S1

ELECTRONIC SUPPORTING INFORMATION (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. Bharateb,d,* and Ram A. Vishwakarmaa,b,d,*

aNatural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,

Jammu-180001, India.

bAcademy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative

Medicine, Canal Road, Jammu-180001, India.

cCancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,

Jammu-180001, India

dMedicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-

180001, India.

ePreformulation Laboratory, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-

180001, India.

*E-mail: ram@iiim.ac.in (R.A.V.); sbharate@iiim.ac.in (S.B.B.); Fax: +91-191- 2586333; Tel: +91-

191- 2585006 (Extn. 345).

† IIIM Publication number IIIM/1616/2013.

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2015Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2016

S2

Contents

Fig. S1. 1H NMR Spectrum of 3 in CDCl3 ..........................................................................................S5

Fig. S2. 13C NMR Spectrum of 3 in CDCl3 .........................................................................................S5

Fig. S3. DEPT-135 Spectrum of 3 in CDCl3 .......................................................................................S6

Fig. S4. HRMS Spectrum of 3.............................................................................................................S6

Fig. S5. HPLC chromatogram of 3 ......................................................................................................S7

Fig. S6. 1H NMR Spectrum of 4a in CDCl3 ........................................................................................S7

Fig. S7. 13C NMR Spectrum of 4a in CDCl3 .......................................................................................S8

Fig. S8. DEPT-135 Spectrum of 4a in CDCl3 .....................................................................................S8

Fig. S9. HRMS Spectrum of 4a...........................................................................................................S9

Fig. S10. HPLC chromatogram of 4a ..................................................................................................S9

Fig. S11. 1H NMR Spectrum of 4b in CDCl3 ....................................................................................S10

Fig. S12. 13C NMR Spectrum of 4b in CDCl3 ...................................................................................S10

Fig. S13. DEPT-135 Spectrum of 4b in CDCl3 .................................................................................S11

Fig. S14. HRMS Spectrum of 4b.......................................................................................................S11

Fig. S15. HPLC chromatogram of 4b................................................................................................S12

Fig. S16. 1H NMR Spectrum of 4c in CDCl3 ....................................................................................S12

Fig. S17. 13C NMR Spectrum of 4c in CDCl3 ...................................................................................S13

Fig. S18. DEPT-135 Spectrum of 4c in CDCl3 .................................................................................S13

Fig. S19. HRMS Spectrum of 4c .......................................................................................................S14

Fig. S20. HPLC chromatogram of 4c ................................................................................................S14

Fig. S21. 1H NMR Spectrum of 4d in CDCl3 ....................................................................................S15

Fig. S22. 13C NMR Spectrum of 4d in CDCl3 ...................................................................................S15

Fig. S23. DEPT-135 Spectrum of 4d in CDCl3 .................................................................................S16

Fig. S24. HRMS Spectrum of 4d.......................................................................................................S16

Fig. S25. HPLC chromatogram of 4d................................................................................................S17

Fig. S26. 1H NMR Spectrum of 4e in CDCl3 ....................................................................................S17

Fig. S27. 13C NMR Spectrum of 4e in CDCl3 ...................................................................................S18

Fig. S28. DEPT-135 Spectrum of 4e in CDCl3 .................................................................................S18

Fig. S29. HRMS Spectrum of 4e .......................................................................................................S19

Fig. S30. HPLC chromatogram of 4e ................................................................................................S19

Fig. S31. 1H NMR Spectrum of 4f in CDCl3 .....................................................................................S20

Fig. S32. 13C NMR Spectrum of 4f in CDCl3....................................................................................S20

Fig. S33. DEPT-135 Spectrum of 4f in CDCl3 ..................................................................................S21

S3

Fig. S34. HRMS Spectrum of 4f........................................................................................................S21

Fig. S35. HPLC chromatogram of 4f.................................................................................................S22

Fig. S36. 1H NMR Spectrum of 4g in CD3OD ..................................................................................S22

Fig. S37. 13C NMR Spectrum of 4g in CDCl3 ...................................................................................S23

Fig. S38. DEPT-135 Spectrum of 4g in CDCl3 .................................................................................S23

Fig. S39. HRMS Spectrum of 4g.......................................................................................................S24

Fig. S40. HPLC chromatogram of 4g ................................................................................................S24

Fig. S41. 1H NMR Spectrum of 4h in CDCl3 ....................................................................................S25

Fig. S42. 13C NMR Spectrum of 4h in CDCl3 ...................................................................................S25

Fig. S43. DEPT-135 Spectrum of 4h in CDCl3 .................................................................................S26

Fig. S44. HRMS Spectrum of 4h.......................................................................................................S26

Fig. S45. HPLC chromatogram of 4h................................................................................................S27

Fig. S46. 1H NMR Spectrum of 4i in CDCl3 .....................................................................................S27

Fig. S47. 13C NMR Spectrum of 4i in CDCl3 ....................................................................................S28

Fig. S48. DEPT-135 Spectrum of 4i in CDCl3 ..................................................................................S28

Fig. S49. HRMS Spectrum of 4i........................................................................................................S29

Fig. S50. HPLC chromatogram of 4i .................................................................................................S29

Fig. S51. 1H NMR Spectrum of 4j in CDCl3 .....................................................................................S30

Fig. S52. 13C NMR Spectrum of 4j in CDCl3....................................................................................S30

Fig. S53. DEPT-135 Spectrum of 4j in CDCl3 ..................................................................................S31

Fig. S54. HRMS Spectrum of 4j........................................................................................................S31

Fig. S55. HPLC chromatogram of 4j.................................................................................................S32

Fig. S56. 1H NMR Spectrum of 4k in CDCl3 ....................................................................................S32

Fig. S57. 13C NMR Spectrum of 4k in CDCl3 ...................................................................................S33

Fig. S58. DEPT-135 Spectrum of 4k in CDCl3 .................................................................................S33

Fig. S59. HRMS Spectrum of 4k.......................................................................................................S34

Fig. S60. HPLC chromatogram of 4k................................................................................................S34

Fig. S61. 1H NMR Spectrum of 4l in CD3OD ...................................................................................S35

Fig. S62. 13C NMR Spectrum of 4l in CD3OD ..................................................................................S35

Fig. S63. DEPT-135 Spectrum of 4l in CD3OD ................................................................................S36

Fig. S64. HRMS Spectrum of 4l........................................................................................................S36

Fig. S65. HPLC chromatogram of 4l .................................................................................................S37

Fig. S66. 1H NMR Spectrum of 4m in CDCl3 ...................................................................................S37

Fig. S67. 13C NMR Spectrum of 4m in CDCl3..................................................................................S38

S4

Fig. S68. DEPT-135 Spectrum of 4m in CDCl3 ................................................................................S38

Fig. S69. HRMS Spectrum of 4m......................................................................................................S39

Fig. S70. HPLC chromatogram of 4m...............................................................................................S39

Fig. S71. 1H NMR Spectrum of 4n in CDCl3 ....................................................................................S40

Fig. S72. 13C NMR Spectrum of 4n in CDCl3 ...................................................................................S40

Fig. S73. DEPT-135 Spectrum of 4n in CDCl3 .................................................................................S41

Fig. S74. HRMS Spectrum of 4n.......................................................................................................S41

Fig. S75. HPLC chromatogram of 4n................................................................................................S42

Fig. S76. 1H NMR Spectrum of 4o in CDCl3 ....................................................................................S42

Fig. S77. 13C NMR Spectrum of 4o in CDCl3 ...................................................................................S43

Fig. S78. DEPT-135 Spectrum of 4o in CDCl3 .................................................................................S43

Fig. S79. HRMS Spectrum of 4o.......................................................................................................S44

Fig. S80. HPLC chromatogram of 4o ................................................................................................S44

Fig. S81. 1H NMR Spectrum of 4p in CDCl3 ....................................................................................S45

Fig. S82. 13C NMR Spectrum of 4p in CDCl3 ...................................................................................S45

Fig. S83. DEPT-135 Spectrum of 4p in CDCl3 .................................................................................S46

Fig. S84. HRMS Spectrum of 4p.......................................................................................................S46

Fig. S85. HPLC chromatogram of 4p................................................................................................S47

Fig. S86. 1H NMR Spectrum of 5a in CDCl3 ....................................................................................S47

Fig. S87. 13C NMR Spectrum of 5a in CDCl3 ...................................................................................S48

Fig. S88. DEPT-135 Spectrum of 5a in CDCl3 .................................................................................S48

Fig. S89. HRMS Spectrum of 5a.......................................................................................................S49

Fig. S90. HPLC chromatogram of 5a ................................................................................................S49

Fig. S91. 1H NMR Spectrum of 5b in CDCl3 ....................................................................................S50

Fig. S92. 13C NMR Spectrum of 5b in CDCl3 ...................................................................................S50

Fig. S93. DEPT-135 Spectrum of 5b in CDCl3 .................................................................................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

S5

Fig. S1. 1H NMR Spectrum of 3 in CDCl3

Fig. S2. 13C NMR Spectrum of 3 in CDCl3

NH

O

H3CO

H3COH3CO

O

Cl

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1413

S6

Fig. S3. DEPT-135 Spectrum of 3 in CDCl3

Fig. S4. HRMS Spectrum of 3

S7

Fig. S5. HPLC Chromatogram of 3

Fig. S6. 1H NMR Spectrum of 4a in CDCl3

NH

O

H3CO

H3COH3CO

N

O

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1' 2'

3'4'5'

6'

S8

Fig. S7. 13C NMR Spectrum of 4a in CDCl3

Fig. S8. DEPT-135 Spectrum of 4a in CDCl3

S9

Fig. S9. HRMS Spectrum of 4a

Fig. S10. HPLC Chromatogram of 4a

S10

Fig. S11. 1H NMR Spectrum of 4b in CDCl3

Fig. S12. 13C NMR Spectrum of 4b in CDCl3

NH

O

H3CO

H3COH3CO

N

O

1'2'

3'4'

5'

12

3

44a

5 6

77a

89

1011

12

12a12b

13 14

S11

Fig. S13. DEPT-135 Spectrum of 4b in CDCl3

Fig. S14. HRMS Spectrum of 4b

S12

Fig. S15. HPLC Chromatogram of 4b

Fig. S16. 1H NMR Spectrum of 4c in CDCl3

NH

O

H3CO

H3COH3CO

N

O

O

1' 2'

3'

4'5'

6'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S13

Fig. S17. 13C NMR Spectrum of 4c in CDCl3

Fig. S18. DEPT-135 Spectrum of 4c in CDCl3

S14

Fig. S19. HRMS Spectrum of 4c

Fig. S20. HPLC Chromatogram of 4c

S15

Fig. S21. 1H NMR Spectrum of 4d in CDCl3

Fig. S22. 13C NMR Spectrum of 4d in CDCl3

NH

O

H3CO

H3COH3CO

N

O

NH

1' 2'

3'4'5'

6'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S16

Fig. S23. DEPT-135 Spectrum of 4d in CDCl3

Fig. S24. HRMS Spectrum of 4d

S17

Fig. S25. HPLC Chromatogram of 4d

Fig. S26. 1H NMR Spectrum of 4e in CDCl3

NH

O

H3CO

H3COH3CO

N

O

N

1' 2'

3'4'5'

6'

7'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S18

Fig. S27. 13C NMR Spectrum of 4e in CDCl3

Fig. S28. DEPT-135 Spectrum of 4e in CDCl3

S19

Fig. S29. HRMS Spectrum of 4e

Fig. S30. HPLC Chromatogram of 4e

S20

Fig. S31. 1H NMR Spectrum of 4f in CDCl3

Fig. S32. 13C NMR Spectrum of 4f in CDCl3

NH

O

H3CO

H3COH3CO

N

O

NH2

1' 2'

3'4'5'

6'

12

3

4 4a 5 6

77a

8

9

1011

12

12a

12b

1314

S21

Fig. S33. DEPT-135 Spectrum of 4f in CDCl3

Fig. S34. HRMS Spectrum of 4f

S22

Fig. S35. HPLC Chromatogram of 4f

Fig. S36. 1H NMR Spectrum of 4g in CD3OD

NH

O

O

H3CO

H3COH3CO

N

N

O O

1' 2'

3'4'5'

6'

7'

8' 9'

10'11'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S23

Fig. S37. 13C NMR Spectrum of 4g in CDCl3

Fig. S38. DEPT-135 Spectrum of 4g in CDCl3

S24

Fig. S39. HRMS Spectrum of 4g

Fig. S40. HPLC Chromatogram of 4g

S25

Fig. S41. 1H NMR Spectrum of 4h in CDCl3

Fig. S42. 13C NMR Spectrum of 4h in CDCl3

NH

O

H3CO

H3COH3CO

N

O

1' 2'

3'4'5'

7' 8'

9'

10'

12'

11'

6'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S26

Fig. S43. DEPT-135 Spectrum of 4h in CDCl3

Fig. S44. HRMS Spectrum of 4h

S27

Fig. S45. HPLC Chromatogram of 4h

Fig. S46. 1H NMR Spectrum of 4i in CDCl3

NH

O

O

H3CO

H3COH3CO

N1'

2'

3'

OH4'

5'6'

12

3

44a

5 6

77a

89

1011

12

12a

12b

13 14

S28

Fig. S47. 13C NMR Spectrum of 4i in CDCl3

Fig. S48. DEPT-135 Spectrum of 4i in CDCl3

S29

Fig. S49. HRMS Spectrum of 4i

Fig. S50. HPLC Chromatogram of 4i

S30

Fig. S51. 1H NMR Spectrum of 4j in CDCl3

Fig. S52. 13C NMR Spectrum of 4j in CDCl3

NH

O

O

H3CO

H3COH3CO

N1'2'

3'4'

5'

12

3

4 4a5 6

77a

89

101112

12a

12b

1314

S31

Fig. S53. DEPT-135 Spectrum of 4j in CDCl3

Fig. S54. HRMS Spectrum of 4j

S32

Fig. S55. HPLC Chromatogram of 4j

Fig. S56. 1H NMR Spectrum of 4k in CDCl3

NH

O

H3CO

H3COH3CO

NH2

O

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S33

Fig. S57. 13C NMR Spectrum of 4k in CDCl3

Fig. S58. DEPT-135 Spectrum of 4k in CDCl3

S34

Fig. S59. HRMS Spectrum of 4k

Fig. S60. HPLC Chromatogram of 4k

S35

Fig. S61. 1H NMR Spectrum of 4l in CD3OD

Fig. S62. 13C NMR Spectrum of 4l in CD3OD

6'1'

2'

3'4'

5'

7'8'

9'10'

11'

12'

12

3

4 4a5 6

77a

8

910

11

12

12a

12b

13 14NH

O

N

H3COH3CO

H3CO

HN

O

S36

Fig. S63. DEPT-135 Spectrum of 4l in CD3OD

Fig. S64. HRMS Spectrum of 4l

S37

Fig. S65. HPLC Chromatogram of 4l

Fig. S66. 1H NMR Spectrum of 4m in CDCl3

NH

O

H3CO

H3COH3CO

NH

O

FCl

1'

2'3'4'

5'

6'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S38

Fig. S67. 13C NMR Spectrum of 4m in CDCl3

Fig. S68. DEPT-135 Spectrum of 4m in CDCl3

S39

Fig. S69. HRMS Spectrum of 4m

Fig. S70. HPLC Chromatogram of 4m

S40

Fig. S71. 1H NMR Spectrum of 4n in CDCl3

Fig. S72. 13C NMR Spectrum of 4n in CDCl3

NH

O

H3CO

H3COH3CO

N

O

N

OO

1'2'

3'4'5'

6'

7'8'

9'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S41

Fig. S73. DEPT-135 Spectrum of 4n in CDCl3

Fig. S74. HRMS Spectrum of 4n

S42

Fig. S75. HPLC Chromatogram of 4n

Fig. S76. 1H NMR Spectrum of 4o in CDCl3

NH

O

H3CO

H3COH3CO

NH

O

ClCF3

1'

2'3'4'

5'

6'

7'

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S43

Fig. S77. 13C NMR Spectrum of 4o in CDCl3

Fig. S78. DEPT-135 Spectrum of 4o in CDCl3

S44

Fig. S79. HRMS Spectrum of 4o

Fig. S80. HPLC Chromatogram of 4o

S45

Fig. S81. 1H NMR Spectrum of 4p in CDCl3

Fig. S82. 13C NMR Spectrum of 4p in CDCl3

NH

O

H3CO

H3COH3CO

NH

O

1'

2'3'

4'

5'

6'

7'

OO

12

3

44a

5 6

77a

8

9

1011

12

12a

12b

1314

S46

Fig. S83. DEPT-135 Spectrum of 4p in CDCl3

Fig. S84. HRMS Spectrum of 4p

S47

Fig. S85. HPLC Chromatogram of 4p

Fig. S86. 1H NMR Spectrum of 5a in CDCl3

N

O

O

H3CO

H3COH3CO

OH

O

1'2'

3' 4'

5'6'7'

8'

12

3

44a

5 6

77a

89

1011

12

12a

12b

13 14

S48

Fig. S87. 13C NMR Spectrum of 5a in CDCl3

Fig. S88. DEPT-135 Spectrum of 5a in CDCl3

S49

Fig. S89. HRMS Spectrum of 5a

Fig. S90. HPLC Chromatogram of 5a

S50

Fig. S91. 1H NMR Spectrum of 5b in CDCl3

Fig. S92. 13C NMR Spectrum of 5b in CDCl3

N

O

O

H3CO

H3COH3CO

O

O

O

1'2'

3' 4'

5'

6'7'

1''2''

3'' 4''5''

6''7''

12

3

44a

5 6

77a

89

1011

12

12a

12b

1314

S51

Fig. S93. DEPT-135 Spectrum of 5b in CDCl3

Fig. S94. HRMS Spectrum of 5b

S52

Fig. S95. HPLC Chromatogram of 5b

S53

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.

S54

Figure S97. Proposed mechanism for formation of amino-linked products 4a-p from colchicine (1)

NH

O

OOCH3

H3COH3CO

H3CO HNR1

R2

NH

O

N

OCH3

H3COH3CO

H3CO

R2

R1

NH

O

NOCH3

H3COH3CO

H3CO

R2

R1

NH

O

N

OCH3

H3COH3CO

H3CO

R2

R1

NHR1

R2

NH

O

N

OCH3

H3COH3CO

H3CO

R2

R1NR2

R1

-H2O

..

NH

O

NH3CO

H3CO

H3CO

R2

R1NR2

R1H2O

NH

O

NH3COH3CO

H3CO

R2

R1

NR2

R1

OH

H

NH

O

NH3COH3CO

H3CO

R2

R1

NR2

R1

OH

H

..

NH

O

NHH3COH3CO

H3CO

R2R1

NR2

R1O

H..

NH

O

H3COH3CO

H3CO

N

O

R1R2

NHR1

R2

1

4a-p

I II III

VVIVII IV

VIII

MeOH