Lignans and Lactone From Phyllanthus Anisolobus

7/29/2019 Lignans and Lactone From Phyllanthus Anisolobus

1/3

Phytochemstry, Vol. 33, No. 1, pp. 189-191, 1993 003 -9422/93 $6.00 + 0.00Printed n Great Britain. Q 1993 PergamonPress Ltd

LIGNANS AND LACTONES FROM PH YLLANTHUS ANISOLOBUSTORBEN L. BACHMANN, FELIPE GHIA* and KURT B. G. TORSSELL~

Department of Organic Chemistry, University of Aarhus, 8000 Aarhus C, Denmark; *Department of Chemical Engineering, Instituteof Technology, Quito, Ecuador(Received 14 September1992)

Key Word Index-Phyllanthus anisolobus; Euphorbiaceae; menisdaurilide; aquilegiolide; phyllanthos-tatin A; justicidin B; exiton coupling.

Abstract-The ichtyotoxic lignan justicidin B, ca 0.6% of the dry weight, and the lignan-glucoside phyllanthostatin Awere isolated from the chloroform extract of Phyllanthus anisoZobus.Two y-lactones, menisdaurilide and aquilegiolide,were also isolated and their absolute stereostructures determined by the CD allylic benzoate method.

Phyllanthus anisolobus is used by the Colorados, Quai-quer and Cayapas of western Ecuador as a fish poisonCl]. Recent reviews describe the pharmacological use ofPhyllunthus species C2-41. While thorough phytochemi-cal investigations have been undertaken on several otherPhyllanthus species, no reports have been publishedon P. anisolobus.

RESULTS AND DISCUSSIONThe leaves of P. anisolobus were extracted with hexane,chloroform and methanol. The hexane extract showedthe presence of long chain hydrocarbons, fatty acids,glycerides, squalene, polyprenols, /?-sitosterol, stigmas-terol and chlorophylls; there were no more unusualcompounds as judged from chromatographic analysisand preliminary H NMR studies of the various fractions.The chloroform extract was chromatographed on a

silica gel column using a CHCl,-EtOAc-MeOH gra-dient. The known ichtyotoxic lignan justicidin B (l), pre-viously isolated from Justicia species [S] was isolatedfrom the more apolar fractions of the chloroform extract.Characterization was carried out using NMR techniquesand all data were in agreement with earlier reports [6].Justicidin B was also found in considerable amounts inthe methanol extract. The combined yield constituted0.6% of the dry weight. Phyllanthus anisolobus wouldappear to be the richest known source of this hgnan.Two butenolides were isolated as a mixture from themedium polar fractions of the chloroform extract. It waspossible to separate the two compounds using HPLC andtheir structures were determined by mass, IR and NMRspectroscopy. The data were in good agreement withthose reported from two known compounds menisdauril-

tAuthor to whom correspondence should he addressed.

ide (2) and the stereoisomer aquilegiolide (3). There ishowever a discrepancy between our value of the opticalrotation of 3, [a&,+ 108.5, and the value reported, [x]n-419.6 for aquilegiolide [7,8]. The absolute configura-tions of aquilegiolide and menisdaurilide are unknown.The CD allylic benzoate method [9] has been de-veloped into a technique for determining the absolute

configuration of cyclic allylic alcohols on the basis ofchiral exiton coupling between benzoate and alkenegroups. Theory predicts that a clockwise relationshipbetween the two functions gives a positive Cotton effect,whereas a negative Cotton effect correlates with a counterclockwise relationship, 5 and 6. The benzoates 7 and 8were prepared and their CD spectra recorded. Menis-daurilide benzoate, having a negative first CD wave closeto 250 nm, be = - 7.5, thus has the absolute stereostruc-ture 7; structure 8 was derived for aquilegiolide benzoateby analogy. Menisdaurilide (2) was previously isolatedfrom Menispermum dauricum [7], Coccolus lauriforius[lo] (Menispermaceae) and Aquilegia strata [S] (Ran-unculaceae).A lignan-glucoside was isolated from the highly polarpart of the chloroform extract. On acid hydrolysis it gaveacetic acid, glucose and justicidin B and NMR spectralanalysis showed that it was identical to phyllanthostatinA (4), previously isolated from Phyllanthus aeuminatusClll.The three crude extracts were tested for pharmacolo-gical activity against fungi, bacteria and insects but nosignificant activity was found. We tested the ichtyotoxiceffect of justicidin B on guppies. A concentration of 1 ppmwas lethal; at 0.1 ppm the swimming ability was stronglyaffected but the dose was not lethal.Alkaloid tests on the crude chloroform and methanolextracts showed a weak to negative reaction with theMayer, Hager, Sonnenschein, Wagner and Dragendorffreagents.

189


2/3

190 T. L. BACHMANNet al.Me0

Me01;; 0Tc 0O-J1

0

O IHII,dHo; 35 Positive chirality 6 Negative chirality

EXPERIMENTALGeneral. H and 3C NMR were recorded at 200 and

50 MHz, respectively. Mps are uncorr.Plant material. Pkyllanthus anisolobus Muell. Arg. wascollected in March 1988 near the village of Pedro Vicentede Maldonado (Province of Pichincha) Ecuador. Vou-cher specimens are deposited at the Escuela PolitecnicaNational, Quito and at the Department of OrganicChemistry, Aarhus University.Isolation. Dried and ground leaves and twigs (1.5 kg)were extracted with hexane in a Soxhlet apparatus andthen submitted to a cold extraction with CHCl, andMeOH. The CHCl, extraction yielded 58 g of extract.The CHCl, extract (13.3g) was submitted to flash CC onsilica gel. Elution with CH,Cl, containing increasing

amounts of EtOAc and finally MeOH yielded 70 frs(100 ml each). Frs 2&24 (2.9 g) contained justicidin B co-

0

13

0 8

occurring with chlorophyll. Repeated recrystallizationfrom EtOAc and MeOH yielded 2 g of pure justicidin B,mp 246248 (lit. 247) [S]. Frs 41-44 (425 mg) containedcompounds 2 and 3. Repeated purification by prep. TLC(CH,Cl,-EtOAc, 1: 1) gave a product which according toHNMR consisted mainly of a mixt. of the two com-pounds (60 mg). Final sepn was accomplished by HPLC(RP-18, MeCN-H,O, 1:24) yielding compound 3(30 mg), mp 71, and compound 2 (23 mg), mp 86. Frs61-62 (1.1 g) contained the lignan-glycoside phyllanthos-tatin A (4). Repeated purification by prep. TLC (MeOH-CH,Cl,, 1:9; MeOH-CH,Cl,, 1: 11 and MeOH-EtOAc,1: 19) yielded somewhat impure 4 (136 mg) which wasfurther purified by HPLC (RP-18 H,O-MeOH, 1:2.3)yielding 90 mg of pure 4 as an amorphous solid.

Menisdaurilide (2). Crystals, mp 86 (lit. 113) [7]. [aIn-30.6 (MeOH: ~0.041) (lit. [a&, -31.4(MeOH; c 1.00))


3/3

Constituents of Phyllanthus anisolobus 191[7]. uv AM, nm: 253. IR #A\: cm-: 3400, 1740, 1641. (1H,d,J=10.0Hz),5.90(1H,m),5.89(1H,brs),5.00(1H,HNMR (200 MHz, CDCl,) 6: 6.52 (lH, dd, J= 10.0, ddd, 5=13.7, 5.4, 1.3 Hz), 3.04 (lH, ddd, 5=11.0, 5.5,2.5 Hz), 6.31 (lH, d, J= 10.0 Hz), 5.75 (lH, br s), 4.85 (lH, 5.5 Hz), 1.91 (lH,ddd,5=13.5, 11.0, ll.OHz).Compoundddd, J= 13.3, 5.0, 1.8 Hz), 4.58 (lH, ddd, J= 10.7, 5.4, 2.6 8. Oil. HNMR (CDCl,) 6: 8.20 (2H, d, 5=7.5 Hz), 7.59Hz), 3.43 (lH, br s), 2.86 (lH, ddd, 5=10.7, 5.4, 5.0 Hz), (lH, t, 5=7.5 Hz), 6.43 (2H, t, 5=7.5 Hz), 6.75 (lH, d, J1.61 (lH, ddd, 5=13.1, 10.7, 10.7 Hz). 13CNMR = lO.OHz), 6.44(1H, dd,J= 10.0,5.5 Hz), 5.81 (lH, ddd, J(50 MHz, CDCI,) 6: 174.6, 164.2, 144.8, 120.2, 111.6, 78.7, =4.5,4.5,2.0Hz),5.32(1H,ddd,J=13.5,5.5,2.0Hz),2.7866.9, 40.1. (lH, ddd, 5=13.4, 5.6, 1.0 Hz), 2.00 (lH, m).Aquilegiolide (3). White crystals, mp 71 (lit. 9698)[S]. [@ID+ 108.5 (MeOH; c 0.031) (lit. [a&, -419.6(MeOH; c 1.00)) [8]. UV c.$ nm: 253. IR YE; cm-:3400, 1740, 1642. HNMR (200 MHz, CDCl,) 6: 6.58(lH,d,J=9.7 Hz), 6.30(1H, dd,J=9.7,5.0 Hz), 5.78 (lH,brs), 5.28(1H,ddd,J=12.6,5.1, 1.9 Hz),4.62(1H,m),2.65(lH, br s), 2.60 (lH, ddd, 5=12.6, 5.1, 1.1 Hz), 1.76 (lH,ddd, J= 12.6,12.6,4.1 Hz). 13C NMR (50 MHz, CDCI,) 6:174.4, 163.6, 138.2, 122.4, 112.8, 76.8, 64.6, 37.4.

Phyllanthostatin A (4). Amorphous solid. HNMR(2OOMHz,CDCl3)6:7.51(1H,s),7.03(1H,s),6.77(1H,s),6.70-6.86 (3H, m), 5.80-5.93 (2H, m), 5.48 (lH, d, J=7.5 Hz), 5.00-5.31 (2H, m), 3.95 (3H, s), 3.65 (3H, s),3.2e3.70 (6H, m), 1.95 (3H, s). CNMR (50 MHz,CDCl,) 6: 172.8/172.7 (C-12)*, 168.2 (C-14), 151.2 (C-6),150.9 (C-7), 148.0 (C-4), 147.9/148.1 (C-3)*, 137.7/137.9(C-l)*, 131.5/131.6 (C-l)*, 130.2 (C-lo), 129.0/128.8 (C-2)*, 128.1/128.0 (C-3)*, 128.0 (C-9), 127.4 (C-4),124.2/124.4 (C-6)*, 111.0/l 11.2 (C-2)*, 108.7/108.8 (C-5)*, 106.8 (C-5), 105.8/105.8 (C-8)*, 101.8 (C-15), 65.7 (C-11), 56.2 (C-17), 56.0 (C-16), 21.2 (C-13). Glucose moiety:94.8 (C-l), 76.8 (C-5)t. 76.7 (C-3), 72.7 (C-2), 69.6 (C-4), 61.6 (C-6). *Signals doubled due to hindered rota-tion around Cl-Cl axis. tSigna1 covered by CDCl,signal and identified by 2D HETCOR.

Acknowledgements-We are greatly indebted to Dr 0.Proaiio, Technical University of Quito, Ecuador (EPN)for putting laboratory and transportation facilities at ourdisposal. We thank Dr Niels Jacobsen, Cheminova,Lemvig for carrying out the bactericidal, fungicidal,insecticidal and ichtyotoxic tests and Dr H. Balslev foridentifying our plant material. We also thank Dr S. E.Harnung, University of Copenhagen, for recording theCD spectra with a circular dichroism spectrometer builtthrough a grant from the Danish Natural Science Re-search Council. Financial support was provided by Sta-tens Naturvidenskabelige Forskningsrid and ArhusUniversitets Forskningsfond.

REFERENCES

1. Kvist, L. P. and Helm-Nielsen, L. B. (1986) OperaBotanica 92, 83.

Hydrolysis of phyllanthostatin A. Phyllanthostatin A(14 mg) was heated at reflux in 5% aq. H,SO, (0.5 ml) for3 hr. After cooling, the pH was adjusted to 3.5 withKHCO, and CH,Cl, (2 ml) was added. The phases weresepd and the organic phase was dried over Na,SO, andevapd in NKIJO.The H NMR spectrum of the residue wasidentical to that of justicidin B. D-Glucose was identifiedin the aq. phase by TLC as silica gel.

2. Unander, D. W., Webster, G. L. and Blumberg, B. S.(1990) J. Ethnopharmacol. 30, 233.3. Unander, D. W., Webster, G. L. and Blumberg, B. S.(1991) J. Ethnopharmacol. 34,97.4. Unander, D. W., Webster, G. L. and Blumberg, B. S.

(1992) J. Ethnopharmacol. 36, 103.5. Munakata, K., Marumo, S., Ohta, K. and Chen, Y. L.(1967) Tetrahedron Letters 3821.6. Okigawa, M., Maeda, T. and Kawano, N. (1970)

Tetrahedron X,4301.7. Takahashi, K., Matsuzawa, S. and Takani, M. (1978)

Chem. Pharm. Bull. 26, 1677.8. Guerriero, A. and Pietra, F. (1984) Phytochemistry 23,

2394.Benzoylation t$2 and 3. Compounds 2 and 3 (10 mg)were benzoylated by benzoyl chloride (10 mg), triethyla-mine (10 mg) in CHCl, (1 ml) with stirring at 25 for 2 hr.The soln was washed with HZ0 (1 ml), dried over MgS04,evapd in uucuo and the residue was purified by TLC (silicagel, CHCl,). Compound 7. Mp 105-107. HNMR(CDCI,) 6: 8.02(2H,d,J=7.5 Hz), 7.58(1H, &J-7.5 Hz),7.43 (2H, t, 5=7.5 Hz), 6.69 (lH, dd, J= 10.0,2.0 Hz), 6.31

9. Harada, H. and Nakanishi, K. (1983) CircularDichroic Spectroscopy. Exiton Coupling in OrganicStereochemistry. University Science Books. OxfordUniversity Press, Oxford, U.K.

10. Yogo, M., Ishiguro, S., Murata, H. and Furakawa, H.(1990) Chem. Pharm. Bull. 38,225.11. Pettit, G. R. and Schaufelberger, D. E. (1988) J. Nat.Prod. 51,1104.

Lignans and Lactone From Phyllanthus Anisolobus

Documents