This document presents a proposal to synthesize analogous compounds of thalidomide for inhibiting TNF-α without thalidomide's teratogenic effects. The proposal involves modifying thalidomide's structure through Michael additions and testing the new compounds for TNF-α inhibition, stereochemistry, viability, and presence of secondary products, as measured by assays like IC-50, ELISA, trypan blue, and HPLC. Similar or better TNF-α inhibition is expected compared to thalidomide due to retention of the inhibitory pharmacophore, without the enantiomer responsible for teratogenicity.

1. Synthesis of Thalidomide analogous
compounds for the inhibition of
TNF-α
Félix Vallés
RISE Program
UPR Cayey

2. Introduction
• Thalidomide ((R,S)-2-(2,6-dioxopiperidin-3il)isoindol-1,3-dione)
– Drug that served as a sedative and nausea reliever
mostly used by with child women.
– However, during the 1960’s, the teratogenic
effects(malformations in newborns’ extremities) of this
compound were noted on a large scale[1].
– After several investigations were performed, scientists
discovered that the compound’s S enantiomer was
responsible for these effects[2].

3. Introduction
• Thalidomide ((R,S)-2-(2,6-dioxopiperidin-3-il)isoindol1,3-dione)
– Despite this, studies have revealed that Thalidomide is an
excellent inhibitor for TNF-α(tumor necrosis factor), a
cytosine group protein that stimulates the acute phase of
the inflammatory reaction in the human body.
– Therefore, Thalidomide is an excellent candidate for the
treatment of inflammatory diseases, such as arthritis
reumatoidea.
– Thalidomide has an advantage over other drugs used for
the treatment of arthritis, given that it does not have their
adverse effects on B cells; which are essential for antibody
production [3]. Among these drugs, Embrel®, Humira®,
and Remicade® can be mentioned.

4. Introduction
• Thalidomide ((R,S)-2-(2,6-dioxopiperidin-3il)isoindol-1,3-dione)
– Substitution of Thalidomide’s 2 - (2,6-dioxopiperidin-3-yl) for
1-methyl-2 ,5-pyrrolidinedione, 1-ethyl-2 ,5-pyrrolidinedione,
and 1-phenyl-2 ,5-pyrrolidinedione is proposed, given that the
rest of the molecule is responsible for its inhibitory effects of
TNF-alpha.
– This proposal is based on the Lima et al publication results [4].

5. Hypothesis:
• Synthesis of a Thalidomide analogous
compound will allow for the inhibition of TNFα without the teratogenic side effects of the
molecule’s S enantiomer. This is expected
given that this specific enantiomer will not be
present in the new products.

6. Objectives (Specific Aims):
• Modify Thalidomide to obtain new analogous
compounds with similar TNF-α inhibitory effects.
• Check for successfulness of the reactions through
spectroscopic techniques.
• Utilize a polarimeter to verify stereochemistry of
product.
• Detect the presence of secondary products
through HPLC.
• Test functionality, efficiency, and viability of the
product through IC-50, Trypan Blue, and Elisa.

7. Experimental Design:
• Analogous compounds synthesis:[5]
– Carry out the synthesis via Michael’s reaction.
+
NaOH/ H2O
+
+

8. Experimental Design:
• HPLC: (high performance liquid
chromatography)
– Determine the presence of secondary products and purify the
products.

9. Experimental Design:
• Spectroscopic techniques:
– Determine the successfulness of the reactions and structures of
secondary products if any.

10. Experimental Design:
• Stereochemistry:
– Utilize a Polarimeter to determine the presence of enantiomers
in each of the products.

11. Experimental Design:
• IC-50:[6]
– Determine the minimum concentration required of each
synthesized compound to achieve TNF-alpha inhibition.
TNF-alpha Inhibition
Analogue 1
Analogue 2
Analogue 3
Thalydomide

12. Experimental Design:
• Elisa(Enzyme-linked immunosorbent assay):[7]
– Check for inhibition of TNF-α.

13. Experimental Design:
• Trypan Blue:[8]
– Measure viability of each synthesized analogous compound.

14. Discussion and Expected Results:
• The synthesized compounds will be compared to each other and
Thalidomide(control) for their functionality, efficiency, inhibitory
activity, and viability, which will be measured and checked for via
the tests viewed earlier( IC-50, Trypan Blue, Elisa).
• Similar or better inhibition of TNF-α is expected from the
synthesized compounds, based on Lima et, al publication results.
(9)
• Since each analogous compound will contain the fragment in
Thalidomide responsible for the inhibitory effects, it is expected
that each one will retain the effects.
• Since the specific enantiomer S that causes the teratogenic effects
will not be present, these effects are not expected.
• Secondary products are most likely expected.
• Viability is expected due to the similar polarities and properties.

15. References:
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[1]Vianna, L.F.;Lopez-Camelo, J, S.;Louguercio,C. PLoS One. 2011, accessed
june2013.
[2] "Thalidomide & The Voice" lsc.ucdavis.edu, accessed june2013.
[3] Singhal, S.; Mehta, J. Biomedicine and Pharmacotherapy.2002, 56, 4-12,
accessed july2013.
[4] Lima, L. M.; Castro, P. "Synthesis and Anti- Inflammatory Activity of Phthalimide
Derivatives, Designed as New as New Thalidomide Analogues" Bioorganic
&Medicinal Chemistry. 2002, 10, 3067- 3073, accessed june2013.
[5] Klein, David R.. "Alpha Carbon Chemistry: Enols and Enolates." Organic
chemistry. Hoboken, N.J.: John Wiley, 2012. 1030-1040. Print.
[6]http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides
_Protocol_for_Performing_a_Trypan_Blue_Viability_Test__Technical_Reference_
Guide.pdf, accessed june2013
[7] http://www.piercenet.com/files/TR0065-ELISA-guide.pdf, accessed june2013
[8] biosupport.licor.com/docs/ICW_U0126_11454.pdf, accessed june2013.