Thiothalidomides: Novel Isosteric Analogues of Thalidomide with Enhanced TNF-α Inhibitory Activity

Article abstract of DOI:10.1021/jm030152f

Thalidomide is being increasingly used in the clinical management of a wide spectrum of immunologically-mediated and infectious diseases, and cancers. However, the mechanisms underlying its pharmacological action are still under investigation. In this regard, oral thalidomide is clinically valuable in the treatment of erythema nodosum leprosum (ENL) and mutiple myeloma and effectively reduces tumor necrosis factor-α (TNF-α) levels and angiogenesis in vivo. This contrasts with its relatively weak effects on TNF-α and angiogenesis in in vitro studies and implies that active metabolites contribute to its in vivo pharmacologic action and that specific analogues would be endowed with potent activity. Our focus in the structural modification of thalidomide is toward the discovery of novel isosteric active analogues. In this regard, a series of thiothalidomides and analogues were synthesized and evaluated for their TNF-α inhibitory activity against lipopolysacharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC), This was combined with a PBMC viability assay to differentiate reductions in TNF-α secretion from cellular toxicity. Two isosteric analogues of thalidomide, compounds 15 and 16, that mostly reflect the parent compound, together with the simple structure, dithioglutarimide 19, potently inhibited TNF-α secretion, compared to thalidomide, 1. The mechanism underpinning this most likely is posttranscriptional, as each of these compounds decreased TNF-α mRNA stability via its 3′-UTR. The potency of 19 warrants further study and suggests that replacement of the amide carbonyl with a thiocarbonyl may be beneficial for increased TNF-α inhibitory action. In addition, an intact phthalimido moiety appeared to be requisite for TNF-α inhibitory activity.

Full text of DOI:10.1021/jm030152f

5222
J . Med. Chem. 2003, 46, 5222-5229
Th ioth a lid om id es: Novel Isoster ic An a logu es of Th a lid om id e w ith En h a n ced
TNF -r In h ibitor y Activity†
Xiaoxiang Zhu,‡,X Tony Giordano,§,∆ Qian-sheng Yu,‡ Harold W. Holloway,‡ Tracy Ann Perry,‡
Debomoy K. Lahiri,| Arnold Brossi, and Nigel H. Greig*,‡
Drug Design & Development Section, Laboratory of Neurosciences, Gerontology Research Center (4E02),
Intramural Research Program, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr.,
Baltimore, Maryland 21224-6825, Message Pharmaceuticals, 30 Spring Mill Drive, Malvern, Pennsylvania 19355,
Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine,
Indianapolis, Indiana 46202, and Scientist Emeritus, National Institutes of Health, & School of Pharmacy,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7361
Received April 4, 2003
Thalidomide is being increasingly used in the clinical management of a wide spectrum of
immunologically-mediated and infectious diseases, and cancers. However, the mechanisms
underlying its pharmacological action are still under investigation. In this regard, oral
thalidomide is clinically valuable in the treatment of erythema nodosum leprosum (ENL) and
mutiple myeloma and effectively reduces tumor necrosis factor-R (TNF-R) levels and angio-
genesis in vivo. This contrasts with its relatively weak effects on TNF-R and angiogenesis in
in vitro studies and implies that active metabolites contribute to its in vivo pharmacologic
action and that specific analogues would be endowed with potent activity. Our focus in the
structural modification of thalidomide is toward the discovery of novel isosteric active analogues.
In this regard, a series of thiothalidomides and analogues were synthesized and evaluated for
their TNF-R inhibitory activity against lipopolysacharide (LPS)-stimulated peripheral blood
mononuclear cells (PBMC), This was combined with a PBMC viability assay to differentiate
reductions in TNF-R secretion from cellular toxicity. Two isosteric analogues of thalidomide,
compounds 15 and 16, that mostly reflect the parent compound, together with the simple
structure, dithioglutarimide 19, potently inhibited TNF-R secretion, compared to thalidomide,
1. The mechanism underpinning this most likely is posttranscriptional, as each of these
compounds decreased TNF-R mRNA stability via its 3′-UTR. The potency of 19 warrants further
study and suggests that replacement of the amide carbonyl with a thiocarbonyl may be beneficial
for increased TNF-R inhibitory action. In addition, an intact phthalimido moiety appeared to
be requisite for TNF-R inhibitory activity.
In tr od u ction
Thalidomide (N-R-phthalimidoglutarimide (1) (Figure
1)) is a glutamic acid derivative that was introduced
onto the market as a sedative hypnotic in 1956, but was
withdrawn in 1961 due to the development of severe
F igu r e 1. The structure of thalidomide.
congenital abnormalities in babies born to mothers
using it for morning sickness.1 Interest in the agent was
lowers its synthesis and secretion.6,7 Further studies
reawakened after thalidomide was found clinically
have defined it to be a costimulator of both CD8+ and
effective in the treatment of erythema nodosum lepro-
CD4+ T cells,8 an inhibitor of angiogenesis via its
sum (ENL)2 and in the treatment of HIV wasting
inhibitory actions on basic fibroblast growth factor
syndrome and various cancers.3,4 Mechanistic studies
(bFGF) and vascular endothelial growth factor
of its ENL activity demonstrated an anti-TNF-R action.5
(VEGF),9,10 and an inhibitor of the transcription factor,
Specifically, thalidomide enhances the degradation of
NFkB.6,7 However, it is the action of thalidomide on
tumor necrosis factor-R (TNF-R) RNA and, thereby,
TNF-R that provides the focus of the current study.
TNF-R and family members play pivotal roles in a
variety of physiological and pathological processes,
which include cell proliferation and differentiation,
apoptosis, the modulation of immune responses, and
induction of inflammation. TNF-R acts via two recep-
tors, TNFR1 and 2. The former is expressed in all
tissues and is the predominant signaling receptor for
TNF-R. The latter is primarily expressed on immune
cells and mediates more limited biological responses.
The exposure of cells to TNF-R can result in activation
† Dedicated to our wonderful collaborator, mentor, and friend, Arnold
Brossi, on his 80th birthday.
* To whom correspondence should be addressed. Phone: 410-558-
8278. Fax: 410-558-8323. E-mail: GreigN@vax.grc.nia.nih.gov.
‡ National Institute on Aging, National Institutes of Health.
§ Message Pharmaceuticals.
| Indiana University School of Medicine.
National Institutes of Health, and School of Pharmacy, University
of North Carolina at Chapel Hill.
X Currently at Amicus Therapeutics, NJ .
∆ Currently at Department of Biochemistry & Molecular Biology,
Louisiana State University Health Sciences Center School of Medicine,
Shreveport, LA 71130.
10.1021/jm030152f CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/16/2003

Novel Isosteric Analogues of Thalidomide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5223
of a caspase cascade leading to cell death via apoptosis.
Indeed, major cell surface molecules capable of initiating
apoptosis are members of the TNF family of ligands and
receptors. For example, death-inducing members of the
TNF receptor family each contain a cytoplasmic ‘death
domain’ (DD), which is a protein-protein interaction
motif critical for engaging downstream components of
the signal transduction machinery.11 Recently, TRAIL,
the tumor necrosis factor-related apoptosis-inducing
ligand, has been shown to selectively induce apoptosis
of tumor cells, but not most normal cells.12 It is sug-
gested that TRAIL mediates thymocyte apoptosis and
is important in the induction of autoimmune diseases.
More often, however, TNF-R receptor binding induces
the activation of transcription factors, AP-1 and NFkB,
that thereafter induce genes involved in acute and
chronic inflammatory responses.13,14 Overproduction of
TNF-R has thus been implicated in many inflammatory
diseases, such as rheumatoid arthritis, graft-versus-host
disease, and Crohn’s disease, and it additionally exac-
erbates ENL, septic shock, AIDS, and dementia associ-
ated with Alzheimer’s disease (AD). In this context, our
present work, which examines novel analogues of tha-
lidomide with enhanced TNF-R inhibitory activity,
assumes significance.
F igu r e 2. The structures of thiothalidomides and analogues.
mides and analogues were designed to explore their
action on inhibition of TNF-R (Figure 2).
Ch em istr y
Monothiothalidomide 5 was prepared as shown in
Scheme 1. tert-Butoxycarbonyl-L-glutamine 2 was re-
fluxed with carbonyldiimidazole (CDI) in THF and
cyclized to afford imide 3.21 Imide 3 then was treated
with trifluoroacetic acid in CH2Cl2 to remove the protec-
tive group to generate aminoglutarimide trifluoroacetate
4. Without further purification, compound 4 was reacted
with phthalic anhydride in refluxing THF in the pres-
ence of triethylamine to produce thalidomide 1 in the
total yield of 31% from compound 2. Thalidomide 1 was
thionated with Lawesson’s reagent (LR)28 to generate
a single new product, whose structure was identified
as 6′-thiothalidomide 5 by MS and 1D and 2D NMR
spectra. The position of thiocarbonyl group was estab-
lished from the HMBC cross-peak of H-5′/C-6′ (see
Supporting Information: HMBC spectrum of compound
5).
The synthesis of 3-thiothalidomide 12 is shown in
Scheme 2. N-Phthaloyl-L-glutamic acid 6 was esterified
to afford diester 7. Compound 7 was thionated with LR
at 110 °C to give compound 8 as a major product.
Concurrently, compound 9 was separated as a minor
product by chromatography. However, no thioesters
were separated from the reaction mixture, as any ester
reacts with LR at 130 °C.27 Furthermore, we failed to
obtain 3-thiothalidomide, 12, through the cyclization of
compound 8 with ammonia or amine, as ammonia reacts
with the thioamide; reaction of compound 8 with ben-
zylamine produced the unexpected compound 10. In an
alternative approach, compound 8 was hydrolyzed under
acidic conditions to give diacid 11. Compound 11 was
then reacted with trifluoroacetamide to generate 1-thio-
thalidomide 12 in the presence of 1-hydroxybenzotria-
zole (HOBT) and 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide hydrochloride (EDCI).29
The mechanisms underlying thalidomide’s diverse
actions, together with identification of the active species,
remains an area of intense research. The compound is
more active in vivo than would be predicted from its
potency in in vitro studies.15,16 This suggests that active
metabolites largely account for its in vivo activity17 and
that specific analogues of thalidomide would be endowed
with high potency. Indeed, seldom has such a simple
molecule had such a controversial history and held so
much therapeutic promise.3,4,10,18 The focus of our
research has been two pronged: (i) to elucidate the
action of thalidomide on the regulation of TNF-R levels,
and (ii) to develop analogues of clinical potential that
potently inhibit TNF-R secretion.
A number of papers have been published on the
design and synthesis of thalidomide analogues opti-
mized to reduce TNF-R synthesis.19-25 These have
primarily focused on exploring structural modifications
of the phthaloyl ring or glutarimide ring of thalidomide
and have described N-phthaloyl 3-amino-3-arylpropionic
acid derivatives, amino-phthaloyl-substituted and tet-
rafluorophthaloyl-substituted analogues of thalidomide,
and N-substituted phthalimides with a simplified glu-
tarimide moiety. In addition, following the demonstra-
tion that the antiangiogenic property of thalidomide is
associated with its hydroxylated metabolites, the open
ring metabolites,15,26 syntheses of the hydroxylated and
hydrolysis metabolites as inhibitors of angiogenesis or
tumor metastasis have been reported.23,24,27 In the
current paper we focus on the synthesis and evaluation
of novel sulfur analogues of thalidomide with enhanced
biological activity.
Although extensive studies exist regarding the struc-
ture-activity relationships between thalidomide and
TNF-R, very little is known about the contribution of
four amide carbonyl groups of thalidomide to its biologi-
cal activity. The isosteric replacement of carbonyl group
by a thiocarbonyl group provides isosteric analogues of
thalidomide: thiothalidomides. A series of thiothalido-
In the synthesis of dithiothalidomide, our initial
attempt involved the reaction of monothiothalidomide
with LR at reflux in toluene. Under such conditions,
2′,6′-dithiothalidomide was obtained in a yield of less
than 2% (Scheme 3(a)). Such a yield was so low that

5224 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Zhu et al.
Sch em e 1a
a
Reagents: (a) CDI/THF; (b) CF3COOH/CH2Cl2; (c) phthalic anhydride, Et3N/THF; (d) Lawesson’s reagent/toluene.
Sch em e 2a
a
Reagents: (a) Lawesson’s reagent/toluene; (b) benzylamine; (c) HCl/HOAc; (d) F3CCONH2, HOBT, EDCI, Et3N/CH2Cl2.
Sch em e 3a
a
Reagents: (a) Lawesson’s reagent/toluene; (b) Lawesson’s reagent, pyridine/toluene; (c) Lawesson’s reagent, morpholine/toluene.
Sch em e 4. Mechanism of Catalysis for Lawesson’s
Reagent
concentration of the ylide 14. When pyridine was used
as a catalyst for thionation, monothiothalidomide 5 was
thionated with LR to produce two dithiothalidomides,
13 and 15, in the yields of 45% and 31%, respectively
(Scheme 3b,c). Dithiothalidomide 13 could be further
thionated with LR in the presence of the stronger base,
morpholine, to give trithiothalidomide 16 in the yield
of 65%.
improvement was clearly essential and was undertaken
by modifying the reaction conditions. It is believed that
the mechanism underlying the reaction between LR and
a carbonyl moiety is that a highly reactive dithiophos-
phine ylide 14, rather than LR itself, likely is the active
thionating agent (Scheme 4).28 The Lewis base should
be able to increase the reactivity of LR as the base may
drive the unfavorable equilibrium and elevate the
Glutarimide 17 was thionated with LR in THF at
room temperature to afford compound 18 as a major
product.30 Glutarimide 17 also was refluxed with LR in
toluene to produce dithioglutarimide 19 (Scheme 5).31
Reaction of potassium phthalimide with 3-bromocyclo-
hexene underwent the Gabriel reaction to give com-
pound 21. Thereafter, thionation of compound 21 with

Novel Isosteric Analogues of Thalidomide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5225
Sch em e 5a
were greater than 12 (>30 µM) possessing a normal
glutarimide ring.
In this regard, thalidomide is composed of two distinct
moieties: the glutarimide and phthalimide rings. Thio-
glutarimides and thiophthalimides were thus synthe-
sized and evaluated to assess the effect of thio-analogues
of these two moieties on TNF-R levels. Monothiogluta-
rimide 18 minimally inhibited TNF-R secretion at a
concentration of 30 µM; however, dithioglutarimide 19
exerted a potent inhibitory effect with an IC50 of 8 µM
and a lack of toxicity. An intact phthaloyl ring is
believed to be required for the inhibition of TNF-R
secretion.19 Surprisingly, such a simple structure, dithio-
glutarimide 19, proved to be 25-fold more active than
1. In contrast, 2′,6′-dithiothalidomide 13, a phthalimido-
substituted dithioglutarimide, is less active than dithio-
glutarimide 19 and induces toxicity at high concentra-
tion. Monothiophthalimide 25 showed a marginal TNF-R
activity at a concentration of 30 µM without toxicity.
Interestingly, however, dithiophthalimide 24 was found
to possess potent activity with an IC50 of 3 µM. Although
it was associated with toxicity at 30 µM, its inhibition
of TNF-R occurred at a log lower concentration that was
well tolerated.
a
Reagents: (a) Lawesson’s reagent/THF, rt; (b) Lawesson’s
reagent, reflux/toluene.
LR afforded compounds 22 and 23 (Scheme 6). Com-
pounds 24 and 25 were prepared in a similar procedure
to the preparation of compounds 22 and 23.32
Resu lts a n d Discu ssion
The action of the described thiothalidomide analogues
to inhibit TNF-R secretion was assessed in human
peripheral blood mononuclear cells (PBMC) and is
shown in Table 1. Thalidomide, 1, entirely lacked
activity at 30 µM. A concentration of 100 µM was
required for significant activity. The monothiothalido-
mides, 6′-thiothalidomide 5, and 3-thiothalidomide 12
showed only marginal activity at 30 µM with 31% and
23% inhibition of TNF-R secretion, respectively. In
contrast, the dithiothalidomides, including 2′,6′-dithio-
thalidomide 13 and 3,6′-dithiothalidomide 15, exhibited
more potent inhibitory activities with IC50 values of 20
µM and 11 µM, respectively. However, assessment of
cell viability by MTS assay showed that 13 induced
increasing cytotoxicity at higher concentrations. Trithio-
thalidomide 16 inhibited TNF-R production with an IC50
of 6 µM without accompanying toxicity. Compared with
thalidomide, 1, with an IC50 of ∼200 µM for the
inhibition of TNF-R synthesis, trithiothalidomide 16 is
over 30-fold more active. Hence, successive replacement
of a carbonyl with a thiocarbonyl group led to improved
inhibitory activity compared to 1, unassociated with
toxicity. In this regard, the synthesized thiothalidomides
possessed TNF-R lowering potency in the following
decreasing order: trithiothalidomide 16 > dithiothali-
domide 15 and 13 > monothiothalidomides 5 and 12 >
thalidomide, 1.
A comparison of the physical properties of thalido-
mide, 1, and thiothalidomides shows that they have
similar van der Waals radii and bond angles, although
the CdS bond is slightly longer than the CdO bond. A
possible explanation accounting for the elevated potency
of the thiothalidomides is that their enhanced lipophi-
licity and loss of hydrogen bond acceptor capability
potentially allows the attainment of higher intracellular
drug levels. Interestingly, compounds 8, 9, and 11 are
thio analogues of hydrolysis metabolites of thalidomide.
Assessment of their TNF-R inhibitory action determined
that the monothio analogue, 8, has an IC50 of 20 µM
without toxicity; demethylation (11) lowered potency.
The dithio analogue, 9, proved 2-fold more potent still
than 8, but induced cellular toxicity at lower concentra-
tions. Intriguingly, thio analogues 22 and 23, with a
simplified glutarimide ring, were found to be active
TNF-R inhibitors, albeit with some toxicity at 30 µM,
with IC50 values (15 µM and 16 µM, respectively) that
As described, compounds 15, 16, and 19 potently
inhibited TNF-R secretion without toxicity. As a conse-
quence, additional studies were undertaken to elucidate
the mechanism underpinning this action. Gene and
protein expressions are controlled at the level of tran-
scription, posttranscription, RNA stability, and transla-
tion under different physiological stimuli. Recently,
posttranscriptional pathways have been recognized to
provide a major means of regulating eukaryotic gene
expression. In this regard, TNF-R and other cytokines
and protooncogenes are known to be regulated at the
posttranscriptional level.6,7,33,34 Multiple proteins, in-
cluding the four cloned proteins AUF1, HuR, TTP, and
HuD have been shown to bind to a region of the mRNA
that contains adenylate/uridylate (AU)-rich elements
(AREs) in the 3′-untranslated region (UTR). These
proteins mediate RNA turnover and decay and hence
translational efficiency.35,36 The stability of TNF-R
mRNA is largely regulated at its 3′-UTR, which contains
a well characterized ARE. Although AREs are found in
a number of different cytokine and protooncogene RNAs,
the pathways by which they induce degradation are
highly specific for a given ARE, suggesting some cellular
specificity. When the AREs from different cytokines are
complexed with AUF1, different binding affinities are
observed. Notably, however, the highest affinity for
AUF1 is to human and then mouse TNF-R.
To determine the involvement of the 3′-UTR in the
action of our thalidomide analogues, their ability was
assessed to inhibit reporter gene activity in cells con-
taining the TNF-R 3′-UTR versus a control vector
(Figure 3). Specifically, this cell-based assay comprised
of two stably transfected cell lines derived from the
mouse macrophage line, RAW264.7. One line, desig-
nated “luciferase only”, expressed a luciferase reporter
construct without any UTR sequences, whereas the
other, designated “luciferase + TNF UTRs”, expressed
a luciferase reporter construct with the entire 3′- UTR
of human TNF-R inserted directly downstream of the
luciferase coding region. Compounds 15, 16, and 19

5226 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Zhu et al.
Sch em e 6a
a
Reagents: (a) 3-bromocyclohexene/DMF; (b) Lawesson’s reagent/toluene.
Ta ble 1. Inhibition of LPS-Induced TNF-R Production in
UTR, located 200 nucleotides downstream from the stop
codon.37 This 3′-UTR element acts as an mRNA desta-
bilizer whose function can be inhibited by the presence
of growth factors. In contrast, different cytokines,38
including TNF-R39 and iron,40 can up regulate APP
protein synthesis at the level of its 5′-UTR, whereas,
interestingly, the anticholinesterase, phenserine, that
is currently in clinical trials for AD lowers APP protein
levels with concurrent maintenance of mRNA steady-
state levels through translational modification within
the same 5′-UTR element.41 A further example is that
of the human immunodeficiency virus 1 (HIV-1) Trans-
activating transduction (tat) protein, which binds trans-
activation-responsive region (TAR) RNA.42 Tat is brought
into contact with the transcription machinery after
binding the TAR element, which is a 59-residue stem-
loop RNA found at the 5′ end of all HIV-1 transcripts.42
Finally, thalidomide (1) has been reported to lower
cyclooxygenase-2 (Cox-2) biosynthesis via its 3′-UTR43
that appears to likewise contain an ARE that can
regulate Cox-2 mRNA stability.44 Our studies of ana-
logues 15, 16, and 19 confirm the regulation of TNF-R
protein levels by thalidomide (1) via its 3′-UTR, whether
the 5′-UTR contains a similar element that is accessible
to pharmacological manipulation remains to be deter-
mined, as does action against Cox-2.
PBMC and Cell Viabilitya
cell viability
% inhibition
compd
at 30 µM
IC50 (µM) at 30 µM at 3 µM at 0.3 µM
5
8
9
31
56
85
20
23
52
61
79
15
75
86
85
95
34
>30
20
10
>30
>30
20
>100
93
90
99
86
93
100
87
87
86
84
98
94
89
83
94
96
96
89
93
94
94
94
90
86
99
96
99
83
94
57
86
94
69
11
12
13
15
16
18
19
22
23
24
25
11
6
>100
94
>30
>100
>100
50
57
54
>100
8
15
16
3
>30
a
Thalidomide (1) totally lacked activity at 30 µM (IC50 ∼ 200
µM).
Our future studies are aimed at assessing whether
the in vitro activity of the described thalidomide ana-
logues translates into pertinent in vivo models, such as
in mice challenged with LPS.33 In terms of the mecha-
nism of TNF-R signaling, there is recent in vivo evidence
showing that the AU-rich 3′-UTRs of TNF-R and IL-6
are downstream to MAPKAP kinase 2 (MK2) signaling.
MK2 is an essential component of mechanisms that
regulate biosynthesis of IL-6 at the level of mRNA
stability, and of TNF-R mainly through TNF-ARE-
dependent translational control.45 In addition, future in
vitro studies involving the induction of other cytokines
and deletion of the ARE will define the specificity of the
most potent compounds described herein, as well as
ARE-independent actions. Knockout mice for the TNF
ARE or the destabilizing protein TTP both have elevated
levels of TNF-R and increased inflammation. Hence, the
interaction between the TNF-R ARE and the cognate
RNA binding proteins appears to represent an attractive
target for regulation at the posttranscriptional level.
F igu r e 3. The action of compounds 15, 16, and 19 in cells
(mouse macrophage cell line, RAW264.7.) possessing a lu-
ciferase reporter element plus the 3′-UTR of human TNF-R
compared to cells lacking the 3′-UTR. All agents lowered
luciferase reporter activity in cells stably expressing the 3′-
UTR. Thalidomide 1 lacked activity at 50 µM.
exerted differential effect on the two cell lines in a dose-
dependent manner, consistent with their ability to
inhibit TNF-R production via the 3′-UTR.
As TNF-R protein levels changed without significant
alterations in mRNA levels (data not shown), protein
expression is presumably regulated via translational
control (at the posttranscriptional level). There is pre-
cedence for translational (protein) control through either
the 3′- or 5′-UTR regions of a number of critical proteins
that are current drug targets. For example, levels of the
â-amyloid precursor protein (APP) that is central to the
development of AD can be regulated by either UTR.
Turnover and translation of APP mRNA is regulated
by a 29-nucleotide instability element within the 3′-
In conclusion, we have synthesized a novel series of
thiothalidomide analogues that are more potent inhibi-
tors of TNF-R production in LPS-induced human PB-
MCs than thalidomide, 1. The isosteric replacement of
successive carbonyl groups by a thiocarbonyl leads to
an increasing inhibition with the number moieties
replaced (trithiothalidomide 16 > dithiothalidomide 15
and 13 > monothiothalidomides 5 and 12 > thalidomide
1). Preliminary studies indicate that the induced TNF-R
inhibition involved posttranscriptional mechanisms and

Novel Isosteric Analogues of Thalidomide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5227
d ith ioxo-2H-isoin d ol-2-yl)p en ta n ed ioa te (9). A mixture of
compound 7 (144 mg, 0.47 mmol) and LR (191 mg, 0.47 mmol)
in toluene was stirred in a 110 °C oil bath for 10 h. The solvent
was then evaporated, and the residue was purified by column
chromatography (silica gel) using CH2Cl2 as the eluent, to
obtain compound 9 (17 mg, 11%) as a dark red oil. Thereafter,
using CH2Cl2:EtOAc (10:1) as the eluent the more polar
component 8 (105 mg, 70%) was obtained as a red oil.
Compound 8: 1H NMR (CDCl3) δ 7.98-7.96 (m, 1H), 7.81-
7.70 (m, 3H), 5.53 (dd, J ) 5.1 Hz, J ) 10 Hz, 1H), 3.70 (s,
3H), 3.59 (s, 3H), 2.76-2.56 (m, 2H), 2.40-2.33 (m, 2H); MS
(CI/CH4) m/z 321 (M+).
Compound 9: 1H NMR (CDCl3) δ 7.87-7.84 (m, 2H), 7.73-
7.68 (m. 2H), 6.09 (dd, J ) 5 Hz, J ) 10 Hz, 1H), 3.70 (s, 3H),
3.58 (s, 3H), 2.81-2.63 (m, 2H), 2.40-2.24 (m, 2H); MS (DEI)
m/z 337 (M+); HRMS (DEI) calcd for C15H15NO4S2 337.0442
(M+), found 337.0449.
occurred via its 3′-UTR. Like thalidomide 1, 15, and 16
effectively inhibited angiogenesis, whereas the potent
TNF-R inhibitor, dithioglutarimide 19, was inactive in
angiogenesis assays (data not shown); suggesting that
different structure/activity relations are responsible for
these pharmacological actions. TNF-R has been vali-
dated as a drug target with Remicade (Cetocor, Malvern,
PA; Schering-Plough, Orange, NJ ) and Enbrel (Amgen,
Thousand Oaks, CA; Wyeth-Ayerst, Princeton, NJ ) on
the market; however, both are large macromolecules
and hence require injection. The development of small,
druglike molecules to potently and safely inhibit TNF-R
likely will be of significant clinical value.
Exp er im en ta l Section
Gen er a l. Melting points were determined with a Fisher-
J ohns apparatus and are uncorrected. 1H NMR, 13C NMR, and
2D NMR were recorded on a Bruker AC-300 spectrometer.
Mass spectra and high-resolution mass spectra (HRMS) were
recorded on a VG 7070 mass spectrometer and a Agilent
Technologies 5973N GC-MS (CI). All exact mass measure-
ments show an error of less than 5 ppm. Elemental analyses
were performed by Atlantic Microlab, Inc., Norcross, GA.
3-(ter t-Bu toxyca r bon yla m in o)-2,6-p ip er id in ed ion e (3).
A mixture of N-(tert-butoxycarbonyl)-L-glutamine (4.92 g, 20
mmol) and carbonyl diimidazole (3.24 g, 20 mmol) in THF (100
mL) was refluxed for 16 h. Thereafter, solvent was removed,
and the crude product was recrystallized from hot EtOAc to
give compound 3 (2.04 g, 45%) as white crystals: mp 214-
2-(1,3-Dih yd r o-1-oxo-3-t h ioxo-2H -isoin d ol-2-yl)p e n -
ta n ed ioic Acid (11). Compound 8 (350 mg, 1.09 mmol) was
stirred with a 1:1 mixture of acetic acid glacial and concen-
trated HCl in a 100 °C oil bath for 2.5 h. Ethyl acetate (100
mL) and ice-water (30 mL) was added. The ethyl acetate layer
was separated, washed with ice-water, dried over Na2SO4,
and concentrated. The resulting syrup was crystallized with
ether to afford compound 11 as red crystals (253 mg, 79%);
1
mp 157 °C; H NMR (DMSO-d6) δ 8.04-7.96 (m, 1H), 7.91-
7.74 (m, 3H), 5.43 (dd, J ) 5.1 Hz, J ) 9.6 Hz, 1H), 2.42-2.33
(m, 2H), 2.30-2.26 (m, 2H); MS (DEI) m/z 293 (M+); HRMS
(DEI) calcd for C13H11NO5S 293.0358 (M+), found 293.0363;
Anal. (C13H11NO5S) H, N; C: calcd, 53.24; found, 53.88.
2,3-Dih yd r o-3-t h ioxo-2-(2,6-d ioxo-3-p ip er id in yl)-1H -
isoin d ol-1-on e (12). A mixture of compound 8 (81 mg, 0.276
mmol), trifluoroacetamide (57 mg, 0.50 mmol), 1-hydroxyben-
zotriazole (145 mg, 1.07 mmol), 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide hydrochloride (200 mg, 1.04 mmol), and
triethylamine (0.21 mL, 1.51 mmol) in CH2Cl2 (1.5 mL) was
stirred at ambient temperature for 3 days. Water (10 mL) and
CH2Cl2 (10 mL) were added. The dichloromethane layer was
separated, washed with water, dried over Na2SO4, and evapo-
rated under reduced pressure. Purification by chromatography,
with EtOAc:CH2Cl2 (1:10) as the eluent, gave compound 12
1
215 °C; H NMR (DMSO-d6) δ 4.22 (dd, J ) 6.2 Hz, J ) 11.0
Hz, 1H), 2.77-2.65 (m, 1H), 2.45 (m, 1 H), 1.96-1.87 (m, 2H),
1.40 (s, 9H); MS (CI/CH4) m/z 227 [M - 1]+.
2-(2-Oxo-6-th ioxo-3-p ip er id in yl)-1H-isoin d ole-1,3(2H)-
d ion e (5). Compound 3 (1.14 g, 5 mmol) was suspended in
CH2Cl2 (100 mL). To the mixture was added CF3COOH (10
mL), and this then was stirred at room temperature for 4 h.
The solvent was evaporated to give crude 4 (1.25 g): 1H NMR
(DMSO-d6) δ 11.42 (s, 1H), 8.70 (br, 2H), 4.31 (dd, J ) 5.4 Hz,
J ) 13 Hz), 2.88-2.72 (m, 2H), 2.25-2.09 (m, 2H). A mixture
of crude 4 (1.25 g), phthalic anhydride (0.89 g, 6 mmol), and
Et3N (1.39 mL, 10 mmol) in THF (150 mL) was refluxed for 2
days. The reaction mixture was concentrated, and the residue
was crystallized from ethyl acetate to give thalidomide (1) (0.89
g, 69%) as white crystals; mp 276 °C (lit. 276-279 °C). The
mixture of thalidomide 1 (258 mg, 1 mmol) and Lawesson’s
reagent (222 mg, 0.55 mmol) in toluene (50 mL) was stirred
at reflux for 12 h; thereafter, solvent was removed under
vacuum. The resulting residue was purified by column chro-
matography using CH2Cl2 as the eluent to afford compound 5
(200 mg, 73%) as a yellow solid: mp 225-226 °C; 1H NMR
(DMSO-d6) δ 12.83 (s, 1H, NH), 8.00-7.92 (m, 4H, Ph), 5.32
(dd, J ) 5.6 Hz, J ) 12.9 Hz, 1H, H-3′), 3.28-3.25 (m, 2H,
H-5′), 2.60-2.54 (m, 1H, H-4′), 2.17-2.10 (m, 1H, H-4′); 13C
NMR (DMSO-d6) δ 208.7(C-6′), 165.3(C-2′), 165.2(C-1 and C-3),
133.1(C-5 and C-6), 129.3 (C-3a, C-7a), 121.7 (C-4 and C-7),
46.9 (C-3′), 38.9 (C-5′), 21.79 (C-4′); MS (CI/CH4) m/z 274 (M+);
Anal. (C13H10N2O3S) C, H, N.
1
(48 mg, 63%) as a red solid: mp 255 °C; H NMR (CDCl3) δ
8.00-7.98 (m, 1H), 7.80-7.71 (m, 3H), 5.63 (br, 1H), 2.98-
2.70 (m, 3H), 2.18-2.15 (m, 1H); MS (CI/CH4) m/z 274 (M+);
Anal. (C13H10N2O3S) C, H, N.
2-(2, 6-Dith ioxo-3-p ip er id in yl)-1H-isoin d ole-1,3(2H)-d i-
on e (13) a n d 2,3-Dih yd r o-3-th ioxo-2-(2-oxo-6-th ioxo-3-
p ip er id in yl)-1H-isoin d ol-1-on e (15). The mixture of 5 (146
mg, 0.533 mmol), LR (108 mg, 0.267 mmol), and pyridine (21
µL) in toluene was stirred at 110 °C under an atmosphere of
N2 for 12 h. Thereafter, additional LR (108 mg, 0.267 mmol)
and pyridine (21 µL) were added, and the reaction mixture
was stirred for a further 12 h. The solvent was removed under
vacuum, and the residue was purified by column chromatog-
raphy with CH2Cl2:petroleum ether (2:1, 10:1) and then CH2-
Cl2:EtOAc (10:1) as eluents to afford 13 (30 mg, 45%), 15 (21
mg, 31.5%), and starting material 5 (83 mg).
Compound 13 (yellow solid): mp 263-265 °C; 1H NMR
(CDCl3) δ 7.78-7.74 (m, 2 H), 7.66-7.63 (m, 2 H), 5.00 (dd, J
) 4.9 Hz, 11.9 Hz, 1 H), 3.43-3.35 (m, 1 H), 2.95-2.84 (m, 2
H), 2.08-2.06 (m, 1 H); MS (DEI) m/z 290 (M+); HRMS (DEI)
calcd for C13H10N2O2S2 290.0184 (M+), found 290.0185; Anal.
(C13H10N2O2S2) C, H, N.
Dim et h yl 2-(1,3-d ih yd r o-1,3-d ioxo-2H -isoin d ol-2-yl)-
p en ta n ed ioa te (7). To a solution of N-phthaloyl-L-glutamic
acid (200 mg, 0.72 mmol) in methanol (10 mL) was added,
dropwise, thionyl chloride (1 mL). The reaction mixture was
refluxed for 6 h. The solvent was removed under reduced
pressure, dissolved in ethyl acetate (100 mL), and then washed
with saturated aqueous Na2CO3 solution (2 × 30 mL) and
water (2 × 30 mL). The ethyl acetate layer was dried over Na2-
SO4 and then evaporated, leaving an oil, which upon purifica-
tion by silica gel chromatography, using CH2Cl2:EtOAc (1:1)
Compound 15 (red solid): mp 240-242 °C; 1H NMR (CDCl3)
δ 9.44 (s, 1 H), 8.05-8.02 (m, 1 H), 7.86-7.76 (m, 3 H), 5.75-
5.64 (m, 1 H), 3.57-3.52 (m, 1 H), 3.09-2.99 (m, 2 H), 2.19-
2.12 (m, 1 H). 13C NMR (DMSO): 208.16, 207.98, 166.10,
165.39, 134.32, 133.11, 132.42, 124.30, 122.15, 121.11, 49.64,
21.29; MS (DEI) m/z 291 (MH+); HRMS (DEI) calcd for
1
as the eluent, gave compound 7 (161 mg, 73%) as an oil; H
C
13H11N2O2S2 291.0262 (M+), found 291.0264; Anal. (C13H10
N2O2S2‚0.5H2O) C, H, N.
-
NMR (CDCl3) δ 7.87-7.84 (m, 2H), 7.75-7.72 (m 2H), 4.91
(dd, J ) 5 Hz, J ) 9 Hz, 1H), 3.73 (s, 3H), 3.62 (s, 3H), 2.67-
2.56 (m, 1H), 2.51-2.44 (m, 1H), 2.41-2.35 (m, 2H).
Dim eth yl 2-(1,3-Dih yd r o-1-oxo-3-th ioxo-2H-isoin d ol-2-
yl)p en ta n ed ioa te (8) a n d Dim eth yl 2-(1,3-Dih yd r o-1,3-
2,3-Dih yd r o-3-th ioxo-2-(2,6-d ith ioxo-3-p ip er id in yl)-1H-
isoin d ol-1-on e (16). The mixture of compound 13 (29 mg, 0.1
mmol), LR (22 mg, 0.054 mmol), and morpholine (9 µL, 0.1

5228 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Zhu et al.
mmol) in toluene (10 mL) was stirred at reflux under an
atmosphere of N2 for 16 h. The solvent was removed under
vacuum, and the residue was purified by column chromatog-
raphy using CH2Cl2:petroleum ether (1:1) as the eluent to
afford compound 16 (20 mg, 65%) as a red solid: mp 244 °C;
1H NMR (CDCl3) δ 10.81 (s, 1H), 8.05-8.01 (m, 1H), 7.91-
7.75 (m, 3H), 5.92 (m, 1H), 3.57-3.52 (m, 1H), 3.13-2.97 (m,
2H), 2.18-2.15 (m, 1H); MS(DEI) m/z 306 (M+); HRMS (DEI)
calcd for C13H10N2OS3 305.9955 (M+), found 305.9951; Anal.
(C13H10N2OS3‚0.5H2O) C, H, N.
6-Th ioxo-2-p ip er id in on e (18). The mixture of glutarimide
(0.45 g, 4 mmol) and LR (0.809 g, 2 mmol) in THF (30 mL)
was stirred at room temperature for 2 days. The solvent was
evaporated under vacuum, and the residue was purified by
column chromatography using petroleum ether:EtOAc (1:1) as
the eluent to give compound 18 as a yellow solid (0.361 g,
70%): mp 135 °C; 1H NMR (CDCl3) δ 2.96 (t, J ) 5.7 Hz, 2 H),
2.58 (t, J ) 5.8 Hz, 2 H), 1.96 (m, 2 H); MS (CI/CH4) m/z 129
(M+); Anal. (C5H7NOS) C, H, N.
2,6-P ip er id in ed ith ion e (19). The mixture of glutarimide
(0.34 g, 3 mmol) and LR (1.22 g, 3 mmol) in toluene (30 mL)
was stirred at reflux for 3 h. The solvent was evaporated under
vacuum, and the residue was purified by column chromatog-
raphy using petroleum ether:EtOAc (20:1) as the eluent to give
compound 19 as a yellow solid (0.286 g, 66%): mp 103 °C; 1H
NMR (CDCl3) δ 3.02 (t, J ) 6.3 Hz, 4H), 1.98 (t, J ) 6.3 Hz,
2H); MS (CI/CH4) m/z 145 (M+); Anal. (C5H7NS2) C, H, N.
2-(3-Cycloh exen yl)-1H-isoin d ole-1,3(2H)-d ion e (21). A
mixture of potassium phthalimide (1.85 g, 3 mmol) and
3-bromocyclohexene (1.79 g, 3 mmol) in DMF (15 mL) was
stirred in a 100 °C oil bath for 12 h. The cooled reaction
mixture was poured into ice-water. The solid was collected
by filtration and purified by flash chromatography with CH2-
Cl2 as the eluent to afford compound 21 (1.6 g, 72%) as pink
crystals; mp 114 °C; 1H NMR (CDCl3) δ 7.73-7.69 (m, 2H),
7.62-7.58 (m, 2H), 5.85-5.82 (m, 1H), 5.47-5.44 (m, 1H),
4.80-4.78 (m, 1H), 2.14-2.00 (m, 3H), 1.86-1.78 (m, 2H),
1.64-1.58 (m, 1H).
lipopolysaccharide (LPS)(100 ng/mL in supplemented medium)
or vehicle was added to induce stimulated and unstimulated
cells, respectively, and the cells were incubated overnight.
Sixteen hours later, supernatants were collected for quanti-
fication of TNF-R levels by ELISA assay (Pierce-Endogen
human TNF-R minikit, Rockford, IL) and the use of specific
capture and detection monoclonal antibodies, M303E and
M302B (Pierce-Endogen), respectively. ELISA plates were read
at 450 nm λ, and TNF-R levels were determined from a six-
point calibration curve that was run concurrently with the test
samples. The effect of test drug concentrations on the cellular
viability of PBMCs was assessed by MTS assay (Promega,
Madison, WI) of the cells that provided the supernatant
samples assayed for TNF-R levels, described above.
Assessm en t of Com p ou n d s on TNF -r/Lu cifer a se Ac-
tivity. This cell-based assay utilized two stably transfected
cell lines derived from the mouse macrophage line, RAW264.7.
One line, designated “luciferase only” expressed a luciferase
reporter construct without any UTR sequences. The other line,
designated “luciferase + TNF-R UTR” expressed a luciferase
reporter construct with the entire 3′-UTR of human TNF-R
inserted directly downstream of the luciferase coding region.
Compounds were added in a concentration-dependent manner,
as above, and at the end of the incubation period (16 h, 37 °C,
5% CO2) the media was removed, cells were lysed, and
luciferase activity was assayed with Steady-glo luciferase assay
reagent (Promega) according to the supplier’s directions.
Background was subtracted, and data from this assay was
expressed as a ratio of the +3′-UTR to -3′-UTR (control) values
and was expressed as a percent as shown in Figure 3. In this
manner, compounds that show a differential effect on the two
cell lines, with and without a 3′-UTR, are highlighted.
Ack n ow led gm en t. The authors wish to thank Drs.
Gordon Powers and Gretchen Temeles (Message Phar-
maceuticals, Malvern, PA) for the numerous assays and
valued input that they each provided into this research
project.
2-(3-Cycloh exen yl)-1H-isoin d ol-1,3(2H)-d ith ion e (22)
a n d 2,3-Dih yd r o-3-th ioxo-2-(3-cycloh exen yl)-1H-isoin d ol-
1-on e (23). The mixture of compound 21 (68 mg, 0.3 mmol)
and LR (121 mg, 0.3 mmol) in toluene was refluxed under N2
for 10 h. The solvent was removed under vacuum, and the
residue was purified by column chromatography using petro-
leum ether as the eluent to obtain compound 22 (37 mg, 48%)
as a dark green solid. Then, using CH2Cl2:petroleum ether (1:
1) as the eluent, the more polar component 23 (23 mg, 32%)
was obtained as a red solid.
Su p p or tin g In for m a tion Ava ila ble: HMBC spectrum of
5. This material is available free of charge via the Internet at
http://pubs.acs.org.
Refer en ces
(1) Eger, K.; J alalian, B.; Verspohl, E. J .; Lupke, N.-P. Synthesis,
central nervous system activity and teratogenicity of a homotha-
lidomide. Arzneim. Forsch. 1990, 40, 1073-1075.
(2) Sheskin, J . Thalidomide in the treatment of lepra reaction. Clin.
Pharmacol. Ther. 1965, 6, 303.
Compound 22: mp 93 °C; 1H NMR (CDCl3) δ 7.65-7.60 (m,
2H), 7.49-7.42 (m, 2H), 5.92-5.88 (m, 1H), 5.66-5.63 (m, 1H),
5.47-5.43 (m, 1H), 2.40-2.35 (m, 1H), 1.99-1.95 (m, 2H),
(3) Kumar, S.; Witzig, T. E.; Rajkumar, S. V. Thalidomide as an
anti-cancer agent. J . Cell Mol. Med. 2002, 6, 160-174.
(4) Richardson, P.; Hideshima, T.; Anderson, K. Thalidomide:
emerging role in cancer medicine. Annu. Rev. Med. 2002, 53,
628-657.
1.75-1.59 (m, 3H); MS (CI/CH4) m/z 259 (M+); Anal. (C14H13
NS2) C, H, N.
-
Compound 23: mp 67-68 °C; 1H NMR (CDCl3) δ 7.94-7.91
(m, 1H), 7.73-7.64 (m, 3H), 5.92-5.88 (m, 1H), 5.60-5.51 (m,
2H), 2.27-2.10 (m, 3H), 1.96-1.76 (m, 2H), 1.81-1.70 (m, 1H);
MS (CI/CH4) m/z 243 (M+); Anal. (C14H13NOS) C, H, N.
Assessm en t of Com p ou n d s on th e Secr etion of TNF -r
by P BMCs. Freshly prepared PBMCs were utilized in all
studies. Blood, 40 mL, was drawn from a volunteer, im-
mediately mixed with 50 U/mL Na heparin and was diluted
to 50 mL total volume with sterile PBS. Samples, 20 mL, of
this preparation then was layered on 20 mL of Ficoll-Paque
and was centrifuged (800g, 20 min). The Ficoll/serum interface,
containing PBMCs, was collected, diluted to 200 mL with PBS,
and then was centrifuged (800g, 15 min) to pellet the cells.
Thereafter, the recovered pellet was resuspended in 37 °C
tissue culture medium (RPMI/1 mM Sodium pyruvate/10%
heat inactivated FBS/2 mM Glutamax) and placed on ice.
Recovered cells were counted, pipetted (200 µL of 5 × 105/
mL) into 96-well plates, and incubated for 1 h (37 °C, 5% CO2).
Thereafter, appropriate concentrations of test compounds or
vehicle (10 µL of DMSO) were added to duplicate wells.
Following a further hour of incubation, a 10 µL sample of
(5) Sampaio, E. P.; Sarno, E. N.; Gallily, R.; Cohn, Z. A.; Kaplan,
G. Thalidomide selectively inhibits tumor necrosis factor
a
production by stimulated human monocytes. J . Exp. Med. 1991,
173, 699-703.
(6) Kruys, V.; Marinx, O.; Shaw, G.; Deschamps, J .; Huez, G.
Translational blockade imposed by cytokine-derived UA-rich
sequences. Science 1989, 245 (4920) Aug 25, 1989, 852-855.
(7) Moreira, A. L.; Sampaio, E. P.; Zmuidzinas, A.; Frindt, P.; Smith,
K. A.; Kaplan, G. Thalidomide exerts its inhibitory action on
tumor necrosis factor a by enhancing mRNA degradation. J . Exp.
Med. 1993, 177, 1675-1680.
(8) Haslett, P.; Corral, L.; Albert, M.; Kaplan, G. Thalidomide
costimulates primary human
T lymphocytes, preferentially
inducing proliferation, cytokine production, and cytotoxic re-
sponses in the CD8+ subset. J . Expl. Med. 1998, 187, 1885-
1889.
(9) D′Amato, R. J .; Loughnan, M. S.; Flynn, E.; Folkman, J .
Thalidomide in an inhibitor of angiogenesis. Proc. Natl. Acad.
Sci. 1994, 91, 4082-34085.
(10) Figg, W. D.; Kruger, E. A.; Price, D. K.; Kim, S.; Dahut, W. D.
Inhibition of angiogenesis: Treatment options for patients with
metastatic prostate cancer. Invest. New Drugs 2002, 20, 183-
194.
(11) MacFarlane, M. TRAIL-induced signaling and apoptosis. Toxicol.
Lett. 2003, 139, 89-97.

Novel Isosteric Analogues of Thalidomide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5229
(12) Lamhamedi-Cherradi, S. E.; Zheng, S. J .; Maguschak, K. A.;
Peschon, J .; Chen, Y. H. Defective thymocyte apoptosis and
accelerated autoimmune diseases in TRAIL-/- mice. Nat. Im-
munol. 2003, 4, 207-208.
(32) Cherkasov, R. A.; Kutyrev, G. A.; Pudovik, A. N. Organothio-
phosphorus reagents in organic synthesis. Tetrahedron 1985, 41,
2567-2624.
(33) Iyer, S.; Kontoyiannis, D.; Chevrier, D.; Woo, J .; Mori, N.;
Cornejo, M.; Kollias, G.; Buelow, R. Inhibition of tumor necrosis
factor mRNA translation by a rationally designed immunomodu-
latory peptide. J . Biol. Chem. 2000, 275, 17051-17057.
(34) Prichett, W.; Hand, A.; Shields, J .; Dunnington, D. Mechanism
of action of bicyclic imidazoles defines a translational regulatory
pathway for tumor necrosis factor-R. J . Inflammation 1995, 45,
97-105.
(35) Kruys, V.; Huez, G. Translational control of cytokine expression
by 3′ AU-rich sequences. Biochemie 1994, 76, 862-866.
(36) Chen, C.-Y. A.; Shyu, A.-B. AU-rich elements: characterization
and importance in mRNA degradation. Trends Biol. Sci. 1995,
20, 465-470.
(13) Thanos, D.; Maniatis, T. NFkB: a lesson in family values. Cell
1995, 80, 529-532.
(14) Begg, A. A.; Baltimore, D. An essential role of NFkB in
preventing TNF-R-induced cell death. Science 1996, 274, 782-
784.
(15) Bauer, K. S.; Dixon, S. C.; Figg, W. D. Inhibition of angiogenesis
by thalidomide requires metabolic activation, which is species-
dependent. Biochem. Pharmacol. 1998, 55, 1827-1834.
(16) Meierhofer, C.; Wiedermann, C. J . New insights into the
pharmacological and toxicological effects of thalidomide. Curr.
Opin. Drug Discovery Dev. 2003, 6, 92-99.
(17) Price, D. K.; Ando, Y.; Kruger, E.; Weiss, M.; Figg, W. D. 5′-OH-
thalidomide, a metabolite of thalidomide, inhibits angiogenesis
Ther. Drug Monit. 2002, 24, 104-110.
(18) Kling, J . Redeeming thalidomide. Modern Drug Discovery 2000,
3, 35-36.
(37) Rajagopalan, LE.; Malter, J . S. Growth factor-mediated stabi-
lization of amyloid precursor protein mRNA is mediated by a
conserved 29-nucleotide sequence in the 3′-untranslated region.
J . Neurochem. 2000, 74, 52-59.
(19) Muller, G. W.; Corral, L. G.; Shire, M. G.; Wang, H.; Moreira,
A.; Kaplan, G.; Stirling, D. I. Structural modifications of thali-
domide produce analogues with enhanced tumor necrosis factor
inhibitory activity. J . Med. Chem. 1996, 39, 3238-3240.
(20) Muller, G. W.; Shire, M. G.; Wong, L. M.; Corral, L. G.; Patterson,
R. T.; Chen, Y.; Stirling, D. I. Thalidomide analogs and PDE4
inhibition. Bioorg. Med. Chem. Lett. 1998, 8, 2669-2674.
(21) Muller, G. W.; Chen, R.; Huang, S.-Y.; Corral, L. G.; Wong, L.
M.; Patterson, R. T.; Chen, Y.; Kaplan, G.; Stirling, D. I. Amino-
substitutes thalidomide analogs: potent inhibitors of TNF-R
production. Bioorg. Med. Chem. Lett. 1999, 9, 1625-1630.
(22) Hashimoto, Y. Structural development of biological response
modifiers based on thalidomide. Bioorg. Med. Chem. 2002, 10,
461-479.
(38) Lahiri, D. K.; Chen, D.; Vivien, D.; Ge, Y.-W.; Greig, N. H.;
Rogers, J . T. Role of cytokines in the gene expression of amyloid
§-protein precursor: identification of a 5′-UTR-binding nuclear
factor and its implications in Alzheimer’s disease. J . Alzheimer’s
Dis. 2003, 5, 81-90.
(39) Rogers, J . T.; Leiter, L.; Mcphee, J .; Cahill, C. M.; Zhan, S. S.;
Potter, H.; Nilsson, L. N. Translation of the Alzheimer amyloid
precursor protein mRNA is upregulated by interleukin 1 through
5′-untranslated region sequences. J . Biol. Chem. 1999, 274,
6421-6431.
(40) Rogers, J . T.; Randall, J . D.; Cahill, C.; Eder, P. S.; Huang, X.;
Gunshin, H.; Sarang, S.; Utsuki, T.; Greig, N. H.; Lahiri, D. K.;
Tanzi, R. E.; Bush, A. I.; Giordano, T.; Gullans, S. R. An iron-
responsive element type II in the 5′ untranslated region of the
Alzheimer’s amyloid precursor protein transcript. J . Biol. Chem.
2002, 277, 45518-45528.
(41) Shaw, K. T. Y.; Utsuki, T.; Rogers, J . T.; Yu, Q. S.; Sambamurti,
K.; Brossi, A.; Ge, Y. W.; Lahiri, D. K.; and Greig, N. H.
Phenserine regulates translation of b-amyloid precursor mRNA
by a putative interleukin-1 responsive element, a target for drug
development. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7605-7610.
(42) Dingwall, C.; Ernberg, I.; Gait, M. J .; Green, S. M.; Heaphy, S.;
Karn, J ,; Lowe, A. D.; Singh, M.; Skinner, M. A.; Valerio, R.
(23) Teubert, U.; Zwingenberger, K.; Stephan, W.; Eger, K. 5′-
Substituted thalidomide analogs as modulators of TNF-R Arch.
Pharm. Pharm. Med. Chem. 1998, 331, 7-12.
(24) Luzzio, F. A.; Thomas, E. M.; Figg, W. D. Thalidomide metabo-
lites and analogs. part 2: cyclic derivatives of 2-N-phthalimido-
2S,3S (3-hydroxy) ornithine. Tetrahedron Lett. 2000, 41, 7151-
7155.
(25) Niwayama, S.; Turk, B. E.; Liu, J . O. Potent inhibition of tumor
necrosis factor
a production by tetrafluorothalidomide and
tetrafluorophthalimides. J . Med. Chem. 1996, 39, 3044-3045.
(26) Weinz, C.; Blaschke, G. Investigation of the in vitro biotrans-
formation and simultaneous enantioselective separation of tha-
lidomide and its neutral metabolites by capillary electrophoresis.
J . Chromatogr., B: Biomed. Sci. Appl. 1995, 674, 287-292.
(27) Shan, J . H.; Swartz, G. M.; Papathanassiu, A. E.; Treston, A.
M.; Fogler, W. E.; Madsen, J . W.; Green, S. J . Synthesis and
enantiomeric separation of 2-phthalimido-glutaric acid ana-
logues: potent inhibitors of tumor metastasis. J . Med. Chem.
1999, 42, 3014-3017.
(28) Cava, M. P.; Levinson, M. I. Thionation reaction of Lawesson’s
reagents. Tetrahedron 1985, 41, 5061-5087.
(29) Flaih, N.; Pham-Huy, C.; Galons, H. An expeditious synthesis
of cyclic imides. Tetrahedron Lett. 1999, 40, 3697-3698.
(30) Milewska, M. J .; Bytner, T.; Polonski, T. Synthesis of lactams
by regioselective reduction of cyclic dicarboximides. Synthesis
1996, 12, 1485-1488
Human immunodeficiency virus
1 tat protein binds trans-
activation-responsive region (TAR) RNA in vitro. Proc. Natl.
Acad. Sci. U.S.A. 1989, 86, 6925-6929.
(43) Fujita, J .; Mestre, J . R.; Zeldis, K.; Subbaramaiah, K.; Dannen-
berg, A. J . Thalidomide and its analogues inhibit lipopolysa-
charide-mediated induction of cyclooxygenase-2. Clin. Cancer
Res. 2001, 7, 3349-3355.
(44) Sheng, H.; Shao, J .; Dixon, D. A.; Williams, C. S.; Prescott, S.
M.; DuBois, R. N.; Beauchamp, R. D. Tranforming growth factor-
â1 enhances Ha-ras-induced expression of cyclooxygenase-2 in
intestinal epithelial cells via stablization of mRNA. J . Biol.
Chem. 2000, 275, 6628-6635.
(45) Neininger, A.; Kontoyiannis, D.; Kotlyarov, A.; Winzen, R.;
Eckert, R.; Volk, H. D.; Holtmann, H.; Kollias, G.; Gaestel, M.
MK2 targets AU-rich elements and regulates biosynthesis of
tumor necrosis factor and interleukin-6 independently at dif-
ferent posttranscriptional levels. J . Biol. Chem. 2002, 277,
3065-3068.
(31) Gossauer, A.; Roebler, F.; Zilch, H. Einige Pra¨prrative Anwen-
dungen der reaktion von N-(thioacyl)urethanen und deren
vinylogen mit resonanzstabilisierten phosphor-yliden. Liebigs
Ann. Chem. 1979, 9, 1309-1321.
J M030152F