1
Supporting Information
Discovery, Total Synthesis and SAR of Anaenamides A and B: Anticancer Cyanobacterial Depsipeptides with a Chlorinated Pharmacophore
David A. Brumley,†,§ Sarath P. Gunasekera,‡ Qi-Yin Chen,†,§ Valerie J. Paul,‡ and Hendrik Luesch†,§,*
†Department of Medicinal Chemistry and §Center for Natural Products, Drug Discovery and
Development (CNPD3), University of Florida, 1345 Center Drive, Gainesville, Florida 32610, United States
‡Smithsonian Marine Station at Ft. Pierce, 701 Seaway Drive, Ft. Pierce, FL 34949, United States
Contents Page
General Experimental Procedures 4
Collection, Extraction and Isolation 4
Methylation of anaenoic acid (2) 5
Structural elucidation of anaenamide A (1a), B (1b), and anaenoic acid (2) 6 Table S1. Tabulated NMR Data for Anaenamides A (1a) and B (1b) 7 Table S2. Tabulated NMR Data for Anaenoic acid (2) 8
Acid Hydrolysis and Enantioselective HPLC analysis 9
Scheme S1. Proposed Anaenamide Biosynthetic Pathway 9
General Cell Culture Procedure 9
Cell Viability Assay (MTT) 10
Total Synthesis of 1a–1c and 2 10
Supplementary Reference 18
Figure S1. 1H NMR spectrum of OBn-12 in CDCl3 (600 MHz) 19 Figure S2. 13C NMR spectrum of OBn-12 in CDCl3 (150 MHz) 20 Figure S3. HSQC spectrum of OBn-12 in CDCl3 (600 MHz) 21 Figure S4. 1H NMR spectrum of 12 in CDCl3 (600 MHz) 22
2
Figure S5. 13C NMR spectrum of 12 in CDCl3 (150 MHz) 23
Figure S6. HSQC spectrum of 12 in CDCl3 (600 MHz) 24
Figure S7. 1H NMR spectrum of OBn-2 in CDCl3 (600 MHz) 25 Figure S8. 13C NMR spectrum of OBn-12 in CDCl3 (150 MHz) 26 Figure S9. 1H NMR spectrum of synthetic 2 in CDCl3 (600 MHz) 27 Figure S10. 13C NMR spectrum of synthetic 2 in CDCl3 (150 MHz) 28
Figure S11. HSQC spectrum of synthetic 2 in CDCl3 29
Figure S12. 1H NMR spectrum of Boc-3a in CDCl3 (600 MHz) 30 Figure S13. 13C NMR spectrum of Boc-3a in CDCl3 (150 MHz) 31 Figure S14. 1H NMR spectrum of Boc-3b in CDCl3 (600 MHz) 32 Figure S15. 13C NMR spectrum of Boc-3b in CDCl3 (150 MHz) 33 Figure S16 Comparisons of 1H NMR data for synthetic 1a with isolated material 34 Figure S17 Comparisons of 13C NMR data for synthetic 1a with isolated material 37 Figure S18 Comparisons of 1H NMR data for synthetic 1b with isolated material 40 Figure S19 Comparisons of 13C NMR data for synthetic 1b with isolated material 43 Figure S20 Comparisons of 1H NMR data for synthetic 2 with isolated material 46 Figure S21 Comparisons of 13C NMR data for synthetic 2 with isolated material 49 Figure S22. 1H NMR spectrum of 1c in CDCl3 (600 MHz) 52 Figure S23. 13C NMR spectrum of 1c in CDCl3 (150 MHz) 53
Figure S24. HSQC spectrum of 1c in CDCl3 54
Figure S25 Determination of compound purity for 1a 55
Figure S26 Determination of compound purity for 1b 56
Figure S27 Determination of compound purity for 2 57
Figure S28 Determination of compound purity for 1c 58 Figure S29. 1H NMR spectrum of natural 2 in CDCl3 (600 MHz) 59 Figure S30. 13C NMR spectrum of natural 2 in CDCl3 (150 MHz) 60
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Figure S32. HSQC spectrum of natural 2 in CDCl3 62
Figure S33. HMBCspectrum of natural 2 in CDCl3 63 Figure S34. 1H NMR spectrum of natural 1a in CDCl3 (600 MHz) 64 Figure S35. 13C NMR spectrum of natural 1a in CDCl3 (150 MHz) 65 Figure S36. COSYspectrum of natural 1a in CDCl3 66
Figure S37. HSQC spectrum of natural 1a in CDCl3 67
Figure S38. HMBCspectrum of natural 1a in CDCl3 68
Figure S39. NOESYspectrum of natural 1a in CDCl3 69
Figure S40. 1H NMR spectrum of natural 1b in CDCl3 (600 MHz) 70 Figure S41. 13C NMR spectrum of natural 1b in CDCl3 (150 MHz) 71 Figure S42. COSYspectrum of natural 1b in CDCl3 72
Figure S43. HSQC spectrum of natural 1b in CDCl3 73
Figure S44. HMBCspectrum of natural 1b in CDCl3 74
4 General Experimental Procedures
1
The optical rotations were recorded on a Rudolph Research Analytical Autopol III automatic 2
polarimeter or a Perkin-Elmer 341 polarimeter (Na D line) using a microcell of 1 dm path length. 3
UV spectrophotometric data was acquired on a Shimadzu PharmaSpec UVvisible 4
spectrophotometer. NMR data were collected on a JEOL ECA-600 spectrometer or a Bruker 5
Avance II 600 MHz, high-resolution 5-mm cryoprobe spectrometer operating at 600 MHz for 1H 6
and 150 MHz for 13C. The edited-HSQC experiment was optimized for JCH = 140 Hz and the 7
HMBC spectrum was optimized for 2/3JCH = 8 Hz. 1H NMR chemical shifts (referenced to residual 8
CHCl3 observed at 7.26) were assigned using a combination of data from 2D DQF COSY and 9
multiplicity-edited HSQC experiments. Similarly, 13C NMR chemical shifts (referenced to CDCl3 10
observed at 77.16 were assigned on the basis of multiplicity-edited HSQC experiments. The 11
HRMS data were obtained using a Q Exactive Focus with electrospray ionization (ESI) at UF’s 12
Center for Natural Products, Drug Discovery and Development (CNPD3). Silica gel 60 (EMD 13
Chemicals, Inc. 230400 mesh) was used for column chromatography. All solvents were HPLC 14
or LCMS grade (Fisher Scientific). 15
16
Collection, Extraction, and Isolation
17
The sample of green filamentous cyanobacterial assemblage (VPG16-5) was collected on 18
November 30, 2016 from Anae Island, Guam and processed for DNA sequencing of 16S rDNA as 19
previously detailed in Brumley et al.1 A BLASTn search of the resulting sequence indicated a 20
complete match (i.e., 100% identity) to VPG16-59 (accessioned as MH578561). A voucher 21
specimen (VPG16-5) is maintained at the Smithsonian Marine Station, Fort Pierce, FL, and the 22
sequence was deposited in GenBank as MT218338. 23
The freeze-dried material (7.121 g) was first extracted with EtOAc−50% MeOH followed by 24
MeOH−10% H2O. The combined lipophilic (0.090 g) and the polar extract (0.935 g) was 25
partitioned between EtOAc and H2O. Concentration of the extracts furnished 0.315 g of EtOAc-26
soluble fraction and 0.670 g of water-soluble fraction. The EtOAc-soluble fraction (0.315 g) was 27
chromatographed on a column of SiO2 (15 g) using a step gradient system of hexanesEtOAc, 28
EtOAc, EtOAcMeOH and MeOH to give five fractions (a –e). The first two sub-29
fractions (a, b) were eluted with hexanesEtOAc. A portion (10 mg ) of sub-fraction c (93 30
mg) eluted with EtOAc on further purification by reversed-phase HPLC (semi-prep 250 x 10 mm, 31
5 m, RP-18, flow 3.0 mL/min) using MeOH15% H2O yielded anaenoic acid (2) (1.0 mg, tR = 32
15.7 min, yield, 0.13% dry wt, based on 10% yield of 2 in entire 93 mg of fraction c). The sub-33
fractions b (0.020 g) was further chromatographed on a column of C18 (5 g) using a step gradient 34
system of MeOHH2O, MeOHH2O and MeOHH2O to give 3 sub-fractions (f – 35
h), respectively. The sub-fraction g (0.012 g) eluted with MeOHH2O was further purified 36
by reversed-phase HPLC (semi-prep 250 x 10 mm, 5 m, RP-18, flow 3.0 mL/min) using 37
5
MeOH10% H2O to give anaenamide A (1a) (4.1 mg, tR = 11.5 min, yield, 0.05% dry wt) and 1
anaenamide B (1b) (0.9 mg, tR = 12.5 min, yield, 0.01% dry wt). 2
3
Anaenamide A (1a): colorless, solid; []25
D 46 (c 0.29, CHCl3); UV (MeOH) max (log )
4
225sh (3.98), 276 (2.97) nm; 1H and 13C NMR data, see Table S1, assignments were made by 5
interpretation of 2D DQF COSY, edited-HSQC, HMBC and NOESY data; HRMS (ESI) m/z
6
[M+H]+ calcd for C27H39NO835/37Cl, 540.2364/542.2334; found 540.2349/542.2322. 7
Anaenamide B (1b): colorless, solid; []25
D 38 (c 0.06, CHCl3); UV (MeOH) max (log )
8
225sh (3.82), 276 (2.97) nm; 1H and 13C NMR data, see Table S1, assignments were made by 9
interpretation of 2D DQF COSY, edited-HSQC and HMBC data; HRMS (ESI) m/z [M+H]+ calcd 10
for C27H39NO835/37Cl, 540.2364/542.2334; found 540.2351/542.2330. 11
Anaenoic acid (2): colorless, solid; []25
D 35 (c 0.15, CHCl3); UV (MeOH) max (log ) 203
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(4.39), 276 (3.29) nm; 1H and 13C NMR data, see Table S2, assignments were made by 13
interpretation of 2D DQF COSY, edited-HSQC, HMBC and NOESY data; HRMS (ESI) m/z
14
[M+Na]+ calcd for C22H32O7Na, 431.2045; found 431.2041. 15
Methylation of Anaenoic acid (2)
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Compound 2 (1.5 mg) was dissolved in 1.0 mL of MeOH and cooled to 4 oC. The cooled solution 17
was treated with 50 L of 2.0 M trimethylsilyl diazomethane in diethyl ether. Evaporation of 18
MeOH and excess trimethylsilyl diazomethane gave an oily product. This product was purified by 19
reversed-phase HPLC (semi-prep 250 x 10 mm, 5 m, RP-18, flow 3.0 mL/min) using 20
MeOH10% H2O to give anaenoic acid methyl ester (1.5 mg, tR = 12.7 min). 21
Anaenoic acid methyl ester: colorless, solid; []25
D (c 0.05, CHCl3); UV (MeOH) max 22 (log ) 276 (3.45) nm; 1H NMR (CDCl3, 600 MHz) 7.27 (1H, t, J = 8.2 Hz, H-5), 6.82 (1H, d, J 23 = 8.2 Hz, H-6), 6.74 (1H, d, J = 8.2 Hz, H-4), 5.32 (1H, d, J = 4.1 Hz, H-2’), 5.22 (1H, q, J = 6.8 24 Hz, 2”), 3.78 (3H, s, H-13), 3.75 (3H, s, OCH3-1”), 2.63 (2H, t, J = 7.5 Hz, 8), 2.09 (1H, m, H-25 3’), 1.59 (2H, m, H-9), 1.56 (1H, m, H-4’), 1.52 (3H, d, J = 6.8 Hz, H-3”), 1.36 (1H, m, H-4’), 26 1.28 (2H, m, H-10), 1.28 (2H, m, H-11), 0.99 (3H, d, J = 6.8 Hz, H-6’), 0.97 (3H, t, J = 7.5 Hz, 27 H-5’), 0.85 ( 3H, m, H-12); 13C NMR (CDCl3, 150 MHz) 170.7 (C, C-1”), 169.5 (C, C-1’), 28 168.0 (C, C-1), 156.6 (C, C-3), 142.1 (C, C-7), 130.4 (CH, C-5), 123.1 (C, C-2), 121.5 (CH, C-6), 29 108.3 (CH, C-4), 75.3 (CH, C-2’), 69.3 (CH, C-2”), 55.7 (CH3, C-13), 52.4 OCH3, C-1”), 37.1 30 (CH, 3’), 33.2 (CH2, 8), 31.7 (CH2, 10), 31.1 (CH2, 9), 25.8 (CH2, 4’), 22.6 (CH2, C-31 11), 17.1 (CH3, C-3”), 14.4 (CH3, C-6’), 14.1 CH3, C-12), 11.6 (CH3, C-5’). 32 33
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Structure Elucidation of Anaenamides A (1a) and B (1b) and Anaenoic acid (2)
1
Anaenoic acid (2) was obtained as an optically active colorless solid. HRESIMS data supported 2
the molecular formula of C22H32O7. Following edited HSQC experiment (Table S2) the 1H and 3
13C NMR signals were assignable to five methylene groups (C-8 to C-11 and C-4’), three methine 4
groups (C-2’, C-3’ and C-2”), four methyl groups (C-12, C-5’, C-6’ and C-3”), three contiguous 5
aromatic signals (C-4 to C-6) and one OMe group (C-13). In addition, the 13C NMR spectrum 6
indicated signals for the presence of three non-protonated aromatic carbon atoms (C 156.6. C-3: 7
141.9, C-7; 122.8, C-2) and three carbonyl groups (C 168.1, C-1; 169.4, C-1’; 175.3, C-1”). The 8
COSY and HMBC NMR data (Table S2) was analyzed. The combination of COSY and HMBC 9
correlations established the planar structure. Strong NOESY correlations (Table S2) between the 10
13-OMe (H 3.77) to 4-H (H 6.74), and 8-methylene (H 2.62) to 6-H (H 6.82) further confirmed 11
the substitution pattern in the aromatic ring. Methylation of compound 2 with CH2N2 gave the 12
methyl ester thus confirming the presence of a free carboxylic group. The methyl ester showed the 13
presence of NMR signals for an additional methoxy group (H 3.75, C 52.4). These data 14
established the planar structure for anaenoic acid (2). The related compounds 1a and 1b were 15
obtained as colorless solids. HRESIMS data gave the same molecular formula for compounds 1a
16
and 1b: HRMS (ESI) m/z [M+H]+ calcd for C27H39NO835/37Cl, 540.2364/542.2334; found 17
540.2349/542.2322 (for 1a) and 540.2351/542.2330 (for 1b). Following edited HSQC experiment 18
(Table S1), the 1H and 13C NMR signals for 1a and 1b were assignable to six methylene groups 19
(C-8 to C-11, C-4’ and C-4”’), three methine groups (C-2’, C-3’ and C-2”), four methyl groups 20
(C-12, C-5’, C-6’ and C-3”), three contiguous aromatic signals (C-4 to C-6), one isolated olefinic 21
proton, and two OMe groups (C-13 and C-1”’). In addition, the 13C NMR spectrum indicated 22
signals for the presence of four non-protonated sp2 carbon atoms C-2, C-3, C-7 and C-2”’) and 23
four carbonyl groups (C 169.4, C-1; 169.0 C-1’; 170.5, C-1” and 162.3, C-1”’). Based on similar 24
HSQC, COSY, HMBC, and NOESY arguments (Table S1) the planar structures left of the amide 25
bond was established for 1a and 1b. Based on characteristic isotope abundance it was determined 26
that both structures contained a chloride atom. Attaching the chloride to the vacant position on the 27
quaternary olefinic carbon satisfied the molecular formulas of 1a and 1b. The C-terminal methyl 28
ester(s) for both compounds were first inferred based on (1) the relatively low carbonyl chemical 29
shifts (1aC 162.3; 1b C 162.7) and (2) characteristic OMe resonances in the 1H NMR spectra 30
(1a H 3.71; 1b H 3.76). Strong HMBC correlations from the N-H proton to their respective
31
carbonyls bridged the two partial structures. The 1a double bond was assigned to the Z
32
configuration based on a weak NOESY correlation between H-3”’(H 7.01) and terminal OMe 33
group (H 7.01). Notable shielding of the 1b (H 6.43 vs 7.01) vinyl proton was attributed to facial
34
proximity (i.e., vicinal-cis) to the chloride, and confirmed the E configuration for 1b. 35
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Table S1. NMR Spectroscopic Data (600 MHz, CDCl3) for Anaenamides A (1a) and B (1b)
Unit C/H No C mult. J in Hz) COSYa HMBCb NOESYc C mult. J in Hz)
Anaenamide A (1a) Anaenamide B (1b)
Alkyl salicylic 1 169.4, C 169.4, C acid 2 121.1, C 122.2, C 3 156.7, C 156.7, C 4 108.5, CH 6.76, d (8.2) 5 1, 2, 3 5, 13 108.4, CH 6.76, d (8.2) 5 130.8, CH 7.29, t (8.2) 4, 6 3, 7, 2 4, 6 130.8, CH 7.29, t (8.2) 6 121.8, CH 6.84, d (8.2) 5 2, 4, 8 5, 8, 9 121.7, CH 6.84, d (8.2) 7 142.2, C 142.0, C 8 33.1, CH2 2.57, t (7.5) 9 6, 7, 9 9, 10 33.1, CH2 2.58, t (7.5) 9 31.3, CH2 1.59, m 8, 10 7, 11 6, 8 31.2, CH2 1.59, m 10 31.6, CH2 1.29, m 9, 11 12 8 31.7, CH2 1.29, m 11 22.6, CH2 1.29, m 10, 12 10, 12 12 22.5, CH2 1.29, m 12 14.0, CH3 0.85, m 11 10, 11 11 14.1, CH3 0.86, m 13 55.9, CH3 3.79, s 3 4 55.9, CH3 3.79, s Hmpa 1’ 169.0, C 169.0, C 2’ 76.8, CH 4.93, d (5.5) 3’ 1, 4’, 6’ 3’, 5’ 76.8, CH 4.97, d (5.5) 3’ 36.3, CH 2.01, m 2’, 4’, 6’ 1’, 5’, 6’ 2’, 5’, 6’ 36.3, CH 2.02, m 4’ 25.3, CH2 1.55, m 3’, 5’ 5’, 6’ 5’ 25.3, CH2 1.55, m 1.32, m 3’, 5’ 5’, 6’ 3’, 5’ 1.32, m 5’ 11.2, CH3 0.96, t (7.5) 4’ 3’ 3’, 4’ 11.3, CH3 0.97, t (7.5) 6’ 14.7, CH3 1.02, d (6.8) 3’ 2’, 4’ 3’ 14.7, CH3 1.01, d (6.8) Lactic acid 1” 170.5, C 170.4, C 2” 70.7, CH 5.40, q (6.8) 3” 1’, 1” 3” 70.8, CH 5.38, q (6.8) 3” 17.6, CH3 1.53, d (6.8) 2” 2”, 1” 2” 17.6, CH3 1.53, d (6.8) amino ester 1’” 162.3, C 162.7, C 2’” 125.3, C 123.9, C 3’” 138.5, CH 7.01, t (6.8) 4”’ 1’”, 2’” 4’” 142.2, CH 6.43, t (6.2) 4’” 38.6, CH2 4.17, ddd 3”’, NH 1”, 2’”, 3’”, NH 38.7, CH2 4.34, ddd (16.5, 6.9, 6.1) (16.5, 6.9, 6.1) 1’” 53.1, CH3 3.71, s 1”’ 3”’ 53.0, CH3 3.76, s, -OMe 4’”-NH 7.09, t (6.2) 4”’ 1”, 4’” 4’” 7.13, t (6.2)
a 1H-1H COSY correlations are from proton(s) stated to the indicated proton(s). b HMBC correlations,
optimized for 2/3J
CH = 8 Hz, are from proton(s) stated to the indicated carbon. c NOESY correlations are from proton(s) stated to the indicated proton(s).
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Table S2. NMR Spectroscopic Data (600 MHz, CDCl3) for Anaenoic acid (2)
unit C/H No C mult. J in Hz) COSYa HMBCb NOESYc
Alkyl salicylic acid 1 168.1, C 2 122.8, C 3 156.6, C 4 108.3, CH 6.74, d (8.2) 5 1, 2, 3 5, 13 5 130.4, CH 7.26, t (8.2) 4, 6 3, 7, 2 4, 6 6 121.4, CH 6.82, d (8.2) 5 2, 4, 5, 7, 8 5, 8, 9 7 141.9, C 8 33.1, CH2 2.62, t (7.5) 9 6, 9 9, 10 9 31.1, CH2 1.59, m 8, 10 7, 10, 11 6, 8 10 31.5, CH2 1.28, m 9, 11 12 8 11 22.5, CH2 1.28, m 10, 12 10, 12 12 12 13.9, CH3 0.85, m 11 10, 11 11 13 55.7, CH3 3.77, s 3 4 Hmpa 1’ 169.4, C 2’ 75.3, CH 5.31, d (4.1) 3’ 1, 3’, 4’, 6’ 3’, 5’ 3’ 37.0, CH 2.09, m 2’, 4’, 6’ 1’, 4’, 5’, 6’ 2’, 5’, 6’ 4’ 25.7, CH2 1.56, m 3’, 5’ 5’, 6’ 5’ 1.36, m 3’, 5’ 5’, 6’ 3’, 5’ 5’ 11.5, CH3 0.96, t (7.5) 4’ 3’ 3’, 4’ 6’ 14.3, CH3 0.99, d (6.8) 3’ 2’, 4’ 3’ Lactic acid 1” 175.3, C 2” 68.7, CH 5.24, q (6.8) 3” 1’, 1” 3” 3” 16.8, CH3 1.57, d (6.8) 2” 2”, 1” 2”
a 1H-1H COSY correlations are from proton(s) stated to the indicated proton(s). b HMBC correlations,
optimized for 2/3J
CH = 8 Hz, are from proton(s) stated to the indicated carbon. c NOESY correlations are from proton(s) stated to the indicated proton(s).
9
Acid Hydrolysis and Enantioselective HPLC analysis
Compounds 1a, 1b, and 2 (0.1 mg) were suspended in 6 N HCl (0.5 mL) and heated at 110 ºC for 12 h in separate sealed tubes. The hydrolysates were concentrated to dryness. The residue was reconstituted in 0.1 mL of H2O and analyzed by enantioselective HPLC using two separate conditions and compared the retention times with those of authentic standards. Condition 1: [Phenomenex Chirex (D) Penicillamine, 4.6 x 250 mm, 5 m column]; solvent 5% CH3CN in 2.0
mM CuSO4 in water); detection at 254 nm, with a flow rate of 1.0 mL/min, the retention times (tR min) for authentic standards were L-lactic acid (16.2) and D-lactic acid (20.3). Condition 2: [Diacel
Chemical Laboratories, Ltd. Chiralpak MA (+) (4.6 x 50 mm) column]; solvent 15% CH3CN in 2.0 mM CuSO4 in H2O; detection at 254 nm, with a flow rate of 1.0 mL/min, the retention times (tR min) for authentic standards were (2R,3S)-Hmpa (13.2), (2R,3R)-Hmpa (15.4), (2S,3R)-Hmpa (19.9), (2S,3S)-Hmpa (24.3). The absolute configurations of the hydroxy acid moieties in the three hydrolysates were confirmed by comparing the retention times in their respective chromatograms as L-lactic acid (16.2) and (2R,3S)-Hmpa (13.2).
Scheme S1. Proposed Anaenamide Biosynthetic Pathway
Proposed biosynthetic pathway leading to 1a and 1b starting from a fatty acid precursor (R1). Domain notation: FAAL: Fatty acid AMP ligase; KS: Ketosynthase; AT: Acyl transferase; ACP: Acyl carrier protein; PCP: Peptidyl carrier protein A: Adenylation; KR: Ketoreductase; C: Condensation; T: Thiolation. Magenta regions denote bioinformatic handles for ongoing gene cluster annotation efforts.
General Cell Culture Procedure
HCT116 human colorectal carcinoma cells were purchased from the American Type Culture Collection (ATCC). Cryo-preserved cell stocks were generated at early cell passages to ensure cell competency. In order to confirm purity and correct morphology, cell cultures were observed under a microscope. Cells were cultured in Dulbecco’s Modified Eagle Medium (Invitrogen, Mar. Drugs 2019, 17, 83 12 of 14 Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS,
10
Sigma–Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Invitrogen) under a humidified environment with 5% CO2 at 37 oC.
Cell Viability Assay (MTT)
HCT116 cells were seeded at a density of 10,000 cells per well in 96-well plates. After 24 h incubation, the cells were treated with varying doses of anaenoic acid (2) and anaenamides (1a,
1b, and 1c; n=3, technical replicates), and positive control, taxol (50 nM; 30% cell viability). Cells were then incubated with compounds for 48 h, followed by addition of the MTT reagent (Promega, Madison, WI, USA). In order to establish maximum viability and background absorbance the appropriate negative (cells + medium + 0.1% DMSO) and media (media + 0.1% DMSO) controls were included for all experiments. Cell viability was measured according to the manufacturer’s instructions, recorded on SpectraMax M5, and the subsequent data was processed via GraphPad Prism 6.
Total synthesis of 1a–c and 2 Synthesis of 7
To a round bottom flask containing solid (2R,3S)-2-amino-3-methylpentanoic acid (2.0 g, 15.2 mmol, 1.0 eq) 10 mL of 1.25 M H2SO4 was added and the solution was chilled to 0 Co over 15 minutes. A 2.9 mM solution of NaNO2 (8 mL, 22.8 mmol, 1.5 eq) was added dropwise to the reaction vessel and allowed to stir for 2 h at 0 oCand at room temperature for 16 h. The reaction was diluted with de-ionized water, pH adjusted to 2.0 with 1.0 M HCl (aq), exhaustively extracted with Et2O, and the combined organic extracts were dried over MgSO4 and reduced in vacuo. The resulting crude residue (1.75 g) was carried forward without purification.
In a round bottom flask crude residue (1.75 g) enriched in (2R,3S)-2-hydroxy-3-methylpentanoic acid was re-suspended in 33 mL DMF (aq) (5% H2O) and chilled to 0 oC. The flask was charged with K2CO3 (3.66 g, 26.5 mmol; 2.0 eq), benzyl bromide (2.72 g, 15.9 mmol, 1.2 eq), and stirred
H2SO4(aq) 0 oC 15 min;
NaNO2 (aq) 0 oC 2 h; r.t. O/N
H2N O OH H HO O OH H K2CO3, BnBr 0 oC to r.t O/N DMF (15% aq) HO O OH H HO O OBn H 7
11
at room temperature overnight. The reaction was quenched with a solution of 90% aqueous methanol saturated with K2CO3, and extracted thrice with EtOAc (80 mL). The combined organic layers were washed with brine, dried over MgSO4, and reduced in vaccuo. The resulting crude was adsorbed onto loose SiO2 and purified via automated normal phase chromatography (50 g SiO2 column; 10% to 25% EtOAc in hexanes) yielding 7 (2.22 g, 66% yield over 2 steps; white solid 1H NMR (600 MHz, CDCl3) δ 7.41 – 7.32 (m, 5H), 5.27 – 5.17 (m, 2H), 4.23 (d, J = 2.9 Hz, 1H), 2.66 (s, 1H), 1.88 – 1.78 (m, 1H), 1.57 – 1.49 (m, 1H), 1.36 – 1.26 (m, 1H), 0.94 (t, J = 7.4 Hz, 3H), 0.78 (d, J = 6.8 Hz, 3H). CAS: 113956-82-4
Synthesis of 10
In a flame dried round bottom flask, charged with argon, 1-(3-methoxyphenyl)-N,N -dimethylmethanamine (5.0 g, 30.3 mmol, 1.0 eq) was dissolved in anhydrous THF (30 mL), and cooled to 0 oC. To the chilled reaction, 1.6 M nBuLi (20.8 mL, 33.3 mmol, 1.10 eq) was added dropwise, and the mixture was stirred for 1 hour to ensure deprotonation. The reaction was further cooled to -78 oC followed by dropwise addition of ethyl chloroformate (7.5 mL, 78.7 mmol, 2.6 eq), and the reaction was stirred at room temperature overnight. The reaction was quenched/diluted with de-ionized water, extracted with EtOAc, the combined organic layers were washed with brine, further dried over MgSO4, and reduced in vaccuo. The crude reaction was purified via normal phase automated fractionation (100g SiO2; 15% EtOAc in hexanes) yielding 10 (3.14 g, 35% yield, yellow liquid). 1H NMR (600 MHz, CDCl3) δ 7.35 (t, J = 8.0 Hz, 1H), 7.03 (d, J = 7.7 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.60 (s, 2H), 4.43 (q, J = 7.1 Hz, 2H), 3.84 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). CAS: 81625-30-1
Synthesis of 11
In a dry 350 mL pressure tube, 10 (3.14 g, 10.9 mmol, 1.0 eq), and triphenylphosphine (3.4 g; 13 mmol; 1.2 eq) were dissolved in 20 mL of dry toluene, and the reaction was refluxed for 3 days at
12
90 oC via oil bath. The resulting white solid/crystals were filtered and washed with cold toluene yielding pure ethyl 2-methoxy-6-((triphenylphosphoranylidene)methyl)benzoate (4.85 g, 96% yield, white solid). 1H NMR (600 MHz, DMSO-d6) δ 7.93 – 7.87 (m, 3H), 7.76 – 7.69 (m, 6H), 7.55 (dd, J = 8.3, 7.4 Hz, 6H), 7.29 (t, J = 8.1 Hz, 1H), 7.12 (dd, J = 8.4, 1.5 Hz, 1H), 6.56 (dd, J
= 7.8, 2.5 Hz, 1H), 4.96 (d, J = 15.1 Hz, 2H), 3.98 (m, 2H), 3.74 (s, 3H), 1.04 (t, J = 7.1 Hz, 3H).
In a dried round bottom flask charged with argon, ethyl 2-methoxy-6-((triphenylphosphoranylidene)methyl)benzoate (5.22 g, 11.5 mmol, 1.0 eq) was suspended in 15 mL of dry THF. The suspension was cooled to -78 oCand freshly prepared LDA (8 mL, 12.1 mmol, 1.05 eq) was added dropwise over 15 minutes. The reaction was warmed to -48 oC, and allowed to stir for an additional 20 minutes, followed by dropwise addition of crotylaldehyde (1.04 mL, 12.7 mmol, 1.05 eq). The reaction was allowed to warm to room temperature over 3.5 hours. Upon completion the reaction was quenched with D.I water, thoroughly extracted with Et2O, and the combined organic layers washed with brine. The pooled organic phase was dried over MgSO4 and reduced in vaccuo. The crude diene mixture was subjected to automated normal phase fractionation (100 g SiO2; 5% to 10% EtOAc in hexanes) yielding an isomeric mixture of benzoate products (2.39 g, 84% yield).
Synthesis of 8
In a dry round bottom flask containing 11 (2.39 g; 9.70 mmol was charged with 10% Pd/C (5 %wt) and suspended in absolute ethanol. The flask was flushed with H2 and stirred overnight at room temperature. Upon completion the reaction was filtered over a celite plug, and purified via automated normal phase chromatography (50 g SiO2; 10% to 30% EtOAc in hexanes) yielding 2.0
Ph3P O OEt O LDA -78 oC 15 min; O -48 oC to r.t. 3.5 h THF OMe OEt O 11 OMe OEt O Pd/C (10%), H2 EtOH r.t. O/N OMe OEt O 11
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g (83 %yield) of ethyl 2-methoxy-6-pentylbenzoate. 1H NMR (500 MHz, CDCl3) δ 7.26 (t, J = 8.0 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 3.81 (s, 3H), 2.59 – 2.51 (m, 2H), 1.64 – 1.54 (m, 2H), 1.38 (t, J = 7.1 Hz, 3H), 1.36 – 1.25 (m, 4H), 0.94 – 0.82 (m, 3H). CAS: 208586-08-7
In a pressure tube compound 11 (2.0 g; 8.1 mmol; 1.0 eq) was suspended in DMSO (30 mL) and 15% NaOH solution (30 mL). The vessel was heated overnight (oil bath) at reflux, diluted with excess water, adjusted to pH 2 with 6N HCl and exhaustively extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4 and reduced under vacuum. The reaction crude was subjected to automated normal phase chromatography (100 g SiO2; 5 to 20% EtOAc in hexanes modified with 1% formic acid) yielding 1.5 g (83 %yield) of 2-methoxy-6-pentylbenzoic acid. 1H NMR (600 MHz, CDCl3) δ 7.31 (t, J = 8.0 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 3.89 (s, 3H), 2.76 – 2.71 (m, 2H), 1.66 – 1.60 (m, 2H), 1.37 – 1.32 (m, 4H), 0.92 – 0.86 (m, 3H). CAS: 183621-71-8
Synthesis of 12
In a dry round bottom flask charged with argon 8 (500 mg, 2.25 mmol, 1.0 eq) was suspended in DCM (10 mL) and stirred at 0 oC for 10 minutes. Trifluoroacetic anhydride (567 mg, 2.7 mmol, 1.2 eq) was added drop wise and the anhydride was allowed to form over 20 minutes. To the chilled solution 7 (500 mg, 2.25 mmol, 1.0 eq) was added, and the reaction was allowed to come to room temperature over 1 hour. Upon completion the reaction was quenched with NaHCO3, extracted thrice with DCM, the combined organic layers were washed with brine, and dried over MgSO4. The dried organic phase was reduced under pressure and purified via automated normal phase
OMe OH O HO O OBn H + DCM TFAA 0 oC 20 min; r.t. 1 h 7 8 H O OBn OMe O O OBn-12
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fractionation (50 g SiO2; 5% to 25% EtOAc in hexanes) yielding OBn-12 (740 mg, 77% yield). [α]25D = -26 (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.40 – 7.31 (m, 5H), 7.28 (t, J = 8.0 Hz, 1H), 6.83 (d, J = 7.8, 0.8 Hz, 1H), 6.75 (d, J = 8.3, 0.8 Hz, 1H), 5.33 (d, J = 3.6 Hz, 1H), 5.25 (s, 2H), 3.76 (s, 3H), 2.67 – 2.59 (m, 2H), 2.13 – 2.03 (m, 1H), 1.62 – 1.49 (m, 4H), 1.38 – 1.28 (m, 5H), 0.96 (t, J = 7.4 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.89 – 0.85 (m, 3H). 13C NMR (151 MHz, CDCl3) δ 169.8, 168.2, 156.6, 142.1 x 2, 135.6, 130.3, 128.6 x 2 , 128.3 x 2, 123.1, 121.5, 108.2, 75.2, 66.8, 55.6, 36.9, 33.2, 31.7, 31.2, 25.8, 22.6, 14.6, 14.1, 11.6. HRMS (ESI) m/z
[M+H]+ calcd for C26H35O5 427.2484; found 427.2477.
A dry bottom flask containing OBn-12 (740 mg, 1.74 mmol, 1.0 eq) was charged with 10% Pd/C (5% wt; 36 mg), and the contents were suspended in absolute ethanol (20 mL) at room temperature. The flask was flushed with H2 and stirred overnight. Upon completion the reaction was filtered over a celite plug and purified via automated normal phase chromatography (50 g SiO2; 10 to 50% EtOAc in hexanes) yielding 12 (512 mg; 87% yield). [α]25D = -22 (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.32 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 5.53 (d, J = 3.1 Hz, 1H), 3.85 (s, 3H), 2.75 – 2.65 (m, 1H), 2.63 – 2.55 (m, 1H), 2.23 – 2.15 (m, 1H), 1.67 – 1.55 (m, 1H), 1.55 – 1.48 (m, 1H), 1.41 – 1.33 (m, 1H), 1.33 – 1.27 (m, 4H), 1.02 – 0.95 (m, 6H), 0.91 – 0.82 (m, 4H). 13C NMR (151 MHz, CDCl3) δ 173.8, 167.3, 156.3, 142.6 x 2, 130.8, 122.2, 108.5, 74.7, 56.0, 37.0, 33.3 x 2, 31.8, 31.4, 25.9, 22.5, 14.4 x 2, 11.6. HRMS (ESI) m/z
[M+Na]+ calcd for C19H28O5Na, 359.1834; found 359.1823.
Synthesis of Anaenoic acid (2) OMe O O O H O OH O H O OBn Pd/C (10%), H2 EtOH r.t. O/N OMe OBn-12 12 O O OBn DCM + OMe O O H O OH HO OBn O DCC, DMAP 0 oC 10 min; r.t. 4 h OMe O O H O 12 6 OBn-2
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In a dry round bottom flask charged with argon 12 (100 mg, 0.30 mmol, 1.0 eq) was dissolved in DCM (AH) (5 mL), stirred at 0 oC for 10 minutes, followed by addition of: DCC (123 mg, 0.59 mmol, 2.0 eq), DMAP (7.3 mg, 0.06 mmol, 0.2 eq), and 6 (59 mg, 0.33 mmol, 1.1 eq). The reaction was allowed to warm to room temperature over 4 hours, diluted with chilled EtOAc, filtered, and condensed under reduced pressure. The crude reside was purified via automated normal phase chromatography (10 g SiO2; 5% to 25% EtOAc in hexanes) yielding OBn-2 (121 mg, 82% yield) [α]25D = -30 (c 0.02, CHCl3) 1H NMR (600 MHz, CDCl3) δ 7.35 – 7.29 (m, 5H), 7.32 – 7.23 (m, 2H), 6.82 (d, J = 7.8, 0.8 Hz, 1H), 6.73 (d, J = 8.3, 0.8 Hz, 1H), 5.33 (d, J = 3.5 Hz, 1H), 5.25 (q, J = 7.0 Hz, 1H), 5.22 – 5.13 (m, 2H), 3.76 (s, 3H), 2.62 (dt, J = 1.7, 7.3 Hz, 2H), 2.12 – 2.03 (m, 1H), 1.63 – 1.53 (m, 3H), 1.52 (d, J = 7.0 Hz, 3H), 1.40 – 1.30 (m, 1H), 1.32 – 1.22 (m, 4H), 0.99 – 0.90 (m, 6H), 0.88 – 0.81 (m, 3H). 13C NMR (151 MHz, CDCl3) δ 170.1, 169.6, 168.0, 156.7, 142.1 x 2, 135.4, 130.5, 128.7, 128.5 x 2, 128.3, 123.1, 121.5, 108.4, 75.3, 69.5, 67.2, 55.7, 37.2, 33.3, 31.7, 31.2, 25.9, 22.7, 17.1, 14.5, 14.1, 11.7. HRMS (ESI) m/z [M+Na]+ calcd for C29H38O7Na, 521.2515; found 521.2504.
A dry bottom flask containing OBn-2 (44 mg; 0.09 mmol; 1.0 eq) was charged with 10% Pd/C (5% wt; 4 mg), and the contents were suspended in absolute ethanol (1 mL) at room temperature. The flask was flushed with H2 and stirred for 1 hour. Upon completion the reaction was filtered over a Celite plug, and purified via preparative normal phase TLC (SiO2; 1:1 EtOAc:hexanes + 1.0% formic acid) yielding anaenoic acid (2) (21.8 mg,59% yield). [α]25D -37 (c 0.06, CHCl3) 1H NMR (600 MHz, CDCl3) δ 7.28 (t, J = 8.0 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 5.31 (d, J = 3.6 Hz, 1H), 5.25 (q, J = 7.2 Hz, 1H), 3.78 (s, 3H), 2.63 (m, 2H), 2.14 – 2.07 (m, 1H), 1.64 – 1.51 (m, 6H), 1.41 – 1.31 (m, 1H), 1.32 – 1.28 (m, 4H), 1.00 (d, J = 6.8 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H), 0.91 – 0.82 (m, 3H). 13C NMR (151 MHz, CDCl3) δ 175.6, 169.5, 168.3, 156.8, 142.1, 130.6, 123.0, 121.6, 108.5, 75.6, 69.3, 55.8, 37.1, 33.3, 31.7, 31.2, 25.9, 22.7, 17.1, 14.5, 14.1, 11.7. HRMS (ESI) m/z [M+Na]+ calcd for C22H32O7Na, 431.2045; found 431.2041.
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Synthesis of 5
In a round bottom flask a solution of 5.25% NaClO (aq) (610 mg, 8.30 mmol, 5.0 eq) was titrated to pH 7.0 with 3 N HCl, and chilled to 0 oC for 10-15 minutes. Methyl 2 (dimethoxyphosphoryl)acetate (300 mg, 1.64 mmol, 1.0 eq) was added dropwise to the solution, and stirred at room temperature for an additional five minutes. The aqueous mixture was exhaustively extracted with hexanes, and the combined organic layers were dried over MgSO4. The dried organic phase was evaporated under reduced pressure yielding pure methyl 2,2-dichloro-2-(dimethoxyphosphoryl)acetate (289 mg, 70% yield).
Methyl 2,2-dichloro-2-(dimethoxyphosphoryl)acetate (144 mg, 0.58 mmol, 1.0 eq) was transferred to a round bottom flask, suspended in methanol, and chilled to 0 oC over 10 minutes. A 0.26 mM solution of Na2SO4 (4.5 mL, 2.0 eq) was added dropwise over 15 min with vigorous stirring. The reaction was further stirred for 20 minutes at room temperature, exhaustively extracted with CHCl3, the combined organic layers were dried over MgSO4 and evaporated under reduced vacuum pressure. The resulting crude residue was resuspended in hexanes and exhaustively extracted with 0.1 M NaHCO3. The aqueous layer was then back extracted with CHCl3 and the combined organic phase was reduced under pressure. The crude residue was purified via normal phase chromatography (10 to 50% EtOAc in hexanes) yielding pure 5 (100 mg, 70% yield).
Synthesis of 3a and 3b
Compound 5 (100 mg, 0.63 mmols, 1.0 eq) was suspended in 2 mL of THF (AH) in a dry round bottom flask charged with argon. The solution was chilled to -78 oC and 1.6 M nBuLi (0.63 mmols,
H O MeO P OMe O O OMe Cl THF -78 oC 2 h BocHN Cl OMe O nBuLi -78 oC 1 h; NHBoc 4 Boc-3a BocHN Cl O OMe Boc-3b 5
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1.0 eq) was added dropwise over 10 minutes. The reaction was stirred for 1 hour followed by addition of aldehyde 4 dissolved in THF. The solution was further stirred at -78 oC for 2 hours, quenched with saturated NH4Cl, and extracted with EtOAc. The combined organic layers were dried over MgSO4 and evaporated under reduced vacuum pressure. The crude residue was purified via automated normal phase chromatography (10 g SiO2; 5% to 25% EtOAc in hexanes) yielding a 6:1 mixture of E/Z isomers (147.8 mg; 50% yield). In order to characterize both isomers a 17 mg aliquot of the crude reaction was purified via normal phase preparative TLC (25% EtOAc in hexanes) yielding pure E (6 mg) and Z (1 mg) isomers of methyl 4-((tert -butoxycarbonyl)amino)-2-chlorobut-2-enoate. E-Isomer: 1H NMR (600 MHz, CDCl3) δ 6.53 (t, J = 6.5 Hz, 1H), 4.93 (s, 1H), 4.16 (t, J = 6.5 Hz, 2H), 3.84 (s, 3H), 1.45 (s, 9H). HRMS (ESI) m/z [M+Na]+ calcd for C10H16N35/37ClO4Na, 272.0665/274.0636; found 272.0656/274.0636. Z-Isomer: 1H NMR (600 MHz, CDCl3) δ 7.05 (t, J = 5.9 Hz, 1H), 4.79 (s, 1H), 4.04 (s, 2H), 3.84 (d, J = 4.1 Hz, 3H), 1.46 (s, 9H). HRMS (ESI) m/z [M+Na]+ calcd for C10H16N35/37ClO4Na, 272.0665/274.0636; found 272.0656/274.0637.
In a dry round bottom flask a mixture of E/Z methyl 4-((tert -butoxycarbonyl)amino)-2-chlorobut-2-enoate (23 mg, 0.09 mmol, 1.0 eq) was dissolved in 1 mL of DCM:TFA (1:2, v/v) at stirred at room temperature for 30 minutes. The reaction was diluted with toluene (AH) and reduced under pressure. In order to remove any excess TFA this process was repeated 3 times, and the crude TFA salts were carried forward to the next step without purification.
Synthesis of Anaenamide A (1a) and B (1b)
A dry reaction vessel containing dry 2 (22 mg; 0.05 mmol; 1.0 eq) was purged with argon, and the residue suspended with 3 mL DMF (AH). The reaction was chilled to 0 oC, and charged with EDC.HCl (13 mg; 0.10 mmol; 2.0 eq), HOBt (15 mg, 0.11 mmol, 2.1 eq), DIEA (19.4 mg, 0.15
OMe O O H O O OH O
EDC, HOBt, DIEA DMF (0 oC - r.t.) H3N Cl OMe O (E/Z) + OMe O O H O O N H O Cl (E/Z) OMe O CF3CO2 2 3a and 3b 1a and 1b
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mmol, 3.0 eq), and 3a/3b (0.09 mmol; 1.8 eq). The reaction was allowed to stir over night at room temp, evaporated under reduced pressure, and purified via normal phase TLC (SiO2; 1:1 EtOAc:hexanes) yielding 20 mg of a mixture of E/Z isomers. The mixture was further purified via semipreparative reversed phase HPLC (Phenomenex Synergi Hydro 250 x 10mm) using an isocratic method of 85% aqueous methanol over 35 minutes yielding pure Z (5.29 mg) and E (9.03 mg) natural products (53% yield). Anaenamide A: []25
D (c 0.20 , CHCl3) HRMS (ESI) m/z
[M+H]+ calcd for C27H39NO835/37Cl, 540.2364/542.2334; found 540.2349/542.2329. Anaenamide B: []25
D (c 0.20 , CHCl3) HRMS (ESI) m/z [M+H]+ calcd for C27H39NO835/37Cl,
540.2364/542.2334; found 540.2351/542.2332.
Synthesis of 1c
A dry reaction vessel containing 2 was purged with argon, and the residue suspended with 3 mL DMF (AH). The reaction was chilled to 0 oC, and solutions of EDC. HCl (9.4 mg; 0.05 mmol; 2.0 eq), HOBt (6.9 mg, 0.05 mmol, 2.1 eq), DIEA (9.5 mg, 0.07 mmol, 3.0 eq) and 3c (0.044 mmol; 1.8 eq). The reaction was stirred over night at room temperature, evaporated under reduced under pressure, and purified via normal phase preparative TLC (SiO2; 1:1 EtOAc:Hexanes) yielding pure
1c (9.8 mg, 80% yield). 13C NMR (151 MHz, CDCl3) δ 173.8, 170.3, 169.2, 169.0, 156.7, 142.3, 130.8, 122.5, 121.8, 108.5, 70.9, 56.0, 51.7, 38.9, 36.5, 33.3, 31.8, 31.5, 31.4, 25.5 x 2, 24.6, 22.7, 17.9, 14.8, 14.2, 11.4. 1H NMR (600 MHz, CDCl3) δ 7.30 (t, J = 8.0 Hz, 1H), 6.87 – 6.79 (m, 2H), 6.79 – 6.74 (m, 1H), 5.36 (q, J = 6.9 Hz, 1H), 5.03 (d, J = 5.3 Hz, 1H), 3.80 (s, 2H), 3.61 (s, 3H), 3.38 – 3.24 (m, 2H), 2.59 (t, J = 8.0 Hz, 2H), 2.34 (t, J = 7.4 Hz, 2H), 2.08 – 1.99 (m, 1H), 1.90 – 1.79 (m, 2H), 1.64 – 1.51 (m, 5H), 1.39 – 1.31 (m, 1H), 1.33 – 1.25 (m, 4H), 1.04 – 0.95 (m, 6H), 0.87 (m, 3H). HRMS (ESI) m/z [M+H]+ calcd for C27H42 NO8, 508.2910; found 508.2900.
Supplementary Reference
1. Brumley, D.; Spencer, K. A.; Gunasekera, S. P.; Sauvage, T.; Biggs, J.; Paul, V. J.; Luesch, H. Isolation and Characterization of Anaephenes A–C, Alkylphenols from a Filamentous Cyanobacterium (Hormoscilla sp., Oscillatoriales). J. Nat. Prod. 2018, 81 (12), 2716– 2721. OMe O O H O O OH O
EDC, HOBt, DIEA DMF (0 oC - r.t.) + OMe O O H O O N H O OMe O H2N HCl O OMe 2 3c 1c
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Figure S21 Comparison of 13C NMR data for synthetic 2 (top panel) with the isolated material (bottom panel) (150 MHz CDCl3).
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