Eremophila spp. by Combined use of Dual High-Resolution PTP1B and α- Glucosidase Inhibition Profiling and HPLC-HRMS-SPE-NMR

Supporting Information for: Identification of PTP1B and α-glucosidase Inhibitory Serrulatanes from Eremophila spp. by Combined use of Dual High-Resolution PTP1B and α- Glucosidase Inhibition Profiling and HPLC-HRMS-SPE-NMR Sileshi G. Wubshet, Yousof Tahtah, Allison Maree Heskes, Kenneth Thermann Kongstad, Irini Pateraki, Björn Hamberger, Birger Lindberg Møller, and Dan Staerk,* Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark and Center for Synthetic Biology biosynergy, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark 1

Table S1: Retention time, HRMS and 1 H NMR data of peaks 1-7 from Eremophila gibbosa, 8-13 from Eremophila glabra and 14-27 from Eremophila aff. drummondii 'Kalgoorlie'. Peak Name RT (min) m/z (MF, ppm) 1 Dinatin 14.9 301.0708 [M + H] + (C 16 H 13 O 6 +, ΔM 0.4) 2 Unidentified 19.6 317.1027 [M + H] + (C 17 H 17 O 6 +, ΔM 2.2) 3 Sakuranetin 23.5 287.0920 [M + H] + 4 8,16-dihydroxyserrulat-14- en-19-oic acid (C 16 H 15 O 5 +, ΔM 2.1) 27.5 333.2062 [M + H] + (C 20 H 29 O 4 +, ΔM 0.4) 5 Unidentified 28.6 335.2213 [M + H] + 6 8-hydroxy-16-oxoserrulat- 14-en-19-oic acid (C 20 H 31 O 4 +, ΔM 1.1) 31.0 331.1910 [M + H] + (C 20 H 27 O 4 +, ΔM 2.0) 7 8-hydroxy-16- acetoxyserrulat-14-en-19- oic acid 36.2 375.2155 [M + H] + (C 22 H 31 O + 5, ΔM 2.8) 8 Verbascoside 17.8 625.2121 [M + H] + 9 8,16-dihydroxyserrulat-19- oic acid (C 29 H 37 O 15 +, ΔM 1.0) 30.2 335.2215 [M + H] + (C 20 H 31 O 4 +, ΔM 0.6) 1 H NMR δ (nh, m, J (in Hz)) a 7.84 (BB', 2H, H-3'/5'), 6.92 (AA', 2H, H-2'/6'), 6.58 (s, 1H, H-3), 6.56 (s, 1H, H-8), 3.88 (s, 3H, 6- OCH 3 ). Not available 7.31 (2H, BB', H-3'/5'), 6.82 (2H, AA', H-2'/6'), 6.04 (1H, d, J = 2.2, H-8), 6.03 (1H, d, J = 2.2 Hz, H-6), 5.36 (1H, dd, J = 3.1, 12.9, H-2), 3.13 (1H, dd, J = 12.9, 17.1, H-3a), 2.73 (1H, dd, J = 3.1, 17.1, H-3b), 3.80 (3H, s, 7-OCH 3 ). 7.38 (1H, br, H-5), 7.23 (1H, br, H-7), 5.25 (1H, t(br), J = 6.9, H-14), 3.87 (2H, s, H-16), 3.19 (1H, m, H-1), 2.63 (1H, m, H-4), 2.03 (1H, m, H-13a), 1.93 (1H, m, H-11), 1.89 (1H, m, H-2a), 1.88 (1H, m, H- 3a), 1.86 (1H, m, H-13b), 1.72 (1H, m, H-3b), 1.57 (3H, s, H-17), 1.49 (1H, m, H-2b), 1.28 (1H, m, H- 12a), 1.18 (3H, d, J = 6.9, H-20), 1.13 (1H, m, H-12b), 1.13 (3H, d, J = 6.8, H-18). Not available Refer to Table 1 Refer to Table 1 3-Hydroxytyrosol: 6.70 (1H, d, J = 2.0, H-2), 6.68 (1H, d, J = 8.0, H-5), 6.57 (1H, dd, J = 8.0, 2.0, H-6), 4.05 (1H, m, H-8a), 3.73 (1H, m, H-8b), 2.80 (2H, d, J = 15.8, H-7); Caffeoyl: 7.59 (1H, d, J = 15.8, H- 8), 7.06 (1H, d, J = 2.0, H-2), 6.96 (1H, dd, J = 8.3, 2.0, H-6), 6.79 (1H, d, J = 8.3, H-5), 6.27 (1H, d, J = 15.8, H-7); Glucose: 4.92 (1H, dd, J = 9.2, 9.2, H-4), 4.38 (1H, d, J = 7.9, H-1), 3.82 (1H, dd, J = 9.2, 9.2, H-3), 3.63 (1H, dd, J = 4.7, 14.5, H-6A), 3.54 (1H, m, H-5), 3.39 (1H, dd, J = 7.9, 9.2, H-2), 3.54 (1H, m, H-6B); Rhamnose: 5.18 (1H, d, J = 1.6, H-1), 3.92 (1H, dd, J = 3.2, 1.6, H-2), 3.58 (1H, m, H- 5), 3.57 (1H, m, H-3), 3.29 (1H, m, H-4), 1.09 (3H, d, J = 6.2, H-6). 7.37 (1H, br, H-5), 7.23 (1H, d, J = 1.5, H-7), 3.35 (1H, dd, J = 5.9, 10.7, H-16a), 3.26 (1H, dd, J = 6.6, 10.7, H-16b), 3.20 (1H, m, H-1), 2.64 (1H, m, H-4), 1.97 (1H, m, H-2a), 1.92 (1H, m, H-3a), 1.89 (1H, m, H-11), 1.77 (1H, m, H-3b), 1.53 (1H, m, H-2b), 1.48 (1H, m, H-15), 1.34 (1H, m, H-13a), 1.28 (1H, m, H-14a), 1.25 (1H, m, H-12a), 1.19 (3H, d, J = 6.8, H-20), 1.18 (1H, m, H-13b), 1.11 (1H, m, H-12b), 2

10 8-hydroxyserrulat-14-en-19- oic acid 35.7 317.2105 [M + H] + (C 20 H 29 O 3 +, ΔM 2.1) 11a 8-hydroxy-16-[4- methylpent-3- enoyloxy]serrulat-19-oic acid 37.8 431.2806 [M + H] + (C 26 H 39 O + 5, ΔM 3.3) 11b 8-hydroxy-16-37.8 467.2774 [M + H] + hydrocinnamoyloxyserrulat- (C 29 H 39 O + 5, ΔM 3.8) 19-oic acid 12 8-hydroxy-16-38.3 465.2614 [M + H] + cinnamoyloxyserrulat-19- (C 29 H 37 O + 5, ΔM 4.7) oic acid 13 Unidentified 43.3 317.2111 [M + H] + (C 20 H 29 O 3 +, ΔM 4.7) 14 Dinatin 12.2 301.0709 [M + H] + (C 16 H 13 O 6 +, ΔM -0.9) 15 Jaceosidin 12.9 331.0816 [M + H] + (C 17 H 15 O 7 +, ΔM -1.3) 16 3,7,8-trihydroxy-16- oxoserrulat-14-en-19-oic acid 14.8 363.1809 [M + H] + (C 20 H 27 O + 6, ΔM -1.8) 17 Unidentified 18.3 393.1914 [M + H] + 18 3,7,8-trihydroxyserrulat-14- en-19-oic acid 19 8,16-dihydroxyserrulat-19- oic acid (C 21 H 29 O 7 +, ΔM -1.5) 26.0 349.2014 [M + H] + (C 20 H 29 O 5 +, ΔM -1.2) 26.8 335.2221 [M + H] + (C 20 H 31 O 4 +, ΔM -1.1) 1.01 (1H, m, H-14b), 0.96 (3H, d, J = 6.8, H-18), 0.84 (3H, d, J = 6.7, H-17). 7.37 (1H, br, H-5), 7.23 (1H, d, J = 1.5, H-7), 4.96 (1H, t(br), J = 7.1, H-14), 3.20 (1H, m, H-1), 2.64 (1H, m, H-4), 1.98 (1H, m, H-13a), 1.95 (1H, m, H-11), 1.94 (1H, m, H-2a), 1.83 (1H, m, H-13b), 1.92 (1H, m, H-3a), 1.75 (1H, m, H-3b), 1.63 (3H, s, H-16), 1.52 (3H, s, H-17), 1.51 (1H, m, H-2b), 1.25 (1H, m, H-12a), 1.19 (3H, d, J = 6.9, H-20), 1.12 (1H, m, H-12b), 0.98 (3H, d, J = 6.8, H-18). Refer to Table 2 Refer to Table 2 Refer to Table 2 Not available 7.84 (BB', 2H, H-3'/5'), 6.92 (AA', 2H, H-2'/6'), 6.58 (s, 1H, H-3), 6.56 (s, 1H, H-8), 3.88 (s, 3H, 6- OCH 3 ). 7.52 (1H, dd, J = 1.8, 8.4, H-6'), 7.50 (1H, d, J = 1.8, H-1'), 6.95 (1H, d, J = 8.4, H-1'), 6.65 (s, 1H, H-3), 6.59 (s, 1H, H-8), 3.97 (s, 3H, 3'-OCH 3 ), 3.89 (s, 3H, 6-OCH 3 ). Refer to Table 1 Not available Refer to Table 1 7.37 (1H, br, H-5), 7.23 (1H, d, J = 1.5, H-7), 3.35 (1H, dd, J = 5.9, 10.7, H-16a), 3.26 (1H, dd, J = 6.6, 10.7, H-16b), 3.20 (1H, m, H-1), 2.64 (1H, m, H-4), 1.97 (1H, m, H-2a), 1.92 (1H, m, H-3a), 1.89 (1H, m, H-11), 1.77 (1H, m, H-3b), 1.53 (1H, m, H-2b), 1.48 (1H, m, H-15), 1.34 (1H, m, H-13a), 1.28 (1H, m, H-14a), 1.25 (1H, m, H-12a), 1.19 (3H, d, J = 6.8, H-20), 1.18 (1H, m, H-13b), 1.11 (1H, m, H-12b), 3

20 7,8,16-trihydroxyserrulat- 19-oic acid 30.5 351.2169 [M + H] + (C 20 H 31 O 5 +, ΔM -0.7) 21 7,8-dihydroxy-16- caffeoyloxyserrulat-19-oic acid 36.1 513.2474 [M + H] + (C 29 H 37 O + 8, ΔM 1.7) 22 ( )-7-hydroxycalamenene 37.7 219.1745 [M + H] + 23 7,8-dihydroxy-16- feruloyloxyserrulat-19-oic acid 24 7,8-dihydroxyserrulat-14- en-19-oic acid (C 15 H 23 O +, ΔM -0.7) 38.6 527.2638 [M + H] + (C 30 H 39 O 8 +, ΔM 0.3) 40.5 333.2058 [M + H] + (C 20 H 29 O 4 +, ΔM 0.8) 1.01 (1H, m, H-14b), 0.96 (3H, d, J = 6.8, H-18), 0.84 (3H, d, J = 6.7, H-17). 7.20 (1H, s, H-5), 3.36 (1H, dd, J = 5.7, 10.6, H-16a), 3.27 (1H, dd, J = 6.6, 10.6, H-16b), 3.23 (1H, m, H-1), 2.53 (1H, m, H-4), 1.94 (1H, m, H-2a), 1.86 (1H, m, H-3a), 1.84 (1H, m, H-11), 1.70 (1H, m, H- 3b), 1.50 (1H, m, H-15), 1.48 (1H, m, H-2b), 1.34 (1H, m, H-13a), 1.28 (1H, m, H-14a), 1.26 (1H, m, H- 12a), 1.19 (3H, d, J = 7.0, H-20), 1.18 (1H, m, H-13b), 1.09 (1H, m, H-12b), 1.02 (1H, m, H-14b), 0.93 (3H, d, J = 6.9, H-18), 0.85 (3H, d, J = 6.8, H-17). Refer to Table 2 6.86 (1H, s, H-5), 6.52 (1H, s, H-8), 2.73 (1H, m, H-1), 2.49 (1H, m, H-4), 2.18 (1H, m, H-10), 2.12 (3H, s, H-13), 1.76 (1H, m, H-2a), 1.72 (1H, m, H-3a), 1.61 (1H, m, H-3a), 1.60 (1H, m, H-2a), 1.21 (3H, d, J = 7.1, H-9), 1.00 (3H, d, J = 7.0, H-11), 0.74 (3H, d, J = 6.8, H-12). Refer to Table 2 Refer to Table 1 25 7,8-dihydroxy-16-41.6 421.2586 [M + H] + Refer to Table 2 butanoyloxyserrulat-19-oic acid (C 24 H 37 O + 6, ΔM -0.4) 26 Unidentified 42.9 447.2745 [M + H] + Not available (C 26 H 39 O + 6, ΔM -1.0) 27 7,8-dihydroxy-16-[3- methylbutanoyloxy]serrulat- 19-oic acid 43.2 435.2744 [M + H] + (C 25 H 39 O + 6, ΔM -0.5) Refer to Table 2 a 1 H resonance frequency 600.13 MHz. Spectra acquired at 300 K in methanol-d 4. 4

Figure S1: 1 H spectrum of compound 6 from E. gibbosa Figure S2: COSY spectrum of compound 6 from E. gibbosa Figure S3: NOESY spectrum of compound 6 from E. gibbosa 5

Figure S4: HSQC spectrum of compound 6 from E. gibbosa Figure S5: HMBC spectrum of compound 6 from E. gibbosa 6

Figure S6: 1 H spectrum of compound 7 from E. gibbosa Figure S7: COSY spectrum of compound 7 from E. gibbosa Figure S8: NOESY spectrum of compound 7 from E. gibbosa 7

Figure S9: HSQC spectrum of compound 7 from E. gibbosa Figure S10: HMBC spectrum of compound 7 from E. gibbosa Figure S11: 13 C spectrum of compound 7 from E. gibbosa 8

Figure S12: 1 H spectrum of compound 11a and 11b from E. glabra Figure S13: COSY spectrum of compound 11a and 11b from E. glabra Figure S14: NOESY spectrum of compound 11a and 11b from E. glabra 9

Figure S15: HSQC spectrum of compound 11a and 11b from E. glabra Figure S16: HMBC spectrum of compound 11a and 11b from E. glabra Figure S17: 13 C spectrum of compound 11a and 11b from E. glabra 10

Figure S18: 1 H spectrum of compound 12 from E. glabra Figure S19: COSY spectrum of compound 12 from E. glabra Figure S20: NOESY spectrum of compound 12 from E. glabra 11

Figure S21: HSQC spectrum of compound 12 from E. glabra Figure S22: HMBC spectrum of compound 12 from E. glabra Figure S23: 13 C spectrum of compound 12 from E. glabra 12

Figure S24: 1 H spectrum of compound 16 from E. aff. drummondii 'Kalgoorlie' Figure S25: COSY spectrum of compound 16 from E. aff. drummondii 'Kalgoorlie' Figure S26: NOESY spectrum of compound 16 from E. aff. drummondii 'Kalgoorlie' 13

Figure S27: HSQC spectrum of compound 16 from E. aff. drummondii 'Kalgoorlie' Figure S28: HMBC spectrum of compound 16 from E. aff. drummondii 'Kalgoorlie' 14

Figure S29: 1 H spectrum of compound 18 from E. aff. drummondii 'Kalgoorlie' Figure S30: COSY spectrum of compound 18 from E. aff. drummondii 'Kalgoorlie' Figure S31: NOESY spectrum of compound 18 from E. aff. drummondii 'Kalgoorlie' 15

Figure S32: HSQC spectrum of compound 18 from E. aff. drummondii 'Kalgoorlie' Figure S33: HMBC spectrum of compound 18 from E. aff. drummondii 'Kalgoorlie' Figure S34: 1 H spectrum of compound 21 from E. aff. drummondii 'Kalgoorlie' 16

Figure S35: 1 H spectrum of compound 23 from E. aff. drummondii 'Kalgoorlie' Figure S36: COSY spectrum of compound 23 from E. aff. drummondii 'Kalgoorlie' Figure S37: NOESY spectrum of compound 23 from E. aff. drummondii 'Kalgoorlie' 17

Figure S38: HSQC spectrum of compound 23 from E. aff. drummondii 'Kalgoorlie' Figure S39: HMBC spectrum of compound 23 from E. aff. drummondii 'Kalgoorlie' 18

Figure S40: 1 H spectrum of compound 24 from E. aff. drummondii 'Kalgoorlie' Figure S41: COSY spectrum of compound 24 from E. aff. drummondii 'Kalgoorlie' Figure S42: NOESY spectrum of compound 24 from E. aff. drummondii 'Kalgoorlie' 19

Figure S43: HSQC spectrum of compound 24 from E. aff. drummondii 'Kalgoorlie' Figure S44: HMBC spectrum of compound 24 from E. aff. drummondii 'Kalgoorlie' 20

Figure S45: 1 H spectrum of compound 25 from E. aff. drummondii 'Kalgoorlie' Figure S46: COSY spectrum of compound 25 from E. aff. drummondii 'Kalgoorlie' Figure S47: NOESY spectrum of compound 25 from E. aff. drummondii 'Kalgoorlie' 21

Figure S48: HSQC spectrum of compound 25 from E. aff. drummondii 'Kalgoorlie' Figure S49: HMBC spectrum of compound 25 from E. aff. drummondii 'Kalgoorlie' 22

Figure S50: 1 H spectrum of compound 27 from E. aff. drummondii 'Kalgoorlie' Figure S51: COSY spectrum of compound 27 from E. aff. drummondii 'Kalgoorlie' Figure S52: NOESY spectrum of compound 27 from E. aff. drummondii 'Kalgoorlie' 23

Figure S53: HSQC spectrum of compound 27 from E. aff. drummondii 'Kalgoorlie' Figure S54: HMBC spectrum of compound 27 from E. aff. drummondii 'Kalgoorlie' 24

Figure S55. IC 50 curves for compound 12, 20, 25 and 27. 25