5'-Substituted thalidomide analogs as modulators of TNF-alpha.

Article abstract of DOI:10.1002/(SICI)1521-4184(199801)331:1<7::AID-ARDP7>3.0.CO;2-N

The synthesis of 5'-substituted thalidomide analogs is described. The amino acids 2 necessary to synthesize the target compounds were prepared by Michael reaction. Condensation of 2 with phthalic anhydrides followed by reaction with urea yielded 4 as diastereomeric mixtures. Furthermore glutethimide (5) was brominated by an improved method and the resulting compound 6 was reacted in several steps with sodium azide, hydrogen, and phthalic anhydride to give 8. In a similar manner, 6 was reacted with sodium azide and various phthalic anhydrides to give 9, 10, and 11. All final compounds were tested in vitro for their inhibitory activity on the release of TNF-alpha, using stimulated peripheral mononuclear blood cells (PBMCs). Compounds with an additional aromatic substituent in position 5' of the thalidomide molecule were more active than thalidomide. Compound 11 was able to reduce increased levels of IL-2 in vitro.

Full text of DOI:10.1002/(SICI)1521-4184(199801)331:1<7::AID-ARDP7>3.0.CO;2-N

5’-Substituted Thalidomide Analogs
7
5′-Substituted Thalidomide Analogs as Modulators of TNF-α✩
a)
b)
c)
a)
Uwe Teubert , Kai Zwingenberger , Stephan Wnendt , and Kurt Eger*
a) Universität Leipzig, Institut für Pharmazie, Brüderstr. 34, D-04103 Leipzig, Germany
b) Medical Scientific Division and c) Molecular Pharmacology Department, Grünenthal GmbH, Zieglerstr. 8, D-52068 Aachen, Germany
Key Words: Thalidomide; TNF-α; 5′-substituted analogs
Besides teratogenicity and association with neuropathy,
thalidomide was found to have some properties of pharma-
cological interest, e.g. it suppresses the release of tumor
necrosis factor-α (TNF-α) produced by monocytes/macro-
Summary
The synthesis of 5′-substituted thalidomide analogs is described.
The amino acids 2 necessary to synthesize the target compounds
were prepared by Michael reaction. Condensation of 2 with
phthalic anhydrides followed by reaction with urea yielded 4 as
diastereomeric mixtures. Furthermore glutethimide (5) was bromi-
nated by an improved method and the resulting compound 6 was
reacted in several steps with sodium azide, hydrogen, and phthalic
anhydride to give 8. In a similar manner, 6 was reacted with sodium
azide and various phthalic anhydrides to give 9, 10, and 11. All
final compounds were tested in vitro for their inhibitory activity
on the release of TNF-α, using stimulated peripheral mononuclear
blood cells (PBMCs). Compounds with an additional aromatic
substituent in position 5′ of the thalidomide molecule were more
active than thalidomide. Compound 11 was able to reduce in-
creased levels of IL-2 in vitro.
[4]
[5]
phages
and it inhibits angiogenesis in animal models
.
The immunomodulating and antiinflammatory properties of
the drug are used in clinical studies in a variety of diseases
including graft-versus-host-disease (GvHD) following bone
marrow transplantation, rheumatoid arthritis, Behçet’s dis-
ease, cachexia in AIDS, and several dermatological diseases
[6–9]
. It is supposed, that overexpression of the cytokine
TNF-α plays a major role in many of these inflammatory
manifestations. Substances which can effectively modulate
the production of TNF-α may be of benefit for the treatment
of disorders with excessive levels of TNF-α. Using thalido-
mide as a lead structure several analogs with structural modi-
fications of the molecule have been described. N-substituted
phthalimides with a simplified glutarimide moiety were re-
ported by Hashimoto et al. Most of the synthesized com-
[10–13]
pounds enhanced the release of TNF-α in vitro
. It was
Introduction
also found that the modulation of TNF-α release depends on
Thalidomide 1, developed as a sedative, was introduced on
the market in 1956. The drug quickly found broad acceptance
because of its quality to induce a “physiological” sleep with-
out the so called “hangover” syndrome which was sometimes
observed with other sedatives such as derivatives of barbitu-
ric acids. Due to an apparent lack of severe toxicity, the drug
was regarded as particularly safe. During this time, malfor-
mations were observed in newborn children which hitherto
the inducing agent, the used cell line and additionally on the
[14,15]
optical properties of chiral compounds
. Muller et al.
synthesized phthalimido β-amino acid derivatives as analogs
of thalidomide which were effective in inhibition of TNF-α
[16]
in vitro
. The influence of halogenation of the phthalimide
moiety of thalidomide and several alkylated N-phenyl-
[17]
phthalimide analogs was investigated by Liu et al.
.
We also used thalidomide as lead structure and were espe-
cially interested in structural modifications at the position 5′
of the molecule. The synthesis of various derivatives with
enhanced inhibition of release of TNF-α is described herein.
To our knowledge, only two 5′-substituted derivatives of
thalidomide are described in the literature. The first one,
5′-hydroxy thalidomide could be a metabolite of thalidomide
since it was found in traces after purification of a mixture of
thalidomide with rat liver microsomes. The structure of 5′-
hydroxy thalidomide was elucidated by chromatographic and
[1]
[2]
had occurred only in rare cases. Lenz and McBride were
the first to suspect a link between these malformations and
the use of thalidomide by pregnant women. Subsequently all
preparations containing thalidomide were withdrawn from
[1]
the market
.
[18]
spectroscopic methods . The authors did not synthesize the
compound therefore we synthesized the compound and
evaluated its activity. The second 5′-substituted thalidomide
[23]
analog is a derivative of glutethimide (9)
(see below).
Chemistry
However, some time later it was noted that the drug was
very effective for treatment of erythema nodosum leprosum
(ENL), an acute inflammatory manifestation of lepromatous
The 5′-monosubstituted thalidomide analogs 4 were pre-
pared by conventional methods as shown in Scheme 1. The
required substituted glutamic acids 2 were synthesized by
[3]
leprosy . Today, thalidomide is the drug of choice for treat-
ment of this condition also known as type II leprosy reaction. Michael reaction of acetamidomalonate with α-substituted
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/0101/0007 $17.50 +.50/0

8
Teubert, Zwingenberger, Wnendt, and Eger
alkyl acrylates. Subsequent hydrolysis in concentrated hydro-
chloric acid gave the amino acids 2 in good yields as dia-
stereomeric mixtures, which were used without further
[19]
separation. γ-Phenylglutamic acid 2b
was first synthe-
sized by this method.
Scheme 1. Reagents: (a) 1. phthalic anhydride, pyridine, reflux, 2. acetic
anhydride, reflux; (b) urea, 200 °C.
Scheme 2. Reagents: (a) Br2, HOAc, reflux; (b) NaN3, DMSO, H2O, 100 °C;
(c) 1. H2, 10% Pd/C, EtOH, 2. phthalic anhydride, HOAc, reflux , (d) HOAc,
reflux.
Phthalic anhydride was condensed with 2 in the presence of
pyridine and, after elimination of the solvent, treated with
acetic anhydride to give the anhydrides 3. Final conversion
of the anhydrides 3 to the imides 4 was accomplished by
treating with urea at about 200 °C. The products were ob-
tained as diasteromeric mixtures in a ratio of about 4:1 (4a)
and about 6:1 (4b). The strong biological activity of 4b (see
below) prompted us to elucidate the relative configuration.
Compound 4b was tested as a mixture of diastereomers and
it was assumed that the major diastereomer of 4b was respon-
the saturated compound 8 and the desired unsaturated deriva-
tives 9, 10, and 11. Catalytic hydrogenation of 7 using palla-
dium/charcoal followed by treatment with phthalic anhydride
1
in acetic acid yielded 8. The H-NMR data of 8 indicated the
existence of only one diastereomer. It was assumed to be the
cis-configuration (rac-cis) comparable to the major dia-
stereomer of compound 4b (rac-cis). To elucidate the correct
relative configuration of the substance, further analyses using
chromatographic methods are in preparation. Some reports in
the literature showed the nitrogen of the amino group of 7 to
1
sible for the inhibitory activity. The H-NMR data proved the
cis-configuration (R,R;S,S) of the substituents at position 3′
and 5′ and a diequatorial conformation in the glutarimide
moiety ( Figure 1).
be sufficiently nucleophilic to react with several electrophilic
[21,23]
substances
al.
. For instance 7 was treated by Casini et
[23]
with phthalic anhydride at high temperatures without
solvent to give 9. We found an improved method by reacting
7 with various phthalic anhydrides in refluxing acetic acid.
The products obtained (9, 10, and 11) crystallized spontane-
ously in high purity after cooling of the reaction mixtures.
To investigate the influence of the polar hydroxy group in
position 5′ of the thalidomide molecule 5′-hydroxy thalido-
mide 16 was synthesized as shown in Scheme 3.
We started with the preparation of γ-hydroxyglutamic acid
[24]
as described
. From the mixture of the diastereomers, the
threo-form (rac-trans) was separated as the protected lactone
Figure 1. Conformation of the major diastereomer of 4b (rac-cis).
[25]
12
. Subsequent synthetic steps were carried out only with
this threo-form. 12 was reacted with a saturated solution of
dry ammonia in methanol to give 13 which was cyclized to
14 as nearly a single diastereomer (rac-trans 14). Deprotec-
tion of the amino group followed by reaction with phthalic
anhydride in acetic acid yielded 15 as nearly a single dia-
stereomer (rac-cis 15) together with a mixture of diastereom-
ers in a ratio of about 1:2. The single diastereomer15 (rac-cis)
was subsequently heated with p-toluenesulfonic acid in dry
methanol to give 16 (rac-cis). The relative configuration of
It was assumed that the diequatorial position of the substi-
tuents bears importance for the inhibitory activity. To stabi-
lize the substituents in such a position we decided to establish
a double bond between C-3′ and C-4′ of the molecule. The
easily available glutethimide (5) was utilized as educt. The
synthetic route started with the bromination of 5 (Scheme 2).
[20,21]
The known procedures
to prepare 6 were improved
by treatment of 5 with bromine in refluxing acetic acid.
Further reaction of6 with sodium azide in hot aqueous DMSO
[22]
according to ref.
yielded 7, which was utilized to prepare 16 was elucidated by NMR experiments.
Arch. Pharm. Pharm. Med. Chem. 331, 7–12 (1998)

5’-Substituted Thalidomide Analogs
9
All tested compounds were substi-
tuted in position 5′ of the original tha-
lidomide molecule. Substitution of
one proton by a methyl group (4a)
decreased slightlythe inhibitory activ-
ity. On the other hand, the introduc-
tion of a polar hydroxy group in the
glutarimide moiety (16) decreased the
activity compared to thalidomide. It
can be concluded that the metabolic
hydroxylation in position 5′ of the
molecule had no influence on the re-
lease of TNF-α by thalidomide. Com-
pared with 16, acetylation of the
hydroxy group (compound 15) im-
proved the activity only slightly. 5′-
Phenyl thalidomide 4b was found to
be 5-fold more active than thalido-
mide in inhibition of TNF-α release.
Since 4a and 4b have two chiral carb-
on atoms they can occur in four
Scheme 3. Reagents:(a) ammonia, methanol, (b) acetic anhydride, reflux, (c) 1. H2/10% Pd/C, ethanol,
2. phthalic anhydride, acetic acid, reflux, (d) p-toluenesulfonic acid, methanol, reflux.
stereoisomers in form of two diastereomers. The ratio be-
tween the diastereomers possibly depends on the steric prop-
erties of the substituents. In every case, we isolated a mixture
of diastereomers with an excess of one diastereomer. In
compound 4b the diastereomeric excess was slightly higher
than in 4a. We assume, that the major diastereomer (rac-cis)
of 4b was responsible for the strong activity. The preferred
conformation of this diastereomer could be important for the
biological activity. The separation of the major diastereomer
to examine the inhibitory activity is under way.
Table 1. TNF-α inhibition by 5′-substituted thalidomide analogs.
——————————————————————————————
Inhibition of release
Compound 8 has an additional ethyl and phenyl group
located at the position 5′ of the original thalidomide molecule.
of TNF-α
Value (%)
at 50 µg/ml
——————————————————————————————
IC50 (µM)
The IC of 11 µM is comparable to compound 4b. It is
50
No. R1
R2
R3
R4
R5
X-Y
therefore to be concluded that an additional aromatic system
in position 5′ of thalidomide enhances the inhibitory activity.
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH–CH2
CH–CH2
CH–CH2
CH–CH2
C=CH
65
48
69
80
42
0
39–58
n.d.a
8.7
3
As a result of the introduction of a ∆ double bond in com-
4a
4b
8
H
Me
Ph
Ph
Ph
Ph
Ph
pounds 9, 10, and 11 the hybridization of the carbon atom at
H
3
2
position 3′ has changed from sp to sp . At the same time, the
molecules are less flexible in the glutarimide moiety and,
additionally, the compounds have only one chiral center in
comparison to 4a and 4b, which occur as diastereomeric
mixtures. 9 without any substitution at the phthalimide ring
enhanced the release of TNF-α. 10 is symmetrically substi-
tuted by two methoxy groups in position 5 and 6 of the
phthalimide moiety and it showed no inhibitory activity. A
small change in the substitution pattern by the two methoxy
groups in 11, showed a dramatic increase of the activity. 11
was found to be nearly 25-fold more active than thalidomide
Et
Et
Et
11.0
n.d.
n.d.
2.0
9
10
OMe OMe
C=CH
11
15
16
H
H
H
OMe OMe Et
C=CH
90
36
22
H
H
H
H
H
H
OAc CH–CH2
OH CH–CH2
n.d.
n.d.
——————————————————————————————
a n.d.: not determined.
Results and Discussion
with a IC of 2 µM. Obviously, the inhibitory activity of the
prepared glutethimide derivatives 9–11 depends strongly on
the substitution pattern in the phthalimide moiety.
Compound 11 was also found to inhibit the release of IL-2
in vitro. Human PBMCs were stimulated by monoclonal
antibodies CD2/CD28 and staphylococcal TSST-1. At a con-
centration of 119 µM, 11 decreased the concentration of IL-2
by 86±6% (CD2/CD28) and 77±20% (TSST-1). In this assay
thalidomide showed only weak activity.
50
Inhibition of release of TNF-α was measured in the super-
natant of human PBMCs stimulated with LPS. All substances
were screened in the first line using an initial concentration
of 50 µg/ml. Compounds 4a and 4b were tested as dia-
stereomeric mixtures. If a compound showed a better inhibi-
tory activity than thalidomide, the IC values were
50
determined by using different concentrations of the sub-
stance. The results are shown in Table 1.
Arch. Pharm. Pharm. Med. Chem. 331, 7–12 (1998)

10
Teubert, Zwingenberger, Wnendt, and Eger
3.28–3.33 (m, 1H, 5′-H), 5.54 (dd, J = 13.0, 5.7 Hz, 1H, 3′-H), 7.89–7.97 (m,
4H, aromatic H).– 13C-NMR δ = 15.12 (CH3), 28.15 (C-4′), 35.07 (C-5′),
48.26 (C-3′), 123.61, 131.04, 135.04 (C-aromatic), 165.74, 166.59, 169.29
(C=O).– Anal. (C14H11NO5)
Conclusions
Using thalidomide as a lead structure, we have prepared
new analogs which inhibit TNF-α production to varying
degrees in LPS-stimulated human PBMCs. All synthesized
compounds were substituted in position 5′ of the original
thalidomide molecule. Only compounds with an aromatic
substitution showed enhanced inhibitory activity on the re-
lease of TNF-α by stimulated PBMCs. The ability of com-
pound 11 to decrease enhanced IL-2 concentrations can be of
value with regard to the role of this cytokine in inflammation
and immune response.
2-(5-Phenyl-2,6-dioxo-tetrahydro-pyran-3-yl)-1H-isoindole-1,3[2H]-dione
(3b)
A mixture of 3.00 g (12.40 mmol) of 2b and 2.10 g (14.30 mmol) of
phthalic anhydride was refluxed in 40 ml of pyridine for 6 h. The hot solution
was filtered and evaporated in vacuo to dryness. The residue was dissolved
in 50 ml of hydrochloric acid (5%) and extracted three times with 50 ml of
ethyl acetate. The combined organic layers were washed withwater and dried
over sodium sulfate. The solvent was evaporated in vacuo and the residue
was refluxed in 40 ml of acetic anhydride for 1 h. The solvent was removed
in vacuo to a residue of about 5 ml which was diluted with 30 ml of ether.
The precipitated product was filtered off, washed with 100 ml of ether and
recrystallized from toluene to yield 2.60 g (63%) with mp 163–165 °C.–
(FT-IR) ν = 1816, 1774, 1716, 1392, 1040.– 1H-NMR (major diastereomer)
δ = 2.34–2.38 (m, 1H, 4′-H), 2.93–2.98 (m, 1H, 4′H), 4.64 (dd, J= 13.7 ,
4.9 Hz, 1H, 5′-H), 5.73 (dd, J = 13.1 , 5.4 Hz, 1H, 3′-H), 7.33–7.38 (m, 5H,
aromatic H phenyl), 7.89–7.94 (m, 4H, aromatic H phthalimide).– 13C-NMR
(major diastereomer) δ = 28.61 (C-4′), 46.55 (C-5′), 48.45 (C-3′), 123.64,
131.07, 135.07 (C-aromatic phthalimide), 127.73, 128.58, 128.67, 136.68
(C-aromatic phenyl), 165.38, 166.58, 167.49 (C=O).– Anal. (C19H13NO5)
Acknowledgment
Thanks are due to the Verband der Chemischen Industrie, Fonds der
Chemischen Industrie for supporting this work.
Experimental section
Melting points were determined on a Boetius melting point apparatus and
are uncorrected. Infrared spectra were recorded on a Perkin-Elmer FT-IR PC
16 spectrophotometer and NMR data were obtained with a Varian Gemini
300 spectrometer using tetramethylsilane as an internal standard. The IR and
NMR spectra of each compound were consistent with the assigned structure.
Elemental analysis were performed at the Institute of Organic Chemistry,
University Leipzig and were within ± 0.4 % of the theoretical values. Mass
spectra were obtained on a Hewlett Packard LC/MS (LC: HP 1050, MS:
HP-MS-Engine 5989 A). Thin layer chromatography was performed using
silica gel 60 F254 (Merck). Solvents were dried by conventional methods.
α-Methylglutamic acid 2a was synthesized according to ref.[26], diethyl
acetamidomalonate was used as reactant.
2-(5-Methyl-2,6-dioxo-piperidin-3-yl)-1H-isoindole-1,3[2H]-dione (4a)
A mixture of 2.00 g (7.30 mmol) of 3a and 0.23 g (3.80 mmol) of dried
urea was heated to 185–190 °C for 30 min. After cooling, the residue was
first refluxed in 5 ml of acetic anhydride for 5 min. Then 5 ml of ethanol was
added to the suspension at about 80 °C and the mixture was refluxed for
10 min. The precipitated product was filtered off and recrystallized from
DMF/H2O to yield 1.35 g (68%) as diastereomeric mixture with mp 270–
272 °C.– (FT-IR) ν = 1778, 1728, 1386, 1228.– 1H-NMR (major dias-
tereomer) δ = 1.16 (d, J = 6.8 Hz, 3H, CH3), 2.11–2.16 (m, 1H, 4′-H),
2.28–2.41 (m, 1H, 4′-H), 2.93–3.02 (m, 1H, 5′-H), 5.24 (dd, J = 12.9, 5.2 Hz,
1H, 3′-H), 7.89–7.94 (m, 4H, aromatic-H), 11.11 (s, 1H, NH).– 13C-NMR
(major diastereomer) δ = 14.80 (CH3), 30.08 (C-4′), 35.39 (C-5′), 49.40
(C-3′), 123.25, 131.18, 134.85(C-aromatic), 167.11, 170.02, 175.21 (C=O).–
MS(EI); m/z = 258 (30) [M]+.– Anal. (C14H12N2O4)
γ-Phenylglutamic acid (Diastereomeric mixture) (2b)
A suspension of 18.90 g (87.10 mmol) diethyl acetamidomalonate in a
solution of 0.20 g (8.70 mmol) of sodium in 50 ml of ethanol was stirred for
15 min. A solution of 17.00 g (91.60 mmol) of ethyl α-phenylacrylate in
20 ml of ethanol was added dropwise to the orange coloured suspension to
keep the mixture gently boiling. After 2.5 h the reaction was stopped with
5 ml glacial acetic acid and the solution was evaporated to dryness. The
residue was refluxed in 80 ml of concentrated hydrochloric acid for 16 h.
After cooling, the solution was extracted with 50 ml of ethyl acetate and the
aqueous layer was evaporated to dryness. The residuewascoevaporatedthree
times with 100 ml of water and then dissolved in 50 ml of water. The solution
was adjusted to pH 3 with ammonia solution (25%). The precipitated product
was filtered off and washed with 200 ml of ice cold water to yield 10.80 g
(51%) as raw product which was used without further purification.
Recrystallisation from water yielded a single diastereomer with mp 220–
223 °C.– FT-IR(KBr) ν = 3538, 2938, 1696, 1624, 1600, 1532, 1446, 1358,
1292, 1252.– 1H-NMR δ = 2.03–2.27 (m, 2H, CH2), 2.99–3.04 (m, 1H, 4-H),
3.60–3.65 (m, 1H, 2-H), 7.35–7.44 (m, 5H, aromatic H).– 13C-NMR δ =
39.02 (C-3), 51.92 (C-4), 54.62 (C-2), 127.03, 128.30, 128.92, 141.08
(C-aromatic), 182.28, 183.39 (C=O).– Anal. (C14H11NO5 • H2O)
2-(5-Phenyl-2,6-dioxo--piperidin-3-yl)-1H-isoindole-1,3[2H]-dione (4b)
The procedure followed the synthesis of 4a. From 2.00 g (6.00 mmol) of
3b was yielded 0.80 g (40%) 4b as diastereomeric mixture with mp 228–
231 °C.– (FT-IR) ν = 3088, 1780, 1760, 1392, 1220.– 1H-NMR (major
diastereomer) δ = 2.25–2.89 (m, 2H, 4′-H), 4.34 (dd, J = 13.6 , 4.7 Hz, 1H,
5′-H), 5.40 (dd, J = 12.9, 5.2 Hz, 1H, 3′-H), 7.22–7.39 (m, 5H, aromatic H
phenyl), 7.87–7.79 (m, 4H, aromatic H phthalimide), 11.35 (s, 1H, NH).–
13C-NMR (major diastereomer) δ = 30.39 (C-4′), 47.25 (C-5′), 49.51 (C-3′),
123.27, 123.58, 131.20, 134.84, 134.92 (C-aromatic phthalimide), 127.11,
128.43, 128.68, 138.25 (C-aromatic phenyl), 166.99, 167.17, 169.76, 173.46
(C=O).– MS(EI); m/z = 334 (57) [M]+.– Anal. (C19H14N2O4)
2-(5-Ethyl-5-phenyl-2,6-dioxo-piperidin-3-yl)-1H-isoindole-1,3[2H]-dione
(8)
No attempt was made to isolate the other diastereomer.
The solution of 1.00 g (4.30 mmol) of 7 in 40 ml of ethanol was vigorously
stirred under a atmosphere of hydrogen in the presence of 0.10 g of palla-
dium/charcoal (10%) until the starting material disappeared (TLC). The
catalyst was filtered off and the solution evaporated to dryness. The residue
was refluxed with0.70g(4.80mmol) ofphthalicanhydride in40ml ofglacial
acetic acid for 4 h. After cooling, the solvent was removed in vacuo and the
residue was recrystallized from ethanol to yield 0.99 g (63%) with mp
174–177 °C.– (FT-IR) ν = 3226, 1720, 1668, 1392.– 1H-NMR δ = 0.76 (t, J
= 7.3 Hz, 3H, CH2CH3), 1.85–1.95 (m, 2H, CH2CH3), 2.75–2.88 (m, 2H,
4′-H), 4.59 (dd, J = 13.1, 4.9 Hz, 1H, 3′-H), 7.37–7.51 (m, 5H, aromatic H
phenyl), 7.89–7.92 (m, 4H, aromatic H phthalimide), 11.55 (s, 1H, NH).–
13C-NMR δ = 8.86 (CH3), 30.24 (C-4′), 32.66 (CH2), 47.22 (C-3′), 51.74
(C-5′), 123.37, 123.61, 131.04, 131.07, 134.95, 135.03 (C-aromatic
2-(5-Methyl-2,6-dioxo-tetrahydro-pyran-3-yl)-1H-isoindole-1,3[2H]-dione
(3a)
A mixture of 2.00g(11.20mmol)of2a and 1.95 g (13.20 mmol) of phthalic
anhydride was refluxed in 15 ml of pyridine for 6 h. The solvent was removed
in vacuo and the residue was refluxed in acetic anhydride for 1 h. After
cooling, the precipitated product was collected by filtration and washed with
50 ml of ether. A second fraction was obtained by evaporation of the mother
liquor to dryness and dilution of the residue with 20 ml of ether. The
combined crude products were recrystallized from toluene to yield 2.00 g
(65%) with mp 223–225 °C.– FT-IR (KBr) ν = 1814, 1764, 1712, 1394, 1090,
1030.– 1H-NMR δ = 1.27 (d, J = 6.8 Hz, 3H, CH3), 2.15–2.48 (m, 2H, 4′-H),
Arch. Pharm. Pharm. Med. Chem. 331, 7–12 (1998)

5’-Substituted Thalidomide Analogs
11
phthalimide), 125.87, 127.78, 129.18, 138.73 (C-aromatic phenyl), 167.02,
phthalic anhydride for 6.5 h. After cooling, the dark solution was stirred over
night at room temperature. The precipitated product was collected by filtra-
tion and resolved in acetone. Addition of petroleum ether (bp 68–80 °C)
yielded 1.20 g (40%) as single diastereomer with mp 253–256 °C. From the
mother liquor 0.62 g (21%) was isolated as mixture of diastereomers.–
(FT-IR) ν = 3216, 3100, 1720, 1394, 1224.– 1H-NMR (single diastereomer)
δ = 2.12 (s, 3H, CH3), 2.38–2.43 (m, 1H, 4-H), 2.70–2.75 (m, 1H, 4-H), 5.49
(dd, J = 13.1 , 5.4 Hz, 1H, 5-H), 5.86 (dd, J = 13.0 , 5.7 Hz, 1H, 3-H), 7.91
(s, 4H, aromatic H), 11.54 (s, 1H, NH).– 13C-NMR (single diastereomer) δ =
20.44 (CH3), 27.60 (C-4), 47.69 (C-5), 67.37 (C-3), 123.40, 131.15, 134.95
(C-aromatic), 166.37, 167.11, 169.17, 169.90 (C=O).– MS (TSP); m/z = 334
(100) [M+18]+.– Anal. (C15H12N2O4)
168.87, 174.59 (C=O).– MS(EI); m/z = 362 (43) [M]+.– Anal. (C21H18N2O4)
2-(5-Ethyl-5-phenyl-2,6-dioxo-1,2,5,6-tetrahydro-pyridin-3-yl)-
1H-isoindole-1,3[2H]-dione (9)
A mixture of 0.50 g (2.20 mmol) of 7 and 0.40 g (2.70 mmol) of phthalic
anhydride was refluxed in 15 ml of glacial acetic acid for 6 h. After cooling
the precipitated product was collected by filtration, washed with 20 ml glacial
acetic acid and 20 ml of ether to give after drying in vacuo an analytically
pure product. Yield was 0.62 g (78%) with mp 273–274 °C (ref.[15] 275–
276.5 °C) (FT-IR) ν = 3186, 3072, 1724, 1690, 1662, 1378, 1238.– 1H-NMR
δ = 0.92 (t, J = 7.2 Hz, 3H, CH2CH3), 2.03–2.10 (m, 1H, CH2CH3), 2.55–2.62
(m, 1H, CH2CH3), 7.36–7.54 (m, 6H, aromatic H phenyl, 4′-H), 7.93–8.02
(m, 4H, aromatic H phthalimide), 11.72 (m, 1H, NH).– 13C-NMR δ = 9.34
(CH3), 30.10 (CH2), 55.02 (C-5′), 122.83 (C-3′), 123.78, 131.28, 135.16
(C-aromatic phthalimide), 126.83, 128.15, 128.91, 138.91 (C-aromatic
phenyl), 150.51 (C-4′), 161.50, 166.33, 166.58, 173.71 (C=O).– MS(EI); m/z
= 360 (28) [M]+.– Anal. (C21H16N2O4)
2-(5-Hydroxy-2,6-dioxo-piperidin-3-yl)-1H-isoindole-1,3[2H]-dione (16)
A solution of 1.00 g (3.20 mmol) of 15 (single diastereomer) and 0.30 g
(1.60 mmol) of p-toluenesulfonic acid was refluxed in 30 ml of methanol for
5 h. After cooling, the precipitated product was filtered off and recrystallized
from acetone/petroleum ether (bp 60–80 °C) or acetonitrile to yield 0.52 g
(60%) withmp195–230°C.–(FT-IR) ν = 3410, 1786, 1394, 1224.– 1H-NMR
δ = 2.27–2.53 (m, 2H, 4′-H), 4.53–4.57 (m, 1H, 5′-H), 5.29 (dd, J = 13.1,
5.2 Hz, 1H, 3′-H), 5.82 (d, J = 6.0 Hz, 1H, OH), 7.90–7.94 (m, 4H, aromatic
H), 11.22 (s, 1H, NH).– 13C-NMR δ = 31.03 (C-4′), 48.22 (C-3′), 66.33
(C-5′), 123.29, 131.19, 138.88 (C-aromatic), 166.86, 176.17, 169.70, 174.71
(C=O).– MS(EI); m/z = 274 (13) [M]+.– Anal. (C13H10N2O5)
2-(5-Ethyl-5-phenyl-2,6-dioxo-1,2,5,6-tetrahydro-pyridin-3-yl)-
5,6-dimethoxy-1H-isoindole-1,3[2H]-dione (10)
Preparation followed the synthesis of 9, from 0.45 g (1.90 mmol) of 7 and
0.45 g (2.10 mmol) of 4,5-dimethoxyphthalic anhydride in a yield of 0.54 g
(65%) with mp 288–290 °C.– (FT-IR) ν = 3436, 1716, 1372, 1310, 1224.–
1H-NMR δ 0.95 (t, J = 7.4 Hz, 3H, CH2CH3), 2.06–2.11 (m, 1H, CH2CH3),
2.51–2.59 (m, 1H, CH2CH3), 3.96 (s, 6H, OCH3), 7.44–7.54 (m, 8H, aro-
matic H, 4′-H), 11.61 (s, 1H, NH).– 13C-NMR δ = 9.31 (CH3), 30.20 (CH2),
55.06 (C-5′), 56.54 (OCH3), 106.15, 124.69, 154.22 (C-aromatic
phthalimide),123.15 (C-3′), 126.84, 128.06, 128.85, 139.08 (C-aromatic
phenyl), 150.14 (C-4′), 161.57, 166.58, 173.68 (C=O).– MS(EI); m/z = 420
(51) [M]+.– Anal. (C23H20N2O6)
Assay for inhibition of TNF-α synthesis by human PBMCs
PBMCs isolated from three healthy human donors were cultured in RPMI
1640 (containing 10% fetal calf serum, 100 µM β-mercaptoethanol, 50 µg/ml
penicillin, 2 mM glutamine and 50 µg/ml streptomycin) at a density of 1–3 ×
106 cells/ml at 37 °C, 5% CO2 in presence of test compounds in 24-well-
plates (Sigma, Deisenhofen, FRG). Test compounds were dissolved in di-
methylsulfoxide (DMSO, Merck, Darmstadt, FRG) and routinely applied
1 hour before release of TNF-α was stimulated by addition of lipopolysac-
charide from Escherichia coli serotype 0127:B8 (Sigma, Deisenhofen, FRG)
at a final concentration of 2.5 µg/ml. The final concentration of DMSO was
adjusted to 0.1 % for each culture sample. After incubation for 20 h at 37 °C
and 5% CO2 the TNF-α concentration of all culture supernatants was
determined using a commercial TNF-α-ELISA (Boehringer-Mannheim).
The percent inhibition of release of TNF-α was calculated relative to cultures
treated with DMSO alone. The IC50-values were calculated by linear regres-
sion analysis.
2-(5-Ethyl-5-phenyl-2,6-dioxo-1,2,5,6-tetrahydro-pyridin-3-yl)-
4,5-dimethoxy-1H-isoindole-1,3[2H]-dione (11)
Preparation followed the synthesis of 9 using 0.45 g (1.90 mmol) 7 and
0.45 g (2.10 mmol) of 3,4-dimethoxyphthalic anhydride. After recrystallisa-
tion from ethanol 0.55 g (67%) of 11 was yielded with mp 203–205 °C.–
(FT-IR) ν = 3218, 1724, 1496, 1374, 1276.– 1H-NMR δ = 0.91–0.97 (m, 3H,
CH2CH3), 2.07–2.09 (m, 1H, CH2CH3), 2.57–2.59 (m, 1H, CH2CH3), 3.95
(s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.31–7.70 (m, 8H, aromatic H, 4′-H),
11.69 (s, 1H, NH).– 13C-NMR δ = 9.34 (CH3), 29.98 (CH2), 55.02 (C-5′),
56.75 (OCH3), 61.85 (OCH3), 117.72, 120.29, 122.99, 123.36, 146.73,
158.02 (C-aromatic phthalimide), 121.78 (C-3′), 126.83, 128.12, 128.90,
138.95 (C-aromatic phenyl), 150.42 (C-4′) 161.54, 164.44, 165.73, 173.74
(C=O).– MS(EI); m/z = 420 (15) [M]+.– Anal. (C23H20N2O6)
Assay for inhibition of IL-2 synthesis by human PBMCs
PBMCs were isolated and cultured as described above. Test compounds
were dissolved in dimethylsulfoxide (DMSO, Merck, Darmstadt, FRG) and
1 µl solution was added to 1 ml culture and incubated for 1 h at 37 °C and
5% CO2. PBMCs were stimulated with 0.1 µg/ml monoclonal antibodies
(Klon-Nr. AICD2.M1 Prof. Dr. Meurer; anti CD-28, CLB, Amsterdam) and
0.1 µg/ml staphylococcal-superantigen (Escherichia coli serotype
0127:B8,TSST-1, Sigma, Deisenhofen, FRG, ) respectively. After incuba-
tion for 20 h at 37 °C and 5% CO2 the IL-2 concentration of all culture
supernatants was determined using a commercial TNF-α-ELISA (Boehrin-
ger-Mannheim). The percent inhibition of IL-2 synthesis was calculated
relative to cultures treated with DMSO alone.
Acetic acid 5-benzyloxycarbonylamino-2,6-dioxo-piperidin-3-yl ester (14)
A solution of 10.00 g (31.90 mmol) of 13 [17] was refluxed in 100 ml of
acetic anhydride for 3 h. The solution was evaporated to dryness. The residue
was dissolved in 10 ml of ethanol and diluted with 200 ml of water. The
precipitated product was filtered off and recrystallized from H2O to yield
7.50 g (73%) with mp 149–150 °C.– (FT-IR) ν = 3332, 1746, 1728, 1260.–
1H-NMR δ = 2.10 (s, 3H, CH3), 2.21–2.28 (m, 2H, 4-H), 4.38–4.42 (m, 1H,
3-H), 5.07 (s, 2H, CH2O), 5.54 (dd, J = 6.9 , 5.6 Hz, 1H, 5-H), 7.31–7.37 (m,
5H, aromatic H), 7.97 (d, J = 8.5 Hz, 1H, CONH), 11.27 (s, 1H, 1-H).–
13C-NMR δ 20.46 (CH3), 30.09 (C-4), 48.20 (C-3), 65.83 (CH2O), 66.72
(C-5), 127.81, 127.88, 128.35, 136.70 (C-aromatic), 156.09 (CONH),
168.90, 169.22, 170.75 (C=O).– MS(TSP); m/z = 338 (64) [M+18]+, 353
(100) [M+33]+.– Anal. (C15H16N2O6)
References
✩
Dedicated to Professor P. Welzel, Universität Leipzig, Institut für
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Received: August 18, 1997 [FP247]
Arch. Pharm. Pharm. Med. Chem. 331, 7–12 (1998)