Graduate School of Nutritional and Environmental Sciences, University of Shizuoka,

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1 Identification of Indole Alkaloid Structural Units Important for Stimulus Selective TRPM8 Inhibition: SAR Study of Naturally Occurring Iboga Derivatives Yuko Terada,, Mariko Kitajima,, Fuyumi Taguchi, Hiromitsu Takayama, Syunji Horie, and Tatsuo Watanabe Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 5-1 Yada, Suruga-ku, Shizuoka 4-856, Japan Department of Biofunctional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana, Chuo-ku, Chiba 6-86, Japan Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo Togane, Chiba , Japan These authors contributed equally to this work 1

2 A CAP only µm of 1- only (TRPV1 activity) + BCTC TREx HEK * B CAP 6 1 C CAP % Activity to.1 µm CAP 1 CAP only + BCTC + µm of 1-9 (TRPV1 inhibition) * ** n. s. CAP ** ** D Agonist concentration (log M) Supplemental figure 1. TRPV1 activation (A, B) and inhibition (C, D) by administration of compunds 1-. (A and C) Gray colored column, response by.1 μm CAP; slash striped column, inhibition by 1 μm BCTC; filled column, response by compounds 1- only (A), and response by.1 μm CAP after pre-incubation of μm 1-9 (C); unfilled column, response by compounds, 6, 1, and in T-REx HEK cells, not expressing TRPV1 (A). (B) Concentration response curves of CAP and, 6, 1, and. X-axis: agonist concentration (log M). (D) Comparison of IC values of compounds 1-9 with those of BCTC and CPZ against.1 μm CAP. X-axis: antagonist concentration (log M). Each data point represents the mean ± SEM, n = 4-8. Data values are expressed as a percent response to 5 μm ionomycin (A, B) and.1 μm CAP (C, D). represents p <.5, ** represents p <.5, * represents p <.5, and n. s. indicates not significant, respectively (unpaired t-test). % Activity to.1 µm CAP 1 + BCTC + CPZ Antagonist concentration (log M)

3 A AITC only + HC-331 TREx HEK 3 µm of 1- only µm of 1- only (TRPA1 activity) C 1 AITC only + HC , 6 (TRPA1 inhibition) % Activity to 1 µm AITC B 1 AITC AITC AITC Agonist concentration (log M) Supplemental figure. TRPA1 activation potency (A, B) and inhibitory potency (C) of compounds 1-. (A) Gray colored column, response by 1 µm AITC; slash striped column, TRPA1 antagonism by 3 μm HC-331; unshaded column, responses in T-REx HEK cells not expressing TRPA1; horizontal striped column, response by μm and 7. (B) Concentration response curves for AITC and 1-. X-axis: agonist concentration (log M). (C) Gray colored column, response by 1 µm AITC only; slash striped column, inhibition by 3 μm HC-331; filed column, AITC-induced response cotreatment with μm 5 and 6. Each data point represents the mean ± SEM, n = 4-8. Data values are expressed as a percent response to 5 μm ionomycin (A, B) and 1 μm AITC (C). represents p <.5 (unpaired t-test). 3

4 Supplemental method Preparation of Voacanginol (7) from Voacangine (1). To a stirred solution of 1 (. mg,.136 mmol) in dry THF (1.3 ml) was added Super hydride (1. M in THF, 1.36 ml, 1.36 mmol) at C, and the mixture was stirred for h at C and then for 4 h at room temperature under Ar atmosphere. After addition of Super hydride (1. M in THF, 1.36 ml, 1.36 mmol), the reaction mixture was heated at 6 C for 1 h. The reaction was quenched by adding MeOH at C and the reaction mixture was dlluted with H O and then extracted three times with CHCl 3. The combined organic layers were washed with brine, dried over MgSO 4, filtered, and evaporated. The residue was purified by silica gel flash column chromatography (% AcOEt/n-hexane - AcOEt) to afford 7 (7.1 mg, 59 %) as oil. Voacanginol (7): UV (MeOH) λ max 8.,. (sh), 4. nm; 1 H NMR (CDCl 3, 4 MHz) δ 8.8 (1H, br s, NH), 7.18 (1H, d, J=8.8 Hz, H-1), 6.94 (1H, d, J=. Hz, H-9), 6.8 (1H, dd, J=8.8,. Hz, H-), 3.89 (1H, d, J=.4 Hz, CH OH), 3.86 (3H, s, 1-OMe), 3.77 (1H, d, J=.4 Hz, CH OH), 3.35 (1H, m, H-5), 3.1 (1H, br s, H-1), (4H, overlapped, H-3, H-5, H -6),.84 (1H, br d, J=8.4 Hz, H-3), (3H, overlapped, H-14, H -17), 1.68 (1H, m, H-15), (3H, overlapped, H -19, H-), 1.18 (1H, m, H-15),.9 (3H, dd, J=7.3, 7.3 Hz, H 3-18). 4

5 Preparation of Dihydrocatharanthine (1) from Catharanthine (). A mixture of (1. mg,.3 mmol) and Pd(OH) (% on carbon, 4. mg,.6 mmol) in dry MeOH (.3 ml) was stirred for 74 h at room temperature under H atmosphere. The reaction mixture was filtered through Celite and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (1% AcOEt/n-hexane) to afford 1 (4.3 mg, 4%) as oil. Dihydrocatharanthine (1): 1 H NMR (CDCl 3, 4 MHz) δ 7.69 (1H, br s, NH), 7.47 (1H, br d, J = 7.9 Hz, H-9), 7.3 (1H, d, J = 7.9 Hz, H-1), 7.14 (1H, ddd, J = 7.9, 7.1, 1.1 Hz, H-), 7.8 (1H, ddd, J = 7.9, 7.1, 1.1 Hz, H-), 3.79 (1H, d, J = 3. Hz, H-1), 3.65 (3H, s, CO Me), 3.56 (1H, m, H-5), (4H, overlapped, H-3, H-5, H -6),.8 (1H, d, J = 9. Hz, H-3),.6 (1H, dd, J = 14., 3. Hz, H-17),.7 (1H, m, H-), (3H, overlapped, H-14, H-15, H-17), 1.94 (1H, br s, H-14), 1.33 (1H, m, H-19), 1.16 (1H, br d, J =.8 Hz, H-15), 1.1 (1H, m, H-19),.9 (3H, dd, J = 7.3, 7.3 Hz, H 3-18); 13 C NMR (CDCl 3, MHz) δ 1.6 (CO Me), (C-), (C-13), 18.5 (C-8),.9 (C-), 9. (C-1), 8.4 (C-9),.4 (C-1),.3 (C-7), 56.3 (C-1), 53.1 (C-16), 5.4 (C-5), 5. (CO Me), 51.3 (C-3), 43.9 (C-), 37. (C-17), 31.5 (C-15), 7.4 (C-14), 7.3 (C-19), 1.6 (C-6), 1.6 (C-18); ESIMS m/z 339 [M+H] +. 5