Four Diphenylpropanes and a Cycloheptadibenzofuran from Bussea sakalava from the Madagascar Dry Forest

Abstract

Investigation of the endemic Malagasy plant Bussea sakalava Du Puy & R. Rabev. (Fabaceae) for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of the four new diphenylpropanes 1–4 and the new cycloheptadibenzofuran 5; compound 5 has a previously unreported natural product skeleton. The structure elucidation of these compounds was based on the analysis of their 1D and 2D NMR and mass spectroscopic data. Compounds 1–5 were tested for antiproliferative activity against the A2780 human ovarian cancer cell line.

In our continuing search for biologically active natural products from tropical rainforests as part of an International Cooperative Biodiversity Group (ICBG) program, we obtained an ethanol extract from the roots of a plant identified as Bussea sakalava Du Puy & R. Rabev. (Fabaceae) from Madagascar. This extract showed moderate antiproliferative activity against the A2780 human ovarian cancer cell line with an IC₅₀ value of 10 µg/mL. The extract was selected for examination on the basis of this activity and the absence of previous phytochemical studies of the species.

Previous studies on the genus Bussea indicated the presence of azetidine-2-carboxylic acid and 3-hydroxyproline in seeds of different Bussea species,^2,3 and the cytotoxicity and high trypanocidal activity of a methanol extract of stem bark of Bussea occidentalis has been reported.⁴

Fractionation of a dichloromethane fraction of an ethanol extract of B. sakalava by C-18 open column and high performance liquid chromatography (HPLC) yielded four new diphenylpropanes named bussealins A–D (1 – 4) and a cycloheptadibenzofuran derivative named bussealin E (5). Herein we report the structural elucidation of these new compounds and their antiproliferative properties against the A2780 human ovarian cancer cell line.

Results and Discussion

Bussealin A (1) was obtained as an off-white amorphous solid. Its positive ESI-MS revealed a pseudomolecular ion peak at m/z 321.1338 [M + H]⁺ corresponding to the molecular formula C₁₇H₂₁O₆. The IR spectrum showed absorptions of OH (3367 cm⁻¹) and aromatic groups. The ¹H NMR spectrum (Table 1) exhibited a singlet at δ_H 6.18 (2H, s) corresponding to a pair of aromatic protons of an A₂ system, two aromatic doublets [δ_H 6.50 (d, J = 8.4) an 6.38 (d, J = 8.4)] of an AB system, two OCH₃ groups [δ_H 3.75 (s) and 3.78 (s)], and a multiplet and two triplet methylene groups at δ_H 1.79 (2H, m), 2.52 (2H, t, J = 7.7) and 2.41 (2H, t, J = 7.7) respectively. The ¹³C NMR spectrum of 1 exhibited signals for 17 carbons, including three methylene carbons (δ_C 36.5, 33.0, and 30.6), two OCH₃ groups (δ_C 56.5 and 60.8), and twelve aromatic carbons assignable to two isolated aromatic rings. Six of the aromatic carbons were oxygenated, as shown by their deshielded carbon chemical shifts (Table 1) and were consistent with the molecular formula. The above data suggested that 1 had a diphenyl propane skeleton. The complete ¹H and ¹³C NMR assignments and the connectivities were determined from analysis of a combination of COSY, HMQC, and HMBC data. Three mutually coupled methylene groups were revealed by the cross peaks observed in the COSY spectrum. In the HMBC spectrum, H-1 (δ_H 2.41) showed correlations with C-2 (δ_C 33.0), C-3 (δ_C 30.6), C-1' (δ_C 140.2), and with C-2' and C-6', both of which had the same chemical shifts (δ_C 108.7). The A₂ substitution pattern of the A ring of 1 was established by HMBC correlations from the signal at δ_H 6.18 (H-2' and H-6') to C-1 (δ_C 36.5), C-1' (δ_C 140.2), C-3' (δ_C 151.3), C-4' (δ_C 134.7) and C-6' and C-2' (δ_C 108.7), as well as the correlation from one OCH₃ group at δ_H 3.75 to C-4' (δ_C 134.7). The proton substitutions on the B ring were assigned based on the ³J HMBC correlations between H-3 (δ_H 2.52) and C-6" (δ_C 120.5), and between H-5" (δ_H 6.38) and C-1" (δ_C 123.4). Moreover, the H-5" proton showed HMBC correlations to C-6" (δ_C 120.5), C-4" (δ_C 147.8) and C-3" (δ_C 134.9). The location of the remaining OCH₃ group was at C-4", as deduced from the HMBC correlation between the signal at δ_H 3.78 and that of C-4". On the basis of the molecular formula of 1, the remaining four OH groups were located at C-2" (δ_C 144.7), C-3" (δ_C 134.9), C-3' (δ_C 151.3), and C-5' (δ_C 151.3). Bussealin A is thus assigned the structure 3',5',2",3"-tetrahydroxy-4',4"-dimethoxy-1,3-diphenylpropane (1).

Table 1.

¹H and ¹³C NMR data for Bussealin A–D (1–4)^a

position	1		2		3		4
	1H (J, Hz)	13C	1H (J, Hz)	13C	1H (J, Hz)	13C	1H (J, Hz)	13C
1	2.41 t (7.9)	36.5	2.50 t (7.8)	30.4	2.49 t (7.9)	36.1	2.55 t (7.7)	30.4
2	1.79 m	33.0	1.76 m	31.9	1.81 m	33.2	1.79 m	31.8
3	2.52 t (7.7)	30.6	2.54 t (7.8)	30.7	2.54 t (7.7)	30.6	2.55 t (7.7)	30.6
1'		140.2		124.7		137.2		124.3
2'	6.18 s	108.7		152.2	6.64 d (2.0)	116.5		153.3
3'		151.3	6.58 s	99.4		147.3	6. 61 s	99.6
4'		134.7		147.2		147.0		149.1
5'		151.3		141.0	6.79 d (8.2)	112.9		144.1
6'	6.18 s	108.7	6.60 s	117.9	6.60 dd (8.2, 2.0)	120.6	6.75 s	116.3
1"		123.4		123.7		123.5		123.6
2"		144.7		144.7		144.7		144.7
3"		134.9		134.9		135.0		134.9
4"		147.8		147.8		147.8		147.8
5"	6.38 d (8.4)	103.8	6.39 d (8.4)	103.8	6.39 d (8.4)	103.9	6.39 d (8.4)	103.9
6"	6.50 d (8.4)	120.5	6.51 d (8.4)	120.4	6.50 d (8.5)	120.5	6.51 d (8.3)	120.4
2'-OMe			3.76 s	56.8			3.78 s	56.6
4'-OMe	3.75 s	60.8	3.83 s	57.0	3.80 s	56.6	3.82 s	56.8
5'-OMe							3.76 s	57.6
4"-OMe	3.78 s	56.5	3.80 s	56.6	3.80 s	56.6	3.80 s	56.9

^aIn CD₃OD; δ (ppm) 500 MHz for ¹H and 125 MHz for ¹³C; multiplicities; J values (Hz) in parentheses.

Bussealin B (2) was obtained as an off-white amorphous solid. Its positive ESI-MS revealed a pseudomolecular ion peak at m/z 335.1512 [M + H]⁺ corresponding to molecular formula C₁₈H₂₃O₆. The ¹H NMR spectrum (Table 1) showed two singlets of an AX system at δ_H 6.58 (s) and 6.60 (s), two aromatic doublets of an AB system at δ_H 6.51 (d, J = 8.4) and 6.39 (d, J = 8.4), three OCH₃ groups [δ_H 3.76 (s), 3.80 (s) and 3.83 (s)], and one multiplet and two triplet methylene groups at δ_H 1.76 and 2.54 (t, J = 7.8) and 2.50 (t, J = 7.8). Inspection of the ¹H and ¹³C NMR spectra of 2 revealed close similarities with those of 1, except for the presence of an additional OCH₃ signal and the chemical shifts of the AX system of ring A. The fact that the chemical shifts of the carbons of ring B of compounds 1 and 2 were superimposable (Table 1) indicated the presence of a 2",3"-dihydroxy-4"-methoxyphenyl group in 2. Interpretation of HMBC and NOESY experiments allowed us to determine the location of the OCH₃ groups to be at 2', 4', and 4". The two singlet aromatic protons on ring A were assigned according to the observation of ³J HMBC correlations from H-6' (δ_H 6.60) to C-1 (δ_C 30.4) and from H-3' (δ_H 6.58) to C-1' (δ_C 124.7). Moreover, the proton signal of H-1 (δ_H 2.50) showed HMBC correlations with C-1' (δ_C 124.7), C-6' (δ_C 117.9) and the methoxylated carbon at C-2' (δ_C 152.2). This indicated that the third OCH₃ group must be at C-4' or C-5'. NOESY correlations from H-3' (δ_H 6.58) to 2'-OMe (δ_H 3.76) and to 4'-OMe (δ_H 3.83) established the location of the methoxy group at C-4' and the hydroxy group at C-5'. The structure of bussealin B was thus assigned as 5',2",3"-trihydroxy-2',4',4"-trimethoxy-1,3-diphenylpropane.

Bussealin C (3) was obtained as an off-white amorphous solid. Its positive ESI-MS revealed a pseudomolecular ion peak at m/z 305.1384 [M + H]⁺ corresponding to molecular formula C₁₇H₂₁O₅. Its ¹H NMR and ¹³C NMR spectra (Table 1) indicated that 3 is also a diphenylpropane with a 2",3"-dihydroxy-4"-methoxyphenyl group substituted at C-3. The 1,3,4-trisubstituted A ring was determined by the proton coupling constants and HMBC correlations from H-2' (δ_H 6.64) and H-6' (δ_H 6.60) to C-1 (δ_C 36.1), and COSY correlations between H-5' (δ_H 6.79) and H-6' (δ_H 6.60). Furthermore, the HMBC spectrum showed a ³J correlation from H-6' to the methoxylated carbon at C-4' (δ_C 147.0), which was confirmed by NOESY correlations between H-5' (δ_H 6.79) and 4'-OMe (δ_H 3.80). The above data coupled with the molecular formula led to assignment of the structure of bussealin C as 3', 2",3"-trihydroxy-4',4"-dimethoxy-1,3-diphenylpropane.

Bussealin D (4) was obtained as an off-white amorphous solid. The positive ESI-MS exhibited a pseudomolecular ion peak at m/z 349.1648 [M + H]⁺ corresponding to the molecular formula C₁₉H₂₅O₆. The ¹H NMR and ¹³C NMR spectra (Table 1) indicated that 4 had the same tetrasubstituted B ring with an OCH₃ group at C-4" as in compounds 1–3. In its ¹H NMR spectrum, the coupling patterns and the locations of the aromatic proton resonances of ring A were very similar to those of 2. The presence of three OCH₃ groups and the substitution pattern of ring A of compound 4 were deduced by interpretation of the 1D and 2D NMR data. The HMBC spectrum of 4 showed correlations from H-1 (δ_H 6.79) to C-1' (δ_C 124.3), C-6' (δ_C 116.3) and to the methoxylated carbon at C-2' (δ_C 153.3). Furthermore, a clear ³J long-range correlation from the singlet proton H-3' (δ_H 6.61) to C-1' (δ_C 124.3) was also observed. Thus, the two remaining OCH₃ groups were determined to be at C-4' (δ_C 149.1) and C-5' (δ_C 144.1). The structure of bussealin D was thus determined to be 2",3"-dihydroxy-2',4',5',4"-tetramethoxy-1,3-diphenylpropane.

The positive ESI-MS of bussealin E (5) displayed a pseudomolecular ion peak at m/z 331.1181 [M + H]⁺ corresponding to the molecular formula C₁₈H₁₉O₆. The ¹H NMR spectrum in CDCl₃ showed signals for a singlet aromatic proton at δ_H 6.70, two OH groups (δ_H 5.75 and 5.69), three OCH₃ groups at δ_H 4.24, 4.24, and 4.01, and three methylene groups as multiplets at δ_H 3.13, 3.12 and 2.17. The ¹³C NMR spectrum of 5 exhibited 18 signals, assigned to three methylene (δ_C 35.5, 28.7, 24.3), three OCH₃ (δ_C 60.8, 60.8 and 61.7), and twelve aromatic carbons of two isolated aromatic rings. Seven of the aromatic carbons were oxygenated, based on their deshielded chemical shifts (Table 2). The ten degrees of unsaturation implied by the molecular formula C₁₈H₁₈O₆ required two additional rings. Interpretation of ¹H-¹H COSY, HMQC, HMBC and NOESY spectra allowed assignment of the locations of the functionalities present in 5. In the COSY spectrum, the three methylene groups were mutually coupled. The assignment of a singlet aromatic proton was substantiated by the observation of HMBC correlations from H-1 (δ_H 6.70) to C-10 (δ_C 35.5), C-3b (δ_C 118.2), and two oxygenated aromatic carbons at C-2 (δ_C 146.5) and C-3 (δ_C 129.7). HMBC correlations from the signal at δ_H 5.69 to C-1 (δ_C 110.1), C-2 (δ_C 146.5) and the methoxylated carbon at C-3 (δ_C 129.7) were observed, substantiating the location of a hydroxy group at C-2. The other hydroxy group was assigned to position 7 based on the observation of HMBC correlations from the signal at δ_H 5.75 to the carbon signals at C-6 (δ_C 136.5), C-7 (δ_C 142.3) and C-7a (δ_C 115.0). In addition, the signal at δ_H 5.75 showed NOESY correlations to H-8 (δ_H 3.13) and 6-OMe (δ_H 4.01). These observations required that the remaining OCH₃ group be placed at C-5. Furthermore, the HMBC correlations observed from H-10 (δ_H 3.12) to C-1 (δ_C 110.1), C-10a (δ_C 131.7), C-3b (δ_C 118.2), C-8 (δ_C 28.7) and C-9 (δ_C 24.3) confirmed the location of the cycloheptadiene ring. The above data confirmed the cycloheptadibenzofuran skeleton of 5. Assignments of the ¹³C NMR signals of C-3a, C-4a and C-4b were made by comparing the measured data with those calculated by ACD/ChemSketch version 11.01. The calculated shifts were in excellent agreement with the observed values, and were all within the standard deviation of the software (5 ppm), except for C-7a. Therefore, the structure of 5 was assigned as 9,10-dihydro-2,7-dihydroxy-3,5,6-trimethoxy-8H-cyclohepta[klm]dibenzofuran.

Table 2.

¹H and ¹³C NMR Data for Bussealin E (5)

position	1Ha	13Ca	13Cb	1Hc
1	6.70 s	110.1	110.0	6.59 s
2		146.5	149.1
3		129.7	131.3
3a		146.2	148.7
3b		118.2	113.3
4a		140.7	139.7
4b		120.6	117.4
5		135.6	137.0
6		136.5	137.9
7		142.3	145.1
7a		115.0	109.7
8	3.13 m	28.7	28.9	3.08 m
9	2.17 m	24.3	24.2	2.12 m
10	3.12 m	35.5	34.5	3.07 m
10a		131.7	128.6
3-OCH3	4.24 s	60.8	61.5	4.18 s
2-OH	5.69 s
5-OCH3	4.24 s	60.8	61.6	4.08 s
6-OCH3	4.01 s	61.7	61.0	3.90 s
7-OH	5.75 s

^aIn CDCl₃; δ (ppm) 600 MHz for ¹H and 150 MHz for ¹³C; multiplicities; J values (Hz) in parentheses.

^bCalculated using ACD/ChemSketch version 11.01.

^cIn CD₃OD; δ (ppm) 600 MHz for ¹H; multiplicities; J values (Hz) in parentheses.

It is noteworthy that bussealin E is the first cycloheptadibenzofuran isolated from natural sources, and the cycloheptadibenzofuran skeleton is rare among synthetic compounds. The only simple synthetic compound with this ring system is 9,10-dihydro-1-methyl-8H-cyclohepta[ klm]dibenzofuran (6) and its 8-keto derivative.⁵

The presence of diphenylpropanes in B. sakalava suggests that bussealin E is biosynthesized by oxidative coupling of an appropriate precursor diphenylpropane. This could be followed by nucleophilic attack from a phenolate anion on a carbonyl group followed by dehydration to afford the new cycloheptadibenzofuran skeleton (5) as indicated in Scheme 1.

Scheme 1.

Possible biosynthesis of cycloheptadibenzofuran 5 in B. sakalava

The bioactivity of diphenylpropanes has been widely studied. The diphenylpropane broussonin A inhibited respiratory syncytial-virus (RSV) more effectively than the standard antiviral drug ribavirin,⁶ and its anti-aromatase activity has also been evaluated.⁷ Broussonin B moderately inhibited a chymotrypsin-like activity of the proteasome.⁸ The anti-inflammatory,^9,10 antifungal,¹¹ antivascular,¹² adipogenic,¹³ and anti-hCNT3 (human concentrative nucleoside transporter 3)¹⁴ activities of diphenylpropane analogues have also been reported. Since there have been no previous studies on the properties of diphenylpropanes on human ovarian cancer cells, we investigated the antiproliferative activity of diphenylpropanes 1–4 against the A2780 human ovarian cancer cell line. Bussealins A–D (1–4) showed only weak antiproliferative activities, with IC₅₀ values of 36, 24, 36, and 40 µM, respectively. Bussealin E (5), with a new chemical skeleton, was also tested against the A2780 cell line, but it also only exhibited weak activity with an IC₅₀ value of 45 µM. The new skeleton of bussealin E thus does not appear to confer any novel antiproliferative activity beyond that which is normal for diphenylpropanes.

Experimental Section

General Experimental Procedures

UV and IR spectra were measured on a Shimadzu UV-1201 spectrophotometer and a MIDAC M-series FTIR spectrophotometer, respectively. NMR spectra were recorded in CD₃OD or CDCl₃ on either JEOL Eclipse 500 or Bruker Avance 600 spectrometers. The chemical shifts are given in δ (ppm) and coupling constants (J) are reported in Hz. Mass spectra were obtained on an Agilent 6220 LC-TOF-MS. HPLC was performed on a Shimadzu LC-10AT instrument with a semi-preparative C18 Varian Dynamax column (5 µm, 250 × 10 mm).

Antiproliferative Bioassays

Antiproliferative activities were obtained at Virginia Polytechnic Institute and State University against the drug-sensitive A2780 human ovarian cancer cell line as previously described, except that the samples were added in 1 µL 100% DMSO per well instead of 20 µL of 1:1 DMSO:H₂O; paclitaxel (IC₅₀ 0.017 µM) was used as a positive control.¹⁵ The A2780 cell line is a drug-sensitive ovarian cancer cell line.¹⁶

Plant Material

A sample of root of Bussea sakalava Du Puy & R. Rabev. (Fabaceae) was collected on January 25, 2007, near Ambolobozobe, Madagascar at coordinates 12°31'26"S 49°31'29"E, at an elevation of 20 m. Its assigned collection number is Rakotonandrasana et al. 1079. The genus Bussea Harms is a small genus including 7 species (5 from Tropical Africa and 2 from Madagascar). B. sakalava is endemic to deciduous forest from western to northern Madagascar. The hard wood of this species is used in construction and as firewood.¹⁷ Voucher specimens have been deposited at the Parc Botanique and Zoologique de Tsimbazaza (TAN) and at the Centre National d'Application des Recherches Pharmaceutiques (CNARP) in Antananarivo, Madagascar; the Missouri Botanical Garden in St. Louis, Missouri (MO); and the Muséum National d'Histoire Naturelle in Paris, France (P).

Extraction and Isolation

Dried roots of B. sakalava (275 g) were ground in a hammer mill, then extracted with ethanol by percolation for 24 hours at room temperature to give the crude extract MG 4273 (14.4 g), of which 3.0 g was shipped to Virginia Polytechnic Institute and State University (VPISU) for bioassay-guided isolation. Sample MG 4273 (IC₅₀ 9.6 µg/mL, 2.1 g) was suspended in aqueous MeOH (MeOH-H₂O, 9:1, 100 mL) and extracted with hexane (3 × 100 mL portions). The aqueous layer was then diluted to 60% MeOH (v/v) with H₂O and extracted with CH₂Cl₂ (3 × 150 mL portions). The hexane extract was evaporated in vacuo to leave 227 mg with an IC₅₀ value of 19 µg/mL. 102.9 mg of residue from the CH₂Cl₂ extract had an IC₅₀ of 10 µg/mL. The aqueous MeOH extract (1.7 g) was inactive. The CH₂Cl₂ extract was selected for fractionation using an SPE cartridge over C-18, and two fractions were collected. Fractions I and II (70.2 mg and 26.8 mg) had IC₅₀ values of 8.6 and 15 µg/mL, respectively. Fraction I was separated by C-18 HPLC (65% MeOH-H₂O), and compounds 1 (3.3 mg t_R 12.5 min), 2 (1.7 mg t_R 18.6 min), 3 (2.0 mg t_R 22.0 min), 4 (1.1 mg t_R 29.5 min) and 5 (1.1 mg t_R 26.5 min) were isolated.

3',5',2",3"-Tetrahydroxy-4',4"-dimethoxy-1,3-diphenyl-propane (1)

off-white amorphous solid; UV (MeOH) λ_max nm (log ε) 218 (4.40), 267 (3.69), 294 (3.52); IR ν_max cm⁻¹: 3367, 1648, 1450, 1115, 1024. ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD), see Table 1; ESI-MS m/z 321.1338 [M + H]⁺ (calcd for C₁₇H₂₁O₆, 321.1338).

5',2",3"-Trihydroxy-2",4',4"-trimethoxy-1,3-diphenyl-propane (2)

off-white amorphous solid; UV (MeOH) λ_max nm (log ε) 214 (4.25), 229 (sh) (4.10), 290 (3.59) nm; IR ν_max cm⁻¹: 3332, 1599, 1444, 1095, 1032; ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD), see Table 1; ESI-MS m/z 335.1512 [M + H]⁺ (calcd for C₁₈H₂₃O₆, 335.1495).

3',2",3"-Trihydroxy-4',4"-dimethoxy-1,3-diphenyl-propane (3)

off-white amorphous solid; UV (MeOH) λ_max nm (log ε) 208 (4.15), 267 (3.54), 289 (3.47) nm; IR ν_max cm⁻¹: 3338, 1656, 1450, 1115, 1024; ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD), see Table 1; ESI-MS m/z 305.1384 [M + H]⁺ (calcd for C₁₇H₂₁O₅, 305.1389).

3',2",3"-Trihydroxy-4',4"-dimethoxy-1,3-diphenyl-propane (4)

off-white amorphous solid; UV (MeOH) λ_max nm (log ε) 210 (4.21), 229 (sh) (4.01), 289 (3.48) nm; IR ν_max cm⁻¹: 3350, 1602, 1450, 1115, 1026; ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR (125 MHz, CD₃OD), see Table 1; ESI-MS m/z 349.1648 [M + H]⁺ (calcd for C₁₉H₂₅O₆, 349.1651).

9,10-Dihydro-2,7-dihydroxy-3,5,6-trimethoxy-8H-cyclohepta[klm]dibenzofuran (5)

off-white amorphous solid; UV (MeOH) λ_max nm (log ε) 218 (4.25), 270 (3.78), 294 (3.73), 316 (3.47)) nm; IR ν_max cm⁻¹: 3332, 1567, 1449, 1115, 1024; ¹H NMR (600 MHz, CD₃OD and CDCl₃) and ¹³C NMR (150 MHz, CD₃OD), see Table 2; ESI-MS m/z 331.1181 [M + H]⁺ (calcd for C₁₈H₁₉O₆, 331.1182).

Figure 1.

COSY, HMBC and NOESY correlations of 5