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Thalidomide as a Multi-Template for Development of Biologically Active Compounds.

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536
Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
Review
Thalidomide as a Multi-Template for Development of
Biologically Active Compounds
Yuichi Hashimoto
Institute of Molecular & Cellular Biosciences, The University of Tokyo, Tokyo, Japan
Thalidomide is a teratogenic/hypnotic/sedative agent which elicits a wide range of pharmaceutical/biological activities. The diversity of its biological activities suggested that the drug might be
useful as a multi-template for development of various kinds of biologically active compounds.
We adopted two strategies for the structural development of thalidomide. The first was to
develop the structure of the drug based on the target molecules to which thalidomide itself and/
or its metabolites directly bind, or the assay systems in which thalidomide itself and/or its
metabolites exhibit activity. Based on this strategy, tumor necrosis factor-a production-regulating agents, cyclooxygenase inhibitors, nitric oxide synthase inhibitors, histone deacetylase
inhibitors, anti-angiogenic agents, and tubulin polymerization inhibitors have been created.
The second was to develop the structure of thalidomide based on hypothetical target molecule(s)/biological response(s) which might be relevant to the pharmacological effects elicited by
thalidomide. Based on this strategy, androgen antagonists, progesterone antagonists, cell differentiation inducers, aminopeptidase inhibitors, thymidine phosphorylase inhibitors, l-calpain
inhibitors, a-glucosidase inhibitors and nuclear liver X receptors (LXRs) antagonists have been
created. Our structural development studies on thalidomide are reviewed focusing on recent
development of tubulin polymerization inhibitors, a-glucosidase inhibitors, and nuclear liver X
receptors antagonists.
Keywords: Biological response modifier / Enzyme inhibitor / Metabolites / Nuclear receptor ligand / Thalidomide /
Received: October 22, 2007; accepted: January 2, 2008
DOI 10.1002/ardp.200700217
Introduction
Small molecules with a wide range of biological activities
are likely to be superior seed compounds for creating
novel biologically active compounds, even if their biological activities include both favorable and unfavorable
ones. One example is thalidomide 1 (Fig. 1), a hypnotic /
sedative drug, which was launched in the 1950's, but was
Correspondence: Yuichi Hashimoto, Institute of Molecular & Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 1130032, Japan
E-mail: hashimot@iam.u-tokyo.ac.jp
Fax: +81 3 5841-8495
Abbreviations: nuclear liver X receptors (LXRs); multiple myeloma
(MM); cyclooxygenase (COX); nitric oxide synthase (NOS); histone deacetylase (HDAC); all-trans retinoic acid (ATRA); 1-deoxynojirimycin
(dNM);
2-(2,6-diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione
(5HPP-33)
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Figure 1. Structure of thalidomide.
withdrawn from the market in the 1960's because of
severe teratogenicity [1 – 5]. Despite this, thalidomide 1
has been established to be useful for the treatment of
Hansen's disease and multiple myeloma (MM), and the
drug was formally approved by the FDA (USA) for the former purpose in 1998 and for the latter purpose in 2006,
under critical control. Many reports have appeared on its
therapeutic usefulness in other various diseases, includ-
Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
Thalidomide as a Multi-Template
537
Table 1. Typical pharmacological effects elicited by thalidomide and their putative /
hypothetical target phenomena / molecules in relation to cancer chemotherapy.
ing various cancers, rheumatoid arthritis, graft-versushost diseases, acquired immunodeficiency syndrome
(AIDS), and others [1 – 5]. The anti-MM activity of thalidomide 1 is a particular focus of investigation, because thalidomide 1 overcomes the drug resistance of human MM
cells to conventional therapy [6]. Although various pharmacological effects elicited by thalidomide 1, including
tumor necrosis factor (TNF)-a production-regulating
activity and anti-angiogenic activity [1 – 5], have been
reported, the mechanism of its anti-MM activity remains
unclear.
On the other hand, pharmacological applications of
thalidomide 1 have been widely investigated. The beneficial pharmacological effects elicited by thalidomide 1
include anticachexia activity, anti-inflammatory activity,
antitumor-promoting activity, anti-angiogenic activity,
tumor cell invasion-inhibiting activity, antiviral activity,
and hypoglycemic effect (Table 1) [1, 5]. The prevailing
hypothesis had been that all of the beneficial effects of
thalidomide 1 are elicited through regulation of TNF-a
production. However, the TNF-a production-regulating
activity elicited by thalidomide 1 has been found to be bidirectional, and structural development studies of thalidomide 1 have indicated that there is no relationship
between TNF-a production-regulating activity and other
thalidomidal activities, including anti-angiogenic activity [1, 3 – 5]. These findings suggested that thalidomide 1
is a multi-target drug, and this, in turn, implied the possible usefulness of thalidomide 1 as a template for development of various kinds of medicaments. In fact, various
biologically active compounds, such as TNF-a production
regulators (including bi-directional ones, pure inhibitors, and pure enhancers) [7, 8], androgen antagonists
[9 – 11], aminopeptidase inhibitors [12 – 15], a-glucosidase
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inhibitors [16 – 18], thymidine phosphorylase inhibitors
[19], cyclooxygenase (COX) inhibitors [20 – 22], nitric
oxide synthase (NOS) inhibitors [23, 24], histone deacetylase (HDAC) inhibitors [25 – 27], anti-angiogenic agents
[28, 29], tubulin polymerization inhibitors [30 – 32], cell
differentiation inducers [33], and liver X receptor (LXR)
antagonists [34, 35] have been developed based on the
structure of thalidomide 1 (Fig. 2 and 3). In this article,
we review tubulin polymerization inhibitors [29 – 32], aglucosidase inhibitors [16 – 18], and LXR antagonists
[34, 35], as well as the recent progress of our related
research.
Tubulin polymerization inhibitors derived
from metabolites of thalidomide, 5-hydroxythalidomide, and N-hydroxythalidomide
As mentioned above, thalidomide 1 affords a superior
scaffold for development of various kinds of biologically
active compounds (Fig. 2 and 3) [1, 3 – 5, 7 – 35]. However,
thalidomide 1 itself elicits only some of the biological
activities listed in Fig. 2 and Fig. 3, i.e. TNF-a productionregulating activity [36, 37], COX-inhibiting activity [20],
NOS-inhibiting activity [23], and anti-angiogenic activity
[29]. In addition, thalidomide 1 is a labile compound
both metabolically and chemically. Therefore, metabolic
activation of thalidomide 1 may occur. On this basis,
known and/or possible thalidomide metabolites have
been comprehensively prepared [38, 39]. Among them,
two major thalidomide metabolites, 5-hydroxythalidomide (5-OH-thalidomide 9), and N-hydroxythalidomide
(N-OH-thalidomide 10) (Fig. 4) have been well investigated. During the course of these studies, enhancing
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Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
Figure 2. Typical enzyme inhibitors derived from thalidomide.
Figure 3. Typical biological response modifiers (nuclear receptor ligands and others) derived from thalidomide.
effects of thalidomide 1 and its metabolites (9 and 10) on
all-trans retinoic acid (ATRA)-induced granulocytic differentiation of human leukemia HL-60 cells were found
(Fig. 4) [40].
As shown in Fig. 4, thalidomide 1, 5-OH-thalidomide 9,
and N-OH-thalidomide 10 have no HL-60 cell differentiation-inducing activity by themselves. However, they
induce HL-60 cell differentiation in a dose-dependent
manner in the presence of a physiological concentration
of ATRA. Under the experimental conditions, HL-60 cell
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differentiation-inducing activity of ATRA alone was
scarcely detectable at the physiological concentration of
2 nM, but addition of 100 lM thalidomide 1 or its metabolites, 5-OH-thalidomide 9, and N-OH-thalidomide 10,
resulted in an increase of the proportion of differentiated, NBT (nitroblue tetrazolium)-positive cells to 22%
and 32%, respectively (Fig. 4). These NBT-positive cell percentage values correspond to a greater HL-60 cell differentiation-inducing activity than that elicited by 5 nM
ATRA (15% NBT-positive cells). Differentiation-inducing
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Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
Thalidomide as a Multi-Template
539
Figure 4. Effects of thalidomide 1, 5-OH-thalidomide 9, and N-OH-thalidomide 10 on ATRA-induced HL-60 cell differentiation.
Panels a – c: Percentage of NBT-positive cells treated with the indicated concentration of thalidomide 1, 5-OH-thalidomide 9, or NOH-thalidomide 10 in the presence or absence of 2 nM ATRA.
activity in the presence of 2 nM ATRA could be observed
at a concentration as low as 1 lM thalidomide 1, 5-OHthalidomide 9, or N-OH-thalidomide 10. Cell morphological analysis and studies using flow cytometry suggested
that the three compounds enhanced ATRA-induced HL60 cell differentiation [40].
Our previous studies had indicated that enhancing
effects on chemically induced cell differentiation are one
of general features of tubulin polymerization/depolymerization disruptors [41]. This led us to examine the effects
of the three compounds on tubulin polymerization/depolymerization [30]. Investigations using porcine tubulin
fraction revealed that 5-OH-thalidomide 9 and N-OH-thalidomide 10 possess moderate tubulin polymerizationinhibiting activity (Fig. 5A) [30, 32].
Structural development of tubulin polymerization
inhibitors based on 5-OH-thalidomide
Based on the discovery of the tubulin polymerizationinhibiting activity of thalidomide metabolites, 5-OH-thalidomide 9 and N-OH-thalidomide 10, structural development studies to create superior tubulin polymerization
inhibitors were initiated. As mentioned above, we have
obtained various kinds of biologically active derivatives
of thalidomide 1 (Fig. 2 and 3) [1, 3 – 5, 7 – 35]. In the
course of those studies, we noticed that the o,o9-diisopropylphenylphthalimide skeleton can often replace the
thalidomide skeleton. This led us to focus on 2-(2,6-diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP33) 5 (Fig. 3), which possesses a hydroxyl group at the
position corresponding to that of 5-OH-thalidomide 9
[30 – 32]. As expected, 5HPP-33 5 possesses a potent tubu-
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Figure 5. Effects of 5HPP-33 5 on tubulin polymerization/depolymerization.
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Y. Hashimoto et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
lin polymerization-inhibiting activity with the IC50 value
of 6.9 lM. Its isoelectronic analog, 5APP-33 6 (Fig. 3) was
also a potent tubulin polymerization inhibitor. The structural requirement for the activity seems to be critical,
because other analogs investigated, including a non-substituted analog (PP-33; 4) (Fig. 3), regioisomers, and derivatives of 5HPP-33 5/5APP-33 6 with less bulky alkyl groups
showed no or only slight tubulin-polymerization-inhibiting activity. Nitro-substituted analogs are also inactive.
The results suggest that the tubulin-polymerizationinhibiting activity of phenylphthalimide analogs is a specific feature of the 2,6-diisopropylphenylphthalimide
structure substituted with an electron-donating group at
the 5-position.
cell differentiation of HL-60 at 5 lM [33], and inhibits
tube formation (angiogenesis) of human umbilical vein
endothelial cells (HUVEC) [29]. Although it remains to be
investigated whether these biological effects of 5HPP-33 5
are elicited by tubulin polymerization inhibition, the
lack of teratogenicity of the compound, as far as investigated, suggests that it may be useful as a non-teratogenic
substitute for thalidomide 1.
Mode of tubulin polymerization-inhibition by 5HPP-33
As mentioned above, 5HPP-33 5 was found to be a very
potent tubulin polymerization inhibitor. Its activity is
comparable to that of the known potent tubulin polymerization inhibitor, rhizoxin [30 – 32]. Depolymerization of tubulin was induced by cooling of the tubulin
fraction polymerized at 378C to 08C, or by addition of
4 mM CaCl2 at 378C. As shown in Fig. 5, once-polymerized, tubulin was depolymerized by cooling to 08C. The
tubulin depolymerized by cooling could be re-polymerized again by warming at 378C. The addition of 5HPP-33 5
to polymerized tubulin at 378C did not affect the coolinginduced depolymerization step. As expected, the tubulin
depolymerized by cooling did not re-polymerize as effectively upon being warmed in the presence of 5HPP-33 5
(at least, the repolymerization was not enhanced). Thus,
5HPP-33 5 showed no inhibitory effect on tubulin depolymerization, and does not itself induce tubulin depolymerization, at least in our system. Our findings suggest that
5HPP-33 5 binds only to free a,b-tubulin heterodimer protein to inhibit tubulin polymerization, but not to polymerized tubulin. In fact, studies using surface plasmon
resonance (SPR) measurement techniques suggested that
5HPP-33 5 binds directly to a,b-tubulin heterodimer protein with the calculated association constant of
4.56106 M – 1 under our experimental conditions [32].
The calculated value is in good agreement with the IC50
value of 5HPP-33 5 for inhibition (6.9 lM) of tubulin polymerization. To examine the binding site(s) on tubulin of
our novel polymerization inhibitors, we performed binding competition studies using commercially available
radio-labeled colchicine and vinblastine, but no binding
competition was observed. The results suggested that
5HPP-33 5 does not bind to the site at which colchicine or
vinblastine binds. 5HPP-33 5 effectively induces apoptosis
of human leukemia cell lines HL-60, THP-1, and human
myeloma cells IM9 at 10 lM [30], induces granulocytic
Not all the biological activities of thalidomide 1 (Table 1)
can be interpreted as a result of TNF-a production-regulating activity alone (vide supra). We have created various
kinds of biologically active thalidomide-based compounds, as listed in Fig. 2 and 3 [1, 3 – 5, 7 – 35]. Among
the activities shown in Fig. 2 and 3, only TNF-a production-regulating activity, COX-inhibiting activity, NOSinhibiting activity, anti-angiogenic activity, HDAC-inhibiting activity, and tubulin polymerization-inhibiting
activity are elicited by thalidomide 1 or thalidomide
metabolites. Other biologically active compounds were
derived based on hypothetical target molecules / biological responses which might be relevant to each biological
effect listed in Table 1. Firstly, we identified pharmacological and biological effects of thalidomide. We then
formed a hypothesis as to the molecular target or target
phenomenon which might be relevant to each pharmacological / biological effect (Table 1). It is important to
note that it does not matter whether thalidomide 1 itself
really binds to the hypothetical molecular target. The
aim is simply to reproduce the relevant pharmacological / biological effect specifically by using newly prepared
compounds. The third step is the creation of potent and
specific compounds based on each biological assay system independently. Compounds thus prepared showed
the corresponding single biological activity. This means
that their overall structures are quite different from each
other. They mimic thalidomide's pharmacological / biological effects, but might have no relation to thalidomide
at the molecular mechanistic level. Nevertheless, we
believe that, by preparing compounds that mimic the
pharmacological/biological effects elicited by thalidomide 1 (even if the molecular mechanism is different
from that of thalidomide), and using combinations of
the prepared compounds, we will be able to reproduce or
reconstruct the spectrum of pharmacological / biological
effects of thalidomide 1.
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a-Glucosidase inhibitors and liver X receptor
antagonists derived from thalidomide,
structural development based on
hypothetical target molecules/phenomena
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Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
a-Glucosidase inhibitors derived from thalidomide
Among the pharmacological effects of thalidomide 1
shown in Table 1, i. e. anticachexia effect, anti-inflammatory effect, antitumor promotion effect, anti-angiogenic
effect, anti-cell invasion effect, antiviral effect, and hypoglycemic effect, only the anticachexia effect and antiinflammatory effect can be definitely interpreted in
terms of TNF-a production-regulating activity. The antitumor promotion effect can also be partly interpreted in
terms of the same activity, but is more likely to be mainly
due to anti-androgenic activity, especially in the case of
prostate cancer, and COX-2-inhibiting activity. The latter
activity should be related to the anti-inflammatory effect.
The anti-angiogenic effect can be interpreted partly in
terms of TNF-a production-regulating activity and, partly,
in terms of thymidine phosphorylase-inhibiting activity.
The latter activity might also play a role in the antiviral
effect. The antiviral effect, especially against immunodeficiency virus (HIV), might be partly explained by TNF-a
production-regulating activity. The anti-cell invasion
effect can be interpreted in terms of puromycin-sensitive
aminopeptidase (PSA)-inhibiting activity [12].
As for the remaining hypoglycemic effect, and, in part,
antiviral activity, we suspected that a-glucosidase-inhibiting activity might be important. a-Glucosidase is an
enzyme which catalyzes the final step in the digestion of
carbohydrates. Inhibitors of this enzyme may retard the
uptake of dietary carbohydrates and suppress post-prandial hyperglycemia, and could be useful in the treatment
of diabetes, obesity, and certain forms of hyperlipoproteinemia. They also have potential as antiviral agents controlling viral infectivity through interference with the
normal biosynthesis of N-linked oligosaccharides by glycosidation of viral coat/envelope glycoproteins and are
being investigated for the treatment of both cancer and
AIDS. A well-established classical a-glucosidase inhibitor
is 1-deoxynojirimycin (dNM). Some derivatives of dNM
have been shown to be effective against AIDS and B- and
C-type viral hepatitis. Our structural development studies based on a-glucosidase-inhibiting activity yielded tetrachlorophthalimide derivatives (Table 2) [16]. As shown
in Table 2, all of the tetrachlorophthalimide derivatives
(2, 3, 11 – 15) showed more potent a-glucosidase-inhibiting activity (IC50 of 2.0 – 10.9 lM) than dNM (IC50 =
47.6 lM). The tetrachlorophthalimide structure seems to
be necessary for a potent activity, because the corresponding unsubstituted phthalimide derivatives showed
no a-glucosidase-inhibiting activity.
Comparison of the a-glucosidase-inhibiting activity
(IC50 values; Table 2) of the methylene-spacered tetrachlorophthalimide derivatives (3, 11 – 15: n = 1 – 6;
Table 2) showed a clear tendency for potency to be
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Thalidomide as a Multi-Template
541
Table 2. a-Glucosidase inhibitory activity of CPnP (2, 3 and 11 –
15).
Figure 6. Lineweaver – Burk plot analysis of [A] CP0P 2 and [B]
CP4P 3.
&: in the absence of inhibitor; &: in the presence of inhibitor;
Vertical scale: 1/[V] (6105 M – 1 min); Horizontal scale: 1/[S]
(6103 M – 1).
dependent on the length of the methylene spacer (number n, Table 2); the potency increased in the order of
CP1P (11: n = 1) a CP2P (12: n = 2) a CP3P (13: n = 3) a CP4P
(3: n = 4) and decreased in the order of CP4P (3: n = 4)
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Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
Figure 7. LXR antagonistic activities of a-glucosidase inhibitors (CP0P 2 and CP4P 3) measured by means of reporter gene assay.
Various concentration of the test compounds were added in the presence of 0.1 lM T0901317. The relative luciferase activity (vertical
scale) induced with 0.1 lM T0901317 alone was defined as 100%.
A CP5P (14: n = 5) A CP6P (15: n = 6) and the maximum
potency was found for CP4P (3: n = 4).
The compound without a methylene spacer, CP0P (2:
n = 0; Table 2), showed exceptional characteristics. Its
potent a-glucosidase-inhibiting activity deviates from the
general trend of the activity of methylene-spacered tetrachlorophthalimide derivatives 3, 11 – 15, i. e. CP0P 2 possesses potent inhibitory activity which is comparable to
that of CP4P 3. This suggests that the mechanism of a-glucosidase inhibition elicited by CP0P 2 is different from
that elicited by CP4P 3. In fact, Lineweaver – Burk plot
analysis (Fig. 6) indicated that CP0P 2 inhibits the enzyme
non-competitively (the inhibition curve crosses the horizontal axis), while CP4P 3 inhibits it competitively (the
inhibition curve crosses the vertical axis), as dNM does.
The results suggest that CP4P 3 (and possibly other
methylene-spacered tetrachlorophthalimide derivatives
11 – 15) inhibits a-glucosidase by binding to its catalytic
site in a mutually competitive manner with the cognate
substrate, which implies that the methylene-spacered tetrachlorophthalimide group could be a sugar mimic [16].
Liver X receptor antagonists derived from
thalidomide-related a-glucosidase inhibitors
Liver X receptors (LXRa and LXRb) are members of the
nuclear receptor superfamily (a family of ligand-dependent transcription factors which modulate specific gene
expression and thereby influence diverse biological processes, including cell growth, differentiation, and metabolism) [42, 43]. The physiological ligands of LXRs are considered to be oxysterols, such as 24(S),25-epoxycholesterol (EPC) [42, 43]. Several synthetic agonists, including
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GW3965 [44] and T0901317 [45] and an antagonist, the
natural product riccardin C [46], have been reported.
LXRs function as heterodimers with other nuclear receptors, the retinoid X receptors (RXRa, RXRb, and RXRc), to
regulate important aspects of cholesterol homeostasis by
controlling the expression of their target genes, including the ATP binding cassette ABCA1 and CYP7A genes
[47, 49]. LXRs also regulate the expression of several genes
involved in glucose metabolism [49, 50]. Thus, LXRs have
been regarded as members of the metabolic subfamily of
nuclear receptors, participating in the regulation of both
lipid and sugar metabolism. An undesirable effect
observed with oxysterol-type LXR agonists, including
24(S),25-epoxycholesterol and 22-(R)-hydroxycholesterol,
was a significant increase in serum and liver triglyceride
levels via the up-regulation of SREBP-1c and other lipogenic genes in the liver. In addition, LXR-mediated lipotoxicity in pancreatic b-cells, i. e. induction of pancreatic
b-cell apoptosis through hyperactivation of lipogenesis
caused by chronic activation of LXR, has been also
reported [51]. Consequently, LXR antagonists might be
candidate therapeutic agents for the treatment of atherosclerosis. In fact, it has been reported that LXR antagonists reduce lipid formation and increase glucose metabolism in myotubes from lean, obese, and type 2 diabetic
individuals [52].
On the other hand, Mitro et al. reported that LXRs act
as glucose sensors, i. e. D-glucose and D-glucose-6-phosphate act as ligands for LXRs and activate their transcription activity with EC50 values of 3141 lM for LXRa and
308 lM for LXRb [53]. This finding indicates that LXRs recognize both oxysterols and glucose derivatives as their
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Thalidomide as a Multi-Template
543
Table 3. a-Glucosidase inhibitory activity of 1-deoxynojirimaycin
and typical LXR ligands.
Table 4. LXR antagonistic and a-glucosidase inhibitory activities
of riccardin C derivatives.
physiological ligands. As mentioned above, we have
developed a series of potent a-glucosidase inhibitors,
including CP0P 2 and CP4P 3 (Fig. 2, Table 2) [16]. CP0P 2
is a non-competitive inhibitor of a-glucosidase, whereas
CP4P 3 is a competitive inhibitor. The competitive inhibition of a-glucosidase by CP4P 3 indicated that this compound might be a structural mimic of glucose. On this
basis, we considered that CP4P 3might be recognized as a
glucose mimic by LXRs (vide supra), and might act as a
ligand for LXRs.
Although neither CP0P 2 nor CP4P 3 showed LXRs-agonistic activity, the competitive inhibitor, CP4P 3, showed
dose-dependent antagonistic activity toward both LXRa
and LXRb, as shown in Fig. 7 [34]. A non-competitive aglucosidase inhibitor, CP0P 2, did not show LXR-antagonistic activity (Fig. 7). CP4P 3 seems to be an almost nonselective (very slightly LXRa-selective) LXR antagonist
with calculated IC50 values of 88 lM for LXRa and 101 lM
for LXRb. LXR-antagonistic activity is not correlated with
a-glucosidase-inhibitory activity, because the non-competitive a-glucosidase inhibitor CP0P 2 did not show LXRantagonistic activity. The finding that the competitive a-
glucosidase inhibitor, CP4P 3, shows LXR antagonistic
activity suggests that they are indeed recognized by LXRs
as glucose mimics, as we had expected (vide supra) [34].
Although the precise mode of molecular recognition of
glucose and CP4P 3 by a-glucosidase/LXRs is not known at
this stage, the results imply some similarity between the
modes of molecular recognition by a-glucosidase and
LXRs. In fact, we found that typical LXR ligands, GW3965,
T0901317, and riccardin C, possess a-glucosidase-inhibiting activity with IC50 values of 4.8, 100, and 9.9 lM,
respectively (Table 3) [54]. Similarly, known a-glucosidase
inhibitors, especially competitive inhibitors, might act
as LXRs ligands. These activities are expected to be separable by appropriate structural development, especially for
riccardin C derivatives (Table 4) [54].
The CP0P 2 is inactive towards both LXRa and LXRb, as
mentioned above. However, insertion of a spacer with
one to four methylene units between the tetrachlorophthalimide moiety and the phenyl moiety resulted in the
appearance of antagonistic activity (Table 5), as is the
case for a-glucosidase-inhibiting activity (Table 2), i. e.
CP1P 11, CP2P 12, CP3P 13, and CP4P 3 at 100 lM showed
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Table 5. LXR-antagonistic activities of CPnP (2, 3, 11 – 14 and 16), 5CP4P (17) and 56CP4P (18) measured by means of reporter
gene assay. The test compounds (100 lM) were added in the presence of 0.1 lM T0901317. Inhibition (%) of the relative luciferase
activity induced by 0.1 lM T0901317 alone is presented.
Figure 8. LXR antagonistic activities of PP2P (7), PP50 (23), and 5CPPSS-50 (8) measured by means of reporter gene assay.
Various concentration of the test compounds were added in the presence of 0.1 lM T0901317. The relative luciferase activity induced
with 0.1 lM T0901317 alone was defined as 100%.
30 – 60% inhibition of T0901317-induced transcriptional
activation of both LXRa and LXRb [34]. Although no clear
structure-activity relationship concerning the methylene
spacer length was observed, the compounds with an odd
number of methylene units (CP1P 11 and CP3P 13)
seemed to be more potent than those with an even number of methylene units (CP2P 12 and CP4P 3) (though the
difference is not large). However, CP5P 14, a derivative
with a five-methylene unit spacer, showed weaker antagonistic activity toward LXRs as compared with CP4P 3.
This result suggests that insertion of an over-long spacer
causes disappearance of the activity. A derivative with a
ten-methylene unit spacer, CP10P 16, was inactive
towards both LXRa and LXRb (Table 5).
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The 5CP4P 17 and 56CP4P 18 are dechlorinated derivatives of CP4P 3. 5CP4P 17 and 56CP4P 18 showed similar
antagonistic activity towards both LXRa and LXRb, with
the former being slightly more potent (Table 5). The
results indicate that a phenylphthalimide skeleton can
be regarded as a novel scaffold for LXR antagonists. The
reported crystal structures of the ligand binding
domains of LXRs suggest that the ligand-binding pocket
is larger than that of retinoic acid receptors (RARs) and
smaller than that of peroxisome proliferator-activated
receptors (PPARs) [55, 56]. The amino acid sequences of
LXRs and the results of molecular evolutional analysis
suggest that LXRs are evolutionally close to PPARs, which
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Table 6. LXR antagonistic activities of 29-alkylated phenylphthalimide 19 – 25 and PP2P (7) in the presence of 0.1 lM T0901317.
Thalidomide as a Multi-Template
545
design and prepare 5CPPSS-50 (8) (Fig. 3). As expected,
5CPPSS-50 (8) was found to be the most potent LXR antagonist among the prepared compounds, with IC50 values of
9 – 14 lM for both LXRa and LXRb (Fig. 8) [35].
Conclusions
ing pocket of approximately 1300 – 1400 3 [57]. Based on
these findings, we designed Y-shaped, branched, 29-substituted phenylphthalimide derivatives, PPn0 19 – 25 and
PP2P (7) (Table 6 and Fig. 8).
As expected, PP2P (7) was a potent LXR antagonist
(Table 6). The IC50 values of PP2P (7) in the presence of
0.1 lM T0901317 were 9.8 lM for LXRa and 44 lM for
LXRb (Fig. 8) [34]. As shown in Table 4, the effect of the 29alkyl chain length on the LXR-antagonistic activity of the
compounds was clear. The compound with a methyl
group, PP-10 19, showed only very weak antagonistic
activity toward both LXRa and LXRb. The ethyl analog,
PP-20 20, showed also very weak antagonistic activity
toward LXRb, but it had moderate antagonistic activity
toward LXRa. The antagonistic activity of compounds
with a 29-alkyl chain longer than a methyl group
increased in the order of: PP-20 (20) a PP-30 (21) a PP-40
(22) a PP-50 (23), for both LXRa and LXRb. Further elongation of the 29-alkyl chain, i. e. PP-60 24 and PP-70 25,
scarcely affected (in the case of LXRa), or seemed to
slightly decrease (for LXRb) the activity. Thus, the 29-npentyl group (PP-50; 23) seemed to be the best substituent
for LXR-antagonistic activity. The substituent effect on
antagonistic activity seemed to be greater for LXRa than
for LXRb. The IC50 values of PP-50 23 were calculated to be
42 – 45 lM and 69-82 lM for LXRa and LXRb, respectively
(Fig. 8) [35].
Further structural development studies revealed that
5-chlorination and / or thiocarbonylation of PP-50 23
enhanced its LXR-antagonistic activity, which led us to
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
In this article, our structural development studies of thalidomide 1 focused on tubulin polymerization inhibitors, a-glucosidase inhibitors and LXR antagonists were
reviewed. Tubulin polymerization inhibitors were
derived from a thalidomide metabolite (5-OH-thalidomide 9), for which its tubulin polymerization-inhibiting
activity had been found. Other biologically active compounds, including TNF-a production regulators, COXinhibitors, NOS-inhibitors, HDAC-inhibitors, anti-angiogenic inhibitors (Figs. 2 and 3), were derived from thalidomide 1 and/or its metabolites for which the corresponding activity was observed. On the other hand, a-glucosidase inhibitors were derived from thalidomide 1
based on its hypothetical target molecule. A similar strategy was effective to create various biologically active
compounds, including puromycin-sensitive aminopeptidase (PSA) inhibitors, thymidine phosphorylase inhibitors, l-calpain inhibitors, DPP-IV-inhibitors, androgen
antagonists, progesterone antagonists, cell invasion
inhibitors, cell differentiation inducers, and retinoids.
Further hypothetical target molecules/biological
responses, including inhibition of phosphodiesterases
and transcription factor NF-kB, are also considered to be
signposts for further structural development studies of
thalidomide 1. LXR antagonists were derived from a-glucosidase inhibitors based on the hypothesis that both aglucosidase and LXRs recognize phthalimide derivatives
as glucose mimics.
Thalidomide 1 can be regarded as a multi-target drug,
though its pharmaceutically favorable effects have not
been completely interpreted at this stage. In addition,
thalidomide 1 is unstable both metabolically and chemically [38, 39]. Structural development based on active thalidomide metabolites as well as chemical modification to
endow stability of the compounds, would be useful to
investigate the molecular basis of thalidomidal action.
One example of the latter would be lenalidomide/CC1053/revlimid which is under clinical development for
the treatment of the myelodysplastic syndromes [2].
Structurally, lenalidomide/CC-1053/revlimid is a derivative of thalidomide 1 with 4-amination and 3-decarbonylation. These modifications of thalidomide 1 would be
expected to make it quite stable chemically. In fact, lenalidomide/CC-1053/revlimid is more than 103-fold stable
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546
Y. Hashimoto et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 536 – 547
Figure 9. Typical biologically active compounds based on a 3,3-diphenylpentane skeleton.
under the physiological conditions both in hydrolysis
and racemization. It can be also regarded as a mimic of
thalidomide metabolite 5-OH-thalidomide 9, because
both hydroxyl and amino group are electron donating
groups. In fact, lenalidomide/CC-1053/revlimid elicits
biological activities which are observed for thalidomide
1 and its metabolites, including COX-inhibiting and
tubulin polymerization-inhibiting activities.
All of these findings strongly suggest the value of thalidomide 1 as a multi-template for creation of various biologically active compounds. Another example of the
multi-template strategy is the application of a 3,3-diphenylpentane skeleton as a steroid skeleton substitute [58 –
62]. There exist various kinds of steroidal biologically
active compounds, including steroid hormones and neurosteroids. If the steroid and/or seco-steroid skeleton
could be replaced with another simple skeleton(s), such a
skeleton(s) would be a superior multi-template for creation of various biologically active compounds whose
activities mimic those of steroidal molecules. Such skeletons would also afford scaffolds for creating inhibitors of
enzymes whose substrates are steroidal/secosteroidal
compounds, including steroid biosynthetic/converting
enzymes. In fact, we have prepared various nuclear receptor ligands, including nuclear vitamin D3 receptor agonists [56 – 58], nuclear androgen receptor antagonists
[58 – 60], progesterone receptor antagonists, nuclear peroxisome proliferators-activated receptor (PPAR) agonists
[61], and nuclear farnesoid X receptor (FXR) agonists [61],
based on a 3,3-diphenylpentane skeleton as a steroid skeleton substitute (Fig. 9). The same strategy was successfully applied to create inhibitors of 5a-reductase, an
enzyme which converts testosterone to its active metabolite, dihydrotestosterone (Fig. 9) [62]. Overall, the author
would like to emphasize that this multi-template strat-
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
egy has already proved to be a very fruitful method for
drug design/development.
The authors have declared no conflict of interest.
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