Synthesis of Isatin Based Caspase Inhibitors

Synthesis of Isatin Based Caspase Inhibitors DESIGN AND SYNTHESIS OF ISATIN BASED CASPASE INHIBITORS FOR RUTHENIUM CAGING APPLICATIONS KASUN CHINTHAKA RATNAYAKE ABSTRACT Apoptosis is the energy dependent programmed cell death. Improper function of apoptosis could lead to diseases such as cancers, strokes, alziemer’s disease. Caspases are the enzymes involved in the later stage of this process. Peptidyl and non-peptidyl caspase inhibitors have been synthesized recently. One of these non-peptidyl compound classes which consist of pyrrolidinyl-5-sulfo isatins have showed a greater potency against executioner caspases, caspase-3 and -7. According to literature and for further caging studies, two compounds were designed, synthesized and evaluated their inhibition against caspase-3 in this study. 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 cyanoethyl group (8). Thus, the compound  7  was selected for further caging studies with ruthenium. Chapter 1: Introduction 1.1 Apoptosis and Caspases Apoptosis is the process of programmed cell death. This is a significant cellular process which is directly co-related with embryogenesis, immune system, ageing and various diseases including cancers, stroke, myocardial infarction and neurodegenerative disorders.1 Caspases (cysteinyl dependent aspartate directed 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 signaling 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 signaling cascade.2The caspases 1, 4 and 5 are found to be non-active in the cell death process. 1.2 Caspase inhibition and modified isatin sulfonamides as caspase inhibitors Caspases play a significant role in both inflammation and apoptosis. Extensive researches have been conducted on caspases and their functions because they act as potential targets in drug discovery. 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 used for different types of caspases. Isatin sulfonamides have showed inhibition on executioner caspases (caspase-3 and -7) in recent studies. In 2000, Lee and researchers reported the x-ray structure 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 effect 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 structure activity relationships and performed these biological assays. The following isatin sulfonamides have shown to be inhibit caspase-3. The stereochemistry of substituted pyrrolidine moiety, cyclic vs acyclic ring structures and ring sizes have been examined for these inhibition studies (figure 3).5 1.3 Ruthenium complexes for caging applications Ruthenium compounds have been reported as significant candidates for caging applications. Light activation of these metal complexes has been extensively studied. Recently, neuroactive biomolecules as well as small molecular enzyme inhibitors have been reported to be caged with these ruthenium complexes. Spatial and temporal release of these caged molecules upon light activation gives insight to develop new tools that could be used to treat various diseases in biological systems. In this study Ruthenium polypyridyl compounds are used in future studies since they have been considered as excellent candidates for caging application of small molecules. Chapter 2: Results and Data 2.1 General considerations All reagents were purchased from commercial suppliers and used as received. Varian FT-NMR Mercury-400 Spectrometer was used to record all NMR spectra. IR spectra were recorded on High resolution mass spectra were recorded on.Melting points were recorded on .Enzyme inhibition assays were done on 2.2 Designing of Caspase inhibitors Recent 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. Use of specific stereochemistry in the pyrrolidine moiety is important since S-alkoxypyrrolidine is more potent than its R-stereoisomer which shows almost no potency against caspase-3. It is reported that methoxymethyl pyrrolidinyl analogs show higher cell toxicity 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. Therefore, 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 th e 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 analogs The designed analogs were synthesized using literature 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 Assay Caspase-3 inhibition assay was performed for compounds 6 and 7 according to the literature 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 Experimental 2.5.1 Sodium 2,3-dioxoindoline-5-sulfonate (1) Isatin (10 g, 0.068 mol) was added carefully to a stirred solution of 20% SO3/H2SO4 (20 mL) at -15 °C. The reaction mixture was gently warmed up to 70  °C with stirring. Reaction mixture was stirred at 70  °C for another 15-20 min. The reaction mixture was carefully poured on to crushed ice 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 precipitation of the desired product. The solid was filtered, washed with ice-cold water and dried to give red-orange crystalline solid. The 1H-NMR data was compared and matched with 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 stirred for 3 h. The reaction was cooled to room temperature and kept in an ice bath. Then 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 oil) (960 mg, 40.0 mmol) was added and stirred for 10 min. Then methyl iodide (2.65 mL, 42.5 mmol) was added dropwise and reaction was stirred 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 over anhyd. Na2SO4 and concentrated to give a pale yellow oil and purified with petroleum ether: ether (9:1) 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, 100%) 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 1:1 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 1:1 EtOAc:Petroleum 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 saturated NaCl (20 mLÃâ€"3). The organic layer was dried over anhyd. Na2SO4 and concentrated in vacuo. The crude product was crystallized using EtOAc:Hexanes and isolated as a yellow solid. Yield: 85.8 mg (0.207 mmol, 40%) mp = 172-174  °C, 1H NMR (400 MHz, CDCl3): ÃŽ ´ 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): ÃŽ ´ 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): ÃŽ ´ 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, 24.1 IR (ÃŽ ½max) (KBr): 3443, 2929, 2361, 2342, 1747, 1616, 1478, 1450, 1417, 1365, 1344, 1330, 1199, 1181, 1154, 1130, 1115, 1070, 1041, 994 MS (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 min at RT. Then acrylonitrile (45  µL, 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 mLÃâ€"3). The combined organic layer was washed with 10% NaCl (20 mLÃâ€"3). The organic layer was dried over anhyd. Na2SO4 and concentrated in vacuo. The crude product was purified with CH2Cl2: MeOH (99:1) to afford yellowish-orange solid. Yield: 63.6 mg (0.169 mmol, 27%) mp = 134-138  °C, 1H NMR (400 MHz, CDCl3): ÃŽ ´ 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): ÃŽ ´ 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, 16.7 IR (ÃŽ ½max) (KBr): 3422, 2921, 2852, 2361, 2251, 1742, 1717, 1647, 1612, 1558, 1542, 1508, 1475, 1456, 1418, 1373, 1364, 1340, 1314, 1268, 1234, 1195, 1175, 1153, 1133, 1063, 1046, 991, 970, 905, 877 MS (HRMS): 470 (M+Na+MeOH)+ Chapter 3: Conclusion and Future directions The 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.