linear regression model (Microsoft Excel).
Compounds 6t and 25c are Highly Active Against Drug Resistant Tumor Cell Lines. One
major limitation of anticancer drug therapy is intrinsic and acquired multidrug resistance. Tumor
expression of the ATPꢀdependent drug efflux pump, Pꢀglycoprotein (Pgp), is associated with
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