Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2011.05.029

Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16and 18were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 μM, compounds 12, 17and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 μM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease.

Full text of DOI:10.1016/j.bmc.2011.05.029

Bioorganic & Medicinal Chemistry 19 (2011) 3965–3972
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological assessment of novel N-substituted
3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted
2,6-dioxopiperidines for TNF-a inhibitory activity
Weiming Luo a, Qian-sheng Yu a, Isidro Salcedo a, Harold W. Holloway a, Debomoy K. Lahiri b,
Arnold Brossi c, , David Tweedie a, , Nigel H. Greig a, ,
⇑
a Drug Design & Development Section, Laboratory of Neurosciences, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
b Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA
c School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted
3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11–14 and 3-substituted 2,6-dioxopiperidines 16and 18were
Received 9 April 2011
Revised 10 May 2011
Accepted 17 May 2011
Available online 23 May 2011
synthesized as tumor necrosis factor-a (TNF-a) synthesis inhibitors. Synthesis involved utilization of a
novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimi-
nation, to generate the phthalimidine ring required for the creation of compounds 9–14. Agents were,
thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-
charide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells.
a in a lipopolysac-
Keywords:
Thalidomide
Revlimid
N-substituted EM-12
Dithiocarbamates
Whereas compounds 9, 14 and 16 exhibited potent TNF-
a
lowering activity, reducing TNF- by up to
a
48% at 30 M, compounds 12, 17and 18 presented moderate TNF-
l
a
inhibitory action. The TNF-a lowering
properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In partic-
ular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the
3-Substituted 2,6-dioxopiperidines
Iminium rearrangement
Neurodegenerative diseases
greatest potency of the analogs to reduce TNF-
a synthesis, but achieved this with minor cellular toxicity
at 30 M. The pharmacological focus of the presented compounds is towards the development of well-
l
TNF-a inhibition
tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegener-
ative disorders, epitomized by Alzheimer’s disease and Parkinson’s disease.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
mary intrinsic immune-competent cell type is the microglial cell.
In the normal, inactivated state microglial cells display a ramified
Inflammation of the central nervous system, neuroinflamma-
tion, is now recognized as a key feature of all neurodegenerative
disorders that, with the progressively aging population, are becom-
ing increasingly prominent.1 Alzheimer’s disease (AD) and Parkin-
son’s disease (PD) are prime examples of debilitating disorders that
impact over 5 million and one million Americans, respectively.
Neuroinflammation incorporates a wide spectrum of complex cel-
lular responses that often contribute to the pathogenesis and can
ultimately drive the progression of a neurological disorder.2a–c
The normal adult brain possesses low or undetectable levels of
peripheral inflammatory cell subtypes. In the mature brain a pri-
morphology.2c However, during either chronic or acute neuroin-
flammation, microglia activation rapidly occurs that results in an
alteration in morphology and a subsequent release of many inflam-
matory mediators.2a–c Key among these is the pro-inflammatory
cytokine, tumor necrosis-alpha (TNF-a), a potent activator of the
immune system that can induce immune cell activation leading
to the rapid recruitment of neighboring resting microglia or astro-
cytes within the adjacent brain microenvironment.2b,c
Pharmacological interventions that are able to reset the fine and
often lost balance between resting and activated glial cells could be
of significant clinical benefit.2c TNF-
a protein is a key candidate
therapeutic target as—based on data from many clinical, cell biol-
ogy and animal studies—abnormal regulation of this protein is
strongly associated with an unregulated glial cell activity and
resulting neuroinflammation.2b The identification of novel agents
which can restore the normal function of activated CNS glial cells
⇑
Corresponding author. Address: Drug Design and Development Section, Labo-
ratory of Neurosciences, Intramural Research Program, National Institute on Aging,
BRC Room 05C220, 251 Bayview Blvd., Baltimore, MD 21224, USA.
E-mail addresses: greign@grc.nia.nih.gov, GreigN@vax.grc.nia.nih.gov (N.H.
Greig).
by means of reducing the pro-inflammatory effects of TNF-
a pro-
tein within brain represent a viable mechanism of action for the
management of clinical disease.2c
These three authors contributed equally to this work as senior authors.
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.05.029

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W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
Figure 1. Known thalidomide, EM-12, revlimid and sulfur containing analogs possessing TNF-a action.
Thalidomide (1) (Fig. 1) is effective in alleviating erythema
atom was introduced into the thalidomide pharmacophore, a more
potent antitumor activity was detected.9 Up to now the biological
properties of N-dithiocarbamate substituted 3-(phthalimidin-2-
yl)-2,6-dioxopiperidines have not been reported. Based on our
unpublished observations and a structural analysis of specific
nodosum leprosum associated with leprosy and aphthous ulcers
in AIDS patients.3a–d Although the mechanism via which this drug
ameliorates these inflammatory diseases remains to be fully eluci-
dated, thalidomide is known to induce a wide range of immuno-
modulatory actions. Among this diversity of activities is a TNF-
lowering action, mediated by destabilizing TNF- mRNA levels at
the level of its 30-untranslated region to reduce the rate of synthe-
sis of TNF-
protein.4a,b As the close thalidomide analog, (revlimid
(3) (Fig. 1)), likewise possesses TNF-
action,3c,d the backbone
holds promise as a multi-template for development of biologically
active compounds. In addition, as thalidomide (1) displays a mod-
erate degree of lipophilicity (C log D value ꢀ0.83)6a that allows it
access to the brain, analogs bearing equivalent physicochemical
properties may provide promise in the treatment of neurodegener-
ative diseases exacerbated by neuroinflammation. Additionally, as
a
known compounds possessing TNF-
ated two novel N-dithiocarbamate substituted 3-(phthalimidin-2-
yl)-2,6-dioxopiperidines, 9 and 10, as likely TNF-
(Scheme 1).
a inhibitory activity, we gener-
a
a inhibitors
a
a
Compounds 11–14 were synthesized as shown in Scheme 1.
The rationale underpinning the conception of these agents is based
on green chemistry,10 whereby two potentially active agents may,
under appropriate conditions, derive from a single compound;
thereby providing a form of ‘medicinal economy’. Specifically, un-
der physiological conditions, the use of a methylene linkage to con-
nect two nitrogen atoms, as in 11–14, may permit the occurrence
of simple metabolism to allow generation of the corresponding
2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 (2) (Fig. 1), glu-
tarimide, dithioglutarimide, phthalimide or dithiophthalimide.
the TNF-a activity of 1 is not particularly potent, requiring admin-
istration of high doses that are often both sedative and associated
with adverse effects,5 the synthesis of novel and more effective
TNF-
a
lowering agents is a worthy goal. In this regard, several
Importantly, these latter agents all possess a degree of TNF-a low-
N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and
ering potency.6a Previously, we and others have demonstrated that
3-substituted 2,6-dioxopiperidines are described that demonstrate
2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 (2) generates
promising anti-TNF-
a
potency.
TNF-a
inhibitory activity with a low cellular toxicity6c,e and that
dithiophthalimide is likewise active and well-tolerated.6a As illus-
trated in Table 1, compound 14, containing such a methylene
linkage that simultaneously connects the 2-(2,6-dioxopiperidin-
3-yl)phthalimidine EM-12 and dithiophthalimide moieties, proved
2. Results and discussion
2.1. Chemistry
to be the most potent TNF-
analyzed compounds.
a lowering candidate among the eleven
Prior studies have identified a number of thalidomide analogs
with improved TNF-
and CNS neuroinflammation. In this regard, compounds in Figure
a, lowering properties in models of cellular
For the syntheses of compounds 9–14, we adopted our own no-
vel condensation approach to allow formation of the phthalimidine
ring. This one-pot reaction involved addition, rearrangement and
elimination. A potential mechanism underpinning this is shown
in Fig. 2. The process proved to be simple and the product yield
was good (ranging from 52% to 89%), thereby providing a shortcut
for synthesis of 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12
analogs. In chemistry more generally, such dehydration spanning
a nitrogen atom to form a lactam is, likewise, interesting and has
1 have demonstrated encouraging results regarding anti-TNF-
a po-
tency associated with tolerable cellular toxicity.6a–d
N-substituted thalidomide derivatives have been reported with
potent antitumor activities.7 Indeed, several N-alkylated thalido-
mide analogs have been screened in cell culture and then further
evaluated in animals, in which a higher antitumor activity than
that of thalidomide was observed.8a–f In general, when a sulfur

W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
3967
Scheme 1. Reagents and conditions: (i) formaldehyde (37% solution in water), N2, reflux, 0.5 h; (ii) thionyl chloride, DMF, 0 °C, 1 h; (iii) trifluoroacetic acid, CH2C12, N2, rt,
22.5 h; (iv) phthaldialdehyde, THF, N2, rt, 71 h; (v) carbon disulfide, cyclohexylamine (for 9), piperidine (for 10), CH3CN, N2, rt, 42–46 h; (vi) glutarimide (for 11),
dithioglutarimide (for 12), KOH, CH3CN, N2, rt, 16–18 h; (vii) phthalimide (for 13), dithiophthalimide (for 14), KOH, CH3CN, N2, rt, 18 h.
Table 1
Inhibition of LPS-induced TNF-a production in RAW 264.7 cells, cell viability and calculated lipophilicity of assayed compounds 9–14, 16–20 are shown. The anti-TNF-a
properties of the analogs are compared with those of revlimid 3
Compd
TNF-
a
activity (30
l
M) Control = 100%
Cell viability (30
% Control
l
M) Control = 100%
C log Da
Fold to 1 anti-TNF-a
activityb
% Control
P value
P value
3
9
109 10
0.4482
<0.001
0.0734
0.0087
0.0184
0.0571
0.0009
0.0092
0.0154
0.0043
0.4214
0.5184
106
103
112
102
94
98
82
109
116
84
3
2
1
2
3
2
6
3
2
2
5
4
0.1252
0.1555
<0.0001
0.2028
0.1488
0.4333
0.0427
0.0762
0.0163
0.0018
0.5793
0.6212
ꢀ1.31
+1.39
+0.65
ꢀ1.66
ꢀ0.72
ꢀ0.48
+0.27
ꢀ1.75
ꢀ1.98
ꢀ2.13
ꢀ1.93
ꢀ2.04
1
45
4
55
96
79
63
88
52
55
61
67
104
96
3
1
3
2
2
2
4
5
5
3
2
10
11
12
13
14
16
17
18
19
20
21
37
12
48
45
39
33
ꢀ4
4
95
96
Presented values are mean S.E.M. of n = 3 measurements. A students t-test was used to assess for statistically significant changes; P <0.05 was considered significant.
a
C log D—calculated log D values were determined at pH 7.0 (CompuDrug, Pallas).
b
Fold to thalidomide (1) anti-TNF-
a activity was defined as (100 ꢀ TNF-a activity %change of every compound)/(100 ꢀ TNF-a activity %change contrasted with 1), TNF-a
activity %control for thalidomide was equal to 99% of levels achieved in the presence of vehicle. The TNF-activity of compound 17 compares favorably with that of A10 in
Ref. 6c.

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W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
Figure 2. Possible mechanism forming intermediate 8.
not previously been reported. By contrast, previously published
synthetic routes to yield EM-12 are greater than a single step, as
is evident from the work of Luzzio and colleagues6f where synthe-
sis was achieved in six steps in a yield of 25%. Other reports de-
scribe the use of a radical inducing reagent or UV light. In our
one-pot reaction, however, synthesis is undertaken at room tem-
perature and a yield up to 80% is achievable, even if undertaken
in neutral or weakly basic reaction mediums. Our experiment
showed that the compound 12 in solution existed in tautomerism.
Consequent to the introduction of hetero-atoms, such as sulfur
and nitrogen, into thalidomide that provided significant
ities of the above analogs were compared to those of revlimid (3).
In addition to thalidomide (1), 3 is a credible TNF-
inhibitor,11a,b
a
and is both approved for and effective in the for treatment of mul-
tiple myeloma and specific myelodysplastic syndromes.12a–f Here-
in, compounds 9, 12, 14 and 16–18 possessed more potent TNF-
a
inhibitory activity than that of revlimid (3) as well as thalidomide
(1) in our assay model, which has now been extensively character-
ized.6c Indeed, compounds 9, 14 and 16 not only showed the most
potency as TNF-a inhibitors among all eleven assayed compounds
(contrasting markedly with revlimid (3)) but appeared well toler-
ated, albeit 14 was associated with a mild decline in cell viability
TNF-a
inhibitory activity, we designed compound 16, in which
at 30 lM. Parenthetically, the TNF-a activity of compound 17,
the 1,3-carbonyl groups of thalidomide are replaced by sulfonyl
groups, and compounds 17a and 18, in which the 1,3-dioxo and
1-oxo of thalidomide were substituted by imino groups, respec-
tively (Scheme 2, Fig. 3).
whose chemistry is reported for the first time herein, compares
favorably to that reported by Tweedie et al.,6c (agent A10), demon-
strating the consistency of the assay across time.
The C log D values of our analogs, interestingly, ranged from
lipophilic (9: +1.39)to water-soluble (16: ꢀ1.75) (Table 1), suggest-
For the synthesis of 17a, as illustrated in Figure 3, the principal
product is 17.6c This can be attributed to the presence of a 1, 3-H
transfer, as depicted in Figure 3, under the reaction conditions indi-
cated. Two primary amino groups may prove more advantageous
to support the intramolecular elimination of ammonia. The con-
densation of reactants 3-iminoisoindolinone and 15 normally
afforded product 18, which has been confirmed by two correlative
peaks of 30-H/1-C and 30-H/3-C in the (1H-detected) heteronuclear
multiple-bond correlation (HMBC) spectrum. Syntheses of com-
pounds 19 and 20 proved straightforward using the methods de-
fined in Scheme 2, and the yield of 20 proved to be high (93%).
ing that their potency as TNF-a inhibitors related more to their
structural configuration rather than to a physicochemical charac-
teristic, such as lipophilicity, that would be predicted to augment
cellular uptake. Clearly, structural configuration together with
physicochemical properties impact the ability of a compound to
suitably orient, dock and then appropriately interact with a re-
quired target, such as one regulating TNF-
and are thereby fundamental to its TNF- lowering effects. How-
ever, regulation of TNF- synthesis by thalidomide (1) and analogs
a protein synthesis,
a
a
is not mediated via a classical receptor or enzyme-based interac-
tion for which structure–activity relations are generally available
but, instead, appears to involve complex post-transcriptional regu-
2.2. TNF-a inhibitory activity
latory actions mediated at the level of the 30-UTR of TNF-
a
Inhibition of LPS-induced TNF-
a
production in RAW 264.7 cells,
mRNA.4a,b
cell viability and computed lipophilicity (C log D value) of assessed
compounds 9–14, 16–20 are shown in Table 1. The biological activ-
In general, mRNAs are amenable to several forms of post-tran-
scriptional regulation, which include pre-mRNA splicing and mat-

W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
3969
ization and translation.13a–c Two major kinds of trans acting factors
that recognize specific cis elements (RNA sequences) within the
target mRNA are involved in the regulation of these steps,
RNA-binding proteins (RBPs) and microRNAs. For TNF-
a gene
expression, the role of RBPs has been widely explored, whereas
the part of microRNAs remains to be more fully characterized.13c,d
For inflammatory cytokines like TNF-
a, in particular, but also for
IL-2, IL-6 and interferon- , synthesis is tightly regulated at the
c
level of mRNA stability, thereby permitting rapid responses to
external stimuli, as occurs for LPS. The presence of adenylate–
uridylate-rich elements (AREs) within the 30- UTR of TNF-
a mRNA
plays a primary role in post-transcriptional repression, targeting it
for rapid degradation or inhibition of translation.13a,b p38 MAPK
has been implicated as a major signaling cascade facilitating the
stability of TNF-a
via its 30-UTR ARE cis elements, which have been
shown to be mediated through interactions with RBPs.13a–d In par-
ticular, proteins such as HuR, whose translocation to the cytoplasm
is potently induced by oxidative stress and other aberrant stim-
uli,13c have been associated with promoting transcript stabiliza-
tion. Upon export to the cytoplasm, HuR binds and stabilizes
ARE-containing transcripts and conveys them to translational
machinery. Conversely, RNA-binding proteins such as tristetrapro-
lin (TTP) and related proteins (e.g., butyrate response factor 1 and
2) have a major regulatory role in accelerating the degradation of
bound mRNAs, albeit the precise mechanisms accounting for this
degradation are incompletely understood but they include the
action of several cellular structures known to breakdown labile
mRNAs, the proteasome, exosome and RNA processing-body.13a–c
It widely accepted that LPS challenge of RAW 264.7 cells
extends the half-life of TNF-a mRNA, allowing release of its trans-
lational repression. By contrast, administration of thalidomide (1)
induces an increase in translational blockade and a shortening of
the TNF-
a
mRNA half-life from 30 to 17 min.4a,14a,b Although yet
to be elucidated, interactions between thalidomide analogs and
the binding of HUR as well as TTP related RBPs with 30-UTR cis ele-
ments likely underpins alterations in the rate of TNF-
a synthesis.
In this regard, small chemical inhibitors that disrupt HuR: ARE
binding have been described.14c,d As yet, insufficient compounds
have been assessed to provide structure–activity relations, but
such a target, or a strengthening of TTP:ARE binding, represents a
potential means via which thalidomide analogs may effectively
Scheme 2. Reagents and conditions: (i) 1,2-benzenedisulfonyl dichloride, Et3N,
THF, N2, reflux, 24 h; (ii) 1,3-diiminoisoindoline, Et3N, THF, N2, reflux, 98 h; (iii)
3-iminoisoindolinone, Et3N, THF, N2, reflux, 72 h; (iv) 2,3-pyridinedicarboxylic
anhydride, AcOH, N2, reflux, 7.5 h; (v) 3,4-pyridinedicarboxylic anhydride, AcOH,
N2, reflux, 6.5 h.
regulate TNF-a protein levels.
These same properties of thalidomide analogs may or may not be
vital for pharmacological interactions with other targets, such as ni-
tric oxide,15a,b known to be regulated by thalidomide (1) and are a fo-
cus of current studies. In the event that the inhibitory activity of
uration (30polyadenylation and 50 capping), mRNA nuclear export
to the cytoplasm, appropriate sub-cytoplasmic localization, stabil-
Figure 3. Possible mechanism forming compound 17.

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W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
designed compounds proves ideal, in part or in whole, it not only
provides a basis to manipulate TNF- levels to define its pharmaco-
4.1.3. 3-Amino-1-chloromethyl-2,6-dioxopiperidine
trifluoroacetate (7)
a
logical role in health and disease, but also affords structural informa-
Trifluoroacetic acid (28.4 mL) was dropwise added into a solu-
tion to allow investigation of the target and its regulatory elements.
tion of compound
6 (4.0 g, 14.5 mmol) in dichloromethane
In this regard, these TNF-
a
lowering data are sufficiently prom-
(274 mL) at room temperature. The mixture was reacted for
22.5 h under an atmosphere of nitrogen at the same temperature.
Thereafter, it was concentrated and precipitated with ether. Iso-
lated solid was dried overnight to afford product 7 (4.05 g, 96.2%)
as a purplish salt; 1H NMR (DMSO-d6) d 8.80 (s, 3H, NH3+), 5.56
and 5.50 (AB system, J = 9.2 Hz, 2H, CH2Cl), 4.51–4.28 (m, 1H, C3-
H), 3.01–2.71 (m, 2H) and 2.31–1.90 (m, 2H) ppm; 13C NMR
(DMSO-d6) d 170.2, 169.2, 159.0, 158.5, 49.9, 48.1, 30.6 and
21.3 ppm; MS (CI/CH4), m/z 288 (M-2).
ising to indicate that further study of the pharmacology and biol-
ogy of these agents is warranted in models of neurodegenerative
diseases where a component of the disease pathology is associated
with neuroinflammation.
3. Conclusion
Novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithio-
carbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-
dioxopiperidines 11–14 and 3-substituted 2,6-dioxopiperidines 16
4.1.4. 2-(1-Chloromethyl-2,6-dioxopiperidin-3-
yl)phthalimidine (8)
and 18 were designed, prepared and assessed for TNF-a lowering
A mixture of phthaldialdehyde (2.74 g, 20.4 mmol) and com-
pound 7 (5.94 g, 20.4 mmol) in THF (1.7 L) was stirred under an
atmosphere of nitrogen at room temperature for 71 h. Thereafter,
solvent was removed, and the crude product was purified with
chromatography on silica gel (CH3CN/CH2Cl2 = 1/4) to afford prod-
uct 8 (3.1 g, 51.7%) as a white solid: mp 180.5–181.0 °C; 1H NMR
(CDCl3) d 7.94–7.43 (m, 4H, Ar-H), 5.59 and 5.51 (AB system,
J = 8.1 Hz, 2H, CH2Cl), 5.30–5.19 (m, 1H, C30-H), 4.50 and 4.36 (AB
system, J = 16.5 Hz, 2H, C3-H), 3.15–2.85 (m, 2H) and 2.46–2.13
(m, 2H) ppm; 13C NMR (CDCl3) d 169.4, 169.3, 168.6, 141.4,
132.1, 131.3, 128.3, 124.2, 122.9, 52.5, 47.2, 47.1, 31.9 and
22.3 ppm; MS (CI/CH4), m/z 294 (M+2). Anal. Calcd for
activity. Synthesis involved a novel condensation approach for the
generation of analogs of 2-(2,6-dioxopiperidin-3-yl)phthalimidine
EM-12 (2). In addition to N-substituted 3-(phthalimidin-2-yl)-2,6-
dioxopiperidines 14 and 9, 3-(o-benzenedisulfonimid-2-yl)-2,6-
dioxopiperidine 16 proved to be a potent anti-TNF-
a candidate
agent. These novel compounds thereby provide potential promise
as immunomodulatory drugs candidates whose biological and phar-
macological actions warrant further assessment in animal models of
inflammatory disorders. Particularly relevant would be a neuroin-
flammatory focus, as invariably occurs in neurodegenerative condi-
tions, exemplified by Alzheimer’s disease and Parkinson’s disease.
C14H13ClN2O3: C, 57.44; H, 4.48; N, 9.57. Found: C, 57.37; H,
4. Experimentals
4.1. Chemistry
4.46; N, 9.17.
4.1.5. [3-(1-Oxo-1,3-dihydro-1H-isoindol-2-yl)-2,6-
dioxopiperidin-1-yl]methylcyclohexyldithiocarbamate (9)
A mixture of compound 8 (37.0 mg, 0.126 mmol), carbon disul-
fide (19.2 mg, 0.252 mmol) and cyclohexylamine (25.1 mg,
0.253 mmol) in acetonitrile (7 mL) was reacted for 42.5 h under
an atmosphere of nitrogen at room temperature. After removing
solvent, the residues were separated with chromatography on sil-
ica gel (MeOH/CH2Cl2 = 1/20) to afford product 9 (36.0 mg, 66.7%)
as a yellow crystals: mp 128.0–129.0 °C; 1H NMR (CDCl3) d 8.75
(s, 1H, NH), 7.95–7.38 (m, 4H, Ar-H), 5.31–5.01 (m, 3H, C30-H and
CH2S), 4.49–4.25 (m, 2H, C3-H) and 3.14–1.05 (m, 15H, C5,4-H
and cyclohex-H) ppm; 13C NMR (CDCl3) d 191.2, 171.3, 170.2,
169.3, 141.3, 132.1, 131.3, 128.3, 124.2, 122.9, 55.8, 52.6, 47.4,
43.3, 31.8, 31.4, 25.3, 24.7 and 22.3 ppm; MS (CI/CH4), m/z 432
(MH+). Anal. Calcd for C21H25N3O3S2: C, 58.44; H, 5.84; N, 9.74.
Found: C, 58.05; H, 5.88; N, 9.47.
Melting points (uncorrected) were measured with a Fisher–Johns
apparatus. 1H NMR, and 13C NMR were recorded on a Bruker (Bellev-
ica, MA) AC-300 spectrometer. MS (m/z) data were measured on an
Agilent 5973 GC–MS (CI). Elemental analyzes were performed by
Atlantic Microlab, Inc. (Norcross, GA). All reactions involving non-
aqueous solutions were performed under an inert atmosphere.
4.1.1. 1-Hydroxymethyl-3-(tert-butoxycarbonylamino)-2,6-
dioxopiperidine (5)
A
mixture of 3-(tert-butoxycarbonylamino)-2,6-dioxopiperi-
dine 4 (7.10 g, 31.1 mmol) and formaldehyde (37% solution in
water, 37.2 ml) was refluxed under an atmosphere of nitrogen
for 0.5 h. After cooling, isolated precipitate was recrystallized with
acetone to afford product 5 (5.85 g, 72.9%) as white crystals: mp
216.5–217.5 °C; 1H NMR (DMSO-d6) d 7.20 (d, J = 6.0 Hz, 1H, NH),
6.04 (t, J = 10.5 Hz, 1H, OH), 5.05–4.92 (m, 2H, CH2OH), 4.38–4.22
(m, 1H, C3-H), 2.89–2.59 (m, 2H), 1.99–1.83 (m, 2H) and 1.41 (s,
9H, CH3) ppm; 13C NMR (DMSO-d6) d 172.1, 171.9, 155.8, 78.5,
62.7, 51.3, 31.6, 28.5 and 23.6 ppm; MS (CI/CH4), m/z 257 (M-1).
4.1.6. [3-(1-Oxo-1,3-dihydro-1H-isoindol-2-yl)-2,6-
dioxopiperidin-1-yl]methyl piperidin-1-carbodithioate (10)
A mixture of compound 8 (33.0 mg, 0.113 mmol), carbon disul-
fide (17.2 mg, 0.226 mmol) and piperidine (19.2 mg, 0.225 mmol)
in acetonitrile (6 mL) was reacted for 46.0 h under an atmosphere
of nitrogen at room temperature. After removing solvent, the resi-
dues were separated with chromatography on silica gel (MeOH/
CH2Cl2 = 1/15) to afford product 10 (33.5 mg, 71.0%) as a white
powder: mp 180.0–181.5 °C; 1H NMR (CDCl3) d 7.90–7.41 (m, 4H,
Ar-H), 5.79 and 5.68 (AB system, J = 14.5 Hz, 2H, CH2S), 5.29–5.18
(m, 1H, C30-H), 4.49 and 4.30 (AB system, J = 16.5 Hz, 2H, C3-H),
4.24 (s, br, 2H, pip-C2,6-H), 3.81 (s, br, 2H, pip-C2,6-H), 3.09–
2.81 (m, 2H), 2.41–2.11 (m, 2H) and 1.79–1.54 (m, 6H, pip-
C3,4,5-H) ppm; 13C NMR (CDCl3) d 192.7, 170.3, 169.2, 141.4,
131.9, 131.4, 128.1, 124.0, 122.8, 52.4, 51.6, 47.1, 46.4, 32.0, 25.8,
24.1 and 22.5 ppm; MS (CI/CH4), m/z 258 (M-159). Anal. Calcd
for C20H23N3O3S2: C, 57.53; H, 5.55; N, 10.06. Found: C, 57.80; H,
5.38; N, 9.95.
4.1.2. 1-Chloromethyl-3-(tert-butoxycarbonylamino)-2,6-diox-
opiperidine (6)
Thionyl chloride (7.35 g, 61.8 mmole) was dropwise added to a
solution of compound 5 (5.83 g, 22.6 mmole) in DMF (17 mL) at
0 °C. The mixture was reacted for 1 h maintained at the same tem-
perature. Thereafter, it was poured onto ice (60 g). Precipitate was
isolated and was washed with ice water to pH 7 ca. The crude
product was recrystallized with acetone to afford 6 (5.7 g, 90.5%)
as a white solid: mp 134.0–135.0 °C; 1H NMR (DMSO-d6) d 7.31
(d, J = 6.1 Hz, 1H, NH), 5.49 (s, 2H, CH2Cl), 4.58–4.30 (m, 1H, C3-
H), 3.08–2.67 (m, 2H), 2.10–1.87 (m, 2H) and 1.48 (s, 9H,
CH3) ppm; 13C NMR (DMSO-d6) d 171.5, 171.1, 155.8, 78.7, 51.3,
48.5, 31.4, 28.5 and 23.3 ppm; MS (CI/CH4), m/z 276 (M+).

W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
3971
4.1.7. N-(1,3-dioxopiperidin-2-yl)methyl-3-(1-oxo-1,3-dihydro-
1H-isoindol-2-yl)-2,6-dioxopiperidine (11)
A mixture of glutarimide (36.0 mg, 0.318 mmol) and potassium
hydroxide (42 mg, 0.750 mmol) in acetonitrile (42 mL) was stirred
(m, 1H, C30-H), 4.49 and 4.32 (AB system, J = 16.3 Hz, 2H, C3-H),
3.09–2.79 (m, 2H) and 2.49–2.07 (m, 2H) ppm; 13C NMR (CDCl3)
d 197.0, 170.4, 169.3, 169.1, 141.5, 134.8, 133.5, 132.0, 131.6,
128.2, 124.2, 123.6, 123.0, 52.5, 49.1, 47.1, 32.2 and 22.7 ppm;
MS (CI/CH4), m/z 383 (M-52). Anal. Calcd for C22H17N3O3S21/3
H2O: C, 59.87; H, 4.03; N, 9.52. Found: C, 59.91; H, 3.71; N, 9.17.
for an hour at room temperature. Thereafter, compound
8
(93.0 mg, 0.318 mmol) was added to the reaction system and stirred
for a further 17 h under an atmosphere of nitrogen at the same tem-
perature. After removing solvent, the residues were separated with
chromatography on silica gel (CH3CN/CH2Cl2 = 1/3) to afford prod-
uct 11(68.0 mg, 57.9%) as a white solid: mp 216.5–217.0 °C; 1H
NMR (CDCl3) d 7.88 (d, J = 9.0 Hz, 1H, C7-H), 7.57 (d, J = 8.5 Hz, 1H,
C4-H), 7.46 (t, J = 9.0 Hz, 2H, C5,6-H), 5.85 (s, 2H, CH2N2), 5.19–
5.10 (m, 1H, C30-H), 4.52 and 4.35 (AB system, J = 16.5 Hz, 2H, C3-
H), 3.08–1.81 (m, 10H, C50,40-H and glu-H) ppm; 13C NMR (CDCl3)
d 171.9, 170.3, 169.4, 169.3, 141.6, 132.0, 131.7, 128.2, 124.1,
123.0, 52.8, 47.4, 45.0, 32.9, 32.2, 22.8 and 17.0 ppm; MS (CI/CH4),
m/z 300 (M-69). Anal. Calcd for C19H19N3O5H2O: C, 58.91; H, 5.46;
N, 10.85. Found: C, 59.17; H, 5.38; N, 10.61.
4.1.11. 3-(o-Benzenedisulfonimid-2-yl)-2,6-dioxopiperidine
(16)
A
mixture of 1,2-benzenedisulfonyl dichloride (1.00 g,
3.63 mmol), 3-amino-2,6-dioxopiperidine trifluoroacetate 15
(0.88 g, 3.63 mmol) and triethylamine (1.10 g, 10.87 mmol) in
anhydrous THF (100 mL) was first reacted for 1.5 h at room tem-
perature under an atmosphere of nitrogen, and thereafter, it was
refluxed for 24 h under an atmosphere of nitrogen. Following cool-
ing, a gray crude product was filtered. This was recrystallized from
hot acetone to provide product 16 (0.77 g, 64.2%) as a purplish
powder: mp 253 °C (dec.); 1H NMR (DMSO-d6) d 11.08 (s, 1H,
NH), 8.40–8.01 (m, 4H, Ar-H), 5.51–5.40 (m, 1H, C30-H), 2.96–
2.51 (m, 2H) and 2.50–2.30 (m, 2H) ppm; 13C NMR (DMSO-d6) d
172.5, 169.1, 137.4, 135.8, 122.3, 59.0, 31.5 and 24.7 ppm; MS
(CI/CH4), m/z 331 (MH+). Anal. Calcd for C11H10N2O6S2: C, 39.99;
H, 3.05; N, 8.48. Found: C, 40.57; H, 3.22; N, 8.21.
4.1.8. N-(1,3-dithioxopiperidin-2-yl)methyl-3-(1-oxo-1,3-
dihydro-1H-isoindol-2-yl)-2,6-dioxopiperidine (12)
A mixture of dithioglutarimide (46.2 mg, 0.318 mmol), potas-
sium hydroxide (42 mg, 0.750 mmol) and compound 8 (93.0 mg,
0.318 mmol) in acetonitrile (42 mL) was stirred for 16 h under an
atmosphere of nitrogen at room temperature. After removing sol-
vent, the residues were separated with chromatography on silica
gel (CH3CN/CH2Cl2 = 1/3) to afford product 12 (33.0 mg, 25.8%) as
a yellow solid: mp 218.0–220.0 °C; 1H NMR (CDCl3) d 7.91–7.43
(m, 4H, Ar-H), 5.22–5.12 (m, 1H, C30-H), 5.07 and 4.94 (AB system,
J = 10.1 Hz, 2H, CH2N2), 4.50 and 4.39 (AB system, J = 16.5 Hz, 2H,
C3-H), 3.09–1.89 (m, 10H, C50,40-H and thioglu-H) ppm; 13C NMR
(CDCl3) d 201.7, 170.9, 169.8, 169.5, 141.6, 132.2, 131.6, 128.4,
124.4, 123.1, 53.1, 48.1, 43.6, 37.6, 32.0, 22.6 and 21.2 ppm; MS
(CI/CH4), m/z 404 (M+3). Anal. Calcd for C19H19N3O3S2H2O: C,
54.40; H, 5.04; N, 10.01. Found: C, 54.08; H, 4.42; N, 9.48.
4.1.12. 3-(1,3-Diimino-1,3-dihydro-2H-isoindol-1N-yl)-2,6-
dioxopiperidine (17)
A mixture of 1,3-diiminoisoindoline (0.50 g, 3.44 mmol), 3-ami-
no-2,6-dioxopiperidine trifluoroacetate 15 (0.83 g, 3.43 mmol) and
triethylamine (0.95 g, 9.39 mmol) in THF (200 mL) was refluxed for
98 h under an atmosphere of nitrogen. After cooling, a gray crude
product was filtered. This was recrystallized from hot acetone to
afford product 17 (0.67 g, 76.1%) as a gray powder: mp 246 °C
(dec.); 1H NMR (DMSO-d6) d 10.72 (s, 1H, CONH), 8.51 and 8.39
(sh, 2H, CCNH and C@NH), 7.90–7.50 (m, 4H, Ar-H), 5.01 (t,
J = 5.5 Hz, 1H, C30-H), 2.61 (m, 2-H) and 2.04 (m, 2-H) ppm; 13C
NMR (DMSO-d6) d 173.8, 173.5, 170.6, 168.5, 139.8, 136.1, 131.1,
130.3, 121.5, 121.0, 59.2, 30.3 and 26.3 ppm; MS (CI/CH4), m/z
257 (MH+). Anal. Calcd for C13H12N4O2: C, 60.93; H, 4.72; N,
21.86. Found: C, 60.89; H, 4.82; N, 21.58.
4.1.9. N-Phthalimidomethyl-3-(1-oxo-1,3-dihydro-1H-isoindol-
2-yl)-2,6-dioxopiperidine (13)
A mixture of phthalimide (46.8 mg, 0.318 mmol) and potassium
hydroxide (42 mg, 0.750 mmol) in acetonitrile (42 mL) was stirred
for an hour at room temperature. Thereafter, compound
8
4.1.13. 3-(1-Oxo-3-imino-1,3-dihydro-2H-isoindol-2-yl)-2,6-
dioxopiperidine (18)
(93.0 mg, 0.318 mmol) was added to the reaction system and stir-
red for another 17 h under an atmosphere of nitrogen at the same
temperature. After removing solvent, the residues were separated
with chromatography on silica gel (CH3CN/CH2Cl2 = 1/3) to afford
product 13 (67.0 mg, 52.2%) as a white solid: mp 209.5–210.5 °C;
1H NMR (CDCl3) d 7.95–7.40 (m, 8H, Ar-H), 5.83 and 5.72 (AB sys-
tem, J = 13.5 Hz, 2H, CH2N2), 5.39 -5.23 (m, 1H, C30-H), 4.54 and
4.34 (AB system, J = 16.6 Hz, 2H, C3-H), 3.10–2.82 (m, 2H) and
2.46–2.11 (m, 2H) ppm; 13C NMR (CDCl3) d 170.3, 169.6, 169.3,
167.0, 141.7, 134.3, 132.0, 131.7, 131.6, 128.2, 124.2, 123.6,
123.0, 52.5, 47.1, 43.6, 32.1 and 22.7 ppm; MS (CI/CH4), m/z 390
(M-13). Anal. Calcd for C22H17N3O5: C, 65.50; H, 4.25; N, 10.42.
Found: C, 65.32; H, 4.12; N, 10.21.
A mixture of 3-iminoisoindolinone (0.50 g, 3.42 mmol), 3-amino-
2,6-dioxopiperidine trifluoroacetate 15 (0.83 g, 3.43 mmol) and tri-
ethylamine (0.94 g, 9.29 mmol) in THF (200 mL) was refluxed for
72 h under an atmosphere of nitrogen. After removing solvent,
crude product was recrystallized from hot acetone to afford product
18 (0.38 g, 43.2%) as a white powder: mp 267 °C (dec.); 1H NMR
(DMSO-d6) d 11.15, 10.90 (2s, 2H, 2NH), 7.88–7.68 (m, 4H, Ar-H),
4.46 (t, J = 7.6 Hz, 1H, C30-H), 2.71–2.59 (m, 2H) and 2.22–2.08 (m,
2H) ppm; 13C NMR (DMSO-d6) d 173.4, 172.0, 169.2, 152.5, 136.6,
133.9, 132.5, 131.5, 123.1, 122.3, 59.5, 30.4 and 26.1 ppm; MS (CI/
CH4), m/z 257 (M+). Anal. Calcd for C13H11N3O3: C, 60.70; H, 4.31;
N, 16.33. Found: C, 60.67; H, 4.38; N, 16.20.
4.1.10. N-Dithiophthalimidomethyl-3-(1-oxo-1,3-dihydro-1H-
isoindol-2-yl)-2,6-dioxopiperidine (14)
4.1.14. 3-(4-Aza-1,3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-2,6-
dioxopiperidine (19)
A mixture of dithiophthalimide (57.0 mg, 0.318 mmol) and
potassium hydroxide (42 mg, 0.750 mmol) in acetonitrile (42 mL)
was stirred for an hour at room temperature. Thereafter, com-
pound 8 (93.0 mg, 0.318 mmol) was added to the reaction system
and stirred for another 17 h under an atmosphere of nitrogen at the
same temperature. After removing solvent, the residues were sep-
arated with chromatography on silica gel (CH3CN/CH2Cl2 = 1/3) to
afford product 14 (17.0 mg, 12.3%) as a yellow gum. 1H NMR
(CDCl3) d 7.98–7.39 (m, 8H, Ar-H), 6.47 (s, 2H, CH2N2), 5.31–5.19
A mixture of 2, 3-pyridinedicarboxylic anhydride (1.0 g,
6.71 mmol) and 3-amino-2,6-dioxopiperidine trifluoroacetate 15
(1.63 g, 6.73 mmol) in acetic acid (51 mL) was refluxed under an
atmosphere of nitrogen for 7.5 h. After removing solvent, crude
product was recrystallized from acetone to afford product 19
(0.18 g, 10.3%) as white needle crystals: mp 266.0–267.5 °C; 1H
NMR (CDCl3) d 8.95 (s, br, 1H, C5-H), 8.13 (d, J = 9.3 Hz, 1H,
C7-H), 8.00 (s, 1H, NH), 7.61 (s, br, 1H, C6-H), 5.11–4.91 (m, 1H,
C30-H), 2.99–2.65 (m, 3H) and 2.21–2.09 (m, 1H) ppm; 13C NMR

3972
W. Luo et al. / Bioorg. Med. Chem. 19 (2011) 3965–3972
(CDCl3) d 170.2, 167.2, 165.2, 165.0, 155.7, 151.3, 131.6, 127.7,
127.2, 49.6, 31.3 and 22.5 ppm; MS (CI/CH4), m/z 259 (M+). Anal.
Calcd for C12H9N3O4: C, 55.60; H, 3.50; N, 16.21. Found: C, 55.03;
H, 3.62; N, 15.74.
References and notes
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4.1.15. 3-(5-Aza-1,3-dioxo-1, 3-dihydro-2H-isoindol-2-yl)-2,6-
dioxopiperidine (20)
A
mixture of 3, 4-pyridinedicarboxylic anhydride (1.0 g,
6.71 mmol) and 3-amino-2,6-dioxopiperidine trifluoroacetate 15
(1.63 g, 6.73 mmol) in acetic acid (51 mL) was refluxed under an
atmosphere of nitrogen for 6.5 h. After removing solvent, crude
product was recrystallized from acetone to afford product 20
(1.62 g, 93.1%) as white crystals: mp 234.0–235.5 °C; 1H NMR
(DMSO-d6) d 11.18 (s, 1H, NH), 9.29–9.10 (m, 2H, C4-H, C6-H),
7.99 (d, J = 7.8 Hz, 1H, C7-H), 5.31–5.15 (m, 1H, C30-H), 3.05–2.39
(m, 3H) and 2.19–2.02 (m, 1H) ppm; 13C NMR (DMSO-d6) d
173.0, 169.9, 166.8, 166.4, 156.7, 144.7, 139.1, 125.7, 117.5, 49.6,
31.2 and 22.1 ppm; MS (CI/CH4), m/z 259 (M+). Anal. Calcd for
C12H9N3O4: C, 55.60; H, 3.50; N, 16.21. Found: C, 55.65; H, 3.56;
N, 15.99.
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RAW 264.7 cells were cultured and treated with thalidomide
analogs and LPS as has been previously described.6c In a manner
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vate TNF-a levels in animals—both systemically as well as within
the brain, and to be associated with neurodegenerative disease on-
set.16a,b The CellTiter 96 Aqueous One Solution Cell Proliferation
Assay (Promega, Madison, WI) is a commercially used assay, that
is, widely used to quantitatively determine cell proliferation, and
this assay was utilized in our studies in line with the manufac-
turer’s guidelines. Changes in cellular health status can be deter-
mined by use of indirect measures related to the formation of a
colored tetrazolium dye product that can be measured spectropho-
tometrically at 490 nm. An increase in absorbance at 490 nm is
indicative of an increase in cell numbers and thus cell prolifera-
tion; and a decrease in absorbance is indicative of cell death. Data
are expressed as a percentage change in Optical Densities (O.D.)
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compared to the appropriate control values. TNF-
were measured in culture media by use of an ELISA specific for
mouse TNF- protein (BioLegend MAX™ Mouse TNF- ELISA Kit,
a protein levels
a
a
BioLegend, San Diego, CA) and are expressed as a percentage
change from the appropriate control.
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Acknowledgments
This work was supported in part by the Intramural Research
Program of the National Institute on Aging, National Institutes
of Health (NIH) and by NIH Grants (AG18379 and AG18884) to
D.K.L. The authors declare that they have no conflicts of interest
regarding the contents of this manuscript, and are grateful to
Dr. Amy Newman and colleagues of the Medicinal Chemistry Sec-
tion, National Institute on Drug Abuse, NIH, for use of NMR
equipment.