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1 Natural product-derived Transient Receptor Potential Melastatin (TRPM8) channel modulators Christina M. LeGay, a Evgueni Gorobets, a Mircea Iftinca, b Rithwik Ramachandran, c Christophe Altier, b and Darren J. Derksen *a a. Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada, T2N 1N4. b. Department of Physiology & Pharmacology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada, T2N 4N1. c. Department of Physiology & Pharmacology, Schulich Medicine & Dentistry, University of Western Ontario, 1151 Richmond St, London, Ontario, N6A 3K7. * dderksen@ucalgary.ca Supporting Information I. General considerations S2 II. Abbreviations S2 III. Experimental section S3 IV. Biological evaluation methods S17 V. References S18 VI. Electrophysiology Graphs S19 VII. Spectral data S21 [S1]

2 I. General considerations Unless otherwise noted, all reagents and solvents were purchased from commercial sources and used without further purification. Anhydrous solvents were prepared by standard methods (Na/benzophenone for THF and CaH 2 for DCM). DriSolv EMD Millipore grade DMF was used. Column chromatography was preformed using silica gel (technical grade, pore size 60 Å, mesh particle size, µm particle size). 1 H and 13 C NMR spectra were obtained on a Bruker Avance III (RDQ) 400 MHz ( 1 H), 100 MHz ( 13 C) or Bruker DMX 300 MHz ( 1 H), 75 MHz ( 13 C) spectrometer with tetramethylsilane as an internal standard. 1 H and 13 C NMR spectra were obtained in CDCl 3 and the chemical shifts (in ppm) are relative to the CHCl 3 peak (7.27 ppm for 1 H and 77.0 ppm for 13 C). Coupling constants (J values) are reported in Hz. Infrared (IR) spectra were recorded on a Nexus 470 FT-IR spectrometer. Solid samples for IR were handled as pressed KBr pellets or as CCl 4 thin films while liquid samples were analyzed neat between KBr plates. GenTech 5890 Series II SSQ 7000 and Agilent Technologies 6520 Accurate-Mass Q-TOF LC/MS instruments were used for low-resolution mass spectra (LRMS) and high resolution mass spectra (HRMS) analyses respectively and were accomplished by Mr. Wade White at the University of Calgary. Elemental analyses (EA) were performed on PerkinElmer Series II CHNS/O 2400 instrument by Mr. Jian Jun Li at the University of Calgary. Melting points were obtained using a melting point apparatus and are uncorrected. Optical rotation data was obtained on a polarimeter Rudolph Autopol IV using a quartz cell with a 10 cm path length. New compounds were characterized by their melting point, optical rotation, 1 H NMR, 13 C NMR, IR, LRMS, HRMS, and elemental analysis data. II. Abbreviations Abbreviation CI 3-CPBA EI ESI DCM DIBAL DMAP DMF EtOAc LDA pyr rt TBAF TBS TESCl THF TLC TMSCl Meaning chemical ionization 3-chloroperoxybenzoic acid electron impact ionization electron spray ionization dichloromethane diisobutylaluminium hydride 4-dimethylaminopyridine dimethylformamide ethyl acetate lithium diisopropylamide pyridine room temperature tetra-n-butylammonium fluoride tert-butyldimethylsilyl chlorotriethylsilane tetrahydrofuran thin layer chromatography trimethylsilyl chloride [S2]

3 III. Experimental Section (2R,5R)-5-isopropenyl-2-methyl-cyclohexanone (7) O This compound 1 was prepared by reduction of commercially available (R)-( )-carvone with sodium dithionite under phase transfer conditions according to the well-elaborated procedure [S-1]. 79% yield of the target product was recovered after separation from another diastereomer by silica gel column chromatography (5% EtOAc in hexanes). 1 H NMR (300 MHz, CDCl 3 ) δ 4.84 (1 H, m, =CHH), 4.70 (1 H, s, =CHH), (4 H, m, CH, CH 2 ), (1 H, m, CH), (1 H, m, CH), 1.71 (3 H, s, CH 3 ), (1 H, m, CH), 1.33 (1 H, dq, J=12.1, 1.7 Hz, CH), 1.01 (3 H, d, J=6.5 Hz, CH 3 ). All other spectral data is in agreement with the literature [S-2]. (2R,5R)-5-isopropyl-2-methyl-cyclohexanone (8) 7 O 8 A solution of dihydrocarvone 7 (6.37 g, 41.9 mmol) in ethanol (abs., 250 ml) was stirred with PtO 2 2 (75 mg) under hydrogen (1 atm) at rt until the starting compound was consumed (ca. 1 h, monitoring by 1 H-NMR 3 ). The majority of the solvent was removed in vacuo and the flask was flushed with nitrogen. The residue was diluted with EtOAc (200 ml) and the catalyst was removed by vacuum filtration. The filtrate was concentrated in vacuo and the product was forwarded to SiO 2 (200 g, 10% EtOAc in hexanes) column chromatography to acquire the target ketone 8 (6.01 g, 94%). 1 H NMR (300 MHz, CDCl 3 ) δ 2.40 (1 H, dt, J=12.8, 3.1 Hz, CH), 2.33 (1 H, sept, J=6.5 Hz, CH), (2 H, m, CH), (1 H, m, CH), (2 H, m, CH 2 ), 1.44 (1 H, dq, J=12.2, 3.2 Hz, CH), 1.30 (1 H, dq, J=12.5, 3.3 Hz, CH), 1.01 (3 H, d, J=6.5 Hz, CH 3 ), 0.90 and 0.88 (3 H, d, J=6.5 Hz, CH 3 ). All other spectral data is in agreement with the literature [S-3 - S-5]. 1 (+)-Dihydrocarvone (8) can be purchased as an 8:2 mixture of its two diastereomers from Sigma-Aldrich (catalog number ML) and separated relatively easily by silica gel column chromatography eluting with 5% EtOAc in hexanes. 2 Exploiting Pd/C instead of PtO 2 gives a substantial amount (up to 20%) of another diastereomer formation owing to the double bond migration in course of the hydrogenation. 3 Monitoring the progress of the reaction using 1 H-NMR (checking an aliquot every 15 min) reduces the chance of forming the unwanted corresponding secondary alcohol after completion of the target double bond hydrogenation. [S3]

4 (5S,6S)-5-isopropenyl-2-methyl-6-trimethylsilyloxy-cyclohex-2-en-1-one (intermediate 6a) O OTMS int. 6a This compound was prepared from (R)-(+)-carvone according to the two-step procedure described for producing its (5R,6R)-enantiomer from (S)-(+)-carvone [S-6]. The separation of 6a from the diastereomer formed during the Rubottom oxidation (7:1 ratio) was successfully executed by silica gel column chromatography (5% EtOAc in hexanes), furnishing the desired product 6a (56%). 1 H NMR (300 MHz, CDCl 3 ) δ 6.64 (1 H, m, CH=), 4.83 (2 H, m, =CH 2 ), 4.12 (1 H, d, J=12.2 Hz, CHOTMS), 2.74 (1 H, dt, J=12.3, 5.1 Hz, CH), (2 H, m, CH), 1.77 (3 H, d, J=1.3 Hz, CH 3 ), 1.74 (3 H, s, CH 3 ), 0.10 (9 H, s, CH 3 ). All other spectral data is in agreement with the literature [S-6]. [(1S,6S)-6-isopropenyl-3-methyl-2-methylene-cyclohex-3-en-1-yl]oxy-trimethyl-silane (9) OTMS 9 To a suspension of methyltriphenylphosphonium bromide (2.4 g, 6.73 mmol) in THF (15 ml), 1.6 M n-buli solution in hexane (3.93 ml, 6.3 mmol) was added at 0 o C over 5 min. The mixture was stirred at 0 o C for an additional 30 min, after which it was cooled to -78 o C and a solution of intermediate 6a (1.0 g, 4.2 mmol) in THF (3 ml) was added to it over 2 min. After 1 h the dry ice bath was removed and the reaction mixture was left to warm up to rt overnight. A solution of NH 4 Cl (sat. aq., 10 ml) and Et 2 O (10 ml) was added to the rt reaction mixture and allowed to stir for 10 min. The organic layer was separated and the aqueous layer was extracted with Et 2 O (2x15 ml). The combined organic extracts were washed with brine, dried over Na 2 SO 4, filtered, and concentrated in vacuo. The resulting residue was dissolved in CHCl 3 (7 ml) and diluted with a 9:1 hexane/etoac mixture (50 ml). After stirring for 15 min the mixture was forwarded to the fridge for 30 min, and the precipitated Ph 3 P(O) was removed via vacuum filtration. The filtrate was concentrated in vacuo and the residue was forwarded to column chromatography (60 g SiO 2, 5% EtOAc in hexanes) to give the desired 9 as a colorless oil (0.81 g, 81%). [α] 20 D (c 1.025, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.58 (1 H, m, CH=), 5.11 and 5.00 (1 H, s, =CH 2 ), 4.82 and 4.79 (1 H, s, =CH 2 ), 4.20 (1 H, d, J=9.5 Hz, CHOTMS), (1 H, m, CH), 2.23 (2 H, m, CH 2 ), 1.80 (3 H, s, CH 3 ), 1.73 (3 H, s, CH 3 ), 0.10 (9 H, s, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (C), (C), (CH), (CH 2 ), (CH 2 ), 74.6 (CH), 49.5 (CH), 29.9 (CH 2 ), 21.9 (CH 3 ), 19.7 (CH 3 ), 0.41 (CH 3 ); IR (film, cm -1 ) 2960, 2894, 1642, 1605, 1439, 1246, 1143, 1111, 1077, 905, 892, 841, 745; LRMS (EI) 236 (M +, 2%), 221 (12), 195 (76), 179 (13), 146 (32), 131 (89), 115 (10), 105 (29), 91 (32), 73 (100), 50 (10); LRMS (ESI) 237 (M+H) + ; HRMS (ESI) calcd for C 14 H 25 OSi , found [S4]

5 (1S,6S)-6-isopropenyl-3-methyl-2-methylene-cyclohex-3-en-1-ol (10) OH 10 To a solution of 9 (0.74 g, 3.13 mmol) in THF (15 ml) at 5 o C (ice water bath) was added 1 M TBAF solution in THF (3.5 ml, 3.5 mmol) over 1 min. After 1 h at this temperature the reaction mixture was quenched with the addition of water (5 ml) and stirred for 5 min. The majority of the solvent was removed in vacuo and the residue was extracted with Et 2 O (3x25 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The residue was subjected to column chromatography (30g SiO 2, 20% EtOAc in hexanes) to furnish 10 as a viscous oil (0.46 g, 90%). [α] D (c 0.96, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.60 (1 H, m, CH=), 5.33 and 5.06 (1 H, s, =CH 2 ), 4.93 (1 H, m, =CHH), 4.87 (1 H, s, =CH 2 ), (1 H, m, CHOTMS), (1 H, m, CH), 2.23 (2 H, m, CH 2 ), 1.84 (3 H, d, J=4.3 Hz, CH 3 ), 1.82 (3 H, d, J=1.7 Hz, CH 3 ), 1.75 (3 H, s, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (C), (C), (CH), (CH 2 ), (CH 2 ), 70.5(CH), 50.3 (CH), 30.0 (CH 2 ), 19.4 (CH 3 ), 19.0 (CH 3 ); IR (film, cm -1 ) 3428, 2970, 2924, 2891, 1639, 1599, 1436, 1373, 1107, 1040, 898; LRMS (EI) 164 (M +, 3%), 146 (15), 131 (100), 121 (30), 106 (100), 91 (75), 79 (65), 67 (25); LRMS (ESI) 165 (M+H) + ; HRMS (ESI) calcd for C 11 H 17 O , found trimethyl(((3r,6r)-6-methyl-3-(prop-1-en-2-yl)cyclohex-1-en-1-yl)oxy)silane (intermediate 7a) OTMS int. 7a To a solution of LDA (2 M in THF/heptane/ethylbenzene, 3.1 ml, 6.21 mmol) diluted with THF (10 ml) was added TMSCl (0.90 g, 1.05 ml, 8.3 mmol) at -78 o C over 1 min. After 5 min, a solution of the dihydrocarvone 7 (0.63 g, 4.14 mmol) in THF (3 ml) was added to the mixture at the same temperature over 30 min using a syringe-pump. After 1 h the reaction mixture was allowed to warm up to rt for 1 h and stirred 2 h more. The most part of the solvent and other volatile compounds were removed in vacuo and the residue was diluted with dry THF (10 ml), cooled down with an ice bath and stirred with sat. aq. NaHCO 3 (5 ml) for 15 min. The organic layer was separated and the aqueous layer was extracted with Et 2 O (2x15 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (35 g SiO 2, 2.5% EtOAc in hexanes, 0.01% NEt 3 ) of the residue gave 0.64 g (68%) of the corresponding silyl enol ether (R f 0.6) free of its minor regioisomer (R f 0.5) (9:1 ratio by 1 H NMR in the reaction mixture before chromatography). 1 H NMR (300 MHz, CDCl 3 ) δ (3 H, m, CH=, CH 2 =), (1 H, m, CH), (1 H, m, CH), (1 H, m, CHH), (1 H, m, CHH), 1.69 (3 H, s, CH 3 ), (2 H, m, CH 2 ), 1.01 (3 H, d, J=6.5 Hz, CH 3 ), 0.10 (9 H, s, CH 3 ). 4 4 This set of data is in agreement with the literature [S-7]. [S5]

6 (2S,3S,6R)-6-methyl-3-(prop-1-en-2-yl)-2-((trimethylsilyl)oxy)cyclohexanone (intermediate 7b) O OTMS int. 7b To a solution of intermediate 7a (0.63 g, 2.81 mmol) in DCM (15 ml) was added powdered KHCO 3 (1.40 g, 14 mmol), the mixture was cooled down to 0 o C and treated with 3-CPBA (77%, 0.99 g, 4.4 mmol) while stirring rigorously. After 45 min (100% conversion by TLC) the reaction mixture was filtered and the filtrate was stirred with a 1:1 mixture of sat. aq. Na 2 S 2 O 3 (5 ml) and water (5 ml) for 10 min, and then with sat. aq. NaHCO 3 (10 ml) for another 10 min. The organic solution was dried over Na 2 SO 4, concentrated in vacuo, and the target Rubottom oxidation product was isolated from the crude mixture by column chromatography (40 g SiO 2, 5% EtOAc in hexanes, 0.01% NEt 3 ). Intermediate 7b (0.41 g, 61%) was free of an epimer formed in course of the Rubottom oxidation (3:1 dr) and was carried through to the next step (Wittig olefination) without any additional purification. 1 H NMR (300 MHz, CDCl 3 ) δ (2 H, m, CH 2 =), 4.06 (1 H, d, J=11.2 Hz, CHOTMS), (2 H, m, CH), (1 H, m, CH), (2 H, m, CH 2 ), 1.72 (3 H, s, CH 3 ), (1 H, m, CH), 1.04 (3 H, d, J=6.5 Hz, CH 3 ), 0.07 (9 H, s, CH 3 ). [(1S,3R,6S)-6-isopropenyl-3-methyl-2-methylene-cyclohexoxy]-trimethyl-silane (11) OTMS 11 Intermediate 7b was smoothly transformed into the diene 11 (0.34 g, 84%) according to the same procedure that was used for the transformation of 6a into 9. [α] D (c 0.78, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.02 (1 H, d, J=1.4 Hz, CHH=), (3 H, m, =CHH, =CH 2 ), 3.86 (1 H, d, J=10.2 Hz, CHOTMS), (2 H, m, CH), (3 H, m, CH, CH 2 ), 1.71 (3 H, s, CH 3 ), 1.06 (3 H, d, J=6.5 Hz, CH 3 ), 0.98 (1 H, dq, J=12.5, 3.9 Hz, CH), 0.06 (9 H, s, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (C), (CH 2 ), (CH 2 ), 76.8 (CH), 55.2 (CH), 36.7 (CH), 36.0 (CH 2 ), 30.2 (CH 2 ), 21.3 (CH 3 ), 18.2 (CH 3 ), 0.14 (CH 3 Si); IR (film, cm -1 ) 2960, 2924, 2854, 1645, 1459, 1257, 1130, 1091, 1051, 921, 888, 838, 748; LRMS (EI) 238 (M +, 45%), 223 (28), 195 (21), 181 (16), 157 (13), 148 (12), 133 (30), 105 (25), 91 (20), 73 (100); LRMS (ESI) 239 (M+H) + ; HRMS (ESI) calcd for C 14 H 27 OSi , found [S6]

7 (1S,3R,6S)-6-isopropenyl-3-methyl-2-methylene-cyclohexanol (12) OH g (86%) of 12 was smoothly prepared from 0.24 g of the diene 11 according to the same procedure that was used for transformation 9 into 10. [α] D (c 0.68, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.12 (1 H, d, J=1.4 Hz, =CH 2 ), 4.90 (1 H, d, J=1.3 Hz, =CH 2 ), 4.85 (1 H, s, =CH 2 ), 4.76 (1 H, d, J=1.4 Hz, =CH 2 ), 3.87 (1 H, d, J=10.4 Hz, CHOH), (2 H, m, CH), 1.89 (1 H, br s, OH), (3 H, m, CH, CH 2 ), 1.74 (3 H, s, CH 3 ), 1.07 (3 H, d, J=6.5 Hz, CH 3 ), 1.03 (1 H, dq, J=12.4, 3.8 Hz, CH); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (C), (CH 2 ), (CH 2 ), 72.9 (CH), 56.0 (CH), 36.4 (CH), 35.8 (CH 2 ), 29.6 (CH 2 ), 18.8 (CH 3 ), 17.9 (CH 3 ); IR (film, cm -1 ) 3445, 2960, 2927, 2851, 1635, 1453, 1403, 1376, 1114, 1034, 898; LRMS (EI) 166 (M +, 35%), 151 (36), 133 (28), 123 (75), 109 (100), 95 (90), 81 (85), 67 (80), 55 (78), 41 (70); LRMS (ESI) (M+H) + ; HRMS (ESI) calcd for C 11 H 19 O , found [((3S,6R)-3-isopropyl-6-methylcyclohex-1-en-1-yl)oxy]trimethylsilane (intermediate 8a) 12 OTMS int. 8a To solution of LDA (2 M in in THF/heptane/ethylbenzene, 1.95 ml, 3.9 mmol) diluted with THF (15 ml) at -78 o C was added TMSCl (0.65 g, 0.76 ml, 6 mmol) over 1 min. After 5 min, a solution of the tetrahydrocarvone 8 (0.45 g, 3.0 mmol) in THF (3 ml) was added to the mixture over 30 min at the same temperature using a syringe-pump. After 1 h the reaction mixture was allowed to warm up to rt over 1 h and stirred 2 h more. The most part of the solvent along with other volatile compounds were removed in vacuo. The residue was diluted with dry THF (10 ml), cooled down with an ice bath and stirred with sat. aq. NaHCO 3 (5 ml) for 15 min. The organic layer was separated and the aqueous layer was extracted with Et 2 O (2x15 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (35 g SiO 2, 2.5% EtOAc in hexanes, 0.01% NEt 3 ) of the residue gave 0.47 g (69%) of the corresponding silyl enol ether (R f 0.6) free of its minor regioisomer (R f 0.5) (9:1 ratio by 1 H NMR in the reaction mixture before the chromatography). 1 H NMR (300 MHz, CDCl 3 ) δ 4.75 (1 H, br s, CH=), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (1 H, m, CHH), (1 H, m, CHH), (2 H, m, CH 2 ), 1.01 (3 H, d, J=6.5 Hz, CH 3 ), 0.88 and 0.86 (3 H, d, J=6.5 Hz, CH 3 ), 0.21 (9 H, s, CH 3 ). [S7]

8 (2S,3S,6R)-3-isopropyl-6-methyl-2-trimethylsilyloxy-cyclohexanone (intermediate 8b) O OTMS int. 8b To a solution of intermediate 8a (0.47 g, 2.0 mmol) in DCM (10 ml) was added powdered KHCO 3 (1.0 g, 10 mmol). This mixture was cooled to 0 o C and treated with 3-CPBA (77%, 0.49 g, 2.2 mmol) while stirring rigorously. After 45 min (100% conversion by TLC) the reaction mixture was filtered and the filtrate was stirred with a 1:1 mixture of sat. aq. Na 2 S 2 O 3 (4 ml) and water (4 ml) for 10 min and then with sat. aq. NaHCO 3 (10 ml) for an additional 10 min. The organic solution was dried over Na 2 SO 4, concentrated in vacuo, and the target Rubottom oxidation product (0.30 g, 62%) was isolated from the crude mixture by column chromatography (30 g SiO 2, 10% EtOAc in hexanes, 0.01% NEt 3 ). [α] D (c 0.55, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 3.97 (1 H, d, J=11.3 Hz, CHOTMS), 2.30 (1 H, sept, J=5.3 Hz, CH), (2 H, m, CH), 1.72 (1 H, dq, J= Hz, CH), (1 H, m, CH), 1.40 (1 H, dq, J=12.5, 3.4 Hz, CH), 1.23 (1 H, dq, J=12.9, 3.4 Hz, CH), 1.02, 0.92 and 0.81 (3 H, d, J=6.5 Hz, CH 3 ), 0.10 (9 H, s, CH 3 ). 13 C NMR (75 MHz, CDCl 3 ) δ (C), 78.7 (CH), 52.5 (CH), 43.5 (CH), 34.7 (CH 2 ), 26.1 (CH), 22.2 (CH 2 ), 20.9 (CH 3 ), 15.2 (CH 3 ), 14.1 (CH 3 ), 0.13 (CH 3 Si); IR (film, cm -1 ) 2960, 2934, 2868, 1728, 1452, 1250, 1147, 1130, 1014, 921, 874, 838, 749; LRMS (EI) 242 (M +, 25%), 227 (70), 199 (15), 171 (30), 143 (80), 73 (100); HRMS (EI) calcd for C 13 H 26 O 2 Si , found [(1S,3R,6R)-6-isopropyl-3-methyl-2-methylene-cyclohexoxy]-trimethyl-silane (13) OTMS g (84%) of this alkene was obtained from 0.30 g of the ketone 8b by following the procedure used for the transformation of intermediate 6a into 9. [α] 20 D (c 0.53, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.00 (1 H, d, J=1.4 Hz, CHH=), 4.69 (1 H, d, J=1.3 Hz, CHH=), 3.78 (1 H, d, J=10.1 Hz, CHOTMS), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (2 H, m, CH 2 ), 1.09, 0.91, and 0.80 (3 H, d, J=6.5 Hz, CH 3 ), (1 H, m, CH), 0.15 (9 H, s, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (CH 2 ), 76.6 (CH), 52.3 (CH), 36.8 (CH), 36.3 (CH 2 ), 25.6 (CH), 22.7 (CH 2 ), 21.3 (CH 3 ), 18.2 (CH 3 ), 15.6 (CH 3 ), 0.16 (CH 3 Si); IR (film, cm -1 ) 2954, 2933, 2870, 1655, 1456, 1379, 1254, 1124, 1084, 1041, 911, 894, 845, 752; LRMS (EI) 240 (M +, 45%), 225 (95), 197 (40), 183 (13), 157 (22), 150 (25), 135 (25), 107 (55), 93 (20), 73 (100); LRMS (ESI) 263 (M+Na) + ; HRMS (ESI) calcd for C 14 H 28 NaOSi , found [S8]

9 (1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexanol (4) OH g (84%) of this allyl alcohol was obtained from 0.24 g of 13 by following the procedure used for the transformation of 9 into H NMR (300 MHz, CDCl 3 ) δ 5.03 (1 H, s, CHH=), 4.74 (s, CHH=), 3.84 (1 H, m, CHOH), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), 1.49 (1 H, d, J=5.6 Hz, OH), (2 H, m, CH 2 ), 1.10, 0.93, and 0.87 (3 H, d, J=6.6 Hz, CH 3 ), (1 H, m, CH). All other spectral data are in agreement with the literature (S-8). [(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexyl] acetate (14) O O To a solution of the allyl alcohol 4 (0.15 g, 0.89 mmol), pyr (1 ml) and DMAP (5 mg) in DCM (5 ml) was added Ac 2 O (1 ml) and the mixture was stirred at rt overnight. The solvents were removed in vacuo and the residue was forwarded to column chromatography (25 g SiO 2, 10% EtOAc in hexanes) to isolate the acetate 14 as a non-viscous oil (0.15 g, 81%). [α] D (c 0.63, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 5.04 (1 H, d, J=11.1Hz, CHOAc), 4.69 (1 H, s, CHH=), 4.64 (1 H, d, J=0.8 Hz, CHH=), 2.11 (3 H, s, CH 3 ), (1 H, m, CH), (2 H, m, CH), 1.69 (1 H, dq, J=16.8, 3.5 Hz, CH), 1.47 (1 H, dt, J=14.9, 3.4 Hz, CH), 1.27 (1 H, dq, J=12.8, 3.6 Hz, CH), 1.07, 0.88, and 0.78 (3 H, d, J=7.0 Hz, CH 3 ), 0.96 (1 H, dq, J=12.6, 3.6 Hz, CH); 13 C NMR (100 MHz, CDCl 3 ) δ (C), (C), (CH 2 ), 75.9 (CH), 49.2 (CH), 36.7 (CH), 36.6 (CH 2 ), 26.9 (CH), 23.2 (CH 2 ), 20.8 (CH 3 ), 20.6 (CH 3 ), 17.9 (CH 3 ), 16.1 (CH 3 ); IR (film, cm -1 ) 2963, 2930, 2871, 1745, 1648, 1379, 1240, 1027, 892; LRMS (EI) 150 (M +, 50%), 135 (48), 107 (100), 93 (28), 79 (26), 43 (45); LRMS (ESI) 233 (M+Na) + ; HRMS (ESI) calcd for C 13 H 22 O 2 Na , found [(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexyl] benzoate (15) 14 O O To a solution of the allyl alcohol 4 (0.147 g, 0.88 mmol) and DMAP (5 mg) in pyr (5 ml) was added BzCl (0.140 g, 1.0 mmol) and the mixture was stirred at rt overnight. The solvents were removed in vacuo and the residue was diluted with xylene (10 ml) and concentrated in high vacuo. 5 The residue was forwarded to column chromatography (25 g SiO 2, 50% CHCl 3 in hexanes) to obtain 5 To remove excess BzCl. 15 [S9]

10 the purified benzoate 15 as an oil (0.214 g, 89%). [α] D (c 0.40, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 8.16 (2 H, d, J=8.0 Hz, CH), 7.58 (1 H, dt, J=8.0, 1.2 Hz, CH), 7.48 (2 H, t, J= 8.0 Hz, CH), 5.38 (1 H, d, J=10.8 Hz, CHOBz), 4.84 (1 H, s, CHH=), 4.72 (1 H, s, CHH=), (1 H, m, CH), 2.04 (1 H, d quint, J=7.0, 2.7 Hz, CH), 1.90 (1 H, dq, J=12.6, 3.5 Hz, CH), (2 H, m, CH), 1.40 (1 H, dq, J=12.7, 3.7 Hz, CH), 1.14, 0.95, and 0.87 (3 H, d, J=6.9 Hz, CH 3 ), 1.08 (1 H, dq, J=12.6, 3.7 Hz, CH); 13 C NMR (100 MHz, CDCl 3 ) δ (C), (C), (CH), (C), (CH), (CH), (CH 2 ), 76.5 (CH), 49.5 (CH), 36.7 (CH), 35.7 (CH 2 ), 27.1 (CH), 23.4 (CH 2 ), 20.7 (CH 3 ), 18.0 (CH 3 ), 16.3 (CH 3 ); IR (film, cm -1 ) 2954, 2924, 2871, 1721, 1652, 1456, 1277, 1114, 1071, 1027, 901, 709; LRMS (EI) 150 (M +, 40%), 135 (30), 105 (100), 93 (15), 77 (35); LRMS (ESI) 290 (M+NH 4 ) + ; HRMS (ESI) calcd for C 18 H 28 O 2 N , found (S)-(1S,3R,6S)-6-isopropyl-3-methyl-2-methylenecyclohexyl propanoate (intermediate 16a) 2-((tert-butyldimethylsilyl)oxy) O int. 16a A solution of (2S)-2-[(tert-butyldimethylsilyl)oxy]lactic acid 6 (0.61 g, 2.05 mmol) and DMF (2 drops) in DCM (10 ml) was cooled to 0 o C and a solution of (COCl) 2 (0.45 g, 0.31 ml, 3.53 mmol) in DCM (1.5 ml) was added dropwise over 15 min. The mixture was stirred for 20 min at 0 o C and then for 1 h at rt. DCM and other volatile compounds in the reaction mixture were removed in vacuo. The residue was dissolved in DCM (10 ml) and concentrated in vacuo again. The residue was dissolved again in DCM (3 ml) and it was treated via slow addition (over 10 min at rt) with a solution of the alcohol 4 (0.35 g, 2.05 mmol) and DMAP (5 mg) in a 50% pyr/dcm mixture (8 ml). After 2 h at rt, the solvent was removed in vacuo and the resulting residue was submitted to high vacuo for 15 min. The residue was forwarded to column chromatography (50g SiO 2, 5% EtOAc in hexanes) to obtain 0.46 g (63%) of the corresponding intermediate ester 16a containing ca. 5% of a non-identifiable contamination. 1 H NMR (400 MHz, CDCl 3 ) δ 5.10 (1 H, d, J=11.2Hz, CHOTBS), 4.74 (1 H, s, CHH=), 4.66 (1 H, s, CHH=), 4.41 (1 H, q, J=6.9 Hz, CHOH), (1 H, m, CH), (2 H, m, CH), (2 H, m, CH), (1 H, m, CH), 1.47 (3 H, d, J=6.9 Hz, CH 3 ), 1.30 (1 H, dq, J=12.8, 3.6 Hz, CH), 1.08, 0.90 and 0.80 (3 H, d, J=7.0 Hz, CH 3 ), 0.98 (1 H, dq, J=12.6, 3.6 Hz, CH), 0.91 (9 H, s, CH 3 ), 0.10 (6 H, d, J=12 Hz, CH 3 ). O OTBS 6 It was made in two steps from (S)-ethyl lactate according to the known procedure [S-9]. [S10]

11 [(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexyl] (2S)-2-hydroxypropanoate (16) 16 To a solution of the ester intermediate 16a (0.43 g, 1.21 mmol) in THF (15 ml) was added TBAF (1 M in THF, 1.40 ml, 1.40 mmol) at 0 o C over 1 min. After 2 h of stirring at that temperature water (5 ml) was added to the reaction mixture and after 10 min the majority of the solvent was removed in vacuo and the residue was extracted with CHCl 3 (3x20 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (50 g SiO 2, 20% EtOAc in hexanes) of the residue gave the target ester 16 as a white crystalline solid (0.27 g, 93%). The product can be recrystallized from EtOAc/hexanes to get analytical-grade crystals. mp o C (EtOAc/hexanes); [α] 20 D (c 0.24, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 5.17 (1 H, d, J=11.1Hz, CHOLact), (2 H, m, CH 2 =), 4.38 (1 H, dq, J=6.9, 5.4 Hz, CHOH), 2.86 (1 H, dd, J=5.2, 4.0 Hz, OH), (1 H, m, CH), (2 H, m, CH), 1.75 (1 H, dq, J=13.5, 3.5 Hz, CH), (1 H, m, CH), 1.54 (3 H, d, J=6.9 Hz, CH 3 ), 1.32 (1 H, dq, J=12.8, 3.6 Hz, CH), 1.10, 0.91, and 0.81 (3 H, d, J=7.0 Hz, CH 3 ), 1.01 (1 H, dq, J=12.6, 3.6 Hz, CH); 13 C NMR (100 MHz, CDCl 3 ) δ (C), (C), (CH 2 ), 66.7 (CH), 49.3 (CH), 36.7 (CH), 35.5 (CH 2 ), 26.9 (CH), 23.1 (CH 2 ), 20.6 (CH 3 ), 20.5 (CH 3 ), 17.9 (CH 3 ), 16.0 (CH 3 ); IR (film, cm -1 ) 3455, 3412, 2961, 2928, 2864, 1742, 1466, 1396, 1240, 1007, 1127, 994, 895; LRMS (EI) 150 (M +, 100%), 135 (48), 107 (82), 95 (41), 79 (18), 45 (34); LRMS (CI) 258 (M+NH 4 ) + ; EA calcd for C 14 H 24 O 3 : C, 69.96; H, Found: C, 70.03, H, Ethyl 2-[(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexoxy]acetate (int. 17a) O O OH O int. 17a To a solution of the allyl alcohol 4 (0.152 g, 0.90 mmol) in DCM (5 ml) was added [Rh(OAc) 2 ] 2 (11.9 mg, mmol) and after 5 min of stirring at rt, a solution of ethyl diazoacetate (85 %, g, ml, 0.69 mmol) 7 in DCM (1 ml) was added dropwise over 2 h. The reaction mixture was diluted with a mixture of 50% EtOAc in hexanes (7 ml) and concentrated in vacuo to ca. 3 ml volume. This dilution-concentration procedure was repeated once more and after an additional dilution the solution was passed through a short plug of SiO 2 (5 g) to eliminate the leftover catalyst. The filtrate was concentrated in vacuo and the residue forwarded to column chromatography (30 g SiO 2, 10% to 20% EtOAc in hexanes) to afford g (59% conversion) of unreacted 5 and g (51% yield) of the target 17a as colorless oil. [α] D (c 0.25, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 4.91 (1 H, d, J=1.2 Hz, CHH=), 4.71 (1 H, d, J=1.4 Hz, CHH=), 4.17 (2 H, q, J=7.2 Hz, CH 2 O), 4.09 (2 H, dd, J=56.5, 16.0 Hz, CH 2 O), 3.48 (1 H, d, J=10.3 Hz, CHOCH 2 ), 2.30 (1 H, d quint, J=6.9 Hz, 3.0 Hz, CH), (1 H, m, CH), 1.75 (1 H, dq, J=8.7, 3.8 Hz, CH), 1.64 (1 H, dq, J=9.7, 3.5 Hz, CH), 1.40 (1 H, tt, J=10.4, 3.4 Hz, CH), 1.24 (3 H, t, J=7.1 Hz, CH 3 ), 1.27 (1 H, O O 7 The reaction was not run to full conversion of the alcohol 5 in order to minimize formation of the cyclopropane containing side products that can result from unwanted addition of the intermediate carbene to the double bond. [S11]

12 dq, J=12.8, 3.6 Hz, CH), (1 H, m, CH), 1.01, 0.86, and 0.81 (3 H, d, J=7.0 Hz, CH 3 ), (1 H, m, CH); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (C), (CH 2 ), 83.9 (CH), 67.6 (CH 2 ), 60.6 (CH 2 ), 50.6 (CH), 36.8 (CH), 35.9 (CH 2 ), 25.8 (CH), 22.9 (CH 2 ), 20.9 (CH 3 ), 17.9 (CH 3 ), 16.1 (CH 3 ), 14.1 (CH 3 ); IR (film, cm -1 ) 2961, 2931, 2871, 1768, 1735, 1648, 1446, 1383, 1283, 1203, 1127, 1027, 905; LRMS (EI) 254 (M +, 50%), 239 (52), 211 (100), 171 (21), 167 (30), 150 (20), 135 (30), 107 (28), 93 (12); LRMS (ESI) 255 (M+H) + ; HRMS (ESI) calcd for C 15 H 27 O , found [(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexoxy]ethanol (17) O OH To a solution of the ester int. 17a (0.136 g, 0.54 mmol) in THF (3 ml) was added DIBAL (1.5 M in toluene, 0.80 ml, 1.19 mmol) at rt over 1 min. After 1 h the mixture was cooled to 0ºC and treated with EtOAc (1 ml). After 30 min, half of the solvents were removed in vacuo and the residue was diluted with Et 2 O (10 ml) and stirred with a Rochelle salt solution (sat. aq., 2 ml) for 30 min to get a biphasic liquid system with a clear organic layer. The organic phase was separated and the aqueous phase was extracted with Et 2 O (4x10 ml). The combined organic solution was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (30 g SiO 2, 10% to 25% EtOAc in hexanes) of the residue gave the desired alcohol 17 as a viscous oil (0.90 g, 78%). [α] D (c 0.27, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 4.94 (1 H, d, J=1.2 Hz, CHH=), 4.70 (1 H, d, J=1.4 Hz, CHH=), (3 H, m, CH 2 O, CHO), (2 H, m, CH 2 O), 2.40 (1 H, br s, OH), 2.20 (1 H, d quint, J=6.9 Hz, 3.0 Hz, CH), (1 H, m, CH), 1.77 (1 H, dq, J=8.6, 3.8 Hz, CH), 1.64 (1 H, dq, J=9.6, 3.5 Hz, CH), 1.34 (1 H, tt, J=10.4, 3.4 Hz, CH), 1.19 (1 H, dq, J=12.8, 3.6 Hz, CH), (1 H, m, CH), 1.05, 0.87, and 0.81 (3 H, d, J=7.0 Hz, CH 3 ); 13 C NMR (100 MHz, CDCl 3 ) δ (C), (CH 2 ), 83.1 (CH), 71.5 (CH 2 ), 62.2 (CH 2 ), 50.9 (CH), 36.8 (CH), 36.1 (CH 2 ), 26.2 (CH), 23.1 (CH 2 ), 20.8 (CH 3 ), 18.0 (CH 3 ), 16.0 (CH 3 ); IR (film, cm -1 ) 3386, 2961, 2931, 2868, 1652, 1449, 1383, 1366, 1120, 1041, 905; LRMS (EI) 212 (M +, 12%), 197 (46), 169 (50), 150 (75), 135 (100), 123 (78), 107 (50), 95 (30), 81 (42); LRMS (ESI) 235 (M+Na) + ; HRMS (ESI) calcd for C 13 H 24 O 2 Na , found [(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexoxy]-4-oxo-butanoic acid (18) 17 O O 18 To a solution of the allyl alcohol 4 (0.16 g, 0.95 mmol), NEt 3 (0.20 ml, g, 1.44 mmol), and DMAP (0.128 g, 1.06 mmol) in DCM (7 ml) was added succinic anhydride (0.240 g, 0.95 mmol) and the mixture was stirred at rt overnight. The solvents were removed in vacuo, the residue was subjected to high vacuo for 15 min, dissolved in CHCl 3 (20 ml) and stirred with 4% aq. HCl (3 ml) for 15 min. The organic layer was separated and the aqueous layer extracted with CHCl 3 (15 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The residue was forwarded to column chromatography (30 g SiO 2, 5% to 10 % MeOH in O OH [S12]

13 CHCl 3 ) to get crystalline 18 (0.24 g, 92%). Recrystallization from CHCl 3 was used to obtain an analytical grade sample. mp o C (CHCl 3 ); [α] D (c 0.63, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 5.10 (1 H, d, J=11.2Hz, CHOR), 4.74 (1 H, s, CHH=), 4.69 (1 H, s, CHH=), 2.75 (4 H, s, CH 2 ), (1 H, m, CH), (2 H, m, CH), 1.73 (1 H, dq, J=13.4, 3.4 Hz, CH), 1.46 (1 H, dt, J=11.4, 3.5 Hz, CH), 1.31 (1 H, dq, J=12.9, 3.6 Hz, CH), 1.09, 0.91, and 0.80 (3 H, d, J=7.0 Hz, CH 3 ), 1.00 (1 H, dq, J=12.5, 3.6 Hz, CH); 13 C NMR (100 MHz, CDCl 3 ) δ (C), (C), (C), (CH 2 ), 76.5 (CH), 49.2 (CH), 36.7 (CH), 35.5 (CH 2 ), 28.9 (CH 2 ), 28.8 (CH 2 ), 26.8 (CH), 23.1 (CH 2 ), 20.6 (CH 3 ), 17.9 (CH 3 ), 15.9 (CH 3 ); IR (film, cm -1 ) 3356, 2954, 2924, 2864, 1735, 1712, 1648, 1409, 1383, 1264, 1173, 954, 898; LRMS (EI) 150 (M +, 80%), 135 (45), 107 (100), 101 (48), 93 (35), 79 (35); LRMS (ESI) 291 (M+Na) + ; EA calcd for C 15 H 24 O 4 : C, 67.14; H, Found: C, 67.12, H, [(1S,3R,6S)-6-isopropyl-3-methyl-2-methylene-cyclohexyl] 4-amino-4-oxo-butanoate (19) To a solution of the acid 18 (0.20 g, 0.75 mmol) and NEt 3 (0.16 ml, g, 1.15 mmol) in DCM (3.0 ml) was added a solution of EtOC(O)Cl (0.10 ml, g, 1.05 mmol) in DCM (1 ml) at rt over 10 min. After 30 min the mixed anhydride solution was cooled to 0 o C and treated with (CH 3 ) 2 NHxHCl (0.102 g, 1.25 mmol), followed by NEt 3 (0.20 ml, 1.50 mmol). After 1 h the ice bath was removed and the reaction mixture was stirred at rt overnight. The solvent and other volatile compounds were removed in vacuo and two consecutive column chromatography purifications (20 g SiO 2, 50% to 100% EtOAc in hexanes and 20 g SiO 2, 5% MeOH in CHCl 3 ) gave g (78%) of the amide 19 as an oil. [α] D (c 0.29, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 5.01 (1 H, d, J=11.2 Hz, CHOR), 4.73 (1H, s, CHH=), 4.61 (1 H, s, CHH=), 2.96 (3 H, s, NCH 3 ), 2.88 (3 H, s, NCH 3 ), (4 H, m, CH 2 ), (1 H, m, CH), 1.85 (1 H, d quint, J=7.0, 2.7 Hz, CH), 1.76 (1 H, dq, J=12.6, 3.5 Hz, CH), 1.65 (1 H, dq, J=13.3, 3.5 Hz, CH), 1.46 (1 H, dt, J=11.7, 3.5 Hz, CH), 1.23 (1 H, dq, J=12.9, 3.6 Hz, CH), 1.01, 0.83, and 0.73 (3 H, d, J=7.0 Hz, CH 3 ), 0.93 (1 H, dq, J=12.5, 3.6 Hz, CH); 13 C NMR (100 MHz, CDCl 3 ) δ (C), (C), (C), (CH 2 ), 76.0 (CH), 49.1 (CH), 36.9 (CH), 36.6 (CH), 35.5 (CH 2 ), 35.4 (CH), 28.3 (CH 2 ), 27.9 (CH 2 ), 26.7 (CH), 23.2 (CH 2 ), 20.6 (CH 3 ), 17.8 (CH 3 ), 16.0 (CH 3 ); IR (film, cm -1 ) 2957, 2927, 2870, 1735, 1658, 1456, 1406, 1373, 1210, 1174, 1137, 1001, 905; LRMS (EI) 150 (M +, 48%), 146 (50), 128 (100), 100 (20); LRMS (ESI) 296 (M+H) + ; HRMS (ESI) calcd for C 17 H 29 O 3 N , found (2S,3R,6R)-3-isopropyl-6-methyl-2-triethylsilyloxy-cyclohexanone (20) O O 19 O O NMe 2 OTES 20 To prepare 20, the same procedure sequence was exploited as for making int. 8a from 8 with the only difference being the usage of TESCl instead of TMSCl on the first step g (78%) of the corresponding intermediate silyl enol ether (structure not shown) was obtained from 0.45 g of the [S13]

14 tetrahydrocarvone 8. The product was contaminated with ca. 5% of a non-separable impurity and was forwarded to the second step without additional purification. Rubottom oxidation furnished 0.37 g (66%) of the target 20 from 0.54 g of the intermediate silyl enol ether. [α] D (c 0.53, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 4.04 (1 H, dd, J=11.3, 1.4 Hz, CHOSi), 2.35 (1 H, sept, J=5.3 Hz, CH), (1 H, m, CH), (1 H, m, CH), 1.74 (1 H, dq, J=19.3, 3.4 Hz, CH), (1 H, m, CH), 1.43 (1 H, dq, J=12.7, 3.5 Hz, CH), 1.25 (1 H, dq, J=12.8, 3.4 Hz, CH), 1.05, 0.94, and 0.85 (3 H, d, J=6.5 Hz, CH 3 ), 0.97 (9 H, t, J=7.7 Hz, CH 3 ), 0.64 (6 H, dq, J=7.6, 3.1 Hz, SiCH 2 ). 13 C NMR (75 MHz, CDCl 3 ) δ (C), 78.9 (CH), 52.9 (CH), 43.5 (CH), 34.8 (CH 2 ), 25.9 (CH), 22.1 (CH 2 ), 21.0 (CH 3 ), 15.1 (CH 3 ), 14.1 (CH 3 ), 6.8 (CH 3 ), 5.0 (CH 2 Si); IR (film, cm -1 ) 2960, 2874, 1741, 1463, 1240, 1160, 1127, 1008, 808, 791, 742; LRMS (EI) 255 (M +, 100%), 199 (5); LRMS (ESI) 307 (M+Na) + ; HRMS (EI) calcd for C 16 H 32 NaO 2 Si , found (1S,2S,3R,6R)-3-isopropyl-6-methyl-2-triethylsilyloxy-cyclohexanol (intermediate 20a) OH OTES int. 20a To a solution of ketone 20 (0.50 g, 1.76 mmol) in THF (25 ml) was added LiAlH(Ot-Bu) 3 (0.90 g, 3.52 mmol) in one portion at 0 o C under stirring. After 15 min a second portion of LiAlH(Ot-Bu) 3 (0.90 g, 3.52 mmol) was added and after an additional 15 min the reaction mixture was allowed to warm up to rt and stirred at this temperature until completion (ca. 5 hs, monitoring the progress by TLC). The reaction mixture was cooled down to 0 o C and any remaining reducing agent was quenched by the addition of acetone (2 ml) followed by stirring at rt for 15 min. The most part of the solvent was removed in vacuo and the residue was diluted with Et 2 O (30 ml) and stirred with a Rochelle salt solution (sat. aq., 25 ml) for 30 min resulting in a biphasic liquid system with a clear organic layer. The phases were separated and the aqueous one was extracted with Et 2 O (4x35 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (120 g SiO 2, 5% to 7.5% EtOAc in hexanes) of the residue gave the desired mono-protected diol 20a as a viscous oil (0.44 g, 88%). [α] 20 D (c 0.27, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 3.23 (1 H, dd, J=10.3, 8.2 Hz, CHOH), 2.93 (1 H, ddd, J=10.2, 8.3, 3.1 Hz, CHOSi), (2 H, m, CH), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (2 H, m, CH 2 ), 1.03, 0.88, and 0.73 (3 H, d, J=6.9 Hz, CH 3 ), 0.96 (9 H, t, J=7.9 Hz, CH 3 ), (1 H, m, CH), 0.62 (6 H, q, J=7.8 Hz, SiCH 2 ); 13 C NMR (75 MHz, CDCl 3 ) δ 81.7 (CH), 78.5 (CH), 48.6 (CH), 37.5 (CH), 32.4 (CH 2 ), 25.0 (CH), 22.0 (CH 2 ), 21.4 (CH 3 ), 18.5 (CH 3 ), 15.7 (CH 3 ), 7.0 (CH 3 ), 5.6 (CH 2 Si); IR (film, cm -1 ) 3635, 3502, 2951, 2874, 1629, 1463, 1373, 1243, 1094, 1017, 795, 739; LRMS (EI) 199 (M +, 8%), 137 (100), 103 (1), 95 (18), 81 (26), 75 (20); LRMS (CI) 287 (M+H) + ; HRMS (CI) calcd for C 16 H 35 O 2 Si , found [S14]

15 (1S,2S,3S,6R)-3-isopropyl-6-methyl-cyclohexane-1,2-diol (21) OH OH 21 To a solution of intermediate 21a (0.53 g, 1.85 mmol) in THF (20 ml) was added TBAF (1 M in THF, 2.0 ml, 2.0 mmol) over 1 min at 0 o C. After 1 h stirring at 0 o C water (5 ml) was added to the reaction mixture and after 5 min the majority of the solvent was removed in vacuo and the residue was extracted with Et 2 O (3x30 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (75 g SiO 2, 50% EtOAc in hexanes) of the residue gave the target diol 21 as a white crystalline solid (0.32 g, 99%) forming cotton-like crystals when crystallized from EtOAc. mp o C (EtOAc); [α] 20 D (c 0.21, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 3.17 (1 H, dd, J=10.3, 8.3 Hz, CHOH), 2.94 (1 H, dd, J=10.3, 8.9 Hz, CHOH), 2.55 and 2.49 (1 H, br s, OH), 2.10 (1 H, d quint, J=6.9, 2.8 Hz, CH), (1 H, m, CH), (1 H, m, CH), (2 H, m, CH), 1.02, 0.88 and 0.79 (3 H, d, J=6.9 Hz, CH 3 ), (1 H, m, CH); 13 C NMR (75 MHz, CDCl 3 ) δ 81.1 (CH), 76.1 (CH), 47.6 (CH), 37.6 (CH), 32.5 (CH 2 ), 25.6 (CH), 22.3 (CH 2 ), 21.0 (CH 3 ), 18.5 (CH 3 ), 15.7 (CH 3 ); IR (film, cm -1 ) 3336, 2961, 2867, 1629, 1463, 1386, 1370, 1071, 1051, 998,868; LRMS (EI) 154 (M +, 13%), 139 (100), 121 (30), 111 (30), 97 (18), 84 (26), 71 (30), 55 (48); LRMS (CI) 190 (M+NH 4 ) + ; EA calcd for C 10 H 20 O 2 : C, 69.72; H, Found: C, 69.78, H, (1R,2S,3S,6R)-3-isopropyl-6-methyl-2-triethylsilyloxy-cyclohexanamine (int. 20b) NH 2 OTES int. 20b To a solution of ketone 20 (0.28 g, 1.76 mmol) in ethanol (abs, 5 ml) was added 7 M NH 3 in MeOH (1.4 ml, 10 mmol) followed by Ti(OiPr) 4 (0.57 g, 0.59 ml, 2 mmol) and the mixture was stirred at rt overnight. NaBH 4 (60 mg, 1.5 mmol) was added to the mixture and after 2 h at rt the majority of the solvent was removed in vacuo. The residue was diluted with EtOAc (10 ml) and treated with 2 M NH 3 in water (2 ml) while stirring. After 10 min the reaction mixture was diluted with EtOAc (10 ml) and insoluble Ti-containing compounds were removed via filtration. The organic phase was concentrated in vacuo and water (5 ml) and CHCl 3 (25 ml) were added to the residue. After 10 min of stirring the organic layer was separated and the aqueous one was extracted with CHCl 3 (15 ml). The combined organic extract was washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. Column chromatography (30 g SiO 2, 5% to 10% MeOH in CHCl 3 ) of the residue resulted in the purified target amine (0.24 g, 81%). [α] D (c 0.35, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 3.40 (1 H, dd, J=10.4, 3.7 Hz, CHOSi), 2.93 (1 H, dd, J=3.7, 3.3 Hz, CHNH 2 ), (1 H, m, CH), (3 H, m, CH, CH 2 ), (4 H, m, NH 2, CH), 0.96, 0.87, and 0.71 (3 H, d, J=6.9 Hz, CH 3 ), 0.94 (9H, t, J=7.8 Hz, CH 3 ), (2 H, m, CH), 0.59 (6 H, q, J=7.8 Hz, SiCH 2 ); 13 C NMR (75 MHz, CDCl 3 ) δ 75.4 (CH), 57.2 (CH), 41.3 (CH), 35.2 (CH), 26.8 (CH 2 ), 24.8 (CH), 22.4 (CH 2 ), 21.2 (CH 3 ), 18.5 (CH 3 ), 15.5 (CH 3 ), 6.8 (CH 3 ), 5.1 (CH 2 Si); IR (film, cm -1 ) 3379, 2950, 2930, 2877, 1618, 1466, 1386, 1240, 1100, 1081, 1051, 1008, 801, 749, 726; LRMS [S15]

16 (EI) 285 (M +, 20%), 256 (100), 242 (10); LRMS (CI) 286 (M+H)+; HRMS (ESI) calcd for C 16 H 36 NOSi , found (1S,2R,3R,6S)-2-amino-6-isopropyl-3-methyl-cyclohexanol (22) NH 2 OH 22 To a solution of the amine intermediate 20b (0.23 g, 0.81 mmol) in dry DMF (10 ml) was added EtOH (abs, 0.5 ml) followed by CsF (0.74 g, 4.9 mmol) and the mixture was stirred at rt for 6 h. The solvents were removed in high vacuo, CHCl 3 (25 ml) was added to the residue and after 15 min of stirring all inorganic Cs-containing compounds were removed via filtration. The organic solution was concentrated and column chromatography of the residue (30 g SiO 2, 10% MeOH in CHCl 3 to 10% (7 M NH 3 in MeOH) in CHCl 3 ) furnished the target amino alcohol 22 as an oil that becomes crystalline at rt (0.13 g, 99%). mp o C. [α] 20 D (c 0.68, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ 3.29 (1 H, dd, J=10.8, 4.1 Hz, CHOH), 2.90 (1 H, t, J=3.4 Hz, CHNH 2 ), 2.18 (1 H, d sept, J=7.0, 2.7 Hz, CH), (2 H, m, CH, CH 2 ), 1.41 (1 H, dq, J=13.4, 3.5 Hz, CH 2 ), 1.16 (1 H, dt, J=11.3, 2.9 Hz, CH), 1.03 (1 H, dq, J=12.8, 3.1 Hz, CH), 0.95, 0.91, and 0.81 (3 H, d, J=7.0 Hz, CH 3 ), (1 H, m, CH); 13 C NMR (100 MHz, CDCl 3 ) δ 72.2 (CH), 56.5 (CH), 43.2 (CH), 35.4 (CH), 27.1 (CH 2 ), 25.7 (CH), 22.5 (CH 2 ), 21.0 (CH 3 ), 18.4 (CH 3 ), 15.9 (CH 3 ); IR (film, cm -1 ) 3292, 2957, 2921, 2870, 1632, 1618, 1539, 1442, 1383, 1047; LRMS (EI) 171 (M +, 50%), 156 (33), 128 (38), 110 (18), 70 (100); EA calcd. for C 10 H 21 NO: C, 70.12; H, Found: C, 70.03, H, Triethyl-[(1S,3R,6R)-6-isopropyl-3-methyl-2-methylene-cyclohexoxy]silane (23) OTES g (87%) of this alkene was obtained from 0.29 g of the ketone 20 by following the same procedure that was used for the transformation of intermediate 6a into 9. [α] 20 D (c 0.56, CHCl 3 ); 1 H NMR (300 MHz, CDCl 3 ) δ 5.01 (1 H, d, J=1.4 Hz, CHH=), 4.65 (1 H, d, J=1.6 Hz, CHH=), 3.78 (1 H, d, J=9.7 Hz, CHOTMS), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (1 H, m, CH), (2 H, m, CH 2 ), 1.06, 0.87 and 0.77 (3 H, d, J=7.0 Hz, CH 3 ), 0.94 (9 H, t, J=7.9 Hz, CH 3 ), (1 H, m, CH), 0.62 (6 H, q, J=7.8 Hz, SiCH 2 ); 13 C NMR (75 MHz, CDCl 3 ) δ (C), (CH 2 ), 75.8 (CH), 52.7 (CH), 37.0 (CH), 36.3 (CH 2 ), 25.5 (CH), 22.7 (CH 2 ), 21.4 (CH 3 ), 18.2 (CH 3 ), 15.8 (CH 3 ), 7.0 (CH 3 ), 5.1 (CH 2 Si); IR (film, cm -1 ) 2957, 2927, 2871, 1655, 1466, 1390, 1247, 1121, 1081, 1048, 1008, 908, 828, 745; LRMS (EI) 282 (M +, 12%), 267 (22), 253 (100), 239 (10), 169 (10), 103 (13); HRMS (ESI) calcd for C 17 H 34 OSi , found [S16]

17 IV. Biological evaluation techniques Chemicals All stock solutions were prepared in ethanol. Ethanol did not have an effect on TRPM8 current at the concentrations used (data not shown). Chemicals used in this study had the following concentrations: menthol 0.01 µm, 0.1 µm, 1 µm, 10 µm, 50 µm, 100 µm and 200 µm; allyl alcohol µm, 0.01 µm, 0.1 µm, 1 µm, 10 µm, 50 µm, 100 µm and 200 µm; benzoate µm, 0.1 µm, 1 µm, 10 µm, 50 µm, 100 µm and 200 µm; and diol µm. Cell culture and transient transfection Maintenance and transfection of human embryonic kidney cells (HEK293) cells were performed as previously described [S-10]. Briefly, HEK cells were grown to 80% confluence at 37 C (5% CO 2 ) in Dulbecco s Modified Eagle s Medium (+10% fetal bovine serum, 200 units/ml penicillin and 0.2 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA)). For electrophysiology, cells were plated on glass coverslips and transfected, using the calcium phosphate method with 2 µg of TRPM8 and 0.3 µg of egfp in a 60 mm dish. Electrophysiological recordings were conducted 24 h to 48 h after transfection. For the calcium assays, cells were plated in a 100 mm dish and transfected with 7 µg of total cdna. NOTE: Both the calcium imaging experiments (data not shown) and the electrophysiology measurements were carried out as blind experiments: the experimenter carrying out those procedures was unaware of the structures and names of the compounds being used. Calcium imaging Cells transiently expressing TRPM8 were loaded with Fluo-4 (0.5 µm for 30 min, Invitrogen) in extracellular solution containing 135 mm NaCl, 5 mm KCl, 3 mm CaCl 2, 2 mm MgCl 2, 10 mm D- Glucose, and 10 mm HEPES; ph adjusted to 7.3 or in HBSS supplemented with 1.5 mm each CaCl 2 and MgCl 2. We added the compound of interest ( µm) followed by menthol (100 µm, Sigma) to examine TRPM8 activity. To assess calcium signals we used an Aminco Bowman Series 2 fluorescence spectrometer (Thermo Fisher Scientific) controlled by the AB2 system software. We expressed a change in fluorescence as a fold-change from baseline normalized to background fluorescence (ΔF/F0) [S-11]. Electrophysiological measurements For recordings, cells were placed into a 2 ml bath solution containing: 140 mm NaCl, 1.5 mm CaCl 2, 2 mm MgCl 2, 5 mm KCl, 10 mm HEPES, and 10 mm D-glucose (ph adjusted to 7.4 with NaOH). GFP-transfected cells were identified with an inverted epi-fluorescence microscope (Olympus IX51, Olympus America Inc., USA). Cells were voltage-clamped and TRPM8 currents were measured using the conventional whole-cell patch clamp method. Borosilicate glass (Harvard Apparatus Ltd., UK) pipettes were pulled and polished to 2-5 MΩ resistance with a DMZ-Universal Puller (Zeitz-Instruments GmbH., Martinsried, Germany). Pipettes were filled with an internal solution containing: 120 mm CsCl, 10 mm EGTA, 10 mm HEPES, 3 mm MgCl 2, 2 mm ATP and 0.5 mm GTP (ph adjusted to 7.2 with CsOH). Recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). All recordings were performed at room temperature (22±2ºC). Membrane potential was held at 0 mv and currents were elicited by 200 ms steps between 100 mv and +100 mv at 5 s interval. Drugs were delivered using a gravitational [S17]

18 perfusion system (ALA-VM8, Scientific Instruments) at a rate of 3-4 ml/min. During perfusion of drugs in the recording bath, membrane potential was held at 0 mv and currents were elicited by a ramp protocol from 100 mv to +100 mv at 2 s interval until the current reached the peak. Voltage clamp protocols were applied using a pclamp 10.4 software (Axon Instruments). Data were filtered at 1 khz (8-pole Bessel) and digitized at 10 khz with a Digidata 1440 A converter (Axon Instruments). For whole-cell recordings the series resistance was compensated by 65-80%. The average cell capacitance of the HEK cells was 25.76± 2.03 pf. Statistics Data analysis and offline leak subtraction were completed in Clampfit 10.4 (Axon Instruments), and all the curves were fitted using Origin 7.0 analysis software (OriginLab, Northampton, MA, USA). The drug dose-response relationships were fitted with the Hill equation: n I x I min [ x1] = n n I max EC50 + x where I x is the steady state TRPM8 current in the presence of menthol at concentration [x], I min and I max are the current amplitudes in the absence and presence of a saturating concentration of menthol, respectively, EC 50 is the menthol concentration at which activation is half-maximal, and n is the slope factor. All averaged data are plotted as mean ±SEM and numbers in parentheses reflect the number of cells (n). Statistical analyses were completed with Origin 7.0 analysis software (OriginLab, Northampton, MA, USA), using paired Student s t-test for all the results obtained before and after drug in the same cells. P values < 0.05 were considered statistically significant and n.s. denominates a non-significant finding. IV. References S-1. Camps, F.; Coll, J.; Guitart, J. Tetrahedron. 1986, 42, S-2. Gabriels, S.; Van Haver, D.; Vandewalle, M.; De Clercq, P.; Viterbo, D. Eur. J. Org. Chem. 1999, 8, S-3. Schneider, D. F.; Viljoen, M. S. Tetrahedron. 2002, 58, S-4. Hamada, H. Bull. Chem. Soc. Jpn. 1988, 61, S-5. Takaki, K.; Okada, M.; Yamada, M.; Negoro, K. J. Org. Chem. 1982, 47, S-6. Hong, S.; Lindsay, H. A.; Yaramasu, T.; Zhang, X.; McIntosh, M. C. J. Org. Chem. 2002, 67, S-7. Baudouy, R.; Maliverney, C. Tetrahedron. 1988, 44, S-8. Hodgson, D. M.; Salik, S.; Fox, D. J. J. Org. Chem. 2010, 75, S-9. Mayer, S. C.; Ramanjulu, J.; Vera, M. D.; Pfizenmayer, A. J.; Joullie, M. M. J. Org. Chem. 1994, 59, S-10. Flynn, R.; Chapman, K.; Iftinca, M.; Aboushousha, R.; Varela, D.; Altier, C. J. Biol. Chem. 2014, 289, S-11. Ramachandran, R.; Mihara, K.; Chung, H.; Renaux, B.; DeFea, K.; Bouvier, M.; Hollenberg, M. D. J. Biol. Chem. 2011, 286, VI. Calcium assays an [S18]

19 VI. Electrophysiology Graphs Figure S1 Effect of allyl alcohol 4 and ( )-menthol on TRPM8 current. (A) Representative whole-cell patch clamp menthol-induced (100 µm) currents recorded from HEK cells transfected with TRPM8 in the absence and presence of 4 (top) and ( )-menthol (bottom). Both drugs were used at a concentration of 100 µm. B) Current (I) - Voltage (V) relations for 4- and ( )-menthol -induced (both at 100 µm) normalized TRPM8 currents. (C) 4 (top) and ( )-menthol (bottom) dose-response curves from HEK cells transfected with TRPM8 (n=5 for each data point). Curves represent fits of a Hill equation. EC50 values for 4 and ( )-menthol were 11±1 µm and 33±3 µm, respectively. Data are expressed as means ± SEM. *p<0.05, (paired t-test). [S19]

20 Figure S2. Effect of 15 on menthol induced TRPM8 current. (A) Representative whole-cell patch clamp menthol-induced (100 µm) currents recorded from HEK cells transfected with TRPM8 in the absence (left) and presence (right) of 15 (100 µm). B) Current (I) - Voltage (V) relations of normalized TRPM8 currents. Pre-incubation with 15 completely reverses the menthol-induced current. (C) 15 dose-response curve from HEK cells transfected with TRPM8 (n=5 for each point). Curves represent fits of a Hill equation. IC50 value was 2±1 µm. Data are expressed as means ± SEM. *p<0.05, (paired t-test). [S20]

21 Figure S3. Effect of diol 21 on menthol-induced TRPM8 current. (A) Representative time-course of a menthol-evoked (100µM) current, at a holding potential of +100 mv, in the absence (top) and presence (bottom) of 21. (B) Current (I)- Voltage (V) relations for menthol-induced TRPM8 currents in the absence (top) and presence (bottom) of 21. The n values are in parentheses. (C) Percentage increase in peak current, obtained by a step to +100 mv from a holding potential of 0 mv, following the 1st and 2nd menthol challenges in the absence and presence of 21 compound. Data are expressed as means ± SEM. *p<0.05, (paired t-test). VII. Spectral data - Copies of 1 H and 13 C NMR [S21]

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