Design and synthesis of thalidomide-deoxyribonucleoside chimeras

Article abstract of DOI:10.1246/cl.2009.1046

Novel thalidomide-deoxyribonucleoside chimeras have been synthesized as potentially safe, thalidomide-based antitumour agents. Copyright

Full text of DOI:10.1246/cl.2009.1046

1046
Chemistry Letters Vol.38, No.11 (2009)
Design and Synthesis of Thalidomide–Deoxyribonucleoside Chimeras
Shigeru Suzuki,1 Takeshi Yamamoto,1 Etsuko Tokunaga,1 Shuichi Nakamura,1
Motohiro Tanaka,2 Takuma Sasaki,2 and Norio Shibataꢀ1
1Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology,
Gokiso, Showa-ku, Nagoya 466-8555
2School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650
(Received July 16, 2009; CL-090667; E-mail: nozshiba@nitech.ac.jp)
thalidomide part
Novel thalidomide–deoxyribonucleoside chimeras have
been synthesized as potentially safe, thalidomide-based antitu-
mour agents.
O
X
O
O
X
N
O
NH
N
H
O
H
NH
N
O
O
HO
O
O
thalidomide: X=H
5-hydroxythalidomide: X=OH
Discovery of a variety of biological effects of thalidomide
such as neurotrophic and neurotoxic effects, anti-inflammatory
and immunomodulatory activity, has prompted recent re-evalu-
ation of its medical applications, despite its notorious drug dis-
aster half a century ago.1 Particularly, due to its strong antiangio-
genesis property,2 thalidomide has served as a new lead in anti-
cancer drug design.1a,1b However, the potent teratogenic side
effect tends to preclude its development as a revival drug; there-
fore its clinical use has continued to be a matter of controversy in
recent years.1c To circumvent the lethal drawback associated
with thalidomide, the development of thalidomide analogues
that have increased efficacy and no teratogenicity has been im-
portant. When it was discovered that the teratogenic effect was
associated only with its S enantiomer,3 it was believed that the
tragedy could have been avoided if only the R isomer had been
administered. However, the tendency of thalidomide to epimer-
ize under physiological conditions4 has raised questions about
the assumption whether thalidomide is stereospecifically tera-
togenic and therefore, the development of nonracemizable, chi-
ral analogues of thalidomide has attracted much attention.5–7 Al-
though the mechanism of action of thalidomide is not fully un-
derstood, a recent biological study revealed that both teratoge-
nicity and antitumor activity of thalidomide are presumably
based on its antiangiogenic action.1,8 This suggests that develop-
ment of safe thalidomide drugs, which are not teratogenic while
retaining their promising antitumor activity, seems to be impos-
sible even if chiral, nonracemizable thalidomide analogues were
found. Searching for an alternative to thalidomide-based cancer
drugs, we are devising a new strategy based on tumor-specific
drug delivery, instead of the nonracemization strategy. The
drug-delivery system approach is well known in the treatment
of cancer, and a variety of drug conjugates with suitable carriers
are being evaluated as drug-delivery systems in anticancer ther-
apy. Therefore, tumor-specific thalidomide delivery could be ac-
complished by conjugation of thalidomide with a carrier which
is selectively taken up by tumor cells. Thalidomide–deoxyribo-
nucleoside chimera 1a is designed as a tumor-selective agent
with reduced potential teratogenicity. We are also interested
in 5-hydroxythalidomide–deoxyribonucleoside chimera 1b,
because 5-hydroxythalidomide, a major metabolite of thalido-
mide,7d,9 has an active antiangiogenic effect in the cell adhesion
molecule assays10 and therefore it is concluded that a metabolite
of thalidomide, not thalidomide itself, is responsible for its anti-
angiogenic effects. The structures of chimeras designed are
shown in Figure 1.
OH
deoxyribonucleoside part
1a: X=H, 1b: X=OH
Figure 1. Structures of thalidomide, 5-hydroxythalidomide,
and thalidomide–deoxyribonucleoside chimeras 1.
H
A)
D)
H
N
C)
O
O
O
N
O
Me
N
N
O
H
H
N
H
N
N
H
N
Me
H
O
H
N
H
H
H
O
N
H
N
N
N
O
N
N
Me
Me
H
Me
H
Me
H
N
N
N
N
N
O
O
H
H
H
B)
N
N
N
H
N
N
N
O
H
H
H
O
N
H
Me
Me
N
Me
N
N
N
Me
H
Figure 2. Dioxopiperidinyl moiety (enol tautomer) present in
thalidomide could act as a hydrogen-bonding partner towards
C or GC base pair.
Since tumor cells generally divide faster than normal cells,
they require more nucleic acid. As a result, the drug-nucleic acid
conjugate is more likely to be taken up into tumor cells. For this
reason, conjugates with nucleoside-related molecules such as ur-
acil mustard and nimustine are being developed as drugs for the
treatment of cancer.11 In addition to this tumor-specific drug de-
livery, the nucleoside carrier has added another advantage to the
chimera, that it can be useful as an unnatural nucleoside building
block for the synthesis of oligonucleotides. A DNA-like binding
interaction exists between nucleobases and the dioxopiperidinyl
moiety present in thalidomide.12 Indeed, our preliminary theo-
retical calculation study based on HF/6-31+Gꢀꢀ with 3-methyl-
piperidine-2,6-dione as the thalidomide model and methylated
nucleobases in DNA indicated that both the carbonyl and the
NH of the dioxopiperidinyl moiety present in thalidomide act
as hydrogen-bonding partners towards C or the GC base pair.
Possible examples are shown in Figure 2. It is interesting to note
that hydrogen bonding was observed theoretically only for the
enol tautomer of 3-methylpiperidine-2,6-dione, but not for 3-
methylpiperidine-2,6-dione itself, and it is as strong as the can-
onical Watson–Crick type (19.8 kcal molꢁ1 for model A, while
23.6 kcal molꢁ1 for a GC base pair B) or Hoogsteen type hydro-
gen bonding (40.9 kcal molꢁ1 for model C, while 63.1
kcal molꢁ1 for C:G:Cþ triplex D). The possibility of DNA inter-
calation of thalidomide is also proposed as one of the mecha-
nisms of its anticancer and antiangiogenic actions1d although
the supporting results to the hypothesis are insufficient. There-
fore, thalidomide-containing oligonucleotides could be key bio-
Copyright ꢀ 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.11 (2009)
1047
O
thalidomide and 5-hydroxythlidomide. The activity of 1a and
1b was increased relative to their parent compounds (KB cell
viability (%) at 100 mg mLꢁ1; thalidomide: 98.7%; 5-hydroxy-
thalidomide: 118.1%; 1a: 82.0%; 1b: 65.3%). A further biolog-
ical assay and chain assembly of the chimeras as DNA building
blocks for oligomers will be reported in due course.
O
O
H
X
X
X
O
N
O
N
NH
N
N
NH
NH
N
O
iii or iv
RO
O
O
O
O
O
TBSO
TBSO
O
O
OR
2: R=H, X=Br
i
OTBS
OTBS
5a: X=H
5b: X-OH
6a: X=H
6b: X-OH
3: R=TBS, X=Br
ii
4: R=TBS, X=NH2
This study was financially supported in part by Grants-in-
Aid for Scientific Research (B) No. 21390030 (JSPS).
Scheme 1. Reaction conditions: i) TBSCl (3 equiv), pyridine,
40 ꢂC, 12 h; ii) ammonia gas, 1,4-dioxane, 60 ꢂC, 5 d, 72% from
2; iii) N-(ethoxycarbonyl)phthalimide (2 equiv), toluene, reflux,
3 h, 49% (5a); iv) 4-hydroxyphthalic anhydride (1.2 equiv),
HMDS (3 equiv), ZnCl2 (1.5 equiv), benzene, reflux, 2 h, 66%
(5b).
References and Notes
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Med. 1999, 5, 489. d) T. D. Stephens, C. J. Bunde, B. J. Fillmore,
Biochem. Pharmacol. 2000, 59, 1489.
O
H
H2N
NH
N
O
TBSO
i
ii or iii
iv
1a
1b
6a: X=H
6b: X-OH
4
O
2
3
4
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OTBS
7
Scheme 2. Reaction conditions: i) H2 (3 atm), 5% Rh/Al2O3
(0.01 equiv), MeOH, rt, 90 h, 70%; ii) N-(ethoxycarbonyl)phthal-
imide (2.5 equiv), benzene, reflux, 12 h, quant. (6a); iii) 4-hy-
droxyphthalic anhydride (1.2 equiv), HMDS (3 equiv), ZnCl2
(1.5 equiv), benzene, reflux, 12 h, 98% (6b); iv) AcOH/H2O
(3/1), rt, 1–2 d, 84% (1a), 78% (1b).
5
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materials to elucidate the mechanism of action in thalidomide
teratogenicity in a suitable antisense or antigene approach.
Synthesis of chimeras 1a and 1b started with the tert-butyl-
dimethylsilyl (TBS) protection of 5-bromouridine (2) in pyridine
which gave the disilyl ether 3. The bromo function of 3 was then
replaced with ammonia in dioxane at 60 ꢂC for 5 days to furnish
TBS-protected amino uridine 4. Using the same precursor for 1a
and 1b, we performed the total synthesis of both chimeras. The
phthalimide of the amino moiety in 4 was converted using N-
(ethoxycarbonyl)phthalimide in toluene at reflux for 3 h to give
5a in 49% yield. The function of 5-hydroxyphthaloyl was intro-
duced by our silicon-induced phthaloylation using 4-hydroxy-
phthalic anhydride/ZnCl2/hexamethyldisilazane (HMDS) in re-
fluxing benzene7d to afford 5b in 66% yield. However, the hy-
drogenolysis of the conjugated olefins at the uridine moiety in
5 to 6 proved to be difficult: usual methods such as Rh/
Al2O3/H2,13 or Pd–C/H2 failed completely (Scheme 1). This
was presumably due to the steric hindrance around the olefin
substituents; alternatively, the hydrogenolysis of the double
bond was first adopted by Rh/Al2O3/H2 followed by phthaloy-
lation by means of 4-hydroxyphthalic anhydride/ZnCl2/HMDS
to afford the core structure 6 (Scheme 2). Finally the TBS deriv-
atives 6a, 6b can be fully deprotected by treatment with AcOH/
H2O at rt for 1–2 d to give target chimeras 1a and 1b in 84 and
78% yield, respectively.14
6
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In conclusion, we present here a design and synthesis of tha-
lidomide–deoxyribonucleoside chimeras 1a and 1b as potential
candidates of tumor-selective thalidomide drug. They are ex-
pected not to be teratogenic while retaining their promising anti-
tumor activity. To our knowledge, these are the first thalido-
mide–nucleic acid conjugates bearing thalidomide embedded
in the backbone of DNA. The preliminary cytotoxicity assays
of 1a and 1b were carried out against human nasopharynx carci-
noma KB cell lines in RPMI-1640 medium as compared with
14 Supporting Information is available electronically on the CSJ-
Journal Web site, http://www.csj.jp/journals/chem-lett/index.
html.
Published on the web (Advance View) October 3, 2009; doi:10.1246/cl.2009.1046