Structure and Absolute Configuration of Methyl (3R)-Malonyl-(13S

Mar 21, 2013 - Akahori , A.; Yasuda , F.; Okanishi , T. Chem. Pharm. Bull. 1971, 19, 2409– 2411. [Crossref], [CAS]. 7. Steroidal components of domes...
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Structure and Absolute Configuration of Methyl (3R)‑Malonyl(13S)‑hydroxycheilanth-17-en-19-oate, a Sesterterpene Derivative from the Roots of Aletris farinosa Victoria L. Challinor,†,§ Sonet Chap,†,§ Reginald P. Lehmann,‡ Paul V. Bernhardt,† and James J. De Voss*,† †

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia Integria Healthcare, Brisbane 4113, Australia

‡

S Supporting Information *

ABSTRACT: We report the isolation and structure elucidation of a new cheilanthane sesterterpene from the roots of Aletris farinosa that possesses an unusual malonate half-ester functional group. The structure of 1 was determined via mass spectrometry and 1D and 2D NMR spectroscopy, while its absolute configuration was determined via X-ray crystallographic analysis performed on its methyl ester derivative 2.

Aletris farinosa L. (Nartheciaceae), commonly known as “true unicorn”, is a medicinal herb native to North America that is used as a tonic and for the treatment of colic, indigestion, and female reproductive health problems.1 The morphological resemblance of A. farinosa to Chamaelirium luteum (L.) A.Gray (“false unicorn”), another medicinal herb with an overlapping geographical distribution and similar indications, has resulted in these plants sometimes being used interchangeably in herbal remedies.2 Though the phytochemistry of A. farinosa has received only limited attention, we have identified the major constituents of C. luteum as open-chain steroidal glycosides derived from the helogenin and (23R,24S)chiograsterol B aglycones.3−5 Early phytochemical investigations suggested that steroidal glycosides are also present in A. farinosa. Marker obtained the steroidal aglycone diosgenin from the roots of A. farinosa,6 while Akahori et al. reported the presence of diosgenin, isonarthogenin, and bethogenin aglycones in the related Aletris spicata (Thunb.) Franch. along with the sterols β-sitosterol and stigmasterol.7 We thus chose to investigate whether these or any related steroidal glycosides are present in A. farinosa and whether there is a chemical basis for the substitution of C. luteum with A. farinosa in herbal remedies. A crude methanolic extract of the roots of A. farinosa was prepared according to procedures used previously for the isolation of steroidal saponins.5 However, analytical reversedphase (RP) HPLC under standard conditions5 revealed that the major component 1 had a retention time inconsistent with that of a steroidal saponin, suggesting it is a much more hydrophobic compound. The hydrophobic nature of 1 allowed © XXXX American Chemical Society and American Society of Pharmacognosy

it to be substantially purified by partitioning into ethyl acetate, though it proved difficult to purify further by normal-phase (NP) HPLC. We hypothesized that a carboxylic acid group may be present and thus undertook esterification with diazomethane to yield 2; an increase of 14 mass units in both positive- (1 m/z 529 [M + Na]+; 2 m/z 543 [M + Na]+) and negative-ion (1 m/z 505 [M − H]−; 2 m/z 519 [M − H]−) ESIMS indicated addition of one methyl group. The methyl ester derivative 2 was then readily purified via semipreparative NPHPLC (isocratic conditions of 50% ethyl acetate in hexane over 30 min, 2 mL/min).

Compound 2 was isolated as a white solid [mp 84−86 °C; [α]22D −13.7 (c 0.20, CHCl3)], and positive-ion HRESIMS Received: January 14, 2013

A

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Table 1. 1H and 13C NMR Spectroscopic Data for 1 and 2 1a,b 1

H [δ, mult, J (Hz)]

1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22 23 24 25 26 1′ 2′ 3′ 4′ a

1.44 1.17 1.88 1.65 4.67

e

m me me m m

1.27 m 1.35−1.44 me 1.35−1.44 me 1.78 me 1.08 m 0.97 m 1.57 1.21 1.83 1.49

m me me me

1.24 1.44 1.44 2.93 2.14

me me me td (11.1, 5.7) td (11.1, 7.3)

5.64 s 1.89 1.16 0.74 0.78 0.84 0.86 3.64

d (1.1) s s s s s s

3.41 br s

2a,b 13

33.5, CH2 22.7, CH2 CH C CH CH2

40.7, CH2 39.1, 59.8, 37.1, 19.0,

C CH C CH2

42.5, CH2 74.6, C 61.5, CH 24.1, CH2 37.5, CH2 162.1, C 115.1, CH 167.1, C 25.7, CH3 23.9, CH3 16.6, CH3 16.0, CH3 27.8, CH3 21.4, CH3 51.2, CH3 166.9, C 41.6, CH2 169.5, C

e

1.47 1.14 1.89 1.65 4.66

m me me dq (15.1, 3.3) t (2.7)

1.24 1.44 1.39 1.80 1.05

me me me me td (12.6, 3.9)

0.92 dd (12.0, 1.9) 1.59 1.24 1.84 1.43

m me me me

1.18 1.46 1.46 2.92 2.19

me me me td (11.0, 5.8) td (11.2, 7.6)

33.5, CH2 22.7, CH2 80.0, 36.8, 50.3, 17.6,

CH C CH CH2

40.9, CH2 39.1, 60.2, 37.1, 19.0,

C CH C CH2

73.7, C 61.9, CH 24.1, CH2 37.5, CH2 162.0, C 115.1, CH 167.0, C 25.7, CH3 24.1, CH3 16.7, CH3 16.0, CH3 27.8, CH3 21.5, CH3 51.1, CH3 166.0, C 41.8, CH2 167.2, C 52.4, CH3

5.64 br s 1.90 1.16 0.76 0.80 0.84 0.87 3.65

C

43.0, CH2

d (1.3) s s s s s s

3.39 s 3.74 s

b

13

H [δ, mult, J (Hz)]

C

80.1, 36.8, 50.1, 17.5,

2c,d

1

1

H [δ, mult, J (Hz)] e

1.38 1.23 1.86 1.72 4.86

m td (13.3, 3.7) me me t (2.6)

1.40 1.42 1.35 2.04 1.18

me me me me td (12.5, 3.5)

1.02 dd (12.2, 1.8) 1.54 1.26 2.02 1.70

me qd (13.0, 3.0) dt (12.6, 3.0) td (13.1, 3.7)

1.37 1.86 1.58 3.05 2.91

t (3.7) me me td (11.9, 5.7) td (11.7, 4.6)

5.78 m 1.90 1.33 0.83 0.77 0.91 0.81 3.62

d (1.3) s s s s s s

3.76, 3.77 ABq (15.8) 3.65 s

13

C

34.0, CH2 23.1, CH2 79.9, 37.0, 50.7, 18.0,

CH C CH CH2

41.5, CH2 39.7, 60.8, 37.4, 19.7,

C CH C CH2

44.8, CH2 73.1, C 62.8, CH 24.5, CH2 37.5, CH2 162.4, C 115.5, CH 166.9, C 25.3, CH3 24.4, CH3 16.9, CH3 16.2, CH3 28.0, CH3 21.6, CH3 50.8, CH3 166.5, C 42.1, CH2 167.7, C 52.2, CH3

Acquired in CDCl3. Recorded at 500 MHz for H NMR and 125 MHz for C NMR. Acquired in C5D5N. dRecorded at 750 MHz for 1H NMR and 188 MHz for 13C NMR. eIndicates overlapping signals. 1

13

c

19), and 167.7 ppm (C-3′) along with two olefinic signals at δC 115.5 (C-18) and 162.4 ppm (C-17) accounted for four of the seven double-bond equivalents provided by the molecular formula for 2. Examination of COSY, TOCSY, HSQC, HMBC, and DEPT spectra allowed the complete assignment of 2 (Table 1). In particular, a series of 1D TOCSY experiments along with HSQC and HMBC spectra revealed a number of isolated spin systems, allowing the identification and linkage determination of the structural fragments in 2. A geminal dimethyl group was identified via the HMBC correlations of H3-25 (δH 0.81) and H3-24 (δH 0.91), which showed mutual three-bond couplings with δC 28.0 (C-24) and 21.6 ppm (C25), respectively, as well as shared correlations with signals at δC 37.0 (C-4), 50.7 (C-5), and 79.9 ppm (C-3). A 1D TOCSY spectrum acquired with irradiation of H-3 (δH 4.86) then revealed the chemical shifts of H-2a/H-2b (δH 1.86 and 1.72) and H-1a/H-1b (δH 1.38 and 1.23), allowing the assignment of C-2 (δC 23.1) and C-1 (δC 34.0) via the HSQC spectroscopic

provided a formula of C30H48O7 from an ion at m/z 543.3288 [M + Na]+ (calculated for C30H48NaO7, m/z 543.3292). Inspection of the 1H NMR spectrum of 2 (C5D5N, 750 MHz) revealed signals for six methyl groups attached to quaternary carbons, at δH 0.77 (s, H3-23), 0.81 (s, H3-25), 0.83 (s, H3-22), 0.91 (s, H3-24), 1.33 (s, H3-21), and 1.90 ppm (d, J = 1.3 Hz, H3-20), as well as two OCH3 groups at δH 3.62 (s, H3-26) and 3.65 ppm (s, H3-4′). These signals were correlated in the HSQC spectrum of 2 with signals at δC 16.2 (C-23), 21.6 (C25), 16.9 (C-22), 28.0 (C-24), 24.4 (C-21), 25.3 (C-20), 50.8 (C-26), and 52.2 ppm (C-4′), respectively. Along with the molecular formula for 2, the presence of six methyl singlets in the 1H NMR spectrum strongly suggested that a terpenoid, but not a steroid-derived metabolite, was the major constituent of A. farinosa. The 13C NMR spectrum of 2 (C5D5N, 188 MHz) displayed 30 signals, including eight methyl groups, nine methylene, five methine, and eight quaternary carbons. The presence of three carbonyl signals at δC 166.5 (C-1′), 166.9 (CB

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supported the chosen enantiomer, but the lack of any atoms heavier than oxygen raised the uncertainty in the absolute structure parameter. More conclusive evidence of absolute structure was obtained from the Bijvoet analysis of Hooft et al.8 implemented in PLATON,9 where a probability of 1.0 was obtained for the correct choice of chirality from a twoenantiomer model. The absolute configuration of the chirotopic atoms in 2 are as follows: C-3 (R); C-5 (R); C-8 (R); C-9 (R); C-10 (R); C-13 (S); and C-14 (S). Compound 2 was thus elucidated as methyl (3R)-malonyl(13S)-hydroxycheilanth-17-en-19-oate, a cheilanthane sesterterpene. Cheilanthane natural products are common in marine organisms but have been reported only rarely from plants.10 The NMR structure determination of 2 was conducted in pyridine-d5, a common solvent for the steroidal saponins that were expected in A. farinosa. However, we also report the subsequent 1H and 13C NMR assignment of 2 in CDCl3 (Table 1) to allow comparison of spectroscopic data with similar compounds in the literature. Compound 2 is the first cheilanthane sesterterpene reported to date having oxygenation at position C-3. In addition, although other terpenoids have been reported with a malonyl substituent,11−13 the compounds reported here represent to our knowledge the only example from the sesterterpene class. While the structure and stereochemistry of 2 had been unambiguously determined, it remained unclear which position was esterified in the monoacid natural product 1 isolated from A. farinosa. We thus isolated the major constituent of A. farinosa (as approximately 0.3% dry weight of plant material) via semipreparative RPHPLC performed on a crude methanolic extract of plant material (gradient of 20% to 100% CH3CN over 60 min, 2 mL/min). Compound 1 was isolated as an offwhite solid [mp 78−81 °C; [α]25D −48.7 (c 0.22, CHCl3)], and negative-ion high-resolution ESIMS provided a molecular formula of C29H46O7 from a quasi-molecular ion at m/z 505.3182 [M − H]− (calcd for C29H45O7, m/z 505.3171). The 1 H (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spectra of 1 were largely identical to those of 2 in CDCl3, although only one OCH3 signal was present (δH 3.64; δC 51.2). As above, a combination of COSY, HSQC, HMBC, and DEPT experiments was used to assign the 1H and 13C NMR spectra of 1 (Table 1). Importantly, HMBC correlations from both H-18 (δH 5.64) and H3-26 (δH 3.64) to C-19 (δC 167.1) revealed that 1 possesses a methyl ester at C-19 and thus a malonate halfester at C-3. During the course of this work, the isolation from A. farinosa of a compound with essentially identical spectroscopic properties to 1 was reported in the patent literature; however, neither the relative nor absolute stereochemistry of this compound was provided.14 Despite coincident NMR spectroscopic data with 1 for positions 14−20 and 26 (ΔδH ≤ 0.02 ppm; ΔδC ≤ 0.1 ppm),14 the geometry of the double bond was represented as E not as Z as shown in this work by 1D NOESY experiments and X-ray crystallography. We propose that the reported compound14 is identical to 1 and should be corrected. The major phytochemical constituent 1 of A. farinosa was identified as a new cheilanthane sesterterpene with an unusual malonate half-ester substituent. In light of this work, the substitution of C. luteum with A. farinosa in herbal remedies is not justified chemically, as the former contains predominantly steroidal saponins.3−5

data. Further HMBC correlations from H3-22 (δH 0.83) and H3-21 (δH 1.33), in combination with HSQC, COSY, and TOCSY data, facilitated the assignment of the remainder of the tricarbocyclic core of 2 and revealed a tertiary hydroxy group located at position C-13 (δC 73.1). Similarly, the long-range correlations of H3-20 (δH 1.90) completed the assignment of the C25 pentaprenyl skeleton of 2 and established the presence of a Δ(17)18-unsaturated methyl ester at position C-19. Finally, the HMBC correlations of H-3, H2-2′, and H-4′ allowed assignment of C-1′ (δC 166.5) and C-3′ (δC 167.7) of a malonate methyl ester moiety linked at position C-3. The relative configuration of the ring junctions and substituents in 2 was assigned via examination of the 1D NOESY spectra. Correlations between H3-23 (δH 0.77) and both H3-22 (δH 0.83) and H3-25 (δH 0.81) indicated the β orientation of the C-22, C-23, and C-25 methyl groups, while a correlation between H3-22 and H3-21 (δH 1.33) revealed the α orientation of the C-13 hydroxy group. An NOE correlation between H-9 (δH 1.02) and H-14 (δH 1.37), along with the absence of correlations between H-9 and H3-22/H3-23, suggested the β orientation of the C-14 side chain and the trans fusion of the B and C rings. Similarly, the absence of an NOE correlation between H3-23 and H-5 (δH 1.40) suggested a trans A/B ring fusion. The presence of an NOE correlation between H-3 (δH 4.86) and both H3-24 (δH 0.91) and H3-25 (δH 0.81) supported the equatorial disposition of H-3 and thus the α orientation of the malonyl group. The coupling pattern of H-3 (t, J = 2.6 Hz) was also consistent with the axial orientation of the C-3 substituent. Finally, an NOE correlation between H18 (δH 5.78) and H3-20 (δH 1.90) indicated the Z configuration of the double bond. The absolute configuration of 2 was determined via X-ray crystallography. Crystals suitable for X-ray work were grown from an ethyl acetate/hexane solution. The structure obtained agreed with that deduced from NMR data above (Figure 1). In

Figure 1. ORTEP view of 2. Ellipsoids are drawn at the 30% level of probability.

particular the malonic ester chain is present in its diketo form in the solid state, with both C−C bonds ∼1.50 Å and both methylene protons on the central C atom visible from difference electron density maps during refinement. The absolute structure was deduced from anomalous dispersion analysis using Cu Kα radiation and with a high degree of redundancy (full sphere of data collected on an orthorhombic crystal system). The traditional Flack parameter (0.04 ± 0.16) C

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ASSOCIATED CONTENT

* Supporting Information S

General experimental methods, experimental procedures for the isolation and purification of 1 and the synthesis of 2, X-ray crystallographic data for 2, and 1H and 13C NMR spectra of 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. A voucher specimen of A. farinosa was collected in the Midlands Wildlife Area, Delaware, USA (September 2011), and has been deposited at the Medicinal Plant Herbarium, Southern Cross University, Lismore, Australia (accession number PHARM-110043), where it was identified by Dr. H. Wohlmuth. Crystal data for 2 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 916952). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]).

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61 (7) 3365 3825. Fax: +61 (7) 3365 4299. E-mail: j. [email protected]. Author Contributions §

V.L.C. and S.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank A. Pengelly (Tai Sophia Institute, Maryland, USA) for collection of the voucher specimen and L. Lambert (Centre for Advanced Imaging, The University of Queensland) and are grateful for an Australian Postgraduate Award to V.L.C.

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REFERENCES

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