Phenylbutenoid dimers from the rhizomes of Zingiber cassumunar

VoL 32,No. 2, pp.351-363,1993 Printedin GreatBritain.

00314422/93 $6.00 + 0.00 0 1993PersamonPressLtd

Phytochemistry,

PHENYLBUTENOID DIMERS FROM THE RHIZOMES OF ZINGIBER CASSUMUNAR AKIKO JITOE, TOSHIYA MASUDA*

and NOBUJI NAKATANI

Laboratory of Food Chemistry, Faculty of Science of Living, Osaka City University, Sumiyoshi, Osaka 558, Japan (Received in revised form 17 June 1992)

Key Word Index-Zingiber

cussumunar, Zingibcraceae; rhizomes; phenylbutenoid;

dimer.

Abstract-Two new phenylbutenoid dimers, (~truns-3-(2,4,5-t~methoxyphenyl)-4-C(cyclohexene and cis-1,2-bis[(E)-3,4-dimethoxystyryl]cyclobutane, have been isolated from the fresh rhizomes of Zingiber cassumunur along with the two known phenylbutenoid dimers. Their structures were elucidated by spectroscopic and chemical methods. The stereochemistry of these cyclohexene compounds was clarified on the basis of ‘H NMR data of their derivatives. The substituted positions for the 3,4-dimethoxystyryl groups of the cyclobutane compound were confumed from a Cope rearrangement product in the pyrolysis of the cyclobutane, and the stereochemistry of the cyclobutane was confirmed by ‘H NMR evidence.

INTRODUCTION

As a part of our investigation of tropical gingers [l-4], we have now studied the rhizomes of Zing&r cassumunar Roxb. which has been used in traditional tropical medicine [S]. Our studies have indicated that the extract of the rhizomes has potent antioxidant and anti-inflammatory activity [6]. We now report the isolation and structural elucidation of four phenylbutenoid dimers, l-4, from the rhizomes of this plant. RESULTS AND DIsCUsSION

The acetone extract of the fresh rhizomes of Zingiber was suspended in water and successively extracted with n-hexane and methylene chloride. The methylene chloride fraction was purified by repeated silica gel column chromatography and preparative TLC, yielding four phenylbutenoid dimers (l-4). Compounds 2 and 3 showed the same molecular ion peak at m/z 380 and similar iH and 13C NMR spectral data. Compounds 2 and 3 were identified to be the stereoisomers of (k)-3-(3,4dimethoxyphenyl)-4-[@J-3,4dimethoxystyryllcyclohexene by spectroscopic methods, which have been reported by Amatayakul et al. [7] and Kuroyanagi et al. [8] respectively, to occur in this plant. Compound 4 has the molecular formula Cz5HJ00) (HR-mass spectrometry; m/z 410.2115 [Ml’, calcd for C,,H,,O,: 410.2092). Although the ‘H and ‘“CNMR spectral data were similar to those of 2 and 3, one additional methoxy signal was observed in the data (Tables 1 and 2). In the ‘HNMR spectrum of 4, signals cassumunar

*Author to whom correspondence should be addressed. 357

due to five methoxyl groups [S 3.71 (3H, s), 3.84 (3H, s), 3.85 (6H, s), 3.88 (3H, s)], 1,3,4_trisubstituted benzene cS6.76 (lH, d, 5=8.6 Hz), 6.79 (lH, dd, 3=8.6, 1.8 Hz), 6.82 (lH, d, J= 1.8 Hz)], and 1,2,4,5_tetrasubstituted benzene [S 6.45 (lH, s), 6.74 (lH, s)] were observed. The homo spin decoupling technique and the 13C-lH COSY spectrum revealed the presence of a 3-phenyl-4-styrylcyclohexene skeleton similar to 2 and 3. The substituted position of the methoxyl groups was determined to be at 2’,4’,5’,3”’and 4”’ based on NOE differential and NOESY spectra as shown in Fig 1. Olefinic proton signals assignable to the C-l” and C-2” positions have the same chemical shift at 6 6.07 in chloroform-d, preventing the determination of the geometry of the double bond. To clarify the geometry by ‘HNMR, solvent etfects were investigated. Only in dimethylsulphoxide-d, out of the various solvents tested, which included chloroform-d benzene-d,, acetone-d,, methanol-d, pyridine-d, and dimethylsulphoxide-d,, were the H-l” and H-2” signals separated [S 6.04 (lH, dd, J= 15.9, and 7.3 Hz), 6.12 (lH, d, J = 15.9 Hz), respectively]. The geometry of the double bond was then determined to be trans on the basis of their coupling constant (.I = 15.9 Hz). Compound 4 did not show optical rotation, indicating 4 is present as a racemate. Thus, compound 4 is ( f )-3-(2,4,5-trimethoxyphenyl)4-[IQ-3,4dimethoxystyryl]cyclohexene. As for the stereochemistry of such cyclohexene compounds (2-4), Amatayakul et al. [7j estimated the relative configuration of 2 to be cis by comparing the coupling constant between H-3 and H-4 with that of the cis-hexamethoxyl analogue, the stereochemistry of which was confirmed by X-ray analysis. However, they could not determine the stereochemistry, because the coupling constant was influenced by the number of methoxyl

A. JITOErt al.

358

R,+

Rl* tlWlS

CiS

5

2’3

-o_rrx

$ OCH,

groups. In the case of 3, Kuroyanagi et al. [g] suggested the stereochemistry to be trans for the reason that 3 was the isomer of 2. Thus, the stereochemistry of these cyclohexene compounds was regarded as not having been confirmed. We clarified the stereochemistry of 4, together with 2 and 3, by a simple chemical and spectroscopic method as follows. Compounds 2-4 were hydrogenated to give 5-7, respectively. As shown in Fig. 2, the

’

%CH 3 3

H-3 coupling pattern of 5 was dt (d, J = 12 Hz, t, f = 3 Hz), ax-ax, ox-eq and ax-eq coupling, indicating 2 was the cisisomer, and that of 6 was dt (d, J = 3 Hz, t, J = 12 Hz), axax, ax-ax and ax-eq coupling, indicating 3 was the transisomer. In the case of 7, the H-3 signal was broad for the same ‘H NMR condition (Table 2). To afford a coupling pattern for H-3 of 7, the ‘HNMR spectrum of 7 was measured at elevated temperature considering the influ-

359

Phenylbutenoids from Zingiber caswmunar Table 1. ‘H NMR spectral data of compounds 2-4 [a (ppm) in CDCI, (400 MHz)] H

2 5.96, 5.78, 3.49, 2.69,

lH, lH, lH, lH,

br d (10) dt (10, 2) m m

5

1.60,2H, m

6 2 3 5 6 1” 2” 2”’ 5” 6”’ OMe

2.21, 2H, m 6.67-6.79, 1H -

3

4

5.89, lH, dd (10, 3) 5.68, lH, dd (10, 2) 3.18, lH, m 2.35, lH, m 1.68, lH, m 1.92, lH, m 2.21, 2H, m 6.70-6.81, 1H

5.89, lH, 5.60, lH, 3.72, lH, 2.35, lH, 1.69, lH, 1.88, lH, 2.20, 2H, -

6.67-6.79, 2H

6.70-6.81, 2H

5.57, lH, dd (16, 9) 6.23, lH, d (16)

6.01, lH, dd (16, 7) 6.09, lH, d (16)

6.67-6.79, 3H

6.70-6.81, 3H

3.73, 3.81, 3.83, 3.84,

3H, 3H, 3H, 3H,

s s s s

3.82, 3.85, 3.85, 3.87,

3H, 3H, 3H, 3H,

s s s s

dd (10, 3) dd (10, 2) m m m m m

6.45, lH, s 6.74, lH, s 6.07, 2H, m 6.82, lH, 6.76, lH, 6.79, lH, 3.71, 3H, 3.84,3H, 3.85, 6H, 3.88, 3H,

d (2) d (9) dd (9, 2) s s s s

Coupling constants (J in Hz) are given in parentheses.

Table 2. ‘H NMR spectral data of compounds S-7 [a (ppm) in CDCl, (400 MHz)] H*

5

1 2 3 4 5 6 2 3 5 6 1”

1.20-1.92, 2H

2” 2” 5” 6” OMe

6

7

1.20-1.92, 4H

1.03-1.85, 4H

1.05-1.83, 4H

278, lH, dt (12, 3)

213, lH, dt (3, 12)

2.73, lH, br m

1.20-1.92, 5H

1.03-1.85, 5H

1.05-1.83, 5H

6.44, lH, d (2r

6.45, lH, d (2) -

6.76, lH, d (8)b 6.48, lH, dd (8, 2)

6.74, lH, d (8)b 6.50, lH, dd (8, 2) 2.03, lH, br d (15) 1.03-1.85, 1H 2.29, lH, ddd (14, 8, 6) 2.53, lH, ddd (14, 9, 5) 6.58, lH, d (2)’ 6.69, lH, d (9r 6.61, lH, dd (9, 2) 3.76, 3H, s 3.80, 3H, s 3.81, 3H, s 3.83, 3H, s

2.12, 1H ddd (14, 8, 6) 2.46, lH, ddd (14,9,5) 6.64-6.67, 1H’ 6.68, lH, d (8)b .6.64-6.67, 1H’ 3.76, 3H, s 3.81, 6H, s 3.84, 3H, s

6.48, lH, s 6.54, lH, s 2.03, lH, br d (15) 1.05-1.83, 1H 2.30, lH, m 2.54, lH, m 6.48, lH, s 6.69, lH, d (8) 6.51, lH, dd (8, 2) 3.37, 6H, s 3.77, 3H, s 3.81, 3H, s 3.85, 3H, s

Coupling constants (J in Hz) are given in parentheses. *Numbering is tentative. ‘-‘Values are interchangeable within each column.

ence of OMe-2’ on the homogeneity of the compound. The H-3 signal showed dt (d, J = 3 Hz, t, J = 12 Hz) at 70” in pyridine-d,, similar to that of 6, indicating 4 was the trans isomer (Fig. 2). From these results, the three cyclohexene compounds were determined to he (+)-cis-3-(3,4-dim&oxyphenyl)-

4-C(E)-3$-dimethoxystyryl]cyclohexene (2), ( f )-wan+ 3-(3,4-dimethoxypheny1)-4-[(&-3,4-dimethoxystyry1]cyclohexene (3), and ( f )-brans-3-(2,4,Strimethoxyphenyl)-4-C(E)-3.4~dimethoxystyryl]cyclohexene (4). Compound 1 showed a molecular ion peak at m/z 380 and the main fragment ion peak at m/z 190 in the El-mass

360

A. JITOEet

al.

spectrum. In the HR-mass spectrum, m/z 190.1034 (calcd for C,2H,,0,: 190.0993) was obtained and the molecular formula was determined to be Cz4H3s04. Both ‘H and 13C NMR spectra showed half the signals for the molecular formula, indicating 1 has a symmetrical structure. The ‘H NMR spectrum of 1 showed signals due to two 1,3,4trisubstituted benzenes [~56.80 (2H, d, J=8.5 Hz), 6.89 (2H, dd, J=8.5, 1.8 Hz) and 6.92 (2H, d, J= 1.8 Hz)], four phenolic methoxyl groups [S 3.87 (6H, s) and 3.90 (6H, s)] and two conjugated trans-double bonds [S 6.20 (2H, dd, J = 15.9 and 6.1 Hz) and 6.30 (2H, d, J= 15.9 Hz)], which

Fig. 1. NOES of compound 4 obtained by NOESY and NOE difference spectra.

suggested

the presence

of two dimethoxystyryl

12 Hz 3 Hz

5 (27’C,

CDC13)

I

I

1

2.5

ppm

I 12 Hz 3 Hz

6 (27OC,

CDC13) I

I

I

I

2.5

I

2.0

ppm

7 (27”C,

C5D5N)

(6O”c,

C5D5N)

I, I

I

12 Hz 3 Hz

( 70°C,

C5D5N)

I

I

3.0

2.5

I

ppm

Fig. 2. ‘A NMR spectra (400 MHz) in the benzyl proton region for compound8 5-7. *Numbering is tentative.

groups.

Phenylbutenoids from Zingiber cassumunar

The homo spin decoupling technique revealed that the u-position of the styryl group was coupled to methine [62.91(2H, m), 845.81 and that the methine and methylene [b 1.89 (2H, m), 2.09 (2H, m), 6 25.31 formed a cyclobutane, which was also suggested by the chemical shift of the methylene proton. Thus, compound 1 was the dimerized compound at the terminal double bond of dimethoxyphenylbutadiene 11. The substituted positions of the methoxyl groups in the styryl moiety were determined to be at the C-3 and C-4 positions by comparing the “C NMR chemical shifts with calculated values for three patterns of dimethoxy styrene [9]. The substituted positions of the dimethoxystyryl groups were confirmed by a chemical reaction. The cyclooctadiene 12, the structure of which was confirmed by homo decoupling technique in ‘H NMR, was obtained by pyrolysis of 1, indicating that the dimethoxystyryl groups were substituted to the C-l and C-2 positions of the cyclobutane ring by considering the reaction mechanism (Cope rearrangement) [ 10,l l] as shown in Scheme 1. As for the stereochemistry on the cyclobutane, Berson et al. [ 1l] reported the chemical shift proton of cis-l&trans,transof the methine

361

dipropenylcyclobutane for 6 2.96 and tram-1,Z trans,truns-dipropenylcyclobutane for 6 2.50. This suggested that 1 had to be a c&compound. Thus, compound 1 is cis-1,2-bis[(E)-3,4-dimethoxystyryl]cyclobutane. Compound 1 was also synthesized from 3,4dimethoxybenzaldehyde (8) via photo-cycloaddition reaction of crystalline 3,4_dimethoxybutadiene (11) [12] (Scheme 2).

EXPERIMENTAL

General. Mps: uncorr. NMR spectra: ‘H at 400 or 60 MHz, “C at 100 MHz. Mass spectra: EI and high resolution MS, 70 eV, SI-MS, glycerin matrix. Plant material. Rhizomes of Zingiber cassumunar Roxb. cultivated in Tabanan Village (Bali, Indonesia) were collected in April 1990 and identified by Dr I. G. P. Tengah of Udayana University. Voucher specimens are deposited at the Herbarium of the Research Institute of Tropical Agriculture, University of the Ryukyus, Okinawa, Japan.

1-a R

R

transitionstate

1

12

Scheme 1. Mechanism for the pyrolysis of 1-12 via Cope rearrangement. R = 3J-dimethoxyphenyL

CH,O

NaOH

Scheme 2. Synthesis of compound 1 from 3+dimethoxybenzaldehy& dimethoxyphenylbutadiene (11).

(8) via photodimerization of 3’,4’-

A. JITOEeta[.

362

Extraction and isolation. Fresh rhizomes of Zingiber c~sa~u~r (573 g) were crushed and soaked in Me&O (11 x 2) for 18 days at room temp. After filtration, the solvent was removed under red. pres. to give a brownish residue (13.3 g). The residue was suspended in H,O and extracted x 3 each with n-hexane and CH,CI, successively and coned. The CH&l,-soluble fraction (2.7 g) was sepd into 20 frs by silica gel CC eluted with a mixt. of EtOAc and n-hexane. Fr. 11 was crystallized with EtOH and H,O to give a powder, which was purified by silica gel flash CC and silica gel prep. TLC (both CH,Cl,-nhexane, 2: 1) and the mother liquor was purified by silica gel CC (CH,Cl,-n-hexane, 2: 1) to give 1(8 mg), 2 (29 mg) and 3 (32 mg). Fr. 12 was purified by silica gel CC ~Me~CO-Irene, I : 20 and EtOA~-~-hexane, 1:2) to give 4 (8 mg). ( f )-cis-l2-bist(E)-3,4-dimethoxystyryrjCyclobutane (1). Needles (EtOH), mp 76”, [ali f0” (CHCl,; c 0.49), EI-MS m/z (rel. int.) 380 [M]’ (13), 190 (lOO), 175 (16), 159 (68), HR-MS m/z 190.1034 (caicd for C,2H,,02: 190.099). ‘H NMR (CDCl,) S: 1.89 (ZH, m, H-3, H-4), 2.09 (2H, m, H-3, H-4), 2.91 (2H, m, H-l, H-2), 3.87, 3.90 (6H, each s, OMe x 6), 6.20(2H, dd, J= 1X9,6.1 Hz, H-l’), 6.30 (2H, d, J= 15.9 Hz, H-2’), 6.80 (2H, d, 5=8.5 Hz, H-5”), 6.89 (2H, dd, J=8.5, 1.8 Hz, H-6”), 6.92 (2H, d, J= 1.8 Hz, H-2”). 13CNMR (CDCI,) 6: 25.3 (C-3, C-4), 45.8 (C-l, C-2), 55.9 (OMe x 2), 55.9 (OMe x 21, 108.8 (C-2“), 111.4

Table 3. 13C NMR spectral data ofcompounds2-4 [S CDCl, (100 MHz)] C 1 2 3 4 5 6 1’ 2 3 4 5 6’ PI 1 2” f‘, 1 111 2 111 3 4” 5” 6’” OMe

(ppm) in

2

3

4*

128.0 129.2 45.8 42.6 24.4 24.8 133.9 113.8 147.6p 148.2’ 110.5 122.0 132.4 128.5 131.2 108.9 148.3a 149.P 111.3 118.8 55.8 55.9 55.9

127.6 130.3 48.1 45.4 27.8 24.5 137.6 ill.9 147.51 148.4p 111.1 120.5 132.2 128.3 131.1 108.9 148.T 149.0” Ill.3 118.8 55.9

127.6 130.4 39.8 45.0 27.9 24.4 125.1 l43.2’L 97.9 147.8” 148.2’ 113.1 132.7 128.1 131.4 108.9 149.08 151.6= 111.3 118.8 55.8 55.9 56.2 56.6 56.8

*Assignments were based on “C-‘H COSY spectra. ‘Values are interchan~able within each column.

(C-5”), 119.1 (C-6”), 128.3 (C-2’), 130.8 (C-l”), 131.6 (C-l’), 148.5 (C-Q”), 149.1 (C-3”). (f)-cis-3-(3,4-dimethoxyp~e~y~-4-[(E)-3;4styryl]CycIohexene (2). Needles (EtOH), mp 99.0-99.5”, [a]k2 -+O”(CHCI,; c l.O), EI-MS m/z (rel. int.) 380 CM] + (27), 190 (lOO), 175 (28), 159 (91). ‘HNMR SW Table 1. ’ 3C NMR see Table 3. (~)-trans-3~3,4-dimer~o~yp~e~y~-4-[(E)-3,4-d~~t~oxys~yryl]CycZohexe~ (3). Powder (EtOH), mp 78’, [ol]&’ f 0” (CHCI,; c 1.O),EI-MS (rel. int.) 380 [M] + (47), 190 (lOO), 175 (45), 159 (92). ‘HNMR see Table 1. ’ 3C NMR see Table 3. (+)-trans-3-(~4,5-trimethoxy~~e~y~-[(E)-3,4-d~~~~oxystyryllcyclohexene (4). Powder (MeOH), mp 127”, [or];’ &O”(CHCI,; c 0.24), EI-MS m/z (rel. int.) 410 [M] + (20), 220 (IOO), 189 (24), HR-MS m/z 410.2115 (calcd for C&H&,; 410.2092). ‘HNMR see Table 1. 13CNMR see Table 3. NOESY and NOE difference spectra see Fig. 1. Synthesjs of ~~)-~s-l,2-b~s[(E)-3,4-d~met~o~ystyry~cyclobutane

(1) from

3,4-d~merhoxybenzuldehyde

(8).

(i) 3’,4’-DimethoxyphenyI-(l E)-butene-3-one (9) from compound 8. To a soln of 8 (2 g) in Me,CO (80 ml) was added 10% NaOH (60 ml). After stirring for 30 min at room temp., the mixt. was acidified to pH 6 with 1 N HCl, and extracted with CH,Cl, (250 ml x 2). The CH,Cl, layer was dried over Na,SO, and coned under red. pres. The residue was crystallized with EtOH to give 9 (1.28 g) as pale yellow plates. Mp 83.5-84.0”; ‘HNMR (60 MHz, CDCl,) 6: 2.36 (3H, s, -COMe), 3.92 (6H, s, OMe x 2), 6.60, 7.50 (lH, each d, J= 15 Hz, -CH=CH-), 6.75-7.30 (3H, m, Ar). (ii) 3’,4’-i)imethoxyphenyl-(lE)-butene-3-ol (10) from compuund 9. To a soln of 9 (1.28 g) in MeOH (25 ml), NaBH, was added at room temp. The mixt. was poured into H,O (35 ml), and extracted with CH,Cl, (30 ml x 5). The CH,Cl, layer was dried over Na2S0.,, and coned under red. pres. to give 10 as an oil. ‘HNMR (60 MHz, CDCl,) S: 1.38 (3H, d, J=6 Hz, Me), 3.86 (6H, s, OMe x 2), 4.48 (lH, m, >CHOH), 6.10-7.22 (SH, Ar and -CH=CH-). (iii) (3,4-dimethoxyphenyl)-(lE,3E)-ButudieBe (11)from compound 10. To a soln of 10(1.05 g) in dioxane (210 ml), 50% H,SO, (1 ml) was added at room temp. After stirring for 25 min at 40”, the mixture was poured into satd NaHCO, soln, and extracted with CH,Cl, (250 ml x4). The CH,CI, layer was dried over Na,SO,, and coned under red. pres. The residue was purified by silica gel flash CC (EtOAc-n-hexane, 1:9) to give 11 as an oil. The oil was crystallized with n-hexane at 0” to give needles. (0.23 g), mp 37.0-37.5”, EI-MS m/z 190 [Mf’. ‘H NMR (400MHz, CDCI,) S: 3.89(3H, s, OMe), 3.91 (3H,s,OMe),5.~3{lH,d,~=l6.5Hz,H-4),5.3O(lH,d,~ ==9.2Hz, H-4), 6.44-6.54(2H, H-l, H-3), 6.68 (LH, d, J =15.3, ll.OHz, H-2), 6.82 (lH, d, J=7.9 Hz, H-S),

6.93-6.96 (2H, H-2’, H-6’). (iv) Photochemical reaction of compound 11to compound 1. Crystals of 11(14 mg) sealed in a Pyrex tube under Nz were irradiated with sunlight at 0” for 11 hr. The reaction mixt. was purified by silica gel prep. TLC (EtOAc-n-

Phenylbutenoids from Zingiber cassumunar hexane, 1: 4) to give 1 (3 mg, 21%) and recovered 11 (5 mg, 36%). Catalytic hydrogenation of compounds 2-4. A soln of 2 (1 mg) in MeOH (1 ml) was stirred over PtO, (ca 0.5 mg) under H, for 10 min at room temp. The mixt. was filtered with CH,Cl, and coned under red. pres. to give 5. EI-MS mfz 384 [M] +. ‘H NMR see Table 2. Similar treatment of 3 (1 mg) provided 6. EI-MS m/z 384 [Ml’. ‘H NMR see Table 2. Similar treatment of 4 (1.5 mg) provided 7. EIMS m/z 414 [M] +. ‘HNMR see Table 2. Pyrolysis ofI. A soln of l(9.5 mg) in o-dichlorobenxene (2.8 ml) was heated at 120” for 20 min. After removing the solvent in uacuo, the mixt. was purified by silica gel TLC (CH,Cl,) to give 3,4-bis(3,4_dimethoxyphenyl)-1,5octadiene (12, 1.5 mg, 15% yield), 2 (1.9 mg 20% yield), 3 (2.3 mg, 24% yield), and recovered 1 (3.9 mg, 41% yield). Compound 12:needles (EtOH), mp 141.5-142”. ‘H NMR (CDCl,) 6: 2.30 (2H, m, H-7 and H-8), 2.89 (2H, m, H-7 and H-8), 3.66 (6H, s, OMe), 3.83 (6H, s, OMe), 4.33 (2H, br d, J=6.6 Hz, H-3 and H-4), 5.58 (2H, br dd, J= 11.6 and 6.6 Hz, H-2 and H-5), 5.73 (2H, br dd, J = 11.6 and 6.6 Hz, H-l and H-6), 6.30 (2H, d, J= 1.8 Hz, H-2’), 6.42 (2H, dd, J=8.5 and 1.8 Hz, H-6’), 6.68 (2H, d, 3=8.5 Hz, H-5’), SI-MS m/z 381 CM+ 13’.

Acknowledgements-We thank Dr I. G. P. Tengah (Udayanana University, Bali, Indonesia) and Dr S. Yomenori (University of Ryukyus, Okinawa, Japan) for the plant identification. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.

363 REFERENCES

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