Norcolocynthenins A and B, Two Cucurbitane 3-nor-Triterpenoids

from Citrullus colocynthis and Their Cytotoxicity

Yushuang Liua,b,d, Lu Zhanga, Jingjing Xuec, Kaibo Wangd, Huiming Huac and Tao

Yuana,b,

aThe Laboratory of Effective Substances of Jiangxi Genuine Medicinal Materials, College of Life Sciences, Jiangxi Normal University, Nanchang, 330022, ChinabThe Key Laboratory of Plant Resources and Chemistry of Arid Zone, State Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, ChinacKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, ChinadDepartment of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States

Corresponding author. Tel.: +86-791-8814-0829; fax: +86-791-8814-0829; e-mail: tyuan@jxnu.edu.cn

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Content

1. NMR and ECD calculations....................................................................................................

2. NMR, HRESIMS, and IR spectra of compounds 1 and 2...................................................

3. Flow chart of Extraction and Isolation.............................................................................

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1.NMR and ECD calculations 13C and 1H NMR shielding constants of compounds 1-2 were calculated with the

GIAO method at the MPW1PW91-SCRF/6-31G(d,p) level in MeOH with PCM

(Fig.3, Fig. S1. The shielding constants so obtained were converted into chemical

shifts by referencing to TMS at 0 ppm (δcalcd = σTMS – σcalcd), where the σTMS

was the shielding constant of TMS calculated at the same level. The parameters a and

b of the linear regression δcalcd = aδexpt + b; the correlation coefficient, R2; the

mean absolute error (MAE) defined as Σn |δcalcd – δexpt|/n; the corrected mean

absolute error (CMAE), defined as Σn |δcorr – δexpt|/n, where δcorr = (δcalcd – b)/a

and therefore corrects for systematic errors were presented.

The relative configuration of compounds 1-2 was determined by a combination of

1D NMR chemical shifts and 2D NOESY spectrum. However, the absolute

configuration of compounds 1-2 remains unclear. Quantum chemical TDDFT

calculations of their theoretical ECD spectra were then carried out to determine their

absolute configuration. Firstly, conformational analysis of compounds 1-2 was carried

out via xtb and creat programs1. In the relative energy window of 0-4 kcal/mol, the

result showed 22 and 16 lowest energy conformers for compounds 1-2 respectively.

The conformers were then reoptimized using DFT at the M062X/def2svp level in

vacuum in the Gaussian 09 program2. The M062X/def2svp harmonic vibrational

frequencies were further calculated to confirm their stability. Seven conformers for 1

and three ones for 2 whose relative Gibbs free energies and geometry in the range of

0-0.15 kcal/mol and 0-0.15 angstrom, respectively, were refined and considered for

next step. The energies, oscillator strengths, and rotational strengths of the first 30

electronic excitations were calculated using the TDDFT methodology at the Cam-

B3LYP/def2svp level in vacuum. The ECD spectra were simulated by the overlapping

Gaussian function ( 0.30 eV for 1 and 2). To get the conformationally averaged

ECD spectra, the simulated spectra of the lowest energy conformers were averaged

according to the Boltzmann distribution theory and their relative Gibbs free energy

(G) using Specdis software3.

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1. Pracht, P.; Bohle, F.; Grimme, S. Automated exploration of the low-energy chemical space with fast quantum

chemical methods. Physical Chemistry Chemical Physics. 2020, DOI: 10.1039/C9CP06869D

2. Gaussian 09, Revision A.1, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;

Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.;

Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.;

Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery,

Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.;

Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;

Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;

Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma,

K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.;

Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc., Wallingford CT, 2009.

3. Stephens, P. J.; Harada, N. ECD cotton effect approximated by the Gaussian curve and other methods. Chirality

2010, 22, 229–233.

Conformers

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Fig. S1. M062X/def2svp optimized lowest energy conformers for 1.

Fig. S2. M062X/def2svp optimized lowest energy conformers for 2.

Table 1. Experimental Circular Dichroism data of 1 and 2 (in MeOH)

Wavelength

Compound 1Repeat

Compound 2Repeat

Wavelength

Compound 1Repeat

Compound 2Repeat

400 -0.567244 -0.194434 299 23.7674 18.4989399 -0.54659 0.0323133 298 25.4047 19.3248398 -0.741081 0.00208027 297 26.7 20.2212397 -0.232955 0.15375 296 28.1209 21.0372396 -0.472615 0.260591 295 28.0791 21.7923395 -0.753779 0.419187 294 28.4659 22.4699394 -0.279626 0.23853 293 28.8612 23.0811393 -0.544925 0.180034 292 29.3801 23.549392 -0.901375 0.165739 291 29.9565 23.9816391 -0.500327 0.0594636 290 30.0479 24.3099390 -0.76863 0.024687 289 31.123 24.6936389 -1.18486 0.0487586 288 32.0744 25.1521388 -1.02518 -0.00650595 287 32.0171 25.5665387 -1.13377 0.043374 286 32.3162 25.7809386 -1.3562 0.122269 285 31.0852 26.0649385 -1.24479 0.139085 284 30.5949 26.3612384 -1.39478 0.136453 283 29.3637 26.5578383 -1.25071 0.0930378 282 27.6018 26.6327

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382 -1.50845 0.00011519 281 25.4113 26.543381 -1.59518 -0.0490849 280 23.2582 26.4966380 -2.13779 0.0605077 279 21.2585 26.4747379 -1.6722 0.143227 278 20.332 26.4955378 -1.58316 0.17961 277 18.8144 26.4362377 -2.01623 0.21938 276 17.0071 26.1483376 -1.84618 0.141806 275 15.6824 25.7567375 -1.94161 0.119285 274 13.8457 25.3794374 -2.32592 0.208327 273 12.3016 24.9998373 -2.18274 0.284799 272 10.8915 24.5997372 -2.43228 0.309359 271 8.76876 24.0013371 -2.30428 0.444413 270 6.52249 23.3446370 -2.40536 0.331767 269 4.39888 22.7831369 -2.80396 0.201642 268 2.75198 22.1497368 -3.35559 0.0784416 267 1.80858 21.4706367 -2.98015 0.0297998 266 0.414162 20.6852366 -2.95109 0.112086 265 -0.983323 19.6983365 -3.28154 0.285994 264 -1.15129 18.8402364 -3.07424 0.126631 263 -1.35951 18.1283363 -3.39959 0.0982639 262 -1.19601 17.2347362 -3.8515 0.0225147 261 -0.747502 16.2326361 -3.47524 -0.113073 260 -0.225255 15.2376360 -4.05111 -0.043929 259 1.54286 14.2786359 -4.19759 -0.017399 258 2.9239 13.4033358 -4.14227 -0.0921139 257 4.82824 12.4458357 -4.32237 -0.0925425 256 6.48007 11.3563356 -4.21029 -0.162305 255 7.57387 10.4688355 -4.53898 -0.26712 254 9.32111 9.77013354 -4.42111 -0.244647 253 11.199 9.35299353 -4.18831 -0.199111 252 12.4694 8.85487352 -4.32771 -0.111856 251 14.2053 8.15321351 -4.16558 0.000129064 250 14.7132 7.60078350 -4.32549 -0.00220762 249 16.5924 7.11442349 -4.27065 -0.027515 248 17.7109 6.74688348 -4.48471 -0.137422 247 18.8944 6.35418347 -4.24905 -0.260664 246 19.2007 5.85707346 -4.46877 -0.379761 245 20.0667 5.45646345 -4.48186 -0.416656 244 21.1273 5.44653344 -4.40032 -0.462131 243 21.8857 5.40322343 -4.461 -0.470078 242 22.4919 5.36672342 -4.35155 -0.377007 241 23.0134 5.12548341 -4.31589 -0.269191 240 23.2613 5.07998340 -3.86534 -0.317034 239 22.918 4.87864339 -3.96098 -0.317019 238 22.9651 4.93311

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338 -4.21431 -0.462489 237 21.8501 4.75031337 -3.76671 -0.546526 236 22.3539 4.76603336 -3.90038 -0.495024 235 20.8219 4.58268335 -3.21914 -0.399487 234 19.5746 4.67138334 -3.41948 -0.428037 233 17.1783 4.34649333 -3.14255 -0.311625 232 15.421 4.3077332 -2.92517 -0.320011 231 12.134 4.15343331 -2.15533 -0.13597 230 8.55318 4.13301330 -1.86876 0.102809 229 4.80951 4.08223329 -1.68497 0.302044 228 1.81971 4.09621328 -1.31674 0.541977 227 -1.13054 3.6842327 -1.26702 0.781283 226 -4.69645 3.2976326 -0.65967 0.94361 225 -7.17952 2.87774325 -0.500066 1.1158 224 -9.44489 1.80223324 -0.15818 1.38131 223 -11.7282 1.24852323 0.109433 1.64978 222 -14.1565 0.620111322 0.728997 2.01415 221 -16.2757 0.212546321 1.08345 2.30876 220 -16.9597 -0.105924320 1.41777 2.66862 219 -18.0311 -0.593356319 1.94135 3.06265 218 -18.9535 -1.31159318 2.45333 3.49038 217 -19.5286 -1.96707317 2.98282 4.06915 216 -19.487 -2.88096316 3.25126 4.6402 215 -20.5415 -3.77173315 3.87928 5.16054 214 -20.7045 -4.78399314 3.99622 5.80372 213 -21.2939 -6.15524313 4.54796 6.44688 212 -22.0505 -7.2111312 4.81061 7.02178 211 -21.8424 -8.88316311 5.50225 7.72729 210 -20.6816 -10.3076310 6.06208 8.54657 209 -20.931 -12.1185309 7.0532 9.36155 208 -20.0604 -13.8886308 8.49964 10.2349 207 -19.1672 -15.7969307 9.4237 11.0469 206 -17.7078 -17.5578306 10.789 11.9111 205 -17.0957 -19.7748305 12.7266 12.756 204 -15.5392 -21.7166304 14.3559 13.6807 203 -15.1495 -24.2281303 15.7228 14.6744 202 -13.842 -26.913302 17.886 15.7199 201 -14.0697 -29.7717301 20.1314 16.7075 200 -13.6566 -32.4339300 21.9465 17.686

Table 2. Experimental and Calculated 13C (101 MHz) NMR data of 1 (δ in ppm)

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gaussian_num

real_num

C_experimental

C_calculated

delta correction

delta_correction

1 5 165.6 165.6 0 166 0.42 10 168.6 170.8 2.2 171.4 2.83 6 123.3 121 2.3 120.7 2.64 7 200.2 197.1 3.1 198.2 25 8 60.5 62.4 1.9 61.1 0.66 9 51 54.9 3.9 53.5 2.57 14 49.7 51.8 2.1 50.3 0.68 13 50.4 52.3 1.9 50.8 0.49 12 51.3 54 2.7 52.5 1.2

10 11 210.5 212.9 2.4 214.1 3.611 15 45.9 47.1 1.2 45.5 0.412 16 71.4 72.1 0.7 71 0.413 17 61.1 61 0.1 59.7 1.414 20 80.2 79 1.2 78 2.215 22 205.4 202.1 3.3 203.2 2.216 23 122.7 124.8 2.1 124.6 1.917 24 151.9 154.8 2.9 155.1 3.218 25 81.2 80.6 0.6 79.7 1.519 26 27 27.5 0.5 25.6 1.420 27 26.6 27.3 0.7 25.4 1.223 19 29.3 31.2 1.9 29.4 0.124 18 21.3 24.4 3.1 22.5 1.225 30 20.9 24.3 3.4 22.3 1.426 21 25.9 26.7 0.8 24.8 1.130 31 172 171 1 171.6 0.431 32 22 24.2 2.2 22.3 0.333 1 130.6 129.1 1.5 129 1.634 2 208.9 205.6 3.3 206.8 2.135 4 47.8 51.4 3.6 49.9 2.138 28 22.1 23.1 1 21.2 0.939 29 23.4 24.5 1.1 22.6 0.8

Table 3. Experimental and Calculated 1H (400 MHz) NMR data of 1 (δ in ppm)

gaussian_num temp_num

real_num

H_experimental

H_calculated

delta correction

delta_correction

42 42 6 6.28 6.24 0.04 6.12 0.1640 40 8 2.85 2.7 0.15 2.69 0.1643 43 12 2.8 2.94 0.14 2.92 0.1244 44 12 3.05 3.08 0.03 3.06 0.0146 46 15 2.33 2.23 0.1 2.23 0.145 45 15 1.27 1 0.27 1.04 0.23

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47 47 16 4.58 4.06 0.52 4.01 0.5741 41 17 2.5 2.57 0.07 2.56 0.0648 48 23 6.85 7.06 0.21 6.92 0.0749 49 24 6.99 7.49 0.5 7.34 0.35

50, 51, 52 50 26 1.55 1.53 0.02 1.55 053, 54, 55 53 27 1.57 1.55 0.02 1.57 056, 57, 58 56 19 1.45 1.57 0.12 1.59 0.1459, 60, 61 59 18 1 1.06 0.06 1.1 0.162, 63, 64 62 30 1.16 1.32 0.16 1.35 0.1965, 66, 67 65 21 1.41 1.52 0.11 1.54 0.1370, 71, 72 70 31 2.02 2.09 0.07 2.1 0.08

73 73 1 6.02 6.2 0.18 6.08 0.0674, 75, 76 74 28 1.24 1.13 0.11 1.16 0.0877, 78, 79 77 29 1.23 1.16 0.07 1.2 0.03

Table 4. Experimental and Calculated 13C (101 MHz) NMR data of 2 (δ in ppm)

gaussian_num

real_num

C_experimental

C_calculated

delta correction

delta_correction

1 5 134.3 133.5 0.8 135.4 1.12 10 154.7 153.9 0.8 156.6 1.93 6 132.1 131.1 1 132.9 0.84 7 26 32 6 29.8 3.85 8 44.1 48 3.9 46.4 2.36 9 52 56.6 4.6 55.5 3.57 14 49.7 49.4 0.3 47.9 1.88 13 50 56.5 6.5 55.3 5.39 12 51.6 54.7 3.1 53.4 1.810 11 213.1 210.2 2.9 215.2 2.111 15 45.6 48.5 2.9 47 1.412 16 71.8 73.6 1.8 73.1 1.313 17 60.7 62.7 2 61.8 1.114 20 80.3 77.8 2.5 77.4 2.915 22 205.3 198.1 7.2 202.5 2.816 23 122.7 124.3 1.6 125.8 3.117 24 151.9 152.5 0.6 155.1 3.218 25 81.2 81.2 0 81 0.219 26 27 26.5 0.5 24.1 2.920 27 26.6 28.1 1.5 25.8 0.823 19 25.1 24.7 0.4 22.3 2.824 18 20.8 23.1 2.3 20.6 0.225 30 19.6 21.6 2 19 0.626 21 25.7 26.1 0.4 23.7 230 31 172 168.9 3.1 172.1 0.1

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31 32 22 23.6 1.6 21.1 0.933 1 111.8 109.7 2.1 110.7 1.134 2 166.3 156.3 10 159 7.336 4 83.9 81 2.9 80.8 3.138 28 27.9 30.3 2.4 28.1 0.239 29 29.7 27.9 1.8 25.6 4.1

Table 5. Experimental and Calculated 1H (400 MHz) NMR data of 2 (δ in ppm)

gaussian_num

temp_num

real_num

H_experimental

H_calculated

delta correction

delta_correction

42 42 6 6.34 6.4 0.06 6.37 0.0343 43 7 2.4 2.5 0.1 2.54 0.1444 44 7 2.64 2.51 0.13 2.56 0.0840 40 8 2.29 2.09 0.2 2.14 0.1545 45 12 2.4 2.64 0.24 2.68 0.2846 46 12 3.07 2.85 0.22 2.88 0.1948 48 15 1.93 1.73 0.2 1.78 0.1547 47 15 1.44 1.19 0.25 1.26 0.1849 49 16 4.56 4.05 0.51 4.06 0.541 41 17 2.56 2.3 0.26 2.34 0.2250 50 23 6.85 6.83 0.02 6.79 0.0651 51 24 6.99 7.37 0.38 7.32 0.33

52, 53, 54 52 26 1.56 1.57 0.01 1.63 0.0755, 56, 57 55 27 1.58 1.54 0.04 1.6 0.0258, 59, 60 58 19 1.32 1.49 0.17 1.55 0.2361, 62, 63 61 18 0.92 0.97 0.05 1.04 0.1264, 65, 66 64 30 1.2 1.23 0.03 1.29 0.0967, 68, 69 67 21 1.41 1.47 0.06 1.53 0.1272, 73, 74 72 32 2.02 2.05 0.03 2.1 0.08

75 75 1 5.32 5.42 0.1 5.41 0.09

2. NMR, HRESIMS, and IR spectra of compounds 1 and 2

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Fig. S3. 13C NMR spectrum of compound 1 in methanol-d4

Fig. S4. 1H NMR spectrum of compound 1 in methanol-d4

11

Fig. S5. 1H-1H COSY spectrum of compound 1 in methanol-d4

12

Fig. S6. HSQC spectrum of compound 1 in methanol-d4

Fig. S7. HMBC spectrum of compound 1 in methanol-d4

13

Fig. S8. HMBC spectrum of compound 1 in methanol-d4

Fig. S9. HMBC spectrum of compound 1 in methanol-d4

14

Fig. S10. NOESY spectrum of compound 1 in methanol-d4

15

Fig. S11. HRESIMS spectrum of compound 1

Fig. S12. IR spectrum of compound 1

Fig. S13. 13C NMR spectrum of compound 2 (101 MHz, methanol-d4)

16

Fig. S14. 1H NMR spectrum of compound 2 (101 MHz, methanol-d4)

Fig. S15. 1H-1H COSY spectrum of compound 2 in methanol-d4

17

Fig. S16. HSQC spectrum of compound 2 in methanol-d4

Fig. S17. HMBC spectrum of compound 2 in methanol-d4

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Fig. S18. HMBC spectrum of compound 2 in methanol-d4

19

Fig. S19. HMBC spectrum of compound 2 in methanol-d4

Fig. S20. NOESY spectrum of compound 2 in methanol-d4

20

Fig. S21. HRESIMS spectrum of compound 2

Fig. S22. IR spectrum of compound 2

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3. Flow chart of Extraction and Isolation

Fig. S23. Flow chart of 3 extraction and isolation

22