1

Synthesis of Ambrox

from ()-nidorellol

Sunisa Suwancharoena, Surachai Pornpakakul

a,b* and Nongnuj Muangsin

a

aResearch Centre for Bioorganic Chemistry, Department of Chemistry, Faculty of Science,

Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand.

bCenter for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Bangkok

10330, Thailand.

psuracha@chula.ac.th

Contents:

1. Experimental details 1-11

2. Figures of GC/MS spectra 12-16

3. Spectroscopic data of compounds 1-10 17-52

Experimental Section

General remarks. Optical rotations were determined at 589 nm on a Perkin-Elmer

Model 341 polarimeter. The melting points were recorded on a MEL-TEMP melting point

apparatus. IR spectra were obtained on a Thermo Scientific Model Nicolet 6700 spectrometer.

The 1H and

13C nuclear magnetic resonance were recorded at 400 and 100 MHz, respectively, on

Varian Model Mercury (400 MHz) and Bruker Model AVANCE (400 MHz) spectrometers,

respectively, in deuterated chloroform (CDCl3). Chemical shifts were reported relative to

residual solvents peaks (H 7.26 ppm, C 77.16 ppm). HRMS were recorded on a Bruker Model

micrOTOF spectrometer and Mass Spectrometer LCT, Micromass UK Limited. X-ray

crystallographic analysis data were collected on a Bruker SMART CCD diffractometer. GC/MS

analysis was performed on a Varian 320-Triple Quadrupole MS, using a VF-5MS column (30 m

x 0.25 mm ID x 0.25 m) and temperature program of 50°C for 0.5 min increasing at 10°C/min

to 210°C and then hold for 5 min. Merck’s TLC Aluminium sheet backed TLC plates coated

with 0.2 mm silicagel 60 F254 (Merck) were used to monitor the reaction course and products.

For chromatographic separations, 230-400 mesh ASTM silica gel (Merck) and 40-60 µm silica

gel 60 RP-18 (Merck) were used as the adsorbent for normal and reverse phase column

chromatography, respectively. Ozone was generated by an OZZON Model 6501T.

2

Isolation of ()-Nidorellol (2)

Croton oblongifolius bark samples were collected from Prachuap Khiri Khan Province in

the southern part of Thailand. Botanical identification was achieved through comparison with a

voucher specimen No. BKF 084729 in the herbarium collection of the Royal Forest Department

of Thailand. The powdered, air-dried stem bark (380 g) of C. oblongifolius was then extracted

with hexane (500 ml x 6) at rt to give a yellowish brown oil (24.9 g). This crude extract was

fractionated by silica gel column chromatography eluting with a hexaneEtOAc gradient in a

stepwise fashion to give 12 combined fractions. The combined fraction 9, eluted with

EtOAchexane (3:7) was re-chromatographed on a reverse phase column using H2OMeOH

(2:8) as mobile phase to afford 2 as a white solid in 0.42% yield.

Compound 2. White solid: Rf = 0.26 (EtOAchexane, 1:1); mp. 78-

80C; []D20

= -23.2o (c = 0.40, CHCl3); IR (KBr) max 3363, 3090,

2920, 1632, 1601, 1387, 1077 cm-1

; 1H NMR (CDCl3, 400 MHz)

0.80 (3H, s, H-19), 0.84 (3H, s, H-20), 0.88 (3H, s, H-18), 0.89 (1H,

m, H-1a), 1.03 (1H, dd, J=1.6, 12.4 Hz, H-5), 1.12 (1H, m, H-3b),

1.14 (3H, s, H-17), 1.26 (1H, t, J = 5.6, H-9), 1.29 (1H, br q, J = 12.0 Hz, H-6b), 1.38 (1H, m, H-

3a), 1.42 (1H, m, H-2b), 1.58 (1H, m, H-2a), 1.60 (1H, m, H-1b), 1.78 (3H, s, H-16), 1.85 (1H,

ddd, J = 1.6, 4.4, 12.8 Hz, H-6a), 2.20 (1H, dt, J = 6.2, 16.0 Hz, H-11a), 2.39 (1H, dt, J = 6.6,

16.0 Hz, H-11b), 3.51 (1H, dd, J = 4.8, 12.0 Hz, H-7), 4.91 (1H, d, J = 10.8 Hz, H-15b), 5.06

(1H, d, J = 17.2 Hz, H-15a), 5.55 (1H, t, J = 7.1 Hz, H-12), 6.33 (1H, dd, J = 10.8, 17.2 Hz, H-

14) ppm; 13

C NMR (CDCl3, 100 MHz) 12.0 (C-16), 15.7 (C-20), 18.0 (C-17), 18.6 (C-2), 21.7

(C-19), 23.6 (C-11), 27.8 (C-6), 33.4 (C-4), 33.6 (C-18), 39.4 (C-10), 39.9 (C-1), 41.7 (C-3),

53.7 (C-5), 60.3 (C-9), 78.2 (C-8), 80.4 (C-7), 110.7 (C-15), 132.8 (C-13), 135.7 (C-12), 141.6

(C-14) ppm; HRESIMS m/z 329.2450 [M+Na]+ (calcd for C20H34O2Na 329.2451).

Cyclization of ()-nidorellol.

To a solution of 2 (353.9 mg, 1.15 mmol) in dichloromethane (50 ml), 20% mol of p-

TsOHH2O was added. After stirring at rt for 3 h, the reaction mixture was transferred into a

separating funnel and sequentially washed with saturated NaHCO3 (2 x 20 ml) and water (3 x 20

ml). The organic layer was separated and dried over anhydrous Na2SO4. Removal of

dichloromethane by evaporation gave a viscous light yellow residue. 1H NMR of the residue

3

showed a 3:1 mixture of 3 and 4. The mixture was isolated on silica gel preparative TLC using

EtOAchexane (3:7) as the mobile phase to give 3 and 4 in 14% and 8% yield, respectively.

Compound 3. Colorless crystals: Rf = 0.41 (EtOAchexane, 3:7); mp. 140-142C; []D20

=

+31.0o (c = 0.29, CHCl3); IR (KBr) max 3353, 2951, 2920, 1051 cm

-1;

1H NMR (CDCl3, 400 MHz) 0.81 (3H,s, H-19), 0.83 (3H, s, H-20),

0.87 (3H, s, H-18), 0.99 (1H, td, J = 4.0, 12.8 Hz, H-1a), 1.04 (1H, dd, J

= 2.0, 13.2 Hz, H-5), 1.13 (3H, s, H-17), 1.16 (1H, td, J = 4.4, 14.4 Hz,

H-3a), 1.32 (1H, q, J = 12.0 Hz, H-6b), 1.42 (1H, m, H-3b), 1.44 (1H,

m, H-2a), 1.46, (1H, m, H-9), 1.48 (1H, m, H-1b), 1.59 (3H, s, H-16),

1.60 (3H, d, J = 6.8 Hz, H-15), 1.66 (1H, m, H-11a), 1.67 (1H, m, H-

2b), 1.80 (1H, dt, J = 5.6, 11.1 Hz, H-11b), 1.89 (1H, ddd, J = 2.0, 4.8, 13.2 Hz, H6-a), 2.51

(1H, br s, OH) 3.68 (1H, dd, J = 4.8, 11.2 Hz, H-7), 4.27 (1H, dd, J = 6.4, 9.2 Hz, H-12), 5.57

(1H, br q, J = 6.8 Hz, H-14) ppm; 13

C NMR (CDCl3, 100 MHz) 12.6 (C-16), 13.2 (C-15), 16.0

(C-20), 18.6 (C-2), 19.7 (C-17), 21.2 (C-19), 28.2 (C-11), 29.2 (C-6), 33.3 (C-4), 33.6 (C-18),

36.0 (C-10), 40.0 (C-1), 42.3 (C-3), 56.0 (C-5), 59.3 (C-9), 79.5 (C-7), 83.6 (C-12), 84.5 (C-8),

119.0 (C-14), 136.4 (C-13) ppm; HRESIMS m/z 329.2459 [M+Na]+ (calcd for C20H34O2Na

329.2457).

Compound 4. White solid: Rf = 0.36 (EtOAchexane, 3:7); mp. 120-

122C; []D20

= -5.7o (c = 0.35, CHCl3); IR (KBr) max 3422, 2950, 2920,

1046 cm-1

; 1H NMR (CDCl3, 400 MHz) 0.83 (6H, s, H-19 and H-20),

0.89 (3H, s, H-18), 0.96 (1H, m, H-1a), 1.05 (1H, dd, J = 1.6, 12.8 Hz, H-

5), 1.15 (3H, s, H-17), 1.16 (1H, m, H-3a), 1.32 (1H, br q, J = 12.4 Hz,

H-6b), 1.37 (1H, m, H-9), 1.41 (1H, m, H-1b), 1.42 (2H, m, H-2a and H-

3b), 1.52 (1H, ddd, J = 2.4, 7.2, 10.4 Hz, H-11a), 1.58 (3H, s, H-16), 1.60 (3H, d, J = 6.8 Hz, H-

15), 1.63 (1H, m, H-2b), 1.90 (1H, m, H-6a), 1.96 (1H, br q, J = 12.0 Hz, H-11b), 3.71 (1H, dd, J

= 4.8, 11.2 Hz, H-7), 4.42 (1H, dd, J = 2.0, 9.2 Hz, H-12), 5.53 (1H, q, J = 6.8 Hz, H-14) ppm;

13C NMR (CDCl3, 100 MHz) 11.8 (C-16), 13.2 (C-15), 15.0 (C-20), 16.3 (C-17), 18.5 (C-2),

21.3 (C-19), 27.7 (C-11), 28.7 (C-6), 33.2 (C-4), 33.7 (C-18), 36.0 (C-10), 39.7 (C-1), 42.3 (C-

3), 56.1 (C-5), 58.1 (C-9), 78.7 (C-7), 81.2 (C-12), 84.7 (C-8), 120.6 (C-14), 136.5 (C-13) ppm;

HRESIMS m/z 329.2449 [M+Na]+ (calcd for C20H34O2Na 329.2451).

4

X-ray crystallography of 3. A colorless crystal was obtained from the combined fraction

obtained from column chromatography eluted with EtOAchexane (3:7) as mobile phase. The

data set was collected at 293(2) K. Cell parameters: a = 12.0921(6) Å, b = 11.7469(6) Å, c =

13.6286(7) Å, V = 1863.68(16) Å3, Z = 4, Dc = 1.092 Mg/m

3, space group = monoclinic, P21/c,

crystal size = 0.30 x 0.25 x 0.18 mm, F(000) = 680. Refinement method was Full-matrix least-

squares on F2. Crystallographic data for the structure of 3 have been deposited at the Cambridge

Crystallographic Data Centre with the deposition number 823857. Copies of this information

may be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union

Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk or

http://www.ccdc.cam.ac.uk).

Synthesis of 5.

To a solution of a 3:1 mixture of 3 and 4 (50.3 mg, 0.16 mmol) in 5% (v/v) H2O in

acetone (20 ml) at -78oC, an O3 stream was bubbled for 3 h. Then, the solution was bubbled with

N2 for 30 min, diluted with water (20 ml) and extracted with dichloromethane (3 x 10 ml). The

organic phase was washed successively with saturated NaHCO3 (10 ml) and water (2 x 10 ml)

and dried over anhydrous Na2SO4. The solvent was removed by evaporation to give a mixture of

ketones 5 (5a and 5b) and lactone 6 as a colorless viscous oil. The mixture of 5a, 5b and 6 was

then subjected to silica gel column chromatography and eluted with EtOAchexane (2:3) to

afford a white solid of 6 in 19% yield, a colorless oil of 5a in 15% yield and a colorless oil of 5b

in 14% yield.

Compound 5a. Colorless viscous oil: Rf = 0.16 (EtOAchexane, 4:6);

[]D20

= -14.7o (c = 0.38, CHCl3); IR (KBr) max 3432, 2923, 1715, 1044

cm-1

; 1

H NMR (CDCl3, 400 MHz) 0.83 (3H, s, H-19), 0.84 (3H, s, H-

20), 0.89 (3H, s, H-18), 0.95 (1H, br t, J = 12.8 Hz, H1a), 1.04 (1H, br d,

5

J = 12.8 Hz, H-5), 1.15 (1H, m, H-3a), 1.17 (3H, s, H-17), 1.25 (1H, dd, J = 7.6, 12.4 Hz, H-9),

1.34 (1H, br q, J = 12.8 Hz, H-6b), 1.42 (1H, m, H-3b), 1.43 (1H, m, H-2a), 1.44 (1H, m, H-1b),

1.65 (1H, m, H-2b), 1.91 (1H, m, H-11a), 1.93 (1H, m, H-6a), 2.10 (1H, br q, J = 12.0 Hz, H-

11b), 2.21 (3H, s, H-14), 3.75 (1H, dd, J = 3.6, 11.2 Hz, H-7), 4.41 (1H, br d, J = 10.4 Hz, H-12)

ppm; 13

C NMR (CDCl3, 100 MHz) 15.1 (C-20), 16.3 (C-17), 18.4 (C-2), 21.2 (C-19), 26.7 (C-

11), 26.9 (C-14), 29.1 (C-6), 33.2 (C-4), 33.6 (C-18), 36.0 (C-10), 39.7 (C-1), 42.2 (C-3), 56.0

(C-5), 57.7 (C-9), 78.7 (C-7), 81.3 (C-12), 86.2 (C-8), 210.4 (C-13) ppm. HRESIMS m/z

317.2089 [M+H]+ (calcd for C16H27O3 317.2087).

Compound 5b. Colorless viscous oil: Rf = 0.16 (EtOAchexane, 4:6);

[]D20

= +4.0o (c = 0.28, CHCl3); IR (KBr) max 3425, 2927, 1715, 1047

cm-1

; 1H NMR (CDCl3, 400 MHz) 0.83 (6H, s, H-19 and H-20), 0.89

(3H, s, H-18), 1.00 (1H, m, H-1a), 1.04 (1H, dd, J = 2.4, 12.8 Hz, H-5),

1.08 (3H, s, H-17), 1.17 (1H, m, H-3a), 1.32 (1H, m, H-6b), 1.43 (1H,

m, H-3b), 1.44 (1H, m, H-9), 1.46 (2H, m, H-2), 1.49 (1H, m, H-1b),

1.89 (1H, m, H-11b), 1.92 (1H, m, H-6a), 2.08 (1H, ddd, J = 6.4, 8.0, 12.0 Hz, H-11a), 2.25 (3H,

s, H-14), 3.70 (1H, dd, J = 4.8, 11.2 Hz, H-7), 4.32 (1H, dd, J = 8.0, 8.8 Hz, H-12) ppm; 13

C

NMR (CDCl3, 100 MHz) 15.5 (C-20), 17.8 (C-17), 18.4 (C-2), 21.2 (C-19), 25.9 (C-11), 27.0

(C-14), 29.3 (C-6), 33.2 (C-4), 33.6 (C-18), 36.0 (C-10), 39.7 (C-1), 42.2 (C-3), 55.9 (C-5), 59.9

(C-9), 78.9 (C-7), 82.9 (C-12), 85.9 (C-8), 211.1 (C-13) ppm; HRESIMS m/z 317.2096 [M+H]+

(calcd for C16H27O3 317.2087).

Synthesis of 7-hydroxy-sclareolide (6)

(i) via ozonolysis of a mixture of 3 and 4.

To a solution of the mixture of 3 and 4 (351.8 mg, 1.15 mmol) in 5% (v/v) H2O in

acetone (50 ml) at -78oC, an O3 stream was bubbled for 3 h. Then, the solution was bubbled with

N2 for 30 min, diluted with water (30 ml) and extracted with dichloromethane (3 x 20 ml). The

organic phase was washed successively with saturated NaHCO3 (10 ml) and water (2 x 20ml)

and dried over anhydrous Na2SO4. The solvent was removed by evaporation to give a colorless

viscous oil (285 mg). The viscous oil was exposed to air at ambient temperature for 14 days and

6

compound 6 self crystallized. After recrystallization from acetone, colorless crystals of 6 were

obtained in 90% yield.

(ii) via ozonolysis of ()-nidorellol.

To a solution of 2 (50.1 mg, 0.16 mmol) in 5% (v/v) H2O in acetone (25 ml) at -78oC, an

O3 stream was bubbled for 3 h. Then, the solution was bubbled with N2 for 30 min, diluted with

water (20 ml) and extracted with dichloromethane (3 x 10 ml). The organic phase was washed

successively with saturated NaHCO3 (10 ml) and water (2 x 10 ml) and dried over anhydrous

Na2SO4. The solvent was removed by evaporation to give a colorless viscous oil (46.3 mg). The

viscous oil was exposed to air at ambient temperature for 3 weeks during which time white solids

appeared in the viscous oil. Isolation on silica gel column chromatography eluting with

EtOAchexane (3:7) as the mobile phase led to white solids of 6 in 47% yield.

Compound 6: Rf = 0.19 (EtOAchexane, 3:7); mp. 144-146C; []D20

=

-25.0o (c = 0.30, CHCl3); IR (KBr) max 3408, 2953, 1781, 1133, 1053

cm-1

; 1H NMR (CDCl3, 400 MHz) 0.84 (3H, s, H-19), 0.90 (3H, s, H-

18), 0.91 (3H, s, H-20), 1.00 (1H, br t, J = 11.2 Hz, H-1a), 1.11 (1H, br

d, J = 13.2 Hz, H-5), 1.17 (1H, td, J = 4.0, 14.0 Hz, H-3a), 1.32 (3H, s,

H-17), 1.39 (1H, br q, J = 12.8 Hz, H-6b), 1.42 (1H, m, H-1b), 1.44 (1H, m, H-3b), 1.46 (1H, m,

H-2a), 1.66 (1H, m, H-2b), 1.82 (1H, dd, J = 6.6, 14.8 Hz, H-9), 2.01 (1H, br d, J = 13.6 Hz, H-

6a), 2.26, (1H, dd, J = 6.6, 16.0 Hz, H-11a), 2.48 (1H, dd, J = 15.2, 16.0 Hz, H-11b), 3.88 (1H,

dd, J = 4.7, 11.0 Hz, H-7) ppm; 13

C NMR (CDCl3, 100 MHz) 15.2 (C-20), 16.5 (C-17), 18.2

(C-2), 21.0 (C-19), 28.4 (C-11), 29.0 (C-6), 33.2 (C-4), 33.3 (C-18), 35.6 (C-10), 39.3 (C-1),

42.0 (C-3), 55.4 (C-5), 57.0 (C-9), 76.8 (C-7), 89.7 (C-8), 176.3 (C-12) ppm; HRESIMS m/z

267.1952 [M+H]+ (calcd for C16H27O3 267.1955).

X-ray crystallography of 6. A colorless crystal of 6 was obtained from acetone. The data set

was collected at 293(2) K. Cell parameters: a = 6.2810(5) Å, b = 35.810(4) Å, c = 7.3289(9) Å, V

= 14925(3) Å3, Z = 4, Dc = 1.181 Mg/m

3, space group = monoclinic, P2(1), crystal size = 0.18 x

0.20 x 0.30 mm, F(000) = 580. Refinement method was Full-matrix least-squares on F2.

Crystallographic data for the structure of 6 have been deposited at the Cambridge

Crystallographic Data Centre with the deposition number 820557. Copies of this information

7

may be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union

Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk or

http://www.ccdc.cam.ac.uk).

Synthesis of 7. To a suspension of LiAlH4 (449.5 mg) in dried THF (10 ml) a solution of

7-hydroxy sclareolide 6 (262.9 mg, 0.99 mmol) in dried THF (10 ml) was slowly added at 0oC.

The reaction mixture was stirred for 5 h under argon at rt. Then, an aqueous solution of 5% (v/v)

HCl (15 ml) was added and then extracted with Et2O (4 x 10 ml). The organic layer was washed

with brine (20 ml) and dried over anhydrous Na2SO4. Removal of the solvent afforded a white

solid residue. The residue was crystallized from acetone with a small amount of water to give a

white solid of 7 in 91% yield:

Rf = 0.24 (EtOAc); mp. 210-211C; []D20

= +1.6o (c = 0.31, MeOH); IR

(KBr) max 3295, 2947, 1386, 1097, 1050,1014 cm-1

; 1H NMR (CDCl3 and

2 drops of MeOD, 400 MHz) 0.76 (3H, s, H-20), 0.77 (3H, s, H-19), 0.85

(3H, s, H-18), 0.86 (1H, m, H-1a), 1.00 (1H, dd, J = 1.6, 12.4 Hz, H-5),

1.09 (3H, s, H-17), 1.11 (1H, m, H-3a), 1.15 (1H, m, H-9), 1.26 (1H, q, J =

12.4 Hz, H-6a), 1.37 (1H, m, H-3b), 1.40 (1H, m, H-2a), 1.57 (1H, m, H-2b), 1.58 (1H, m, H-

1b), 1.63 (2H, m, H-11), 1.81 (1H, ddd, J = 1.6, 4.0, 12.8 Hz, H-6b), 3.37 (1H, td, J = 4.0, 10.0

Hz, H-12b), 3.48 (1H, dd, J = 4.4, 11.6 Hz, H-7), 3.73 (1H, dt, J = 4.4, 10.0 Hz, H-12a) ppm; 13

C

NMR (CDCl3 and 2 drops of MeOD, 100 MHz) 15.5 (C-20), 18.0 (C-17), 18.3 (C-2), 21.5 (C-

19), 27.1 (C-11), 27.9 (C-6), 33.3 (C-4), 33.4 (C-18), 39.1 (C-10), 39.2 (C-1), 41.8 (C-3), 53.8

(C-5), 57.4 (C-9), 63.7 (C-12), 76.6 (C-8), 80.3 (C-7) ppm; HRESIMS m/z 293.2094 [M+Na]+

(calcd for C16H30O3Na 293.2087).

8

Synthesis of 7-hydroxy-ambrox 8.

Triol 7 (8.6 mg, 0.032 mmol) was stirred with pyridine (0.8 ml) at rt for 20 min. To this solution

TsCl (18.2 mg) was added and the mixture was continually stirred for 3 h under argon. The

reaction mixture was diluted with water (10 ml) and then extracted with Et2O (4 x 10 ml). The

organic phase was washed successively with 2 M HCl (2 x 10 ml), saturated NaHCO3 (2 x 10

ml) and water (20 ml). Removal of the solvent afforded a crude product as a white solid.

Purification on silica gel column chromatography eluting with CH2Cl2EtOAc (5:5) as the

mobile phase led to colorless needles of 7-hydroxy-ambrox 8 in 90% yield:

Rf = 0.33 (EtOAchexane, 1:1); mp. 136-138C; []D20

= +31.7o (c = 0.34,

CHCl3); IR (KBr) max 3425, 2917, 1383, 1034, 1001 cm-1

; 1H NMR

(CDCl3, 400Mz) 0.83 (3H, s, H-19), 0.84 (3H, s, H-20), 0.89 (3H, s, H-

18), 0.98 (1H, td, J = 3.6, 13.2 Hz, H-1a), 1.04 (1H, dd, J = 2.8, 12.8 Hz, H-

5), 1.09 (3H, s, H-17), 1.16 (1H, td, J = 4.4, 13.6 Hz, H-3a), 1.30 (1H, m, H-

9), 1.33 (1H, q, J = 12.8 Hz, H-6a), 1.42 (1H, m, H-3b), 1.43 (1H, m, H-2a), 1.48 (1H, m, H-1b),

1.65 (1H, m, H-2b), 1.78 (1H, m, H-11a), 1.80 (1H, m, H-11b), 1.89 (1H, ddd, J = 2.8, 4.8, 13.6

Hz, H-6b), 3.64 (1H, dd, J = 4.8, 11.2 Hz, H-7), 3.82 (1H, q, J = 8.4 Hz, H-12a), 3.93 (1H, td, J

= 3.6, 8.8 Hz, H-12b) ppm; 13

C NMR (CDCl3, 100 MHz) 15.2 (C-20), 15.9 (C-17), 18.5 (C-2),

21.3 (C-19), 22.5 (C-11), 29.1 (C-6), 33.2 (C-4), 33.7 (C-18), 35.9 (C-10), 39.8 (C-1), 42.3 (C-

3), 56.1 (C-5), 58.6 (C-9), 65.5 (C-12), 78.9 (C-7), 83.8 (C-8) ppm; HRESIMS m/z 253.2154

[M+H]+ (calcd for C16H29O2 253.2162).

Synthesis of 7-oxo-ambrox (9).

To the solution of 7-hydroxy-ambrox 8 (28.3 mg, 0.22 mmol) in dried CH2Cl2 (5 ml) was

added PCC (169.2 mg) and continually stirred for 4 hours under argon. The mixture was diluted

with CH2Cl2 (5 ml), filtered through silica gel and flushed with Et2O (5 ml x 2). The filtrate was

evaporated under reduced pressure and colorless needles of 7-oxo-ambrox 9 were obtained in

98% yield:

Rf = 0.42 (EtOAchexane, 1:1); mp. 130-132C; []D20

= +112.9o (c = 0.35,

CHCl3); IR (KBr) max 2923, 1721, 1070, 1007 cm-1

; 1H NMR (CDCl3, 400

MHz) 0.86 (3H, s, H-19), 0.87 (3H, s, H-18), 1.05 (3H, s, H-20), 1.06 (1H,

9

m, H-1a), 1.18 (1H, td, J = 4.5, 13.7 Hz, H-3a), 1.32 (3H, s, H-17), 1.35 (1H, dd, J = 2.8, 14.0

Hz, H-5), 1.47 (1H, m, H-3b), 1.50 (1, m, H-2a), 1.60 (1H, br d, J = 13.2 Hz, H-1b), 1.70 (1H, m,

H-2b), 1.71 (1H, dd, J = 6.0, 12.8 Hz, H-9), 1.88 (2H, m, H-11), 2.39 (1H, dd, J = 2.8, 13.6 Hz,

H-6a), 2.51 (1H, t, J = 13.6 Hz, H-6b), 3.88 (1H, q, J = 8.4 Hz, H-12a), 3.98 (1H, td, J = 3.0, 8.7

Hz, H-12b) ppm; 13

C NMR (CDCl3, 100 MHz) 14.7 (C-20), 18.3 (C-2), 20.2 (C-17), 20.8 (C-

19), 22.0 (C-11), 33.2 (C-18), 33.9 (C-4), 36.2 (C-10), 37.2 (C-6), 39.6 (C-1), 41.9 (C-3), 59.3

(C-5), 60.8 (C-9), 65.4 (C-12), 86.2 (C-8), 209.4 (C-7) ppm; HRESIMS m/z 251.2006 [M+H]+

(calcd for C16H27O2 251.2005).

Treatment of 7-oxo-ambrox (9) with p-Toluenesulfonylhydrazine.

To the solution of 7-oxo-ambrox 9 (26.5 mg, 0.11 mmol) in dried THF (5 ml) was added

TsNHNH2 (59.1 mg) and refluxed for 5 h. The reaction mixture was cooled to rt and evaporated

under reduced pressure to give a white solid residue. Purification on silica gel column

chromatography and eluted with EtOAchexane (3:7) as the mobile phase afforded 10 as

colorless needles in 91% yield:

Rf = 0.40 (EtOAchexane, 3:7); mp. 190-192C; []D20

= +62.0o (c =

0.30, CHCl3); IR (KBr) max 3169 (NH), 3060, 2927, 1329, 1160,

1017 cm-1

; 1H NMR (CDCl3, 400 MHz) 0.80 (3H, s, H-19), 0.84

(3H, s, H-18), 0.87 (3H,s, H-20), 0.90 (1H, m, H-1a), 0.97 (1H, br d,

J = 13.2 Hz, H-5), 1.10 (1H, td, J = 3.6, 14.8 Hz, H-3a), 1.16 (3H, s, H-17), 1.42 (1H, m, H-3b),

1.44 (1H, m, H-2a), 1.45 (1H, m, H-9), 1.48 (1H, m, H-1b), 1.64 (1H, m, H-2b), 1.68-1.75 (2H,

m, H-11), 2.06 (1H, t, J = 14.2 Hz, H-6a), 2.42 (3H, s, 4′-CH3), 2.42 (1H, br d, J = 13.2 Hz, H-

6b), 3.86 (1H, q, J = 8.4 Hz, H-12a), 3.96 (1H, br dd, J = 7.5, 13.1 Hz, H-12b), 7.28 (2H, d, J =

8.0 Hz, H-3′,H-5′), 7.79 (2H, d, J = 8.0 Hz, H-2′, H-6′), 10.37 (1H, s, -NH) ppm; 13

C NMR

(CDCl3, 100 MHz) : 14.5(C-20), 18.3 (C-2), 20.1 (C-17), 20.8 (C-19), 21.1 (C-11), 21.7 (4′-

CH3), 31.0 (C-6), 33.3 (C-18), 33.3 (C-4), 35.6 (C-10), 39.4 (C-1), 42.1 (C-3), 57.5 (C-5), 60.5

(C-9), 66.1 (C-12), 85.7 (C-8), 127.9 (C-2′ and C-6′), 129.4 (C-3′ and C-5′), 136.5 (C-4′), 143.3

(C-1′), 158.0 (C-7) ppm; HRESIMS m/z 441.2183 [M+Na]+ (calcd for C23H34N2O3Na 441.2182).

10

Synthesis of Ambrox

.

(i) via reduction of 10 with NaBH4.

To the solution of 10 (31.9 mg, 0.07 mmol) in dried THF (5 ml) was added NaBH4 (57.6

mg) and refluxed overnight. After cooling to rt the mixture was diluted with Et2O (10 ml). The

solution was then sequentially washed with water (10 ml), saturated NaHCO3 (10 ml), 2 M HCl

(10 ml) and water (10ml). The organic layer was dried over anhydrous Na2SO4 and evaporated

under reduced pressure to give a white solid residue. Purification on silica gel column

chromatography eluted with EtOAchexane (1:4) as the mobile phase afforded ()-ambrox as

colorless needles in 33% yield.

(ii) via reduction of 10 with NaBH3CN

Tothe solution of 10 (20.1 mg, 0.05 mmol) in dried DMF (1ml) and sulfolane (1ml) was

added p-TsOH (12.9 mg) and NaBH3CN (31.4 mg) and continually stirred under argon for 4 h.

After cooling to rt the mixture was diluted with water (10 ml) and extracted with Et2O (3 x 10

ml). The organic phase was washed successively with water (2 x 10 ml) and brine (10 ml), dried

over anhydrous Na2SO4 and evaporated under reduced pressure to give a viscous oil residue.

Purification on silica gel column chromatography eluted with EtOAchexane (1:4) as the mobile

phase afforded ()-ambrox as colorless needles in 15% yield.

(iii) Via Wolff-Kishner reduction of 9.

To a solution of compound 9 (30.1 mg, 0.12 mmol) in diethylene glycol (3 ml) was added

KOH (41.1 mg) and 98% hydrazine hydrate (0.1 ml) and the mixture was then refluxed for 1 h at

160C followed by 3 h at 200C. After cooling to rt, the mixture was diluted with water (10 ml),

neutralized with 2 M HCl and extracted with EtOAc (4 x 10 ml). The organic layer was dried

over anhydrous Na2SO4 and evaporated under reduced pressure to give a viscous oil residue.

Purification on silica gel column chromatography eluted with EtOAc-hexane (1:4) as the mobile

phase afforded ()-ambrox as colorless needles in 74% yield:

11

Rf = 0.59 (EtOAc-hexane, 3:7); mp. 72-74C []D20

= +23.0o (c = 0.25,

CHCl3); IR (KBr) max 2920, 1456, 1376, 1004 cm-1

; 1H NMR (CDCl3, 300

MHz) 0.83 (6H, s, H-19 and H-20), 0.87 (3H, s, H-18), 0.96 (1H, dd, J =

2.0, 12.4 Hz, H-5), 1.03 (1H, td, J = 2.0, 12.8 Hz, H-1b), 1.08 (3H, s, H-

17), 1.18 (1H, td, J = 4.0, 13.6 Hz, H-3a), 1.29 (1H, td, J= 2.0, 12.8 Hz, H-

6a), 1.38 (1H, m, H-7b), 1.40 (1H, m, H-9), 1.41 (1H, m, H-3a), 1.42 (1H,

m, H-2b), 1.47 (1H, m, H-1a), 1.66 (1H, m, H-2a), 1.68-1.76 (2H, m, H-11), 1.75 (1H, m, H-6b),

1.94 (1H, dt, J = 3.2, 11.2 Hz, H-7a), 3.82 (1H, br q, J = 8.2 Hz, H-12b), 3.91 (1H, m, H-12a)

ppm; 13

C NMR (CDCl3, 100 MHz) 15.2 (C-20), 18.6 (C-2), 20.8 (C-6), 21.3 (C-17), 21.3 (C-

19), 22.8 (C-11), 33.2 (C-4), 33.7 (C-18), 36.3 (C-10), 39.9 (C-7), 40.1 (C-1), 42.6 (C-3), 57.4

(C-5), 60.3 (C-9), 65.1 (C-12), 80.0 (C-8) ppm; HRESIMS m/z 259.2045 [M+Na]+ (calcd for

C16H28ONa 259.2032).

()-ambrox

12

Figure 1. GC/MS spectra of acetic acid in a sealed vials showing (a) the GC/MS result analyzed

by using headspace technique and (b-d) selected ion peaks corresponding to acetic acid and its

fragmentation at (b) m/z 43.0 [CH3CO+], (c) 45.0 [M-CH3]

+ and (d) 60.0 [M

+].

a.

b.

c.

d.

13

Figure 2. GC/MS spectra of a sealed blank vial showing (a) the GC/MS result analyzed by using

headspace technique and (b-d) selected ion peaks corresponding to acetic acid and its

fragmentation at (b) m/z 43.0 [CH3CO+], (c) 45.0 [M-CH3]

+ and (d) 60.0 [M

+].

a.

b.

c.

d.

14

Figure 3. GC/MS spectra of the volatile product from air-auto oxidation of ketone 5a in a sealed

vial showing (a) the GC/MS result analyzed by using headspace technique and (b-d) selected ion

peaks corresponding to acetic acid and its fragmentation at (b) m/z 43.0 [CH3CO+], (c) 45.0 [M-

CH3]+ and (d) 60.0 [M

+].

a.

b.

c.

d.

15

Figure 4. GC/MS spectra of the volatile product from air-auto oxidation of ketone 5b in a sealed

vial showing (a) the GC/MS result analyzed by using headspace technique and (b-d) selected ion

peaks corresponding to acetic acid and its fragmentation at (b) m/z 43.0 [CH3CO+], (c) 45.0 [M-

CH3]+ and (d) 60.0 [M

+].

Scheme shows the proposed mechanism of air oxidative degradation of 5

a.

b.

c.

d.

16

HOH

O

O

HOH

O

O

5 6H

OH

O

O

O

O H

H3C

O

OH+O2

no O2 (stored in desiccator under anaerobic condition)X

12

17

Spectroscopic data of compound 1-10

Compound 1

18

19

20

21

22

23

Compound 2

24

25

26

27

28

29

Compound 3

30

31

32

Compound 4

33

34

35

Compound 5a

36

37

Compound 5b

38

39

40

Compound 6

41

42

43

Compound 7

44

45

46

Compound 8

47

48

49

Compound 9

50

51

Compound 10

52