Isolation and structure elucidation of caryophyllane sesquiterpenoids from leaves of Eremophila

Crude extract of Eremophila spathulata leaves was investigated by semi-preparative scale high-performance liquid chromatography (HPLC), analytical scale HPLC, and hyphenated high-performance liquid chromatography-photodiode array-high-resolution mass spectrometry-nuclear magnetic resonance (HPLC-PDA-HRMS-SPE-NMR), which afforded seven previously unreported caryophyllane sesquiterpenoids. Semi-preparative scale separation of the crude extract afforded (1 R* ,4 R *,9 S* , E )-8-formyl-11,11-dimethylbicyclo[7.2.0]undec-7-ene-4-carboxylic acid ( 5 ) and analytical-scale HPLC separation afforded (1 R* ,4 S* ,7 S *,9 S* )-7-hydroxy-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 1 ), (1 S* ,6 R* ,9 R*,E )-10,10-dimethylbicyclo[7.2.0]undec-2-ene-2,6-dicarboxylic acid ( 2 ), (1 R* ,4 S* ,9 S* )-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid ( 3 ), and (1 R* ,4 R* ,9 S* )-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid ( 4 ). HPLC-PDA-HRMS-SPE-NMR afforded (1 R* ,4 R* ,9 S* )-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 6 ) and (1 R* ,4 S* ,9 S* )-11,11-dimethyl-8-methylenebicyclo[7.2.0]undecane-4-carboxylic acid ( 7 ). The structures of all isolated compounds were established based on HRMS as well as extensive 1D and 2D NMR analysis. Relative configurations were determined by correlations in spectra from rotational Overhauser effect spectroscopy.


Introduction
Eremophila (Scrophulariaceae) is a genus with more than 200 known species of which some have been used by the Australian Aboriginal people for medicinal and ceremonial purposes (Ghisalberti, 1994).Eremophila spp.are also commonly known as fuchsia bush, poverty bush or emu bush and are shrubs or small trees endemic to Australia, and frequently found in Western Australia.They grow in arid and semi-arid areas and the genus is adapted to live under these harsh conditions of the deserts, which the name Eremophila also indicate, being derived from the Greek words eremos meaning 'desert' and phileo meaning 'love'.Many Eremophila species have been biologically and/or phytochemically investigated since the 1950s, and these studies indicate that Eremophila spp.are especially rich in terpenoids, mostly sesquiterpenoids and diterpenoids, but also flavonoids and fatty acids have been isolated (Ghisalberti, 1994;Richmond and Ghisalberti, 1994;Singab et al., 2013;Fowler et al., 2020;Pedersen et al., 2020).In recent years, more than 200 natural products have been isolated from Eremophila, and some of these have shown antimicrobial (Ndi et al., 2007), antiviral (Singab et al., 2013), and cytotoxic properties (Anakok et al., 2011).Other natural products isolated from Eremophila have shown protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase inhibitory activity (Wubshet et al., 2016;Tahtah et al., 2016: Zhao et al., 2019;Kjaerulff et al., 2020), potentially making them drug leads for management of type 2 diabetes.
Caryophyllanes constitute a class of sesquiterpenoids characterized by a unique ring system, in which a dimethylcyclobutane and a cyclononane are fused.The first identified molecule belonging to this compound class was β-caryophyllene that was isolated from Eugenia caryophyllata L. in 1834 (Di Sotto et al., 2020).β-Caryophyllene and β-caryophyllene oxide are the two major compounds in this class and they are frequently isolated from various plants.The caryophyllanes isolated so far have shown many different pharmacological activities, e.
g. anti-inflammatory, antibacterial, antifungal, and chemosensitizing activity (Di Sotto et al., 2020).Eremophila spathulata has not been phytochemically investigated before, and in this paper, we describe the targeted isolation of caryophyllane sesquiterpenoids from leaves of Eremophila spathulata.

Results and discussion
As part of our ongoing research on chemical and pharmacological properties of Eremophila species (Wubshet et al., 2016;Tahtah et al., 2016;Zhao et al., 2019;Pedersen et al., 2020;Gericke et al., 2021), leaf extracts of Eremophila spathulata W.Fitzg. (Scrophulariceae) were investigated using high-performance-liquid chromatography-photodiode array-high-resolution mass spectrometry (HPLC-PDA-HRMS).This indicated the presence of a series of previously unreported sesquiterpenoids, and further investigation of the leaf extracts of E. spathulata were therefore initiated.

Isolation, identification, and structure elucidation
Crude extract of E. spathulata was separated by semi-preparative reversed-phase HPLC, leading to isolation of fractions F1-F4 as well as compound 5 as shown in Fig. 1.Fractions F1-F3 were subsequently subjected to separation by analytical-scale HPLC-PDA, leading to isolation of compounds 1-4, and fraction F4 was subjected to highperformance-liquid chromatography-photodiode array-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC-PDA-HRMS-SPE-NMR) analysis (Schmidt et al., 2014;Wubshet et al., 2016), leading to isolation of compounds 6 and 7 (Fig. 2).Assigned 1 H and 13 C NMR data for all isolated compounds are provided in Tables 1 and 2, selected correlations used for structure elucidation are provided in Fig. 3, 1 H NMR, COSY, HSQC, HMBC, and ROESY spectra are provided in Supplementary data Fig.S1-S35, and correlations observed in these spectra are provided in Supplementary data Tables S1-S7.
The nine-membered ring system was identified based on the H-3 ↔   corresponding to the molecular formula C 14 H 22 O 3 and a hydrogen deficiency index of 4 for both compounds.The COSY spectrum of 3 revealed the H-10 spin system (Supplementary data Table S3 and Fig. 3), and together with HMBC correlations from H-1, H-6, H-7α, H-7β, H-9, H-10α, and H-10β to C-8 at δ 216.5, this showed 3 to contain the basic 11,11-dimethylbicyclo [7.2.0]undecane structure, but having an oxo group at C-8 instead of a branching carbon atom (C-13 in 1 and 2) (Fig. 2).HMBC correlations from H-3β and H-5A/H-5B to the resonance at δ C 180.1 established the position of the carboxylic acid at C-4. ROESY correlations from H-10α to Table 1 1 H and 13 C NMR spectroscopic data of compounds 1-4.H-14 and H-1 showed these three hydrogens to be α-oriented, whereas a ROESY correlation network from H-13 to H-10β to H-9 to H-7β to H-4 showed these hydrogens to be β-oriented.Thus, compound 3 was identified as the previously unreported (1R*,4S*,9S*)-11,11-dimethyl-8-oxobicyclo[7.2.0]undecane-4-carboxylic acid, for which the name spathulatane C is suggested.Compound 4 was shown to have the same basic skeleton as 3 based on correlations found in COSY and HMBC spectra (Supplementary data Table S4 and Fig. 3).However, the ROESY spectrum of 4 showed correlations from H-14 (i.e., H-15 in 1 and 2) to H-1 to H-4, i.e., the same ROESY correlation pattern as seen for 1 and 2, showing 4 to have the same configuration at C-4 as 1 and 2 and thus being the 4-epimer of 3. Thus, compound 4 was identified as the previously unreported (1R*,4R*,9S*)-11,11-dimethyl-8-oxobicyclo[7.2.0] undecane-4-carboxylic acid, for which the name spathulatane D is suggested.
The material eluted with peak 5 showed an It should be noted that a similar structure named 5,6-dihydrocaryophyllen-15-oic acid previously has been proposed, but only isolated as the methyl ester and not providing stereochemistry and NMR data (Bohlmann et al., 1980).The sesquiterpenoids isolated from E. spathulata are characterized by sharing a caryophyllane core structure.Biosynthetically they are envisioned formed from (E,E)-farnesyl pyrophosphate by the action of a sesquiterpene synthase.Sesquiterpene synthases catalyzing conversion of (E,E)-farnesyl pyrophosphate to (E)-β-caryophyllene have been characterized from several plant species (Yoshikuni et al., 2006;Cheng et al., 2007;Köllner et al., 2008;Bleeker et al., 2011;Christianson, 2017).However, some sesquiterpene synthases are able to use (E, E)-farnesyl pyrophosphate as well as (Z,Z)-farnesyl pyrophosphate as substrates (Bleeker et al., 2011;Yoshikuni et al., 2006).Eremophila species are known to produce diterpenoids from nerylneryl pyrophosphate, the (Z,Z) isomer of geranylgeranyl pyrophosphate (Zhao et al.,  2019; Kjaerulff et al., 2020;Gericke et al., 2020Gericke et al., , 2021)).However, none of the sesquiterpenoids isolated from E. spathulata harbors double bonds matching the position of the acyclic C 15 isoprenoid pyrophosphate precursor.It is therefore not possible to determine from the structures produced, whether the caryophyllanes isolated from E. spathulata are formed using (2E,6E)-farnesyl pyrophosphate or (2Z,6Z)-farnesyl pyrophosphate as the substrate, and the substrate preferences of the sesquiterpene synthase in E. spathulata catalyzing caryophyllene formation thus remains unclear A putative biosynthetic pathway based on (2Z,6Z)-farnesyl pyrophosphate is provided in Fig. 4. The formation of a basic caryophyllene skeleton requires formation of two new C-C bonds, and it is envisioned that the sesquiterpene synthase involved positions the acyclic C 15 isoprenoid pyrophosphate in the active site pocket in a reaction-ready conformation (Christianson, 2017).The basic caryophyllene skeleton formed is then decorated by monooxygenation reactions most likely catalyzed by cytochrome P450 enzymes (Hansen et al., 2021).In all isolated sesquiterpenoids, the first double-bond-attached methyl group of a putative isoprenoid diphosphate starter unit (C-12 in 1-7) has been subject to a set of triple hydroxylation reactions, resulting in its conversion into a carboxylic acid, with the possibility of R or S configurations at C-4.In compounds 3 and 4, the vinylic C-13 carbon of the basic caryophyllene skeleton has been lost and replaced by an oxo function.Biosynthetically, this may be envisioned to proceed by consecutive P450-catalyzed monooxygenation reactions, initially resulting in the introduction of an aldehyde function followed by formation of a carboxylic acid group, a subsequent decarboxylation reaction, and finally introduction of an oxo function at C-8 by two monooxygenation reactions.The same series of successive monooxygenations from a putative hydroxyl group at C-13 (compound not isolated) to the aldehyde function (5) and carboxylic acid (2) was observed, likely starting from the loss of the C-7 hydroxyl group of 1.The observed adjacent presence of a double bond or hydroxyl group at C-7 in compounds 2 and 1, respectively, are most likely also introduced by cytochrome P450 oxygenations.The multiplicity of cytochrome P450 catalyzed oxygenation reactions may be carried out by multifunctional cytochromes P450 as observed in other terpenoid pathways (Hansen et al., 2021).
Eremophila spp.are culturally important plants for many of Australia's First Peoples, the Aboriginal peoples.If you use the information here provided to make commercial products, we urge you to strongly consider benefit sharing with the Aboriginal communities or groups in the areas where these species grow.We acknowledge that this work took place on the lands of Aboriginal peoples who are the custodians of this land and acknowledge and pay our respects to their Elders, past and present.
Before trapping the cartridges were conditioned with 1000 μL MeOH at a flow rate of 6 mL/min and equilibrated with 500 μL H 2 O at a flow rate of 1 mL/min.The loaded SPE cartridges were dried with pressurized nitrogen gas for 45 min each before the trapped material was eluted into 1.7-mm NMR tubes with methanol-d 4 , using a Gilson 215 liquid handler equipped with a 1-mm needle.Chromatographic separation and analyte trapping were controlled by Hystar ver.3.2 (Bruker Daltonik, Bremen, Germany), and the elution process was controlled by Prep Gilson ST ver.1.2 (Bruker Biospin, Karlsruhe, Germany).NMR experiments were acquired on a Bruker Avance III 600 MHz NMR spectrometer ( 1 H operating frequency 600.13 MHz) equipped with a Bruker SampleJet autosampler and a 1.7-mm TCI cryoprobe (Bruker Biospin, Karlsruhe, Germany) using standard Bruker pulse sequences.All acquisitions of NMR data were recorded with temperature equilibration to 300 K, optimization of lock parameters, gradient shimming, and setting of receiver gain, all automatically controlled by Topspin ver.3.5 pl 7 and IconNMR ver.5.0.7 (Bruker Biospin, Karlsruhe, Germany).Chemical shifts of 1 H and 13 C were referenced to the residual solvent signals at respectively δ H 3.31 and δ C 49.0.

Plant material and extraction
The leaves of Eremophila spathulata W.Fitzg. (Scrophulariaceae) were collected from several individual plants in May 2017 224 km South of Kumarina, Western Australia, Australia (26 • 24 ′ 15.1 ′ ' S; 118 • 35 ′ 53.8 ′ ' E) and identified by Dr Bevan Buirchell.A voucher specimen was lodged at the University of Melbourne Herbarium, Melbourne, Victoria, Australia (accession number MELUD122714a).The material was stored at − 20 • C until it was extracted.To extract the leaf resins, whole leaves (84.14 g) were submerged in 250 mL of acetonitrile and shaken for 10 min (Ratek, Knox City, VIC, Australia).The extract was then filtered using a glass funnel.The filtrate was dried in vacuo at 45 • C using a IKA RV10 rotary evaporator (IKA Werke, Staufen, Germany).The extract (2.52 g) was re-dissolved into a small volume of methanol transferred in amber vials, dried under a stream of nitrogen gas, and then stored at − 20 • C until use.
Separations were performed at 40 • C with a flow rate of 0.5 mL/min, using a combination of solvent A (H 2 O/MeCN/HCO 2 H, 94.9:5:0.1,v/v) and solvent B (H 2 O/MeCN/HCO 2 H, 5:94.9:0.01,v/v) with the following gradient profile: 0 min, 35 % B; 2 min, 35 % B; 12 min, 50 % B; 13 min, 65 % B; 47 min, 100 % B; 57 min, 100 % B. Mass spectra were acquired in both positive and negative mode and a solution of sodium formate clusters was automatically injected at the beginning of each run to enable internal mass calibration.
For analytical-scale HPLC-separation, the material eluted with fraction F1 was re-dissolved in a 40:60 mixture of water and MeCN to a concentration of 12.5 mg/mL, and separated isocratically at 57 % B to yield 1 (14.1 min, 0.68 mg, more than 95 % purity as determined by 1 H NMR). The material eluted with fraction F2 was re-dissolved in a 40:60 mixture of water and MeCN to a concentration of 12.5 mg/mL, and separated isocratically at 58 % B to yield 2 (15.5 min, 0.39 mg, more than 95 % purity as determined by 1 H NMR) and 3 (16.4min, 0.44 mg, more than 95 % purity as determined by 1 H NMR). The material eluted with fraction 3 was re-dissolved in a 40:60 mixture of water and MeCN to a concentration of 12.5 mg/mL, and separated isocratically at 50 % B to yield 4 (17.3 min, 1.12 mg, more than 95 % purity as determined by 1 H NMR).
For HPLC-PDA-HRMS-SPE-NMR analysis, fraction F4 was redissolved in buffer B to a concentration of 12.5 mg/mL and separated isocratically at 72 % B. Successive trapping of 6 and 7 after three repeated separations of 20-μL injections yielded 0.33 mg of 6 (26.9 min, more than 95 % purity as determined by 1 H NMR) and 0.21 mg of 7 (27.4min, more than 95 % purity as determined by 1 H NMR).
(1H, ddd, J H1,H2β = 11.3,J H1, H9 = 9.2, J H1,H2α = 2 c nH = number of hydrogens, mult.= multiplicity.d Multiplicities reported as apparent splittings: s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, m = multiplet, br = broad.α denotes hydrogen pointing into the plane, β denotes hydrogen pointing out of the plane.A denotes the lowest chemical shift value and B denotes the highest chemical shift value.Coupling constants assigned where possible. 600.13) and 13 C NMR (150.90MHz) data obtained with samples in methanol-d 4 .b Assignments based on HSQC and HMBC experiments.c nH = number of hydrogens, mult.= multiplicity.d Multiplicities reported as apparent splittings: s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, m = multiplet, br = broad.α denotes hydrogen pointing into the plane, β denotes hydrogen pointing out of the plane.A denotes the lowest chemical shift value and B denotes the highest chemical shift value.Coupling constants assigned where possible.E.K. Bredahl et al.

Fig. 3 .
Fig. 3. Key 2D NMR correlations used for the structure elucidation of compounds 1-7.COSY correlations are shown as black bold lines, ROESY correlations are shown as double-headed red arrows, and HMBC correlations are shown as blue arrows pointing from H to C.