5. RESULTS & DISCUSSION

Transcription

1 5. RESULTS & DISCUSSIO 5.1. Synthesis and characterization of 5-(2-bromopyridin-3-yl-amino)-2- alkyl/aryl-isoindoline-1,3-dione (AG 11-20) A variety of novel phthalimide derivatives, 5-(2-bromopyridin-3-yl-amino)-2- alkyl/aryl-isoindoline-1,3-dione (AG 11-20), were synthesized from intermediate bromo substituted phthalimide derivatives, AG (1-10), and 3-amino-2- bromopyridine. In the first step, 5-bromo-2-alkyl/aryl-isoindoline-1,3-dione derivatives AG (1-10), were synthesized by refluxing 4-bromophthalic anhydride (1) and primary amines in the presence of glacial acetic acid and molecular sieves (4Å) at 120 C. To explore the possibility of introducing pyridyl moiety in these derivatives, palladium-catalyzed amination (Buckwald-Hartwig) reaction was carried out. It was found that the use of Pd(OAc) 2 /±BIAP couple in the presence of Cs 2 CO 3 in toluene is an efficient catalytic system to provide a general route (Scheme I) to a range of unknown 5-(2-bromopyridin-3-yl-amino)-2-alkyl/aryl-isoindoline-1,3- dione. In the first phase of screening experiment, 3-amino-2-bromopyridine and 5-bromo-2-methyl-isoindoline-1,3-dione were used as substrates for investigating the effects of various solvents, bases, ligands, and palladium sources. The basic condition for coupling in the presence of aotbu, K 2 CO 3, and K 3 PO 4 was carefully examined. However, no desired product was obtained and no amination occurred in the absence of any ligand. The use of Pd 2 (dba) 3 /dppf in lieu of Pd(OAc) 2 /±BIAP, did not yield the expected product. The amination remained unsuccessful either by decreasing the temperature (<80 C) or by increasing the temperature (>120 C). An attempt was made in microwave conditions at 710 watt for 4h and use of Ace pressure tube for 24h, but it appeared to be ineffective in providing good yield. When the reaction was performed in the presence of catalyst Pd(OAc) 2, ligand ±BIAP, and the base Cs 2 CO 3 in toluene at 110 C, the amination proceeded reasonably well and the expected products were obtained in moderate yield. The reaction never went to completion even after 24h reflux. With the increase in reaction time, the formation of relative impurities was increasing. The reaction 68

2 mixture was, therefore, quenched after 18h and purified using 1% Methanol:DCM in silica gel ( mesh size) based column chromatograpy under gravity. With this promising result in hand, different substituted 5-(2-bromopyridin-3-ylamino)-2-alkyl/aryl-isoindoline-1,3-dione were successfully prepared. All the synthesized compounds AG (1-10) and AG (11-20) were structurally evaluated and confirmed by FTIR, 1 H MR, 13 C MR, and LC/MS spectral analysis. The following is the physical and analytical data of the compounds synthesized using scheme I. AG 1: off-white powder (R f =0.89, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 3H, -CH 3 ), (d, 1H, J=8.1Hz, ArH), (m, 2H, ArH); LC/MS m/z (M+1), (M+3); IR (ν cm -1, KBr): , , (C=O), , , (ArCH=CH); M.p C. AG 2: off-white powder (R f =0.90, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 3H, -CH 3 ), (s, 2H, -CH 2 -), (d, 1H, J=8.1Hz, ArH), (m, 2H, ArH); LC/MS m/z (M+1); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH); M.p C. AG 3: off-white powder (R f =0.6, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, MeOD) δ: (t, 1H, J=8.4Hz), (d, 1H, J=7.8Hz, ArH), (m, 3H, ArH), (d, 1H, J=4.8Hz, ArH), (s, 1H, ArH); LC/MS m/z (M+1), (M+3); IR (ν cm -1, KBr): , , (C=O), , (ArCH=CH); M.p C. AG 4: off-white flakes (R f =0.7, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, MeOD) δ: (m, 4H, ArH), (d, 1H, J=7.8Hz, ArH), (t, 2H, ArH); LC/MS m/z (M+1),

3 (M+3); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH); M.p C. AG 5: grey powder (R f =0.56, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, MeOD) δ: (s, 3H, -CH 3 ), (d, 2H, J=9Hz, ArH), (d, 2H, J=9Hz, ArH), (d, 1H, J=6Hz, ArH), (m, 2H, ArH); LC/MS m/z (M+1), (M+3); IR (ν cm -1, KBr): , , (C=O), , , (ArCH=CH); M.p C. AG 6: off-white powder (R f =0.9, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 5H, ArH), (d, 1H, J=7.8Hz, ArH), (t, 2H, ArH); LC/MS m/z (M + ), (M+1), (M+2), (M+3); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH); M.p C. AG 7: pale yellow powder (R f =0.8, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 6H, 2CH 3 ), (s, 1H, ArH), (d, 1H, J=8.1Hz, ArH), (m, 2H, ArH); LC/MS m/z 333 (M+1), (M+2); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH); M.p C. AG 8: off-white powder (R f =0.5, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 2H, ArH), (d, 1H, J=7.8Hz, ArH), (m, 3H, ArH), (d, 1H, J=1.5Hz, ArH); LC/MS m/z (M + ), (M+1); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH); M.p C. AG 9: off-white powder (R f =0.9, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (t, 3H, -CH 2 ), (m, 5H, ArH), (d, 1H, J=8.4Hz), (m, 2H, ArH); LC/MS m/z 70

4 (M + ), (M+1); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH); M.p C. AG 10: off-white flakes (R f =0.35, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (d, 1H, J=8.1Hz, ArH), (m, 2H, ArH), (s, 1H, ArH), (bs, 1H, H); LC/MS m/z (M + ), (M+2); IR (ν cm -1, KBr): , , (C=O), , (ArCH=CH), (H); M.p >250 C. AG 11: dark yellow powder (R f =0.4, 1%MeOH:DCM, Visualization: U.V.). 1 HMR (400MHz, DMSO-d 6 ) δ: (s, 3H, -CH 3 ), (bs, 1H, H), (s, 1H, ArH), (d, 1H, J= 1.6Hz, ArH), (d, 1H, J=8.0Hz, ArH), (dd, 1H, J=6.4Hz, ArH), (d, 1H, J=2.0Hz, ArH), (d, 1H, J=8.0Hz, ArH); 13 CMR (ppm): , , , , , , , , , , , , , ; LC/MS m/z (M+2), (M+3); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), , (H); U.V. (λmax) 378nm; M.p C. AG 12: dark yellow powder (R f =0.4, 1%MeOH:DCM, Visualization: U.V.). 1 HMR (400MHz, DMSO-d 6 ) δ: (s, 3H, CH 3 ), (s, 2H, -CH 2 ), (bs, 1H, H), (s, 1H, ArH), (d, 1H, J= 1.6Hz, ArH), (d, 1H, J=8.0Hz, ArH), (dd, 1H, J=6.4Hz, ArH), (d, 1H, J=2.0Hz, ArH), (d, 1H, J=8.0Hz, ArH); LC/MS m/z (M), (M+1); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH), (H); M.p C. AG 13: pale yellow powder (R f =0.4, 1%MeOH:DCM, Visualization: U.V.). 1 HMR (400MHz, DMSO-d 6 ) δ: (bs, 1H, H), (d, 1H, J=8.8Hz), (m, 2H, ArH), (m, 3H, ArH), (dd, 1H, J=10.8Hz, ArH), (t, 1H, J=15.2Hz, ArH), (d, 1H, J=8Hz, ArH), 71

5 (d, 1H, J=8.4Hz, ArH); LC/MS m/z (M-H), (M+H), (M+2), (M+4); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH), , (H); M.p C. AG 14: dark yellow powder (R f =0.6, 1%MeOH:DCM, Visualization: U.V.). 1 HMR (400MHz, CDCl 3 ) δ: (s, 3H, CH 3 ), (bs, 1H, H), (m, 6H, ArH), (s, 1H, ArH), (d, 1H, J=10.8Hz, ArH), (d, 1H, J=10.8Hz, ArH), (t, 1H, J=8Hz); LC/MS m/z (M-H), (M+H), (M+2); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), , (H); M.p C. AG 15: dark yellow powder (R f =0.5, 1%MeOH:DCM, Visualization: U.V.). 1 HMR (400MHz, DMSO-d 6 ) δ: (s, 1H, -OCH 3 ), (bs, 1H, H), (s, 1H, ArH), (dd, 1H, J=1.6Hz, ArH), (d, 1H, J=2.0Hz, ArH), (d, 1H, J=6.0Hz, ArH), (m, 4H, ArH), (d, 1H, J=14.8Hz, ArH); 13 CMR (ppm): , , , , , , , , , , , , , , , , , , ; LC/MS m/z (M+H), (M+3); IR (ν cm -1, KBr): , 1767,54 (C=O), , , (ArCH=CH), , (H); M.p C. AG 16: dark yellow powder (R f =0.42, 1%MeOH:DCM, Visualization: U.V.). 1 HMR (400MHz, DMSO-d 6 ) δ: (bs, 1H, H), (d, 1H, J=12Hz, ArH), (m, 6H, ArH), (d, 1H, J=3.6Hz, ArH), (d, 1H, J=1.2Hz, ArH), (dd, 1H, J=6.4Hz, ArH), (s, 1H, ArH); LC/MS m/z (M + ), (M+H), (M+2); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), , (H); M.p C. 72

6 Figure 9. IR Spectra of AG 1 Figure 10. Mass Spectra of AG 1 73

7 Figure HMR Spectra of AG 1 Figure 12. IR Spectra of AG 3 74

8 Figure 13. Mass Spectra of AG 3 Figure HMR Spectra of AG 3 75

9 Figure 15. IR Spectra of AG 4 Figure 16. Mass Spectra of AG 4 76

10 Figure HMR Spectra of AG 4 Figure HMR Spectra of AG 5 77

11 Figure HMR Spectra of AG 7 Figure HMR Spectra of AG 10 78

12 Figure 21. IR Spectra of AG 11 Figure 22. Mass Spectra of AG 11 79

13 Figure HMR Spectra of AG 11 Figure CMR Spectra of AG 11 80

14 Figure 25. DSC Spectra (M.P & Purity) of AG 11 Figure 26. UV Spectra of AG 11 81

15 Figure 27. IR Spectra of AG 12 Figure 28. Mass Spectra of AG 12 82

16 Figure HMR Spectra of AG 12 Figure 30. IR Spectra of AG 13 83

17 Figure 31. Mass Spectra of AG 13 Figure HMR Spectra of AG 13 84

18 Figure 33. I.R Spectra of AG 14 Figure 34. Mass Spectra of AG 14 85

19 Figure HMR Spectra of AG 14 Figure 36. DSC Spectra (M.P & Purity) of AG 14 86

20 Figure 37. IR Spectra of AG 15 Figure 38. Mass Spectra of AG 15 87

21 Figure HMR Spectra of AG 15 Figure CMR Spectra of AG 15 88

22 5.2. Synthesis and characterization of bis-phthalimide derivatives (AG 34-43) A series of novel bis-phthalimide derivatives (AG 34-43) structurally related to thalidomide were synthesized by coupling intermediate 2-(1,3-dioxoisoindolin-2- yl)acetic acid derivatives (AG 24-33) with lenalidomide (AG 23) prepared in-house using a reported procedure (Scheme II). In the first phase of experiments, 2-(1,3-dioxoisoindolin-2-yl)acetic acid derivatives (AG 24-33) were synthesized by refluxing 4-bromophthalic anhydride (1) and phthalic anhydride with primary amines in the presence of glacial acetic acid and molecular sieves (4Å) at 120 C. Further, to obtain the novel bisphthalimide derivatives (AG 34-43), DCC and EDCI.HCl were evaluated as the peptide coupling agents. The coupling agents, DCC and EDCI.HCl were found to be in effective for these reactions in the presence of DCM since all the intermediates were completely insoluble. The reactions moved well to obtain the desired products (AG 34-43) in the presence of EDCI.HCl and DMF in a micro reaction vessel at 0-40 C. The synthesized compounds AG 21, AG (23-33) and AG (34-43) were structurally evaluated and confirmed by FTIR, 1 H MR, 13 C MR, and LC/MS spectral analysis. The following is the physical and analytical data of the compounds under scheme II. AG 21: white powder (R f =0.9, 40% EtOAc:Hexane, Visualization: U.V., I 2 ). 1 HMR (300MHz, MeOD) δ: (m, 10H, CH+Boc), (m, 2H, CH 2 ), (m, 2H, CO(-CH 2 -)), (m, 1H, H), (bs, 1H, CO-H-CO); LC/MS m/z (M-1), (M + ); IR (ν cm -1, KBr): , (C=O), , (CH-CH); M.p C. AG 23: off-white powder (R f =0.4, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (dd, 2H, CH 2 -), (q, 1H, -CH), (s, 2H, H 2 ), (d, 1H, J=7.2Hz), (t, 1H, ArH), (s, 1H, H); LC/MS m/z

23 (M+1); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH), (H), , (H 2 ); U.V. (λmax) nm; M.p C. AG 24: white powder (R f =0.1, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, MeOD) δ: (s, 2H, -CH 2 ), (d, 1H, J= 7.8Hz), (m, 2H, ArH); LC/MS m/z (M-1), (M + ), (M+1); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (OH); M.p C. AG 25: white powder (R f =0.3, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, MeOD) δ: (m, 3H, -CH 3 ), (m, 1H, - CH-), (s, 1H, ArH), (m, 2H, ArH); LC/MS m/z (M- 2), (M+1); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (OH); M.p C. AG 26: off-white powder (R f =0.5, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, MeOD) δ: (m, 3H, -CH 3 ), (m, 3H, -CH 3 ), (m, 1H, -C-CH), (m, 1H, --CH-), (d, 1H, J=7.8Hz, ArH), (m, 2H, ArH); LC/MS m/z (M-2), (M + ), (M+1); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (OH); M.p C. AG 27: white powder (R f =0.65, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, MeOD) δ: (m, 6H, 2CH 3 ), (m, 1H, - CH), (m, 1H, -C-CH), (m, 1H, -C-CH), (m, 1H, --CH-), (d, 1H, J=8.1Hz, ArH), (m, 2H, ArH); LC/MS m/z (M-2), (M + ), (M+1); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (OH); M.p C. 90

24 AG 28: white flakes (R f =0.2, 5% MeOH:DCM, Visualization: U.V., inhydrin, I 2 ). 1 HMR (300MHz, MeOD) δ: (d, 2H, J=12.0Hz, ArH), (d, 2H, J=9.0Hz, ArH), (d, 1H, J=7.8Hz, ArH), (m, 2H, ArH); LC/MS m/z (M + ), (M+1), (M+2); IR (ν cm -1, KBr): , , (C=O), (ArCH=CH), (OH); M.p >300 C. AG 29: off-white powder (R f =0.1, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 2H, -CH 2 -), (s, 4H, ArH), (bs, 1H, -OH); LC/MS m/z (M-1), (M + ); IR (ν cm -1, KBr): , (C=O), , , , (ArCH=CH), (OH); M.p C. AG 30: white powder (R f =0.2, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 3H, -CH 3 ), (s, 1H, - CH-), (s, 4H, ArH), (bs, 1H, OH); LC/MS m/z (M-1), (M + ); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH), (OH); M.p C. AG 31: off-white powder (R f =0.2, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 3H, -CH 3 ), (s, 3H, -CH 3 ), (m, 1H, -CH), (d, 1H, J=6.9Hz, - CH), (s, 4H, ArH), (bs, 1H, -OH); LC/MS m/z (M-1), (M + ); IR (ν cm -1, KBr): , , (C=O), , , (ArCH=CH), , (OH); M.p C. AG 32: off-white powder (R f =0.24, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, DMSO-d 6 ) δ: (s, 6H, 2CH 3 ), (m, 1H, -CH), (m, 1H, -CH), (m, 1H, -CH-), (t, 1H, --CH-), (s, 4H, ArH), (bs, 1H, -OH); LC/MS m/z 91

25 (M-1), , (M + ); IR (ν cm -1, KBr): , (C=O), , , , (ArCH=CH), (OH); M.p C. AG 33: off-white powder (R f =0.34, 5% MeOH:DCM, Visualization: U.V., inhydrin). 1 HMR (300MHz, DMSO-d 6 ) δ: (d, 2H, J=8.4Hz, ArH), (d, 2H, J=3Hz, ArH), (m, 4H, ArH); LC/MS m/z (M-1), (M + ); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (OH); M.p C. AG 34: white powder (R f =0.42, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (400MHz, DMSO-d 6 ) δ: (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 4H, 2CH 2 - ), (q, 1H, -CH), (m, 2H, ArH), (m, 2H, ArH), (m, 2H, ArH), (s, 1H, H), (s, 1H, H); 13 CMR (ppm): , , , , (Aliphatic C), , 122.4, 122.6, , , , , , , , , (Aromatic C), , , , , , (Carbonyl C=O); LC/MS m/z (M+2), (M-1+a + ), (M+1+a + ), (M+2+a + ); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (H); U.V. (λmax) 296.1nm; M.p C (DSC). AG 35: white powder (R f =0.42, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (400MHz, DMSO-d 6 ) δ: (d, 3H, J=7.2Hz, CH 3 ), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 2H, CH 2 -), (q, 1H, -CH), (m, 1H, -CH), (m, 3H, ArH), (d, 1H, J=24Hz, ArH), (m, 2H, ArH), (d, 1H, H, J=6.0Hz), (s, 1H, H); 13 CMR (ppm): (CH 3 ), , , , , (Aliphatic C), , , 122.4, , , , , 92

26 , , , , (Aromatic C), , , , , , (Carbonyl C=O); LC/MS m/z (M + ), (M-1+a + ), (M+1+a + ), (M+2+a + ); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (H); U.V. (λmax) 296.0nm; M.p C (DSC). AG 39: off-white powder (R f =0.41, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (d, 2H, J=7.2Hz, CH 2 -), (s, 2H, CH 2 -), (q, 1H, -CH), (m, 2H, ArH), (dd, 1H, ArH), (m, 4H, ArH), (s, 1H, H), (s, 1H, H); LC/MS m/z (M+1), (M+2), (M+a + ); IR (ν cm -1, KBr): , (C=O), , (ArCH=CH), (H); U.V. (λmax) nm; M.p C (DSC). AG 40: light grey powder (R f =0.41, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (d, 3H, J=6.9Hz, CH 3 ), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 2H, CH 2 -), (q, 1H, -CH), (m, 1H, -CH), (m, 2H, ArH), (dd, 1H, ArH), (m, 4H, ArH), (d, 1H, J=5.4Hz, H), (s, 1H, H); LC/MS m/z (M+1), (M+2), (M+a + ); IR (ν cm -1, KBr): , (C=O), (ArCH=CH), (H); U.V. (λmax) 296.1nm; M.p >300 C. AG 41: light grey powder (R f =0.42, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 6H, 2CH 3 ), (m, 1H, CH), (m, 1H, CH), (m, 1H, CH), (m, 2H, 2CH), (m, 2H, CH 2 -), (m, 2H, 2-CH), (m, 3H, ArH), (m, 4H, ArH), (s, 1H, H), (s, 1H, H); 93

27 LC/MS m/z (M+1); IR (ν cm -1, KBr): (C=O), , (ArCH=CH), (H); U.V. (λmax) 296.0nm; M.p >300 C. AG 42: off-white powder (R f =0.42, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 7H, CH(CH 3 ) 2 ), (m, 2H, CH 2 ), (m, 2H, CH 2 ), (m, 2H, CH 2 ), (t, 2H, CH 2 -), ( q, 1H, -CH), (q, 1H, -CH), (m, 3H, ArH), (m, 4H, ArH), (s, 1H, H), (bs, 1H, H); LC/MS m/z (M+1), (M+2); IR (ν cm -1, KBr): , (C=O), , , (ArCH=CH), (H); U.V. (λmax) 296.0nm; M.p >300 C. AG 43: off-white powder (R f =0.45, 5% MeOH:DCM, Visualization: U.V., I 2 ). 1 HMR (300MHz, DMSO-d 6 ) δ: (m, 2H, CH 2 ), (m, 2H, CH 2 ), (s, 2H, CH 2 -), (q, 1H, -CH), (m, 4H, ArH), (d, 1H, J=7.5Hz, ArH), (m, 4H, ArH), (d, 2H, J=8.1Hz, ArH), (bs, 1H, H), (bs, 1H, H); LC/MS m/z (M+1), (M+2), (M-1+a + ); IR(ν cm -1, KBr): , (C=O), , (ArCH=CH), (H); U.V. (λmax) 296.0nm; M.p >300 C. 94

28 Figure 41. IR Spectra of Intermediate AG 21 Figure HMR Spectra of Intermediate AG 21 95

29 Figure 43. IR Spectra of Intermediate AG 23 (Lenalidomide) Figure HMR Spectra of Intermediate AG 23 (Lenalidomide) 96

30 Figure 45. DSC Spectra (M.P & Purity) of Intermediate AG 23 Figure 46. UV Spectra of Intermediate AG 23 97

31 Figure 47. IR Spectra of AG 24 Figure 48. Mass Spectra of AG 24 98

32 Figure HMR Spectra of AG 24 Figure 50. IR Spectra of AG 27 99

33 Figure 51. Mass Spectra of AG 27 Figure HMR Spectra of AG

34 Figure 53. IR Spectra of AG 30 Figure HMR Spectra of AG

35 Figure HMR Spectra of AG 31 Figure 56. Mass Spectra of AG

36 Figure HMR Spectra of AG 32 Figure 58. IR Spectra of AG

37 Figure 59 IR Spectra of AG 34 Figure 60. Mass Spectra of AG

38 Figure HMR Spectra of AG 34 Figure CMR Spectra of AG

39 Figure 63. DSC Spectra (M.P & Purity) of AG 34 Figure 64. UV Spectra of AG

40 Figure 65. IR Spectra of AG 35 Figure 66. Mass Spectra of AG

41 Figure HMR Spectra of AG 35 Figure CMR Spectra of AG

42 Figure 69. DSC Spectra (M.P & Purity) of AG 35 Figure 70. UV Spectra of AG

43 Figure 71. IR Spectra of AG 39 Figure 72. Mass Spectra of AG

44 Figure HMR Spectra of AG 39 Figure 74. DSC Spectra (M.P & Purity) of AG

45 Figure 75. UV Spectra of AG 39 Figure 76. IR Spectra of AG

46 Figure 77. Mass Spectra of AG 40 Figure HMR Spectra of AG

47 Figure HMR Spectra of AG 41 Figure 80. IR Spectra of AG

48 Figure HMR Spectra of AG 42 Figure 83. IR Spectra of AG

49 Figure 84. Mass Spectra of AG 43 Figure HMR Spectra of AG

50 5.3. Synthesis and characterization of pyrrolo-δ-carbolines; 8-methyl-7,9- dioxo-5h-pyrido[3,2-b]pyrrolo[4,3-f]indole (AG 44) and 2-methyl-1,3-dioxo- 6H-pyrido[3,2-b]pyrrolo[4,3-e]indole (AG 45) To establish the hypothesized scheme 44, compound AG 11 was selected as the substrate for investigating the intramolecular Heck cyclization process towards the synthesis of pyrrolo-δ-carbolines i.e. 8-methyl-7,9-dioxo-5Hpyrido[3,2-b]pyrrolo[4,3-f]indole (AG 44) and 2-methyl-1,3-dioxo-6H-pyrido[3,2- b]pyrrolo[4,3-e]indole (AG 45). The two reported processes (A & B) were adopted for the cyclization process as shown in scheme III. It was observed that method (b) utilizing Pd(OAC) 2, a 2 CO 3, in DMF at 150 C proceeded for a longer duration as compared to method (A) in which a t OBu was used as a base with Pd(OAC) 2 and tri-t-butylphosphonium tetrafluoroborate in 1,4-dioxane. Both the methods yielded the desired product but method (B) worked well with higher (48%) percentage yield. The obtained product was the mixture of two compounds AG 44/45 which was confirmed by DSC and Mass in which two thermogram peaks were observed at different temperature ranges with a single molecular ion base peak at m/z daltons in mass spectroscopy. Further, the FTIR spectroscopy also confirmed the presence of expected functional (C=O & H) groups. The separation of this mixture could not be achieved on column chromatography even in mesh size silica due to similar R f value. The mixture may be separated using preparative HPLC in the future. AG 44/45 Mixture: Pale Yellow powder (R f =0.36, 5% MeOH:DCM, Visualization: U.V., I 2 ). LC/MS m/z (M + ), (M+1), (M-1+a + ), (M+a + ); IR (ν cm -1, KBr): (C=O), , , (ArCH=CH), (H); U.V. (λmax) 380nm; M.p C, C (Two peaks in DSC). 117

51 Figure 86. IR Spectra of a mixture of AG 44 & 45 Figure 87. Mass Spectra of a mixture of AG 44 & 45 Figure 88. DSC Spectra (M.P) of a mixture of AG 44 &

52 5.4. Docking studies: The glide docking of the synthesized molecules was carried out using the previously prepared receptor grid. The favorable interactions between ligand molecules and the receptor were scored using Glide ligand docking program. The validation results of Glide docking procedure for topoisomerase IIα revealed a very good agreement between the localization of ligand upon docking as compared to the crystal structure. The re-docked ligand, AP, showed similar hydrogen bonding interactions with Asn91, Asn120, Ser148, Ser149, Asn150, Arg162, Asn163, Gly164, Tyr165, Gly166, Ala167, Lys168, Gln376, Lys378 and Mg 2+ residues of the protein suggesting the reliability of the Glide docking in reproducing the experimentally observed binding mode for AP. The parameters set for Glide docking are, therefore, reasonable to reproduce the X-ray structure. All the synthesized molecules (AG and AG 34-45) were screened in silico for catalytic inhibition of human topoisomerase IIα (htopoiiα) at the ATP site. The docking scores and the structural descriptors data in extra precision (XP) docking studies revealed that four molecules AG 11, AG 13 (diaryl amines), AG 34 and AG 39 (bis-phthalimides) demonstrated remarkable H-bond interactions with crucial amino acids of the protein including Asn91, Asn95, Ser148, Ser149, Arg162, Asn163, Gly164, Ala167, Lsy168 and catalytic MG 2+. The Glide score (GScore) along with their XP descriptors, H-bond and lipophilic interactions including bond length for the best poses (Figure 89-90) are given in Table 7-8. In comparison with standard molecules, salvicine and ICRF 193, the test molecules, AG 13, AG 34 and AG 39 showed comparative Glide score, better Glide energy, lipophilicity and H-bond score (Table 7-8). The screened molecules AG 44 and AG 45 (pyrrolo-δ-carbolines) were found to be inactive in htopoiiα as compared to the test as well as standard molecules (Figure 91-92). 119

53 AG 11 AG 13 Figure 89. XP Docked pose of AG 11 and AG 13 at the ATP site of htopoiiα (Chain B) 120

54 Figure 90. XP Docked pose of AG 39 at the ATP site of htopoiiα (Chain B) Table 7 Docking parameters of selected ligands Code G.Score G Energy Lipophilicity H-Bond RMSD AG AG AG AG AG AG Salvicine ICRF

55 Salvicine ICRF 193 Figure 91. Salvicine and ICRF-193 docked in the ATP pocket of htopoiiα (Chain B) 122

56 AG 44 AG 45 Figure 92. XP Docked pose of AG 44 and AG 45 at the ATP site of htopoiiα (Chain B) 123

57 Table 8 Hydrogen bond interactions of selected ligands Code (H-bond Donor / Acceptor) Hydrogen bond interaction Amino acids interacting (H-bond Donor / Acceptor) AG 11 (C=O, -H) (-H) Asn 91 (-H) Ala167 (O-H) Ser 148 (O-H) Ser 149 Mg 2+ AG 13 (C=O, -H) (C=O) Asn 95 (-H) Lsy 168 (-H) Gly 164 (-H) Asn 163 (-H) Arg 162 Mg 2+ AG 34 (C=O) (-H) Arg 98 (-H) Arg 98 (O-H) Asn 91 (-H) Asn 91 (-H) Ser 149 (O-H) Ser 148 AG 39 (C=O) (-H) Arg 98 (-H) Arg 98 (O-H) Asn 91 (-H) Asn 91 (-H) Ser 149 (O-H) Ser 148 AG 44 (C=O, -H) (-H) Asn 91 (-H) Ala 167 (O-H) Ser 148 (C=O) Ser 149 AG 45 (C=O) (O-H) Water (O-H) Thr 215 (-H) Asn 120 ICRF 193 (C=O, ) (-H) Arg 162 (-H) Ala 167 (O) Asn 91 (-H) Lsy 168 Salvicine (C=O) (-H) Ser 149 (-H) Ser 149 (-H) Asn 150 (-H) Ala 167 (-H) Lsy 168 Bond length (Å)

58 5.5. In-vitro antineoplastic screening From the results of docking studies, the four compounds, AG 11, AG 13 (diaryl amines), AG 34, AG 39 (bis-phthalimides) were selected for their anti-proliferative potential on human small lung cancer (A549) and larynx epidermoid carcinoma (HEp-2) cell lines using MTT assay. Even though the Glide energy for AG 11 was less, it was selected for in vitro studies since the Glide score was comparable with other test molecules. The structurally similar anticancer drug lenalidomide was used as the standard. To correlate the relationship between in silico and in vitro, two compounds AG 35 (bis-phthalimide derivative) & AG44/45 mixture (pyrrolo-δ-carboline), which did not show promising activity on htopoiiα, was also selected for the study. All the compounds showed dose dependent anti-proliferative pattern against A549 and HEp-2 cell lines (Table 9). As expected from the results of docking studies, the compounds AG 13, AG 34 and AG 39 were found to be cytotoxic and showed potent antiproliferative activity with CTC μg/ml, 120μg/ml and 120μg/ml respectively against A549 cell line (Figure 93). The compounds AG 13, AG 34 and AG 39 also showed potent antiproliferative activity against HEp-2 cell lines with CTC μg/ml, 135μg/ml, 140μg/ml respectively (Table 9). The other two compounds AG 35 and AG 44/45 mixture did not show any prominent activity on both the cell lines in comparison with the standard. Table 9 In-vitro anticancer activity on A549 and HEp-2 cell lines CTC 50 μg/ml Compound Cell Line A549 Cell Line HEp-2 AG AG AG AG AG AG 44/45 Mix Lenalidomide (STD)

59 Figure 93. A. ormal A549 Cell Culture B. 100% Cytotoxicity by AG 13 C. 100% Cytotoxicity by AG

60 5.6. Ligand based molecular modeling studies to identify more potent ligands The hypothetical scheme 44 was working out well for the synthesis of pyrrolo-δcarbolines, therefore, a library of 3000 synthetically accessible novel pyrrolo-δcarbolines, to produce maximum inhibition of htopo IIα enzyme, was designed with different substitutions on the fifth (R) and eighth (R ) position of 7,9-dioxo- 5H-pyrido[3,2-b]pyrrolo[4,3-f]indole and sixth (R) and second (R ) position of 1,3-dioxo-6H-pyrido[3,2-b]pyrrolo[4,3-e]indole (Figure 94). The selection of substituent at both the positions R and R 1 was mainly guided by lipophilicity and electronic charge considerations at the active site of the enzyme. The in silico library of 17,000 tautomers was generated as an output of ligand preparation wizard of 3000 designed ligands. In search of the candidate molecules that can inhibit htopo IIα, the generated library of 17,000 tautomers was subjected to simultaneous HTVS and XP docking studies. On the basis of Glide energy, Docking score, RMSD and Glide rank, eight molecules were selected from XP docking studies (Table 10). The selected eight molecules were further subjected to induced fit docking studies. Docking models for all the ligands selected after in silico experiments was generated to study the interactions at the active site. Of all the initial ligands used for the induced fit docking studies, a total of 4 potential hits (Figure 94) were found and studied in detail. The binding energies of the lowest-energy poses for each of the topoisomerase II inhibitors for the ATP binding site are summarized in Table 11. Docking simulation of the first ranked molecule AG698 into htopo IIα suggested hydrogen bond interactions with nine amino acid residues, Glu87, Asn91, Asn95, Arg98, Arg162, Ala167, Lys168, Gln376, Lys378 (Figure 95A), which are comparable to the original interactions of AP in 1ZXM. The -H groups from the amino acid residues interact with the α- and γ-phosphates of AP by non-bridging oxyanions in 1ZXM similarly, the oxyanion of carboxylic group of AG698 interacts 127

61 with Arg162, Ala167 and Lys168 by hydrogen bond. Although AG698 does not interact with an active site Mg 2+ ion, but it does interact with the δ-1 carbonyl oxygen of Asn-91 which in 1ZXM interacts with both Mg 2+ ion and AP. The highly conserved Lys-378 from the QTK-loop of the transducer domain forms a salt bridge with the γ-phosphate of AP. Similar interaction was also observed in the docking pose of AG698. This salt bridge is believed to stabilize the transition state of the hydrolysis reaction. The key catalytic residue, Glu87, is hydrogenbonded to one of the two water molecules coordinated to Mg 2+ in 1ZXM. Similarly one of the carboxylic acid group at R 1 position in AG698 interacts with Glu87 and a water molecule. The water molecule in turn is bonded to Glu87 and another carboxylic acid group at R 1 position of the AG698 molecule. These interactions suggest that AG698 can be better ATP competitor at the ATPase site of htopo IIα. The second ranked molecule AG2549 showed hydrogen bond interactions with seven amino acid residues, Asp94, Ser149, Asn150, Arg162, Gly164, Tyr165, Lys168 (Figure 95B). The third ranked ligand AG2099 interacted with the residues Asp152, Glu155, Ser149, Arg98 (Figure 95C) and the fourth ranked ligand AG898 interacted with the residues Arg162, Ala167, Lys168, Lys378 (Figure 95D). The Docking results were compared with the already existing ATP competitors, salvicine and ICRF 193 and also ADP at the ATPase site of htopo IIα (Table 11). The interatomic hydrogen bond distances of AG698 with the key amino acids are within the range of 2-3 Å. The glide energy and docking score of AG698 was found to be and respectively which is almost double then ICRF 193 and salvicine. The data was also compared to ADP glide energy and docking score which indicate that the designed molecule AG698 could act as a better competitor. The molecule also satisfies the Lipinski s rule of five with molecular weight 496 daltons, Log P -1.3, CLogP , Total number of hydrogen bond acceptors <10, total number of hydrogen bond donors <5, and molar refractivity cm 3 /mol. It can be predicted that the designed molecule AG698 may have good water solubility and better bioavailability due to 128

62 carboxylic groups and may act as a better ATP competitor at the ATPase site of htopo IIα. O R' O R 7,9-dioxo-5H-pyrido[3,2-b] pyrrolo[4,3-f]indole I O R' O R 1,3-dioxo-6H-pyrido[3,2-b] pyrrolo[4,3-e]indole II O O O H O ICRF 193 H O HO OH O Salvicine O H O OH HO O O O AG698 O OH O H O O O O AG2549 O O O H H O O AG2099 O HO H O O AG898 Figure 94. Standard molecules and selected ligands for Induced fit docking studies. 129

63 An analysis of the in silico data generated from docking studies suggested that, the activity of the designed molecular framework is dependent upon the substituents at the R and R 1 positions and/or their combination. Compounds which have COOH and Morpholine groups at R position were found to be more active in the in silico models than compounds which have H, alkyl or aryl group at R position. Compounds which have tetrazole, triazole, and amino acid residues at R 1 position were found to be more active in the in silico models than compounds which have H, alkyl or aryl group at R 1 position. The combination showed better in silico activity in all the docking modules of glide. In conclusion, the electron withdrawing hydrophilic substitutes are more advantageous at the R and R1 position rather than lipophilic substituents or electronically neutral hydrophilic substitutes for achieving the potent activity data. Table 10 Extra Precision Glide docking and scoring of best fit conformation of selected eight virtual ligands as per their rank Molecules RMSD docking glide glide score gscore energy AG AG AG AG AG AG AG AG

64 Table 11 Docking parameters and amino acid Interactions of four designed virtual ligands as inhibitors of HumanTopoisomerase IIα ATP Site Molecules (Rank) AG698 (Rank 1) AG2549 (Rank 2) AG2099 (Rank 3) AG898 (Rank 4) Salvicine (Std) ICRF 193 (Std) Hydrogen Bond Donor Hydrogen Bond Acceptor Length of Hydrogen Bond (A ) RMSD Docking Score Glide Energy Asn91(D2) AG698 (O) Asn95() AG698(O) Arg98(E) AG698 (O) Arg162() AG698 (O) Ala167() AG698 (O) Lys168(Z) AG698 (O) Gln376(E2) AG698 (O) Lys378(Z) AG698 (O) Lys378(Z) AG698 (O) Ser149(OG) AG2549(O) Asn150(D2) AG2549(O) Asn150(D2) AG2549(O) Gly164() AG2549() Lys168(Z) AG2549(O) Gln376(E2) AG2549() Arg98(E) AG2099 (O) Ser149() AG2099 (O) Ser149(OG) AG2099 (O) AG2099 () Asp152(OD2) AG2099 () Glu155(OE1) Arg162() AG898(O) Ala167() AG898(O) Lys168(Z) AG898(O) Lys378(Z) AG898(O) Ser149() Std(O2) Ser149(O) Std(O1) Asn150 () Std(O1) Ala167() Std(O3) Arg162() Std(O4) Ala167() Std(O3) Std(3) Asn91(O) ADP Asn120() ADP(O) Asn150() ADP(O) Ala167() ADP(O) Gly166() ADP(O) Asn163() ADP(O) Arg162() ADP(O) ADP() Ser148(O)

65 Figure 95. The docked configuration of AG698, AG2549, AG2099, AG898 at the ATP site of Human Topoisomerase IIα (PDB id 1ZXM). (A) The detailed binding interactions between AG698 and residues in ATP binding site of htopo IIα. The dashed lines in yellow represent the hydrogen bonds between the amino acids Asn91, Asn95, Arg98, Arg162, Ala167, Lys168, Glu 87 hydrogen bonded to Water molecule, Gln376, Lys378, and AG698. (B) The detailed binding interactions between AG2549 and residues in ATP binding site of htopo IIα. The dashed lines in yellow represent the hydrogen bonds between the amino acids Asp94, Ser149, Asn150, Arg162, Gly164, Tyr165, Lys168 and AG2549. (C) The detailed binding interactions between AG2099 and residues in ATP binding site of htopo IIα. The dashed lines in yellow represent the hydrogen bonds between the amino acids Asp152, Ser149, Asn91, Arg98 and Glu155. (D) The detailed binding interactions between AG898 and residues in ATP binding site of htopo IIα. The dashed lines in yellow represent the hydrogen bonds between the amino acids Ala167, Ser149, Arg162, Lys168 and Lys378. The pictures were prepared using the PyMol programs ( 132