Majoranolide: A δ-lactone from Persea major

Majoranolide: A δ-lactone from Persea major

Short Reports 2698 2940,2850,1460,1380,730,720. ‘H NMR: sO.90(3H, d, 5=6 Hz, H,-3), 0.80 (6H, t, 5=6 Hz, 2 x terminal Me), 1.54 (lH, m, H-3), 1.25 (...

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2940,2850,1460,1380,730,720. ‘H NMR: sO.90(3H, d, 5=6 Hz, H,-3), 0.80 (6H, t, 5=6 Hz, 2 x terminal Me), 1.54 (lH, m, H-3), 1.25 (54H, brs, +H,),,). MS m/z (rel. int.): 436 [M]’ (C,,H,,)(6), 421 [M-Me]+ (2). 407 (1 I), 393 (3), 379 (8), 365 (9), 3.51 (lo), 99 (25), 85 (50), 71 (62), 57 (lOO), 43 (95). Lupenone (2). The residue (10 mg) from hexaneC,H, (3: 1) frs (71-82) afforded a solid mp 170”, [z]n+58” (CHCI,) identified by co-TLC, IR, MS, mmp. Tritriacontanol(3). Eluates (95-106) from hexane-C,H, (1: 1) fractions afforded a residue, 15 mg, mp 69-71” identified by IR, MS, co-TLC, mmp. Tetratriacontanol (4). Removal of solvent from the hexaneC,H, (1:l) fractions (112-127) afforded a residue which was chromatographed in C,H,-CHCl, (1: l), 10 mg, mp 72-73” identified by IR, MS, mmp, co-TLC. Sitosterol (5). Hexane-C,H, (1:3) frs (131-168) and C,H, fractions (169-185) provided a residue (65 mg), mp 134135” identified by IR. MS, co-TLC, mmp. Triacontanoic acid (6). The residue from the C&H, fractions (186-202) was subjected to prep. TLC in CHCl,-MeOH (99: 1) to provide a fast moving compound, 10 mg, mp 92-93” identified by co-TLC, IR, MS, NMR. Dotriacontanoic acid (7). The slow moving compound from the above prep. TLC gave a residue, 20 mg, mp 96” identified by coTLC, IR, MS.

Echinopsine (8). Fast moving compound from prep. TLC afforded a residue, 25 mg, mp 148-150’ identified by co-TLC, mmp, IR, MS. Echinopsidine (9). The material remaining at the base in the above prep. TLC on further purification yielded a viscous mass, 20 rn& identified as hydrochloride mp 214 , IR, MS, co-TLC.

Acknowledgement-We are grateful Lucknow for MS of the compounds.

REFERENCES

@X&9422/90 %3.00+0.00 Pergamon Press plc

A &LACTONE

W.-w. MA, J. E. ANDERSON, Department

of Medicinal

Chemistry

at C.D.R.I.,

1. (1893-1902) Botanical Surrey oflndia Vol. 1, p. 202. Government of India Printings, Calcutta. 2. Singh, R. P., Pandey, V. B. and Sepulveda. S. (1987) Chem. ind. (Lond.), 828. 3. Bentley, K. W. (1963) Elucidation ofstructure by Physical and Chemical Methods, Part I, p. 291. Interscience. New York. 4. Chaudhuri, P. K. (1987) Phytoshemistry 26, 587. 5. Shukla, Y. N. and Thakur, R. S. (I 984) Phytochemistry 23,799. 6. Mahmood, U., Shukla, Y. N. and Thakur, R. S. (1983) Phytochemistry 23, 167. 7. Mahmood, U., Shukla, Y. N. and Thakur. R. S. (1985) Phytochemistry 24, 1618.

Phytochemistry,Vol. 29, No. 8, pp. 2698-2699, 1990. Printed in Great Britain.

MAJORANOLIDE:

to R.S.I.C.

C-J.

CHANG,

FROM PERSEA

D. L. SMITH and

MAJOR

J. L. MCLAUGHL.IN*

and Pharmacognosy, School of Pharmacy and Pharmacal West Lafayette, IN 47907, U.S.A.

Sciences, Purdue

University.

(Received 10 January 1990)

Key Word Index--Persea shrimp

lethality;

human

major; Lauraceae; bark; &lactone; majoranolide; majorynolide; tumour cell cytotoxicity; inhibition of crown gall tumours.

majorenolide;

brine

Abstract-From the ethanol extract of the bark of Persea major, an additional new bioactive &lactone, majoranolide, has been isolated and characterized. The absolute stereochemistries of majoranolide and two previously isolated d-lactones, majorynolide and majorenolide, have been determined by Horeau’s method and CD spectra. Brine shrimp lethality was used to guide the fractionation, and the new lactone is moderately cytotoxic and inhibits crown gall tumours on potato discs.

INTRODUCTION

In a previous investigation of the bark of Persea major Mill (Lauraceae) as a source of bioactive compounds (antitumour, pesticidal), two Nactones, majorynolide (2) and majorenolide (3), were isolated while using brine shrimp lethality as a bioassay (1). We have now isolated

another new related S-lactone, majoranolide (1). The configuration at C-5 of majoranolide (1) was determined by Horeau’s method [2, 31 as S. That the absolute configurations at C-5 of majorynolide (2) and majorenolide (3) are the same was supported by a close resemblance among the circular dichroism (CD) curves of these three compounds.

Short Reports

H

1 2 3

W&e CcCH CH-CH,

Majoranolide (1) showed bioactivities in the brine shrimp lethality test (LC,, 63.06) (4), the inhibition of crown gall tumours on potato discs (84%) (5), and against human tumour cells: A-549 lung carcinoma @“se 4.98 pg ml-‘), MCF-7 breast carcinoma (ED,, 5.04 pg ml-‘), and HT-29 colon adenocarcinoma (ED,, 3.11 /Ig ml-‘). RESULTSANDDISCUSSION

Majoranolide (1) was isolated by further chromatographic separation of FO03 Cl], monitoring with brine shrimp lethalities [43. Compound 1 was a wax with mp 46”, the HR mass spectrum (CI) showed a peak [M + l] + at 311.2564 in agreement with the formula CigH,,O, (calcd 311.2586). The IR spectrum showed the carbonyl group at 1746cm-‘, an exocyclic double bond at 1680 cm-‘, and the hydroxyl group at 3358 cm-‘. ‘HNMR and 13CNMR spectra indicated the exocyclic a&unsaturated 4-OH-&lactone structure with proton and carbon peaks for the ring similar to those reported for 2 and 3 (1) (see Experimental). The geometry of the exocyclic double bond at H-7 of 1 is also cis to the carbonyl group as its chemical shift is at 66.75 which is same as in 2 and 3 [6]. The proton peak at 60.91(3H, t, .I =7.1 Hz), and the carbon signal at 614.33 indicated a saturated side chain. Considering the integration of protons in the ‘H NMR at 6 1.28 and the M, from the mass spectral data, 1 contains two more methylene groups on the side chain than 2 and 3. The proposed structure was further confirmed by COSY and APT spectra. Compound 1 reacted with racemic 2-phenylbutanoic anhydride to produce an ester and 2-phenylbutanoic acid. The optical rotation of the acid was levorotatory with an optical rotation yield of 19.9%; this application of Horeaus’ method [2,3] permitted the conclusion that the configuration at C-5 of 1 is in the S form. Compound 1 showed a positive Cotton-effect in the CD spectrum. By direct comparison of the CD spectra of 1 with those of 2 and 3 which also showed positive Cotton-effects, all of these three d-lactones are proposed having the same stereochemistry, i.e. S at C-5 EXPERIMENTAL

The CD spectra were measured in MeOH. For the instrumentation source of plant material, separation, and isolation see previous work [l]. Majoranolide (1). Wax (61 mg) was isolated chromatographically from FO03 using silica gel column and silica gel Chro-

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matotron with hexane, CH,Cl, and acetone gradients; mp 46”; C~ID-~” WQ; ~0.1). UVAt:P nm: 229 (logs=3.94). IR vkf; cm-‘: 3358, 2919, 2851, 1746, 1680, 1467, 1214 and 1057. CIMS (isobutane) m/z: 311; FABMS (DTT/DTE) m/z: 311; HRMS (CI): 311.2564 for C,,H,,O, (calcd 311.2586); EIMS (% rel. int.) m/z: 311 (1.43), 293 (1.33), 261 (2.04) 137 (15.36), 121 (15.13), 95 (59.64), 81 (76.87), 67 (100). ‘HNMR (5OOMHz, CDCI,): 66.75 (lH, tt, J=7.6, 2.9 Hz, H-7), 4.63 (lH, dddd, J =8.4, 6.3, 5.3, 3.1 H& H-5), 3.86 (lH, dd, J=12.2, 3.1 Hz, H-6), 3.63 (lH, dd, J=l2.2, 5.3 Hz, H-6’), 2.86 (IH, ddd, J=l6.9, 8.4, 2.9 Hz, H-4), 2.67.(1H, ddd, J= 16.9, 6.3, 2.9 Hz, H-4’) 2.15 (2H, tddd, J=7.6,6.7, 1.6, 1.2 Hz, H-8 and H-8’), 1.92 (br, -OH), 1.45 (2H, ttt, J=7.6,7.6, 7.6 Hz), 1.2-1.3 (20H, m, H-10 to H-19) 0.85 (3H, t, J= 7.1 Hz, H-20); APT- 13CNMR (125.7 MHz, C,D,): 6 170.24 (C-2), 139.71 (C-7), 127.01 (C-3), 76.95 (C-5), 64.10 (C-6), 32.30 (C-8), 30.18, 30.13, 30.10, 30.09, 29.95, 29.81, 29.79, 29.64, 28.34 (C-9 to C-18) 26.54 (C-4), 23.08 (C-19) 14.33 (C-20). Determination of configuration of 1 by Horeau’s method. A mixture of 1 (12 mg), 2-phenylbutanoic anhydride (24 mg), and pyridine (3 ml dried) was allowed to stand at room temp. for 16 hr. H,O (0.3 ml) was added and the mixture left to stand 0.5 hr to hydrolyse the excess anhydride. The acid was then titrated with 0.1 M NaOH soln in the presence of C,H, (3 ml) and a little powdered phenolphthalein; 0.4 ml of base was required for esterification, and hence the yield of ester&cation was 100%. (2-Phenylbutanoic anhydride prepared in our laboratory [3] contained traces of HOAc-Ac,O; the esterification yield was, therefore, based on the comparison of the basic solution needed for a blank control and that for the reaction mixture.) The mixture was then transferred to a separatory funnel, separated, and the pink aqueous basic phase washed with CH,Cl, to remove traces of ester and acidified. The 2-phenylbutanoic acid, thus formed, was extracted with C,H, (3 x 4 ml). The CsHs extract was dried over Na,SO, and the solvent removed by rotary vacuum evapn. The rotation of 2-phenylbutanoic acid (2 mg) was then measured in C,H, (1.2 ml) and gave [alo -4.8”; corresponding to an optical yield of 19.9%. This result indicated that the configuration at C-5 of 1 is thus, the S form. The A szl, of CD spectra were + 1.36, 2.11, and 1.78 for l-3 respectively, showing similar chiralities. Acknowledgement-This

work was supported by ROl grant no. 30909 from the National Institutes of Health, National Cancer Institute. We are grateful to Mr Richard G. Powell (USDA, Peoria), for his cooperation in securing the plant material. REFERENCES

1. Ma, W., Anderson, J. E., Chang, C., Smith, D. L. and McLaughlin, J. L. (1989) J. Nat. Prod. 52, 1263. 2. Horeau, A. and Kagan, H. B. (1964) Tetrahedron 20, 2431. 3. Fiaud, I. C., Horeau, A. and Kagan, H. B. (1977) Stereochemistry Fundamentals and Methods Vol. 3 (Kagan, H. B., ed.), pp. 51-94. Georg Thieme, Stuttgart. 4. Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. E. and McLaughlin, J. L. (1982) PInnta Med. 45, 31. 5. Ferrigni, N. R., Putnam, J. E., Anderson, B., Jacobson, L. B.,

Nichols, D. E., Moore, D. S., McLaughlin, J. L., Powell, R. G. and Smith, Jr. C. R. (1982) J. Nat. Prod. 45, 679. 6. Jacman, L. M. and Sternhell, S. (1969) Apglication of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry 2nd Edn, p. 222. Pergamon Press, Oxford.