Synthesis, configurational stability and stereochemical biological evaluations of (S)- and (R)-5-hydroxythalidomides

Article abstract of DOI:10.1016/j.bmcl.2009.02.108

The first asymmetric synthesis of (S)- and (R)-5-hydroxythalidomides, one of thalidomide's major metabolites, was achieved using HMDS/ZnBr₂-induced imidation as a key reaction. 5-Hydroxythalidomide was found to be configurationally more stable

Full text of DOI:10.1016/j.bmcl.2009.02.108

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3973–3976
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, configurational stability and stereochemical biological evaluations
of (S)- and (R)-5-hydroxythalidomides
a
a
a
Takeshi Yamamoto a, Norio Shibata a, , Daisuke Sukeguchi , Masayuki Takashima , Shuichi Nakamura ,
*
Takeshi Toru a, Nozomu Matsunaga b, Hideaki Hara b, Motohiro Tanaka c, Tohru Obata c, Takuma Sasaki c
a Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso, Showa-ku, Nagoya 466-8555, Japan
b Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
c Department of Bioorganic Chemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The first asymmetric synthesis of (S)- and (R)-5-hydroxythalidomides, one of thalidomide’s major metab-
olites, was achieved using HMDS/ZnBr2-induced imidation as a key reaction. 5-Hydroxythalidomide was
found to be configurationally more stable than thalidomide at physiological pH. Stereochemical biolog-
ical effects of thalidomide and 5-hydroxythalidomide on anti-angiogenesis and antitumor activities were
also investigated using racemic and pure enantiomers.
Received 5 February 2009
Revised 25 February 2009
Accepted 26 February 2009
Available online 3 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Thalidomide
Asymmetric synthesis
Metabolites
Thalidomide is one of the most notorious drugs that caused a
tragic medical disaster in the late 1950s in Germany, England, Can-
ada, Japan and more. It was prescribed as a sleeping drug/sedative
and spread to pregnant women as a remedy for the relief of their
nausea due to morning sickness. However, an epidemic of limb
malformations broke out in these countries and years later it was
ascertained that thalidomide was the cause of these terrible birth
defects. Although thalidomide was banned worldwide in 1962, it
has been licensed again for clinical use in several countries due
to the discovery of thalidomide’s unique multiple pharmacological
actions against a number of intractable diseases, such as leprosy,
human immunodeficiency virus replication in acquired immune
deficiency syndrome, and cancer.1
Thalidomide possesses a single chiral center and was marketed
as a racemate. In 1979, Blaschke et al found that the enantiomers
of thalidomide have different biological properties.2 While
sedative and hypnotic effects are associated with the R-enantio-
mer, S-enantiomer of thalidomide is responsible for the terato-
genic side effect. Nau and co-workers also supported the result
using an enantiomerically pure thalidomide derivative, EM-12.3
However, these reports are still a matter of debate because, under
physiological conditions, optically active thalidomide rapidly
undergoes racemization, making separation of its biological
effects impossible.4
During recent studies on the structure–activity relationships of
thalidomide and its derivatives, thalidomide metabolites have
emerged as biologically significant targets.5 Thalidomide is meta-
bolically labile and several metabolites have been isolated.6 Hence,
the possibility should be considered that the teratogenic side effect
or/and other multiple pharmacological activities might be due to
the metabolites, including their enantiomers, rather than thalido-
mide itself. Two major metabolites of thalidomide are 5-hydroxy-
thalidomide and 50-hydroxythalidomide. Recently, we reported the
synthesis and biological evaluation of optically active (S)- and (R)-
50-hydroxythalidomides.7 As an extension of our work on thalido-
mide research,8 we describe herein the asymmetric synthesis, con-
figurational stability and stereochemical biological evaluation of
(S)- and (R)-5-hydroxythalidomides (2) (Fig. 1).
Many research groups have actively studied the biological activ-
ity of racemic 2.5 For example, Hashimoto and co-workers revealed
that 2 possesses moderate anti-angiogenic activity and tubulin-
polymerization-inhibitory activity.5c,d,f Li and co-workers also re-
ported that 2 is an active anti-angiogenic agent.5b On the other
hand, to our knowledge, biological evaluation of optically active
(S)- and (R)-2 have never been examined, presumably due to both
the possibility of racemization under physiological conditions and
the lack of availability of enantiomerically pure (S)- and (R)-2. Pre-
viously, we reported a three-step synthesis of optically pure (S)-
and (R)-1.8b This synthetic route is simple but fully determined
for avoiding racemization during the sequence of its synthesis.
We therefore examined the synthesis of optically pure (S)- and
(R)-2 by a strategy similar to that of optically active 1 (Scheme
* Corresponding author. Tel./fax: +81 52 735 7543.
E-mail address: nozshiba@nitech.ac.jp (N. Shibata).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.108

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T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3973–3976
4
O
O
O
R
R
H
H
HO
H
3
N
O
N
O
NH
NH
N
O
O
O
O
)-1; R=H
)-2; R=OH
O
2
1
0
NH
(
S
S
)-1; R=H
)-2; R=OH
(
(
R
O
O
(
R
(R)-2:
(black line)
Figure 1. Structure of (S)- and (R)-thalidomides (1) and their metabolites, (S)- and
(R)-5-hydroxythalidomides (2).
210
-1
260
310
360
O
Δ
HO
H
1). First, the synthesis of 4 was attempted by the reaction of (S)-3
with 4-hydroxyphthalic anhydride (5) under heating condition, but
4 was obtained as a racemate. We next examined the reaction of 3
using N,O-bis-Boc-protected 4-hydroxyphthalimide 6 in the pres-
ence of Et3N in CH2Cl2 at room temperature. Unfortunately, again,
product 7 was obtained with a slight degree of racemization (91%
ee). After careful optimization of the introduction of the 4-hydrox-
yphthaloyl group, a combination of hexamethyldisilazane (HMDS)
and ZnBr29 was found to be very effective for the condensation of 3
with 5 to produce (S)-4 in high yield without any loss of enantio-
purity. Namely, (S)-3 was treated with 5 in the presence of Et3N
in 1,4-dioxane at room temperature for 4 h followed by the reac-
tion with HMDS and ZnBr2 in refluxing benzene for 6 h to afford
(S)-4 in high yield with >99% ee. The 5-hydroxyl group of 4 was
protected by the use of O-silylated ketene acetal 810 followed by
oxidation using a catalytic amount of RuO2 in the presence of ex-
cess NaIO4 in a two-phase system to furnish silylated 5-hydroxy-
thalidomide 10. Finally, the treatment of 10 with concn HCl
precipitated enantiomerically pure (S)-2 in high yield with 99%
ee. In a manner similar to that described for the preparation of
(S)-2, (R)-2 was obtained from (R)-3 in five steps in good overall
yield. The CD spectra of (S)-2 and (R)-2 indicated good agreement
with those in the literature (Fig. 2).11
We next investigated the stabilities of 1 and 2 toward racemiza-
tion and hydrolysis (decay). Optically pure (S)-1 and (S)-2 were
incubated at 37 °C and varying pH values, and monitored by HPLC
using a reported protocol.4b,7 CHIRALCEL OJ-H with ethanol was
used for the separation. Three-buffer systems, pH 6.24 (100 mM
sodium phosphate monobasic, 100 mM sodium phosphate diba-
sic), pH 7.23 (100 mM sodium phosphate monobasic, 100 mM so-
dium phosphate dibasic) and pH 8.78 (40 mM tris base, 40 mM
hydrochloric acid) were employed. The results are shown in Figs.
3 and 4. In the racemization study, (S)-1 was racemized and de-
cayed using all buffer solutions; the rate of racemization and
hydrolysis was rather quick in neutral and alkaline buffer solu-
tions, which was consistent with earlier studies by Hashimoto as
well as by us.4b,7 On the other hand, the stability of (S)-2 to racemi-
zation and hydrolysis was higher than (S)-1 under all the condi-
N
O
-2
-3
-4
NH
O
O
(S)-2:
λ [nm]
(gray line)
Figure 2. CD Spectra of (S)-2 and (R)-2.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
Incubation Time (h)
(S)-2
(S)-1
(S)-2
(S)-1
(S)-2
( )-1
pH 6.24
pH 6.24
pH 7.23
pH 7.23
pH 8.78
pH 8.78
S
Figure 3. Racemization studies of (S)-1 and (S)-2.
100
90
80
70
60
50
40
30
20
10
0
HCl
O
O
H2N
O
RO
RO
H
H
a
b
d
N
N
O
N
H
NH
NH
O
O
O
O
t
(
(
S
)-10: R=SiMe2 Bu
)-2: R=H
(
(
(
S
S
S
)-4: R=H
0
10
20
30
40
Incubation Time (h)
50
( )-3
S
e
c
S
)-7: R=Boc
t
(S)-1
(S)-2
(S)-1
(S)-2
(S)-1
pH 6.24
pH 6.24
pH 7.23
pH 7.23
pH 8.78
)-9: R=SiMe2 Bu
(S)-2
pH8.78
O
O
t
HO
BocO
OSiMe2 Bu
Figure 4. Hydrolysis (decay) studies of (S)-1 and (S)-2.
O
N-Boc
OMe
O
O
5
6
8
tions. The half-life to racemization of (S)-1 was estimated by a
plot of the experimental data, t0.5 = R/S = 0.5. While the racemiza-
tion half-lives of (S)-1 were found to be 27.8 h at 6.24 pH, 5.1 h
at 7.23, and 2.6 h at 8.78 in buffer solution at 37 °C, those of (S)-
2 were 53.2 h at 6.24 pH, 26 h at 7.23 and 13 h at 8.78.
Scheme 1. Reagents, conditions and yields: (a) 6, Et3N, CH2Cl2, rt, 10 h, 89%, 91% ee;
(b) (i) 5, Et3N, MS-3 Å, 1,4-dioxane, rt, 4 h; (ii) HMDS, ZnBr2, benzene, reflux, 6 h,
85%, >99% ee; (c) 8, MeCN. rt, 1 h; (d) RuO2, 10% NaIO4 aq AcOEt, CH2Cl2, 40 °C, 12 h,
78% (2 steps); (e) concn HCl aq, MeOH, rt, 1 h, 88%, >99% ee.

T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3973–3976
3975
2.5
2
The tube formation assay, one of the trusted assay systems, was
examined using an angiogenesis assay kit according to the manu-
facturer’s instructions. Human umbilical vein endothelial cells
(HUVECs) co-cultured with fibroblasts were cultivated in the pres-
ence or absence of various concentrations of test drugs plus Vascu-
lar endothelial growth factor-A (VEGF-A, 10 ng/ml).7,13 After
11 days, cells were fixed in 70% ethanol. The cells were incubated
with diluted primary antibody (mouse anti-human CD31, 1:4000)
for 1 h at 37 °C, and with the secondary antibody (goat anti-mouse
IgG alkaline phosphatase-conjugated antibody, 1:500) for 1 h at
37 °C, and visualization was achieved using 5-bromo-4-chloro-3-
indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). Images were
obtained from five different fields (5.5 mm2 per field) for each well,
and tube area was quantified using an angiogenesis image ana-
lyzer. The results are shown in Figure 8. While racemic 1 and (S)-
1 blocked vascular sprout formation in high concentration (tube
area; <60%), (R)-1, racemic 2, (R)-2 and (S)-2 showed weak inhibi-
tory activity (tube area; >70%). Although all the activities are weak,
(R)-2 is slightly more potent than (S)-2.
1.5
1
0.5
0
210 230 250 270 290
310
λ / nm
330 350 370
390 410
Figure 5. UV–vis spectra of 1 in buffer solution: pH 6 (black line), pH 7 (gray line),
pH 8 (black broken line), pH 9 (gray broken line).
Finally in vitro antitumor activities of enantiomers of 1 and 2
(100 lg/ml) were evaluated against KB cells from oral cancer,
It is interesting to note that the stabilization effect is markedly
high in basic conditions; we next investigated the UV–vis spectra
of (S)-1 and (S)-2 in different buffer solutions. The spectra are
shown in Fig. 5 and 6. While (S)-1 showed similar UV–vis spectra
in any pH conditions (Fig. 5), the UV–vis spectra of (S)-2 were pro-
nouncedly red-shifted in basic solution (Fig. 6). These results indi-
cate that the contribution of phenolphthalein-like resonance
structure in phthalimide moiety is responsible for the higher sta-
bility of (S)-2 (Fig. 7).
Biological evaluation of enantiomers of 1 and 2 were investi-
gated next. Among the diverse biological activities of thalidomide
that have been suggested, first, we were interested in anti-angio-
genesis activity of thalidomide. It is hypothesized that the anti-
angiogenic activity is correlated with teratogenicity but not with
the sedative or the mild immunosuppressive properties of thalido-
mide.12 The disruption of blood vessel formation in the fetal limb
bud might be responsible for the teratogenicity of thalidomide.
MCF-7 from breast cancer, and multiple myeloma (MM) cell lines
(IM-9, and Hs Sultan). Tumor cell suspension (5 ꢀ 103 cells/mL)
in RPMI-1640 medium was prepared before treatment. Each sam-
ple was dissolved in DMSO at the concentration of 10 mg/mL. Two
microliters of sample solution and 198 ll of cell suspension were
plated into a 96 well microplate and incubated for 72 h at 37 °C
in a humidified atmosphere of 5% CO2. The surviving cells were
determined by the MTT assay.14 As shown in Table 1 showed weak
inhibitory activity in MCF-7, IM-9, and Hs-Sultan cell lines. On the
120
Tube area
100
**
*
**
**
**
**
80
60
40
20
0
**
**
**
1.2
1
0.8
0.6
0.4
0.2
0
μM
30 100 30 100 30 100 30 100 30 100 30 100
racemic-1 (R)-1
(S)-1 racemic-2 (R)-2
(S)-2
10 ng/ml VEGF
Figure 8. Effects of 1 and 2 on tube area. Comparison of angiogenesis induced by
VEGF: mean S.E.M (VEGF: n = 9, VEGF + compounds: n = 3). *; P < 0.05, **; P < 0.01
versus VEGF (Dunnett’s multiple comparison test).
210
230 250
270
290 310 330 350
370 390 410
λ / nm
Table 1
Figure 6. UV–vis spectra of 2 in buffer solution: pH 6 (black line), pH 7 (gray line),
Antitumor activities of 1 and 2.
pH 8 (black broken line), pH 9 (gray broken line).
Compds
Cell viability (%) at 100 lg/ml
KB
MCF-7
IM-9
Hs-Sultan
O
Racemic-1
(S)-1
(R)-1
Racemic-2
(S)-2
(R)-2
92.2
95.6
96.9
118.1
121.0
151.0
86.5
81.3
89.8
122.1
199.7
204.6
107.0
93.5
87.1
48.2
67.2
57.9
85.9
88.6
75.6
55.6
35.0
46.4
O
O
H
O
H
N
O
N
O
NH
NH
O
O
O
O
Figure 7. Resonance of 2.

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T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3973–3976
other hand, growth of the MM cell lines was markedly inhibited by
2. It is also interesting to note that metabolite 2 possesses a prolif-
erating effect on KB and MCF-7 cells. No significant stereospecific
effect was observed for the antitumor activities between enantio-
mers of 1 and 2.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.108.
In summary, we have reported the first asymmetric synthesis of
(S)- and (R)-5-hydroxythalidomides (2). Racemization was effec-
tively avoided by the use of HMDS/ZnBr2-induced imidation meth-
od. Incubation experiments at physiological pH revealed that
metabolite 2 is configurationally more stable than thalidomide it-
self due to the contribution of phenolphthalein-like resonance
structures. A preliminary tube formation assay to assess the ability
to inhibit angiogenesis suggests that (S)-1 is the most active and its
enantiomer (R)-1, metabolites (R)-2 and (S)-2 are rather weak com-
pared to (S)-1. On antitumor activities, 2 exhibited proliferating ef-
fects in KB and MCF-7 cells while 1 has very weak antitumor
activities. Despite the higher configurational stability of 2, none
of the significant stereochemical effects in the antitumor assays
was observed for enantiomers of 2. It should be noted that the
inhibitory activity against MM cell lines of 2 is much stronger than
that of 1. All the results indicate that the multiple myeloma inhib-
itory activity of thalidomide may be responsible for its metabolite
2, while anti-angiogenesis is responsible for (S)-1. Although it is re-
ported that hydroxylation of 1 results in increased teratogenicity in
experiments using a developing chick embryo,5a our results appear
to indicate that metabolite 2 is slightly safer for the treatment of
multiple myeloma due to its weaker anti-angiogenesis property.
Further detailed studies to investigate the influence of stereochem-
istry and metabolites on biological activities of thalidomide would
give useful information to develop safer non-teratogenic drugs
based on thalidomide’s structure.
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Acknowledgments
Support was provided by JSPS KAKENHI (19390029, 19020024)
and Nagai Foundation Tokyo (Research Grant).