Synthesis of Thalidomide analogous compounds for the inhibition of

TNF-αFélix Vallés

RISE ProgramUPR Cayey

Introduction

• Thalidomide ((R,S)-2-(2,6-dioxopiperidin-3-il)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].

Introduction• Thalidomide ((R,S)-2-(2,6-dioxopiperidin-3-il)isoindol-

1,3-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.

Introduction

• Thalidomide ((R,S)-2-(2,6-dioxopiperidin-3-il)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].

Hypothesis:

• Synthesis of a Thalidomide analogous compound will allow for the inhibition of TNF-α 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.

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.

Experimental Design:

• Analogous compounds synthesis:[5] – Carry out the synthesis via Michael’s reaction.

NaOH/ H2O+

+

+

Experimental Design:

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

products.

Experimental Design:

• Spectroscopic techniques:– Determine the successfulness of the reactions and structures of

secondary products if any.

Experimental Design:

• Stereochemistry:– Utilize a Polarimeter to determine the presence of enantiomers

in each of the products.

Experimental Design:

• IC-50:[6] – Determine the minimum concentration required of each

synthesized compound to achieve TNF-alpha inhibition.

TNF-alpha InhibitionAnalogue 1Analogue 2Analogue 3Thalydomide

Experimental Design:

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

Experimental Design:

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

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.

References:• [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.