Synthesis of racemic cis-5-hydroxy-3-phthalimidoglutarimide. A metabolite of thalidomide isolated from human plasma

Article abstract of DOI:10.1021/jo0514772

A synthesis of the glutarimide-derived metabolite of thalidomide, 5′-hydroxythalidomide (2), is described. The synthesis employed the lactone derivative of N-benzyloxycarbonyl (CBZ)-protected 4-hydroxyglutamic acid 12, which is prepared by a de novo route from diethyl acetamidomalonate. The reaction of 12 with 4-methoxybenzylamine gave the corresponding isoglutamine, which then provided the key CBZ-protected N-PMB-glutarimide 14 after dehydration. Deprotection of both the CBZ and PMB groups followed by phthalimidation and deacetylation of the 3-amino-5-acetoxyglutarimide 16 afforded 2.

Full text of DOI:10.1021/jo0514772

Synthesis of Racemic
cis-5-Hydroxy-3-phthalimidoglutarimide.
A Metabolite of Thalidomide Isolated from
Human Plasma
Frederick A. Luzzio,*,† Damien Y. Duveau,†
Erin R. Lepper,‡ and William D. Figg‡
Department of Chemistry, University of Louisville,
2320 South Brook Street, Louisville, Kentucky 40292, and
Clinical Pharmacology, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892
FIGURE 1. Thalidomide and analogues with varying degrees
of oxygenation.
formation of thalidomide to its oxygenated analogues is
somewhat understood, the complete identification of the
products is further complicated by its hydrolytic and
configurational lability.7 Among the suspected active
components are those which result from metabolic oxy-
genation of the glutarimide (2) or phthalimide rings (3-
5) and have intact imide bonds (Figure 1). The phthal-
imide ring-oxygenated metabolites of thalidomide (3-5)
have been identified since the early investigations of its
embryotoxicity and possess little or no teratogenic activ-
ity. More recently, 5′-hydroxythalidomide (2) was de-
tected in plasma samples after administration of racemic
1 to healthy male volunteers and in incubates of 1 with
human liver homogenate.6a,b,c The metabolic formation
of 2 can result in the diastereoisomeric cis and trans
forms which possess epimerization properties similar to
those of the parent molecule.6c We have explored several
syntheses of 2 with the goals of obtaining sufficient
quantities for biological evaluation and establishing
general stereoselective routes to glutarimide-derived
analogues of 1.5 Our success with the use of 4-methoxy-
benzylamine as an “ammonia equivalent” in glutarimide
formation in the synthesis of the active phthalimidine
analogue EM-12 (6) has prompted our present report on
the synthesis of 2 with full experimental details.8 The
synthesis described herein parallels the first synthesis
of 5′-hydroxythalidomide as reported by Eger in which 2
was of interest as a tumor necrosis factor (TNF-R)
inhibitor.4 The Eger route employed glutarimide forma-
tion from N-benzyloxycarbonyl (CBZ)-protected 4-hy-
droxyisoglutamine 13 followed by CBZ deprotection and
N-phthaloylation as the key steps. Isoglutamine 13 was
prepared by amination of the CBZ-protected lactonic acid
form of the rare racemic 4-hydroxyglutamic acid 9
(Scheme 1).9, 10
faluzz01@gwise.louisville.edu
Received July 16, 2005
A synthesis of the glutarimide-derived metabolite of thali-
domide, 5′-hydroxythalidomide (2), is described. The syn-
thesis employed the lactone derivative of N-benzyloxycar-
bonyl (CBZ)-protected 4-hydroxyglutamic acid 12, which is
prepared by a de novo route from diethyl acetamidoma-
lonate. The reaction of 12 with 4-methoxybenzylamine gave
the corresponding isoglutamine, which then provided the key
CBZ-protected N-PMB-glutarimide 14 after dehydration.
Deprotection of both the CBZ and PMB groups followed by
phthalimidation and deacetylation of the 3-amino-5-acetoxy-
glutarimide 16 afforded 2.
In contrast to its notoriety as a potent embryotoxin,
the diverse biological effects of thalidomide (1) have been
beneficial. The compound is an approved antilepromatic
and together with some of its analogues are active as
immunosuppressants, immunomodulators, antiinflam-
matories, and anticancer agents.1a-d As an experimental
therapeutic, thalidomide and closely related analogues
have gained much attention as angiogenesis inhibitors.1a,e,f
The antiangiogenic activity of 1 is a consequence of
metabolic activation as demonstrated by in vivo studies
in the rabbit and ex vivo studies with human mi-
crosomes.2,3 At present, the identity of the active species
has not been confirmed and is the objective of many
synthetic and biological studies.4-6 While the biotrans-
The synthesis commences with the base-mediated
alkylation of commercially available diethyl acetami-
* To whom correspondence should be addressed. Tel: (502) 852-
7323. Fax: (502) 852-8149.
(4) Teubert, U.; Zwingenberger, K.; Wnendt, S.; Eger, K. Arch.
Pharm. Pharm. Med. Chem. 1998, 331, 7-12.
† University of Louisville.
‡ National Cancer Institute.
(5) Luzzio, F. A.; Mayorov, A. V.; Figg, W. D Tetrahedron Lett. 2000,
41, 2275-2278.
(1) (a) Richardson, P.; Hideshima, T.; Anderson, K. Annu. Rev. Med.
2002, 53, 629-657. (b) Marriot, J. B.; Muller, G.; Stirling, D.; Dalgleish,
A. G. Exp. Opin. Biol. Ther. 2001, 1, 675-682. (c) Raje, N.; Anderson,
K. N. Engl. J. Med. 1999, 341, 1606-1609. (d) Hales, Nat. Med. 1999,
5, 489-490. (e) Luzzio, F. A.; Figg, W. D. Exp. Opin. Ther. Pat. 2004,
14, 215-229. (f) Gasparini, G.; Longo, R.; Fanelli, M.; Teicher, B. A.
J. Clin. Oncol. 2005, 23, 1295-1311.
(6) (a) Eriksson, T.; Bjorkman, S.; Roth, B.; Bjork, H.; Hoglund, P.
J. Pharm. Pharmacol. 1998, 50, 1409-1416 (b) Knoche, B.; Blaschke,
G. Chirality 1994, 6, 221-224 (c) Meyring, M.; Muhlbacher, J.; Messer,
K.; Kastner-Pustet, N.; Bringmann, G.; Mannschreck, A.; Blaschke,
G. Anal. Chem. 2002, 74, 3726-3735 (d) Price, D. K.; Ando, Y.; Kruger,
E.; Weiss, M.; Figg, W. D. Thera. Drug Monit. 2002, 24 104-110.
(7) Schumacher, H.; Smith, R. L.; Williams, R. T. Brit. J. Pharmacol.
1965, 25, 338-351.
(2) D’Amato, R. J.; Loughnan, M. S.; Flynn, E.; Folkman, J. Proc.
Nat Acad. Sci. U.S.A. 1994, 91, 4082-4085.
(3) Bauer, K. S.; Dixon, S. C.; Figg, W. D. Biochem. Pharmacol. 1998,
55, 1827-1834.
(8) Luzzio, F. A.; Mayorov, A. V.; Ng, S. S. W.; Kruger, E. A.; Figg,
W. D. J. Med. Chem. 2003, 46, 3793-3799.
10.1021/jo0514772 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/21/2005
J. Org. Chem. 2005, 70, 10117-10120
10117

SCHEME 1. Retrosynthesis of
5′-Hydroxythalidomide 2
SCHEME 3. Amination of the Lactonic Acid 12
and Glutarimide Formationa
SCHEME 2. Preparation of the N-CBZ-lactonic
Acid 12a
a Reagents and conditions: (a) 4-MeOC6H4CH2NH2/pyr/70 °C/
16 h, 85%; (b) Ac2O/(F3CCO)2O/pyr/70 °C/16 h, 68%; (c) CAN/
CH3CN/H2O/25 °C/3 h, 68%.
the requisite N-CBZ-protected lactonic acid 12. On
exposure to air, 11 becomes a sticky intractable gum
which is not convenient to handle or store. Converting
11 to its corresponding lactonic acid 12 leads to a
crystalline solid and furthermore activates the R-carboxy
group toward regiospecific isoglutamine formation when
treated with amines prior to glutarimide closure. There-
fore, treatment of the N-CBZ-protected 4-hydroxyglutam-
ic acid 11 with boiling 1 N aqueous hydrochloric acid
yielded several crops of the lactonic acid 12 in 71%
combined yield. In contrast to the established use of the
4-methoxybenzyl (PMB) group as a protecting group for
amides,13a it has not been used as a “protecting group”
for imides per se other than for elimination or replace-
ment of the acidic imide proton.13b Amidation of the
lactonic acid 12 with 4-methoxybenzylamine in the
presence of excess anhydrous pyridine furnished the
N-CBZ-protected 4-hydroxyisoglutamine 13 in 85% yield
as a white solid (Scheme 3). Treatment of isoglutamine
13 with acetic anhydride, pyridine, and a catalytic
amount of trifluoroacetic acid resulted in glutarimide
formation and thus provided the 5-acetoxy-(1-N-PMB)-
2-N-CBZ-glutarimide 14 in 68% yield as an inseparable
diastereoisomeric mixture (65:35 cis/trans). Interestingly,
removal of the CBZ group of 14 by catalytic hydrogenoly-
sis could not be effected to furnish the N-PMB-5-ac-
etoxyaminoglutarimide 18. Presumably, the presence of
the PMB group interfered with the CBZ cleavage of 14.
The N-4-methoxybenzyl group was removed from 14
using ceric ammonium nitrate (CAN) in aqueous aceto-
nitrile and provided the N-CBZ-5-acetoxyglutarimide 15
in 68% yield as a mixture of diastereoisomers (70:30 cis/
trans). Deprotection-phthaloylation of the R-nitrogen of
N-CBZ-glutarimide 15 could be accomplished in a com-
bined two-step sequence (Scheme 4). Hydrogenation of
15 with 10% palladium on activated carbon in THF
removed the CBZ group and provided the crude 3-amino-
4-acetoxyglutarimide 16. Immediate admixture of 16
with phthalic anhydride and triethylamine followed by
heating resulted in N-phthaloylation and gave 5′-acetoxy-
thalidomide 17 in 46% yield (from 15) as a chromato-
graphically inseparable mixture of diastereoisomers
(65:35 cis/trans). While N-phthaloylations of aminoglu-
tarimides such as 16 have been accomplished by heating
a Reagents and conditions: (a) NaH/THF/rt then EtOOCCH-
(OAc)CH2Cl/70 °C/2 h, 88%; (b) 4 N HCl/reflux/4 h, 60%; (c)
BnOCOCl/NaHCO3/H2O/20 °C/24 h, 94%; (d) 1 N HCl/100 °C, 71%.
domalonate 7, an operation in which a successful outcome
depended critically on the choice of nonprotic reaction
conditions rather than the usual alcohol/alkoxide protocol
(Scheme 2).11 Following formation of the diester anion
with sodium hydride in tetrahydrofuran, treatment with
ethyl 2-acetoxy-3-chloropropionate gave the crystalline
amidic acetoxy triester 8 in 88% yield as the exclusive
product.9a,10 Exhaustive hydrolysis of the amidic triester
8 with 4 N refluxing HCl for 16 h afforded γ-hydroxy-
glutamic acid 9 as a mixture of diastereoisomers. Inter-
estingly, direct phthalimidation of the hydroxyamino acid
9 using several reagent systems based on either phthaloyl
dichloride or phthalic anhydride under several sets of
conditions yielded no 2-N-phthalolyl-4-hydroxyglutamic
acid 10, the so-called “hydroxy PG acid.”12 Treatment of
9 with benzyl chloroformate (1.5-2 equiv) in the presence
of aqueous sodium bicarbonate provided the N-CBZ-
protected hydroxyglutamic acid 11 in 94% yield as a very
hygroscopic amorphous solid. Glutarimide formation by
amidation-dehydration first required the preparation of
(9) (a) Benoiton, L.; Bouthillier, L. P. Can. J. Chem. 1955, 33, 1474-
1476. (b) Benoiton, L.; Winitz, M.; Birnbaum, S. M.; Greenstein, J. P.
J. Am. Chem. Soc. 1957, 79, 6192-6198. (c) Kaneko, T.; Lee, Y. K.;
Hanafusa, T. Bull. Chem. Soc. Jpn. 1962, 46, 875-878. (d) Lee, Y. K.;
Kaneko, T. Bull. Chem. Soc. Jpn. 1973, 46, 3494-3498. (e) Kusumi,
T.; Kakisawa, H.; Suzuki, S.; Harada, K.; Kashima, C. Bull. Chem.
Soc. Jpn. 1978, 51, 1261-1262. (f) Kristensen, E. P.; Larsen, L. M.;
Olsen, O.; Sorensen, H. Acta Chem. Scand. 1980, 34, 497-504.
(10) The preparation of 8 en route to 9 has been reported. However,
important analytical details, e.g., mp and 1H and 13C NMR, were not
included (ref 9b).
(11) For some examples of recent applications of this rather fickle
reaction, see: Seidel, G.; Laurich, D.; Fuerstner, A. J. Org. Chem. 2004,
69, 3950-3952, Godfrey, A. G.; Brooks, D. A.; Hay, L. A.; Peters, M.;
McCarthy, J. R.; Mitchell, D. J. Org. Chem. 2003, 68, 2623-2632, Witt,
A.; Bergman, J. Tetrahedron 2000, 56, 7245-7253, Hansen, M. M.;
Bertsch, C. F.; Harkness, A. R.; Huff, B. E.; Hutchison, D. R.; Khau,
V. V.; LeTourneau, M. E.; Martinelli, M. E.; Misner, J. W.; Peterson,
B. C.; Rieck, J. A.; Sullivan, K. A.; Wright, I. G. J. Org. Chem. 1998,
63, 775-785.
(12) 2-N-Phthaloyl-4-hydroxyglutamic acid 10, the putative product
of exhaustive in vivo hydrolysis of the glutarimide ring of 2, would
also be the metabolic product of 2-N-phthaloylglutaric acid, an inhibitor
of angiogenesis: Kenyon, B. M.; Brown, F. M.; Folkman, J.; D’Amato,
R. J. Exp. Eye Res. 1997, 64, 971-978.
(13) (a) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 3rd ed.; Wiley: New York, 1999; p 639. (b) Luzzio, F. A.;
Piatt Zacherl, D.; Figg, W. D. Tetrahedron Lett. 1999, 40, 2087-2090.
10118 J. Org. Chem., Vol. 70, No. 24, 2005

SCHEME 4. Conversion of
5-Acetoxy-2-N-CBZ-glutarimide 15 to
5′-OH-thalidomide 2a
glutarimide nitrogen, thereby avoiding the use of metha-
nolic ammonia. Of particular note are the mild conditions
utilized for the removal of protecting groups as well as
their stability. The PMB group, while being stable to
hydrogenolysis conditions, was otherwise cleaved using
ceric ammonium nitrate without affecting the glutarim-
ide ring. The removal of the 5′acetoxy group was facili-
tated with Dowex acid resin without cleavage of the
N-phthaloyl group, glutarimide ring, or decomposition of
the sensitive substrate.
Experimental Section
R-(4-Methoxybenzylamido)-N-benzyloxycarbonyl-γ-hy-
droxyisoglutamine (13). A mixture of N-benzyloxycarbonyl-
γ-hydroxyglutamic acid lactone 12 (141.4 mg, 0.51 mmol) and
4-methoxybenzylamine (125 mg, 0.99 mmol) was stirred in dry
pyridine (2 mL) at 70 °C for 16 h. The reaction mixture was
cooled to rt followed by evaporation of the solvent. The residue
was dissolved in EtOAc (10 mL), and the resulting organic phase
was washed with 5% aqueous HC1 (3 × 10 mL), deionized water,
(10 mL), andbrine (10 mL), dried over sodium sulfate, and
concentrated. Purification of the crude residue by flash column
chromatography (hexane/EtOAc, 4:1) yielded 13 (180 mg, 85%)
as a white hygroscopic solid: Rf 0.67 (n-butanol/methanol/
benzene/water, 2/4/2/2); mp 143-144 °C; 1H NMR (DMSO-d6,
500 MHz) δ 1.84 (m,1H), 1.96 (m, 1H), 3.73 (s, 3H), 3.98 (m,
1H), 4.22 (m, 3H), 5.05 (s, 2H), 6.86 (d, J ) 8.3 Hz, 2H), 7.16 (d,
J ) 8.3 Hz, 2H), 7.37 (m, 5H), 7.48 (d, J ) 8.3 Hz, 1H); 13C NMR
(DMSO-d6, 125 MHz) δ 14.8, 36.6, 42.2, 52.5, 55.7, 66.2, 67.2,
79.9, 114.3, 128.4, 129.0, 132.1, 137.6, 156.7, 158.8; IR (KBr,
cm-1) 3317, 1697, 1651, 1535, 1253, 1100; HMRS (FAB + Na,
70 eV) calculated for C21H24N2O7 ([M + Na]+) 439.1481, found
439.1482.
1-(4-Methoxybenzyl-3-N-benzyloxycarbonyl-5-acetoxy-
piperidine-1,6-dione (14). R-(4-Methoxybenzylamido)-N-ben-
zyloxycarbonyl-γ-hydroxyisoglutamine 13 (244 mg, 0.59 mmol)
was stirred in pyridine (1.2 mL) and acetic anhydride (1.2 mL)
in the presence of trifluoroacetic anhydride (50 µL) at 70 °C for
16 h. The reaction mixture was cooled to room temperature
followed by removal of the solvent. The residue was dissolved
in EtOAc (10 mL), and the resulting solution was washed with
aqueous HC1 (3 × 10 mL), deionized water (10 mL), and brine
(10 mL), dried over sodium sulfate, and concentrated. Purifica-
tion of the resultant crude residue by flash column chromatog-
raphy (hexane/EtOAc, 4:1) yielded 14 (175 mg, 68%; 65:35, cis/
trans) as a hygroscopic white solid: Rf 0.53 (hexane/ethyl acetate,
1:1); mp 46-50 °C; 1H NMR (DMSO)-d6, 500 MHz) δ 2.10 (s,
3H), 2.28 (m, 2H, 4-H), 3.71 (s, 3H), 4.53 (dd, J ) 7.6 Hz, 14.4
Hz, 1H, 3-H trans), 4.75 (m, 3H, CH2 + 3-H cis), 5.07 (s, 2H),
5.70 (dd, J ) 5.5 Hz, 7.0 Hz, 1H, 5-H trans), 582 (dd, J ) 6.8
Hz, 11.7 Hz, 1H, 5-U cis),6.85 (d, J ) 8.3 Hz, 2H), 7.18 (d, J )
8.4 Hz, 2H), 7.37 (m, 5H), 7.82 9 (d, J ) 8.7 Hz, 1H, NH cis),
8.10 (d, J ) 8.9 Hz, 1H, NH trans); 13C NMR (DMSO-d6, 125
MHz) δ 21.1, 29.9, 43.5, 43.5, 49.2, 50.5, 55.71, 55.77, 57.8, 66.3,
67.6, 68.4, 111.0, 128.49, 128.59, 129.09, 129.48, 129.86, 137.5,
156.7, 159.09, 159.11, 169.86, 169.93, 170.0, 171.9; HMRS
(FAB + Na, 70 eV) calculated for C23H24N2O7 ([M + Na]+)
463.1481, found 463.1494.
a Reagents and conditions: (a) H2/Pd/THF/20 °C/3 h, then (b)
phthalic anhydride/TEA/reflux/4 h, 46% (from 15); (c) Dowex-H+/
MeOH/reflux/2 h, quant.
the substrates with phthalic anhydride in acetic acid, the
same reactions in our hands resulted in both decomposi-
tion and mixtures of both 5′-acetoxythalidomide 17 and
2 (19%). The appearance of 2 is presumed to be through
a hydrolytic side reaction during the phthaloylation in
acid and does not proceed to completion. The deacylation
of the 5′-oxygen in either the glutarimide series (such as
17) or the analogous 4-oxygen in the acyclic glutamate
series has been consistently problematic due to the
sensitivity of the N-phthaloyl group to weakly basic
conditions as well as the ease of the system to racemize.5
On a considerably smaller scale than previously re-
ported,4 the methanolic p-toluenesulfonic acid-mediated
hydrolysis of 17 provided 2 in 25% isolated yield along
with decomposition products. Similarly, treatment of 17
with potassium carbonate in methanol provided 2 in 31%
yield along with hydrolysis products. The best results
were obtained by exposing 17 to acidic Dowex resin while
heating in methanol thereby affording quantitative con-
version to 2 in a cis/trans ratio of 28:1 (vide infra).
The propensity of thalidomide to racemize in biological
fluids has been established, and this process, prior to
metabolism, results in a diastereoisomeric mixture of cis-
and trans-5′-hydroxythalidomides. High-performance liq-
uid chromatography has proven effective in earlier
investigations where the glutarimide-derived metabolites
were detected, and more recently, the HPLC separation
and confirmation of the individual diastereoisomers
generated in vitro has been reported.6c The preparation
of 17 consistently provides cis/trans diastereoisomeric
mixtures (2:1) as determined by 1H NMR. However, when
the acid-mediated hydrolysis step in the conversion of 17
to 2 is undertaken under several sets of conditions,
extensive epimerization takes place and the cis/trans
ratio is increased to 28:1 as detected by the HPLC method
that we developed for thalidomide derivatives (see the
Supporting Information).
3-N-Benzyloxycarbonyl-5-acetoxypiperidine-1,6-dione
(15). 1-(4-Methoxybenzyl)-3-N-benzyloxycarbonyl-5-acetoxypi-
peridine-1,6-dione 14 (175.8 mg, 0.4 mmol) was stirred in a
mixture of acetonitrile/water (3:1) and ammonium cerium(IV)
nitrate (859 mg, 1.6 mmol) at rt for 3 h. The reaction mixture
was quenched by addition of saturated aqueous sodium thio-
sulfate (2 mL) and saturated aqueous sodium carbonate (2 mL),
which led to the formation of a thick, white precipitate. The
reaction mixture was then filtered through a short Celite pad
and the filtrate was concentrated to a white/yellow solid residue.
The residue was submitted to flash column chromatography
(hexane/EtOAc, 2:1) and gave 15 as a white solid (87 mg, 68%;
70:30 mixture of cis and trans): Rf 0.23 (hexane/ethyl acetate,
In conclusion, we have outlined an improved synthesis
of the rare thalidomide metabolite 5′-hydroxythalidomide
with full experimental and analytical details. The syn-
thesis utilized a reliable and scalable multigram prepa-
ration of the intermediate N-CBZ-hydroxyglutamic acid
and employed 4-methoxybenzylamine to introduce the
J. Org. Chem, Vol. 70, No. 24, 2005 10119

1
1:1); mp 176-178 °C; H NMR (DMSO-d6, 500 MHz) δ 2.10 (s,
trans diastereoisomers (white solid) (45.6 mg, 46%): Rf 0.32
1
3H), 2.22 (m, 2H, 4-H), 4.38 (dd, J ) 7.7 Hz, 14.2 Hz, 1H, 3-H
trans), 4.62 (m, 1H, 3-H cis), 5.06 (s, 2H), 5.55 (dd, J ) 5.1 Hz,
7.5 Hz, 1H, 5-H trans), 5.69 (dd, J ) 6.0 Hz, 12.6 Hz, 1H, 5-H
cis), 7.36 (m, 5H), 7.69 (d, J ) 8.7 Hz, 1H, CONH cis), 7.98 (d,
J ) 8.8 Hz, 1H, CONH trans); 13C NMR (DMSO-d6, 125 MHz)
δ 20.5, 29.9, 48.2, 49.7, 65.63, 65.83, 66.7, 67.5, 127.79, 127.88,
128.3, 136.73, 136.84, 155.9, 156.0, 169.19, 169.27, 169.83, 170.8,
171.7; IR (KBr, cm-1) 3300, 1740, 1693, 1531, 1373, 1219, 1037;
HMRS (FAB + Na, 70 eV) calcd for C15H16N2O6 ([M + Na]+)
343.0906, found 343.0910.
3-N-Phthaloyl-5-acetoxypiperidine-1,6-dione (5′-Acet-
oxythalidomide) (17). 3-N-Benzyloxycarbonyl-5-acetoxypip-
eridine-1,6-dione 15 (101.3 mg, 0.32 mmol) was stirred in dry
THF (3.3 mL) under hydrogen gas (1 atm) in the presence of
palladium over activated carbon (10.1 mg, 10 wt % of substrate)
for 3 h. The reaction mixture was then filtered through a short
Celite pad followed by concentration of the filtrate to about 3.5
mL. The resultant unprotected aminoglutarimide was then
directly mixed with phthalic anhydride (56.2 mg, 0.38 mmol)
and triethylamine (140 µL, 1.00 mmol) and refluxed (4 h). TLC
(n-butanol/methanol/water/benzene, 2:4:2:2) of the reaction mix-
ture showed full conversion of the unprotected amine to the
chromatographically more mobile product. The reaction mixture
was cooled to room temperature and the solvent was removed.
The crude residue was purified by flash column chromatography
(hexane/EtOAc, 1:1) to yield 17 as a 65:35 mixture of cis and
(hexane/EtOAc, 1:1); mp 251-253 °C; H NMR (DMSO-d6, 500
MHz) δ 2.12 (s, 3H, AcO cis), 2.13 (s, 3H, AcO trans), 2.41 (m,
1H, 4-H), 2.43 (m, 1H, 4-H), 2.67 (m, 1H,- 4-H), 2.70 (m, 1H,4-
H), 5.10 (t, J ) 7.0 Hz, 1H, 5-H trans), 5.48 (dd, J ) 5.4 Hz,
13.2 Hz, 1H, 5-H cis), 5.63 (dd, J ) 5.1 Hz, 7.9 Hz, 1H, 3-H
trans), 5.86 (dd, J ) 5.6 Hz, 13.1 Hz, 1H, 3-H cis), 7.9 (m, 4H),
11.55 (s, 1H, NH cis), 11.65 (s, 1H, NH trans); 13C NMR (DMSO-
d6, 125 MHz) δ 21.1, 28.28, 28.55, 46.5, 48.4, 67.5, 68.1, 124.11,
124.23, 124.40, 131.88, 131.92, 135.7, 167.9, 169.88, 169.92,
170.3; IR (KBr,cm-1) 3800, 1712, 1392, 1227, 1088; HMRS
(FAB + Na, 70 eV) calcd for C15H12N2O6 ([M + Na]+) 339.0593,
found 339.0600.
Acknowledgment. Support of this work by the
Molecular Pharmacology Research Core and the Clinical
Pharmacology Sections of the NCI is gratefully acknowl-
edged.
Supporting Information Available: 1H NMR and 13C
NMR spectra of compounds 8, 9, 11-15, and 17, the HPLC
chromatogram of compound 2, and experimental procedures
for compounds 8, 9, 11, 12, and 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO0514772
10120 J. Org. Chem., Vol. 70, No. 24, 2005