A Meroisoprenoid, Heptenolides, and C-Benzylated Flavonoids from Sphaerocoryne gracilis ssp. gracilis

A new meroisoprenoid (1), two heptenolides (2 and 3), two C-benzylated flavonoids (4 and 5), and 11 known compounds (6–16) were isolated from leaf, stem bark, and root bark extracts of Sphaerocoryne gracilis ssp. gracilis by chromatographic separation. The structures of the new metabolites 1–5 were established by NMR, IR, and UV spectroscopic and mass spectrometric data analysis. (Z)-Sphaerodiol (7), (Z)-acetylmelodorinol (8), 7-hydroxy-6-hydromelodienone (10), and dichamanetin (15) inhibited the proliferation of Plasmodium falciparum (3D7, Dd2) with IC50 values of 1.4–10.5 μM, although these compounds also showed cytotoxicity against human embryonic kidney HEK-293 cells. None of the compounds exhibited significant disruption in protein translation when assayed in vitro.

T he genus Sphaerocoryne (Annonaceae), formerly known as Melodorum, consists of the three species S. f ruticosum, S. gracilis (ssp. engleriana and gracilis), and S. punctulatum. It is represented by S. gracilis ssp. engleriana, native to Angola, the Democratic Republic of Congo, and Zambia, in western Africa, while S. gracilis ssp. gracilis is confined to Kenya, Mozambique, and Tanzania, in eastern Africa. In Tanzania, S. gracilis ssp. gracilis (Engl. & Diels) Verdc. is reported to grow in a few fragmented localities, including forest reserves in the Eastern Arc Mountains and in the Coastal Forests. 1,2 Previous phytochemical studies of the genus revealed the presence of aporphine alkaloids in S. punctulatum (formerly Melodorum punctulatum), a small rain-forest tree endemic to New Caledonia (a French archipelago in the Southwest Pacific Ocean). 3 Further studies revealed that S. f ruticosum (M. f ruticosum), a species native to Southeast Asia, contains novel antitumor heptenolides. 4−6 Phytochemical investigation of the Tanzanian Sphaerocoryne species also yielded heptenolides together with polyhydroxylated C-benzyl flavanones. 7,8 The structures of some of the polyhydroxylated C-benzyl flavonoids have not been fully described, and nor have their bioactivities been reported. 7,8 As part of our continued efforts in the search for bioactive metabolites from Tanzanian flora, S. gracilis spp. gracilis was reinvestigated regarding its activity against the malaria pathogen Plasmodium falciparum and also for cytotoxicity using the human embryonic kidney cell (HEK-293) assay. Some of the isolated metabolites were also screened for their potential translational inhibitory activity by monitoring both cap-dependent and independent translation. 9,10 ■ RESULTS AND DISCUSSION Repeated silica gel column chromatography of the methanolic extracts of the stem and root barks and leaves of Sphaerocoryne gracilis spp. gracilis, followed by gel filtration on Sephadex LH-20 and further purification on HPLC, gave five new metabolites (1−5) and 11 known compounds (6−16, Figure  S1, Supporting Information). The structures of the new meroisoprenoid (1), two heptenolides (2 and 3), and two Cbenzylated flavonoids (4 and 5) were established using NMR spectroscopic and mass spectrometric techniques. The identities of the known compounds 3″-hydroxygracinol (6), 7 (Z)-sphaerodiol (7), 11,12 (Z)-acetylmelodorinol (8), 10,11,13 (Z)-melodorinol (9), 10−12 7-hydroxy-6-hydromelodienone (10), 4 pinocembrin (11), 11,14 5,7-dihydroxyflavone (12), 15 chamanetin (13), 16 isochamanetin (14), 16 dichamanetin (15), 16 and polycarpol (16) 16 were confirmed by comparing their spectroscopic data with those previously reported.
Compound 1 was obtained as a colorless oil and was assigned the molecular formula C 21 H 23 O 6 based on HRESIMS ( Figure S9, Supporting Information) and NMR data (  Figure S7, Supporting Information) these were established as being part of a cinnamoyl moiety. Thus, the β-proton (δ H 7.75) showed 3 J H,C HMBC correlations with the aromatic carbons C-2′/6′ (δ C 129.9) and the carbonyl carbon C-9′ (δ C 167.6) of the cinnamoyl moiety. The aromatic proton signals at δ H 7.39−7.40 were assigned to H-3′/5′ and H-4′, while the signal at δ H 7.54 to H-2′/6′. Moreover, the HSQC spectrum ( Figure S6, Supporting Information) indicated two sets of oxymethylene units with diastereotopic protons. One of these was part of an ABX spin system (COSY) encompassing protons H-1a (δ H 4.69), H-1b (δ H 4.47), and H-2 (δ H 3.96). These diastereotopic protons gave HMBC cross-peaks to C-7″ (δ C 167.3) and were hence linked to the benzoyloxy unit. The second set of diastereotopic protons H-4a (δ H 4.48) and H-4b (δ H 4.20) showed only a geminal coupling, 2 J = 11.5 Hz, and were shown to be linked to the oxygenated quaternary carbon C-3 (δ C 73.4) on one side and to the cinnamoyl unit on the other, as indicated by their HMBC cross-peaks to C-2 (δ C 73.6), C-3 (δ C 73.4), and C-9′ (δ C 167.6). The position of CH 3 -3 (δ H 1.34; δ C 20.0) was deduced based on its HMBC cross-peak to the oxy-quaternary chiral carbon C-3 (δ C 73.4). Hence, compound 1 was concluded to be constituted by an isoprenoyl unit attached to a cinnamoyl unit on one end and a benzoyloxy moiety on the other end.
The relative configurations of C-2 (δ C 73.6) and C-3 (δ C 73.4) might be indicated by the NOESY correlation ( Figure  S5, Supporting Information) of CH 3 -3 (δ H 1.34) with oxymethine proton H-2 (δ H 3.96), suggesting the erythro relationship of the hydoxy groups at C-2 and C-3. As the C-2− C-3 bond freely rotates, the determination of the relative configuration of these carbons cannot be trusted and should be corroborated by other techniques. Based on the above spectroscopic evidence, however, this new natural product, gracidiol (1), isolated from the leaves of S. gracilis ssp. gracilis, was characterized as 4-(cinnamoyloxy)-2β,3β-dihydroxy-3αmethylbutyl benzoate. From the biogenesis point of view, the compound is envisaged to be a meroisoprenoid formed through both mevalonic and shikimate pathways. It is unprecedented in the genus Sphaerocoryne.
Compound 2 was obtained as a colorless oil. Its molecular formula was determined as C 9 H 10 O 5 based on HRESIMS ( Figure S17, Supporting Information) and NMR (Table 2) analyses. Its UV spectrum showed an absorption at λ max 270 nm, consistent with a conjugated system. 16 The broad IR absorption band at 3446 cm −1 is typical of a hydroxy functionality, whereas those at 1743 and 1781 cm −1 are compatible with the α,β-unsaturated carbonyls of butenolides. 11,16 The chemical shifts of the olefinic protons resonating at δ H 6.27 (H-2) and δ H 7.38 (H-3) were also consistent with an α,β-unsaturated carbonyl moiety. The NMR spectroscopic Comparison of its NMR data suggested 2 to be a close derivative of (Z)-sphaerodiol (7), with additional NMR signals observed at δ H 2.11 and δ C 20.9 and δ C 171.2 (OAc-7), suggesting the presence of an additional acetyl group. The position of the acetyl moiety at C-7 was established from the HMBC (Table 2; Figure S16, Supporting Information) crosspeaks of the C-7 methylene protons (δ H 4.24 and δ H 4.18) to δ C 171.2 (OAc-7). NOESY correlations ( Figure S14, Supporting Information) of H-3 (δ H 7.38) to H-5 (δ H 5.33) indicated the exocyclic double-bond geometry is Z. Its configuration at C-6 was the same as in compound 7, based on the comparable sign and magnitude of optical rotation. 10,11 Based on the above evidence, the new compound (Z)-7acetylsphaerodol (2) was characterized as (S,Z)-2-hydroxy-3-(5-oxofuran-2(5H)-ylidene)propyl acetate, the acetylated derivative of (Z)-sphaerodiol (7). 11 Both compounds were obtained from the stem and the root bark methanol extracts of S. gracilis ssp. gracilis. Compound 3 was obtained as a colorless oil and was assigned the molecular formula C 16 H 15 O 7 based on analysis of its HRESIMS ( Figure S25, Supporting Information) and NMR (Table 3, Figures S18−S24, Supporting Information) data. Its IR spectrum showed absorption bands at 3417 and 1639 cm −1 , indicating the presence of O−H and CC functionalities, respectively. 16 The UV absorptions at λ max 205 and 240 nm along with the NMR data were compatible with the presence of a benzene moiety and an α,β-unsaturated carbonyl system, which was corroborated by NMR (vide infra). 16 The 13 C NMR spectrum (Table 3, Figure S19, Supporting Information) showed 16 signals, which were sorted with the aid of the HSQC spectrum ( Figure S23, Supporting Information) into an oxymethine, an oxymethylene, an oxomethyl, three carbonyls, and 10 sp 2 carbons. Six sp 2 carbons were assigned to an aromatic ring and four to two olefinic bonds ( Table 3). The 1 H NMR spectrum of 3 (Table 3, Figure S18, Supporting Information) displayed signals at δ H 4.61 (H-7a), δ H 4.55 (H-7b), δ H 5.31 (H-5), and δ H 6.15 (H-6) corresponding to an ABMX spin system, as revealed by COSY and TOCSY spectra ( Figures S20 and S22, Supporting Information), resembling that observed for compound 2. The chemical shifts of the olefinic protons δ H 6.30 (H-2) and δ H 7.38 (H-3) were also similar to those of 2 and hence compatible with an α,βunsaturated carbonyl moiety. The above spectroscopic features are reminiscent of a butenolide system with an extension of an alicyclic three-carbon skeleton forming a heptenolide moiety, which has previously been reported for similar metabolites. [4][5][6][7]10,11 Overall, the NMR data of 3 resembled that of (Z)-acetylmelodorinol (8) and of related compounds 4−7,10,11 with the only difference being the ortho-hydroxy group substitution of its benzoyloxy group. The placement of this hydroxy group functionality was established by the ABCD coupling pattern of the aromatic signals ( 1 H NMR, COSY, and TOCSY) and by HMBC correlations (Table 2, Figure S24, Supporting Information). NOESY correlations ( Figure S22, Supporting Information) of H-3 (δ H 7.38) and H-5 (δ H 5.31) revealed the Z-geometry of the exocyclic C-4, C-5 double bond, while the NOE of H-5 (δ H 5.31) and H-6 (δ H 6.15) might indicate the β-orientation of OAc-6. Whereas the latter assignment may be corroborated by the configuration of closely related compounds previously isolated from this genus, 4−7,11,17 the C-5 to C-6 bond can freely rotate, making a purely NOE-based assignment unreliable. Nonetheless, the configuration of compound 3 was determined as being similar to those of (Z)-acetylmelodorinol (8) and (Z)-melodorinol (9), which were also isolated in the present study. The absolute configuration of the latter was recently determined by single-crystal X-ray diffraction analysis. 10 Based on the above evidence, this new natural product, (Z)-2′-hydroxyacetylmelodorinol (3), was characterized as (S,Z)-2-acetoxy-3-(5oxofuran-2(5H)-ylidene)propyl 2-hydroxybenzoate.
Compound 4 was obtained as a white powder and was assigned the molecular formula C 22 H 19 O 6 , based on the HRESIMS ( Figure S33, Supporting Information) and NMR (Table 4) data. Its UV absorptions found at λ max 210, 290, and 320 nm are diagnostic of dihydroflavones. 18 The IR absorptions at 3430, 1641, and 1255 cm −1 corresponded to   Figure S26, Supporting Information) along with the COSY spectrum ( Figure S28, Supporting Information) indicated an ABX coupling pattern for H-3α (δ H 3.08), H-3β (δ H 2.84), and H-2 (δ H 5.38), typical of the ring C of flavanones. 17 An AB spin system integrating to two protons at δ 3.87 was assigned to the benzyl methylene protons H-7″ with the help of the HSQC experiment ( Figure  S30, Supporting Information). The signals at δ H 6.74 (C-4″/ C-6″) and δ H 7.05 (C-5″) were deduced as belonging to aromatic protons of a dihydroxybenzyl group. The coupling pattern of the protons of the C-benzyl moiety supported the 2″-and 3″-hydroxy groups as being ortho to each other. 17,18 The linkage of the dihydroxybenzyl group at C-6 instead of C-8 of the dihydroflavone moiety was deduced from the 3 J CH HMBC of H-7″ (δ H 3.87) to C-5 (δ C 160.7) (Table 4, Figure  S31, Supporting Information). In turn, the singlet at δ H 6.03 was assigned to H-8 (instead of C-6 as in 5  Figure S40, Supporting Information) and NMR (Table 5) data. The UV absorption at λ max 280 nm was in agreement with a flavone, 18 while the IR absorptions were consistent with hydroxy (3430 cm −1 ), aromatic (1642 cm −1 ), and C−O (1260 cm −1 ) stretches. 15 The NMR spectroscopic data (Table 5, Figures S33−S39, Supporting Information) were comparable to those of 4, except the absence of an ABX spin system in the 1 H NMR spectrum for ring C. Instead, 5 showed a singlet at δ H 6.69 corresponding to the olefinic H-3 of a flavone moiety. The 3 J H,C HMBC signals (Table 5, Figure S39, Supporting Information) of H-7″ (δ H 4.16) to C-7 and C-8a indicated a dihydroxybenzyl group to be substituted at position C-8, instead of C-6 as in compound 4. The 2D NMR data (Figures S35−S39, Supporting Information) of 5 showed resemblances to those of 4 and were in agreement with the established structure. Based on the above spectroscopic features, the new compound, 2,3-dehydro-3″-hydroxychamanetin (5), was characterized as 8-(2,3-dihydroxybenzyl)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one.
In conclusion, a new meroisoprenoid (1), two new heptenolides (2 and 3), two new C-benzylated flavonoids (4 and 5), and 11 known flavonoids, heptenolides and triterpenoids (6−16), were isolated from S. gracilis ssp. gracilis. The isolation of heptenolides from this plant is of significance, as these compounds have so far been reported to be restricted to the Uvariae tribe of the family Annonaceae. In line with previous reports, some of the isolated heptenolides and flavonoids (7−10 and 15, Table 6) showed antiplasmodial activities that may be interesting for drug development.

■ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined with a Buchi B-545 melting point instrument. The optical rotation and circular dichroism for compounds possessing chiral centers were determined using a 341LC OROT polarimeter (589 nm temp 20.0°C) and a JASCO J-715 spectrometer, respectively. The UV measurements were done using a 264 UV−vis spectrophotometer. A MIR 450FT-IR spectrometer was used to record the IR spectra. NMR spectra were acquired on a Bruker Avance III HD 800 NMR MHz spectrometer and analyzed with the software MestReNova (v10.0.0). Structural assignments were based on 1 H NMR, 13  Extraction and Isolation. Samples of the stem bark, root bark, and leaves were air-dried for 2 weeks and then pulverized to obtain about 2 kg of each. The materials were then separately soaked twice, consecutively, in methanol for 48 h. The extracts were then filtered and concentrated in vacuo on a rotary evaporator, affording 70.0, 43.0, and 73.0 g of leaf, stem bark, and root bark extracts, respectively.
The crude methanol extract of the stem bark (43.0 g) was fractionated chromatographically and purified as described above, leading to the isolation of (Z)-7-acetylsphaerodol (2, 32.0 mg), 3″hydroxygracinol (6, 89.0 mg), (Z)-sphaerodiol (7, 24.  Antiplasmodial and Cytotoxicity Assays. Antiplasmodial activity was determined using a high-content imaging assay, as described previously. 10,24 The cytotoxicity of the antiplasmodial compounds was evaluated against human embryonic kidney cells (HEK-293) following an established protocol. 10,24 Human red blood cells for culture of P. falciparum were provided by the Australian Red Cross Blood Bank in accordance with their routine Material Transfer Agreement (MTA) for nonclinical blood product supply. These bioassay studies were approved by the Griffith University Biosafety and Human Ethics Committee (GU ref no. ESK/03/12/HREC; 03/ 08/11019).
Translation Inhibition Assay. Luciferase inhibition activity assays were performed following standard procedures described previously. 9,10