ent-Labdane Diterpenoids from Dodonaea viscosa - Journal of Natural

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ent-Labdane Diterpenoids from Dodonaea viscosa Wesley J. Olivier,† Nathan L. Kilah,† James Horne,‡ Alex C. Bissember,*,† and Jason A. Smith*,† †

School of Physical Sciences−Chemistry and ‡Central Science Laboratory, University of Tasmania, Hobart, Tasmania, Australia S Supporting Information *

ABSTRACT: Seven new and two known ent-labdane diterpenoids have been isolated from a single plant specimen of Dodonaea viscosa ssp. spatulata, found in Tasmania, Australia. Prior to this study, only seven different labdane diterpenoids had been isolated from D. viscosa. The structures of the natural products were assigned via 1D and 2D NMR spectroscopy and other standard spectroscopic methods. The absolute configuration of three ent-labdane diterpenoids was determined by single-crystal X-ray crystallography of synthetic derivatives. Significantly, the results of this study suggest that the absolute configuration of some known labdane diterpenoids may have been misassigned. known labdane diterpenoids8 and other likely points of revision6b,c are proposed.

Dodonaea Miller (Sapindaceae) is a genus of plants with approximately 70 species that are endemic to Australia.1 Dodonaea viscosa is the most widespread species of Dodonaea and is found throughout Australia and in five other continents.1 Australian D. viscosa have been divided into seven subspecies, and one of these, ssp. spatulata, grows throughout southern Australia, including Tasmania.1,2 Previous chemical studies on D. viscosa led to the isolation of clerodane diterpenoids,3 flavonoids,3d,4 triterpenoids,5 and labdane diterpenoids.6 There are only three reports of the isolation of labdane diterpenoids from D. viscosa (Figure 1).6 These molecules represent a class

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RESULTS AND DISCUSSION D. viscosa ssp. spatulata leaves were soaked in Et2O twice for 0.5 h, and the combined extracts subjected to flash column chromatography on silica gel to afford compounds 6 (0.5% w/ w yield) and 7 (0.3% w/w yield) as the major constituents. This was found to be the most efficient extraction method and, in this case, superior to the recently developed rapid pressurized hot water extraction approach.9 It was recognized that numerous other compounds were present, and a second extraction and isolation targeting these minor components was performed. The Et2O extract was extracted with aqueous NaOH, and the phases were separated to yield a neutral and an alkaline fraction. Flash column chromatography of the Et2O fraction gave ent-labdanes 8−12, along with compounds 6 and 7. The carboxylic acids 13 and 14 were obtained as an inseparable mixture after acidification and extraction of the alkaline extract (Figure 2). These acids were converted to their respective methyl esters 15 and 16 (Figure 3) by Fischer esterification to enable purification via flash column chromatography and characterization. Compound 6 was isolated as a colorless oil (0.5% w/w yield). On the basis of the spectroscopic data (1H and 13C NMR and IR) the molecular formula of C20H30O2 was proposed and was supported by HRESIMS data (m/z calculated [M + Na]+ 325.2138; found 325.2140). The IR spectrum included a broad band at 3348 cm−1 consistent with an OH stretch. The 1H NMR spectrum included resonances at δH 7.35 (H-15), 7.22 (H-16), and 6.28 (H-14), which were consistent with a 3-substituted furan moiety10 and a signal characteristic of an alkenyl proton at δH 5.43 (H-7) (Table 1). This spectrum also featured a resonance at δH 3.85 (H-2) for a methine proton, which was thought to be positioned geminal

Figure 1. Structures assigned to five of the seven labdane diterpenoids previously isolated from D. viscosa.

of bicyclic diterpenoids that have been isolated from various organisms including plants, fungi, and insects and are known to exist in both enantiomeric series. A wide range of useful biological activity has been reported for labdane diterpenoids, including antifungal, antibacterial, antiviral, antiprotozoal, cytotoxic, anti-inflammatory, antihypertensive, and hepatoprotective properties.7 To date seven different labdanes have been isolated from D. viscosa. Herein, the isolation of seven new ent-labdane diterpenoids from a single plant specimen of D. viscosa ssp. spatulata as well as two known natural products that have not previously been isolated from Dodonaea species is reported. In addition, revisions of the absolute configurations of some © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 19, 2016

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DOI: 10.1021/acs.jnatprod.6b00858 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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of a substituted 3-ethylfuran unit to the structure of 6 (Figure 4). The HMBC spectrum revealed that methyl carbon signals at δH 22.9 (C-18) and 33.3 (C-19) correlated to resonances assigned to H-19 (δH 0.92) and H-18 (δH 0.92), respectively, and shared all other correlations, which indicated that the structure of 6 contained geminal-related methyl groups. A resonance for a quaternary carbon at δC 34.9 correlated to signals assigned to H-18 and H-19, which permitted assignment of the former resonance to C-4. The resonance at δH 3.85 (H-2) exhibited tt multiplicity, which indicated that the corresponding methine proton was related vicinally with regard to two methylene groups. This proposition was supported by correlation between the aforementioned signal and resonances for two diastereotopic methylene protons at δH 2.15 (H-1a)/0.86 (H-1b) and 1.75 (H-3a)/1.12 (H-3b) in the COSY spectrum. The absence of further couplings suggested the methylene groups were positioned adjacent to quaternary carbons. The HMBC spectrum contained peaks that correlated the signals at δH 1.75 and 1.12 with resonances assigned to C-4, C-18, and C-19, and these data permitted assignment of the former resonances to H-3a and H-3b. Signals at δH 2.15/0.86 (H-1a/H-1b) were correlated to a resonance for a quaternary carbon at δC 38.7, in the HMBC spectrum. Hence, the latter resonance was assigned to C-10, the quaternary carbon adjacent to C-1. The HMBC spectrum contained strong cross-peaks between the signals assigned to C-18 and C-19 and a resonance for a methine proton at δH 1.16 and, thus, was assigned to H-5. The resonance assigned to H-5 exhibited dd multiplicity, which indicated that H-5 was coupled to protons of a methylene group. This assessment was supported by correlation between the signal assigned to H-5 and resonances for methylene protons at δH 2.00 (H-6a) and 1.84 (H-6b), in the COSY spectrum. Cross-peaks were evident between the signals assigned to H-6a/H-6b and a resonance at δH 5.43, which was thereby assigned to H-7. A resonance at δC 135.0 was assigned to a quaternary alkenyl carbon (C-8) on the basis of the characteristic chemical shift and HMBC cross-peak (consistent with a three-bond separation) with the signals assigned to H-6a/H-6b. Resonances for a methyl group at δC 22.7/δH 1.74 (C-17/H-17) were assigned to the C-17 olefinic methyl group, on the basis of the characteristic chemical shift and an HMBC cross-peak between the carbon signal and the resonance assigned to H-7. A signal corresponding to methyl protons at δH 0.80 was assigned to H3-20 on the basis of an HMBC cross-peak with signals assigned to C-10, C-1 (δC 48.5) and C-5 (δC 49.6). A resonance for a methine carbon at δC 54.5 was correlated to signals assigned to H-1, H-5, H-7, H3-17, and H3-20 in the HMBC spectrum and was therefore assigned to C-9. The latter carbon was designated as the point of connection of the 3ethylfuran fragment to the decalin unit. This was in accordance with cross-peaks between the resonances assigned to H-11a/H11b and a signal at δH 1.75, attributable to either H-9 or H-3a in the COSY spectrum. This was also supported by the HMBC spectroscopic data (Figure 4). The relative configuration of compound 6 was assigned with reference to an HSQC-NOESY spectrum (Figure 4). Crosspeaks related the signals assigned to H-2 and C-20, which was consistent with a cis relationship between H-2 and the C-20 methyl group. Correlation was not evident between H-5/C-5 and H-20/C-20, which was consistent with trans ring fusion of

Figure 2. ent-Labdane diterpenoids (6−14) isolated from D. viscosa ssp. spatulata (carboxylic acids 13 and 14 were isolated as their respective methyl esters after derivatization).

Figure 3. Derivatives synthesized from ent-labdane diterpenoids (15− 19).

with respect to a hydroxy group. Upon examination of the 1H NMR and HSQC spectra, it was proposed that the structure of compound 6 contained four methyl groups, corresponding to singlets at δH 0.92 (H3-18 and H3-19) and 0.80 (H3-20) and a singlet-like resonance embedded in a multiplet at δH1.74 (H317). The HMBC spectrum revealed a correlation between resonances for methylene protons at δH 2.63 and 2.38, respectively, with signals assigned to C-14 (δC 111.1) and C16 (δC 138.9). This supported assignment of the former signals to H-12a and H-12b, respectively. The resonance assigned to H-12b (δH 2.38) exhibited ddd multiplicity, which indicated the presence of a C-11 methylene group. This assertion was supported by correlation between the signals assigned to H12a/H-12b and resonances corresponding to methylene protons at δH 1.70 (H-11a) and 1.47 (H-11b) in the COSY spectrum. Therefore, the 2D NMR data supported assignment B



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Table 1. 1H NMR Chemical Shifts for Compounds 6, 10, 12, and 15 Recorded at 600 MHz and ent-Labdane 7 Recorded at 400 MHz in CDCl3 δ, multiplicity (J, Hz) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMeb a

6 2.15, dt (11.4, 3.4) 0.86, t (11.7) 3.85 tt (11.5, 3.9)

7

10

12

15

1.86, dt (13.4, 3.5) 1.05, td (12.8, 5.1) 1.60a 1.56a 3.22, dd (10.7, 5.3)

2.15, dt (11.9, 2.7) 1.14, t (11.9) 3.85, tt (11.5, 3.9)

2.05, ddd (11.9, 3.5, 2.2) 0.81a 3.91, tt (11.4, 4.2)

2.16, m 0.91a 3.85, tt (11.5, 3.9)

1.75, ddd (12.0, 3.6, 2.7) 0.92a

1.77a 1.10, t (11.9)

1.75, ddd (12.2, 3.8, 2.6) 1.12a

1.19, dd (10.6, 6.4) 1.97, m

0.83a 1.52a

5.42, m

1.17, 1.99, 1.84, 5.40,

1.14a 1.97a 1.83, m 5.39, m

1.67a

1.71a

1.67a 1.46, m 2.62, m 2.37, m

1.61, 1.35, 2.37, 2.08,

6.28, dd (1.7, 0.8)

6.27, m

5.63, t (6.9)

7.35, t (1.7) 7.22, dd (1.4, 0.9) 1.74a 0.92, s 6H 0.92, s 6H 0.80, s

7.35, 7.22, 1.73, 0.85, 0.97, 0.76,

4.20a 4.18a 1.71a 0.92a 0.92a 0.75, s

1.75a 1.12, t (11.9) 1.16, 2.00, 1.84, 5.43,

dd (12.2, 4.7) m m m

1.75a 1.70, 1.47, 2.63, 2.38,

m m m ddd (17.2, 10.4, 6.8)

t (1.5) m s s s s

dd (12.3, 4.7) m m m

m m m m

1.75a 1.46a 0.79a

1.62, brs

1.47a 1.28a 1.36a 1.24a 1.57a 1.63a 1.40a 3.68, m 0.94a 1.14, s 0.87, s 0.94a 1.00, s

1.48a 1.15a 1.52a 1.15a 1.93a 2.33, dd (14.7, 6.0) 2.12, dd (14.7, 8.1) 0.95, d (6.7) 1.66, s 0.91a 0.91a 0.79, s 3.66, s

Multiplicity not reported due to overlap of signals; chemical shifts determined from the HSQC spectrum. bApplicable to methyl ester 15 only.

consistent with an OH stretch. The 1H NMR spectrum included resonances consistent with a 3-substituted furan at δH 7.35 (H-15), 7.22 (H-16), and 6.27 (H-14), an alkenyl proton at δH 5.42 (H-7), and four methyl groups at δH 0.76 (H3-20), 0.85 (H3-18), 0.97 (H3-19), and 1.73 (H3-17). The molecular formula of C20H30O2 was proposed for compound 7 and was supported by HRESIMS data (m/z calculated [M + H]+ 303.2319; found 303.2318). Overall, the spectroscopic data indicated that compound 7 possessed a furanolabdane skeleton related to ent-labdane 6 and was an isomer of this compound. The key difference in the structure assigned to molecule 7, relative to ent-labdane 6, was the position of the hydroxy group. A resonance for a methine proton at δH 3.22 (H-3) was assigned to a proton geminal to a hydroxy group on the basis of the chemical shift. This resonance was correlated to signals assigned to the geminal-related methyl groups C-18 (δC 15.2) and C-19 (δC 28.0) as well as C-4 (δC 49.7) in the HMBC spectrum. In addition, the COSY spectrum revealed that resonances at δH 3.22 (H-3), 1.86 (H-1a), and 1.05 (H-1b) were each correlated to signals for methylene protons at δH 1.60 (H-2a) and 1.56 (H-2b). Collectively, these data supported assignment of the resonance at δH 3.22 to H-3. Thus, a hydroxy group was also assigned to C-3. The relative configuration of compound 7 was assigned with reference to a NOESY spectrum. The spectrum featured crosspeaks that related the signals assigned to H-5 and H-3, which permitted assignment of their cis relationship. The lack of

Figure 4. (A) Selected key COSY (bold red bonds) and HMBC (arrows) correlations for 6. (B) Selected key HSQC-NOESY correlations for diterpenoid 6.

the decalin unit. The spectrum revealed correlation between the resonances assigned to H-5 and C-9, and therefore H-5 and H9 were assigned to be cis-related. Correlation between the signal assigned to H3-20 and a resonance for a methyl carbon at δC 22.9 permitted assignment of the latter resonance to C-18. The absolute configuration of compound 6 was unambiguously assigned by single-crystal X-ray crystallography, after conversion to the (1S)-(−)-camphanoate 17 (Figures 3 and 5). Thus, diterpenoid 6 was defined as a member of the ent-labdane series. Compound 7 was isolated as a colorless gum (0.3% w/w yield). The IR spectrum included a broad band at 3414 cm−1, C



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Figure 5. X-ray crystal structures of (1S)-(−)-camphanoates 17 and 18 synthesized from ent-labdane diterpenoids 6 and 7, respectively. Thermal ellipsoids are drawn at the 30% probability level.

labdane series on the basis of “detailed comparison of the carbon shifts of C-2 and C-3 in similar structures of labdane 3O-β-D-glucoside and ent-labdane 3-O-β-D-glucoside”.6c In 2012, Wabo and co-workers reported the isolation of the labdane natural products 3−5 from a specimen of D. viscosa found in Cameroon.6b The structures and relative configurations of these compounds were assigned via analysis of 1D and 2D NMR spectra. However, the determination of the absolute configuration was not explicitly discussed, and it appears that these natural products were assumed to belong to the normal labdane enantiomeric series. Interestingly, the enantiomers of normal labdanes 4 and 5, previously isolated from Dodonaea microzyga, were unambiguously assigned as ent-labdanes by Jefferies and co-workers.11f Therefore, it is possible that the absolute configurations of labdanes 3−5 may have been incorrectly assigned. Compound 8 was isolated as a colorless oil (0.04% w/w yield). The 1H NMR data showed similarity to the spectra of ent-labdanes 6 and 7 including resonances consistent with a 3substituted furan moiety and an olefinic proton. The spectrum also contained singlets consistent with three tertiary methyl groups. The IR spectrum included an OH stretch, and, as such, molecule 8 was proposed to be an isomer of 6 and 7 (C20H30O2), which was supported by HREIMS data (m/z calculated M+ 302.2246; found 302.2240). The major difference in the NMR spectroscopic data of 8 in relation to diterpenoids 6 and 7 was that the spectra did not contain signals consistent with an allylic methyl group. In addition, the 1 H NMR spectrum of 8 featured doublets at δH 4.19 (H2-17a) and 4.09 (H2-17b), which were not present in the respective spectra of ent-labdanes 6 and 7. These resonances were proposed to correspond to diastereotopic methylene protons located geminal to a hydroxy group at C-17, as was consistent with the aforementioned features of the spectrum. This assignment was supported by correlation between the signals assigned to H2-17a/H2-17b and resonances assigned to C-7 (δC 126.0), C-8 (δC 139.4), and C-9 (δC 51.4) in the HMBC spectrum. The relative configuration of ent-labdane 8 was assigned with reference to a NOESY spectrum. The spectroscopic data for natural product 8 were consistent with those reported for synthesized ent-8,15 and comparison of the specific rotation of compound 8 {[α]20D −3.2 (c 1.6, CHCl3)} with that reported

correlation between the signals assigned to H3-20 and H-5 was consistent with trans fusion of the decalin unit. The absolute configuration of ent-labdane 7 was unambiguously assigned by single-crystal X-ray crystallography after conversion to (1S)(−)-camphanoate 18 (Figures 3 and 5). Interestingly, in 2012, Simpson and co-workers reported the isolation of the enantiomer of diterpenoid 7 from Dodonaea polyandra found in Australia.8 However, the reported absolute configuration was assigned by NMR spectroscopy, and the specific rotation of the compound in question was not reported by the authors.8 The 1H and 13C NMR spectroscopic data we obtained from ent-labdane 7 are consistent with equivalent data published by Simpson. It should be noted that prior to the aforementioned publication by Simpson and co-workers8 the isolation of normal labdane diterpenoids from an Australian Dodonaea species had not been reported. Jefferies and Payne conducted an extensive investigation on the chemical composition of Australian Dodonaea. These authors isolated ent-labdanes from the leaves of several species of Dodonaea11 and, using appropriate evidence, assigned them all to the entlabdane enantiomeric series.11d−g Although it is possible for plants of different genera or species to produce different enantiomers of secondary metabolites,12 and there are examples of plant species producing both enantiomers of labdane-type diterpenoids,13 it is not common for plant species of the same genus to produce both enantiomers of the diterpenoid natural products.12,14 Thus, we suggest that it is more likely that the compound isolated by Simpson and co-workers is ent-labdane 7.8 The above-mentioned observations prompted an examination of the respective methods used to determine the absolute configuration of the labdanes previously isolated from D. viscosa.6a,b In 1991, ent-labdane 1 was isolated from D. viscosa in Mexico by Mata and co-workers (Figure 1).6a The structure and relative configuration of compound 1 was assigned via 1D and 2D NMR spectroscopy, and the absolute configuration was inferred through analysis of the ECD spectrum. In 2012, de Oliveira and co-workers reported the isolation of two entlabdane diterpenoids and a related ent-labdane glucoside 2 from D. viscosa in Brazil.6c The structures and relative configuration of the three compounds were assigned primarily by 1D and 2D NMR spectroscopy. Only the absolute configuration of compound 2 was discussed, and it was assigned to the entD



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for the synthesized enantiomer of 8 {[α]20D +7.2 (c 3.9, CHCl3)} permitted assignment of 8 to the ent-labdane enantiomeric series.15 There is one previous report of the isolation of natural product 8 from Acritopappus morii (Asteraceae); however, the compound was only partially characterized.16 Compound 9 was isolated as a white, amorphous solid (0.3% w/w yield). The structure was assigned as the 2,17-diol based upon similarity of the spectroscopic data to compound 6. A resonance for a methine proton occurring at δH 3.83 was assigned to H-2, and the doublets resonating at δH 4.17/4.01 were assigned to the diastereotopic methylene protons H-17a/ H-17b. These assignments were supported by 2D NMR spectroscopy. The relative configuration of ent-labdane 9 was assigned from an HSQC-NOESY experiment by analogy to entlabdane 6. Compound 10 was isolated as a white, amorphous solid (0.009% w/w yield). The molecular formula of C20H34O3 was proposed for 10, which was supported by HRESIMS data (m/z calculated [M + Na]+ 345.2406; found 345.2399). The NMR spectroscopic data did not indicate the presence of a furan unit as was found in diterpenoids 6−9. However, analysis of the 1D and 2D NMR spectroscopic data resulted in assignment of a hydroxylated decalin unit, by analogy to ent-labdane 6. The 1H NMR spectrum of 10 featured a triplet consistent with an alkenyl proton at δH 5.63 (H-14) and a multiplet comprising two resonances for methylene protons at δH 4.20 (H2-15) and 4.18 (H2-16). The signals at δH 4.20 (H2-15) and 4.18 (H2-16) were proposed to correspond to hydroxymethylene protons. A COSY experiment showed correlation between the signals at δH 4.20 (H-15) and δH 5.63 (H2-14). The triplet multiplicity of the signal at δH 5.63 (H-14) and lack of further COSY correlations indicated that the corresponding olefinic proton was positioned adjacent to a quaternary carbon. Similarly, the signal at δH 4.18 (H-16) lacked correlations in the COSY spectrum, which indicated that the corresponding methylene protons were also positioned adjacent to a quaternary carbon. Further analysis of the 2D NMR spectroscopic data resulted in assignment of a but-2-ene-1,4-diol unit instead of a furan moiety (Figure 6). The relative configuration of compound 10 was assigned with reference to the NOESY spectrum. This spectrum featured cross-peaks between the signals assigned to H-14 and H2-12 and H2-11, and no correlation was evident between the resonances for H-14 and H2-16. Therefore, the NOESY data were consistent with Z-double-bond geometry. Compound 11 was isolated as a white, amorphous solid (0.08% w/w yield). Consistent with the NMR and MS data, the structure was assigned to be an isomer of compound 10 with a hydroxy group attached to C-17. The 1D and 2D NMR spectroscopic data were consistent with the presence of but-2ene-1,4-diol and decalin units as observed for ent-labdanes 8 and 10, respectively. Compound 11 was reported to occur in Achyrocline alata (Asteraceae), but only as the triacetate after derivatization.17 Specifically, tri-O-acetyl-11 was isolated after reaction with acetic anhydride and partially characterized, but no spectroscopic data were reported for the parent compound. ent-Labdane 12 was isolated as a white, amorphous solid (0.06% w/w yield). The structure of compound 12 was assigned to feature a decalin unit with a hydroxy group attached to C-2 through analysis of the 1D and 2D NMR spectroscopic data, as in compounds 6, 9, and 10. However, the 1H NMR spectrum of 12 did not feature resonances downfield of the resonance assigned to H-2 (δH 3.91), which indicated that the

Figure 6. A, C, and E feature selected key COSY (bold red bonds) and HMBC (arrows) correlations for diterpenoids 10, 12, and 15, respectively. B and D feature selected key NOESY correlations for diterpenoids 10 and 12, respectively.

Δ7,8 double bond was absent. The 13C NMR spectrum supported this proposition, as the most deshielded signal resonated at δC 73.3 (Table 2). The molecular formula was postulated to be C20H38O3 (HRESIMS m/z calculated [M + Na]+ 349.2719; found 349.2711), which was consistent with a saturated decalin unit. A tertiary hydroxy group was assigned to be attached to a tertiary carbon resonating at δC 73.3 (C-8). The HMBC spectrum featured a cross-peak between the resonance attributed to C-8 and a resonance corresponding to protons of a methyl group at δH 1.14, which was assigned to H17. HMBC correlations were evident between the signal for H17 and the resonances assigned to the proximal carbon atoms (Figure 6). This supported assignment of an oxygenated decalin system. The side chain consisted of a methyl, a methine, and four methylene carbons, as indicated by the HSQC spectroscopic data. The key resonances in the 1H NMR spectrum assigned to the side chain included a multiplet at δH 3.68 (H-15) and a methyl group resonance at δH 0.94 (H-16). The COSY spectrum indicated that H3-16 was positioned adjacent to the methine H-13 (δH 1.93) (Figure 6). The relative configuration of compound 12 was assigned via NOESY data (Figure 6). A cross-peak between the resonances assigned to H3-17 and H3-20 was not observed; however, a correlation between resonances assigned to H3-17 and H-9 was evident. Thus, the 17-methyl group was assigned to be cis with respect to the axially oriented H-9 and trans with respect to the axially oriented 20-methyl group. The configuration at C-13 has been assigned on the basis of the configuration established for the related carboxylic acid 13 (vide inf ra). ent-Labdane 13 was isolated as the corresponding methyl ester derivative 15 (Figure 3) as a colorless oil (0.25% w/w yield). The IR spectrum contained a strong band at 1732 cm−1, consistent with a CO stretch for an ester carbonyl. The NMR spectroscopic data were consistent with a labdane E



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Table 2. 13C NMR Chemical Shifts for Compounds 6, 10, 12, and 15 Recorded at 150 MHz and ent-Labdane 7 Recorded at 100 MHz in CDCl3 δ, type position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMea a

6 48.5, 65.3, 51.4, 34.9, 49.6, 23.8, 125.4, 135.0, 54.5, 38.7, 28.1, 27.1, 125.3, 111.1, 142.9, 138.9, 22.2, 22.9, 33.3, 14.6,

7 CH2 CH CH2 C CH CH2 CH C CH C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3

37.3, 27.5, 79.2, 38.8, 49.7, 23.6, 122.5, 135.1, 54.3, 36.7, 27.9, 27.0, 125.3, 111.1, 142.9, 138.9, 22.1, 15.2, 28.0, 13.7,

10 CH2 CH2 CH C CH CH2 CH C CH C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3

48.4, 65.3, 51.2, 34.9, 49.6, 23.8, 122.5, 135.0, 54.7, 38.7, 26.1, 38.1, 143.9, 127.0, 58.6, 60.8, 22.1, 22.9, 33.3, 14.6,

CH2 CH CH2 C CH CH2 CH C CH C CH2 CH2 C CH CH2 CH2 CH3 CH3 CH3 CH3

12 48.5, 64.9, 51.3, 35.1, 55.5, 18.3, 42.3, 73.3, 59.4, 40.8, 22.9, 41.4, 30.6, 40.0, 61.3, 19.7, 30.9, 22.7, 33.7, 16.4,

CH2 CH CH2 C CH CH2 CH2 C CH C CH2 CH2 CH CH2 CH2 CH3 CH3 CH3 CH3 CH3

15 48.5, 65.2, 51.4, 34.9, 49.6, 23.8, 122.3, 135.2, 55.4, 38.8, 24.8, 39.4, 31.4, 41.5, 173.8, 20.1, 22.1, 22.9, 33.3, 14.6, 51.5,

CH2 CH CH2 C CH CH2 CH C CH C CH2 CH2 CH CH2 C CH3 CH3 CH3 CH3 CH3 CH3

Applicable to methyl ester 15 only.

skeleton, and analysis of the 2D NMR data resulted in assignment of a hydroxy group attached to C-2 of the decalin substructure, by analogy to compound 6. The side chain attached to C-9 of the decalin unit was assigned upon examination of the 1H, 13C, and 2D NMR spectroscopic data. Signals occurring at δH 2.33 and 2.12 were assigned to H-14a and H-14b, respectively, adjacent to the carbonyl carbon (C15) based on the HMBC cross-peaks between the resonances for H-14a/H14-b and C-15 (δC 173.8). The COSY spectrum revealed correlation between the resonances assigned to H14a/H-14b and a resonance at δH 1.93 (H-13). The latter signal also correlated to a doublet corresponding to protons of a methyl group at δH 0.95 (H3-16). The HMBC spectrum featured cross-peaks between the resonance assigned to C-12 (δC 39.4) and H-14a/H-14b, H3-16, and H-13. Therefore, the 2D NMR spectroscopic data of 15 (Figure 6) supported assignment of a side chain terminating in a hydroxycarbonyl functional group in acid 13. With the exception of C-13, the relative configuration of methyl ester 15 was assigned with reference to an HSQCNOESY spectrum, by analogy to compound 6. The absolute configurations of compounds 13 and 15 were unambiguously determined by single-crystal X-ray crystallography after conversion of methyl ester 15 to p-chlorobenzoate 19 (Figures 3 and 7). ent-Labdane 14 was isolated as the corresponding methyl ester derivative 16 (Figure 3) as a colorless oil (0.05% w/w yield). Analysis of the 1D and 2D NMR spectroscopic data revealed that the decalin unit of ester 16 was structurally related to ent-labdane 8 and included a hydroxy group bonded to C-17. In addition, the side chain of 16 was assigned in a similar fashion to ester 15. Carboxylic acid 14 may have been isolated after derivatization to methyl ester 16 from Haplopappus pectinatus (Compositae).18 However, this report did not assign the configuration at C-13 and did not include characterization

Figure 7. X-ray crystal structure of p-chlorobenzoate 19, synthesized from methyl ester 15. Thermal ellipsoids are drawn at the 30% probability level.

by 13C NMR or complete 1H NMR data. Consequently, it is unclear whether the compound in question was methyl ester 16 or an isomer. In conclusion, extensive chemical investigation of an Et2O extract of the leaves of D. viscosa ssp. spatulata resulted in the isolation of nine ent-labdane diterpenoids, including seven new compounds. Significantly, prior to this study, only seven different labdanes have been isolated from D. viscosa. This suggests that either the spatulata subspecies is a unique source of ent-labdanes among D. viscosa or that some diterpenoids, although present, were not isolated or identified in previous chemical studies of plants of this species. Furthermore, labdane diterpenoids belonging to both enantiomeric series have been reportedly isolated from specimens belonging to Dodonaea. F



Article

Labdanes 6 and 7. Dried whole leaves of D. viscosa ssp. spatulata (100 g) were soaked in Et2O (950 mL) for 0.5 h, the Et2O was decanted, and the procedure was repeated. The combined Et2O extracts were concentrated under reduced pressure. The crude extract (12.6 g) was passed through a plug of silica gel (30% EtOAc/hexanes elution), and fractions were collected (6 × 50 mL). The second and third collected fractions were combined, and the solvent was removed under reduced pressure. Automated flash column chromatography (silica; 0−25% EtOAc/hexanes elution) of the combined fractions provided labdane 6 as a colorless oil (480 mg, 0.5% w/w) and labdane 7 as a colorless gum (276 mg, 0.3% w/w). (2R,4aR,8R,8aR)-8-(2-(Furan-3-yl)ethyl)-4,4,7,8a-tetramethyl1,2,3,4,4a,5,8,8a-octahydronaphthalen-2-ol (6): [α]20D −10.7 (c 1.5, CHCl3); IR (NaCl) 3348, 2959, 2924, 2849, 1464, 1365, 1161, 1049, 1024, 874, 777, 600 cm−1; 1H NMR, see Table 1; 13C NMR, see Table 2; HRESIMS m/z [M + Na]+ calcd for C20H30O2Na 325.2138; found 325.2140. (2R,4aS,5R,8aS)-5-(2-(Furan-3-yl)ethyl)-1,1,4a,6-tetramethyl1,2,3,4,4a,5,8,8a-octahydronaphthalen-2-ol (7): [α]20D −9.5 (c 5.8, CHCl3); IR (NaCl) 3414, 2959, 2928, 2851, 1460, 1442, 1383, 1163, 1061, 1024, 874, 779 cm−1; 1H NMR, see Table 1; 13C NMR, see Table 2; HRESIMS m/z [M + H]+ calcd for C20H31O2 303.2319; found 303.2318. Labdanes 8−14. Dried whole leaves of D. viscosa ssp. spatulata (100 g) were soaked in Et2O (1.20 L) for 0.5 h. The Et2O extract was concentrated under reduced pressure to a volume of ∼200 mL and extracted with aqueous NaOH (∼100 mL of a 1 M solution). The phases were separated, and the aqueous layer was extracted with Et2O (2 × 50 mL). The combined organic extracts were dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was subjected to automated flash column chromatography (silica; 0−100% EtOAc/hexanes elution then 0−10% MeOH/EtOAc elution) to provide a mixture of labdanes 6 and 7 (742 mg), labdane 8 as a colorless oil (40 mg, 0.04% w/w), labdane 9 as a white, amorphous solid (277 mg, 0.3% w/w), labdane 10 as a white, amorphous solid (9 mg, 0.009% w/w), labdane 11 as a white, amorphous solid (83 mg, 0.08% w/w), and labdane 12 as a white, amorphous solid (57 mg, 0.06% w/w). The aqueous fraction, obtained as described above, was carefully adjusted to pH ∼2 using HCl (10 M aqueous solution) and extracted with Et2O (3 × 50 mL). The combined organic extracts were dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was passed through a plug of silica gel, and fractions were collected (5 × 50 mL 5% MeOH/CH2Cl2 elution; 2 × 50 mL 10% MeOH/CH2Cl2 elution; 2 × 50 mL 20% MeOH/CH2Cl2 elution). Fractions 3−8 were combined, and the solvent was removed under reduced pressure. The residue was dissolved in MeOH (18 mL), Amberlyst ion-exchange resin (500 mg) was added, and the mixture was stirred. After 19 h, MeOH (10 mL) and Amberlyst ion-exchange resin (220 mg) were added to the reaction mixture. After an additional 4 h, the mixture was filtered and the filtrate was concentrated under reduced pressure and subjected to automated flash column chromatography (silica; 0−40% EtOAc/hexanes elution) to provide the methyl ester 15 as a colorless oil (254 mg, 0.25% w/w) and the methyl ester 16 as a colorless oil (47 mg, 0.05% w/w). {(1S,4aR,8aR)-1-[2-(Furan-3-yl)ethyl[-5,5,8a-trimethyl1,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl}methanol (8): [α]20D −3.2 (c 1.6, CHCl3); IR (NaCl) 3364, 2922, 2849, 1460, 1441, 1163, 1024, 874, 774 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.34 (t, J = 1.6 Hz, 1H, H-15), 7.23 (m, 1H, H-16), 6.29 (m, 1H, H-14), 5.77 (m, 1H, H-7), 4.19 (d, J = 12.1 Hz, 1H, H-17a), 4.01 (d, J = 12.1 Hz, 1H, H-17b), 2.71 (m, 1H, H-12a), 2.44 (ddd, J = 17.1, 9.9, 7.2 Hz, 1H, H12b), 2.06 (m, 1H, H-6a), 1.96−1.81 (m, 3H, H-1a, H-6b, H-9), 1.71 (m, 1H, H-11a), 1.55−1.38 (m, 4H, H-2a, H-2b, H-3a, H-11b), 1.23− 1.11 (m, 2H, H-3b, H-5), 0.94−0.87 (m, 4H, H-1b, H-18), 0.86 (s, 3H, H-19), 0.75 (s, 3H, H-20) ppm; 13C NMR (100 MHz, CDCl3) δ 142.8 (C-15), 139.4 (C-8), 139.0 (C-16), 126.0 (C-7), 125.5 (C-13), 111.2 (C-14), 66.2 (C-17), 51.4 (C-9), 50.0 (C-5), 42.3 (C-3), 39.2 (C-1), 36.7 (C-10), 33.2 (C-19), 33.1 (C-4), 27.5 (C-11), 26.3 (C12), 23.8 (C-6), 22.0 (C-18), 18.9 (C-2), 13.7 (C-20) ppm; HREIMS m/z M+ calcd for C20H30O2 302.2246; found 302.2240.

However, we suggest that, in some recent reports, the data and/ or rationale used to determine absolute configuration does not permit the unambiguous assignment of absolute configuration. Thus, further investigations should be undertaken to determine if the labdanes previously isolated from Dodonaea actually belong to the ent-labdane enantiomeric series.

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EXPERIMENTAL SECTION

General Experimental Conditions. Unless otherwise specified, reactions were performed under an air atmosphere. Solvents were analytical grade and purified by standard laboratory procedures. Reagents were purchased from Sigma-Aldrich (Sydney, Australia), AK Scientific, and Oakwood and were used without purification. Polarimetry was performed with a Rudolph Research Analytical Autopol III automatic polarimeter with a 0.5 dm cell. Infrared spectrometry was performed on a Shimadzu FTIR 8400s spectrometer. A thin film of material was deposited onto NaCl plates following evaporation of CH2Cl2 and/or CHCl3. NMR experiments were performed either on a Bruker Avance III NMR spectrometer operating at 400 MHz (1H) or 100 MHz (13C) or on a Bruker Avance III NMR spectrometer operating at 600 MHz (1H) or 150 MHz (13C). The deuterated solvents used were either CDCl3 or acetone-d6 as specified. Chemical shifts were recorded in ppm. Spectra were calibrated by assignment of the residual solvent peak to δH 7.26 and δC 77.16 for CDCl3 and δH 2.09 and δC 29.84 for acetone-d6. Coupling constants (J) were recorded in Hz. HRESIMS analyses were conducted on a Thermo-Scientific LTQ-Orbitrap using a syringe pump operated at 3 μL/min, and full scan data acquired in positive ionization mode using an electrospray voltage of 5 kV. HREIMS analyses were performed using a Kratos Analytical Concept ISQ hybrid magnetic sector quadrupole tandem mass spectrometer. MS samples were prepared in CHCl3. X-ray crystallographic data for the structure determinations of compounds 17, 18, and 19 were recorded at 100 K on a Bruker AXS D8 Quest (Cu Kα radiation). Compounds 17 and 19 were solved by charge-flipping methods with Superflip,19 and compound 18 was solved by direct methods with SHELXS,20 with all structures refined using CRYSTALS.21 Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were visible in the diffraction map, but were included at calculated positions riding on the atoms to which they are attached. Refinement of the Flack parameter22 and comparisons with the known stereochemistry of the (1S)-(−)-camphanoates were used to assign the absolute configurations of compounds 17 and 18. The refined Flack parameter for compound 19 was inadequate to confidently assign absolute configuration and prompted further analysis using the likelihood method.23 Analysis of the Bayesian statistics of the Bijvoet pairs was performed in PLATON,24 which indicated the correct assignment of the absolute configuration (see Supporting Information). Molecular graphics were produced with ORTEP-3.25 Crystallographic data for compounds 17, 18, and 19 have been deposited with the Cambridge Crystallographic Data Centre (1488213, 1488214, and 1499108, respectively). TLC was performed using Merck silica gel 60-F254 plates. Developed chromatograms were visualized by UV absorbance (254 nm) or through application of heat to a plate stained with cerium molybdate {Ce(NH4)2(NO3)6, (NH4)6Mo7O24·4H2O, H2SO4, H2O}. Manual flash column chromatography was performed with flash grade silica gel (60 μm) and the indicated eluent in accordance with standard techniques.26 Automated flash column chromatography was performed with a Grace Reveleris X2 flash column chromatography system with 40 μm silica gel cartridges and the indicated eluent gradient. Plant Material. Leaves and stems of D. viscosa ssp. spatulata were obtained from a specimen at the University of Tasmania, Sandy Bay campus (location: 42.904134° S, 147.323871° E), over the period of October 2015 to June 2016. This specimen contained female flowers. A voucher specimen (no. HO581453) has been lodged with the Tasmanian Herbarium and identified by Dr. Miguel de Salas. Leaves were dried overnight in a 40 °C oven prior to extraction. G



Article

(2R,4aR,8S,8aR)-8-[2-(Furan-3-yl)ethyl]-7-(hydroxymethyl)4,4,8a-trimethyl-1,2,3,4,4a,5,8,8a-octahydronaphthalen-2-ol (9): [α]20D ∼0; the value obtained was too small to confidently report (c 2.2, CHCl3); IR (NaCl) 3354, 2957, 2924, 2848, 1460, 1389, 1366, 1159, 1024, 779 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.33 (m, 1H, H-15), 7.22 (m, 1H, H-16), 6.28 (m, 1H, H-14), 5.78 (m, 1H, H-7), 4.17 (d, J = 12.4 Hz, 1H, H-17a), 4.01 (d, J = 12.4 Hz, 1H, H-17b), 3.83 (tt, J = 11.5, 3.8 Hz, 1H, H-2), 2.70 (m, 1H, H-12a), 2.43 (ddd, J = 17.3, 10.3, 7.4 Hz, 1H, H-12b), 2.14 (m, 1H, H-1a), 2.07 (m, 1H, H6a), 1.97−1.83 (m, 2H, H-6b, H-9), 1.77−1.68 (m, 2H, H-3a, H-11a), 1.50 (m, 1H, H-11b), 1.17 (dd, J = 12.1, 4.8 Hz, 1H, H-5), 1.11 (t, J = 11.9 Hz, 1H, H-3b), 0.91 (s, 6H, H-18, H-19), 0.85 (t, J = 11.8 Hz, 1H, H-1b), 0.78 (s, 3H, H-20) ppm; 13C NMR (100 MHz, CDCl3) δ 142.8 (C-15), 138.98 (C-8), 138.96 (C-6), 125.7 (C-7), 125.2 (C-13), 111.1 (C-14), 65.8 (C-17), 65.1 (C-2), 51.5 (C-9), 51.2 (C-3), 49.2 (C-5), 43.8 (C-1), 38.5 (C-10), 34.8 (C-4), 33.2 (C-19), 27.6 (C-11), 26.2 (C-12), 23.6 (C-6), 22.9 (C-18), 14.6 (C-20) ppm; HREIMS m/z M+ calcd for C20H30O3 318.2195; found 318.2196. (Z)-2-{2-[(1R,4aR,7R,8aR)-7-Hydroxy-2,5,5,8a-tetramethyl1,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl]ethyl}but-2-ene-1,4diol (10): [α]20D −7.9 (c 0.31, CHCl3); 1H NMR, see Table 1; 13C NMR, see Table 2; IR (NaCl) 3343, 2957, 2924, 1460, 1437, 1389, 1366, 1022 cm−1; HRESIMS m/z [M + Na]+ calcd for C20H34O3Na 345.2406; found 345.2399. (Z)-2-{2-[(1S,4aR,8aR)-2-(Hydroxymethyl)-5,5,8a-trimethyl1,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl]ethyl}but-2-ene-1,4diol (11): [α]20D −2.4 (c 2.8, CHCl3); IR (NaCl) 3341, 2922, 1460, 1441, 1383, 1366, 999 cm−1; 1H NMR (400 MHz, CDCl3) δ 5.74 (m, 1H, H-7), 5.60 (t, J = 7.0 Hz, 1H, H-14), 4.21−4.10 (m, 5H, H-15, H16, H-17a), 3.98 (d, J = 12.1 Hz, 1H, H-17b), 2.48 (m, 1H, H-12a), 2.11−2.01 (m, 2H, H-6a, H-12b), 1.95−1.82 (m, 3H, H-1a, H-6b, H9), 1.60−1.38 (m, 5H, H-2a, H-2b, H-3a, H-11a, H-11b), 1.22 (dd, J = 11.8, 4.6 Hz, 1H, H-5), 1.16 (td, J = 13.1, 3.9 Hz, 1H, H-3b), 0.98 (td, J = 12.9, 3.9 Hz, 1H, H-1b), 0.88 (s, 3H, H-18), 0.86 (s, 3H, H-19), 0.75 (s, 3H, H-20) ppm; 13C NMR (100 MHz, CDCl3) δ 143.8 (C13), 139.0 (C-8), 127.0 (C-7), 126.4 (C-14), 66.6 (C-17), 60.2 (C16), 58.4 (C-15), 52.0 (C-9), 50.1 (C-5), 42.4 (C-3), 39.2 (C-1), 37.1 (C-12), 36.9 (C-10), 33.2 (C-19), 33.1 (C-4), 25.5 (C-11), 23.9 (C-6), 22.0 (C-18), 18.9 (C-2), 13.7 (C-20) ppm; HRESIMS m/z [M + Na]+ calcd for C20H34O3Na 345.2406; found 345.2402. (1S,2R,4aR,7R,8aR)-1-[(R)-5-Hydroxy-3-methylpentyl]-2,5,5,8atetramethyldecahydronaphthalene-2,7-diol (12): [α]20D −11.9 (c 1.1, CHCl3); IR (NaCl) 3385, 2939, 2868, 1460, 1391, 1366, 1036, 908, 733 cm−1; 1H NMR, see Table 1; 13C NMR, see Table 2; HRESIMS m/z [M + Na]+ calcd for C20H38O3Na 349.2719; found 349.2711. Methyl (R)-5-[(1R,4aR,7R,8aR)-7-hydroxy-2,5,5,8a-tetramethyl1,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl]-3-methylpentanoate (15): [α]20D +7.3 (c 1.1, CHCl3); IR (NaCl) 2953, 2918, 1732, 1437, 1265, 1204, 1153, 1045, 1024, 735, 704 cm−1; 1H NMR, see Table 1; 13 C NMR, see Table 2; HRESIMS m/z [M + Na]+ calcd for C21H36O3Na 359.2557; found 359.2548. Methyl (R)-5-[(1S,4aR,8aR)-2-(hydroxymethyl)-5,5,8a-trimethyl1,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl]-3-methylpentanoate (16): [α]20D +9.2 (c 2.1, CHCl3); IR (NaCl) 2953, 2922, 2849, 1740, 1460, 1437, 1383, 1364, 1207, 1201, 1167, 1155 cm−1; 1H NMR (400 MHz, CDCl3) δ 5.74 (m, 1H, H-7), 4.12 (d, J = 12.2 Hz, 1H, H-17a), 3.96 (d, J = 12.3 Hz, 1H, H-17b), 3.66 (s, 3H, OMe), 2.32 (dd, J = 14.9, 6.4 Hz, 1H, H-14a), 2.15 (dd, J = 14.8, 7.6 Hz, 1H, H-14b), 2.05 (m, 1H, H-6a), 1.97−1.82 (m, 3H, H-1a, H-6b, H-13), 1.77 (m, 1H, H-9), 1.67−1.38 (m, 5H, H-2a, H-2b, H-3a, H-11a, H-12a), 1.22−1.11 (m, 4H, H-3b, H-5, H-11b, H-12b), 1.01−0.91 (m, 4H, H-1b, H-16), 0.88 (s, 3H, H-18), 0.85 (s, 3H, H-19), 0.74 (s, 3H, H-20) ppm; 13C NMR (100 MHz, CDCl3) δ 174.1 (C-15), 139.4 (C-8), 125.7 (C-7), 66.0 (C-17), 52.6 (C-9), 51.5 (C-OMe), 50.1 (C-5), 42.4 (C-3), 41.3 (C-14), 39.2 (C-1), 38.6 (C-12), 36.9 (C-10), 33.2 (C-19), 33.1 (C-4), 31.5 (C-13), 24.4 (C-11), 23.8 (C-6), 22.2 (C-18), 20.1 (C-16), 18.9 (C-2), 13.7 (C-20) ppm; HRESIMS m/z [M + Na]+ calcd for C21H36O3Na 359.2557; found 359.2554. (2R,4aR,8R,8aR)-8-[2-(Furan-3-yl)ethyl]-4,4,7,8a-tetramethyl1,2,3,4,4a,5,8,8a-octahydronaphthalen-2-yl(1S,4R)-4,7,7-trimethyl-

3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (17). Alcohol 6 (63 mg, 0.21 mmol) was dissolved in anhydrous CH2Cl2 (2 mL) under an atmosphere of N2; then (1S)-(−)-camphanic chloride (78 mg, 0.36 mmol), pyridine (30 μL, 0.37 mmol), and a catalytic amount 4dimethylaminopyridine were added and the reaction was stirred for 18 h. CH2Cl2 (10 mL) and H2O (2 mL) were added, and the organic phase was washed once with H2O (10 mL) acidified to pH ∼4 with saturated KHSO4 solution. The organic phase was washed with H2O (10 mL) that had been made alkaline to pH ∼9 with saturated NaHCO3 solution. The organic phase was dried with MgSO4 and filtered, and the solvent was removed under reduced pressure to yield the crude residue (100.7 mg), which was purified with flash column chromatography (silica; 20% EtOAc/hexanes elution) to yield (1S)(−)-camphanoate 17 (14 mg, 0.027 mmol, 13% yield), which was recrystallized by vapor diffusion of Et2O/n-hexane to yield colorless needles suitable for single-crystal X-ray crystallographic analysis. IR (NaCl) 2963, 2932, 1790, 1744, 1724, 1424, 1464, 1317, 1263, 1167, 1105, 1065, 932, 874, 797, 781 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.34 (t, J = 1.4 Hz, 1H), 7.22 (m, 1H), 6.25 (m, 1H), 5.44 (m, 1H), 5.12 (tt, J = 11.9, 3.9 Hz, 1H), 2.63 (m, 1H), 2.44−2.33 (m, 2H), 2.00−1.57 (m, 12H), 1.50 (m, 1H), 1.30 (t, J = 11.9 Hz, 1H), 1.20 (dd, J = 11.9, 4.8 Hz, 1H), 1.12 (s, 3H), 1.06 (s, 3H), 0.99−0.96 (m, 7H), 0.93 (s, 3H), 0.56 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 178.4, 167.1, 142.9, 139.0, 134.8, 125.1, 122.5, 111.1, 91.2, 71.0, 54.9, 54.2, 54.1, 49.6, 46.9, 44.2, 38.7, 34.9, 33.1, 30.7, 29.1, 27.9, 26.8, 23.7, 22.7, 22.2, 17.0, 16.9, 14.4, 9.9 ppm; HRESIMS m/z [M + Na]+ calcd for C30H42O5Na 505.2924; found 505.2914. (2R,4aS,5R,8aS)-5-[2-(Furan-3-yl)ethyl]-1,1,4a,6-tetramethyl1,2,3,4,4a,5,8,8a-octahydronaphthalen-2-yl (1S,4R)-4,7,7-trimethyl3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate (18). Alcohol 7 (58 mg, 0.19 mmol) was placed under an atmosphere of N2 and dissolved in anhydrous CH2Cl2 (2 mL). (1S)-(−)-camphanic chloride (89 mg, 0.41 mmol), pyridine (30 μL, 0.37 mmol), and a catalytic amount of 4dimethylaminopyridine were added, and the mixture was stirred for 18 h. Following the isolation procedure employed for 17, ester 18 was obtained as an off-white solid (101 mg, 0.20 mmol, >95% yield). A portion of the material was further purified by trituration with cyclohexane, then recrystallized from hot n-hexane to provide colorless plates suitable for single-crystal X-ray crystallographic studies. IR (NaCl) 2967, 2930, 1790, 1748, 1460, 1273, 1170, 1107, 1063 cm−1; 1 H NMR (400 MHz, acetone-d6) δ 7.46 (m, 1H), 7.40 (m, 1H), 6.39 (m, 1H), 5.42 (m, 1H), 4.61 (dd, J = 11.2, 4.8 Hz, 1H), 2.65 (m, 1H), 2.53−2.40 (m, 2H), 2.08−1.92 (m, 4H), 1.80−1.67 (m, 6H), 1.62 (m, 1H), 1.49 (m, 1H), 1.36 (t, J = 8.5 Hz, 1H), 1.10 (td, J = 13.3, 4.8 Hz, 1H), 1.11 (s, 3H), 1.06 (s, 3H), 1.00 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.83 (s, 3H) ppm; 13C NMR (100 MHz, acetone-d6) δ 178.3, 167.5, 143.7, 140.0, 135.9, 126.0, 122.7, 112.0, 91.9, 83.0, 55.4, 54.53, 54.51, 50.4, 38.3, 37.4, 37.1, 31.4, 29.4 (peak overlapping with residual solvent signal), 28.6, 28.3, 27.4, 24.9, 23.9, 22.3, 17.3, 17.0, 16.8, 14.0, 9.9 ppm; HRESIMS m/z [M + Na]+ calcd for C30H42O5Na 505.2924; found 505.2998. (2R,4aR,8R,8aR)-8-[(R)-5-Methoxy-3-methyl-5-oxopentyl]4,4,7,8a-tetramethyl-1,2,3,4,4a,5,8,8a-octahydronaphthalen-2-yl 4chlorobenzoate (19). A sample of methyl ester 15 (55 mg, 0.16 mmol) was placed under an atmosphere of N2 and then was dissolved in anhydrous CH2Cl2 (2 mL) and cooled to 0 °C. Pyridine (20 μL, 0.25 mmol), p-chlorobenzoyl chloride (30 μL, 0.23 mmol), and a catalytic amount of 4-dimethylaminopyridine were added, and the mixture was allowed to warm to rt and stirred for 17 h. H2O (10 mL) and CH2Cl2 (10 mL) were added, and the organic phase was washed once with H2O, twice with saturated KHSO4, and twice with saturated NaHCO3. The organic phase was dried with MgSO4 and filtered, and the solvent was removed under reduced pressure. The crude residue was subjected to flash column chromatography (silica; 10% hexanes/ CH2Cl2 elution); then select fractions were further purified by flash column chromatography (silica; 12% EtOAc/hexanes elution) to yield p-chlorobenzoate 19 (27 mg, 0.057 mmol, 36% yield) as a white solid. A minor impurity was removed through crystallization from n-hexane; then the title compound was recrystallized from hot EtOH to yield colorless needles suitable for single-crystal X-ray crystallographic H



Article

analysis. IR (NaCl) 2957, 2855, 1736, 1717, 1595, 1460, 1437, 1273, 1115, 1105, 1092, 1015, 762 cm−1; 1H NMR (600 MHz, CDCl3) δ 7.98 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 5.42 (m, 1H), 5.24 (tt, J = 11.8, 3.8 Hz, 1H), 3.62 (s, 3H), 2.33 (dd, J = 14.7, 6.0 Hz, 1H), 2.25 (m, 1H), 2.10 (dd, J = 14.8, 8.1 Hz, 1H), 2.01 (m, 1H), 1.97− 1.86 (m, 3H), 1.68 (s, 3H), 1.53 (m, 1H), 1.47 (m, 1H), 1.38 (t, J = 12.1 Hz, 1H), 1.24 (dd, J = 12.0, 4.3 Hz, 1H), 1.22−1.15 (m, 3H), 1.02 (s, 3H), 0.96−0.94 (m, 6H), 0.89 (s, 3H) ppm; 13C NMR (150 MHz, CDCl3) δ 173.7, 165.4, 139.3, 135.1, 131.1, 129.5, 128.8, 122.3, 70.1, 55.4, 51.5, 49.8, 47.2, 44.4, 41.3, 39.2, 38.9, 34.9, 33.3, 31.4, 24.7, 23.7, 22.7, 22.1, 20.2, 14.5 ppm; HREIMS m/z M+ calcd for C28H39O4Cl 474.2537; found 474.2524.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00858. 1 H, 13C, and 2D NMR spectra (PDF) Compound characterization checklist (XLS) X-ray crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Alex C. Bissember: 0000-0001-5515-2878 Jason A. Smith: 0000-0001-6313-3298 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the School of Physical Sciences − Chemistry and CSIRO for financial support and the Central Science Laboratory at University of Tasmania for access to NMR spectroscopy services. W.J.O. thanks the University of Tasmania for a 2015 Marshall Hughes Honours Scholarship in Chemistry. N.L.K.’s contribution to this research was supported under the Australian Research Council’s Discovery Early Career Research Award funding scheme (project number DE150100263).

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