<p>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</p><p>Li Zhou,a Xu Zhang2 andWen Zhou3*</p><p>1College of Science, Hunan Agricultural University, Furong, Changsha, Hunan Province, China, 410128</p><p>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]</p><p>Table of contents</p><p>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</p><p>-</p><p>1 1</p><p>OH O</p><p>OH O OH</p><p>2 .79</p><p>3.0 0 1 .63 0 .88 0 .62 0.9 8 1 .69</p><p>0.6 9 0.8 1 0 .85</p><p>12 10 8 6 4 2 0 PPM</p><p>Fig. S1 1H NMR Spectra for compound 3</p><p>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</p><p>- 1 1</p><p>OH O</p><p>O</p><p>OH O</p><p>3 .58 3.2 9 3.0 0</p><p>1 .7 0 1 .7 1</p><p>0.73 1.07 0.88</p><p>0.0 5 -0 .1 6</p><p>12 10 8 6 4 2 0 PPM</p><p>Fig. S3 1H NMR Spectra for compound 4</p><p>2 OCH3OCH3</p><p>OCH3OCH3O</p><p>11 .6 7</p><p>6.00</p><p>2 .91</p><p>1 .79 0 .86 0.8 8 0.96</p><p>12 10 8 6 4 2 0 PPM</p><p>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</p><p>1 1 1 1 1 1 1 1 1 1</p><p>OCH3 OCH3</p><p>O</p><p>OCH3 OCH3</p><p>200 150 100 50 0 PPM</p><p>Fig. S5 13C NMR Spectra for compound 5</p><p>3 4 8 200</p><p>7.266</p><p>3.32 6.854 6.837</p><p>170.485</p><p>0.82 6.328 O O 170.401 C C H H</p><p>3 3 6</p><p>Fig. Fig. S5 6.000 O O C C H H 3 3 O O Fig. Fig. S6 153.761 C C</p><p>150 151.611 O H H C</p><p>O 150.725 3 3 O O C</p><p>H 147.142 C C 3 H H 1 3 3 O H H NMR compoundSpectra for A</p><p> c O 13 C</p><p>O 130.850 C C NMR Spectra forC NMR compound Spectra H 3</p><p>122.899 O 120.963 A c</p><p>4 3.941 11.49 3.889 109.146 3.843 108.072 106.110 100</p><p>81.935 77.670 77.248 76.824</p><p>71.124 9 3.1 2.114 2 3.84 1.940 1.908</p><p>62.664 2.91 1.880 58.148 1.856 57.671 1.846</p><p>57.143 6.00 1.412 6</p><p>50 1.399 1.255 6</p><p>37.110</p><p>30.767 26.153 26.033 22.580 21.472 0 0 PPM PPM</p><p>5 10 14 8 0.86 </p><p>1.70 6.974 6.754 12</p><p>6 5.191 5.175</p><p>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</p><p>C C 3.873 C C</p><p>H H 3.864 Fig. S7 Fig. H H</p><p>3 3 3.831 3 3 4 O 8.68 3.677 0.00 2.80 Fig. Fig. S8 H O 1 O</p><p>1.879 8 A H NMR compoundSpectra for H NMR c</p><p>H 1.854 1.828 7.265</p><p>2 1.809</p><p>1 6.994</p><p>H NMR compoundSpectra for H NMR 1.746</p><p>104 1.0 6.945 0.51 0 3.69 </p><p>1.701 .94 1 6.826 0.00 </p><p>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</p><p>0 1.158 0.027 -0.051</p><p>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 </p><p>2.046 2.041 3.22 3 </p><p>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</p><p>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</p><p> c 133.380 8 13 1 6.856 NMR compoundSpectra for C H H NMR compoundSpectra for </p><p>6.829 O 121.517 119.220 2.72 6.361 H O 6.340 H 6.334 0.76 6.315 107.362</p><p>6 6.000 106.698 104.947 100</p><p>3.930 3.882</p><p>4 3.837 11.67</p><p>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</p><p>6.00 1.895 1.878 1.629</p><p>1.614 7 39.103 8 1.183 1.171 0.069 32.063 0 28.694 28.010 -2</p><p>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</p><p>1 6.876 2.35 H NMR compoundSpectra for H NMR 6.828</p><p>0.64 6.372 6.352 6.329 6 109.035 107.922 105.779 5.164 0.62 </p><p>5.143 100 5.123</p><p>4 3.935</p><p>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</p><p>2.56 2.098 57.549</p><p>2 2.044 57.104</p><p>295 2.9 1.662 3.00 </p><p>1.560 8 50 1.279</p><p>9 1.256 1.233</p><p>39.639</p><p>31.314</p><p>0 -0.002 29.693 29.330</p><p>21.554 PPM</p><p>1.229 0 0.206 PPM 8 12 200 10 O O</p><p>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</p><p>O 149.886 A</p><p>8 146.264 Fig. S14 Fig. S14 c</p><p>133.921 0.84 13</p><p>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</p><p>1.61 5.251 105.738 5.230</p><p>5.215 100 5.210</p><p>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</p><p>2 56.776 1.717 56.396 6.00 1.640 50 9 10</p><p>33.913</p><p>0 0.070 25.043 20.610 17.217</p><p>PPM 0.365</p><p>0 0.327 -0.704 PPM Fig. S15 1H NMR Spectra for compound 11</p><p>Fig. S16 13C NMR Spectra for compound 11</p><p>9 Fig. S17 1H NMR Spectra for compound 12</p><p>Fig. S18 13C NMR Spectra for compound 12</p><p>10 Fig. S19 1H NMR Spectra for compound 13</p><p>Fig. S20 13C NMR Spectra for compound 13</p><p>11 12 14 14 12 12</p><p>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 </p><p>O 7.261 7.228</p><p>8 6.792 O 6.787</p><p>8 6.184 7.262 O 1.30 6.173 H</p><p>0.73 0 7.241 6.158 7.209 0.89 6.777 1.61 O 6.775 0.62 </p><p>6.163 6</p><p>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</p><p>4 3.840 2.524 3.817 2.516 2 14a 14b 3.798 2.499 2.96 0.77 </p><p>3.791 2.87 2.476 3.191 3.00 </p><p>3.14 1.686 3.167 1.657 3.147 1.568</p><p>1.72 3.129 1.550 3.120 1.548 201 2.0 2.533 1.302 2.510 1.288 0</p><p>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</p><p>1 8.095 H H NMR compoundSpectra for O 8.092 0.50 8 7.485 7.479 0.63 </p><p>O 7.474 0.68 </p><p>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</p><p>3.966 4</p><p>591 5.9 3.920 14c</p><p>2.634</p><p>1.60 2.617 2.591 2.567 2</p><p>3.15 1.693</p><p>3.00 1.576 1.287 1.263 1.239</p><p>0 0.004 PPM Fig. S 25 MS (ESI) Spectra for compound 7</p><p>Fig. S 26 MS (ESI) Spectra for compound 8</p><p>14 Fig. S 27 MS (ESI) Spectra for compound 9</p><p>Fig. S 28 MS (ESI) Spectra for compound 13</p><p>15 16</p>