Regioselective Semi-Synthesis of 6-Isomers of 5, 8-O-Dimethyl Ether of Shikonin Derivatives

Regioselective semi-synthesis of 6-isomers of 5, 8-O-dimethyl ether of shikonin derivatives via an 'intramolecular ring- closing/ ring-opening' strategy as potent anticancer agents

Li Zhou,a Xu Zhang2 andWen Zhou3*

1College of Science, Hunan Agricultural University, Furong, Changsha, Hunan Province, China, 410128

2College of Forestry and landscape Architecture, South China Agricultural University, 483, Wushan Rd, Guangzhou, Guangdong province, China, 510642. 3 School of Chinese MeteriaMedica, Guangzhou University of Chinese Medicine, E. 232, University town, Waihuan Rd, Panyu, Guangzhou, Guangdong province, China, 510006 *E-mail:[email protected]

Table of contents

1H NMR spectra for compounds 14 a~14c …………………………………………………………..12-13 Chiral HPLC of shikonin and Cycloshikonin…………………………………………………14 MS (ESI) spectra for compounds 7~9, 13………………………………………………………14-16 9 7 3 2 5 8 1 0 0 4 7 2 0 2 5 4 6 5 0 9 7 0 0 2 2 1 8 5 3 5 5 0 9 0 2 2 1 1 2 2 9 9 9 3 3 3 7 6 6 4 0 ...... 7 7 5 4 2 1 2 7 7 5 4 4 2 2 1 0 2

OH O OH

3.0 0 1 .63 0 .88 0 .62 0.9 8 1 .69

0.6 9 0.8 1 0 .85

12 10 8 6 4 2 0 PPM

Fig. S1 1H NMR Spectra for compound 3

1 Fig. S2 MS Spectra for compound 3 3 2 8 5 5 9 3 7 2 8 8 2 4 1 7 7 3 4 0 8 9 6 5 9 1 7 1 5 7 3 1 6 2 1 5 4 6 9 7 2 1 0 9 8 7 6 3 1 0 4 3 2 9 9 8 7 4 0 5 2 1 6 4 4 2 2 1 8 7 7 1 1 0 9 8 7 4 8 5 5 2 1 1 1 6 6 9 9 8 8 8 7 7 7 3 3 2 2 2 1 1 1 1 6 6 8 8 8 8 8 8 7 7 7 7 5 0 ...... 2 2 7 7 7 5 2 2 1 1 1 1 1 1 1 1 1 1 7 7 7 5 5 5 5 2 2 1 1 1 1 1 1 1 1 1 1 1 0

3 .58 3.2 9 3.0 0

1 .7 0 1 .7 1

0.73 1.07 0.88

0.0 5 -0 .1 6

12 10 8 6 4 2 0 PPM

Fig. S3 1H NMR Spectra for compound 4

2 OCH3OCH3

OCH3OCH3O

11 .6 7

1 .79 0 .86 0.8 8 0.96

12 10 8 6 4 2 0 PPM

Fig. S4 1H NMR Spectra for compound 5 2 2 4 0 2 6 2 7 3 0 1 1 2 7 0 7 4 8 4 8 4 1 5 7 4 2 2 8 6 4 2 7 0 5 3 1 8 6 3 1 4 7 2 3 4 3 8 0 6 8 6 8 2 0 4 4 9 3 0 5 5 4 4 2 6 4 9 4 7 1 3 2 ...... 2 0 9 5 3 2 9 7 6 5 6 4 4 8 4 8 7 0 6 6 1 7 6 6 4 3 2 1 5 5 4 0 0 0 8 7 7 7 7 7 6 5 5 5 3 3 2 2

1 1 1 1 1 1 1 1 1 1

OCH3 OCH3

200 150 100 50 0 PPM

Fig. S5 13C NMR Spectra for compound 5

3 4 8 200

3.32 6.854 6.837

170.485

0.82 6.328 O O 170.401 C C H H

Fig. Fig. S5 6.000 O O C C H H 3 3 O O Fig. Fig. S6 153.761 C C

150 151.611 O H H C

O 150.725 3 3 O O C

H 147.142 C C 3 H H 1 3 3 O H H NMR compoundSpectra for A

c O 13 C

O 130.850 C C NMR Spectra forC NMR compound Spectra H 3

122.899 O 120.963 A c

4 3.941 11.49 3.889 109.146 3.843 108.072 106.110 100

81.935 77.670 77.248 76.824

71.124 9 3.1 2.114 2 3.84 1.940 1.908

62.664 2.91 1.880 58.148 1.856 57.671 1.846

57.143 6.00 1.412 6

50 1.399 1.255 6

37.110

30.767 26.153 26.033 22.580 21.472 0 0 PPM PPM

5 10 14 8 0.86

1.70 6.974 6.754 12

6 5.191 5.175

O O 5.166 O O C C 0.84 5.149 C C H H H H 3 3 O O 3 3 10 O O

C C 3.873 C C

H H 3.864 Fig. S7 Fig. H H

3 3 3.831 3 3 4 O 8.68 3.677 0.00 2.80 Fig. Fig. S8 H O 1 O

1.879 8 A H NMR compoundSpectra for H NMR c

H 1.854 1.828 7.265

2 1.809

1 6.994

H NMR compoundSpectra for H NMR 1.746

104 1.0 6.945 0.51 0 3.69

1.701 .94 1 6.826 0.00

6.00 1.676 6.820 1.562 6.791 1.542 072 0.7 6.255 1.533 6.230 1.515 6 6.202 1.171

0 1.158 0.027 -0.051

3.959 3.937 929 9.2 4 3.899 3.02 3 3.855 -2 3.731 2.778 2.753 194 1.9

2.046 2.041 3.22 3

2 2.002 1.681 -4 6.00 6 1.628 1.590 15 1.567 1.521 1.314 7 1.310 1.257 0.002 0 -6 -2 -8 PPM PPM 6 14 200 12 O O O O C C C C H H 10 H H 3 3 O O 3 3

O O 152.387 150 C C Fig. S9 Fig. Fig. S10 Fig. 150.370 C C H H 149.203 H H 3 3 145.272 O 3 3 O H A

c 133.380 8 13 1 6.856 NMR compoundSpectra for C H H NMR compoundSpectra for

6.829 O 121.517 119.220 2.72 6.361 H O 6.340 H 6.334 0.76 6.315 107.362

6 6.000 106.698 104.947 100

3.930 3.882

4 3.837 11.67

76.309 75.885 75.462 69.535 67.978 2.105 2.042 61.696 2.77 1.979 56.774 2 3.20 1.953 56.094 1.935 109 1.0 55.841 50 1.00 1.914

6.00 1.895 1.878 1.629

1.614 7 39.103 8 1.183 1.171 0.069 32.063 0 28.694 28.010 -2

0 -0.135 PPM PPM 7 200 12 10 170.536 O O O O C C C C H H 3 3 H H 3 3 O O C C O O Fig. S11 Fig. H H C C 3 3 H H 3 3 O Fig. S12 Fig. S12 150 O O O 8 O H 13 7.262 C NMR Spectra forC NMR compound Spectra 130.948

1 6.876 2.35 H NMR compoundSpectra for H NMR 6.828

0.64 6.372 6.352 6.329 6 109.035 107.922 105.779 5.164 0.62

5.143 100 5.123

4 3.935

10.59 3.894 3.834 77.646 77.427 77.222 76.896 76.799 76.734 2.599 71.327 1.51 2.576 70.826 2.564 62.743 58.149

2.56 2.098 57.549

2 2.044 57.104

295 2.9 1.662 3.00

1.560 8 50 1.279

9 1.256 1.233

39.639

31.314

0 -0.002 29.693 29.330

21.554 PPM

1.229 0 0.206 PPM 8 12 200 10 O O

C C 169.506 H H O O 3 3 C C H H O O C C 3 3 H H O O 3 3 Fig. S13 Fig. C C 152.634 H H 150 O 150.760 H 3 3

O 149.886 A

8 146.264 Fig. S14 Fig. S14 c

133.921 0.84 13

1.69 7.017 129.995 C compoundNMRfor spectra 6.807 122.060 119.995 118.558 1 H NMR spectra compound H for NMR 6 108.100 107.294

1.61 5.251 105.738 5.230

5.215 100 5.210

3.938 4 11.61 3.921 3.884 76.860 3.747 76.748 76.651 76.525 76.325 2.544 75.902 2.14 2.522 70.214 2.501 61.843 57.256

2 56.776 1.717 56.396 6.00 1.640 50 9 10

33.913

0 0.070 25.043 20.610 17.217

PPM 0.365

0 0.327 -0.704 PPM Fig. S15 1H NMR Spectra for compound 11

9 Fig. S17 1H NMR Spectra for compound 12

10 Fig. S19 1H NMR Spectra for compound 13

11 12 14 14 12 12

Fig. S22 Fig. S21 Fig. O O 10 O O 10 O O C C H H O O C C 3 3 H H O 3 3 1 1 O 7.265 H H NMR compoundSpectra for H NMR compoundSpectra for

O 7.261 7.228

8 6.792 O 6.787

8 6.184 7.262 O 1.30 6.173 H

0.73 0 7.241 6.158 7.209 0.89 6.777 1.61 O 6.775 0.62

6.163 6

0.74 6.158 6.148 6.139 3.964 6.131 0.70 3.955 6 6.122 3.942 4.022 3.925 3.909 0.77 3.993 53.911803 3.898 2.30 2.50 3.891 3.975 4 3.952 3.884 3.928 3.881 3.910 3.869 3.867 3.887 2.578 3.872 035 10.3 2.568 3.864 4.01 2.542

4 3.840 2.524 3.817 2.516 2 14a 14b 3.798 2.499 2.96 0.77

3.791 2.87 2.476 3.191 3.00

3.14 1.686 3.167 1.657 3.147 1.568

1.72 3.129 1.550 3.120 1.548 201 2.0 2.533 1.302 2.510 1.288 0

3.00 2.483 1.273 2.47 2 2.458 1.266 2.435 1.249 2.236 0.874 2.212 0.849 2.207 0.065 -0.006 2.186 2.162 2.141 -2 2.117 1.679 1.537 -0.016 0 PPM PPM 13 12 Fig. S24. Fig. Shikoninand cycloshikonin analysized by chiral HPLC 10 Fig. S23 Fig. O O (A shikoninfor and Bfor cycloshikonin) O O C C H H 3 3 O

1 8.095 H H NMR compoundSpectra for O 8.092 0.50 8 7.485 7.479 0.63

O 7.474 0.68

7.294 7.275 6.795 2.26 6.777 6.776 6.773 0.73 6.293 6.277 6 6.268 6.252 0.60 5.186

3.966 4

591 5.9 3.920 14c

1.60 2.617 2.591 2.567 2

3.15 1.693

3.00 1.576 1.287 1.263 1.239

0 0.004 PPM Fig. S 25 MS (ESI) Spectra for compound 7

Fig. S 26 MS (ESI) Spectra for compound 8

14 Fig. S 27 MS (ESI) Spectra for compound 9

Fig. S 28 MS (ESI) Spectra for compound 13