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

Abstract

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.

The genus Sphaerocoryne (Annonaceae), formerly known as Melodorum, consists of the three species S. fruticosum, 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. fruticosum (M. fruticosum), 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 C-benzylated 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.

Chart 1.

Compound 1 was obtained as a colorless oil and was assigned the molecular formula C₂₁H₂₃O₆ based on HRESIMS (Figure S9, Supporting Information) and NMR data (Table 1, Figures S2–S8, Supporting Information). Its UV spectrum showed absorptions at λ_max 215, 230, and 275 nm, consistent with a conjugated π-system encompassing a benzene ring, whereas its IR spectrum indicated O–H (3424 cm^–1), C=C/C=O (1641 cm^–1), and C–O (1265 cm^–1) stretches.^11,16 The NMR data (Table 1, Figures S2 and S3, Supporting Information) suggested 1 to have two carbonyl functionalities, two aromatic systems, and four oxygen-substituted aliphatic groups. The NMR spectra showed characteristic signals for a benzoyloxy moiety, that is H-2″/6″ (δ_H 8.05, δ_C 129.6), H-3″/5″ (δ_H 7.45, δ_C 128.6), and H-4″ (δ_H 7.56, δ_C 133.4). In addition, the ¹H NMR spectrum indicated the presence of trans-disposed olefinic protons at δ_H 7.75 and δ_H 6.47 (J = 16.0 Hz) with chemical shifts compatible with an α,β-unsaturated carbonyl system. Based on HMBC correlations (Table 1, Figure S7, Supporting Information) these were established as being part of a cinnamoyl moiety. Thus, the β-proton (δ_H 7.75) showed ³J_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, ²J = 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 CH₃-3 (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.

Table 1. NMR Spectroscopic Data (¹H at 800 MHz, ¹³C at 200 MHz, CDCl₃) of Gracidiol (1).

position	δC, type	δH	J (Hz)	HMBC, H→C
1	66.3, H2C–O	4.69	dd (11.8, 2.8)	C-7″
		4.47	dd (11.8, 7.8)	C-2, C-3, C-7″
2	73.6, HC–O	3.96	dd (7.8, 2.8)	C-1, C-3, C-4, CH3-3
OH-2/3		3.49	s
3	73.4, C–O
CH3-3	20.0, CH3	1.34	s	C-2, C-4
4	68.9, H2C–O	4.48	d (11.5)	C-2, C-3, CH3-3, C-9′
		4.20	d (11.5)	C-2, C-3, CH3-3, C-9′
1′	134.2, C
2′/6′	128.4, CH	7.54	m	C-3′/5′, C-4′, C-7′
3′/5′	129.1/130.8, CH	7.39–7.40a	m	C-1′, C-2′/6′
4′	130.8/129.1, CH	7.39–7.40a	m	C-2′/6′
7′	146.4, CH	7.75	d (16.0)	C-1′, C-2′/6′, C-8′, C-9′
8′	117.2, CH	6.47	d (16.0)	C-1′, C-7′, C-9′
9′	167.6, C=O
1″	134.2, C
2″/6″	129.9, CH	8.05	m	C-4″, C-7″
3″/5″	128.6, CH	7.45	m	C-2″/6″, C-4″
4″	133.4, CH	7.56	m	C-2″/6″
7″	167.3, C=O

^aOverlapping signals.

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 CH₃-3 (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-3α- methylbutyl 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 C₉H₁₀O₅ 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 data of 2 (Table 2, Figures S10–S16, Supporting Information) were diagnostic for a heptenolide moiety consisting of ABMX [δ_H 4.24 (H-7a), δ_H 4.18 (H-7b), δ_H 5.03 (H-6), and δ_H 5.33 (H-5)] and α,β-unsaturated [(δ_H 7.38 (H-3) and δ_H 6.27 (H-2)] spin systems, resembling that of compound 7.^11,17 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) cross-peaks 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)-7-acetylsphaerodol (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.

Table 2. NMR Spectroscopic Data (¹H at 800 MHz, ¹³C at 200 MHz, CDCl₃) of 7-Acetylsphaerodol (2).

position	δC, type	δH	J (Hz)	HMBC, H→C
1	168.9, C=O
2	121.1, CH	6.27	d (5.5)	C-1, C-3, C-4
3	143.7, CH	7.38	d (5.5)	C-1, C-2, C-4, C-5
4	150.1, C
5	112.9, CH	5.33	d (8.0)	C-3, C-4, C-6, C-7
6	65.9, C–O	5.03	ddd, (8.0, 6.8, 3.8)	C-4, C-5, C-7
7	67.2, H2C–O	4.24	dd, (11.5, 3.8)	C-5, C-6, O–Ac-7 (C=O)
		4.18	dd, (11.5, 6.8)	C-5, C-6, OAc-7 (C=O)
OAc-7	171.2, C=O
	20.9, CH3	2.11	s	OAc-7 (C=O)

Compound 3 was obtained as a colorless oil and was assigned the molecular formula C₁₆H₁₅O₇ 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 ¹³C 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 sp² carbons. Six sp² carbons were assigned to an aromatic ring and four to two olefinic bonds (Table 3). The ¹H 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,β-unsaturated 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.^4−7,10,11 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 (¹H 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-(5-oxofuran-2(5H)-ylidene)propyl 2-hydroxybenzoate.

Table 3. NMR Spectroscopic Data (¹H at 800 MHz, ¹³C at 200 MHz, CDCl₃) of (Z)-2′-Hydroxyacetylmelodorinol (3).

position	δC, type	δH	m, J (Hz)	HMBC, H→C
1	168.5, C=O
2	121.9, CH	6.30	d (5.5)	C-1, C-3, C-4
3	143.4, CH	7.38	d (5.5)	C-1, C-2, C-4
4	150.9, C
5	108.6, CH	5.31	d (7.9)	C-3, C-4, C-6, C-7
6	67.2, HC–O	6.15	ddd, (7.9, 6.1, 3.9)	C-4, C-5, C-7, OAc-6 (C=O)
OAc-6	169.9, C=O
	21.0, CH3	2.11	s	OAc-6 (C=O)
7	65.0, H2C–O	4.61	dd, (11.7, 3.9)	C-5, C-6, C-7′
				C5, C6, C-7′
		4.55	dd, (11.7, 6.1)	C-5, C-6, C-7′
7′	169.7, C=O
1′	112.0, C
2′	161.9, C–O
OH-2′		10.54	s	C-1′, C-2′, C-3′
3′	117.9, CH	7.00	dd, (8.4, 1.1)	C-1′, C-2′, C-5′
4′	136.3, CH	7.48	ddd, (8.4, 7.2, 1.8)	C-2′, C-6′
5′	119.5, CH	6.90	ddd, (8.0, 7.2, 1.1)	C-1′, C-3′
6′	130.1, CH	7.81	dd, (8.0, 1.8)	C-2′, C-4′, C-7′

Compound 4 was obtained as a white powder and was assigned the molecular formula C₂₂H₁₉O₆, 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 O–H, C=C aromatic, and C–O stretches, respectively.¹⁸ The ¹H NMR spectrum (Table 4, 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 ³J_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). Moreover, HMBC cross-peaks of H-8 (δ_H 6.03) and H-2 (δ_H 5.38) to C-8a (δ_C 161.4) supported the placement of the dihydroxybenzyl moiety at C-6. The overlapping signals at δ_H 7.42–7.43 (4H) were assigned to the ortho (H-2′/6′) and meta (H-3′/5′) protons, and that at δ_H 7.38 (1H) to the para proton (H-4′) of ring B. The signal at δ_H 12.89 signal was ascribed to OH-5, with its chemical shift being deshielded due to intramolecular hydrogen bonding with the nearby carbonyl group. The levorotatory specific rotation and the ECD spectrum (Experimental Section) of this flavonoid suggested the S-configuration at C-2.^19,20 Based on the features mentioned above, the new compound 3″-hydroxyisochamanetin (4) was characterized as (S)-6-(2,3-dihydroxybenzyl)-5,7-dihydroxy-2-phenylchroman-4-one, an isochamanetin derivative.

Table 4. NMR Spectroscopic Data (¹H at 800 MHz, ¹³C at 200 MHz, CDCl₃) for 3″-Hydroxyisochamanetin (4).

position	δC, type	δH	J (Hz)	HMBC, H→C
2	79.4, C–O	5.38	dd, (13.1, 2.8)	C-3, C-4, C-8a, C-1′, C-2′/6′
3	43.3, CH2	3.08	dd, (17.2, 13.1)	C-2, C-4, C-1′
				C4, C10, C1′
		2.84	dd, (17.2, 2.8)	C-4a, C-4, C-1′
4	196.3, C=O
4a	103.1, C
5	160.7, C–O
OH-5		12.89	s	C-5, C-6, C-4a
6	108.2, C
7	163.0, C–O
8	96.3, CH	6.03	s	C-4, C-4a, C-6, C-7, C-8a
8a	161.4, C–O
1′	138.3, C
2′/6′	126.3, CH	7.42a	m	C-2, C-1′, C-2′/6′, C-3′/5′, C-4′
3′5′	129.0, CH	7.42a	m	C-2, C-1′, C-2′/6′, C-3′/5′, C-4′
4′	129.1, CH	7.38	m	C-1′, C-2′/6′, C-3′/5′
1″	126.8, C
2″	141.2, C–O
3″	144.3, C–O
4″	113.2, CH	6.74a	d (4.8)	C-2″, C-6″
5″	123.1, CH	7.05	dd, (4.8, 4.8)	C-1″, C-3″, C-4″, C-6″
6″	121.1, CH	6.74a	d (4.8)	C-1″, C-2″, C-4″, C-5″, C-7″
7″	22.1, CH2	3.87	AB d (14.5)	C-7, C-6, C-5, C-1″, C-2″, C-6″

^aOverlapping signals.

Compound 5 was obtained as a yellow powder. Its molecular formula, C₂₂H₁₇O₆, was determined based on HRESIMS (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 ¹H 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 ³J_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.

Table 5. NMR Spectroscopic Data (¹H at 800 MHz, ¹³C at 200 MHz, CD₃OD) of 2,3-Dehydro-3″-hydroxychamanetin (5).

position	δC, type	δH	J (Hz)	HMBC, H→C
2	165.8, C–O
3	105.6, CH	6.69	s	C-2, C-4, C-4a, C-1′
4	184.3, C=O
4a	105.7, C
5	161.2, C–O
6	99.9, CH	6.36	s	C-4, C-4a, C-5, C-7, C-8
7	164.3, C–O
8	107.4, C
8a	157.0, C–O
1′	132.6, C
2′/6′	127.6, CH	7.80	m	C-2, C-2′/6′, C-4′
3′/5′	130.2, CH	7.47	m	C-1′, C-2′/6′, C-3′/5′
4′	132.9, CH	7.51	m	C-2′/6′
1″	128.3, C
2″	144.2, C–O
3″	146.0, C–O
4″	113.9, CH	6.62	dd, (7.8, 1.6)	C-2″, C-3″, C-5″ C-6″
5″	120.3, CH	6.47	dd, (7.9, 7.8)	C-1″, C-3″, C-4″, C-6″
6″	120.4, CH	6.34	dd, (7.9, 1.6)	C-2″, C-4″, C-7″
7″	22.8, CH2	4.16	s	C-7, C-8, C-8a, C-1″, C-2″, C-6″

Besides the new compounds 1–5, C-benzylated flavonoids 6(7) and 13–15,¹⁵ heptenolides 7,¹²8,¹³9,¹² and 10,⁴ flavonoids 11(14) and 12,¹⁵ and triterpenoid 16(15) were also isolated and identified by comparison of their spectroscopic features to those previously published. The natural occurrence of heptenolides, such as 2, 3, and 7–9, is limited so far only to the members of the Uvariae tribe within the family Annonaceae.¹⁰ Compounds 11 and 12 appear to be biogenetic precursors of 4–6 and 13–15. Flavonoids, such as 4–6 and 13–15, containing one or two hydroxylated C-benzyl groups on ring A as well as hydroxylated C-benzyl units that extend in a linear fashion, have been isolated from numerous species in the Annonaceae.^8,18,21 Heptenolides¹⁰⁻¹³ and C-benzylated flavonoids^8,18,21,22 are known to possess diverse bioactivities.

As part of an ongoing search for bioactive phytochemicals, the compounds isolated from S. gracilis ssp. gracilis were screened for antiplasmodial activity against the chloroquine-sensitive (3D7) and the chloroquine-resistant (Dd2) strains of P. falciparum. Compounds showing promising antiplasmodial activities, <10 μM, were further screened for cytotoxicity using HEK-293 human embryonic kidney cells. Antiplasmodial activities of IC₅₀ 1.4 to >40 μM (Table 6), with 7-hydroxy-6-hydromelodienone (10) being the most active isolated constituent, were observed [IC₅₀ 1.4 (3D7) and 4.1 μM (Dd2)]. However, 10 also showed cytotoxicity against HEK-293 mammalian cells (100% at 40 μM). (Z)-Acetylmelodorinol (8), melodorinol (9), and dichamanetin (15) showed low selectivities. Compounds 4, 5, and 12–14 inhibited plasmodial growth moderately (Table 6) at a 20 μM concentration. All other compounds showed very weak or no antiplasmodial activities at 40 μM. The crude methanol extracts of the leaves, stem bark, and root bark of S. gracilis ssp. gracilis and the isolated 4, 5, 6, 9, 10, and 12 were tested further for the inhibition of cap-dependent and independent translation initiation using a luciferase model,^17,23 but none showed significant translational inhibitory activity.

Table 6. Antiplasmodial and Cytotoxic Activities of Compounds Isolated from Sphaerocoryne gracilis ssp. gracilis.

compound	3D7	Dd2	HEK-293	SIHEK293/3D7
(Z)-sphaerodiol (7)	10.5a	NT	NT
(Z)-acetylmelodorinol (8)	4.8a	6.7a	11.8a	3
(Z)-melodorinol (9)	3.7a	NT	NT
7-hydroxy-6-hydromelodienone (10)	1.4a	4.1a	100%b
dichamanetin (15)	9.3a	6.7a	75%b
pyrimethamine	0.0025		4.2	1688
chloroquine	0.0045	0.046	60%b	>4000b
pyronaridine	0.0036	0.0075	1.8	494.5
puromycin	0.023	0.045	0.36	15.9
artesunate	0.000 48	0.000 44	75%c	>22.000b
DHA	0.000 11	0.000 15	50%c	>22.000b

^aIC₅₀.

^bPercentage growth inhibition at 40 μM. IA = inactive at 40 μM. NT = not tested.

^cPercentage growth inhibition at 20 μM. The inhibitory activities are given as the mean value of at least two independent measurements. IC₅₀ values were determined for one biological replicate in duplicate.

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 Büchi 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 ¹H NMR, ¹³C NMR, COSY, TOCSY, NOESY, HSQC, and HMBC spectra. LC-MS (ESI) spectra were acquired with a PerkinElmer PE SCIEX API 150 EX instrument equipped with a Turbolon spray ion source and a Gemini 5 mm RP-C18 110 Å column, using a gradient of H₂O–CH₃CN (80:20 to 20:80) in the presence of 0.2% HCO₂H and a separation time of 8 min. HRESIMS were obtained with a Q-TOF-LC/MS spectrometer with a lock mass ESI source (Stenhagen Analysis Lab AB, Gothenburg, Sweden), using a 2.1 × 30 mm 1.7 μm RP-C₁₈ column and an elution gradient of H₂O–CH₃CN (5:95 to 95:5, with 0.2% HCO₂H). Analytical TLC was performed on aluminum plates precoated with silica gel 60 F254 (Merck). After development with an appropriate solvent system, the plates were evaluated under UV light (254 and 366 nm) and then sprayed with 4-anisaldehyde reagent, prepared by mixing 3.5 mL of 4-anisaldehyde with 2.5 mL of concentrated H₂SO₄, 4 mL of glacial HOAc, and 90 mL of MeOH. The plates were then heated for the identification of UV-negative compounds and assessment of the color change of the UV-positive spots. Column chromatography was carried out using silica gel 60 (230–400 mesh), and gel filtration was performed over Sephadex LH-20 (Pharmacia) suspended in CH₂Cl₂–MeOH (1:1). Preparative HPLC was performed on a Waters 600E system using Chromulan software (Pikron Ltd.) and an RP-C₈ Kromasil column (250 mm × 25 mm) with a H₂O–MeOH gradient (70:30 to 100:0) for 20–40 min at a flow rate of 7 mL/min.

Plant Material

The stem bark, root bark, and leaves of Sphaerocoryne gracilis ssp. gracilis were collected in June 2014 from Pugu Forest Reserve, Kisarawe District, Pwani Region, Tanzania, at GPS location S 06°53′28.4″, E 039°05′56.3″ at an elevation of 269 m. The plant was identified in the field and authenticated by Mr. F. M. Mbago, a senior taxonomist of the Herbarium, Botany Department of University of Dar es Salaam. The voucher specimen is deposited there with reference number FMM-3670.

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 leaf extract (65.0 g) was adsorbed on silica gel and subjected to gravity column chromatography with an elution gradient from 30% EtOAc–i-hexane to 10% EtOAc–methanol, to obtain 13 fractions of approximately 200 mL each. Fractions 4–6, obtained with 50% EtOAc–i-hexane, were found to consist of the same components identified by TLC analysis. They were therefore combined and subjected to further gravitational column chromatography, using dry packing and elution with 30% EtOAc–i-hexane, resulting in 25 fractions of ∼80 mL each. TLC analysis enabled the combination of fractions 16–21, which were then subjected to separation on Sephadex, eluted with 1:1 MeOH–CH₂Cl₂, to obtain 23 subfractions of ∼2 mL each. After TLC analysis, the combination of subfractions 7–10 gave 1.3 mg of 5,7-dihydroxyflavone (12), and fractions 13–16 gave 5.8 mg of 3″-hydroxyisochamanetin (4). Subfractions 17–20 were combined for further separation on a Sephadex column, also eluted with 1:1 MeOH–CH₂Cl₂, resulting in 32 fractions of ∼1 mL each. TLC analysis also enabled the combination of fractions 15–23, which were then subjected to preparative HPLC, using 70:30 Milli Q water–methanol, to obtain gracidiol (1, 4.5 mg). Preparative HPLC of fractions 24–29, using the same conditions, gave three peaks, representing 7-hydroxy-6-hydromelodienone (10), (Z)-acetylmelodorinol (8), and (Z)-2′-hydroxyacetylmelodorinol (3). The combined fractions 8–10 from the initial column were subjected to gravity column chromatography, eluted with 50% EtOAc–i-hexane. This yielded 12 fractions, of which fraction 6 was purified further on a Sephadex column, MeOH–CH₂Cl₂ (1:1), giving 32 fractions, of which fractions 10–18 gave 75.7 mg of (Z)-melodorinol (9), while fractions 29–32 yielded 1 mg of 5,7-dihydroxy-8-(2,3-dihydroxybenzyl)flavone (5). Fraction 11, obtained from the initial column, was separated over Sephadex by elution with 1:1 MeOH–CH₂Cl₂, yielding 15 fractions. Fractions 9–11 contained complex, inseparable mixtures of compounds, which were not further analyzed, while fractions 12–14 were separated once again, as described above, yielding 186.5 mg of 3″-hydroxygracinol (6).

The root bark extract (68.0 g) was subjected to vacuum liquid chromatography under gradient elution (20% EtOAc–petroleum ether to 10% MeOH–EtOAc) to obtain 10 fractions of ∼500 mL each, which were analyzed with TLC and developed using EtOAc–petroleum ether at ratios of 2:8, 5:5, 7.5:2.5, and 9:1. Fraction 3, obtained at 50% EtOAc–petroleum ether, was further separated on a Sephadex column, as above, giving 22 fractions. Following TLC analysis, fractions 14 and 15 were combined and subjected to gravity column chromatography using an elution gradient from 30% to 50% EtOAc–i-hexane, giving 45 fractions of 10 mL each. Fractions 44 and 45 contained complex, inseparable mixtures of compounds and were not further analyzed. Fraction 4 from the initial column, obtained using 50–75% EtOAc–i-hexane, was subjected to gravity column chromatography, using gradient elution from 40% to 75% EtOAc–i-hexane, yielding pinocembrin (11, 36.7 mg). Fraction 6, obtained from elution with 3% MeOH–EtOAc, underwent successive separation on the Sephadex column (1:1 MeOH–CH₂Cl₂) and silica gel column chromatography (75% EtOAc–i-hexane), yielding (Z)-sphaerodiol (7, 2.1 mg). The polar fraction, 8 (obtained at 10% MeOH–EtOAc), gave (Z)-7-acetylsphaerodiol (2, 19.8 mg).

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, 24.0 mg), (Z)-acetylmelodorinol (8, 63.0 mg), (Z)-melodorinol (9, 15.0 mg), 5,7-dihydroxyflavone (12, 18.7 mg), chamanetin (13, 64.8 mg), isochamanetin (14, 23.6 mg), dichamanetin (15, 126.8 mg), and polycarpol (16, 18.0 mg).

Gracidiol (1):

colorless oil; [α]²⁰_D +52.0 (c 0.15, MeOH); ECD (MeOH, λ_nm (Δε; M^–1 cm^–1) (+9.23)₃₃₁; (−40.16)₂₈₈; (+45.07)₂₂₂; UV (CHCl₃) λ_max (log ε) 275 (4.53), 230 (4.34), 215 (4.72) nm; IR (KBr) ν_max 3424, 1634, 1265, 739 cm^–1; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 393.2 [M + Na]⁺, 388.4, 371.3, 353.6, 329.6, 279.3, 249.5, 235.3, 223.4, 229.7, 205.2, 177.2, 149.5, 137.5, 131.1, 101.1; HRESIMS [M + H]⁺m/z 371.1479 (calcd for C₂₁H₂₃O₆ 371.1495).

(Z)-7-Acetylsphaerodol (2):

colorless oil; [α]²⁰_D +3.0 (c 15.2, MeOH); ECD (MeOH, λ_nm (Δε; M^–1 cm^–1) (+ 2.20)₃₈₉; (−2.77)₃₆₁; (+0.60)₂₈₆; (−4.20)₂₅₁; (+4.15)₂₁₂; UV (MeOH) λ_max (log ε) 270 (4.41) nm; IR (KBr) ν_max 3446, 3056, 1781, 1743, 1423, 1265, 110, 940, 738 cm^–1; ¹H and ¹³C NMR data, see Table 3; ESIMS m/z 221.3 [M + Na]⁺, 207.8, 181.1, 153.2, 139.3, 121.4, 111.2, 93.2, 85.5, 65.0; HRESIMS [M + H]⁺m/z 199.0629 (calcd for C₉H₁₁O₅ 199.0606).

(Z)-2′-Hydroxyacetylmelodorinol (3):

colorless oil; [α]²⁰_D +0.12 (c 16.7, MeOH); UV (MeOH) λ_max (log ε) 240 (2.89), 205 (3.52) nm; IR (KBr) ν_max 3417, 1639, 1265, 740 cm^–1; ¹H and ¹³C NMR data see Table 2; ESIMS m/z 341.2 [M + Na], 336.3, 284.7, 272.6, 259.4, 221.3, 213.1, 177.3, 183.3, 139.4, 149.4, 121.4; HRESIMS [M + H]⁺m/z 319.0801 (calcd for C₁₆H₁₅O₇ 319.0818).

3″-Hydroxyisochamanetin (4):

white powder; mp 196–197 °C; [α]²⁰_D −3.59 (c 4.4, MeOH); CD (MeOH, λ_nm (Δε; M^–1 cm^–1) (−3.56)₃₈₂; (+1.27)₂₈₆; (−2.64)₂₅₁; (+1.36)₂₂₇; UV (MeOH) λ_max (log ε) 320 (4.72), 290 (4.79) nm; IR (KBr) ν_max 3430.9, 1641, 1477, 1255, 739 cm^–1; ¹H and ¹³C NMR data see Table 4; ESIMS m/z 379.3 [M + H]⁺, 289.4, 269.5, 257.5, 241.4, 233.3, 180.7, 175.4, 149.6, 107.3, 91.2, 83.2, 86.2; HRESIMS [M + H]⁺m/z 379.1171 (calcd for C₂₂H₁₉O₆ 379.1182).

5,7-Dihydroxy-8-(2,3-dihydroxybenzyl)flavone (5):

yellow powder; UV (MeOH) λ_max (log ε) 280 (4.16) nm; IR (KBr) ν_max 3430, 1642, 1265, 740 cm^–1; ¹H and ¹³C NMR data, see Table 5; ESIMS m/z 377.5 [M + H]⁺, 376.5, 267.0, 149.4, 111.5, 99.1, 74.1, 69.3; HRESIMS [M + H]⁺m/z 377.1012 (calcd for C₂₂H₁₇O₆ 377.1025).

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

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00721.

• Structures and tables containing ¹H and ¹³C NMR data for the known compounds 6–16; 1D and 2D NMR spectra and HRESIMS for the new compounds 1–5 (PDF)

The authors declare no competing financial interest.

Notes

Original FIDs are available, open access, at Zenodo with DOI: 10.5281/zenodo.3355165.