Tubulin-polymerization inhibitors derived from thalidomide

Article abstract of DOI:10.1016/j.bmcl.2004.10.072

2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), has potent tubulin-polymerization-inhibiting activity. A metabolite of thalidomide, 5-hydroxythalidomide (2) also showed moderate inhibitory activity 2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), which was obtained during our previous structural development studies on thalidomide, was revealed to possess potent tubulin-polymerization-inhibiting activity, comparable to that of the known tubulin-polymerization inhibitors, rhizoxin and colchicine. A major metabolite of thalidomide, 5-hydroxythalidomide, which possesses a hydroxyl group at the position corresponding to that of 5HPP-33, also showed moderate inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2004.10.072

Bioorganic & Medicinal Chemistry Letters 15 (2005) 321–325
Tubulin-polymerization inhibitors derived from thalidomide
b
a,
Shunsuke Inatsuki, Tomomi Noguchi, Hiroyuki Miyachi, Sawako Oda,
a,
a
b
b
a
Toyotaka Iguchi, Masahiro Kizaki, Yuichi Hashimoto and Hisayoshi Kobayashi
a,
*
a
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b
Division of Hematology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Received 6 October 2004; revised 25 October 2004; accepted 25 October 2004
Available online 13 November 2004
Abstract—2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), which was obtained during our previous struc-
tural development studies on thalidomide, was revealed to possess potent tubulin-polymerization-inhibiting activity, comparable to
that of the known tubulin-polymerization inhibitors, rhizoxin and colchicine. A major metabolite of thalidomide, 5-hydroxythalido-
mide, which possesses a hydroxyl group at the position corresponding to that of 5HPP-33, also showed moderate inhibitory activity.
Ó 2004 Elsevier Ltd. All rights reserved.
1
. Introduction
We have demonstrated that the TNF-a production-reg-
ulating activity of thalidomide is bidirectional, and that
2
,4,5
Thalidomide (1: Fig. 1) is a sedative/hypnotic drug
which was once withdrawn from the market in the
960s because of its severe teratogenicity. However,
thalidomide is a multi-target drug.
development studies of thalidomide, we have obtained
In structural
1
,2
1
TNF-a production regulators (including bidirectional
ones, pure inhibitors, and pure enhancers), androgen
7
,8
the drug has been established to be effective for the treat-
ment of various diseases, including leprosy, myeloma,
9
–11
12–14
antagonists,
sidase inhibitors,
aminopeptidase inhibitors,
15,16
a-gluco-
thymidine phosphorylase inhibi-
3
–5
AIDS, and others.
It was approved in the United
1
7
18,19
States for the treatment of leprosy in 1998, and clinical
studies of its use for the treatment of various cancers,
including multiple myeloma, colon cancer, prostate tu-
mor, and breast cancer are on-going. The molecular
mechanisms of the multiple pharmacological actions
elicited by thalidomide are not clear, but its tumor nec-
rosis factor (TNF)-a production-inhibitory activity has
tors, cyclooxygenase (COX) inhibitors,
2
and nitric
In the course of
0,21
oxide synthase (NOS) inhibitors.
those studies, we noticed that some phenylphthalimide
derivatives show potent cell proliferation-inhibiting
activity toward human leukemia cell lines HL-60
(Table 1), THP-1 and IM9. Among the compounds
listed in the table, 2-(2,6-diisopropylphenyl)-5-hydroxy-
1H-isoindole-1,3-dione (5HPP-33: 11), showed the most
potent activity, with the IC50 value of approximately
4
–6
been well-documented.
5
lM. This cell growth-inhibitory activity seemed to be
specific to 5HPP-33, though some features of the struc-
ture–activity relationship could be deduced. For exam-
ple, introduction of a substituent (hydroxyl, amino or
nitro group) at the 5-position seems to generate the
activity [4HPP-00 (6), 4HPP-11 (7), 4APP-33 (12), and
O
R
N
O
NH
O
O
4
5
3
NPP-33 (14) are inactive, while the corresponding
HPP-00 (9), 5HPP-11 (10), 5APP-33 (13), and 5NPP-
3 (15) are active; the exception is 4HPP-33 (8), though
1
2
: R=H, Thalidomide
: R=OH, 5-Hydroxythalidomide
Figure 1. Structures of thalidomide (1) and one of its known
metabolite, 5-hydroxythalidomide (2).
the corresponding 5HPP-33 (11) is far more potent]. De-
tailed observation of cells treated with 5HPP-33 (11)
indicated that the morphological change of the treated
cells is similar with that of cells treated with tubulin-
polymerization inhibitors, such as rhizoxin and colchi-
cines, and we considered that 5HPP-33 might elicit its
Keywords: Thalidomide; Tubulin; Polymerization; Inhibitor.
*
Corresponding author. E-mail: kobayas@imcbns.iam.u-tokyo.ac.jp
The first two authors (S.I. and T.N.) contributed equally to this work.
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.072

322
S. Inatsuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 321–325
Table 1. Cell growth-inhibitory activity of phenylphthalimide derivatives (3–15) toward human leukemia cell line HL60
O
R
4
5
X
N
O
R
Compound
R
X
Growth inhibition (%)
1
0lM
6lM
3lM
PP-00 (3)
PP-11 (4)
PP-33 (5)
H
CH
H
H
H
ꢀ0
ꢀ0
<5
ꢀ0
ꢀ0
8
ꢀ0
ꢀ0
ꢀ0
ꢀ0
ꢀ0
ꢀ0
<5
ꢀ0
72
ꢀ0
ꢀ0
ꢀ0
ꢀ0
ꢀ0
ꢀ0
ꢀ0
ꢀ0
6
3
CH(CH
H
3
)
)
2
2
4HPP-00 (6)
4HPP-11 (7)
4HPP-33 (8)
5HPP-00 (9)
5HPP-11 (10)
5HPP-33 (11)
4APP-33 (12)
5APP-33 (13)
4NPP-33 (14)
5NPP-33 (15)
4-OH
4-OH
4-OH
5-OH
5-OH
5-OH
4-NH
5-NH
CH
3
CH(CH
H
3
34
6
CH
3
CH(CH
CH(CH
CH(CH
CH(CH
CH(CH
3
3
3
3
3
)
)
)
)
)
2
2
2
2
2
ꢀ100
ꢀ0
23
2
2
ꢀ0
<5
ꢀ0
<5
ꢀ0
ꢀ0
ꢀ0
ꢀ0
4-NO
5-NO
2
ꢀ0
16
2
Figure 2. Morphological change of human myeloma cells, IM9, treated with 10lM 5HPP-33 (11) and DNA ladder formation of the treated cells.
Panel a: control IM9 cells. Panel b: IM9 cells treated with 5HPP-33 (11). Panel c: agarose gel electrophoresis of DNA extracted from the treated cells
at the indicated times after the start of incubation with 10lM 5HPP-33 (11). Line M: molecular weight markers.
cell growth-inhibitory activity by cell cycle arrest and
inducing apoptosis of the cells (Fig. 2, detailed will be
published elsewhere). The observations led us suspect
tubulin as a target molecule of 5HPP-33. In fact, Hu
et al. reported briefly that 5HPP-33 inhibits depolymer-
ization of tubulin. In this paper, we report (1) the effect
of 5HPP-33 (11) and its derivatives on tubulin polymer-
ization, and (2) the discovery of tubulin-polymerization-
inhibiting activity of a known thalidomide metabolite, 5-
hydroxythalidomide (2).
mercaptoethanol and 1mM GTP (pH6.5). Although
the quantitative values differed from experiment to
experiment, the results were basically reproducible. Typ-
ical sets of data are presented in Table 2 and Figure 3.
Synthesis of all the compounds listed in Table 1 and
their chemical/physical analytical data have already
22
2
7
been reported.
As shown in Table 2, 5HPP-33 (11) and its isoelectronic
derivative, 5APP-33 (13), showed potent tubulin-polym-
erization-inhibiting activity with efficacy comparable to
that of rhizoxin or colchicine. The structural require-
ment for the activity seems to be critical, because other
analogs investigated, including regio-isomers of 5HPP-
33 (11) and 5APP-33 (13) (4HPP-33: 12 and 4APP-33:
14, respectively), and derivatives of 5HPP-33 (11) with
less bulky alkyl groups (5HPP-00: 9 and 5HPP-11: 10),
showed no (5, 9, 10, 12), or only slight (8) tubulin-
polymerization-inhibiting activity. Nitro-substituted
analogs (4NPP-33: 14 and 5NPP-33: 15) were also inac-
tive. The results suggest that the tubulin-polymerization-
inhibiting activity of phenylphthalimide analogs is a spe-
2
. Results and discussion
First we investigated the effect of thalidomide (1),
5
erization using the method previously described.
HPP-33 (11) and various derivatives on tubulin polym-
2
3,24
Briefly, microtubule protein was prepared from porcine
2
5,26
brain.
Tubulin polymerization was followed by tur-
bidity measurements at 37°C in microtubule assemble
buffer containing 100mM 2-morpholinoethanesulfonic
acid (MES), 1mM EGTA, 0.5mM MgCl , 1mM 2-
2

S. Inatsuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 321–325
323
Table 2. Tubulin-polymerization-inhibiting activity of phthalimide
analogs
this phenomenon, the result was reproducible in re-
peated. 4HPP-33 (8) appears to retard the initiation of
tubulin polymerization, while having little effect on the
final degree of tubulin polymerization.
Compound
Polymerization Compound
a
inhibition (%)
Polymerization
a
inhibition (%)
PP-33 (5)
<10
7
Thalidomide <10
1)
32
Next we investigated the tubulin-depolymerization-
inhibiting activity of 5HPP-33 (11). Hu et al. briefly re-
ported that 5HPP-33 shows inhibitory activity toward
depolymerization of tubulin. Depolymerization of tub-
ulin was induced by cooling the tubulin fraction polym-
(
5-Hydroxy-
4HPP-33 (8)
thalidomide
b
22
(
Rhizoxin
2)
5
5
5
4
5
4
5
HPP-00 (9)
<10
88
78
HPP-11 (10) <10
HPP-33 (11) 83
APP-33 (12) <10
APP-33 (13) 81
Colchicine
erized at 37°C to 0°C, or by addition of 4mM CaCl at
2
3
7°C, and was followed by means of turbidity measure-
23–25
ments as described previously. As shown in Figure 4
(circles), once-polymerized tubulin was depolymerized
NPP-33 (14) <10
NPP-33 (15) <10
by cooling (Fig. 4, black circles) or by addition of CaCl2
(
Fig. 4, gray circles). The tubulin depolymerized by cool-
a
Calculated from the change of the turbidity at 400nm after incuba-
tion in the presence of test compound (20lM) for 30min at 37°C
compared with that after incubation in the absence of the test
compound.
ing could be re-polymerized again by warming at 37°C.
The addition of 10lM 5HPP-33 (11) to polymerized
tubulin at 37°C did not affect the cooling- or CaCl -in-
duced depolymerization step (Fig. 4, gray squares). As
expected, the tubulin depolymerized by cooling did not
re-polymerize by warming on the presence of 5HPP-33
2
b
The concentration of the test compound was changed to 50lM.
(
11) (Fig. 4, gray squares). Thus, 5HPP-33 (11) showed
1
00-
[
[
: control]
: thalidomide (1) ]
no inhibiting effect on tubulin depolymerization, at least
in our system, which is contrary to the result reported by
Hu et al. Our finding indicates that 5HPP-33 (11)
binds only to heterodimeric a/b-tubulin protein, but
not to polymerized tubulin.
9
8
0 -
2
2
0 -
7
0 -
0 -
6
[
: 5-hydroxy-
5
4
3
0-
0 -
0-
0-
thalidomide (2) ]
[
: 4HPP-33 (8) ]
: 5HPP-33 (11) ]
00-
-
1
[
: control]
[
9
8
0 -
[
: CaCl2]
2
1
0 -
(not cooled)
0-
addition of
HPP-33
0 -
0 -
[
: rhizoxin]
7
6
5
0
-
0
10
20
30
cooling (
, )
INCUBATION TIME (min)
or addition of
CaCl2 ( )
5
0-
Figure 3. Time course curves of inhibition of tubulin polymerization
monitored in terms of turbidity (absorbance at 400nm). A tubulin
preparation was incubated at 37°C in the presence or absence (control)
of test compound. The test concentration was 10lM for 4HPP-33 (8),
40 -
°
3
2
0-
0-
37 C
[
:
5HPP-33 (11) ]
5HPP-33 (11) and rhizoxin, and 50lM for thalidomide (1) and 5-
hydroxythalidomide (2).
10-
0
-
0
20
40
60
cific feature of the 2,6-di-isopropylphenylphthalimide
structure substituted with an electron-donating group
at the 5-position.
28 33
cooling period
INCUBATION TIME (min)
Although the tubulin-polymerization-inhibiting activity
of 5HPP-33 (11) is consistent with the cell growth-inhib-
itory activity of the compound, that of 5APP-33 (13) is
not (Table 1). This might be explained by a difference in
the cell membrane permeability of the two compounds
Figure 4. Time course curves of tubulin polymerization/depolymeri-
zation monitored in terms of turbidity (absorbance at 400nm). A
tubulin preparation was incubated at 37°C (starting at time 0min) to
polymerize it in the absence of 5HPP-33 (11), then cooled to 0°C
(
black circles, the cooling was started at time 28min) in the absence
(
black circles) or presence of 10lM 5HPP-33 (11) (gray squares:
(
details will be discussed elsewhere).
5
HPP-33 was added at time 20min). The time course of cooling-
induced depolymerization (cooling period: time 28–33min) was not
monitored (black circles and gray squares). The treated tubulin
fractions were heated again to 37°C at the time 38min. Gray circles:
Time course curves of the inhibition of tubulin polymer-
ization by selected compounds are shown in Figure 3. 4-
HPP-33 (8), a regio-isomer of 5HPP-33 (11), showed an
unusually shaped curve. Though we cannot yet interpret
the polymerized tubulin was treated with 4mM CaCl (added at time
2
28min) without cooling.

324
S. Inatsuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 321–325
1
2
) carbobenzoyloxychloride/NaOH/H20
O
O
O
) Et N/CF CONH /1-hydroxybenzotriazole/CH Cl /
3
3
2
2
2
HO
OH
N-(3-dimethylamino)propyl-N-ethylcarbodiimide
NH
3
) HBr/CH3COOH
H2N
NH2
O
(
or L-glutamic acid)
1
7 (HBr salt)
O
1
2
) Et3N/THF/molecular sieves
) Pd-C/H /AcOEt/CH COOH
O
2
+
17 (HBr salt)
O
O
2
3
16
Figure 5. Preparation of 5-hydroxythalidomide (2).
The potent cell growth-inhibitory activity and tubulin-
polymerization-inhibiting activity of 5HPP-33 (11) led
us suspect a relation between inhibition of tubulin
polymerization and the usefulness of thalidomide (1)
as an anti-tumor agent, especially for multiple myeloma.
Recently, so-called immunomodulatory drugs based on
thalidomide (IMiDs), especially amino-substituted tha-
lidomide analogs, have been reported to be superior can-
at least in part. In this sense, 5-hydroxythalidomde (2)
might be an active form of thalidomide (1), although
the cell growth inhibitory activity of 5-hydroxythalido-
mide (2) is weaker than that of 5HPP-33 (11) [5-
hydroxythalidomide (2) inhibited the cell growth of
HL-60 cells with a potency of 20–25% at 10lM, while
5-HPP-33 (2) inhibited HL-60 cell growth almost com-
pletely at the same concentration]. Possible biological
activities of other thalidomide metabolites remain to
be investigated.
6
didates for anti-tumor agents. IMiDs, as well as
thalidomide itself, were found to possess the ability to
directly induce growth arrest and caspase-dependent
apoptosis of tumor cells, though the activity of thalido-
In conclusion, we have developed a potent tubulin-
polymerization inhibitor, 5HPP-33 (11), by structural
elaboration of thalidomide. Based on the structural rela-
tionship between 5HPP-33 (11) and one of the major
metabolites of thalidomide, 5-hydroxythalidomide (2),
we found that the metabolite also possesses tubulin-
polymerization-inhibiting activity.
6
,28
mide (1) is less potent than those of IMiDs.
These
activities elicited by thalidomide (1) and IMiDs are sim-
ilar to those elicited by 5HPP-33 (11). Though thalido-
mide itself did not show any tubulin-polymerization-
inhibiting activity (Table 2 and Fig. 3), one or more of
its metabolites (or chemical decomposition products)
might possess tubulin-polymerization-inhibiting activ-
ity. Thalidomide (1) is known to be both chemically
and metabolically unstable, and the structures of vari-
Acknowledgements
2
9
ous metabolites have been reported. Because the 5-
hydroxyl group of 5HPP-33 (11) seemed to be critical
for the tubulin-polymerization-inhibiting activity, we fo-
cused on the thalidomide metabolite with a hydroxyl
group at the corresponding position, that is, 5-hydroxy-
thalidomide (2), which is a major microsomal metabolite
The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science,
and Technology, Japan, and the Japan Society for the
Promotion of Science.
2
9
of thalidomide.
5
tion of 4-benzyloxyphthalic anhydride (16) with 3-
-Hydroxythalidomide (2) was prepared by condensa-
3
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0
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1,32
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