Mono- and dihydroxylated metabolites of thalidomide: Synthesis and TNF-α production-inhibitory activity

Article abstract of DOI:10.1248/cpb.54.1709

Mono- and dihydroxylated metabolites of thalidomide were efficiently prepared and characterized, and their inhibitory activity on tumor necrosis factor (TNF)-α production in the human monocytic leukemia cell line THP-1 was evaluated. 5,N-Dihydroxythalidomide was a much more potent TNF-α production inhibitor than thalidomide.

Full text of DOI:10.1248/cpb.54.1709

December 2006
Chem. Pharm. Bull. 54(12) 1709—1714 (2006)
1709
Mono- and Dihydroxylated Metabolites of Thalidomide: Synthesis and
a
TNF- Production-Inhibitory Activity
Takanori NAKAMURA, Tomomi NOGUCHI, Hisayoshi KOBAYASHI, Hiroyuki MIYACHI, and
Yuichi HASHIMOTO*
Institute of Molecular & Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received August 28, 2006; accepted September 13, 2006
Mono- and dihydroxylated metabolites of thalidomide were efficiently prepared and characterized, and
their inhibitory activity on tumor necrosis factor (TNF)-a production in the human monocytic leukemia cell line
THP-1 was evaluated. 5,N-Dihydroxythalidomide was a much more potent TNF-a production inhibitor than
thalidomide.
Key words thalidomide; hydroxythalidomide; metabolite; tumor necrosis factor (TNF)-a modulator
Thalidomide (1) was developed in the 1950’s as a nontoxic gen atom of the imide ring can also be hydroxylated (Fig.
sedative/hypnotic drug, but was withdrawn from the market 1).10) 5-OH-Thal (2a), N-OH-Thal (2c)11—13) and cis-5ꢀ-OH-
in the early 1960’s because of its serious teratogenicity.1—5) Thal (cis-2f)14—16) have been well-characterized and their
However, it was subsequently identified as an effective agent methods of preparation have been reported in detail. For 4-
for the treatment of multiple myeloma (MM), AIDS, OH-Thal (2b), trans-5ꢀ-OH-Thal (trans-2f), and the dihy-
Hansen’s disease, and various cancers.1—5) The US Food and droxylated metabolites, 5,N-di-OH-Thal (2d), 4,N-di-OH-
Drug Administration (FDA) approved it for the treatment of Thal (2e), and 5,5ꢀ-di-OH-Thal (2g), neither spectroscopic
erythema nodosum in Hansen’s disease in 1998, and (in com- data nor any detailed description of their synthesis is avail-
bination with dexamethasone) for the treatment of MM in able in the literature, to our knowledge. In addition, the re-
2006. Official approval for the use of thalidomide (1) to treat ported synthetic method of cis-5ꢀ-OH-Thal (cis-2f) via free
MM has also been applied for in Japan. Thalidomide (1) has g-hydroxyglutamic acid is unsatisfactory, because some of
been discovered to have various biological activities, includ- the intermediates are sticky and intractable, the overall yield
ing inhibition of tumor necrosis factor-a (TNF-a) produc- is not so high, and the stereochemistry is difficult to control.
tion, and anti-inflammatory, anti-angiogenic, and cyclooxy- We therefore sought to establish methods for systematic
genase (COX)-inhibitory activities.1—5) The TNF-a produc- preparation of all of the proposed metabolites, 2a—g. In this
tion-inhibitory activity was initially considered to be one of paper, we describe the synthesis of the mono- and dihydroxy-
the key mechanisms of thalidomide’s actions,1—5) though the lated metabolites of thalidomide (2a—g), as well as the re-
precise molecular mechanism(s) involved remain unclear.
sults of chemical/physical characterization and evaluation of
Recently, we have reported that thalidomide (1) and/or its their TNF-a production-inhibitory activity.
two hydroxylated metabolites 5-OH-Thal (2a) and N-OH-
Thal (2c) show cell differentiation-enhancing, anti-angio- Chemistry
genic and tubulin polymerization-inhibitory activities.6—9)
Hydroxylated metabolites 2a—e were synthesized as
Concerning the former two activities, thalidomide (1), 5-OH- shown in Chart 1.
Thal (2a) and N-OH-Thal (2c) all exhibit comparable activ-
Boc-glutamine 3 was treated with 1,1ꢀ-carbonyldiimida-
ity.7—9) However, tubulin polymerization-inhibitory activity zole (CDI) in the presence of a catalytic amount of 4-di-
was observed only for the hydroxylated metabolites (2a, 2c), methylaminopyridine (DMAP) to afford the cyclic imide 4.18)
and thalidomide (1) lacks this activity.6) These results The Boc group of 4 was removed with 30% HBr/AcOH to
prompted us to investigate comprehensively the biological afford 5 as the HBr salt. Boc-glutamic acid 6 was treated
activities of hydroxylated metabolites of thalidomide.
with O-benzylhydroxylamine in the presence of N-ethyl-Nꢀ-
Thalidomide (1) is metabolically labile, and many metabo- (3-dimethylaminopropyl)carbodiimide (EDCI) and 1-hydroxy-
lites have been identified or proposed,10—17) including seven benzotriazole (HOBt),13) to afford the cyclic imide 7. The
hydroxylated metabolites, 2a—g (Fig. 1). Hydroxylation is Boc group was removed with trifluoroacetic acid (TFA) to af-
reported to occur mainly at the 5-position in the phthaloyl ford 8 as the TFA salt. The hydroxyl groups of 4-hydroxyph-
moiety and the 5ꢀ-position in the glutarimide moiety,10) al- thalic acid (9a) and 3-hydroxyphthalic acid (9b) were pro-
though the 4-position of the phthaloyl moiety and the nitro- tected as the benzyl ether, and the protected compounds were
Fig. 1. Structures of Thalidomide (1) and Its Hydroxylated Metabolites (2)
∗ To whom correspondence should be addressed. e-mail: hashimot@iam.u-tokyo.ac.jp
© 2006 Pharmaceutical Society of Japan

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Vol. 54, No. 12
Chart 1
Chart 2
heated to 200 °C for 2 h without solvent to give the corre- estingly, NaIO4/RuO2 oxidation progressed only in the case
sponding anhydrides 11a and 11b, respectively. These com- of 16 as a substrate (neither the N-Cbz protected derivative of
pounds were treated with 5 or 8 in the presence of EDCI and 16 nor the O-Bn protected derivative of 16 could be oxidized
HOBt to afford 12a—e, except 12c which was prepared from under the same conditions to afford the corresponding pyrog-
13. The benzyl group of 12a—e was removed by catalytic lutamic amide derivatives of 17). The lactam part of 17 was
hydrogenation with H2 gas on 10% Pd/C to afford the hy- hydrolyzed with 1 mol/l LiOH in THF at 0 °C to afford the g-
droxylated thalidomide derivatives 2a—e.
5ꢀ-Hydroxylated metabolites [5ꢀ-OH-Thal (2f) and 5ꢀ,N- uct was formed as a by-product. Although several kinds of
di-OH-Thal (2g)] were synthesized as shown in Chart 2. alkaline hydrolysis were tried, e.g., with KOH and NaOH, the
hydroxyglutamine derivative 18,21,22) but the desilylated prod-
Trans-N-Boc-L-hydroxyproline 14 was treated with aque- by-product was always formed to some extent. Compound 18
ous ammonia (28%) in the presence of EDCI and HOBt as was cyclized using CDI in the presence of a catalytic amount
coupling reagents to give the carboxamide 15.19) Protection of DMAP to afford the glutarimide derivative 19. The Boc
of the hydroxyl group of 15 as the tert-butyldimethylsilyl group was selectively removed in the presence of TBDMS
(TBDMS) ether 16, followed by oxidation with NaIO4 and a ether with TFA/CH2Cl2 to afford 20 as the TFA salt.23) Care-
catalytic amount of RuO2 in AcOEt/H2O at room tempera- ful reaction of 20 and phthalic anhydride in the presence of
ture afforded the pyroglutamic amide derivative 17.20) Inter- EDCI and HOBt at 0 °C for 30 min afforded only the cis-

December 2006
1711
Table 1. TNF-a Production-Inhibitory Activity of Thalidomide (1) and Its
Hydroxylated Metabolites (2a—g) at 30 mM
isomer 21 (epimerization may occur at this step). When
the reaction time was extended to 2 d at room temperature,
epimerization occurred to afford a ca. 1 : 1 mixture of
cis- and trans-isomers. Trans-5ꢀ-OTBDMS-thalidomide 24
was isolated by recrystallization. Interestingly, cis-5ꢀ-ace-
toxythalidomide was reported to be more crystallizable than
trans-5ꢀ-acetoxythalidomide.14) Therefore, the crystallizabil-
ity may depend on the nature of the 5ꢀ-hydroxyl substituent.
Finally, removal of the TBDMS group of 21 with tetra-n-
butylammonium fluoride (TBAF) at 0 °C gave the desired
cis-5ꢀ-OH-Thal (cis-2f) stereoselectively. Trans-5ꢀ-OH-Thal
(trans-2f) was also prepared by means of a procedure similar
to that used for cis-5ꢀ-OH-Thal (cis-2f). Although it was not
clear whether the 3ꢀ or 5ꢀ position was epimerized, probably
Compound
TNF-a production-inhibitory activity (%)
Thalidomide (1)
5-OH-Thal (2a)
64.1
6.2
4-OH-Thal (2b)
N-OH-Thal (2c)
ꢁ2.1
29.7
90.9
69.8
74.0
72.1
53.1
5,N-di-OH-Thal (2d)
4,N-di-OH-Thal (2e)
cis-5ꢀ-OH-Thal (cis-2f)
trans-5ꢀ-OH-Thal (trans-2f)
5,5ꢀ-di-OH-Thal (2g)
the two were not epimerized simultaneously, judging from the glutarimide ring [N-OH-Thal (2c)] decreased the activity,
the large optical rotation values (see Experimental). This though the compound showed weak TNF-a production-in-
synthetic route is economical, and allows stereo-controlled hibitory activity (29.7% inhibition). On the other hand,
preparation of cis-5ꢀ-OH-Thal (cis-2f). 5ꢀ-OH-Thal’s (cis- mono-hydroxylation at the 5ꢀ-position of the glutarimide
and trans-2f) thus obtained were optically unstable, i.e., this moiety [5ꢀ-OH-Thal (cis-2f and trans-2f)] slightly enhanced
compound epimerizes rapidly to a 1 : 1 mixture of cis- and the activity (72.1—74.0% inhibition). The cis- and trans-iso-
trans-isomers under heating at 100 °C (data not shown).
TNF-a Production-Inhibitory Activity TNF-a is one possibly because of easy isomerization (vide supra).
of the cytokines mediating immune regulation, and has a Interestingly, although both mono-hydroxylation at the ph-
mers (cis-2f and trans-2f) showed almost the same efficacy,
wide range of activities extending beyond the well-character- thalimide/aromatic moiety [5-OH-Thal (2a) and 4-OH-Thal
ized pleiotropic pro-inflammatory properties to include di- (2b)] and at the nitrogen atom in the glutarimide moiety [N-
verse signaling for cell differentiation, proliferation and OH-Thal (2c)] caused loss or decrease of the activity, double
death.24,25) The growing understanding of the pathophysio- hydroxylation at these sites [5,N-di-OH-Thal (2d) and 4,N-
logical role of TNF-a in various diseases, including tumors, di-OH-Thal (2e)] retained or enhanced the activity (69.8—
as well as the discovery of the TNF-a production-inhibitory 90.9% inhibition). 5,N-di-OH-Thal (2d) showed the most po-
activity of thalidomide (1),26) has led researchers to regard tent activity among the compounds examined. It was far
this activity as the molecular basis for the pharmacological more potent than thalidomide (1), with an IC50 value of
effects elicited by thalidomide (1).27) Although thalidomide 3.7 mM in our assay system (TNF-a production inhibition
(1) has recently been found to be a multi-target drug,1—5) was almost complete at 30 mM, as shown in Table 1). Another
TNF-a production-regulating activity is still regarded as one di-hydroxylated analog, 5,5ꢀ-di-OH-Thal (2g), showed mod-
of the major molecular mechanisms of thalidomide’s action,1) erate TNF-a production-inhibitory activity (53.1% inhibi-
and the drug is recognized as an immunomodulatory agent. tion), which is weaker than that of thalidomide (1) or cis- and
Because thalidomide (1) is a metabolically labile compound, trans-5ꢀ-OH-Thal (cis- and trans-2f), but more potent than
as mentioned before, the question of whether its metabolites that of 5-OH-Thal (2a).
retain this activity or not is of great interest.
These results indicate that metabolic hydroxylation of
For this reason, we investigated the TNF-a production-in- thalidomide (1) can result in loss, retention, decrease or in-
hibitory activity of the hydroxylated metabolites of thalido- crease of TNF-a production-inhibitory activity. Therefore, it
mide (2a—g) by means of the previously reported method, is not easy to predict the influence of the metabolism of
using a human monocytic leukemia cell line, THP-1.28,29) thalidomide (1) on the TNF-a production-inhibitory activity
THP-1 cells do not produce TNF-a under normal cell culture in particular in vivo situations. Further studies to investigate
conditions, but do produce TNF-a in response to 12-O- the influence of metabolism on other biological activities of
tetradecanoylphorbol 13-acetate (TPA).30,31) Thalidomide (1) thalidomide are under way.
shows moderate TNF-a production-inhibitory activity in this
assay system.1—5,28,29) The TNF-a production-inhibitory ac-
Experimental
General 1H-NMR spectra were obtained on a JEOL JNM-a 500 spec-
tivity of test compounds was measured as described in the
experimental section as %-inhibition values, i.e., 100% and
trometer (500 MHz). Mass spectra were obtained on a JEOL JMA-HX 110
spectrometer with m-nitrobenzyl alcohol. Melting points, determined on a
0% represent complete inhibition and no inhibition, respec-
tively. Of course the %-values were variable from experiment
to experiment, but the order of efficacy was reproducible. A
typical set of data collected at the test compound concentra-
tion of 30 mM is presented in Table 1.
The mother compound, thalidomide (1) showed moderate
TNF-a production-inhibiting activity (64.1% inhibition at
30 mM), in accordance with our previous reports.28,29) Mono-
hydroxylation at the phthalimide/aromatic moiety [5-OH-
Thal (2a) and 4-OH-Thal (2b)] caused loss of the activity
Yanaco MP-J3 micro melting point apparatus are uncorrected. Optical rota-
tion was measured by JASCO DIP-1000. Flash column chromatographies
were performed on silica gel 60 Kanto Kagaku (40—100 mm).
tert-Butyl 2,6-Dioxopiperidin-3-ylcarbamate (4) To a solution of Boc-
Gln-OH 3 (10.0 g, 40.6 mmol) in dry THF (100 ml) was added 1,1ꢀ-car-
bonyldiimidazole (CDI) (7.78 g, 48.0 mmol), 4-dimethylaminopyridine
(DMAP) (1.22 g, 10.0 mmol) and triethylamine (TEA) (4.86 g, 48.0 mmol).
The mixture was stirred at room temperature for 3 d, then H2O was added.
The mixture was extracted with CHCl3 3 times, and the organic solution was
washed with 0.2 mol/l aqueous HCl, and brine, dried over MgSO4, filtered
and concentrated. The residue was purified by recrystallization (CHCl3/iso-
propyl ether) to afford 4 (8.30 g, 36.4 mmol, 90%) as white crystals. 1H-
(ꢁ2.1—6.2%). Mono-hydroxylation at the nitrogen atom of NMR (CDCl3): d: 8.22 (s, 1H), 5.36 (s, 1H), 4.31 (m, 1H), 2.79 (m, 1H),

1712
Vol. 54, No. 12
2.68 (m, 1H), 2.52 (m, 1H), 1.86 (m, 1H), 1.46 (s, 9H). MS (FAB): m/z 229
(MꢂH)ꢂ.
7.34 (t, Jꢃ7.3 Hz, 1H), 5.37 (s, 2H), 5.08 (dd, Jꢃ12.8, 5.4 Hz, 1H), 2.87 (m,
1H), 2.55 (m, 2H), 2.02 (m, 1H). MS (FAB): m/z 365 (MꢂH)ꢂ.
3-Aminopiperidine-2,6-dione Hydrobromide (5)
A mixture of 4
2-(1-Benzyloxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12c)
To a solution of N-phthaloylglutamic anhydride 13 (620 mg, 239 mmol) in
dry CH2Cl2 (20 ml) was added O-benzylhydroxylamine hydrochloride
(495 mg, 3.10 mmol), EDCI·HCl (959 mg, 5.00 mmol), HOBt·H2O
(676 mg, 5.00 mmol), and TEA (967 mg, 9.56 mmol). The reaction mixture
was stirred at room temperature for 1 d, then washed twice with 0.1 mol/l
aqueous HCl and once with brine, dried over MgSO4, filtered, and concen-
trated. The residue was purified by flash column chromatography
(hexane : AcOEtꢃ4 : 3 v/v) to afford 12c (731 mg, 2.01 mmol, 84%) as a
white solid. 1H-NMR (CDCl3): d: 7.88 (m, 2H), 7.76 (m, 2H), 7.53 (m, 2H),
7.35 (m, 3H), 5.04 (s, 2H), 5.00 (dd, Jꢃ15.6, 8.6 Hz, 1H), 2.95 (m, 1H),
2.75 (m, 2H), 2.10 (m, 1H). MS (FAB): m/z 365 (MꢂH)ꢂ.
5-Benzyloxy-2-(1-benzyloxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-
dione (12d) This compound was obtained as a white amorphous solid
(74% yield), in a manner similar to that described for the preparation of 12a
from 11a and 8. 1H-NMR (DMSO-d6): d: 7.87 (d, Jꢃ8.1 Hz, 1H), 7.36—
7.51 (m, 12H), 5.33 (m, 3H), 4.89 (s, 2H), 2.97 (m, 1H), 2.75 (m, 1H), 2.54
(m, 1H), 2.14 (m, 1H). MS (FAB): m/z 471 (MꢂH)ꢂ.
4-Benzyloxy-2-(1-benzyloxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-
dione (12e) This compound was obtained as a white amorphous solid
(30% yield), in a manner similar to that described for the preparation of 12a
from 11b and 8. 1H-NMR (DMSO-d6): d: 7.31—7.59 (m, 13H), 5.38 (s,
2H), 5.21 (dd, Jꢃ11.3, 5.2 Hz, 1H), 4.89 (s, 2H), 2.91 (m, 1H), 2.73 (m,
1H), 2.15 (m, 1H), 2.00 (m, 1H). MS (FAB): m/z 471 (MꢂH)ꢂ.
5-Hydroxy-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2a) (5-
OH-Thal) To a solution of 12a (590 mg, 1.62 mmol) in AcOH (15 ml) and
AcOEt (40 ml) was added 10% Pd–C (260 mg), and the mixture was stirred
at room temperature under an H2 atmosphere for 1 h. The reaction mixture
was filtered through Celite, and the filtrate was concentrated. The residue
was purified by flash column chromatography (hexane : AcOEtꢃ3 : 1 to 0 : 1
v/v) to afford 2a (260 mg, 0.949 mmol, 59%) as a white powder. mp
ꢄ300 °C. 1H-NMR (DMSO-d6): d: 11.06 (s, 1H), 11.01 (s, 1H), 7.73 (d,
Jꢃ8.1 Hz, 1H),7.16 (s, 1H), 7.14 (d, Jꢃ8.1 Hz, 1H), 5.07 (dd, Jꢃ12.6,
5.0 Hz, 1H), 2. 87 (m, 1H), 2.55 (m, 2H), 2.02 (m, 1H). MS (FAB): m/z 275
(MꢂH)ꢂ. Anal. Calcd for C13H10N2O5: C, 56.94%, H, 3.68%, N, 10.22%.
Found: C, 56.82%, H, 3.78%, N, 10.02%.
(200 mg, 0.877 mmol) and 30% HBr/AcOH (2 ml) was stirred at room tem-
perature for 30 min, then AcOEt was added until a precipitate formed. The
precipitate was collected by filtration and washed with AcOEt twice to af-
1
ford 5 (180 mg, 0.861 mmol, 98%) as a white solid. H-NMR (DMSO-d6):
d: 11.27 (s, 1H), 8.38 (br, 3H), 4.21 (dd, Jꢃ13.2, 5.1 Hz, 1H), 2.71 (m, 1H),
2.58 (m, 1H), 2.13 (m, 1H), 1.99 (m, 1H). MS (FAB): m/z 129 (MꢂH)ꢂ.
tert-Butyl 1-Benzyloxy-2,6-dioxopiperidin-3-ylcarbamate (7) To
a
solution of Boc-Glu-OH 6 (3.71 g, 15.0 mmol) in dry CH2Cl2 (100 ml) was
added N-ethyl-Nꢀ-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI·HCl) (11.5 g, 60.0 mmol), 1-hydroxybenzotriazole monohydrate
(HOBt·H2O) (8.11 g, 60.0 mmol), and TEA (9.11 g, 90.0 mmol). The reac-
tion mixture was stirred at room temperature for 3 d, then washed with
0.2 mol/l aqueous HCl, sat. aqueous NaHCO3, and brine, dried over MgSO4,
filtered, and concentrated. The residue was purified by flash column chro-
matography (hexane : AcOEtꢃ3 : 2 to 2 : 3 v/v) to afford
7 (4.35 g,
13.0 mmol, 87%) as a white solid. 1H-NMR (CDCl3): d: 7.36—7.51 (m,
5H), 5.29 (s, 1H), 5.03 (q, Jꢃ5.1 Hz, 2H), 4.31 (dd, Jꢃ15.6, 8.6 Hz, 1H),
2.85 (m,1H), 2.69 (m, 1H), 2.42 (m, 1H), 1.69 (m, 1H), 1.45 (s, 9H). MS
(FAB): m/z 335 (MꢂH)ꢂ.
3-Amino-1-benzyloxypiperidine-2,6-dione Trifluoroacetate (8)
A
mixture of trifluoroacetic acid (TFA) (15 ml) and 7 (1.90 g, 5.69 mmol) was
stirred at room temperature for 30 min. The reaction mixture was concen-
trated to afford 8 (3.90 g, quant.) as a pale yellow oil. This intermediate was
used for the next reaction without purification. MS (FAB): m/z 235
(MꢂH)ꢂ.
4-Benzyloxyphthalic Acid (10a) To a solution of 4-hydroxyphthalic
acid 9a (5.00 g, 27.5 mmol) in 1.7 mol/l aqueous KOH (80 ml, 138 mmol)
was added benzyl chloride (3.80 g, 30.0 mmol). The reaction mixture was
stirred at 120 °C for 12 h. After the reaction mixture had cooled, 2.0 mol/l
aqueous HCl was added to it until the product precipitated. The precipitate
was collected by filtration and washed with toluene twice, affording 10a
(8.23 g, quant.) as a white solid. This was used for the next reaction without
purification. 1H-NMR (DMSO-d6): d: 7.72 (d, Jꢃ9.5 Hz, 1H), 7.38 (m, 7H),
5.20 (s, 2H). MS (FAB): m/z 273 (MꢂH)ꢂ.
3-Benzyloxyphthalic Acid (10b) To a solution of 3-hydroxyphthalic
anhydride 9b (2.00 g, 12.2 mmol) in 2 mol/l aqueous KOH (30 ml, 60 mmol)
was added benzyl chloride (1.65 g, 13.0 mmol). The reaction mixture was
stirred at 120 °C for 10 h. After cooling, the mixture was acidified by addi-
tion of 2.0 mol/l aqueous HCl, and extracted 5 times with CHCl3/MeOH.
The organic layer was washed with brine, dried over MgSO4, filtered and
concentrated to afford 10b (2.54 g, 9.34 mmol, 77%) as a white solid. This
was used for the next reaction without purification. 1H-NMR (DMSO-d6): d:
7.29—7.48 (m, 8H), 5.18 (s, 2H). MS (FAB): m/z 273 (MꢂH)ꢂ.
4-Hydroxy-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2b) (4-
OH-Thal) To a solution of 12b (500 mg, 1.37 mmol) in MeOH (10 ml)
and DMF (10 ml) was added 10% Pd–C (50 mg), and the mixture was stirred
at room temperature under an H2 atmosphere for 4 h, then filtered through
Celite, and concentrated. The residue was purified by flash column chro-
matography (hexane : AcOEtꢃ2 : 3 v/v) to afford 2b (320 mg, 1.17 mmol,
85%) as a yellow powder. mp 281—282 °C. 1H-NMR (DMSO-d6): d: 11.07
(s, 1H), 11.14 (s, 1H), 7.64 (t, Jꢃ7.9 Hz, 1H), 7.31 (d, Jꢃ7.9 Hz, 1H), 7.24
(d, Jꢃ7.9 Hz, 1H), 5.06 (dd, Jꢃ12.6, 5.0 Hz, 1H), 2.87 (m, 1H), 2.52 (m,
2H), 2.01 (m, 1H). MS (FAB): m/z 275 (MꢂH)ꢂ. Anal. Calcd for
C13H10N2O5: C, 56.94%, H, 3.68%, N, 10.22%. Found: C, 56.65%, H,
3.76%, N, 10.06%.
4-Benzyloxyphthalic Anhydride (11a) 10a (2.20 g, 8.09 mmol) was
heated at 200 °C for 1 h. After cooling, the resulting material was purified by
recrystallization (AcOEt/hexane) to afford 11a (1.24 g, 4.88 mmol, 60%) as
1
pale yellow crystals. H-NMR (DMSO-d6): d: 7.99 (d, Jꢃ8.5 Hz, 1H), 7.66
(s, 1H), 7.55 (d, Jꢃ8.5 Hz, 1H), 7.48 (d, Jꢃ7.2 Hz, 2H), 7.41 (t, Jꢃ7.2 Hz,
2H), 7.35 (t, Jꢃ7.2 Hz, 1H), 5.35 (s, 2H). MS (FAB): m/z 255 (MꢂH)ꢂ.
3-Benzyloxyphthalic Anhydride (11b) This compound was obtained
as yellow crystals (80% yield), in a manner similar to that described for the
preparation of 11a from 10b as a starting material. 1H-NMR (DMSO-d6): d:
7.93 (t, Jꢃ7.7 Hz, 1H), 7.68 (d, Jꢃ7.7 Hz, 1H), 7.59 (d, Jꢃ7.7 Hz, 1H), 7.50
(d, Jꢃ7.3 Hz, 2H), 7.42 (t, Jꢃ7.3 Hz, 2H), 7.35 (t, Jꢃ7.3 Hz 1H), 5.41 (s,
2H). MS (FAB): m/z 255 (MꢂH)ꢂ.
5-Benzyloxy-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12a)
To a solution of 11a (270 mg, 1.06 mmol) in dry CH2Cl2 (20 ml) was added
5 (200 mg, 0.960 mmol), EDCI·HCl (767 mg, 4.00 mmol), HOBt·H2O
(540 mg, 4.00 mmol), and TEA (607 mg, 6.00 mmol). The reaction mixture
was stirred at room temperature for 3 d, then washed twice with 0.2 mol/l
aqueous HCl and once with brine, dried over MgSO4, filtered, and concen-
trated in vacuo. The residue was purified by flash column chromatography
(hexane : AcOEtꢃ3 : 2 v/v) to afford 12a (343 mg, 0.942 mmol, 98%) as a
white solid. 1H-NMR (DMSO-d6): d: 11.08 (s, 1H), 7.79 (d, Jꢃ8.1 Hz, 1H),
7.37—7.43 (m, 5H), 7.28 (m, 2H), 5.20 (s, 2H), 4.95 (dd, Jꢃ12.6, 5.1 Hz,
1H), 2.70—2.92 (m, 3H), 2.15 (m, 1H). MS (FAB): m/z 365 (MꢂH)ꢂ.
4-Benzyloxy-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12b)
This compound was obtained as a white solid (89% yield), in a manner simi-
lar to that described for the preparation of 12a from 11b and 5. 1H-NMR
(DMSO-d6): d: 11.09 (s, 1H), 7.82 (t, Jꢃ7.7 Hz, 1H), 7.59 (d, Jꢃ7.7 Hz,
1H), 7.51 (d, Jꢃ7.3 Hz, 2H), 7.46 (d, Jꢃ7.7 Hz, 1H), 7.41 (t, Jꢃ7.3 Hz, 2H),
2-(1-Hydroxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2c) (N-
OH-Thal) This compound was obtained as a white powder (75% yield), in
a manner similar to that described for the preparation of 2b from 12c. mp
250—251 °C. 1H-NMR (DMSO-d6): d: 10.27 (s, 1H), 7.92 (m, 4H), 5.34
(dd, Jꢃ12.8, 5.1 Hz, 1H), 3.02 (m, 1H), 2.79 (m, 1H), 2.56 (m, 1H), 2.07
(m, 1H). MS (FAB): m/z 275 (MꢂH)ꢂ. Anal. Calcd for C13H10N2O5: C,
56.94%, H, 3.68%, N, 10.22%. Found: C, 56.96%, H, 3.76%, N, 10.17%.
2-(1-Hydroxy-2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione
(2d) (5,N-di-OH-Thal) This compound was obtained as a white powder
(88% yield), in a manner similar to that described for the preparation of 2b
1
from 12d. mp 250—251 °C. H-NMR (DMSO-d6): d: 11.08 (s, 1H), 10.26
(s, 1H), 7.75 (d, Jꢃ7.7 Hz, 1H), 7.18 (s, 1H), 7.15 (d, Jꢃ6.9 Hz, 1H), 5.26
(dd, Jꢃ11.3, 5.2 Hz, 1H), 3.00 (m, 1H), 2.75 (m, 1H), 2.53 (m, 1H), 2.03
(m, 1H). MS (FAB): m/z 291 (MꢂH)ꢂ. Anal. Calcd for C13H10N2O6: C,
53.80%, H, 3.47%, N, 9.65%. Found: C, 53.56%, H, 3.56%, N, 9.87%.
2-(1-Hydroxy-2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione
(2e) (4,N-di-OH-Thal) This compound was obtained as a white powder
(81% yield), in a manner similar to that described for the preparation of 2b
1
from 12e. mp 266—267 °C. H-NMR (DMSO-d6): d: 11.19 (s, 1H), 10.25
(s, 1H), 7.64 (t, Jꢃ7.7 Hz, 1H), 7.30 (d, Jꢃ7.7 Hz, 1H), 7.24 (d, Jꢃ7.7 Hz,
1H), 5.24 (dd, Jꢃ13.1, 5.5 Hz, 1H), 3.00 (m, 1H), 2.75 (m, 1H), 2.54 (m,
1H), 2.01 (m, 1H). MS (FAB): m/z 291 (MꢂH)ꢂ. Anal. Calcd for
C13H10N2O6: C, 53.80%, H, 3.47%, N, 9.65%. Found: C, 53.69%, H, 3.58%,
N, 9.54%.

December 2006
1713
(2S,4R)-tert-Butyl-2-carbamoyl-4-hydroxypyrrolidine-1-carboxylate
HCl and brine, dried over MgSO4, filtered, and concentrated. The residue
(15) To a solution of (2S,4R)-1-tert-butoxycar-bonyl-4-hydroxypyrroli- was purified by flash column chromatography (CHCl3 : MeOHꢃ50 : 1 v/v) to
dine-2-carboxylic acid 14 (4.90 g, 21.2 mmol) in dry CH3CN (60 ml) was afford 21 (170 mg, 0.438 mmol, 54%) as a white solid. 1H-NMR (CDCl3): d:
added EDCI·HCl (4.89 g, 25.5 mmol) and HOBt·H2O (3.45 g, 25.5 mmol)
at 0 °C. The mixture was stirred at room temperature for 6 h, then cooled to
7.95 (s, 1H), 7.93—7.76 (m, 4H), 5.08 (dd, Jꢃ13.7, 5.1 Hz, 1H), 4.49 (dd,
Jꢃ12.8, 5.6 Hz, 1H), 2.98 (ddd, Jꢃ13.7, 12.8, 12.8 Hz, 1H), 2.34 (ddd,
0 °C, and 28% aqueous ammonia (6 ml) was carefully added. Stirring was Jꢃ12.8, 5.6, 5.1 Hz, 1H), 0.92 (s, 9H), 0.23 (s, 3H), 0.15 (s, 3H). MS (FAB):
continued at 0 °C for 30 min and then at room temperature for 1 h. Insoluble
material was removed by filtration, then the filtrate was concentrated and pu-
rified by flash column chromatography (CHCl3 : MeOHꢃ20 : 1 to 5 : 1 v/v)
m/z 389 (MꢂH)ꢂ. [a]D22 ꢂ14.6° (cꢃ1.00, CHCl3).
2-(5-tert-Butyldimethylsilyloxy-2,6-dioxopiperidin-3-yl)isoindoline-
1,3-dione (24) (trans-5ꢀ-OTBDMS-Thalidomide) To a solution of 20
(570 mg, 1.53 mmol) in dry CH2Cl2 (50 ml) was added phthalic anhydride
(252 mg, 1.70 mmol), EDCI·HCl (863 mg, 4.50 mmol), HOBt·H2O
1
to afford 15 (4.87 g, 21.2 mmol, 100%) as a white solid. H-NMR (CDCl3):
d: 6.82, 5.95, 5.35 (each br, 2H), 4.51 (m, 1H), 4.42 (br, 1H), 3.53 (m, 2H),
2.40 (br, 1H), 2.11 (br, 1H), 1.47 (s, 9H). MS (FAB): m/z 231 (MꢂH)ꢂ. (610 mg, 4.50 mmol), and TEA (608 mg, 6.00 mmol). The reaction mixture
[a]D22 ꢁ47.5° (cꢃ1.00, MeOH).
(2S,4R)-tert-Butyl-2-carbamoyl-4-tert-butyldimethylsilyloxy-pyrroli- HCl and brine, dried over MgSO4, filtered, and concentrated. The residue
dine-1-carboxylate (16) To a solution of 15 (100 mg, 0.435 mmol) in dry was purified by flash column chromatography (CHCl3 : MeOHꢃ50 : 1 v/v),
THF (10 ml) was added tert-butyldimethylsilyl chloride (TBDMS-Cl) followed by recrystallization (CH2Cl2/hexane) to afford 24 (151 mg,
was stirred at room temperature for 2 d, then washed with 0.5 mol/l aqueous
(73 mg, 0.48 mmol) and imidazole (96 mg, 1.40 mmol) at 0 °C. The mixture
0.389 mmol, 25%) as a white solid (single diastereomer of the trans form).
was stirred at room temperature for 1 d. Insoluble material was removed by 1H-NMR (CDCl3): d: 7.90—7.75 (m, 4H), 7.81 (s, 1H), 5.48 (dd, Jꢃ12.4,
filtration, and the filtrate was concentrated and purified by flash column 5.1 Hz, 1H), 4.50 (dd, Jꢃ1.7, 1.7 Hz, 1H), 2.88 (ddd, Jꢃ13.5, 12.4, 1.7 Hz,
chromatography (hexane : AcOEtꢃ1 : 3 to 0 : 1 v/v) to afford 16 (130 mg,
1H), 2.18 (ddd, Jꢃ13.5, 5.1, 1.7 Hz, 1H), 0.95 (s, 9H), 0.21 (s, 3H), 0.17 (s,
0.378 mmol, 87%) as a white solid. H-NMR (CDCl3): d: 6.83, 5.97, 5.27 3H). MS (FAB): m/z 389 (MꢂH)ꢂ. [a]D22 ꢂ34.4° (cꢃ1.00, CHCl3).
(each br, 2H), 4.35 (m, 2H), 3.50 (m, 2H), 2.30 (br, 1H), 2.05 (br, 1H), 1.47 2-((3S,5R)-5-Hydroxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(s, 9H), 0.87 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H). MS (FAB): m/z 345 (cis-2f) (cis-5ꢀ-OH-Thal) To a solution of 21 (90.0 mg, 0.232 mmol) in
(MꢂH)ꢂ. [a]D22 ꢁ38.5° (cꢃ1.00, MeOH).
dry THF (15 ml) was added tetrabutylammonium fluoride (TBAF) (73.0 mg,
1
(2S,4R)-tert-Butyl-2-carbamoyl-4-tert-butyldimethylsilyloxy-5-oxopyr- 0.280 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C. After
rolidine-1-carboxylate (17) Sodium periodate (6.84 g, 32.0 mmol) was 90 min, CHCl3 was added, and the whole was washed with 0.5 mol/l HCl and
stirred in H2O (150 ml), and ruthenium(IV) oxide hydrate (1.33 g, brine, dried over MgSO4, filtered and concentrated. The crude product was
1.00 mmol) was added under an argon atmosphere. The mixture was stirred purified by preparative thin layer chromatography (CHCl3 : MeOHꢃ10 : 1
at room temperature for 5 min, and then a solution of 16 (3.60 g, 10.5 mmol)
v/v) to afford cis-2f (18.0 mg, 0.0657 mmol, 28%) as a white powder. mp
1
in AcOEt (90 ml) was added, and the reaction mixture was further stirred at 229—231 °C. H-NMR (DMSO-d6): d: 11.22 (s, 1H), 7.96—7.88 (m, 4H),
room temperature for 3 d. The aqueous layer was separated and extracted 5.82 (d, Jꢃ6.0 Hz, 1H), 5.29 (dd, Jꢃ12.8, 5.1 Hz, 1H), 4.54 (ddd, Jꢃ11.6,
with AcOEt. The combined organic layer was washed with saturated 6.0, 5.5 Hz, 1H), 2.49 (m, 1H), 2.28 (ddd, Jꢃ11.0, 5.5, 5.1 Hz, 1H): MS m/z
NaHSO3 and brine, dried over MgSO4, and filtered through Celite, and the
filtrate was concentrated to afford 17 (3.19 g, 8.91 mmol, 84%) as a white
solid. 1H-NMR (CDCl3): d: 6.01, 5.37 (each br, 2H), 4.62 (dd, Jꢃ8.1,
275 (MꢂH)ꢂ. [a]D22 ꢁ11.0° (cꢃ0.34, MeOH).
2-(5-Hydroxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (trans-2f)
(trans-5ꢀ-OH-Thal) To a solution of 24 (90.0 mg, 0.232 mmol) in dry
2.4 Hz, 1H), 4.49 (d, Jꢃ9.2 Hz, 1H), 2.54 (dd, Jꢃ12.8 Hz, 8.1 Hz, 1H), 2.11 THF (15 ml) was added TBAF (73.0 mg, 0.280 mmol) at 0 °C, and the reac-
(ddd, Jꢃ12.8, 9.2, 2.4 Hz, 1H), 1.53 (s, 9H), 0.90 (s, 9H), 0.18 (s, 3H), 0.13 tion mixture was stirred at 0 °C. After 30 min, CHCl3 was added, and the
(s, 3H). MS (FAB): m/z 359 (MꢂH)ꢂ. [a]D22 ꢂ50.0° (cꢃ1.00, MeOH).
whole was washed with 0.5 mol/l HCl and brine, dried over MgSO4, filtered
(1S,3R)-tert-Butyl-3-tert-butyldimethylsilyloxypropyl-carbamate-3- and concentrated. The crude product was purified by recrystallization
carboxylic Acid (18) A solution of 17 (2.00 g, 5.59 mmol) in dry THF
(400 ml) was stirred at 0 °C, and 1.0 mol/l aqueous LiOH (17 ml, 17.0 mmol)
was carefully added. The reaction mixture was stirred at 0 °C for 3 h, then
(CHCl3/MeOH) to afford trans-2f (38.0 mg, 0.139 mmol, 60%) as a white
powder. mp 226—228 °C. 1H-NMR (DMSO-d6): d: 11.27 (s, 1H), 7.94—
7.88 (m, 4H), 6.52 (d, Jꢃ4.9 Hz, 1H), 5.15 (dd, Jꢃ12.0, 5.2 Hz, 1H), 4.30
acidified by addition of 2.0 mol/l aqueous HCl, and extracted 3 times with (ddd, Jꢃ4.9, 3.5, 3.1 Hz, 1H), 2.65 (ddd, Jꢃ13.9, 5.2, 3.1 Hz, 1H), 2.17
CHCl3. The organic layer was washed with brine, dried over MgSO4, filtered (ddd, Jꢃ13.9, 12.0, 3.5 Hz, 1H). MS m/z 275 (MꢂH)ꢂ. [a]D22 ꢂ59.0°
and concentrated to afford 18 (2.30 g, quant.) as a white amorphous solid.
This intermediate was used for the next reaction without purification. 1H-
NMR (DMSO-d6): d: 12.53 (br, 1H), 6.99 (m, 3H), 4.20 (m, 1H), 3.95 (m,
(cꢃ0.36, MeOH).
5-Benzyloxy-2-(5-tert-butyldimethylsilyloxy-2,6-dioxopiperidin-3-
yl)isoindoline-1,3-dione (22) To a solution of 20 (400 mg, 1.08 mmol) in
1H), 2.03 (m, 1H), 1.70 (m, 1H), 1.37 (s, 9H), 0.86 (s, 9H), 0.04 (s, 6H). MS dry CH2Cl2 (70 ml) was added 11a (280 mg, 1.10 mmol), EDCI·HCl
(FAB): m/z 377 (MꢂH)ꢂ. [a]D22 ꢂ5.68° (cꢃ1.00, MeOH).
(575 mg, 3.00 mmol), HOBt·H2O (406 mg, 3.00 mmol), and TEA (506 mg,
(3S,5R)-tert-Butyl-5-tert-butyldimethylsilyloxy-2,6-dioxopiperidin-3- 5.00 mmol). The reaction mixture was stirred at room temperature for 1 d,
ylcarbamate (19) To a solution of 18 (2.30 g, 5.59 mmol) in dry CH2Cl2
then washed with 0.5 mol/l aqueous HCl and brine, dried over MgSO4, fil-
(300 ml) was added CDI (1.05 g, 6.50 mmol), DMAP (73 mg, 0.600 mmol),
tered, and concentrated in vacuo. The residue was purified by flash column
and diisopropylethylamine (DIPEA) (1.55 g, 12.0 mmol) at 0 °C. The mix- chromatography (Hexane : AcOEtꢃ2 : 1 v/v) to afford 22 (240 mg,
ture was stirred at room temperature for 5 d, then washed with 0.5 mol/l 0.486 mmol, 44%) as a white solid. 1H-NMR (CDCl3): d: 7.90 (s, 1H), 7.78
aqueous HCl and brine, dried over MgSO4, filtered and concentrated. The
residue was purified by flash column chromatography (hexane : AcOEtꢃ3 : 2
v/v) to afford 19 (660 mg, 1.84 mmol, 33%, 2 steps) as a white amorphous
(d, Jꢃ8.1 Hz, 1H), 7.43—7.35 (m, 7H), 5.19 (s, 2H), 5.04 (dd, Jꢃ12.8,
4.3 Hz, 1H), 4.48 (m, 1H), 2.90 (m, 1H), 2.25 (m, 1H), 0.91 (s, 9H), 0.23 (s,
3H), 0.15 (s, 3H). MS (FAB): m/z 495 (MꢂH)ꢂ.
5-Benzyloxy-2-(5-hydroxy-2,6-dioxopiperidin-3-yl)-isoindoline-1,3-
dione (23) To a solution of 22 (210 mg, 0.425 mmol) in dry THF (20 ml)
was added TBAF (122 mg, 0.468 mmol) at 0 °C, and the reaction mixture
1
solid. H-NMR (CDCl3): d: 7.83 (s, 1H), 5.27 (br, 1H), 4.41 (m, 2H), 2.65
(m, 1H), 2.04 (m, 1H), 1.47 (s, 9H), 0.92 (s, 9H), 0.20 (s, 3H), 0.14 (s, 3H).
MS (FAB): m/z 359 (MꢂH)ꢂ. [a]D22 ꢂ18.4° (cꢃ1.10, MeOH).
(3S,5R)-3-Amino-5-tert-butyldimethylsilyloxypiperidine-2,6-dione Tri- was stirred at 0 °C for 40 min, and then at room temperature for 2 h. Then
fluoroacetate (20) To a stirred solution of 19 (390 mg, 1.09 mmol) in dry
CHCl3 was added, and the mixture was washed with 0.5 mol/l HCl and
CH2Cl2 (5 ml) was added TFA (5 ml). The reaction mixture was stirred at brine, dried over MgSO4, filtered and concentrated. The crude product was
room temperature for 30 min, then concentrated to afford 20 (490 mg,
quant.) as a pale pink solid. 1H-NMR (DMSO-d6): d: 11.42 (s, 1H), 8.42 (br,
3H), 4.65 (dd, Jꢃ12.2, 4.9 Hz, 1H), 4.34 (dd, Jꢃ12.8, 4.9 Hz, 1H), 2.36 (m,
purified by flash column chromatography (CHCl3 : MeOHꢃ25 : 1 to 10 : 1
v/v) to afford 23 (166 mg, quant.) as a white solid. 1H-NMR (DMSO-d6): d:
11.22 (s, 1H), 7.85 (m, 1H), 7.55—7.33 (m, 7H), 5.80 (d, Jꢃ4.3 Hz, 1H),
1H), 2.06 (ddd, Jꢃ12.8, 12.8, 12.2 Hz, 1H), 0.88 (s, 9H), 0.12 (s, 3H),0.10 5.33 (s, 2H), 5.24 (m, 1H), 4.52 (m, 1H), 2.49 (m, 1H), 2.25 (m, 1H). MS
(s, 3H). MS (FAB): m/z 259 (MꢂH)ꢂ.
m/z 381 (MꢂH)ꢂ.
2-((3S,5R)-5-tert-Butyldimethylsilyloxy-2,6-dioxopiperidin-3-yl)isoin-
5-Hydroxy-2-(5-hydroxy-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
doline-1,3-dione (21) (cis-5ꢀ-OTBDMS-thalidomide) To a solution of 20 (2g) (5,5ꢀ-di-OH-Thal) To a solution of 23 (160 mg, 0.421 mmol) in dry
(300 mg, 0.806 mmol) in dry CH2Cl2 (15 ml) was added phthalic anhydride 1,4-dioxane (20 ml) was added 10% Pd–C (120 mg), and the mixture was
(148 mg, 1.00 mmol), EDCI·HCl (309 mg, 1.61 mmol), HOBt·H2O stirred at room temperature under an H2 atmosphere for 5 h, then filtered
(218 mg, 1.61 mmol), and DIPEA (416 mg, 3.22 mmol) at 0 °C. The reaction
mixture was stirred at 0 °C for 30 min, then washed with 0.5 mol/l aqueous
through Celite, and the filtrate was concentrated in vacuo. The residue was
purified by flash column chromatography (CHCl3 : MeOHꢃ10 : 1 v/v) to af-

1714
Vol. 54, No. 12
ford 2g (120 mg, 0.144 mmol, 98%) as a white powder. mp 258—260 °C.
1H-NMR (DMSO-d6): d: 11.18 (s, 1H), 7.71 (m, 1H), 5.80 (s, 1H), 5.21 (m,
1H), 4.52 (m, 1H), 2.49 (m, 1H), 2.23 (m, 1H). MS (FAB): m/z 291
(MꢂH)ꢂ.
8) Noguchi T., Miyachi H., Katayama R., Naito M., Hashimoto Y.,
Bioorg. Med. Chem. Lett., 15, 3212—3215 (2005).
9) Noguchi T., Fujimoto H., Sano H., Miyajima A., Miyachi H.,
Hashimoto Y., Bioorg. Med. Chem. Lett., 15, 5509—5513 (2005).
Cells and Measurement of TNF-a THP-1 cells were maintained as 10) Lu J., Hesby N., Palmer B. D., Tingle M., Baguley B. C., Ketstell P.,
previously described.28,29) The exponentially growing cells in RPMI1640
medium supplemented with 10% v/v fetal bovine serum (1.0ꢅ106 cells/ml)
were treated or not treated with 10 nM TPA at 37 °C under a 5% CO2 atmo-
Ching L.-M., J. Pharm. Exp. Ther., 310, 571—577 (2004).
11) Lu J., Palmer B. D., Kestell P., Browett P., Baguley B. C., Muller G.,
Ching L.-M., Clin. Cancer Res., 9, 1680—1688 (2003).
sphere for 18 h in 24-well multiplates. To test the effects of compounds, cells 12) Marks M. G., Shi J., Fry M. O., Xiao Z., Trzyna M., Pokala V., Ihnat
were treated with TPA (10 nM) in the presence or absence of a test sample at
30 mM. Then the cells were collected by centrifugation (1500 rpmꢅ10 min).
The amount of TNF-a in the supernatant was measured by the use of TNF-a
Human Biotrak Easy ELISA (Amersham Biosciences, RPN5967) according
to the supplier’s protocol. The concentration of TNF-a produced by THP-1
cells treated with 10 nM TPA alone was approximately 100 pg/ml under these
experimental conditions. The assay was performed at least three times. The
M. A., Li P.-K., Biol. Pharm. Bull., 25, 597—604 (2002).
13) Robin S., Zhu J., Galons H., Pham-Huy C., Vlaude J. R., Tomas A.,
Viossat B., Tetrahedron Assym., 6, 1249—1252 (1995).
14) Teubert U., Zwingenberger K., Wnendt S., Eger K., Arch. Pharm.,
331, 7—12 (1998).
15) Luzzio F. A., Duveau D. Y., Lepper E. R., Figg W. D., J. Org. Chem.,
70, 10117—10120 (2005).
results were basically reproducible and a typical set of data (mean values of 16) Doi M., Ijiri Y., Akagi M., Uenishi M., Urata H., Anal. Sci., 19, x51—
duplicate) obtained at the same time is presented in Table 1.
x52 (2003).
17) Meyring M., Muehlbacher J., Messer K., Kastner-Pustet N., Bring-
mann G., Mannschreck A., Blaschke G., Anal. Chem., 74, 3726—3735
(2002).
Acknowledgments We wish to thank the laboratories of Prof. Dr.
William D. Figg at the National Cancer Institute, and Prof. Dr. Kurt Eger at
Universität Leipzig for providing a sample of of 5ꢀ-hydroxythalidomide 18) Scott M. C., Todd P. H., Milton L. B., Org. Lett., 5, 2865—2867
(racemic cis-isomer). We are also grateful to Assoc. Prof. Dr. Aya Tanatani (2003).
(Ochanomizu University) for helpful discussions on the structural analysis 19) Fukushima H., Hiratake A., Takahashi M., Saito M., Munetomo E.,
of thalidomide metabolites. The work described in this paper was partially
supported by Grants-in-Aid for Scientific Research from The Ministry of
Education, Culture, Sports, Science and Technology, Japan.
Kitano K., Saito H., Takaoka Y., Yamamoto K., Bioorg. Med. Chem.,
12, 6053—6061 (2004).
20) Xiaojun Z., Aaron C. S., Wen J., Tetrahedron Lett., 42, 5335—5338
(2001).
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