Naphthalene Derivatives from the Roots of Pentas parvifolia and

Article pubs.acs.org/jnp

Naphtalene Derivatives from the Roots of Pentas parvifolia and Pentas bussei Negera Abdissa,†,‡,§ Fangfang Pan,⊥ Amra Gruhonjic,‡,∥ Jürgen Graf̈ enstein,‡ Paul A. Fitzpatrick,∥ Göran Landberg,∥ Kari Rissanen,⊥ Abiy Yenesew,*,† and Máté Erdélyi*,‡,# †

Department of Chemistry, University of Nairobi, P.O. Box 30197-00100, Nairobi, Kenya Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden § Department of Chemistry, Jimma University, P.O. Box 378, Jimma, Ethiopia ∥ Sahlgrenska Cancer Centre, University of Gothenburg, Gothenburg SE-405 30, Sweden ⊥ Department of Chemistry, Nanoscience Center, University of Jyvaskyla, P.O. Box 35, FI-40014 Jyvaskyla, Finland # Swedish NMR Centre, University of Gothenburg, Gothenburg SE-405 30, Sweden ‡

S Supporting Information *

ABSTRACT: The phytochemical investigation of the CH2Cl2/MeOH (1:1) extract of the roots of Pentas parvifolia led to the isolation of three new naphthalenes, parvinaphthols A (1), B (2), and C (3), two known anthraquinones, and five known naphthalene derivatives. Similar investigation of the roots of Pentas bussei afforded a new polycyclic naphthalene, busseihydroquinone E (4), a new 2,2′-binaphthralenyl-1,1′dione, busseihydroquinone F (5), and five known naphthalenes. All purified metabolites were characterized by NMR and MS data analyses, whereas the absolute configurations of 3 and 4 were determined by single-crystal X-ray diffraction studies. The E-geometry of compound 5 was supported by DFT-based chemical shift calculations. Compounds 2−4 showed marginal cytotoxicity against the MDA-MB-231 human triple-negative breast cancer cell line with IC50 values ranging from 62.3 to 129.6 μM.

■

T

RESULTS AND DISCUSSION The air-dried roots of P. parvifolia were extracted with CH2Cl2/ MeOH (1:1) by percolation at room temperature. The extract was subjected to column chromatographic separation on oxalic acid impregnated silica gel, providing eight secondary metabolites, of which three (1−3) are new natural products. Compound 1 was obtained as a brown solid and was assigned the molecular formula C14H14O7 based on HRESIMS analysis ([M + H]+ m/z obsd 295.0830, calcd 295.0818) and 13 C NMR data (Table 1, Supporting Information). In its 13C NMR spectrum, 14 carbon signals comprising 10 aromatic carbons, a methyl ester (δC 171.9 and 52.6), and two methoxy functionalities (δC 55.8, 56.3) were observed. The NMR and UV data (λmax 214, 251, 374 nm) were indicative of a naphthalene skeleton, similar to those of busseihydroquinone A.5,10,13 Thus, the two highly deshielded singlets at δH 12.82 (1OH) and 9.54 (8-OH) and the two aromatic singlets at δH 6.84 (H-3) and 7.15 (H-5) were compatible with a naphthalene skeleton substituted with a methyl ester (δC 171.9 CO, δC 52.6 and δH 3.99), two methoxy (δH 4.04, 3.95), and three hydroxy (δH 5.67, 9.54, 12.82) groups (Table 1, Figures S1and

he genus Pentas (Rubiaceae) is a tropical and subtropical African taxon comprising approximately 40 species, a few of which also occur in Saudi Arabia.1,2 In Kenya 11 indigenous Pentas species are known, of which P. suswaensis and P. decora are endemic.1,3 Despite the wide traditional uses of the genus by East African communities for the treatment of various ailments including malaria, gonorrhea, syphilis, and dysentery,3,4 only a few of its species have been investigated for their secondary metabolites and their biological activities. By far the most extensively investigated member of the genus is P. lanceolata,5−8 while limited information is available on P. parvifolia and P. bussei.5,9−11 Most of the compounds reported from this genus are anthraquinone5,10,12 and dihydronaphthoquinone5,10,11 derivatives, of which some possess antimicrobial, antiplasmodial, and cytotoxic activities.5,9,12 As part of an ongoing search for new bioactive natural products from the genus Pentas,5,12 the isolation of three new naphthalenes (1−3) from the roots of P. parvifolia, a new naphthalene (4), and a new 2,2′binaphthralenyl-1,1′-dione (5) from the roots of P. bussei is reported. Five known compounds from the roots of P. parvifolia and five known compounds from the roots of P. bussei were also identified. The cytotoxicity of some of these compounds against the MDA-MB-231 breast cancer cell line is also reported. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 1, 2016

A

DOI: 10.1021/acs.jnatprod.6b00178 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Compound 2 was obtained as a dark red solid. HRESIMS indicated a protonated molecular ion at m/z 265.0726 ([M + H]+, calcd 265.0712), attributed to the molecular formula C13H12O6, which is consistent with eight indices of hydrogen deficiency. Its UV spectrum (λmax 208, 262, 382 nm) was indicative of a naphthalene skeleton.5,9 The NMR spectroscopic data (Table 1) showed that this compound has an identical ring A as compound 1, namely, OH (δH 11.59) at C-1, methoxycarbonyl at C-2 (δC 170.5 for CO; δH 3.93 and δC 52.5 for OCH3), a proton (δH 6.89, H-3), and a methoxy (δH 3.96; δC 56.2) group at C-4 (δC 147.4). The substitution pattern of this ring was confirmed from the HMBC spectrum (Table 1, Figure S7, Supporting Information). A pair of orthocoupled (J = 8.9 Hz) aromatic protons at δH 7.19 (H-7) and 7.69 (H-8) indicated that ring B is disubstituted with two hydroxy groups (δH 9.50 and 9.14) at C-5 (δC 139.5) and C-6 (δC 145.4), respectively. This regiochemistry was supported by the HMBC correlation of 6-OH (δH 9.14) with C-7 (δC 118.5), C-5 (δC 139.5), and C-6 (δC 145.4). The assignment of the mutually coupled aromatic doublets at δH 7.19 to H-7 and δH 7.69 to H-8 was confirmed by the HMBC correlations of H-8 (δH 7.69) with C-1 (δC 155.2) and C-6 (δC 145.4) and the correlation of the hydrogen-bonded hydroxy proton OH-1 (δH 11.59) with C-8a (δC 119.6), C-8 (δC 115.3), C-2 (δC 101.1), and the carbonyl carbon at δC 170.5. The assignments of the vicinal hydroxy groups to C-5 and C-6 and the vicinal aromatic protons to C-7 and C-8 were corroborated by the NOEs observed between H-8 (δH 7.69) and OH-1 (δH 11.59) and between H-7 (δH 7.19) and OH-6 (δH 9.14). The location of the hydrogen-bonded hydroxyl group at C-1 (δH 11.59) was confirmed by its HMBC correlations to C-1 (δC 155.2), C-2 (δC 101.1), and C-8a (δC 119.6). On the basis of the above spectroscopic evidence, the new compound 2, parvinaphthol B, was characterized as methyl 1,5,6-trihydroxy-4-methoxy-2naphthoate. Compound 3 was obtained as a colorless solid. The molecular formula C23H26O7, consistent with 11 indices of

S2, Supporting Information). The shielded aromatic signal at δH 6.84 showed HMBC correlations to two oxygenated tertiary carbons, C-1 (δC 155.4) and C-4 (δC 147.6), and to the ester carbonyl carbon (δC 171.9), allowing its assignment to H-3 of ring A, with the methyl ester positioned at C-2 (δC 102.3). On the basis of the HMBC and NOESY correlations (Figures S7 and S4, Supporting Information), one of the methoxy groups (δH 3.95; δC 55.8) was placed at C-4 of ring A. Ring B is trisubstituted with two hydroxy groups (δH 5.67, 9.54) and a methoxy (δH 4.04; δC 56.3) functionality placed at C-7 (δC 132.5), C-8 (δC 141.7), and C-6 (δC 151.1), respectively. The only aromatic proton (δH 7.15) of this ring was assigned to H-5 based on its HMBC correlation with C-4. The 13C NMR shift of the methoxy carbon (δC 56.3) is incompatible with a diortho-substituted aromatic moiety and suggests its placement at C-6 (δC 151.1) rather than at C-7 (δC 132.5) or C-8 (δC 141.7). This regiochemistry was supported by the NOE correlation of the methoxy protons (δH 4.04) with H-5 (δH 7.15) (Figure S4, Supporting Information). On the basis of the above spectroscopic data, the new compound 1, parvinaphthol A, was characterized as methyl 1,7,8-trihydroxy-4,6-dimethoxy-2naphthoate (1).

Table 1. 1H and 13C NMR Spectroscopic Data of Parvinaphthols A (1) and B (2) Acquired in CDCl3 and DMSO-d6 [δH, Multiplicity (J in Hz)], Respectively 1 position 1 2 3 4 4a 5 6 7 8 8a COOCH3 COOCH3 4-OCH3 6-OCH3 1-OH 5-OH 6-OH 7-OH 8-OH

δC, type 155.4, 102.3, 98.6, 147.6, 125.0, 93.9, 151.1, 132.5, 141.7, 110.0, 171.9, 52.6, 55.8, 56.3,

C C CH C C CH C C C C C CH3 CH3 CH3

δH

2 δC, type

HMBC (H→C)

6.84, s

C-1, C-2, C-4, C-4a, C-5, C-8a, COOCH3

7.15, s

C-1, C-4, C-4a, C-6, C-7, C-8a

3.99, s 3.95, s 4.04, s 12.82, s

C-2, C-3, C-5, C-1,

COOCH3 C-4 C-6 C-2, C-8a COOCH3

5.67, s 9.54, s

C-6, C-7, C-8 C-6, C-7, C-8, C-8a B

155.2, 101.1, 100.0, 147.4, 118.7, 139.5, 145.4, 118.5, 115.3, 119.6, 170.5, 52.5, 56.2,

C C CH C C C C CH CH C C CH3 CH3

δH (J in Hz)

HMBC (H→C)

6.89, s

C-1, C-2, C-4, C-5, C-8a, COOCH3

7.19, d (8.9) 7.69, d (8.9)

C-1, C-5, C-6, C-8a C-1, C-4, C-4a, C-5, C-6

3.93, s 3.96, s

C-2, COOCH3 C-3, C-4

11.59, s 9.50, s 9.14, s

C-1, C-2, C-8a C-5, C-6, C-7



Article

Table 2. 1H and 13C NMR Spectroscopic Data of Parvinaphthols C (3) and D (4) Acquired in CDCl3 (δH, Multiplicity (J in Hz)) 3 position 1 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ COOH 4-OCH3

δC, type 157.0, 102.0, 103.1, 149.7, 130.2, 118.4, 158.7, 121.3, 126.7, 122.5, 60.7, 48.6, 87.4, 28.1, 40.2,

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

25.5, CH2 42.8, 34.2, 105.3, 17.0, 174.9,

CH CH CH CH3 C

55.2, CH3

4

δH (J in Hz)

δC, type

HMBC (H→C)

7.10, s

C-1, C-2, C-4a, C-4, COOH

7.17, d (9.0) 8.39, d (9.0)

C-5, C6, C-8a C-1, C-4, C-4a, C-6

6.52, d (6.0) 2.28, dd (9.6, 6.0)

C-3′, C-4a, C-9′, C-5, C-6 C-3′, C-5′, C-6′, C-7′

1.32, 1.71, 2.30, 1.78, 1.85, 2.43, 2.11, 4.49, 1.15,

C-2′, C-2′, C-2′, C-2′, C-2′, C-5′, C-2′, C-1′, C-7′,

s m m m m m m d (7.8) d (6.9)

3.95, s

C-3′, C-5′ C-3′, C-4′, C-6′, C-7′ C-3′, C-4′, C-5′, C-6′, C-7′ C-3′, C-5′, C-7′ C-3′, C-5′ C-6′, C-8′, C-10′ C-6′, C-7′, C-9′, C-10′ C-10′, 9′-OCH3 C-8′, C-9′

C-4

9′-OCH2CH3 9′-OCH2CH3 9′-OCH3 1-OH

157.0, 101.7, 103.1, 149.5, 129.9, 119.2, 158.9, 121.3, 126.5, 122.3, 60.5, 49.0, 87.4, 27.8, 40.4,


3.32, s 11.28, s

HMBC (H→C)

7.09, s

C-1, C-2, C-4, C-4a, C-5, COOH

7.18, d (8.9) 8.39, d (8.9)

C-4a, C-5, C-6, C-8a C-1, C-4, C-4a, C-5, C-6

6.58, d (6.0) 2.30, dd (9.8, 6.0)

C-3′, C-4a, C-5, C-6, C-9′ C-3′, C-4′, C-5′, C-6′, C-7′

1.30, s 1.71, m 2.23, m 1.79, m 1.87, m 2.43, m 2.1, m 4.61, d (8.0) 1.15, d (8.0)

C-2′, C-3′, C-5′ C-2′, C-3′, C-4′, C-6′, C-7′ C-2′, C-3′, C-7 C-2′, C-3′, C-5′, C-7′ C-2′, C-3′, C-5′, C-7′, C-8′ C-1′, C-2′, C-6′, C-8′, C-9′, C-10 C-10′, C-6′, C-7′, C-2′, C-9′ C-1′, C-10′, 9′-OCH2CH3 C-7′, C-8′, C-9′

55.4, CH3

3.96, s

C-4

62.9, CH2

3.38, m 3.72, m 1.16, m

C-9′, 9′-OCH2CH3 C-9′, 9′-OCH2CH3 9′-OCH2CH3

11.25, s

C-1, C-2, C-4a, C-8a, C-8

25.7, CH2 43.1, 33.9, 103.1, 16.8, 175.2,

CH CH CH CH3 C

15.3, CH3 55.3, CH3

δH (J in Hz)

C-9′

hydrogen deficiency, was suggested based on HRESIMS analysis (m/z 413.1591, calcd 413.1600 for [M − H]−). Its UV spectrum (λmax 216, 276, 362 nm) was compatible with a naphthalene skeleton, similar to those of 1 and 2.5,10,13 Its Aring is substituted with a hydrogen-bonded hydroxy (δH 11.28) at C-1 (δC 157.0), a hydroxycarbonyl (δC 174.9; νmax 3434− 3468 cm−1) at C-2 (δC 102.0), and a methoxy (δH 3.95; δC 55.2) functionality at C-4 (δC 149.7), as established from the HMBC correlations (Table 2) of H-3 (δH 7.10) with two oxygenated tertiary carbons, C-1 (δC 157.0) and C-4 (δC 149.7), and with a carbonyl carbon (δC 174.9). The NOE correlation of H-3 (δH 7.10) with 4-OCH3 (δH 3.95; δC 55.2) confirmed the placement of this substituent. The position of the ortho-coupled (J = 9.0 Hz) aromatic protons H-7 (δH 7.17) and H-8 (δH 8.39) was elaborated based on the HMBC correlations of H-8 (δH 8.39) to C-1 (δC 157.0) and C-4 (149.7). The dihydropyran C-ring, connected to the B-ring at C-5 (δC 118.4) and C-6 (δC 158.7), is proposed to be formed through cyclization of a geranyl group, initially attached to C-5, with the oxygen attached to C-6, similar to the process in the biogenesis of busseihydroquinone C.5 Attachment of ring C to ring B was confirmed by the HMBC correlations of the benzylic oxymethine H-1′ (δH 6.52) to C-5 (δC 118.4), C-4a (δC 130.2), and C-6 (δC 158.7). H-1′ (δH 6.52) coupled (J = 6.0 Hz) to H2′ (δH 2.28), the former proton also showing HMBC correlations to C-3′ (δC 87.4) of ring C and to C-9′ (δC

105.3) of ring E. C-3′ is substituted with a methyl group (δH 1.32; δC 28.1), similar to the structures of busseihydroquinones C and D.5 Accordingly, CH3-4′ (δH 1.32) showed HMBC correlations to C-2′ (δC 48.6), C-3′ (87.4), and C-5′ (40.2) and an NOE correlation to H-5′β (δH 2.30). A further annulation resulting in ring D, similar to that observed for busseihydroquinone D,5 was evident from the HMBC correlations of H2′ (2.28), H-4′ (δH 1.32), H-5′ (δH 1.71 and 2.30), H-6′ (δH 1.78 and 1.85), and H-7′ (δH 2.43), as indicated in Table 2. The dihydropyran E-ring of 3, fused to both rings C and D, was identified based on its sequential COSY correlations (Figure S19, Supporting Information), i.e., H-9′ (δH 4.49) to H-8′ (δH 2.11), H-8′ to H-7′ (δH 2.43), H-7′ to H-2′ (δH 2.28), and H-2′ to H-1′ (δH 6.52). H-9′ (δH 4.49) was attached to a carbon (C9′, δC 105.3) with a diagnostic acetalic carbon chemical shift. HMBC correlations of 9′-OCH3 (δH 3.32) to C-9′ (δC 103.1) as well as that of H-9′ (δH 4.49) to 9′-OCH3 (δC 55.3) indicated the position of the 9′-OCH3 substituent. The scalar coupling (J = 6.9 Hz) between H-10′ (δH 1.15) and H-8′ (δH 2.11) and the HMBC correlations of CH3-10′ to C-7′ (δC 42.8), C-8′ (δC 34.2), and C-9′ (δC 105.3) indicated the attachment of CH3-10′ to C-8′. Ring E is likely formed by cyclization of a side chain at C-7′ in busseihydroquinone D,5 to the C-1′ hydroxy group. The relative configuration of 3 was established from the NOE correlations (Figure 1a) of H-1′ with H-8′ and H-2′, of C



Article

Figure 1. (a) Some of the key NOE correlations (red arrows) observed for compound parvinaphthol C (3) (mixing time 700 ms, CDCl3, 25 °C, 800 MHz) and (b) its X-ray crystal structure (CCDC1452353, Supporting Information), indicating its overall conformation and configuration.

Figure 2. (a) Some of the key NOE correlations (red arrows) observed for compound busseihydroquinone E (4) (mixing time 700 ms, CDCl3, 25 °C, 800 MHz) and (b) its X-ray crystal structure (CCDC-1452354, Supporting Information), indicating its overall conformation and configuration.

H-2′ with H-5′β, H-8′, and CH3-4′, and of CH3-4′ with H-5′β and H-7′, indicating that these protons are cofacial. The 1,2trans-diaxial configuration of H-8′ and H-9′ was revealed by the magnitude of their scalar coupling (J = 7.8 Hz), and, consequently, CH3-10′ and OCH3-9′ are both equatorially oriented in ring E. The small coupling constants and the strong NOE correlations of H-1′ (δH 6.52) with H-2′ (δH 2.28), and H-2′ with H-7′ (δH 2.43), along with the NOE correlation of H-2′ with CH3-4′ (δH 1.32) are consistent with cis-configured C/D, C/E, and D/E ring junctions. The absolute configuration of this compound was established by single-crystal X-ray diffraction analysis (Figure 1b). Accordingly, compound 3, parvinaphthol C, was characterized as (2R,3R,3aR,3a1S,5aS,12cR)-9-hydroxy-2,12-dimethoxy-3,5a-dimethyl-2,3,3a,3a 1 ,4,5,5a,12c-octahydro-1,6dioxabenzo[l]acephenanthrylene-10-carboxylic acid. Additionally, the known compounds busseihydroquinones A,5 B,5 and C,5 rubiadin-1-methyl ether,5 and damnacanthol5 were identified from the root extract of P. parvifolia, by comparison of their observed and reported spectroscopic and physical data. The re-examination of the roots of P. bussei resulted in the isolation of two new compounds, 4 and 5, along with the five known natural products busseihydroquinones A,5 B,5 C,5 and D5 and methyl 5,10-dihydroxy-7-methoxy-3-methyl-3-(4-methyl-3-pentenyl)-3H-benzo[f ]chromene-9-carboxylate.10 Compound 4 was obtained as a colorless crystalline solid. Its HRESIMS at m/z 427.1754 along with its NMR data was compatible with the molecular formula C24H28O7 (calcd 427.1757 for [M − H]−), indicating 11 indices of hydrogen deficiency, whereas its UV (λmax 214, 276, 372 nm) and IR (νmax 3416, 1626, 1512, 1456 cm−1) spectra suggested a naphthalene skeleton.5,10,13 Its 1H and 13C NMR spectra (Table 2) were virtually identical to those of compound 3, except for the 9′-OCH3 signal of 3 being replaced by a set of signals corresponding to an ethoxy group (Table 2). The attachment of this ethoxy functionality to C-9′ (δC 103.4) was confirmed via the HMBC correlation of the OCH2CH3-9′ protons (δH 3.38) with C-9′ (Table 2). It should be noted that ethoxy groups have been reported to be present in the secondary metabolites of the family Rubiaceae14 and are suggested to be formed by complete reduction of the carbonyl carbon of an acetoxy group. The high negative specific rotation, [α]20D −242, and the comparable ECD Cotton effects (Figure S36, Supporting Information) of 4 to those observed for 3 ([α]20D −258) suggested that these two natural products have identical configurations. Analyses of the NOE correlations (Figure 2a) and of the single-crystal X-ray diffraction data

(Figure 2b) confirmed the proposed constitution and configuration. On the basis of the above data, compound 4, busseihydroquinone E, was characterized as (2R,3R,3aR,3a1S,5aS,12cR)-2-ethoxy-9-hydroxy-12-methoxy-3,5a-dimethyl2,3,3a,3a1,4,5,5a,12c-octahydro-1,6-dioxabenzo[l]acephenanthrylene-10-carboxylic acid. Compound 5 was isolated as a blue, amorphous solid, with its UV spectrum showing absorption maxima (MeOH) at 214 252, 373, 486, and 572 nm. The HRESIMS gave a protonated molecular ion at m/z 509.1983 [M + H]+ (calcd 509.1959) corresponding to the molecular formula C32H28O6, suggesting 19 indices of hydrogen deficiency. Its 1H and 13C NMR spectra (Table 3) contained 16 carbon and 14 proton signals, suggesting that this compound is a symmetrical dimer, the monomer of which is similar to busseihydroquinone B.5 Thus, a pair of ortho-coupled (J = 8.5 Hz) aromatic protons, H-7 (δH 6.88) and H-8 (δH 8.05), indicated a disubstituted B-ring. The presence of a 2,2-dimethyl-2H-chromene moiety (ring C) was evident from the cis-olefinic system comprising two doublets, H-1′ (δH 7.46) and H-2′ (δH 5.72) (J = 10.4 Hz), the CH3-4′/ CH3-5′ (δH 1.46) singlet, and the sp3-hybridized oxygenated tertiary carbon C-3′ (δC 76.1). The above data suggest that rings B and C of 5 are identical to that of busseihydroquinone B.5 However, the substitution pattern of ring A is different, with the 1H NMR data of 5 showing only two singlets corresponding to H-3 (δH 8.40) and OCH3-4 (δH 4.03) and lacking signals corresponding to the hydroxycarbonyl and the hydrogenbonded hydroxy (OH-1) groups of busseihydroquinone B. Furthermore, its 13C NMR data indicated a C-1 carbonyl (δC 188.3) and a quaternary aromatic carbon, C-2 (δC 130.8). The HMBC correlation of H-8 (δH 8.05) to the carbonyl (δC 188.3) allowed the determination of the position of the latter on ring A. Moreover, the HMBC correlations of H-3 (δH 8.40) with C1 (δC 188.3), C-2 (δC 130.8), C-4 (δC 158.7), and C-4a (δC 127.9) further facilitated the establishment of the regiochemistry of this ring. Accordingly, C-2 was identified as the linkage position of the second molecular moiety, which was initially indicated by the molecular formula (C32H28O6) having twice as many protons and carbons compared to the number of observed NMR signals and by the deep blue color of 5, consistent with extended π-conjugation. The shielding of H-3 (δH 8.40) compared to that of the corresponding proton of busseihydroquinone B and other structurally closely related naphthalenes (Table 1) corroborated the proposed structure and is explained by the magnetic anisotropy of the conjugated π-system also involving C-2 and the C-1′ carbonyl in an Edimer (Figure 3). D



Article

Table 3. 1H and 13C NMR Spectroscopic Data for Busseihydroquinone F (5) Acquired in CDCl3 and Calculated for Putative E and Z Configurations δC

δH calcd

a

calcd

position

expt

E

Z

type

1/1″ 2/2″ 3/3″ 4/4″ 4a/4″a 5/5″ 6/6″ 7/7″ 8/8″ 8a/8″a 1′/1‴ 2′/2‴ 3′/3‴ 4′/4‴, 5′/5‴ 4/4″-OCH3

188.3 130.8 105.4 158.7 127.9 116.6 159.8 117.8 130.4 126.7 122.1 130.8 76.1 28.0 55.8

186.5 131.0 102.8 163.7 128.6 114.5 160.4 118.7 132.2 124.8 138.9 143.8 80.6 29.0 54.2

141.9 118.5 117.3 156.2 126.7 112.4 154.1 122.2 123.6 119.3 138.4 140.2 80.9 28.9 66.7


expt

E

Z

J (Hz)

8.40

9.26

7.27

s

C-1/1″, C-2/2″, C-4/4″, C-4a/4″a

6.88 8.05

6.95 7.93

7.02 7.89

d (8.5) d (8.5)

C-5/5″, C-6/6″, C-8a/8″a C-1/1″, C-4a/4″a, C-6/6″

7.46 5.72

7.96 6.04

8.27 6.26

d (10.4) d (10.4)

C-3′/3‴, C-6′/6‴ C-3′/3‴, C-4′/4‴, C-5/5‴

1.46 4.03

1.5 3.89

1.46 3.63

s s

C-2′/2‴, C-3′/3‴ C-3/3″, C-4/4″

HMBC (H→C)

Calculated values are estimated according to the procedure described in the Supporting Information.

Figure 3. Computationally (DFT) optimized geometries of the theoretically possible E (left) and Z (right) geometrical isomers of busseihydroquinone F (5).

Table 4. Cytotoxity (LD50)a of Selected Quinones, Isolated from the Roots of P. parvifolia and P. bussei

The structure elucidation of 5 was complemented by densityfunctional (DFT) calculations to define the geometry of the Δ2,2′ olefinic bond and its hypothetical E and Z configurations (Figure 3). The E configuration shows C2 symmetry, whereas the Z configuration is distorted from Cs to C1 symmetry, resulting in an energy decrease of 26.4 kJ/mol. This distortion is probably driven by the short nonbonded distance between O1 and O-1″ (1.445 and 1.453 Å for Cs and C1, respectively). The Gibbs free energy of the E configuration is lower by 247.4 kJ/mol than that of the (C1) Z configuration, which corroborates the conclusion that E is the more stable configuration. Also, the calculated NMR chemical shifts for the E configuration of 5 are in better agreement with experimental data than those for the Z configuration (Table 3). On the basis of the above spectroscopic evidence, compound 5, busseihydroquinone F, was identified as E-10,10′-dimethoxy3,3,3′,3′-tetramethyl-3H,3′H,7H,7′H-[8,8′-bibenzo[f ]chromenylidene]-7,7′-dione. Quinones have elicited considerable toxicological and pharmacological interest, as their core structure is a common element in cancer chemotherapeutic agents such as doxorubicin, mitomycin C, and mitoxantrone. It is presumably capable of modulating oxidative biochemical processes.15 Accordingly, herbal quinones have been reported to possess cytotoxic activity.5,16,17 A selection of the isolated compounds was, therefore, assayed for activity against the MDA-MB-231 ER-negative human breast cancer cell line (Table 4). For comparison, literature data obtained for known compounds on ER-positive MCF-7 cells are also given. Of the tested

LD50 (μM) compound

MDA-MB-231

MCF-7

parvinaphthol B (2) parvinaphthol C (3) busseihydroquinone E (4) busseihydroquinone A busseihydroquinone B busseihydroquinone C busseihydroquinone D rubiadin-1-methyl ether damnacanthol

96.5 129.6 62.3 − − 48.4 − 54.4 >352

− − − 304.15 373.55 >271.65 >262.15 86.45 >3525

a

Data were obtained for at least six independent experiments; details are given in the Experimental Section; “−”, not tested.

compounds, parvinaphthol E (4), busseihydroquinone C, and rubiadin-1-methyl ether possessed the highest cytotoxicities (54.4−62.3 μM), whereas most constituents had low cytotoxicity.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were recorded on a Büchi B-545 melting point apparatus, optical rotations were measured using a PerkinElmer 341 LC polarimeter, UV/vis spectra were obtained on a Cary 100 Bio spectrophotometer, ECD spectra were measured on a JASCO J-715 spectropolarimeter, and IR spectra (KBr disks) were recorded on a Perkin-Elmer 1725 FTIR spectrometer. NMR spectra were obtained on a Varian MR 400 or a E



Article

Bruker Avanace III HD 800 MHz spectrometer, using the residual solvent peaks as reference. Spectra were processed using the software MNova (v10.0.0). Full assignments were made using 1H, 13C, COSY,18 NOESY,14 HSQC,15 and HMBC19 spectra. HMBC spectra were run with delays set to permit observation of 2J,3J,4J couplings of ∼10−4 Hz. LC-MS(ESI) spectra were acquired using a PerkinElmer PE SCIEX API 150 EX instrument equipped with a Turbolon spray ion source and a Gemini 5 mm C-18 110 Å HPLC column using a H2O/ CH3CN gradient (80:20 to 20:80). The spectra were acquired with 30 electronvolt ionization. The HRMS analysis (Q-TOF-MS with a lockmass-ESI source) was done by Stenhagen Analys Lab AB, Gothenburg, Sweden. Column chromatography was carried out on silica gel (0.06−0.2 mm, Merck) impregnated with 3% aqueous oxalic acid. The analytical TLC was performed on Merck precoated silica gel 60 F254 plates. The gel filtration was done on Sephadex LH-20. Preparative HPLC was performed on a Waters 600E system using the Chromulan (Pikron Ltd.) software and an RP-C8Kromasil (250 mm × 25 mm) column with the solvent system H2O/CH3CN (gradient, 90:10 to 5:95, in 30−80 min, flow rate of 8−15 mL/min). Plant Material. The roots of P. bussei and P. parvifolia were collected in May 2013 from Mombasa, the coastal region, and from western Kenya, respectively. The plant materials were identified by Mr. P. C. Mutiso of the University of Nairobi, School of Biological Science Herbarium, where voucher specimens (PCM-2010/048 for P. bussei and PCM-2010/054 for P. parvifolia) have been deposited. Extraction and Isolation. The dried and ground roots of P. parvifolia (620 g) were extracted with CH2Cl2/CH3OH (1:1) three times for 24 h by cold percolation. The combined extract was concentrated using a rotary evaporator to yield a dark brown residue (24 g, 3.9%). A 20 g portion of the crude extract was subjected to column chromatography, using 300 g of oxalic acid impregnated silica gel, with an increasing gradient of EtOAc in isohexane. A total of 35 fractions, ca. 200 mL each, were collected. On the basis of their TLC profile, fractions 4−8 were combined and loaded on a Sephadex LH20 column (60 cm length and 3 cm diameter; eluent: CH2Cl2/MeOH, 1:1, ca. 50 mL subfractions) to give busseihydroquinone A (6.4 mg), busseihydroquinone B (4.3 mg), and 3 (4.7 mg). Fractions 9−15 were combined and purified on column chromatography (column size: 60 cm length and 4 cm diameter) on silica gel impregnated with oxalic acid (200 g) using an increasing gradient of EtOAc in isohexane (0:10, 1:9, 1:4, 2:3, 1:1, 3:2, 0:10, ca. 500 mL each), followed by further purification of the fractions on Sephadex LH-20 (60 cm length and 3 cm diameter; eluent: CH2Cl2/MeOH, 1:1, ca. 50 mL fractions), yielding busseihydroquinone C (7.1 mg), 1 (2.1 mg), and rubiadin-1methyl ether (5.2 mg). Fractions 17−25 were combined and subjected to preparative HPLC (RPC8 250 mm × 25 mm, Kromasil column; H2 O/CH 3 CN eluent), yielding compound 2 (3.8 mg) and damnacanthol (4.0 mg). The air-dried roots of P. bussei (850 g) were extracted and concentrated in the same way as described above to give 39 g (4.6%) crude extract. A 35 g portion of the extract was subjected to column chromatography (column size: 90 cm length and 6 cm diameter) on oxalic acid impregnated silica gel (400 g) eluted with isohexane containing increasing amounts of EtOAc to afford 48 fractions, ca. 200 mL each. On the basis of their TLC profile, fractions 8−10 (4−9% EtOAc in isohexane) were combined and loaded on a Sephadex LH-20 column (60 cm length and 3 cm diameter; eluent: CH2Cl2/MeOH, 1:1, ca. 200 mL fractions) to give busseihydroquinone A (44.3 mg) and busseihydroquinone B (6.7 mg). Fractions 15−19 (10−20% EtOAc in isohexane) were combined and subjected to column chromatography (column size: 60 cm length and 2 cm diameter) on silica gel impregnated with oxalic acid (200 g) using an increasing gradient of EtOAc in petroleum ether as eluent, followed by further purification of the fractions on Sephadex LH-20 (60 cm length and 3 cm diameter) using CH2Cl2/MeOH (1:1) as eluent to yield 4 (6.8 mg), 5 (1.6 mg), and busseihydroquinone C (9.0 mg). Methyl 5,10dihydroxy-7-methoxy-3-methyl-3-(4-methyl-3-pentenyl)-3H-benzo[f ]chromene-9-carboxylate (4.7 mg) and busseihydroquinone D (2.2 mg) were purified from fractions 30−35 (30−40% EtOAc in isohexane) by filtration on Sephadex LH-20 (60 cm length and 3 cm diameter) using

CH2Cl2/MeOH (1:1) as eluent and preparative TLC (20% EtOAc in isohexane). Parvinaphthol A (1): brown solid; mp 226.2 °C; UV (CH3CN) λmax 214, 251, 374 nm; IR (KBr) νmax 3421, 2956, 1672, 1631 cm−1; 1 H and 13C NMR data see Table 1; HRESIMS m/z 295.0830 [M + H]+ (calcd C14H14O7, 295.0818). Parvinaphthol B (2): dark red solid; mp 226.9 °C; UV (CH3CN) λmax 208, 262, 382 nm; IR (KBr) νmax 3418, 2954, 1675, 1627, 1526 cm−1; 1H and 13C NMR data see Table 1; HRESIMS m/z 265.0726 [M + H]+ (calcd C13H12O6, 265.0712). Parvinaphthol C (3): colorless solid; mp 196.2 °C; [α]20D −258 (c 0.5, CH2Cl2); UV (CH3CN) λmax 216, 276, 362 nm; IR (KBr) νmax 3434−3468, 2954, 1655, 1623, 1457 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 413.1591 [M − H]− (calcd C23H26O7, 413.1600). Busseihydroquinone E (4): colorless solid; mp 198.4 °C; [α]20D −242 (c 0.5, CH2Cl2); UV (CH3CN) λmax 214, 276, 372 nm; IR (KBr) νmax 3416, 2959, 1626, 1512,1456, 1401 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 427.1754 [M − H]− (calcd C24H28O7, 427.1757). Busseihydroquinone F (5): blue powder; mp 214.3 °C; UV (CH3CN) λmax, 214 252, 373, 486, 572 nm; 1H and 13C NMR data see Table 3; HRESIMS m/z 509.1983 [M + H]+ (calcd C32H28O6, 509.1959). Cytotoxicity Assays. These were carried out following the reported protocol.20−22 Hence, MDB-MB-231, human breast cancer cells, were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in humidified 5% CO2. For cytotoxicity assays, cells were seeded in 96well plates at optimal cell density (10 000 cells per well) to ensure exponential growth for the duration of the assay. After a 24 h preincubation growth, the medium was replaced with experimental medium containing the appropriate drug concentrations or vehicle controls (0.1% or 1.0% v/v DMSO). After 72 h incubation, cell viability was measured using Alamar Blue reagent (Invitrogen Ab, Lidingö, Sweden) according to the manufacturer’s instructions. Absorbance was measured at 570 nm with 600 nm as a reference wavelength. Results were expressed as the mean ± standard error for six replicates as a percentage of vehicle control (taken as 100%). Experiments were performed independently at least six times. Statistical analyses were performed using a two-tailed Student’s t test. P < 0.05 was considered to be statistically significant. X-ray Crystallography. The single-crystal X-ray data for 3 and 4 were collected at 123 K using an Agilent Super-Nova dual-wavelength diffractometer with a microfocus X-ray source and multilayer optics monochromatized Cu Kα (λ = 1.541 84 Å) radiation. Details of data collection and analyses are given in the Supporting Information. Computations. All calculations were performed with DFT employing the M06 exchange and correlation functional.23 Geometries were optimized using Jensen’s pc-1 basis set.24,25 All structures were characterized by subsequent frequency calculations. NMR chemical shieldings were calculated for the M06/pc-1 geometries with the gauge-invariant atomic orbitals (GIAO)26 approach using Jensen’s pc2 basis set for O atoms and Jensen’s pcS-227 basis set for all other atoms. Solvent effects were modeled with the polarizable continuum model (PCM). In distinction from the experiments, CHCl3 was not employed as a secondary reference. Instead, chemical shifts were calculated relative to the H and C shieldings in tetramethylsilane, which were calculated at the same level of theory. The calculated chemical shifts were corrected for known inconsistencies of DFT28 by the procedure described in the Supporting Information. All calculations were performed with the Gaussian09 program package.29

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00178. F



■

Article

(20) Deyou, T.; Gumula, I.; Pang, F.; Gruhonjic, A.; Mumo, M.; Holleran, J.; Duffy, S.; Fitzpatrick, P. A.; Heydenreich, M.; Landberg, G.; Derese, S.; Avery, V.; Rissanen, K.; Erdelyi, M.; Yenesew, A. J. Nat. Prod. 2015, 78, 2932−2939. (21) Nyandoro, S. S.; Ndanu, J.; Munissi, J. J.; Gruhonjic, A.; Fitzpatrick, P. A.; Landberg, G.; Lu, Y.; Wang, B.; Pan, F.; Rissanen, K.; Erdelyi, M. J. Nat. Prod. 2015, 78, 2045−2050. (22) Aronsson, P.; Munissi, J. J. E.; Gruhonjic, A.; Fitzpatrick, P. A.; Landberg, G.; Nyandoro, S. S.; Erdelyi, M. Diseases 2016, 4, 3. (23) Zhao, Y.; Truhlar, D. G. Theor. Chem. Acc. 2008, 120, 215−241. (24) Jensen, F. J. Chem. Phys. 2001, 115, 9113−9125. (25) Jensen, F.; Helgaker, T. J. Chem. Phys. 2004, 121, 3463−3470. (26) Wolinski, K.; Hinton, J. F.; Pulay, P. J. Am. Chem. Soc. 1990, 112, 8251−8260. (27) Jensen, F. J. Chem. Theory Comput. 2008, 4, 719−727. (28) Olsson, L.; Cremer, D. J. Chem. Phys. 1996, 105, 8995−9006. (29) Frisch, M. J. T.; G, W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2009.

NMR, MS, UV, and CD spectra, data for X-ray crystallography, and cytotoxicity assays (PDF) Crystallographic data (CIF) Crystallographic data (CIF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +254 733 832 576. Fax: +254 20 444 6138. E-mail: [email protected] (A. Yenesew). *Tel: +46 31 786 9033. E-mail: [email protected] (M. Erdélyi). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to Mr. P. C. Mutiso of the University of Nairobi, Kenya, for identification of the plant materials and to Dr. U. Brath (University of Gothenburg) for proofreading the spectroscopic structure identification. The International Science Program (ISP Sweden, grant KEN-02), the Swedish Research Council (2012-6124), and the Academy of Finland (KR, project nos. 263256 and 265328) are thankfully acknowledged for financial support.

■

REFERENCES

(1) Agnew, A. D. Upland Kenya Wild Flowers: A Flora of the Ferns & Herbaceous Flowering Plants of Upland Kenya; Oxford University Press: London, UK, 1974. (2) Verdcourt, B. In Flora of Tropical East Africa, Crown Agents for Oversea Governments and Administrations; Polhill, R. M., Ed.; Whitefriars Press Ltd: London and Tonbridge, UK, 1976; pp 1−213. (3) Beentje, H. J. Kenya Trees: Shrubs and Lianes, National Museums of Kenya; National Museums of Kenya: Nairobi, Kenya, 1994. (4) Kokowaro, J. O. Medicinal Plants of East Africa, 3rd ed.; University of Nairobi Press: Nairobi, Kenya, 2009. (5) Endale, M.; Ekberg, A.; Akala, H. M.; Alao, J. P.; Sunnerhagen, P.; Yenesew, A.; Erdelyi, M. J. Nat. Prod. 2012, 75, 1299−1304. (6) Koch, A.; Tamez, P.; Pezzuto, J.; Soejarto, D. J. Ethnopharmacol. 2005, 101, 95−99. (7) Schripsema, J.; Caprini, G. P.; van der Heijden, R.; Bino, R.; de Vos, R.; Dagnino, D. J. Nat. Prod. 2007, 70, 1495−1498. (8) Nayak, B. S.; Vinutha, B.; Geetha, B.; Sudha, B. Fitoterapia 2005, 76, 671−675. (9) Irungu, B. N.; Rukunga, G. M.; Mungai, G. M.; Muthaura, C. N. S. Afr. J. Bot. 2007, 73, 204−207. (10) Bukuru, J. F.; Van, T. N.; Van Puyvelde, L.; Mathenge, S. G.; Mudida, F. P.; De Kimpe, N. J. Nat. Prod. 2002, 65, 783−785. (11) Bukuru, J.; Van, T. N.; Van Puyvelde, L.; He, W. D.; De Kimpe, N. Tetrahedron 2003, 59, 5905−5908. (12) Endale, M.; Ekberg, A.; Alao, J. P.; Akala, H. M.; Ndakala, A.; Sunnerhagen, P.; Erdelyi, M.; Yenesew, A. Molecules 2013, 18, 311− 321. (13) Silverstein, R. M.; Basler, G. C.; C, M. T. Identification Spectrométrique des Composés Organiques, 5th ed.; De Boeck&Larciers: Paris, France, 1998. (14) Ee, G. C. L.; Wen, Y. P.; Sukari, M. A.; Go, R.; Lee, H. L. Nat. Prod. Res. 2009, 23, 1322−1329. (15) Monks, T. J.; Jones, D. C. Curr. Drug Metab. 2002, 3, 425−438. (16) Huang, Q.; Lu, G.; Shen, H. M.; Chung, M. C. M.; Ong, C. N. Med. Res. Rev. 2007, 27, 609−630. (17) Queiroz, M. L. S.; Valadares, M. C.; Torello, C. O.; Ramos, A. L.; Oliveira, A. B.; Rocha, F. D.; Arruda, V. A.; Accorci, W. R. J. Ethnopharmacol. 2008, 117, 228−235. (18) Wokaun, A.; Ernst, R. R. Chem. Phys. Lett. 1977, 52, 407−412. (19) Hurd, R. E.; John, B. K. J. Magn. Reson. 1991, 91, 648−653. G


Naphthalene Derivatives from the Roots of Pentas parvifolia and

Recommend Documents