Supporting Information for

Diterpenoids from Croton laui and Their Cytotoxic and

Antimicrobial Activities

Cui-Ping Liu, Jin-Biao Xu, Jin-Xin Zhao, Cheng-Hui Xu, Lei Dong, Jian Ding,
and Jian-Min Yue*

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s
Republic of China

S1. Bioassay.
S1.1. Cytotoxicity Assay.
S1.2. Antimicrobial Activity Assay.
Table S1. X-ray crystallographic data for crotonolide H (11)
Figure S1. 1H NMR spectrum of crotonolide A (1) in CDCl3
Figure S2. 13C NMR spectrum of crotonolide A (1) in CDCl3
Figure S3. HSQC spectrum of crotonolide A (1) in CDCl3
Figure S4. 1H1H COSY spectrum of crotonolide A (1) in CDCl3
Figure S5. HMBC spectrum of crotonolide A (1) in CDCl3
Figure S6. ROESY spectrum of crotonolide A (1) in CDCl3
Figure S7. ESI(+)MS spectrum of crotonolide A (1)
Figure S8. HRESI(+)MS spectrum of crotonolide A (1)
Figure S9. IR spectrum of crotonolide A (1)

Figure S10. 1H NMR spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3
Figure S11. 13C NMR spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3
Figure S12. HSQC spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3
Figure S13. HMBC spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3
Figure S14. ROESY spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3

  1
Figure S15. ESI(+)MS spectrum of crotonolide B (2) & isocrotonolide B (3)
Figure S16. HRESI(+)MS spectrum of crotonolide B (2) & isocrotonolide B (3)
Figure S17. IR spectrum of crotonolide B (2) & isocrotonolide B (3)

Figure S18. 1H NMR spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3
Figure S19. 13C NMR spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3
Figure S20. HSQC spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3
Figure S21. HMBC spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3
Figure S22. ROESY spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3
Figure S23. ESI(+)MS spectrum of crotonolide C (4) & isocrotonolide C (5)
Figure S24. HRESI(+)MS spectrum of crotonolide C (4) & isocrotonolide C (5)
Figure S25. IR spectrum of crotonolide C (4) & isocrotonolide C (5)

Figure S26. 1H NMR spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3
Figure S27. 13C NMR spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3
Figure S28. HSQC spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3
Figure S29. HMBC spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3
Figure S30. ROESY spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3
Figure S31. ESI(+)MS spectrum of crotonolide D (6) & isocrotonolide D (7)
Figure S32. HRESI(+)MS spectrum of crotonolide D (6) & isocrotonolide D (7)
Figure S33. IR spectrum of crotonolide D (6) & isocrotonolide D (7)

Figure S34. 1H NMR spectrum of crotonolide E (8) in CDCl3

Figure S35. 13C NMR spectrum of crotonolide E (8) in CDCl3
Figure S36. HSQC spectrum of crotonolide E (8) in CDCl3
Figure S37. HMBC spectrum of crotonolide E (8) in CDCl3
Figure S38. ROESY spectrum of crotonolide E (8) in CDCl3
Figure S39. ESI(+)MS spectrum of crotonolide E (8)
Figure S40. HRESI(+)MS spectrum of crotonolide E (8)
Figure S41. IR spectrum of crotonolide E (8)
  2
Figure S42. 1H NMR spectrum of crotonolide F (9) in CDCl3
Figure S43. 13C NMR spectrum of crotonolide F (9) in CDCl3
Figure S44. HSQC spectrum of crotonolide F (9) in CDCl3
Figure S45. HMBC spectrum of crotonolide F (9) in CDCl3
Figure S46. ROESY spectrum of crotonolide F (9) in CDCl3
Figure S47. ESI(+)MS spectrum of crotonolide F (9)
Figure S48. HRESI(+)MS spectrum of crotonolide F (9)
Figure S49. IR spectrum of crotonolide F (9)

Figure S50. 1H NMR spectrum of crotonolide G (10) in CDCl3

Figure S51. 13C NMR spectrum of crotonolide G (10) in CDCl3
Figure S52. HSQC spectrum of crotonolide G (10) in CDCl3
Figure S53. HMBC spectrum of crotonolide G (10) in CDCl3
Figure S54. ROESY spectrum of crotonolide G (10) in CDCl3
Figure S55. ESI(+)MS spectrum of crotonolide G (10)
Figure S56. HRESI(+)MS spectrum of crotonolide G (10)
Figure S57. IR spectrum of crotonolide G (10)

Figure S58. 1H NMR spectrum of crotonolide H (11) in CDCl3

Figure S59. 13C NMR spectrum of crotonolide H (11) in CDCl3
Figure S60. HSQC spectrum of crotonolide H (11) in CDCl3
Figure S61. HMBC spectrum of crotonolide H (11) in CDCl3
Figure S62. ROESY spectrum of crotonolide H (11) in CDCl3
Figure S63. EIMS spectrum of crotonolide H (11)
Figure S64. HRESI(−)MS spectrum of crotonolide H (11)
Figure S65. IR spectrum of crotonolide H (11)

Figure S66. 1H NMR spectrum of 12-deoxycrotonolide H (12) in CDCl3

Figure S67. 13C NMR spectrum of 12-deoxycrotonolide H (12) in CDCl3
  3
Figure S68. HSQC spectrum of 12-deoxycrotonolide H (12) in CDCl3
Figure S69. HMBC spectrum of 12-deoxycrotonolide H (12) in CDCl3
Figure S70. EIMS spectrum of 12-deoxycrotonolide H (12)
Figure S71. HREIMS spectrum of 12-deoxycrotonolide H (12)
Figure S72. IR spectrum of 12-deoxycrotonolide H (12)

Figure S73. 1H NMR spectrum of crotonolide I (13) in CDCl3

Figure S74. 13C NMR spectrum of crotonolide I (13) in CDCl3
Figure S75. HSQC spectrum of crotonolide I (13) in CDCl3
Figure S76. HMBC spectrum of crotonolide I (13) in CDCl3
Figure S77. ROESY spectrum of crotonolide I (13) in CDCl3
Figure S78. ESI(+)MS spectrum of crotonolide I (13)
Figure S79. HRESI(+)MS spectrum of crotonolide I (13)
Figure S80. IR spectrum of crotonolide I (13)

Figure S81. 1H NMR spectrum of crotonolide J (14) in CDCl3

Figure S81. 13C NMR spectrum of crotonolide J (14) in CDCl3
Figure S83. HSQC spectrum of crotonolide J (14) in CDCl3
Figure S84. HMBC spectrum of crotonolide J (14) in CDCl3
Figure S85. ROESY spectrum of crotonolide J (14) in CDCl3
Figure S86. EIMS spectrum of crotonolide J (14)
Figure S87. HREIMS spectrum of crotonolide J (14)
Figure S88. IR spectrum of crotonolide J (14)

  4
S1. Bioassay.
S1.1. Cytotoxicity Assay.
The cytotoxicities of compounds 114 against HL-60 (human premyelocytic
leukemia) and P-388 (murine leukemia) cells were tested by using the MTT
method,1,2 and with adriamycin as the positive control. Briefly, the cells in 100 L of
culture medium were plated in each well of 96-well plates (Falcon, CA). Cells were
then treated in triplicate with graded concentrations of compounds at 37 °C for 72 h.
A 20 L aliquot of MTT solution (5 mg/mL) was added directly to the appropriate
wells. The cultures were incubated for 4 h, and then 100 L of “triplex solution”
(10% SDS/5% i-BuOH/12 mM HCl) was added. The plates were incubated at 37 °C
overnight and then measured by using a plate reader at 570 nm (VERSA Max,
Molecular Devices). In these tests, all the experiments were performed in triplicate,
and the results were expressed in IC50 as calculated by the Logit method.
S1.2. Antimicrobial Activity Assay.
The in vitro antibacterial activities against Staphylococcus aureus ATCC 25923,
Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus
subtilis ATCC 6633, Escherichia coli ATCC 25922, Shigella flexneri ATCC 12022
and Pseudomonas aeruginosa ATCC 14502 were conducted by using microdilute
method,3 and with ofloxacin as the positive control. The microbial cells were
suspended in Mueller Hinton broth to form a final density of 5×105106 cfu/mL and
incubated at 37 °C for 18 h under aerobic conditions with the respective compounds,
which have been dissolved in DMSO. The blank controls of microbial culture were
incubated with limited DMSO under the same condition. DMSO was determined not
to be toxic at a limited amount under the experimental conditions.
The in vitro antifungal activities against Candida albicans ATCC 1600,
Saccharomyces sake ATCC 26421, Microsporum gypseum ATCC 14683 and
Trichophyton rubrum ATCC 10218) were completed by using agar dilution method,4
and with amphotericin B as the positive control. The fungi were incubated in
Sabouraud dextrose broth at 37 °C for 48 h with the respective compounds and the
positive control dissolved in DMSO. The blank controls of fungi cultures were
  5
incubated with limited DMSO under the same condition.
Table 1. Cytotoxicity of compounds 114 against HL-60 (human premyelocytic
leukemia) and P-388 (murine leukemia) cells.
IC50(M)a
sample HL-60 P-388
1 9.42 7.45
2/3 10 10
4/5 10 10
6/7 10 10
8 10 10
9 10 10
10 10 10
11 10 10
12 10 10
13 10 10
14 10 10
Adriamycinb 0.473 0.036
a
The IC50 10 M was defined to be inactive; adriamycin was used as the positive
b

control.
Table 2. Antimicrobial activities of 114.
MIC(M)a
No. Sab Seb Mlb Bsb Ecb Sfb Pab Cab Ssb Mgb Trb
1 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150
2/3 >150  >150 >150  >150 >150 >150 >150 >150 >150  >150  >150
4/5 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150
6/7 >150  >150 >150  >150 >150 >150 >150 >150 >150  >150  >150
8 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150 >150
9 >150  >150 >150  >150 >150 >150 >150 >150 >150  >150  >150
10 43.4 43.4  43.4  43.4 >150 >150 >150 >150 >150 >150 >150
11 >150 >150 >150 >150 >150 >150 >150 >150 >150  >150  >150
12 >150  >150 >150  >150 >150 >150 >150 >150 >150 >150 >150
13 >150 >150 >150 >150 >150 >150 >150 >150 >150  >150  >150
14 >150  >150 >150  >150 >150 >150 >150 >150 >150 >150 >150
Ac 0.55 0.55 4.32 0.03 0.03 0.14 4.32
Bc 0.42 0.21 3.38 3.38
a
The MIC > 150M was defined to be inactive. bSa, Se, Ml, Bs, Ec, Sf, Pa, Ca, Ss, Mg,
and Tr represented Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis
ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus subtilis ATCC 6633,
Escherichia coli ATCC 25922, Shigella flexneri ATCC 12022, Pseudomonas

  6
aeruginosa ATCC 14502, Candida albicans ATCC 1600, Saccharomyces sake ATCC
26421, Microsporum gypseum ATCC 14683 and Trichophyton rubrum ATCC 10218,
respectively. cA and B representing ofloxacin and amphotericin B, respectively, were
used as the positive controls.

References
(1) Dong, S. H.; Liu, H. B.; Xu, C. H.; Ding, J.; Yue, J. M. J. Nat. Prod. 2011, 74,
25762581.
(2) Dong, S. H.; Zhang, C. R.; Xu, C. H.; Ding, J.; Yue, J. M. J. Nat. Prod. 2011, 74,
12551261.
(3) Yin, S.; Fan, C. Q.; Wang, Y.; Dong, L.; Yue, J. M. Bioorg. Med. Chem. 2004, 12,
43874392.
(4) Yang, S. P.; Dong, L.; Wang, Y.; Wu. Y.; Yue, J. M. Bioorg. Med. Chem. 2003,
11, 45774584.

  7
Table S1. X-ray crystallographic data for crotonolide H (11).a
Identification code cu_dm13583_0m
Empirical formula C20 H33 O4.50
Formula weight 345.46
Temperature 140(2) K
Wavelength 1.54178 Å
Crystal system Orthorhombic
Space group P 21 21 21
Unit cell dimensions a = 10.4173(2) Å a= 90°.
b = 12.2196(2) Å b= 90°.
c = 29.6743(6) Å g = 90°.

Volume 3777.40(12) Å3

Z 8

Density (calculated) 1.215 Mg/m3

Absorption coefficient 0.675 mm-1

F(000) 1512

Crystal size 0.320 x 0.260 x 0.100 mm3

Theta range for data collection 2.978 to 69.252°.

Index ranges -8<=h<=12, -14<=k<=14, -35<=l<=31
Reflections collected 23569
Independent reflections 6755 [R(int) = 0.0415]
Completeness to theta = 67.679° 99.8 %
Absorption correction Semi-empirical from equivalents
Max. and min. transmission 0.7532 and 0.5669

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 6755 / 0 / 464

Goodness-of-fit on F2 1.040

Final R indices [I>2sigma(I)] R1 = 0.0395, wR2 = 0.1065

R indices (all data) R1 = 0.0421, wR2 = 0.1087
Absolute structure parameter -0.06(7)
Extinction coefficient n/a

Largest diff. peak and hole 0.421 and -0.264 e.Å-3

a
Colorless crystals of 11 obtained in MeCN/H2O (2:3).

  8
Figure S1. 1H NMR spectrum of crotonolide A (1) in CDCl3

  9
Figure S2. 13C NMR spectrum of crotonolide A (1) in CDCl3

  10
Figure S3. HSQC spectrum of crotonolide A (1) in CDCl3

  11
Figure S4. 1H1H COSY spectrum of crotonolide A (1) in CDCl3

  12
Figure S5. HMBC spectrum of crotonolide A (1) in CDCl3

  13
Figure S6. ROESY spectrum of crotonolide A (1) in CDCl3

  14
Figure S7. ESI(+)MS spectrum of crotonolide A (1)

  15
Figure S8. HRESI(+)MS spectrum of crotonolide A (1)

  16
Figure S9. IR spectrum of crotonolide A (1)

  17
Figure S10. 1H NMR spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3

  18
Figure S11. 13C NMR spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3

  19
Figure S12. HSQC spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3 

  20
Figure S13. HMBC spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3 

  21
Figure S14. ROESY spectrum of crotonolide B (2) & isocrotonolide B (3) in CDCl3 

  22
Figure S15. ESI(+)MS spectrum of crotonolide B (2) & isocrotonolide B (3) 

  23
Figure S16. HRESI(+)MS spectrum of crotonolide B (2) & isocrotonolide B (3)

  24
Figure S17. IR spectrum of crotonolide B (2) & isocrotonolide B (3)

  25
Figure S18. 1H NMR spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3

  26
Figure S19. 13C NMR spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3

  27
Figure S20. HSQC spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3

  28
Figure S21. HMBC spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3
O
16

13
14
O 12
20
11
17
9
10 8
O 5
6 7
4
19
O
O 18 OH
O
O

  29
Figure S22. ROESY spectrum of crotonolide C (4) & isocrotonolide C (5) in CDCl3

  30
Figure S23. ESI(+)MS spectrum of crotonolide C (4) & isocrotonolide C (5)

  31
Figure S24. HRESI(+)MS spectrum of crotonolide C (4) & isocrotonolide C (5)

  32
Figure S25. IR spectrum of crotonolide C (4) & isocrotonolide C (5)

  33
Figure S26. 1H NMR spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3

  34
Figure S27. 13C NMR spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3

  35
Figure S28. HSQC spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3

  36
Figure S29. HMBC spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3

  37
Figure S30. ROESY spectrum of crotonolide D (6) & isocrotonolide D (7) in CDCl3

  38
Figure S31. ESI(+)MS spectrum of crotonolide D (6) & isocrotonolide D (7)

  39
Figure S32. HRESI(+)MS spectrum of crotonolide D (6) & isocrotonolide D (7)

  40
Figure S33. IR spectrum of crotonolide D (6) & isocrotonolide D (7)

  41
Figure S34. 1H NMR spectrum of crotonolide E (8) in CDCl3

  42
Figure S35. 13C NMR spectrum of crotonolide E (8) in CDCl3

  43
Figure S36. HSQC spectrum of crotonolide E (8) in CDCl3

  44
Figure S37. HMBC spectrum of crotonolide E (8) in CDCl3

  45
Figure S38. ROESY spectrum of crotonolide E (8) in CDCl3

  46
Figure S39. ESI(+)MS spectrum of crotonolide E (8)

  47
Figure S40. HRESI(+)MS spectrum of crotonolide E (8)

  48
Figure S41. IR spectrum of crotonolide E (8)

  49
Figure S42. 1H NMR spectrum of crotonolide F (9) in CDCl3

  50
Figure S43. 13C NMR spectrum of crotonolide F (9) in CDCl3

  51
Figure S44. HSQC spectrum of crotonolide F (9) in CDCl3

  52
Figure S45. HMBC spectrum of crotonolide F (9) in CDCl3

O
16 14
13
12
11
O
9 17
2 10 8
O
3 5 20
4 7
HO
6
19
18

  53
Figure S46. ROESY spectrum of crotonolide F (9) in CDCl3

  54
Figure S47. ESI(+)MS spectrum of crotonolide F (9)

  55
Figure S48. HRESI(+)MS spectrum of crotonolide F (9)

  56
Figure S49. IR spectrum of crotonolide F (9)

  57
Figure S50. 1H NMR spectrum of crotonolide G (10) in CDCl3

  58
Figure S51. 13C NMR spectrum of crotonolide G (10) in CDCl3

  59
Figure S52. HSQC spectrum of crotonolide G (10) in CDCl3

  60
Figure S53. HMBC spectrum of crotonolide G (10) in CDCl3

  61
Figure S54. ROESY spectrum of crotonolide G (10) in CDCl3

  62
Figure S55. ESI(+)MS spectrum of crotonolide G (10)

  63
Figure S56. HRESI(+)MS spectrum of crotonolide G (10)

  64
Figure S57. IR spectrum of crotonolide G (10)

  65
Figure S58. 1H NMR spectrum of crotonolide H (11) in CDCl3

  66
Figure S59. 13C NMR spectrum of crotonolide H (11) in CDCl3

  67
Figure S60. HSQC spectrum of crotonolide H (11) in CDCl3

  68
Figure S61. HMBC spectrum of crotonolide H (11) in CDCl3

  69
Figure S62. ROESY spectrum of crotonolide H (11) in CDCl3

  70
Figure S63. EIMS spectrum of crotonolide H (11)

  71
Figure S64. HRESI(−)MS spectrum of crotonolide H (11)

  72
Figure S65. IR spectrum of crotonolide H (11)

  73
Figure S66. 1H NMR spectrum of 12-deoxycrotonolide H (12) in CDCl3

  74
Figure S67. 13C NMR spectrum of 12-deoxycrotonolide H (12) in CDCl3

  75
Figure S68. HSQC spectrum of 12-deoxycrotonolide H (12) in CDCl3

  76
Figure S69. HMBC spectrum of 12-deoxycrotonolide H (12) in CDCl3

  77
Figure S70. EIMS spectrum of 12-deoxycrotonolide H (12)

  78
Figure S71. HREIMS spectrum of 12-deoxycrotonolide H (12)

  79
Figure S72. IR spectrum of 12-deoxycrotonolide H (12)

  80
Figure S73. 1H NMR spectrum of crotonolide I (13) in CDCl3

  81
Figure S74. 13C NMR spectrum of crotonolide I (13) in CDCl3

  82
Figure S75. HSQC spectrum of crotonolide I (13) in CDCl3

  83
Figure S76. HMBC spectrum of crotonolide I (13) in CDCl3

  84
Figure S77. ROESY spectrum of crotonolide I (13) in CDCl3

16

20 13
15
17 14
12
9

5 7

18

Crotonolide I (13)

  85
Figure S78. ESI(+)MS spectrum of crotonolide I (13)

  86
Figure S79. HRESI(+)MS spectrum of crotonolide I (13)

  87
Figure S80. IR spectrum of crotonolide I (13)

  88
Figure S81. 1H NMR spectrum of crotonolide J (14) in CDCl3

  89
Figure S82. 13C NMR spectrum of crotonolide J (14) in CDCl3

  90
Figure S83. HSQC spectrum of crotonolide J (14) in CDCl3

  91
Figure S84. HMBC spectrum of crotonolide J (14) in CDCl3

  92
Figure S85. ROESY spectrum of crotonolide J (14) in CDCl3

14

12
11
20
9

3 5
19

18

Crotonolide J (14)

  93
Figure S86. EIMS spectrum of crotonolide J (14)

  94
Figure S87. HREIMS spectrum of crotonolide J (14)

  95
Figure S88. IR spectrum of crotonolide J (14)

  96