EVALUATION OF SALICYALDEHYDEDERIVED BAYLIS HILLMAN ADDUCTS AND COUMARIN

EVALUATION OF SALICYALDEHYDE-DERIVED BAYLIS HILLMAN ADDUCTS AND COUMARIN DERIVATIVES AS POTENTIAL ANTISICKLING COMPOUNDS A PAPER PRESENTATION BY SHITTU, TAWAKALIT OMOLARA AT ALIKO-DANGOTE HALL, EDO UNIVERSITY IYAMHO 22/06/2017 EVALUATION OF SALICYALDEHYDE-DERIVED BAYLIS HILLMAN ADDUCTS AND COUMARIN DERIVATIVES AS POTENTIAL ANTISICKLING COMPOUNDS by SHITTU, TAWAKALIT OMOLARA is licensed under a Creative Commons Attribution-Non. Commercial-Share. Alike 4. 0 International License.

OUTLINE v INTRODUCTION: ANTISICKLING AGENTS SICKLE CELL DISEASE AND PATHOPHYSIOLOGY STATEMENT OF RESEARCH PROBLEM BAYLIS-HILLMAN ADDUCTS & COUMARINS v METHODOLOGY v RESULTS & DISCUSSION v RECOMMENDATION v REFERENCES v ACKNOWLEDGEMENT

INTRODUCTION ANTISICKLING AGENTS These are drugs that interfere with the sickling process in Haemoglobin SS via mechanisms such as induction of foetal haemoglobin (Hb. F); Gardos channel inhibition (membrane-active agents); scavenging iron overloads and in vivo nitric oxide (NO) generation (Dean and Schecter, 1978).

Overview of Sickle cell disease (SCD) is one of the most common haemoglobinopathies ravaging the world population with considerable morbidity and mortality (Ingram, 1956; Pauling et al. , 1949). The homozygous carrier state results to production of two or more mutant hemoglobin S (Hb. SS) and suffer clinical symptoms of the disease. It results from a point mutation on the β 6 -globin chain where valine substitutes glutamic acid Sickle cell disease is prevalent in Africa (WHO, 2011). 16% of the population in Africa has a sickle haemoglobinopathy which is the highest proportion worldwide 4

N = neutrophils, Ec = endothelial cells, ISCs = irreversibly sickle cells, R = reticulocytes. Figure 1: The pathophysiology of sickle cell disease (Steinberg and Martin, 1999)

Clinical symptoms of SCD elevation of bilirubin level leading to high incidence of gall stones and impairment of hepatic excretory function Cell apoptosis Spleen sequestration acute chest syndrome, pulmonary hypertension anemia jaundice

Management of sickle cell disease v Curative therapy: Hematopoietic Stem Cell Transplant (HSCT) v Antisickling drugs: § Fetal hemoglobin inducers: § Hb. S modifiers: (De Simon et al. , 1982; Steinberg et al. , 1997, Mehanna, 2001 ). 7

STATEMENT OF RESEARCH PROBLEM • Hydroxyurea, the only chemotherapeutic agent that has proved efficacious in the management of sickle cell disease, is potentially teratogenic, with variable response, and also having issues of noncompliance in severely affected individuals (Saraceno et al. , 2008; Haywood et al. , 2011). Hence, there is urgent need for development of antisickling agents that can be used to manage this disease. 8

RATIONALE FOR THE STUDY Baylis Hillman adducts have become an important class of bioactive compounds with diverse biological activities such as anticancer, antimalarial, molluscidal activities (Dadwal et al. , 2006). Coumarins possess broad spectrum of biological activities such as anticoagulant (Cheng et al. , 1995), anti-HIV (Olomola, et al. , 2014), antiinflammatory, antioxidant, calcium channel blocker (Yamahara et al. , 1989); anti-cancer (Luo et al. , 2011). However, these compounds are yet to be implicated in the management of sickle cell disease. 9

OBJECTIVES OF THE STUDY (a) to synthesize small libraries of Baylis-Hillman adducts and coumarin derivatives (b) carry out nucleophilic substitution on the coumarin substrates with urea derivatives. (c) Characterize these compounds, and (d) Carry out in vitro antisickling assay on the synthesized compound using Hb. SS blood from patients in steady state. 10

METHODOLOGY Figure 2: Synthetic pathway to the target compounds Baylis. Hillman adducts Hydroxyurea analogues coumarins Compounds R 1 R 2 R 3 1 a, b, c, d H H -CSNH 2, -CONH 2, -NHCSNH 2 2 a, b, c, d OEt H - CSNH 2, -CONH 2, NHCSNH 2 3 a, b, c, d H Br CSNH 2, -CONH 2, -NHCSNH 2 4 a, b, c, d H Cl CSNH 2, -CONH 2, NHCSNH 2 11

METHODOLOGY CONT’D Structure elucidation: § 1 H and 13 C Nuclear magnetic spectroscopy § FT-IR • Biological evaluation: All the compounds were screened for in vitro antisickling activities using an established method for reversal and inhibitory assays (Egunyomi et al. , 2009). inhibitory assay Antisickling assay experiments reversal assay Na 2 S 2 O 5 was used as the deoxygenating agent Positive controls are 12

RESULTS AND DISCUSSION 13

SYNTHESIS • Figure 3: IR spectrum of tert-Butyl-3 hydroxy -3 -( 5 chloro-2 -hydroxyphenyl -2 -methylenepropanoate) 4 b 14

Figure 4: 1 H NMR of tert-Butyl-3 hydroxy -3 -( 5 -chloro-2 hydroxyphenyl -2 -methylenepropanoate) 4 b 15

• ANTISICKLING RESULTS

100 90 80 70 60 4 mg/m. L 2 mg/m. L 50 1 mg/m. L 40 0. 5 mg/m. L 30 20 10 0 2 b 3 b 4 b 1 c 2 c 3 c 4 c 4 e Vanillic acid Figure 5: inhibitory activity of the synthesized compounds and standard drug (vanillic acid)

Figure 6: Micrograph showing untreated Hb. SS cells (Negative control) , viewed at X 400 magnification 18

Figure 7 : micrograph showing the inhibitory activity of compound 3 b at 1 mg/m. L, viewed at x 400 magnification 19

CONCLUSION v The reaction between tert-butylacrylate and salicyaldehyde derivatives afforded the corresponding Baylis-Hillman adducts, which were further cyclized regioselectively to 3(chloromethyl)coumarins in yields up to 96% using a mixture of hydrochloric and acetic acids v The compounds showed progressive inhibition against sodium metabisulphite induced sickling from 0. 5 mg/m. L to 4 mg/m. L and their activities were comparable with the standard drugs vanillic acid and para-hydroxybenzoic acid 20

FURTHER STUDIES 1. NMR-based mechanistic studies should be carried out to explore protein-ligand interactions. 2. Toxicological properties of the compounds should also be evaluated 21

REFERENCES • • De Simone, J. , Heller, P. , Hall, L. and Zwiers, D. (1982). 5 -Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons. Proc. Natl. Acad. Sci. , 79: 4428– 4431. • Dean, A. and Schechter, A. N. (1978). Sickle cell anaemia: Molecular and cellular bases of therapeutic approaches. N. Engl. J. Med. , 299: 752 -763 • Haywood, C. , Beach, M. C. , Bediako, S. , Carroll, C. P. , Lattimer, L. , Jarrett, D. and Lanzkron, S. (2011). Examining the characteristics and beliefs of hydroxyurea users and nonusers among adults with sickle cell disease. Am. J. Hematol. , 86 (1): 85– 87. • Ingram, V. M. (1956). A specific chemical difference between the globins of normal human and sickle-cell hemoglobin. Nature, 178: 792– 794 • • Mehanna, A. S. (2001). Sickle cell anemia and antisickling agents then and now. Curr. Med. Chem. , 8(2): 79 -88. • Olomola, T. O. , Klein, R. and Kaye, T. (2014). Towards the synthesis of Coumarin derivative as potential dual-action HIV-1 protease and reverse transcriptase inhibitors. Tetrahedron, 70: 9449 -9455. •

ACKNOWLEDGEMENT Special thanks to Edo University Iyamho for generous financial support which enabled me to present part of this research findings at the TWAS-NYA conference. 23

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