potent in vitro and in vivo anticancer activity and reduced p-glycoprotein induction liability

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1 Electronic Supplementary Material (ESI) for rganic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry ELECTRNIC SUPPRTING INFRMATIN (ESI) Design and synthesis of colchicine derivatives with potent in vitro and in vivo anticancer activity and reduced p-glycoprotein induction liability Baljinder Singh, a,b Ashok Kumar, b,c Prashant Joshi, b,d Santosh K. Guru, c Suresh Kumar, b,c Zahoor A. Wani, c Girish Mahajan, c Aashiq Hussain, c Asif Khurshid Qazi, c Ajay Kumar, c Sonali S. Bharate, e Bishan D. Gupta, a Parduman R. Sharma, c Abid Hamid, b,c Ajit K. Saxena, b,c Dilip M. Mondhe, b,c Shashi Bhushan, b,c Sandip B. Bharate b,d,* and Ram A. Vishwakarma a,b,d, * a Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu , India. b Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu , India. c Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu , India d Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu , India. e Preformulation Laboratory, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu , India. * ram@iiim.ac.in (R.A.V.); sbharate@iiim.ac.in (S.B.B.); Fax: ; Tel: (Extn. 345). IIIM Publication number IIIM/1616/20. S1

2 Contents Fig. S1. 1 H NMR Spectrum of 3 in CDCl 3...S5 Fig. S2. C NMR Spectrum of 3 in CDCl 3...S5 Fig. S3. DEPT-5 Spectrum of 3 in CDCl 3...S6 Fig. S4. HRMS Spectrum of 3...S6 Fig. S5. HPLC chromatogram of 3...S7 Fig. S6. 1 H NMR Spectrum of 4a in CDCl 3...S7 Fig. S7. C NMR Spectrum of 4a in CDCl 3...S8 Fig. S8. DEPT-5 Spectrum of 4a in CDCl 3...S8 Fig. S9. HRMS Spectrum of 4a...S9 Fig. S10. HPLC chromatogram of 4a...S9 Fig. S11. 1 H NMR Spectrum of 4b in CDCl 3...S10 Fig. S12. C NMR Spectrum of 4b in CDCl 3...S10 Fig. S. DEPT-5 Spectrum of 4b in CDCl 3...S11 Fig. S14. HRMS Spectrum of 4b...S11 Fig. S15. HPLC chromatogram of 4b...S12 Fig. S16. 1 H NMR Spectrum of 4c in CDCl 3...S12 Fig. S17. C NMR Spectrum of 4c in CDCl 3...S Fig. S18. DEPT-5 Spectrum of 4c in CDCl 3...S Fig. S19. HRMS Spectrum of 4c...S14 Fig. S20. HPLC chromatogram of 4c...S14 Fig. S21. 1 H NMR Spectrum of 4d in CDCl 3...S15 Fig. S22. C NMR Spectrum of 4d in CDCl 3...S15 Fig. S23. DEPT-5 Spectrum of 4d in CDCl 3...S16 Fig. S24. HRMS Spectrum of 4d...S16 Fig. S25. HPLC chromatogram of 4d...S17 Fig. S26. 1 H NMR Spectrum of 4e in CDCl 3...S17 Fig. S27. C NMR Spectrum of 4e in CDCl 3...S18 Fig. S28. DEPT-5 Spectrum of 4e in CDCl 3...S18 Fig. S29. HRMS Spectrum of 4e...S19 Fig. S30. HPLC chromatogram of 4e...S19 Fig. S31. 1 H NMR Spectrum of 4f in CDCl 3...S20 Fig. S32. C NMR Spectrum of 4f in CDCl 3...S20 Fig. S33. DEPT-5 Spectrum of 4f in CDCl 3...S21 S2

3 Fig. S34. HRMS Spectrum of 4f...S21 Fig. S35. HPLC chromatogram of 4f...S22 Fig. S36. 1 H NMR Spectrum of 4g in CD 3 D...S22 Fig. S37. C NMR Spectrum of 4g in CDCl 3...S23 Fig. S38. DEPT-5 Spectrum of 4g in CDCl 3...S23 Fig. S39. HRMS Spectrum of 4g...S24 Fig. S40. HPLC chromatogram of 4g...S24 Fig. S41. 1 H NMR Spectrum of 4h in CDCl 3...S25 Fig. S42. C NMR Spectrum of 4h in CDCl 3...S25 Fig. S43. DEPT-5 Spectrum of 4h in CDCl 3...S26 Fig. S44. HRMS Spectrum of 4h...S26 Fig. S45. HPLC chromatogram of 4h...S27 Fig. S46. 1 H NMR Spectrum of 4i in CDCl 3...S27 Fig. S47. C NMR Spectrum of 4i in CDCl 3...S28 Fig. S48. DEPT-5 Spectrum of 4i in CDCl 3...S28 Fig. S49. HRMS Spectrum of 4i...S29 Fig. S50. HPLC chromatogram of 4i...S29 Fig. S51. 1 H NMR Spectrum of 4j in CDCl 3...S30 Fig. S52. C NMR Spectrum of 4j in CDCl 3...S30 Fig. S53. DEPT-5 Spectrum of 4j in CDCl 3...S31 Fig. S54. HRMS Spectrum of 4j...S31 Fig. S55. HPLC chromatogram of 4j...S32 Fig. S56. 1 H NMR Spectrum of 4k in CDCl 3...S32 Fig. S57. C NMR Spectrum of 4k in CDCl 3...S33 Fig. S58. DEPT-5 Spectrum of 4k in CDCl 3...S33 Fig. S59. HRMS Spectrum of 4k...S34 Fig. S60. HPLC chromatogram of 4k...S34 Fig. S61. 1 H NMR Spectrum of 4l in CD 3 D...S35 Fig. S62. C NMR Spectrum of 4l in CD 3 D...S35 Fig. S63. DEPT-5 Spectrum of 4l in CD 3 D...S36 Fig. S64. HRMS Spectrum of 4l...S36 Fig. S65. HPLC chromatogram of 4l...S37 Fig. S66. 1 H NMR Spectrum of 4m in CDCl 3...S37 Fig. S67. C NMR Spectrum of 4m in CDCl 3...S38 S3

4 Fig. S68. DEPT-5 Spectrum of 4m in CDCl 3...S38 Fig. S69. HRMS Spectrum of 4m...S39 Fig. S70. HPLC chromatogram of 4m...S39 Fig. S71. 1 H NMR Spectrum of 4n in CDCl 3...S40 Fig. S72. C NMR Spectrum of 4n in CDCl 3...S40 Fig. S73. DEPT-5 Spectrum of 4n in CDCl 3...S41 Fig. S74. HRMS Spectrum of 4n...S41 Fig. S75. HPLC chromatogram of 4n...S42 Fig. S76. 1 H NMR Spectrum of 4o in CDCl 3...S42 Fig. S77. C NMR Spectrum of 4o in CDCl 3...S43 Fig. S78. DEPT-5 Spectrum of 4o in CDCl 3...S43 Fig. S79. HRMS Spectrum of 4o...S44 Fig. S80. HPLC chromatogram of 4o...S44 Fig. S81. 1 H NMR Spectrum of 4p in CDCl 3...S45 Fig. S82. C NMR Spectrum of 4p in CDCl 3...S45 Fig. S83. DEPT-5 Spectrum of 4p in CDCl 3...S46 Fig. S84. HRMS Spectrum of 4p...S46 Fig. S85. HPLC chromatogram of 4p...S47 Fig. S86. 1 H NMR Spectrum of 5a in CDCl 3...S47 Fig. S87. C NMR Spectrum of 5a in CDCl 3...S48 Fig. S88. DEPT-5 Spectrum of 5a in CDCl 3...S48 Fig. S89. HRMS Spectrum of 5a...S49 Fig. S90. HPLC chromatogram of 5a...S49 Fig. S91. 1 H NMR Spectrum of 5b in CDCl 3...S50 Fig. S92. C NMR Spectrum of 5b in CDCl 3...S50 Fig. S93. DEPT-5 Spectrum of 5b in CDCl 3...S51 Fig. S94. HRMS Spectrum of 5b...S51 Fig. S95. HPLC chromatogram of 4a...S52 Fig. S96. Effect of colchicine (1) and its analog 4o on p27 expression in HCT-116 cells...s53 Fig. S97. Proposed mechanism for formation of amino-linked products 4a-p from colchicine (1) S54 S4

5 Fig. S1. 1 H NMR Spectrum of 3 in CDCl a b 12a a 8 9 Cl 14 Fig. S2. C NMR Spectrum of 3 in CDCl 3 S5

6 Fig. S3. DEPT-5 Spectrum of 3 in CDCl 3 Fig. S4. HRMS Spectrum of 3 S6

7 Fig. S5. HPLC Chromatogram of 3 Fig. S6. 1 H NMR Spectrum of 4a in CDCl a b 12a ' N 6' 5' 7a 8 9 4' 2' 3' S7

8 Fig. S7. C NMR Spectrum of 4a in CDCl 3 Fig. S8. DEPT-5 Spectrum of 4a in CDCl 3 S8

9 Fig. S9. HRMS Spectrum of 4a Fig. S10. HPLC Chromatogram of 4a S9

10 Fig. S11. 1 H NMR Spectrum of 4b in CDCl b a 4 5 4a ' N 4' 5' 7 7a 8 9 2' 3' 14 Fig. S12. C NMR Spectrum of 4b in CDCl 3 S10

11 Fig. S. DEPT-5 Spectrum of 4b in CDCl 3 Fig. S14. HRMS Spectrum of 4b S11

12 Fig. S15. HPLC Chromatogram of 4b Fig. S16. 1 H NMR Spectrum of 4c in CDCl a a 12 12b ' N 6' 5' 7 6 4' 7a 8 9 2' 3' 14 S12

13 Fig. S17. C NMR Spectrum of 4c in CDCl 3 Fig. S18. DEPT-5 Spectrum of 4c in CDCl 3 S

14 Fig. S19. HRMS Spectrum of 4c Fig. S20. HPLC Chromatogram of 4c S14

15 Fig. S21. 1 H NMR Spectrum of 4d in CDCl a b 2 7a 1 12a ' N 2' 6' 4' 3' 5' N H Fig. S22. C NMR Spectrum of 4d in CDCl 3 S15

16 Fig. S23. DEPT-5 Spectrum of 4d in CDCl 3 Fig. S24. HRMS Spectrum of 4d S16

17 Fig. S25. HPLC Chromatogram of 4d Fig. S26. 1 H NMR Spectrum of 4e in CDCl a a 12 12b ' 6' 5' 7 6 N 7a 8 9 4' N 2' 7' 3' 14 S17

18 Fig. S27. C NMR Spectrum of 4e in CDCl 3 Fig. S28. DEPT-5 Spectrum of 4e in CDCl 3 S18

19 Fig. S29. HRMS Spectrum of 4e Fig. S30. HPLC Chromatogram of 4e S19

20 Fig. S31. 1 H NMR Spectrum of 4f in CDCl ' 6' 5' N 4 5 4a b 7 2 7a 1 12a ' 4' 3' 2 14 Fig. S32. C NMR Spectrum of 4f in CDCl 3 S20

21 Fig. S33. DEPT-5 Spectrum of 4f in CDCl 3 Fig. S34. HRMS Spectrum of 4f S21

22 Fig. S35. HPLC Chromatogram of 4f Fig. S36. 1 H NMR Spectrum of 4g in CD 3 D a a 12 12b a 8 9 N 2' 1' 6' 3' 5' N 4' 7' 9' 8' 10' 11' 14 S22

23 Fig. S37. C NMR Spectrum of 4g in CDCl 3 Fig. S38. DEPT-5 Spectrum of 4g in CDCl 3 S23

24 Fig. S39. HRMS Spectrum of 4g Fig. S40. HPLC Chromatogram of 4g S24

25 Fig. S41. 1 H NMR Spectrum of 4h in CDCl a a 12 12b ' 6' 5' 7 6 N 7a 8 9 4' 2' 3' 14 12' 11' 7' 8' 9' 10' Fig. S42. C NMR Spectrum of 4h in CDCl 3 S25

26 Fig. S43. DEPT-5 Spectrum of 4h in CDCl 3 Fig. S44. HRMS Spectrum of 4h S26

27 Fig. S45. HPLC Chromatogram of 4h Fig. S46. 1 H NMR Spectrum of 4i in CDCl a a 12 12b 11 5' 4' a 8 9 N 1' 2' 3' 14 6' H S27

28 Fig. S47. C NMR Spectrum of 4i in CDCl 3 Fig. S48. DEPT-5 Spectrum of 4i in CDCl 3 S28

29 Fig. S49. HRMS Spectrum of 4i Fig. S50. HPLC Chromatogram of 4i S29

30 Fig. S51. 1 H NMR Spectrum of 4j in CDCl a b a 7a ' N 2' 5' 4' 3' 14 Fig. S52. C NMR Spectrum of 4j in CDCl 3 S30

31 Fig. S53. DEPT-5 Spectrum of 4j in CDCl 3 Fig. S54. HRMS Spectrum of 4j S31

32 Fig. S55. HPLC Chromatogram of 4j Fig. S56. 1 H NMR Spectrum of 4k in CDCl a a 12 12b a S32

33 Fig. S57. C NMR Spectrum of 4k in CDCl 3 Fig. S58. DEPT-5 Spectrum of 4k in CDCl 3 S33

34 Fig. S59. HRMS Spectrum of 4k Fig. S60. HPLC Chromatogram of 4k S34

35 Fig. S61. 1 H NMR Spectrum of 4l in CD 3 D a 6 12b 1 12a ' HN 12' 11' 7 7a ' N 2' 5' 3' 4' 7' 8' 10' 9' 14 Fig. S62. C NMR Spectrum of 4l in CD 3 D S35

36 Fig. S63. DEPT-5 Spectrum of 4l in CD 3 D Fig. S64. HRMS Spectrum of 4l S36

37 Fig. S65. HPLC Chromatogram of 4l Fig. S66. 1 H NMR Spectrum of 4m in CDCl a 5 F 1 12a 5' 12 4' 12b 11 6' ' 2' 3' Cl 7a S37

38 Fig. S67. C NMR Spectrum of 4m in CDCl 3 Fig. S68. DEPT-5 Spectrum of 4m in CDCl 3 S38

39 Fig. S69. HRMS Spectrum of 4m Fig. S70. HPLC Chromatogram of 4m S39

40 Fig. S71. 1 H NMR Spectrum of 4n in CDCl a a 12 12b 11 6' 10 5' 7 6 N 7a 8 9 1' N 2' 3' 4' 8' 7' 9' 14 Fig. S72. C NMR Spectrum of 4n in CDCl 3 S40

41 Fig. S73. DEPT-5 Spectrum of 4n in CDCl 3 Fig. S74. HRMS Spectrum of 4n S41

42 Fig. S75. HPLC Chromatogram of 4n Fig. S76. 1 H NMR Spectrum of 4o in CDCl Cl 4 4a a 5' 12 4' 12b 11 6' 1' CF 3 7' ' 3' 7a S42

43 Fig. S77. C NMR Spectrum of 4o in CDCl 3 Fig. S78. DEPT-5 Spectrum of 4o in CDCl 3 S43

44 Fig. S79. HRMS Spectrum of 4o Fig. S80. HPLC Chromatogram of 4o S44

45 Fig. S81. 1 H NMR Spectrum of 4p in CDCl a a 5' 4' 12 7' 12b 11 6' ' 6 2' 3' 7a Fig. S82. C NMR Spectrum of 4p in CDCl 3 S45

46 Fig. S83. DEPT-5 Spectrum of 4p in CDCl 3 Fig. S84. HRMS Spectrum of 4p S46

47 Fig. S85. HPLC Chromatogram of 4p Fig. S86. 1 H NMR Spectrum of 5a in CDCl b a 4 5 4a a 8 9 H 14 3' 4' N 1' 5' 2' 6' 7' 8' S47

48 Fig. S87. C NMR Spectrum of 5a in CDCl 3 Fig. S88. DEPT-5 Spectrum of 5a in CDCl 3 S48

49 Fig. S89. HRMS Spectrum of 5a Fig. S90. HPLC Chromatogram of 5a S49

50 Fig. S91. 1 H NMR Spectrum of 5b in CDCl a ' 4' 7 12b N 1' 7a 2' 5' 1 12a ' 6' 9 3'' 4'' '' 1'' 2'' 6'' 7'' Fig. S92. C NMR Spectrum of 5b in CDCl 3 S50

51 Fig. S93. DEPT-5 Spectrum of 5b in CDCl 3 Fig. S94. HRMS Spectrum of 5b S51

52 Fig. S95. HPLC Chromatogram of 5b S52

53 Fig. S96. Effect of colchicine (1) and its analog 4o on p27 expression in HCT-116 cells Untreated Colchicine (1) 25 nm 50 nm 100 nm 4o 25 nm 50 nm 100 nm Effect of colchicine (1) and its analog 4o on p27 expression in HCT-116 cells. Cells were cultured on coverslips. After 24 h, the cells were treated with different concentrations of the compound. Immunocytochemical staining was conducted using p27 antibody and Alexa Flour-488-labeled secondary antibody. Staining was done by using DAPI. The data are representative of three separate sets of experiments. S53

54 Figure S97. Proposed mechanism for formation of amino-linked products 4a-p from colchicine (1) 1 CH 3.. R 2 N R 1 N H R R 1 2 H VII R HN 2 R 1 -H 2 VI H R H 2 3 C 3 C R N 2 H R N 3 C R 2 I 1 R 1 N CH II III 3 CH 3 R 1 CH 3 R 2 R H 1 3 C R 2 MeH N R R 2 1 N R.. 1 R 2 N H R N N R 1 R R 1 2 H R 2 R 2 N 1 H 2 CH V IV 3 R 1 R 2 R. 1 N. R R 1 H 2 VIII R 2 R 1 N R2 4a-p R 1 S54