Design, synthesis and biological evaluation of new thalidomide analogues as TNF-α and IL-6 production inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.12.031

Several thalidomide analogues were synthesized and compared to thalidomide and its more active analogue, lenalidomide, for their ability to inhibit the production of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α and interleukin (IL)-6 by LPS-activated peripheral blood mononuclear cells (PBMCs). Among these compounds, two analogues containing sulfonyl group displayed interesting downregulation of TNF-α and IL-6 production.

Full text of DOI:10.1016/j.bmcl.2010.12.031

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1019–1022
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of new thalidomide analogues
as TNF-a and IL-6 production inhibitors
Charlotte Chaulet a, Cécile Croix a, David Alagille a, Sylvain Normand b, Adriana Delwail b, Laure Favot b,
Jean-Claude Lecron b, Marie-Claude Viaud-Massuard a,
⇑
a UMR CNRS 6239 GICC Génétique-Immunothérapie-Chimie et Cancer, Equipe 5, Laboratoire de Chimie Organique et Thérapeutique, Université de Tours François Rabelais, 31 avenue
Monge, France
b Laboratoire Inflammation, Tissus Epithéliaux et Cytokines EA 4331, Université de Poitiers and Service Immunologie et Inflammation, CHU de Poitiers – Pôle Biologie Santé, 40 avenue
du recteur Pineau, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Several thalidomide analogues were synthesized and compared to thalidomide and its more active
analogue, lenalidomide, for their ability to inhibit the production of the pro-inflammatory cytokine
Received 13 October 2010
Revised 2 December 2010
Accepted 6 December 2010
Available online 10 December 2010
tumour necrosis factor (TNF)-
cells (PBMCs). Among these compounds, two analogues containing sulfonyl group displayed interesting
downregulation of TNF- and IL-6 production.
a and interleukin (IL)-6 by LPS-activated peripheral blood mononuclear
a
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Thalidomide
TNF-a
IL-6
1H-Imidazo[1,5-a]indole-dione
Dihydro-thiadiazolo[2,3-b]-
isoquinolinone-dioxide
1H-Pyrrolo-[2,3-b]pyridine
Thalidomide (2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione) is
a synthetic glutamic acid derivative that was developed and
synthesized by Chemie Grunenthal in 1956.1–3 It was initially mar-
keted as a sedative, its rapid speed of onset, lack of hangover effect,
and apparent safety after overdose making it an attractive alterna-
tive to barbiturates. In addition, it was a powerful antiemetic, and
was widely taken by pregnant women for the treatment of morn-
ing sickness and as an aid to help them sleep. Nearly four years
later, multiple birth defects including phocomelia (absence or
hypoplasia of arms), absence of ears, deafness, defects of the femur
and tibia, as well as malformations of the heart and the bowel were
observed and associated with use of thalidomide in early preg-
nancy. Abnormalities of the alimentary tract, urinary tract and
other defects of internal organs were also reported.4,5 This resulted
in the withdrawal of thalidomide from the world market.
In 1998, thalidomide was reintroduced onto the market, and has
undergone clinical investigation for various immunomodulatory
and anti-inflammatory applications.7,8 Thalidomide selectively
and partially inhibits the production of TNF-a in human monocytes
in a dose-dependent fashion, with no effect on total protein
synthesis or on the production of other cytokines.9 It is the conse-
quence of the half-life reduction of TNF-
a mRNA from approxi-
mately 30–17 min by thalidomide.10 Additional studies have
shown that thalidomide also reduces the synthesis other cytokines,
including IL-6.11
A few years later, immunomodulatory thalidomide analogues
(IMiDs) were designed to optimise some properties, including
anticancer properties, while abolishing or reducing the toxicity
associated with thalidomide. Extensive preclinical testing of this
class of compound, including its pharmacology, pharmacokinetics
and toxicity, has led to the selection of lenalidomide for clinical
development in the oncology setting.12,13
In recent years, scientific interest in thalidomide has been re-
newed, because of its effectiveness in various diseases including
erythema nodosum leprosum, arthritis, tuberculosis and condi-
tions associated with human immunodeficiency virus infections.6
⇑
Corresponding author. Tel.: +33 247367227; fax: +33 247367229.
E-mail address: marie-claude.viaud-massuard@univ-tours.fr (M.-C. Viaud-
Massuard).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.031

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C. Chaulet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1019–1022
situ, to furnish compound 1, and the precursor of compound
2, which after reduction gives the desired product with a good
yield.
In a previous study, Lima et al. demonstrated that a derivative of
sulfonyl-isoindoline was active in modulating TNF- release. They
have shown that compounds including the sulfonamide moiety
a
An analogue of compounds 1 and 2 was developed from the
7-azaindole skeleton, as shown in Scheme 3. The first step was
the sulfonylation at position 1 in order to steer the introduction
of a carboxylic acid group to position 2 in a second step. The aim
was the esterification of the acid group. A classical reaction with
sulfuric acid in ethanol at reflux led to the formation of the ethylic
ester, and at the same time to desulfonylation, with very good
yield. To complete the synthetic pathway to compound 3, a cycli-
sation with phenylisocyanate was performed, which furnished
the derivative with correct yield.
In order to determine the importance of the ring size, another
tricyclic compound was considered with an isoquinoline structure.
The synthetic pathway of compound 4 is depicted in Scheme 4. The
condensation of formaldehyde with phenylalanine at reflux gave
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid with a correct
yield. The carboxylic acid was then esterified, and the amino group
sulfonylated, followed by a cyclisation at room temperature. After
an N-alkylation with a good yield, the ethyl-4-mercaptobenzoate
was introduced. The synthetic route to the compound 4 was
accomplished by the oxidation of the sulfur with mCPBA.
connected to the phthalimide ring of thalidomide are better inhib-
itors of TNF-a 14,15
Taking these results into account, we designed
.
other molecules with structural similarities. The first group include
derivatives containing a tricyclic structure derived from an imi-
dazo-indole-dione, which has a more rigid structure than thalido-
mide, albeit still including the carbonyl-amine-carbonyl sequence,
present in the phthalimide moiety. The second group is composed
of 7-azaindole derivatives, reported to display some of the biolog-
ical activity associated with sulfonamide.
Lenalidomide was synthesized following the synthetic route of
Muller et al.,16 shown in Scheme 1. It was obtained from the
Na-(tert-butoxycarbonyl)-L-glutamine and the methyl-2-methyl-
3-nitrobenzoate in five steps with an overall yield of 18%.
We then developed a series of tricyclic molecules (Group I: com-
pounds 1–4). Compounds 1 and 2 could be easily obtained, with cor-
rect yields, by a synthetic sequence shown in Scheme 2. The
condensation of potassium isocyanate with indoline-2-carboxylic
acid at reflux led to the formation of 9,9a-dihydro-1H-imi-
dazo[1,5-a]indole-1,3(2H)-dione. This derivative was then function-
alised with arylsulfonyl-methylimidazolium triflate, prepared in
Scheme 1. Reagents and conditions: (i) CDI, DMAP, toluene, reflux, 12 h; 65%; (ii) HCl 1 N, THF, rt, 12 h; quant.; (iii) NBS, AIBN, CCl4, reflux, 24 h; 49%; (iv) 3-aminopiperidine-
2,6-dione, TEA, MeCN, 55 °C, 18 h; 57%; (v) H2, Pd/C, MeOH, DMF; quant.
Scheme 2. Reagents and conditions: (i) KNCO, HCl, H2O, reflux, 3 h; 49%; (ii) arylsulfonyl-methylimidazolium triflate, Et3N, THF, rt, 12 h; 63%; (iii) NBS, AIBN, CCl4, reflux, 1 h;
85%.
Scheme 3. Reagents and conditions: (i) NaOH, Bu4NBr, ClSO2Ph, DCM, 0 °C to rt, 2 h; 90%; (ii) (a) LDA, THF, À35 °C, 30 min; (b) CO2, À35 °C to rt, 12 h; 65%; (iii) H2SO4, EtOH,
reflux, 12 h; 80%; (iv) PhNCO, NaOCH3, toluene; 45%.

C. Chaulet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1019–1022
1021
Scheme 4. Reagents and conditions: (i) HCHO, HCl, H2O, reflux, 3 h; 62%; (ii) (a) H2SO4, MeOH, reflux, 12 h; 95%; (b) HCO2H, ClSO2NCO, TEA, DCM, 0 °C, 4 h; 69%; (iii) (a) NaH,
THF, rt, 2 h; 59%; (b) HCHO, H2O, reflux, 1 h; 70%; (iv) (a) SOCl2, Et2O, rt, 48 h; (b) SHPhCO2Et, DBU, CH3CN, rt, 12 h; 67%; (v) mCPBA, DCM, rt, 3 h; 91%.
Scheme 5. Reagents and conditions: (i) PyBOP, DIEA, DMF, rt, 24 h; compound 5, 34%; compound 6, 34%.
Finally, a last series of molecules (Group II: compounds 5–9)
was prepared from the 1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyri-
dine-2-carboxylic acid through a peptide coupling with PyBOP
and various amines including a sulfonamide group. The synthesis
schemes are shown in Scheme 5 and Scheme 6. Only the aniline
coupling led to the formation of the both products sulfonylated
and desulfonylated. The other amines furnished the desulfonylated
compounds.
inhibitors of TNF-
Compound 3, compared to compounds 1 and 2, was less inhibitor
of TNF- and displayed a similar IL-6 regulating activity. Finally
compound 4 at 100 M inhibits significantly TNF- and IL-6 secre-
tion, but is devoid of effect at 10 and 1 M. One of the structural
differences between compound 3 and compounds 1, 2 and 4 is
the absence of the sulfonyl group. The results obtained for this
set of molecules teach us that the presence of a sulfonyl group is
a secretion, but do not affect IL-6 production.
a
l
a
l
Thalidomide and the compounds 1–9 were screened at concen-
important for regulating the activity of TNF-a.
trations of 100, 10 and 1
l
M for their ability to regulate TNF-
a
and
The compounds in the Group II are less rigid. They are built
around a common core, 7-azaindole, and they have for most of
them a sulfonyl group (compounds 5, 7, 8 and 9). Compound 5 in-
IL-6 production.17 Cytokines concentrations measured in 48 h LPS-
stimulated PBMCs supernatants, are summarized in Figure 1a
and b We noticed that none of these products at the concentration
tested in vitro displayed cytotoxic activity for PBMCs, as assayed by
propidium iodure labelling and flow cytometry detection.
duced a dose-dependent inhibition of TNF-
inhibition of IL-6 compared to thalidomide. Compounds 6–9 dis-
played a weak inhibitory effect on TNF- and IL-6 production.
a production and a best
a
We confirmed that thalidomide inhibits TNF-
a
production, but
We also noted that the presence of the thiomorpholine, which
IL-6 production is unchanged. But, as reported in the literature,
emerged as very important in the Lima’s studies, was not relevant
lenalidomide inhibits TNF-
than thalidomide.
In the Group I, we were interested in the rigidity of molecules
with a tricyclic structure, such as an imidazo-indole-dione or
imidazo-pyrrolopyridine-dione. Compounds 1 and 2 are effective
a
and IL-6 2–3 times more effectively
to the downregulation of TNF-
In conclusion, nine thalidomide analogues were synthesized
and evaluated for their ability to inhibit TNF- and IL-6 production
by LPS-activated PBMCs. Our results show the importance of these
new compounds as potential candidates to inhibit the production
a production in our study.
a
Scheme 6. Reagents and conditions: (i) Functionalized piperazines, DCM, rt, 30 min; 93–95%, (ii) SnCl2, 2H2O, DCM, MeOH, rt, 12 h; 94–97%; (iii) PyBOP, DIEA, DMF,
functionalised piperazines, rt, 24 h; 30–34%.

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C. Chaulet et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1019–1022
Figure 1. Effects of thalidomide, lenalidomide and compounds 1–4 (Group I) and 5–9 (Group II) on TNF-
a
(Fig.1a) and IL-6 (Fig. 1b) secretion. Normal PBMCs from three
M of thalidomide, leanlidomide or compounds 1–9. Cytokines
normal donors were cultured for 48 h with 1 g/ml LPS, in the absence (control) or presence of 100, 10, or 1
l
l
were assayed in culture supernatants by ELISA. Each dot represents the mean of three independents experiments. Not indicated for comprehension, variability is in all cases
<20%.
9. Sampaio, E.; Sarno, E.; Galilly, R.; Cohn, Z.; Kaplan, G. J. Exp. Med. 1991, 173, 699.
10. Moreira, A.; Sampaio, E.; Zmuidzinas, A.; Findt, P.; Smith, K.; Kaplan, G. J. Exp.
Med. 1993, 177, 1675.
11. Moreira, A. L.; Wang, J.; Sarno, E. N.; Kaplan, G. Braz. J. Med. Biol. Res. 1997, 30,
1199.
12. Bartlett, J. B.; Dredge, K.; Dalgleish, A. G. Nat. Rev. Cancer 2004, 4,
314.
13. Corral, L. G.; Kaplan, G. Ann. Rheum. Dis. 1999, 58, 107.
14. Montana, J. G.; Buckley, G. M.; Cooper, N.; Dyke, H. J.; Gowers, L.; Gregory, J. P.;
Hellewell, P. G.; Kendall, H. J.; Lowe, C.; Maxey, R.; Miotla, J.; Naylor, R. J.;
Runcie, K. A.; Tuladhar, B.; Warneck, J. B. H. Bioorg. Med. Chem. Lett. 1998, 8,
2635.
of the pro-inflammatory cytokines TNF-
sults indicate that compounds 4 and 5 show promising activity
against TNF- . The tricyclic structure and the sulfonyl group can
be considered to be scaffolds for the future design and develop-
ment of anti-TNF- and IL-6 agents. The importance of these
a and IL-6. The activity re-
a
a
new compounds cannot be ignored, as they are intermediates in
the lead optimization process.
Acknowledgements
15. Lima, L. M.; Castro, P.; Machado, A. L.; Fraga, C. A. M.; Lugnier, C.; Gonçalves de
Moraes, V. L.; Barreiro, E. J. Bioorg. Med. Chem. 2002, 10, 3067.
16. Muller, G. W.; Chen, R.; Huang, S.-Y.; Corral, L. G.; Wong, L. M.; Patterson, R. T.;
Chen, Y.; Kaplan, G.; Stirling, D. I. Bioorg. Med. Chem. Lett. 1999, 9, 1625.
17. Cell culture and reagents. Normal human peripheral blood mononuclear cells
(PBMCs) were isolated by Ficoll Hypaque (Biochrom AG, Berlin, Germany)
centrifugation. For all experiments, PBMCs were grown in culture medium
consisting of RPMI 1640 (Invitrogen Life Technologies, Cergy Pontoise, France)
supplemented with Glutamax I, 10% heat inactivated foetal calf serum (Sigma
The Laboratory of Organic and Therapeutic Chemistry acknowl-
edges the financial support for C. Chaulet and S Normand provided
respectively by the Conseil Régional de la Région Centre and
Région Poitou-Charentes. This work has been also supported in
part by the INCa (Institut National du Cancer; MAb IMPACT net-
work). Thanks also to Monika Ghosh for the translation assistance.
Aldrich, Saint Quentin Fallavier, France), 100 U/ml penicillin and 100
streptomycin (Invitrogen Life Technologies). One million PBMCs were cultured
for 48 h in 1 ml of medium with 1 g/ml E. coli LPS (Sigma Aldrich), in the
absence or presence of 100 M, 10 M, thalidomide, lenalidomide,
lg/ml
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