Inhibition of tubulin polymerization by select alkenyldiarylmethanes

Article abstract of DOI:10.1016/j.bmcl.2007.11.114

During studies on the alkenyldiarylmethane (ADAM) class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), analogues were discovered that exhibit low micromolar and submicromolar cytotoxicities. Since the ADAMs are structurally related to the tu

Full text of DOI:10.1016/j.bmcl.2007.11.114

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 469–473
Inhibition of tubulin polymerization by select alkenyldiarylmethanes
Matthew D. Cullen,a Taradas Sarkar,b Ernest Hamel,b Tracy L. Hartman,c
Karen M. Watson,c Robert W. Buckheit, Jr.,c Christophe Pannecouque,d
Erik De Clercqd and Mark Cushmana,*
aDepartment of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences,
and the Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA
bToxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA
cImQuest Biosciences, Inc., 7340 Executive Way, Suite R, Frederick, MD 21704, USA
dRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 25 October 2007; revised 23 November 2007; accepted 27 November 2007
Available online 4 December 2007
Abstract—During studies on the alkenyldiarylmethane (ADAM) class of non-nucleoside reverse transcriptase inhibitors (NNRTIs),
analogues were discovered that exhibit low micromolar and submicromolar cytotoxicities. Since the ADAMs are structurally related
to the tubulin polymerization inhibitor CC-5079, a set of 14 ADAMs were tested for inhibition of tubulin polymerization in an
attempt to identify the biological target responsible for their cytotoxicity. The results indicate that, overall, the ADAMs are poor
inhibitors of tubulin polymerization. However, the two most cytotoxic compounds, 15 and 16, are in fact active as inhibitors of
tubulin assembly with IC50 values of 3.7 0.3 and 2.8 0.2 lM, respectively, and they both inhibit the binding of colchicine to
tubulin. Both compounds were investigated for anticancer activity in the National Cancer Institute’s panel of 60 human cancer cell
lines, and both compounds consistently displayed submicromolar cytotoxicities with mean-graph midpoint (MGM) values of
0.31 0.08 and 0.47 0.09 lM, respectively.
Ó 2007 Elsevier Ltd. All rights reserved.
Over the past 25 years, infection by the human immuno-
deficiency virus (HIV) has reached pandemic propor-
tions, and an estimated 41 million people are believed
to be carriers of the acquired immune deficiency syn-
drome’s (AIDS) etiological agent.1 HIV has caused the
deaths of more than 25 million people since its first ma-
jor appearance in 1981,1 and developing a cure for HIV-
infection is one of the major challenges currently facing
medical science. Several FDA-approved drugs are avail-
able to combat HIV infections and AIDS progression.
Unfortunately, the rapid mutation rate of HIV allows
the virus to develop resistance to many antiviral agents
as early as 2 months after initial anti-HIV treatment.
Thus, until a cure is discovered, development of antiviral
therapeutics that are active against both the wild-type
and drug-resistant forms of HIV is a primary goal for
AIDS researchers.2–4
The alkenyldiarylmethane (ADAM) class of non-nucle-
oside reverse transcriptase inhibitors (NNRTIs) was dis-
covered over 10 years ago. The lead compounds 1 and 2
retain antiviral activity against the common HIV-1 re-
verse transcriptase (RT) drug-resistance mutations
K103N and Y188C.5–8 For this reason, the development
of the ADAMs as potential antiviral therapeutics has
been pursued. It has been established that the ADAMs
exert their antiviral properties through the allosteric
inhibition of HIV-1 RT. However, the observation that
some ADAM analogues do not inhibit HIV-1 RT and
yet still exhibit anti-HIV activity indicates that, at least,
certain ADAMs interact with another viral or cellular
entity, and this has led us to investigate other molecular
targets.9
Keywords: Cytotoxicity; Tubulin; HIV; Alkenyldiarylmethane;
ADAM; Antiviral; Anticancer; Anti-HIV.
Another series of ADAMs has recently been developed
by scientists at Celgene Corp. as potent inhibitors of
inflammation, phosphodiesterase type 4 activity, and
*
Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494
6790; e-mail: cushman@pharmacy.purdue.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.114

470
M. D. Cullen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 469–473
The structural similarity between Celgene’s inhibitor
CO2CH3 CO2CH3
OCH3
CO2CH3 CO2CH3
OCH3
H3CO
Br
CC-5079 (Chart 1) and our own antiviral agents led us
to consider that the ADAMs may also exhibit one or
more of the properties displayed by the Celgene inhibi-
tors. It is well known that disrupting microtubule
homeostasis causes cells to undergo apoptosis,11 and
the low micromolar toxicity generally displayed by the
ADAM class of NNRTIs led to the hypothesis that
the ADAMs’ cytotoxic properties may originate from
the inhibition of tubulin polymerization by binding to
the colchicine binding site. Additionally, inhibition of
tubulin polymerization may also account for the RT-
independent antiviral activity displayed by many
ADAM analogues. The viral HIV protein Tat is known
to, among other things, stabilize the microtubule frame-
work of HIV-infected cells by binding to microtubule-
associated protein (MAP) binding sites.14 It has been
proposed that the interactions between Tat and microtu-
bules help facilitate the replication of HIV and may also
contribute to the mechanism of HIV-related cell death.14
In light of this information, the inhibition of tubulin
polymerization by a select group of ADAMs was inves-
tigated. Herein we report the syntheses, antiviral activ-
ity, and tubulin inhibitory effects of ADAMs 4–17
(Chart 2).
H3CO
Br
Br
Br
CO2CH3
Et
1
2
O
OCH3
OCH3
H3CO
H3CO
CH3
OCH3
NH
H3CO
O
H3C
O
NC
OCH3
CC-5079
3
Chart 1.
tubulin polymerization, where tubulin inhibition in-
volves binding of the inhibitor to the same site as the
natural antimitotic agent, colchicine (3, Chart 1).10–13
CO2CH3 CO2CH3
CO2CH3 CO2CH3
OCH3
H3CS
H3CO
O
O
SCH3
OCH3
H3CO
X
OCH3
H3CO
H3C
X
CH3
H3C
N
CH3
N
N
O
H3CN
N
N
H3C
R
4
X = Cl, R = CO2CH3
X = Br, R = Ph
X = Cl, R= CH2CH3
X = Br, R = HC=CH2
X = Cl, R = SCH3
10
5
6
7
8
9
OCH3
N
Ar1
Ar2
Cl
CH3
H3CO
O
O
O
N
H3C
n
H3C
Cl
CH3
N
N
n= 1, Ar1 = Isoxlone, Ar2 = m-Nitrile
n= 1, Ar1 = Isox-OMe, Ar2 = Thio
n= 2, Ar1 = Isox-OMe, Ar2 = Thio
n= 1, Ar1 = m-Nitrile, Ar2 = Isox-OMe
n= 1, Ar1 = Isox-OMe, Ar2 = m-Nitrile
n= 1, Ar1 = Isox-OMe, Ar2 = p-Nitrile
11
12
13
14
15
16
N
OCH3
17
COSCH3
OCH3
CH3
CN
CH3
CN
O
O
N
NCH3
CH3
Thioester
OCH3
Isoxazole-OMe
(Isox-OMe)
O
m-Benzonitrile p-Benzonitrile
(m-Nitrile) (p-Nitrile)
Isoxazolone
(Isxlone)
(Thio)
Chart 2.

M. D. Cullen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 469–473
471
pounds. Nevirapine is included for antiviral activity
comparisons, while colchicine and combretastatin A-4
are well-known inhibitors of tubulin polymerization,
with the latter compound being an exceptionally potent
inhibitor of the binding of radiolabeled colchicine to
tubulin. The majority of the compounds studied for
inhibition of tubulin polymerization were chosen on
the basis of their acute cytotoxicity, which one would ex-
pect to correlate with tubulin destabilizing activity. To
our surprise, only 2 of the 14 analogues investigated
were capable of inhibiting tubulin polymerization at
concentrations lower than 40 lM, despite the structural
similarities observed between the compounds and Cel-
gene’s inhibitors of tubulin polymerization. These data
refute the hypothesis that inhibition of tubulin polymer-
ization is the general source of ADAMs’ cytotoxicity.
However, the tubulin IC50s observed for ADAMs 15
and 16 are not substantially greater than that of the po-
tent antimitotic agent combretastatin A-4, indicating the
extreme cytotoxicity observed with these two analogues
most likely results from disruption of the microtubule
network. Inhibitors of tubulin polymerization often in-
duce cell death at concentrations much lower than their
in vitro tubulin IC50 values in cell-free systems, which
accounts for the 1000-fold difference in tubulin inhibi-
tory activity and cytotoxicity observed for ADAMs 15
and 16. The ADAMs 12 and 13, which are structurally
related to 15 and 16, were completely inactive as inhib-
itors of tubulin polymerization. Evidently, the relatively
small structural change involving the movement of the
nitrile from the para position in 16 to the meta position
in 15 only causes a small drop in activity, but the more
drastic alterations embodied in 12 and 13 are not
tolerated.
P
P
SnBu3
Ar1
a,b
+
X
18
N
N
N
O
N
O
20
CH3
H3C
19
P
Q
Ar1
Ar2
c
Q
N
Ar2
N
22
O
X
21
H3C
X = Br or I
CH3
CN
O
N
OCH3
Ar1 and Ar2 =
CN
CH3
O
NCH3
O
Scheme 1. Reagents and conditions: (a) 5 mol% PdCl2(PPh3)2,
10 mol% CuI, Et3N, THF; (b) 2 mol% Pd(PPh3)4, Bu3SnH, THF,
0 °C; (c) 10 mol% Pd(PPh3)4, 20–100 mol% CuI, CsF, DMF, 60 °C.
In order to confirm that ADAMs 15 and 16 are in
fact binding to the colchicine site of tubulin, the
inhibition of colchicine binding to tubulin was deter-
mined at 37 °C in reaction mixtures incubated for
10 min and containing tubulin (1 lM), 15 or 16
(5 lM), and [3H]colchicine (5 lM). ADAMs 15 and 16
inhibited [3H]colchicine binding by 24 1% and
42 3%, respectively. Combretastatin A-4, which was
used as a positive control, inhibited [3H]colchicine bind-
ing by 98 0.3%.
A number of methods have been developed for the syn-
thesis of the ADAM scaffold, and the syntheses of
16
ADAMs 4–10,ꢀ,15 12,15 13,15 and 17 have been pub-
lished. ADAMs 11, 14, 15, and 16 were constructed
via the general cross-coupling route depicted in Scheme
1. Sonogashira coupling of aryl halide 18ꢀ,17 and termi-
nal alkyne 19,15 followed by hydrostannation, affords
stannane intermediate 20. The stannane and aryl halide
21 are coupled via the Stille reaction to obtain the de-
sired analogue.
The ADAMs 15 and 16 were examined for antiprolifer-
ative activity against the human cancer cell lines in the
National Cancer Institute screen, in which the activity
of each compound was evaluated with approximately
55 different cancer cell lines of diverse tumor origins.
The GI50 values obtained with selected cell lines, along
with the mean-graph midpoint (MGM) values, are sum-
marized in Table 2. The MGM is based on a calculation
of the average GI50 for all of the cell lines tested
(approximately 55) in which GI50 values below and
above the test range (10À8 to 10À4 molar) are taken as
the minimum (10À8 molar) and maximum (10À4 molar)
drug concentrations used in the screening test. Both
ADAMs 15 and 16 consistently produced submicromo-
lar GI50 values in these human cancer cell lines, resulting
in MGM values of 0.31 0.08 and 0.47 0.09 lM,
respectively.
The tubulin polymerization inhibitory data18–20 for
ADAMs 4–17 are presented in Table 1, together with
,
6,21–23
the antiviral data*
associated with the com-
ꢀ
Syntheses for aryl iodides corresponding to the isoxazole and
isoxazolone were reported in Ref. 17. The aryl bromides correspond-
ing to the benzonitriles are commercially available.
*
The ability of target compounds to inhibit the enzymatic activity of
recombinant HIV-1 RT (p66/51 dimer) was evaluated as previously
described.6 Evaluation of antiviral activity against HIV-1RF was
performed in infected CEM-SS cells while using the XTT cytopro-
tection assay, as previously described.6,22 Evaluation of antiviral
activity against the HIV-1IIIB and HIV-2ROD strains was performed
in infected MT-4 cells using the previously described MTT assay.20,23

472
M. D. Cullen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 469–473
Table 1. Antiviral and tubulin polymerization inhibitory activities of ADAMs 4–17
a
b
c
d
Compound
HIV-1 RT IC50 (lM)
EC50 (lM)
CC50 (lM)
Tubulin IC50 (lM)
1RF
1IIIB
2ROD
CEM-SS
MT-4
4
71
0.3
2.3
0.013
13
3.2
0.60
N.A.e
2.5
40
32
36
>40
>40
5
160
>198
N.T.f
62
6
>100
N.T.f
99
2.6
29
>200
29
>40
>40
7
N.A.e
8.2
N.T.f
3.0
N.T.f
N.A.e
N.T.f
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.T.f
N.T.f
8
>100
20
33
>40
>40
9
10
100
67
53
N.T.f
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
1.2
N.T.f
2.9
7.0
>40
>40
11
12
40
33
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
N.A.e
0.0015
N.T.f
N.T.f
1.1
1.2
0.97
1.6
>40
>40
13
14
5.2
73
0.78
2.1
1.1
4.2
>40
15
16
93
0.004
0.004
2.9
N.T.f
N.T.f
N.T.f
0.20
0.34
12
3.7 0.3
2.8 0.2
>40
N.T.f
1.2 0.1
3.8 0.1
>100
0.60
0.08424
N.T.f
N.T.f
17
Nevirapine
Combretastatin A-4
Colchicine
0.053
N.T.f
N.T.f
15
N.T.f
N.T.f
a Inhibitory activity versus HIV-1 RT with poly(rC)Æoligo(dG) as the template primer.
b EC50 is the concentration required to inhibit 50% of the cytopathic effect of HIV-1RF in CEM-SS cells, HIV-1IIIB in MT-4 cells, or HIV-2ROD in
MT-4 cells.
c CC50 is the cytotoxic concentration required to cause cell death for 50% of the mock-infected CEM-SS or MT-4 cells.
d The concentration required to inhibit 50% of tubulin polymerization in vitro. The reaction mixtures contained 10 lM tubulin, and the extent of
assembly after 20 min at 30 °C in 0.8 M monosodium glutamate + 0.4 mM GTP was measured.
e Not active.
f Not tested.
Table 2. Cytotoxicities of Alkenyldiarylmethanes 15 and 16
dent antiviral mechanism. Additional studies are required
Cell line or MGMb
Compound GI50 (lM)
a
to elucidate the so far unknown antiviral mechanism. De-
spite the poor tubulin inhibition generally exhibited by
most members of the ADAM class described here, the
highly cytotoxic analogues 15 and 16 were identified as
potent tubulin destabilizing agents with activities not sub-
stantially greater than the activity of the natural antimi-
totic agent combretastatin A-4. In light of this
information, 15 and 16 could potentially be used as leads
in the development of ADAM-based inhibitors of tubulin
polymerization for the treatment of cancer.
15
16
Lung (HOP-62)
Colon (HCT-116)
CNS (SF-539)
0.76
0.49
0.55 0.08
0.46 0.08
0.30 0.04
0.72 0.19
0.20 0.03
0.78 0.32
0.57 0.14
0.19 0.02
0.47 0.09
0.24 0.06
0.59 0.17
0.10 0.06
0.50 0.07
0.32 0.08
0.089 0.051
0.31 0.08
Melanoma (LOX IMVI)
Ovarian (OVCAR-3)
Renal (SN12C)
Prostate (DU-145)
Breast (MDA-MB-435)
MGM
a Concentration for 50% growth inhibition.
b Mean-graph midpoint for growth inhibition of all human cancer cell
lines found to be sensitive.
Acknowledgments
This investigation was made possible by funding from
the National Institutes of Health, DHHS through Grant
RO1-AI-43637 and research was conducted in a facility
constructed with the financial support of a Research
Facilities Improvement Program Grant, No. C06-
14499, also from the National Institutes of Health.
The authors thank Dr. G.R. Pettit, Arizona State Uni-
versity, for a generous gift of combretastatin A-4.
The results with analogues 4, 6, 8, and 10 are consistent
with the conclusion that inhibition of tubulin polymeri-
zation is not required for the ADAMs’ RT-independent
antiviral mechanism. These compounds each exhibited
RT-independent antiviral activity, as can be seen by
comparing their in vitro RT inhibitory activities with
their respective cytoprotective activities; yet, none of
these four analogues had a significant effect on tubulin
polymerization at 40 lM.
Supplementary data
In summary, select ADAMs were evaluated for inhibition
of tubulin polymerization, and the results indicate that
most members of this structural class of compounds are
poor inhibitors. These results do not support the hypoth-
esis that disruption of microtubule stability is the origin of
the ADAMs’ cytotoxicity, nor do the results support an
important role for tubulin in the ADAMs’ RT-indepen-
Physical constants, elemental analysis data, and experi-
mental procedures for the synthesis of compounds 11,
14, 15, and 16 are included in the Supporting Informa-
tion section of this report. Supplementary data associ-
ated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2007.11.114.

M. D. Cullen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 469–473
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