Synthesis of Isatin Based Caspase Inhibitors

Synthesis of Isatin Based Caspase InhibitorsDESIGN AND SYNTHESIS OF ISATIN BASED CASPASE INHIBITORS FOR RUTHENIUM CAGING APPLICATIONSKASUN CHINTHAKA RATNAYAKE nip caspase-mediated prison cell death is the energy dependent programmed cell death. Improper function of apoptosis could lead to diseases such as cancers, strokes, alziemers disease. Caspases ar the enzymes involved in the later stage of this process. Peptidyl and non-peptidyl caspase inhibitors imbibe been synthesized recently. One of these non-peptidyl mix classes which consist of pyrrolidinyl-5-sulfo isatins obligate showed a greater potency against public executioner caspases, caspase-3 and -7. According to writings and for however caging studies, two compounds were knowing, synthesized and evaluated their inhibition against caspase-3 in this memorise. The analog in which its N-1 position alkylated with a 4-methyl pyridine moiety (7) showed a higher inhibition than the analog in which its N-1 alkylated with cya noethyl sort out (8). Thus, the compound7was selected for further caging studies with atomic number 44.Chapter 1 Introduction1.1 Apoptosis and CaspasesApoptosis is the process of programmed cell death. This is a earthshaking cellular process which is promptly co-related with embryogenesis, immune system, ageing and various diseases including cancers, stroke, myocardial infarction and neurodegenerative disorders.1 Caspases (cysteinyl dependent aspartate say specific proteases) are the enzymes involved in the later stage of apoptosis. Caspases are divided to different classes according to their role played in the sign cascade of apoptosis. Caspases 6, 8, 9 and 10 are involved as initiators and caspases 2, 3 and 7 are identified as executioner caspases in the mark cascade.2The caspases 1, 4 and 5 are found to be non-active in the cell death process.1.2 Caspase inhibition and circumscribed isatin sulfa drugs as caspase inhibitorsCaspases play a significant role in both inflammat ion and apoptosis. Extensive researches have been conducted on caspases and their functions because they act as potential targets in drug discoery. Various inhibitors of Caspase have been made. These inhibitors could be categorized as non-peptidyl and peptidyl based compounds. A greater selectivity could be achieved when non-peptidyl inhibitors are apply for different types of caspases.Isatin sulfa drugs have showed inhibition on executioner caspases (caspase-3 and -7) in recent studies. In 2000, Lee and researchers report the x-ray organise of caspase-3 with an isatin analog, 1-methyl-5-(2-phenoxymethyl-pyrrolidine-1-sulfonyl)-1h-indole-2,3-dione (a) bound to the active site of the enzyme (Figure 1).3 Modifying isatin sulfonamide analogues with pyrrolidine groups have shown significant install on caspase inhibition.4 For example, various pyrrolidinyl-5-sulfo isatins have been shown inhibition to caspases, 3 and 7 (Figure 2). These isatin sulfonamide analogs are modified using s tructure activity relationships and performed these biological assays.The followers isatin sulfonamides have shown to be inhibit caspase-3. The stereochemistry of substituted pyrrolidine moiety, cyclic vs acyclic clique structures and ring sizes have been examined for these inhibition studies (figure 3).51.3 Ruthenium complexes for caging applicationsRuthenium compounds have been reported as significant candidates for caging applications. Light activation of these metal complexes has been extensively studied. Recently, neuroactive biomolecules as rise as small molecular enzyme inhibitors have been reported to be caged with these ruthenium complexes. Spatial and temporal release of these caged molecules upon visible light activation get throughs insight to pause new tools that could be used to treat various diseases in biological systems. In this study Ruthenium polypyridyl compounds are used in forthcoming studies since they have been considered as excellent candidates for c aging application of small molecules.Chapter 2 Results and entropy2.1 General conside balancensAll reagents were purchased from commercial suppliers and used as received. Varian FT- nuclear magnetic resonance Mercury-400 Spectrometer was used to record all NMR spectra. IR spectra were recorded on elevated reroot mass spectra were recorded on.Melting points were recorded on .Enzyme inhibition assays were make on 2.2 Designing of Caspase inhibitorsRecent studies show that various 5-pyrrolidinylsulfonyl isatins act as caspase-3 inhibitors. Several factors were considered in the designing process of these analogs. First, higher caspase inhibition was considered. drill of specific stereochemistry in the pyrrolidine moiety is important since S-alkoxypyrrolidine is much potent than its R-stereoisomer which shows close no potency against caspase-3. It is reported that methoxymethyl pyrrolidinyl analogs show higher cell perniciousness than phenoxymethyl pyrrolidines, thus methoxymethyl pyrrolidine analogs were chosen for further studies. When considering the Ruthenium caging studies, the chosen analogs should contain a group which has a higher binding affinitiy towards Ruthenium. because, pyridyl and cyano groups were selected to incorporate in these isatin sulfonamide analogs. These groups are chosen to be attached to N-1 position of isatin sulfonamide analog. It has been reported that higher alkyl chain on N-1 position could increase the inhibition. Therefore 4-methylpyridine and cyanoethyl groups were selected to attach on N-1 position of these analogs and compounds 7 and 8 are designed (Figure 3).2.3 Synthesis of designed isatin sulfonamide analogsThe designed analogs were synthesized using belles-lettres and modified procedures5, 6, 7 (Scheme 1). The compound 5 was synthesized as the precursor for the final analogs 7 and 8. The compounds 7 and 8 were synthesized using modified and optimized procedures (Scheme 2 and Scheme 3).2.4 Enzyme inhibition AssayCasp ase-3 inhibition assay was performed for compounds 6 and 7 according to the lit procedure.2 Compound 6 was found to be more potent (IC50 = .. ) of than compound 7 (IC50 = ..). Thus, compound 6 was selected for further caging studies with Ruthenium bipyridine complexes.2.5 Experimental2.5.1 sodium 2,3-dioxoindoline-5-sulfonate (1)Isatin (10 g, 0.068 mol) was added carefully to a aroused solution of 20% SO3/H2SO4 (20 mL) at -15C. The chemical reception florilegium was gently warmed up to 70 C with stirring. answer mixture was aroused at 70 C for another 15-20 min. The chemical response mixture was carefully poured on to crushed frosting and let ice to melt and then 20% NaOH was added to the reaction mixture (pH=7). The flask containing reaction mixture was kept in an ice bath to induce heedlessness of the desired product. The solid was filtered, washed with ice- raw body of water supply and dried to give red-orange crystalline solid. The 1H-NMR data was compared and matched wi th literature data.Yield 14.48 g (0.051 mol. 75%)2.5.2 2,3-dioxoindoline-5-sulfonyl chloride (2)sodium 2,3-dioxoindoline-5-sulfonate dihydrate (2 g, 70 mmol) was dissolved in tetramethylene sulfone (10 mL) under Argon environment at 60-70 C and phosphorus oxychloride (3.36 mL, ) was added dropwise. The reaction mixture was turned on(p) for 3 h. The reaction was cooled to room temperature and kept in an ice bath. accordingly ice-cold water was added to the reaction mixture carefully. A precipitate was formed, filtered, washed with ice-cold water and dried used without further purification. The desired compound is yielded as a bright yellow solid. The 1H-NMR data was compared and matched with literature data.Yield 1.58 g (64 mmol, 92%).2.5.3 Tert-butyl (S)-2-(methoxymethyl)pyrrolidine-1-carboxylate (3)To a solution of N-Boc-L-prolinol (5.0 g, 25 mmol) in THF (25 mL) at -78 C, Sodium hydride (60% in mineral cover) (960 mg, 40.0 mmol) was added and horny for 10 min. Then methyl iodi de (2.65 mL, 42.5 mmol) was added dropwise and reaction was emotional for 4h at -78 C and additional 16 h at RT. Then NH4Cl was added until all H2 evolved and EtOAc was added. The organic layer was washed with water and sat. NaCl, dried oer anhyd. Na2SO4 and concentrated to give a pale yellow oil and purified with oil colour ether ether (91) to give a colorless oil. The 1H-NMR data was compared and matched with literature data.Yield 4.986 g (23.16 mmol, 92%)2.5.4 (S)-2-(methoxymethyl)pyrrolidine (4)To a solution of tert-butyl (S)-2-(methoxymethyl)pyrrolidine-1-carboxylate (4.98 g, 23.07 mmol) in DCM (40 mL), TFA (25 mL) was added dropwise over 30 min at 0 C. The reaction was warmed to RT and stirred for additional 1.5 h. The reaction mixture was added to 150 mL of 10% NaOH solution and extracted with DCM (50 mL x 3), dried over anhyd. Na2SO4 and concentrated to obtain a pale yellow oil. The 1H-NMR data was compared and matched with literature data.Yield 2.657 g (23.07 mmol, ascor bic acid%)2.5.5 (S)-5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)indoline-2,3-dione (5)The compound (1) was synthesized according to procedure reported by Harvan et al.1 To a stirred solution of 2,3-dioxoindoline-5-sulfonyl chloride (2 g, 8.153 mmol) in 11 THF/CHCl3 (80 mL), a solution of (S)-2-(methoxymethyl)pyrrolidine (1.033 g, 8.968 mmol) and DIPEA (2.84 mL, 16.310 mmol) in CHCl3 was added dropwise under Argon environment and stirred for 1 h at 0 C. The reaction stirred for additional 1 h at RT. The reaction mixture was concentrated and purified using 11 EtOAc rock oil ether and isolated as bright yellow crystals. The 1H-NMR data was compared and matched with literature data.Yield 1.185 g (36.53 mmol, 45%)2.5.6 4-(bromomethyl)pyridine hydrobromide salt (6)Pyridin-4-ylmethanol (5.0 g) was dissolved in 48% HBr (50 mL) and refluxed for 24 h. (Reaction was monitored for completion using TLC). The reaction mixture was concentrated in vacuo until a thick gum appeared and treated with absolute Ethanol at 5 C. The white crystalline solid obtained was filtered and washed thoroughly with cold absolute Ethanol. The 1H-NMR data was compared and matched with literature data.Yield 4.74 g (18.7 mmol, 41%)2.5.7 (S)-5-((2-(methoxymethyl) pyrrolidin-1-yl)sulfonyl)-1-(pyridin-4-ylmethyl)indoline-2,3-dione (7)To a stirred solution of (S)-5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)indoline-2,3-dione (1) (168 mg, 0.518 mmol) in DMF, 60% NaH in mineral oil (51.8 mg, 1.295 mmol) was added at 0 C under Argon atmosphere. The reaction was stirred for 30 min. Then a solution of 4-Bromomethyl pyridine (130.6 mg, 0.518 mmol) in DMF was added dropwise and stirred for 4 h at 0 C. The reaction was diluted with EtOAc and washed with arrant(a) NaCl (20 mL3). The organic layer was dried over anhyd. Na2SO4 and concentrated in vacuo. The crude product was crystallized using EtOAcHexanes and isolated as a yellow solid.Yield 85.8 mg (0.207 mmol, 40%)mp = 172-174 C,1H NMR (400 MHz, CDCl3) 8 .64 (d, 2H, J = 6 Hz), 8.11 (s, 1H), 8.03 (d, 1H, J = 8.4 Hz), 7.27 (d, 2H, J = 3.6 Hz), 6.83 (d, 1H, J = 8.4 Hz), 4.99 (s, 2H), 3.74 (m, 1H), 3.55 (dd, 1H, J = 9.6 Hz, 4 Hz),1H NMR (400 MHz, DMSO) 8.51 (d, 2H, J = ..Hz), 8.01 (d, 1H, J = Hz), 7.84 (s, 1H), 7.46 (d, 2H, J = Hz), 7.07 (d, 1H, J = Hz), 4.99 (s, 2H), 3.67 (m, 1H), 3.41 (dd, 1H), 3.24 (s, 3H), 3.06 (m, 1H), 1.73 (m, 2H), 1.48 (m, 2H)13C NMR (100 MHz, CDCl3) 183.2, 160.8, 152.5, 150.5, 137.5, 134.9, 124.9, 122.1, 117.5, 110.8, 74.8, 59.2, 59.1, 49.3, 43.3, 28.8, 24.1IR (max) (KBr) 3443, 2929, 2361, 2342, 1747, 1616, 1478, 1450, 1417, 1365, 1344, 1330, 1199, 1181, 1154, 1130, 1115, 1070, 1041, 994MS (HRMS) 432 (M+Na+MeOH)+, 400 (M+Na)+2.5.8 (S)-3-(5-((2-(methoxymethyl) pyrrolidin-1-yl)sulfonyl)-2,3-dioxoindolin-1-yl)propanenitrile (8)To a stirred solution of (S)-5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)indoline-2,3-dione (1) (200 mg, 0.620 mmol) in DMF (10 mL), KOH (4 mg, 0.062 mmol) was added and stirred for 10 mi n at RT. Then acrylonitrile (45 L, 0.680 mmol) was added dropwise and stirred for 2 days under Argon environment at RT. The reaction mixture was added to H2O (30 mL), and extracted with EtOAc (20 mL3). The combined organic layer was washed with 10% NaCl (20 mL3). The organic layer was dried over anhyd. Na2SO4 and concentrated in vacuo. The crude product was purified with CH2Cl2 MeOH (991) to afford yellowish-orange solid.Yield 63.6 mg (0.169 mmol, 27%)mp = 134-138 C,1H NMR (400 MHz, CDCl3) 8.15 (d,1H,J=Hz), 8.11(d,1H, J=.Hz), 7.18(d,1H,J=.Hz), 4.10 (t,2H,J=), 3.77(m,2H), 3.57(dd, 2H, J= Hz), 3.43(m,H), 3.40 (s,..H), 3.38(d, H, J=Hz), 3.36 (s,3H,), 3.14(m,H), 2.98,2.96,2.94, 2.86(t,2H, J=Hz), 2.04(s,..H), 1.92(m,H), 1.69 (m,.H), 1.55(s,H)13C NMR (100 MHz, CDCl3) 180.8, 157.8, 152.3, 137.6, 134.7, 124.9, 117.5, 116.8, 110.4, 74.8, 59.3, 59.1, 49.4, 36.8, 28.8, 24.1, 16.7IR (max) (KBr) 3422, 2921, 2852, 2361, 2251, 1742, 1717, 1647, 1612, 1558, 1542, 1508, 1475, 1456, 1418, 1373, 136 4, 1340, 1314, 1268, 1234, 1195, 1175, 1153, 1133, 1063, 1046, 991, 970, 905, 877MS (HRMS) 470 (M+Na+MeOH)+Chapter 3 Conclusion and Future directionsThe compounds 7 and 8 were both potent for caspase-3 but compound 7 show more inhibition than that of compound 8. Thus compound 7 was selected for further ruthenium caging studies. The caged ruthenium complexes could be subjected for light activation experiments where IC50 of this complex under light and dark conditions could be determined and the dark to light inhibition ratio could be explored. Then cell toxicity studies could be done in order to explore the ability of these ruthenium complexes for prevention of apoptosis in biological systems.These combined experiments and results could lead to the final goal of this research study which is the development of novel tools to prevent apoptosis in biological systems.