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: [email protected] (R.A.V.); [email protected] (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
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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