Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.08.056

A novel triazole-containing chemical series was shown to inhibit tubulin polymerization and cause cell cycle arrest in A431 cancer cells with EC ₅₀ values in the single digit nanomolar range. Binding experiments demonstrated that representative

Full text of DOI:10.1016/j.bmcl.2005.08.056

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5154–5159
Synthesis and structure–activity relationships of 1,2,4-triazoles
as a novel class of potent tubulin polymerization inhibitors
Xiaohu Ouyang,a,* Xiaoling Chen,a Evgueni L. Piatnitski,a Alexander S. Kiselyov,a
Hai-Ying He,a Yunyu Mao,a Vatee Pattaropong,a Yang Yu,a Ki H. Kim,a
John Kincaid,a Leon Smith, II,a Wai C. Wong,a Sui Ping Lee,a Daniel L. Milligan,a
Asra Malikzay,b James Fleming,b Jason Gerlak,b Dhanvanthri Deevi,c
Jacqueline F. Doody,b Hui-Hsien Chiang,b Sheetal N. Patel,b Ying Wang,b
Robin L. Rolser,b Paul Kussie,b Marc Labellea and M. Carolina Tumab
aDepartment of Chemistry, ImClone Systems, 710 Parkside Avenue, Suite 2, Brooklyn, NY 11226, USA
bDepartment of Protein Science, ImClone Systems, 180 Varick Street, New York, NY 10014, USA
cDepartment of Experimental Therapeutics, ImClone Systems, 180 Varick Street, New York, NY 10014, USA
Received 16 July 2005; revised 19 August 2005; accepted 22 August 2005
Available online 28 September 2005
Abstract—A novel triazole-containing chemical series was shown to inhibit tubulin polymerization and cause cell cycle arrest in
A431 cancer cells with EC50 values in the single digit nanomolar range. Binding experiments demonstrated that representative active
compounds of this class compete with colchicine for its binding site on tubulin. The syntheses and structure–activity relationship
studies for the triazole derivatives are described herein.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Microtubules (MT) are among the most successful tar-
gets for anticancer therapy.1 Composed of dynamic poly-
mers of tubulin, microtubules are elements of the cell
cytoskeleton that play key roles in cell division and cell
shape. During mitosis, tightly regulated microtubule
dynamics is essential for spindle formation and chromo-
somal separation. Therefore, rapidly dividing cells are
more susceptible to inhibitors of tubulin polymerization
than non-dividing cells. Indeed, targeting tubulin is a
successful strategy for cancer therapy.2 There are three
well-characterized drug binding sites on tubulin: taxol,
vinca, and colchicine sites. Drugs that bind to the first
two sites, such as Paclitaxel (Taxolꢁ) and Vinblastineꢁ,
have been successfully used in clinics as chemotherapeu-
tics to treat various tumor types; however, their use is
limited by multiple drug resistance (MDR). Several
colchicine site binders, on the other hand, have been
shown to inhibit MDR tumors, instilling renewed
interest in the search for novel antimitotic agents that
bind to the colchicine binding site on tubulin.3
Several classes of colchicine site binders are currently in
clinical trials. Candidates T138067, A-289099, and
A-105972 demonstrate antitumor activity against Paclit-
axel and Vinblastineꢁ resistant tumors4–6 with the added
benefit of oral administration. A new approach to the
use of these colchicine site binders is the targeting of
tumor vasculature (vascular disrupting agents or
VDAs). VDAs preferentially disrupt microtubules in
tumor endothelial cells, causing tumor vascular damage
that leads to massive tumor necrosis even when dosed at
a fraction of their maximum tolerated doses (MTD).7,8
Examples of such agents currently undergoing clinical
trials are ZD-6126 and Combretastatin A4P (CA4P),
derived from colchicine and Combretastatin A4 (CA4),
respectively (Fig. 1).9,10
Novel triazole-containing tubulin inhibitors were dis-
covered by screening our internal compound library
using an in vitro tubulin polymerization assay. At
10 lM, compound 1 inhibited bovine brain tubulin
polymerization in vitro by 80% and depolymerized
Keywords: Tubulin inhibitor; Tubulin polymerization; 1,2,4-Triazole;
Vascular disrupting agent; Tumor vasculature; Antimitotic.
*
Corresponding author. Tel.: +1 347 523 0018; e-mail:
shawn_ouyang@yahoo.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.056

X. Ouyang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5154–5159
5155
O
O
NH2
OC2H5
N
H
NH2
N
NH
N
NH
(i)
(ii)
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
A
Figure 1. Structures of compound (1), colchicine and combretastatin.
H
N
H2N
HI
N
OCH3
H2N
OCH3
(iii)
S
S
CH3
microtubules in cultured A431 cells within 20 min
(Fig. 2). Compound 1 also demonstrated antiprolifera-
tive activity against several tumor cell lines (Table 1)
and induced G2/M arrest of A431 human cancer cells.
Given its potency, small molecular weight, and potential
for significant diversity at various regions of the mole-
cule, we proceeded to synthesize analogs of compound
1. Compounds were evaluated for inhibition of tubulin
polymerization in vitro and capacity to arrest cancer
cells during the G2/M phase of the cell cycle for activity
determination and discrimination from general cytotox-
ic compounds. Representative syntheses and SAR stud-
ies of this novel tubulin inhibitor class are reported
herein.
D
E
(iv)
C
+
E
1
Scheme 1. Reagents and conditions: (i) Ethyl-2-chloronicotinate,
DMF, 140 ꢂC, 20 h; (ii) hydrazine monohydrate, i-PrOH, 120 ꢂC,
10 h; (iii) CH3I, acetone, reflux, 4 h; (iv) pyridine, sealed tube, 140 ꢂC,
4 h.
To identify structural elements required for activity, we
generated a set of compounds following Scheme 1 with
varying R1 groups. Compounds that showed inhibition
of tubulin polymerization were then tested for G2/M
block activity in A431 cells (Table 2). By keeping R2 con-
stant, compounds 1 and 2 demonstrated that meta or para
alkoxylanilines in the R1 position are superior in inhibit-
ing tubulin polymerization and cycling cells than an
unsubstituted aniline analog 3. However, the presence
of dimethoxyanilines (4–6) leads to reduced activity. A
similar effect was also observed for anilines substituted
with other functional groups (7–9). It is noteworthy that
compounds 10 and 11, with cyclized alkoxyl moieties,
showed potency similar to that of compound 1 in the
G2/M assay. Cyclized alkoxyl moieties were later found
to be optimal regardless of modifications made to other
Compound 1 was synthesized by a condensation of a
substituted arylhydrazide C with a substituted methylat-
ed thiourea E according to a previously reported proce-
dure11 (Scheme 1). Intermediate C was synthesized by
heating aniline A with ethyl-2-chloronicotinate to yield
B which was reacted further with hydrazine mono hy-
drate to provide C. An intermediate E was formed by
reacting the substituted thiourea D with methyl iodide.
A variety of substituted anilines and amines replaced
aniline A to produce compounds which are listed in
Table 2.
Figure 2. Disruption of A431 cellular microtubules by (1). Left, 0.1% DMSO. Right, 10 lM compound (1). Cells were stained with an a-tubulin
antibody.
Table 1. Antiproliferative activitya by (1) against various tumor cells
Cell line
GI50 (lM)b
A431
0.05
A498
0.060
DLD1
0.07
DU145
0.02
NCI-H460
0.01
Cell line
GI50 (lM)
LoVo
0.02
HCT116
0.40
SNB19
0.15
SKOV3
0.220
NCI/ADR-RESc
0.150
a Cytotoxicity measured after treatment for 24 h.
b GI50 value for each cell line, the concentration of compound that caused 50% reduction in absorbance at 562 nm relative to untreated cells using
sulfurhodamine B assay. Values are the means of three independent determinations (SD < 10%).
c MDR-1 positive breast cell line.

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X. Ouyang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5154–5159
Table 2. Effects of R1 and R2 on tubulin activity
N
N
R1
N
H
N
X
R2
Compound
R1
R2
X
% ITPa
% G2/M blockb
EC50(lM)c
CA4
Colchicine
1
95
88
0.0035
0.0028
0.090
0.083
>10
>10
NT
80
80
80
72
3-Methoxyphenylamino
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3-Methoxyphenyl
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
2
4-Methoxyphenylamino
Phenylamino
73
68
22
3
5
33
4
2,5-Dimethoxyphenylamino
2,4-Dimethoxyphenylamino
3,5-Dimethoxyphenylamino
4-N,N-Dimethylamino-phenylamino
3,5-Dichlorophenylamino
4-Cyanophenylamino
20
5
47
45
17
6
NT
NT
NT
NT
85
NT
7
35
44
NT
NT
8
9
34
87
NT
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
Benzo[1,3]dioxol-5-ylamino
2,3-Dihydro-benzo[1,4]dioxin-6-ylamino
3-Methoxy-benzylamino
0.062
0.117
0.12
NT
95
100
83
71
4-Methoxy-benzylamino
88
97
22
36
(Tetrahydro-furan-2-yl)-methylamino
3-Methoxyphenyl
3-Methoxybenzyl
NT
15
9
NT
NT
NT
NT
NT
NT
NT
NT
70
NT
NT
3-Methoxyphenylamino
9
NT
3-Methoxyphenylamino
3-Methoxyphenylamino
Phenyl
4
NT
NT
3,5-Difluorophenyl
3,5-Difluorophenyl
3-(2-Oxo-pyrrolidin-1-yl)-propyl
2-(Pyridin-3-yl)ethyl
Pyridin-4-yl-methyl
3,4-Difluorobenzyl
0
Benzo[1,3]dioxol-5-ylamino
2,3-Dihydro-benzo[1,4]dioxin-6-ylamino
2,3-Dihydro-benzo[1,4]dioxin-6-ylamino
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
2,3-Dihydro-benzo[1,4]dioxin-6-ylamino
2,3-Dihydro-benzo[1,4]dioxin-6-ylamino
3-Methoxyphenylamino
28
21
NT
NT
23
84
NT
0.066
NT
NT
84
84
23
32
3,4-Difluorobenzyl
4-Methoxybenzyl
68
30
83
NT
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Dimethoxyphenyl
3,5-Difluorophenyl
3,5-Dimethylphenyl
3-Hydroxy-5-methoxyphenyl
3,4,5-Trimethoxyphenyl
3-Dimethylamino-phenyl
Pyridin-3-yl
100 (100d)
98 (91d)
100 (83d)
100 (88d)
99 (73d)
98 (76d)
97 (57d)
87 (51d)
49d
0.021
0.0072
0.010
0.0036
0.023
0.031
NT
Benzo[1,3]dioxol-5-ylamino
3-Hydroxyphenylamino
O
83
O
86
89
2,3-Dihydro-benzo[1,4]dioxin-6-ylamino
3,5-Dimethoxyphenylamino
4-Dimethylamino-phenylamino
4-Cyanophenylamino
O
O
87
86
O
O
27
27
3,5-Difluorophenylamino
Pyridine-3-amino
3-Methoxy-benzylamino
O
NT
O
61
32
0.066
NT
O
100
4-Methoxy-benzylamino
Piperonylamino
O
86
90d
28
24
NT
NT
O
Pyridin-4-yl-methylamino
(3-Methoxy-phenyl)-methyl-amino
(3-Methoxy-benzyl)-methyl-amino
4-Methoxy-benzamido
O
68
44
NT
NT
O
5d
NT
NT
NT
23
O
28d
NT
O
8d
NT
NT
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
Benzo[1,3]dioxol-5-ylamino
O
97d
O
52d
83
83
0.010
0.012
0.016
0.011
NT
O
80d
O
89d
86
63
O
53d
O
43d
21
37
3-Fluorophenyl
O
43d
NT
NT, not tested.
a % ITP = percent inhibition of tubulin polymerization; 30 lM tubulin and 10 lM compounds were used. Measured as percent inhibition of tubulin
assembly compared to that of DMSO control. Average of three experiments with SD < 15%. Compounds that showed >55% inhibition were tested in the
G2/M block assay.
b Induction of cell cycle arrest, expressed as percent of cells in the G2/M phase. A431 cells were treated with 0.1 lM compound for 24 h and then analyzed
by flow cytometry. Average of two experiments with SD < 10%. Only compounds that showed G2/M cell arrest in single point measurements were tested
for EC50s.
c Dose–response induced by cell cycle arrest in the G2/M phase, expressed as the compound concentration that causes 50% cells to arrest. Average of three
experiments with SD < 10%.
d 3.33 lM compounds were used in the assay. Average of three experiments with SD < 10%. At this concentration, only compounds with 40% inhibition or
higher were tested in the G2/M block assay.

X. Ouyang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5154–5159
5157
O
areas of the molecule. In contrast, results of replacing ani-
lines with alkylamines were disappointing. Compounds
12–14 displayed excellent activities in inhibiting tubulin
polymerization in vitro; however, only 12 showed good
cellular activity. Efforts to remove the NH linker or re-
place it with a methylene group in the R1 position resulted
in a loss of activity. For example, compounds 15 and 16,
synthesized by condensing hydrazide C (Scheme 1) with
3-methoxy-benzamidine or 2-(3-methoxy-phenyl)-acet-
amidine respectively, were not active. In conclusion, two
R1 moieties, benzo[1,3]dioxol-5-ylamino and 2,3-dihy-
dro-benzo[1,4]dioxin-6-ylamino, as well as meta and para
mono methoxyphenylamino groups gave us the best
cellular activity.
NHNH2
OH
(iii)
(i) and (ii)
OCH3
N
O
27
H3CO
H3CO
OCH3
F
Scheme 2. Reagents and conditions: (i) Ethyl-2-chloronicotinate,
K2CO3, DMF, 100 ꢂC, 18 h; (ii) hydrazine monohydrate, i-PrOH,
70 ꢂC, 18 h; (iii) E from Scheme 1, pyridine, sealed tube, 140 ꢂC, 4 h.
NH2
N
O
(ii)
(i)
NH2
NH
42
H3CS
H3CS
OCH3
While exploring structural requirements at R2, we found
that a 3,5-dimethoxyphenyl moiety was necessary to
maintain activity (17–20). Alkyl groups at R2 (21–26)
Scheme 3. Reagents and conditions: (i) 4-methoxybenzoyl chloride,
TEA, pyridine, 25 ꢂC, 20 min; (ii) F from Scheme 2, 125 ꢂC, 12 h.
Table 3. Effects of B and C rings on tubulin activitya
C
NH
B
X
O
(CH2)n
O
H3CO
OCH3
Compound
B
C
X
O
n
% ITP
96c
% G2/M block
86
EC50 (lM)
N
N
N
N
N
N
N
N
N
N
N
50
2
2
2
2
1
1
2
2
0.013
N
H
N
N
51
52
53
54
55
56
57
O
O
O
69c
65c
83
82
40
85
84
77
68
0.044
0.0066
0.21
N
N
H
N
N
H
N
59c
N
N
H
N
N
NH
NH
NH
NH
89b
0.017
0.0094
0.0053
0.053
N
N
H
N
N
N
N
100b
100b
100b
N
H
N
N
H
N
N
N
H
N
58
59
60
NH
NH
O
2
2
2
64c
76b
30c
25
0.24
0.13
NT
N
N
CH3
CH3
N
N
N
26
N
N
N
N
NT
N
CH3
a See Table 2 footnotes for the assay description.
b 10 lM compound concentration was used in the polymerization assay.
c 3.33 lM compound concentration was used in the polymerization assay.

5158
X. Ouyang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5154–5159
had little activity, with only benzylamines (23–26) show-
ing marginal effects in arresting cells in the G2/M phase
of the cell cycle. We implemented several strategies for
aniline removal from the lower portion of the molecule.
Replacing NH with an oxygen atom was the most suc-
cessful (X = O, Table 2). The biaryl ether analogs were
synthesized by replacing aniline A with corresponding
phenols in a modification of Scheme 1. In step (i),
K2CO3 was introduced at lower temperatures, in a var-
iation on Scheme 1. Step (ii) was also carried out at re-
duced temperature to minimize the cleavage of the biaryl
ether linkage by hydrazine (Scheme 2). Whilst keeping
R2 constant as a 3,5-dimethoxyphenyl group, com-
pounds 27–35, bearing various aromatic amines at R1,
displayed similar or improved functional activity com-
pared to those of corresponding analogs in Table 2.
However, only the compounds with electron-rich aniline
or phenol groups at R2 caused cells to arrest. A similar
result was observed for the alkylamines at R1 where
compounds 36–39 had no cellular activity. The aniline
NH at R1 appears to be critical for activity, since meth-
ylene (40, 41) or acyl (42) analogs showed no activity.
Compound 42 was prepared according to Scheme 3. In
summary, for the SAR at region R2, only electron-rich
substituents provided good functional and cellular
activities, as demonstrated by compounds 43–49.
dine analogs (50–53 compared to 30; 54 and 55 com-
pared to 10; 56 and 57 compared to 11). Pyrimidine
containing analogs 51 and 57 were produced according
to the general outline in Scheme 4.
Preparation of compounds 50 and 53–56 was achieved
using procedures similar to that described in Scheme 1
where the corresponding commercially available ring B
chloro derivatives were used instead of ethyl-2-chloro-
nicotinate. Compound 52 was synthesized using an
alternative procedure depicted in Scheme 5.
Methyl substitution at two different positions of the tri-
azole ring (58–60) reduced activity, indicating that either
NH groups are critical for binding or steric hindrance
plays a role in this region. The synthesis of compounds
58 and 60 is shown in Scheme 6.
N-Methyltriazole analog 59 was prepared by a slightly
modified procedure of Scheme 1 where hydrazine is
substituted by methylhydrazine.
Given that most of our potent compounds possessed
structural motifs that mimic colchicine and CA4
(i.e., multiple methoxy groups, Fig. 1), we predicted
they would bind to the same tubulin site. Molecular
modeling suggested that the binding mode of com-
pound 10 is similar to that of podophyllotoxin,
another potent colchicine site binder.12 Superposition
of the compounds suggests that the methoxyphenyl
and benzodioxole groups share a similar pharmaco-
phore (Fig. 3). To test our hypothesis, we evaluated
these compounds in a colchicine competition binding
We also investigated the two central ring moieties con-
necting R1 and R2 (Table 3). We demonstrated that
the 2,3-disubstituted pyridine at ring B was not required
for activity, while a regioisomeric pyridine, pyrimidine,
pyrazine or even a phenyl ring was equal to or more
effective than the corresponding 2,3-disubstituted pyri-
O
O
NH2 or OH
N
OH
NH or O
N
OC2H5
NH or O
O
N
H3CS
N
H3CO
OCH3
(ii)
N
OC2H5
(i)
then (iii)
H3CS
N
O
Cl
H3CO
OCH3
H3CO
OCH3
O
H
N
H2N
HI
N
O
NH2
NH2
N
N
(CH2)n
H
SCH3
O
O
N
NH or O
(iv)
then (v)
(vi)
51 or 57
H3CO
OCH3
G
Scheme 4. Reagents and conditions: (i) DMF, K2CO3 if phenol starting material, 105 ꢂC; (ii) Raney-Ni, THF, 50 ꢂC; (iii) 2N LiOH, THF; (iv) DCC,
HOBt, DCM, 25 ꢂC; (v) TFA, DCM, 0 ꢂC; (vi) pyridine, sealed tube, 140 ꢂC.
O
O
OH
O
O
H2N
HI
N
O
O
OCH3
NHNH2
SCH3
O
I
H3CO
OCH3
(iii)
OC2H5
(iv)
(i) and (ii)
52
H3CO
OCH3
H3CO
OCH3
Scheme 5. Reagents and conditions: (i) CuI, Cu, K2CO3, water, ultrasound, 40 min, 25 ꢂC; (ii) 1% H2SO4 in MeOH, 50 ꢂC, 4 h; (iii) N2H4.H2O,
i-PrOH, 8 h, 120 ꢂC; (iv) pyridine, TEA, 4 h, 140 ꢂC.

X. Ouyang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5154–5159
5159
O
O
O
O
O
S
(i)
(ii)
(iii)
SCH3
NHCH3
58 or 60
O
N
NHCH3
NCS
N
H
Scheme 6. Reagents and conditions: (i) CH3NH2, EtOH, 25 ꢂC, 3 h; (ii) CH3I, acetone, 65 ꢂC, 4 h; (iii) G from Scheme 4, pyridine, microwave,
190 ꢂC, 1 h.
tastatin A4 and colchinol, and are currently being
investigated in various in vivo tumor models.
OH
H
O
O
O
H
O
Acknowledgments
H3CO
OCH3
OCH3
We thank Peter Bohlen, Joel Kawakami, and James
Tonra for insightful discussions, and Elizabeth Wu
for technical support.
Podophyllotoxin
Figure 3. Superposition of the suggested binding mode of 10 (green)
and crystal structure of podophyllotoxin (pink) when bound to
tubulin.
Supplementary data
Supplementary data associated with this article can
be found in the online version at doi:10.1016/
j.bmcl.2005.08.056.
assay.13 Indeed, the majority of potent compounds
inhibited [3H]colchicine binding to tubulin. Table 4
summarizes the dose dependent results of colchicine
competitive binding, and G2/M arrest of cells for
selected compounds and standards. Representative tri-
azole derivatives were also evaluated in cytotoxicity
assays with NIH 3T3 fibroblasts and showed reduced
toxicity compared to tumor cell lines (GI50 10–100·
higher, data not shown). The most potent compounds
are highly active in all assays, comparable to CA4 and
colchinol, the active entities of the VDA prodrugs
CA4P and ZD6126, respectively. To evaluate the
potential of the triazole derivatives as drug candidates,
we plan to investigate their effects in vivo, and the
results will be reported in due course.
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In conclusion, a series of triazole derivatives has been
discovered as a novel class of tubulin polymerization
inhibitors that bind to the colchicine site on tubulin.14,15
Structure–activity relationships have been established
for this class of compounds in terms of their capacity
to inhibit tubulin polymerization in vitro and cause
G2/M arrest in tumor cells. Selected compounds showed
biochemical characteristics similar to those of Combre-
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Table 4. Ki for selected compounds and standards
b
Compound
Kia (lM)
EC50 (lM)
CA4
Colchinol
0.03 0.02
1.85 1.68
0.10 0.06
0.06 0.00
0.16 0.13
0.05 0.01
0.01 0.01
0.02 0.01
0.0035 0.002
0.0435 0.033
0.090 0.035
0.062 0.015
0.117 0.007
0.021 0.012
0.0072 0.004
0.0036 0.008
1
10
11
27
28
30
a Colchicine competition binding assay, with tubulin at 40 nM and
[3H]colchicine at 65 nM.
b Compound concentration required for 50% of A431 cells to accu-
mulate at the G2/M phase of the cell cycle.