310 zy zyxw zyxw zyx zyxwv zyxwv Journal of Natural Products Vol. 49, No. 2,pp. 310-312, Mar-Apr 1986 zyxwvu NAPHTHOQUINOIDS FROM LIPPIA SIDOZDES L.M.A. MACAMBIRA’, C.H.S. ANDRADE,F.J . A . MATOS, A.A. CRAVEIRO, Laboratdrio de Produtos Naturais Associado ao CNPq, Departamto de Quimica Orgrinica e lnwgrinira, Centro de Ciinrias da UFC, Caixa Postal, 301 0, Fwtaleza, Ceura, Brazil and R. BRAZ FILHO Universiahk F e h a l Rural ah Rio deJaneiro, Institute de Ciinrias Exatas, Departamento de Quimica, 23460, Sercipedica, Rio deJaneiro, Brazil Lippia sidoides Cham. (Verbenaceae) is an aromatic shrub, widespread in occurrence in the deciduous vegetation “caatinga” of Northeastern Brazil. Our specimen was collected near Mossoro in the state of Rio Grande do Norte. Observations of the use of an aqueous extract of leaves by rural populations verified the presence of healing and antiseptic effects. Some chemical and pharmacological studies of this plant were made previously, including chemical analysis of the essential oils of the leaves, which contain thymol and carvacrol as major constituents ( 2 ) . The essential oil shows bactericidal, bacteriostatic, fungicidal, and fungistatic activities against Staphylococcus aureus and other microorganisms (3). The residual water resulting from the steam distillation process presented several pharmacological effects on isolated organs and promoted arterial hypotension on rats (4). Interesting naphthoquinoids have been reported in plants of the genus Lippia (5,6),but no mention is made of their occurrence in L. sidoides. These observations and demonstration of several biological activities in preparations of this plant stimulated us to reexamine the chemical constituents of the species, especially the nature of the nonvolatile components. The following substances were identified in the methanolic extract of leaves and branches: palmitic acid, stearic acid, behenic acid, arachidic acid, lignoceric ‘This work is part of the M.S. thesis submitted by L.M.A. Macarnbira to Universidade Federal do Ceara. acid, thymol, carvacrol, p-sitosterol, lapachenol, isocatalponol, and a new substance, 6-oxo-3,4,4a,5-tetrahydro3-hydroxy-2,2-dimethylnaphtho-1,2pirane (1). All substances but the last were reported previously in plants of the genus Lippia but not in this species. 1 zy The ir spectrum of the new substance (1) presented an absorption at 3530 cm-’ assignable to a nonassociated OH group as well as absorptions for an aromatic ketone (1670 cm-’), an aromatic double bond (1580 cm- ’), for four adjacent hydrogens in an aromatic ring (775 cm-’), and an indication of a cyclic ether (1280 crn-’) (7). Absorption at 256 nm in the uv spectrum confirmed the presence of a carbonyl group conjugated with the aromatic ring (8). The presence of this carbonyl group is also supported by an absorption at S196.7 in the 13C-nmr spectrum (9).The ms of 1 presented a significant peak at 246 daltons assignable to a molecular ion of C15H1803as a molecular formula. The ‘H-nrnr spectrum of 1 taken at 60 MHz in CDCI, contained the following signals: (a) a multiplet from 7.3 to 8.0 8, corresponding to four aromatic protons; (b) a one hydrogen doublet at 4.56 (J=9.0Hz) in agreement with absorp- zyxw zyxwvuts zyxw zyxwv zyxwvuts zyxwvu Mar-Apr 19861 Macambira et a / .: Naphthoquinoids tion due to a proton on a benzylic carbon bonded to oxygen; (c) a one hydrogen quartet centered at 3.6 SY=5.0 and 12.0 Hz) attributed to a carbinolic proton; (d) a broad absorption at 2.5 6 corresponding to one hydrogen assignable to an axial methynic proton; (e) a two hydrogen multiplet from 2.4 to 2.7 6 corresponding to the methylene protons of carbon five; (0 a multiplet from 1.9 to 2 . 2 6 attributed to two aliphatic protons of carbon four; (g) two 3-hydrogen singlets at 1.3 and 1.4 corresponding to the two gem methyl groups. The PND 13C-nmrspectrum showed the presence of 15 spectral lines in agreement with the proposed molecular formula. In addition to the carbonyl (196.7 6), six aromatic carbons are clearly present (131.0, 144.6, 125.0, 127.4, 126.8, and 134.0 6). Three carbons are bonded tooxygen(76.5,73.4, and70.8 6), one carbon is in the vicinity of a carbonyl (43.1 6), and four aliphatic carbons (16.2, 28.1, 39.8, and 35.0 6) are observed. Specific assignments are given in the Experimental section. The placement of the hydroxyl group at carbon 3 instead of carbon 4 was possible from analysis of 13C-nmrspectrum where the difference in absorptions of the gem methyl groups (28.1 and 16.2 6) can be attributed to the y-effect of the hydroxyl in the vicinity. Furthermore, it is reasonable to propose isocatalponol (2) as the biosynthetic precursor which, by cyclization, produces 1. In fact, treatment of isocaltalponol (2) with mchloroperbenzoic acid in CHCI, followed by reaction with BF,. Et,O gave 1, identical, by tlc in three systems of solvent and 'H-nmr spectrum, with the natural product. The same argument 311 also favors positioning the hydroxyl at position 3. Acetylation of 1 gave a monoacetyl derivative, confirming the presence of a secondary aliphatic hydroxyl. The proposed structure (1)for this naphthoquinoid has no parallel in the literature. as far as we know. EXPERIMENTAL GENERALEXPERIMENTAL PROCEDURES.Mps were determined on a Mettler FP-5/52 and are uncorrected. Spectral data were obtained on the following instruments: ir, Perkin-Elmer Model 283-B; 'H nmr, Varian EM-360; I3C nmr; Varian XL-100; ms, Hewlett, Packard H P 5995A; gc/ms, HP-5933 equipped with datasystem; uv, Varian Model 634-S. Adsorbents for cc and tlc, silicagel 60, 230-400 mesh and silica gel 60 tlc plates 250 mesh were purchased from Merck and Co. zyxwv PLANT MATERIAL.-haves and branches of L. sidoiah were collected around Mossoro, Rio Grande do Norte, in the semiarid area of the Brazilian Northeast in June 1981. Voucher specimens are deposited in the "Herbario Prisco Bezerra" of the Universidade Federal do Ceara, Fortaleza-Cad, Brazil. EXTRACTIONAND PURIFICATION.-Dried parts (23 g) were extracted with MeOH in a Sohxlet apparatus, giving 760 g of a resinous extract. This material (250 g) was adsorbed on silica gel (500 g) and eluted with CHCI,, Me,CO, and MeOH successively to give fractions of 41.9 g, 107.6 g , and 80.6 g. These fractions were submitted to fractionation by repetitive cc to give a fixed oil, an aromatic volatile oil, four known solid substances, and the new naphthoquinone zyxw zyxw 2 (1). The fixed oil was treated with BF,-MeOH complex, and the product was extracted with hexane. Gdms analysis of the hexane extractives gave five peaks which were compared with ms spectra and retention times of standard methyl esters of fatty acids and identified as the methyl esters ofpalmitic, stearic, arachidic, behenic, and lignoceric acids. The volatile oil was obtained by further cc of the Me,CO eluate and is showed by gc/ms analysis two major components that were identified as thymol and carvacrol by comparison of their ms and Kovat's indices with standards. 6,7-Dimethoxy-5,4'dihydroxyflavone was isolated by silica gel cc of the Me,CO eluate and recrystallized from CHCI3-Me,CO to give an amorphous yellow solid, 50 mg, with mp 285288" (10.11). p-Sitosterol was isolated (970 mg) from the CHCI, eluate by further cc and purified zyxwvutsrq zyz 3 12 Journal of Natural Products P o l . 49, No. 2 lowed by workup produced 1(0.0235 g) identical by repetitive precipitations with MeOH until with natural naphthoquinoid (1). ‘H nmr 7.9pure, mp 136-139”. Lapachenol was isolated 7.1 (m, 4, ArH), 4.5 (d, 1.10 Hz, H-la), 3.6 (100.0 mg) from the CHCI, eluate by repetitive (dd,]=5.0 Hz and]=12.0 Hz, 1, H-3), 2.3cc. Recrystallization of the main fraction gave a 2.7 (m, 3, H-4 and H-5), 1.7-2.2 (m, 2, H - 4 , yellow solid, mp 57-60”. Its ir, ’H nrnr, and 1.45 (s, 3, CH,), 1.35 (s, 3, CH3). mass spectra were identical to those obtained from an authentic sample (5). Isocatalponol was ACKNOWLEDGMENTS isolated by cc of the CHCI, eluate followed by deThis work was supported by grants from the colorizing with charcoal and a new cc to give neeBrazilian institutions CNPq and FINEP. We dles (150.0 mg), mp 78-79’, Its mp, ir, ‘H nmr, would like to thank Prisco Bezetra and Afdnio G. and mass spectra were in agreement with those of Fernandes from the Herbarium of Universidade isocatalponol(6,12). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Federal do Cead for collection and botanical iden6-OXO-3,4,4A-5-TETRAHYDRO-3-HYDROXY- tification of the plants. 2,2-DIMETHYLNAPHTHO C1,21 P Y R A N (I).LITERATURE CITED Obtained by cc of the Me,CO eluate. Recrystal1. A.A. Craveiro, J.W. Alencar, F.J.A. lized from C6H6, mp 148-149’, M+ 246. Matos, C.H. S. Andrade, and M. I.L. C,,H1,O3; ir Y max (IU3r) 3530, 3050, 1670, Machado,]. Nat. Prod., 44, 679 (1980). 1580, 1450, 1280, 1250, 1110, 1070, 1050, 2. A.A. Craveiro, A.G. Fernandes, C.H.S. 1020, 775, 640 cm-’; ms m/z (96)246 (M+, l), Andrade, F.J.A. Matos, J.W. Alencar, 228 (l), 188 (lo), 157 (3), 145 (26), 144 (loo), and M.I.L. Machado, “Oleos Essenciais de 129 (4), 116 (23), 115 (28), 105 (2,2), and 91 Plantas do Nordeste,” ed. UFC, 1980, p. (3); ‘H nmr (60 MHz, CDCI,) 6 8.02-7.30 (m, 110. 4,ArH),4.5(d,]=9.0Hz, l,H-la),3.63(dd, 3. M.L.B.A. Aguiar, F.J.A. Matos, and ]=5.0; 12.0 Hz, 1, H-3), 2.50 (m, 1, H-4a), V.L.A. Moura, CiEncia e Cultura, 36 (7), 2.40-2.70 (m, 2, H-5), 1.99-2.20 (m, 2, H-4), Suplemento, 547 (1984). 1.41 (s, 3, CH,), 1.30(s, 3, CH,); 13Cnmr(25.2 4. F.F. Matos, “Efeitosfarmacologicos de LipMHz, CDCI, 6 196.7 (s, C-6), 144.6(s, C-loa), pia sihides Cham.” Disset-racio de Mes134.0 (d, C-9), 131.0 (s, C-7a), 127.4 (d, C-7), trado, Universidade Federal do Ceara, For126.8 (d, C-8), 125.0 (d, C- lo), 76.5 (s, C-2), taleza-ce., 1980. 73.4 (d, C-la), 70.8 (d, C-3), 43.1 (t, C-5), 39.8 5. A.R. Burnett and R.H. Thomson, /. (d, C-4a), 35.0 (t, C 4 ) , 28.1 (q, CH, eq.), and Chem. Soc. (C), 2100 (1967). 16.2 (9, CH, ax.);{CX]*’D +22.46. 6. K. Inowe, H . Inone, T. Taga, R. Fugita, 6-oX0-3,4,4A, 5-TETRAHYDRO-3-HYDROXYK. Osaki, and K. Kukiyama, Chem. 2,2-DIMETHYL-NAPHTHOC1,21PYRAN ACETATE. Phann. Bull., 28, 1224 (1980). -To pyridine (1.0 ml) and Ac20 (2.0 ml) was 7. R.M. Silverstein, G.C. Bassler, and T.C. added l ( 2 5 0 mg). The reaction mixture was kept Morril, “SpectrometryIdentification of Orfor 24 h at room temperature. Usual workup gave ganic Compounds,” 3rd ed.,John Wiley & an oil. ‘H nmr (60 MHz, CDCI,) 6 8.02-7.3 (m, Sons Inc., NewYork, 1974, pp. 73-117. 4, ArH), 4.6 (d, /= 10.0 Hz, H- la), 4.7 (dd, 8. E.M. Peixoto, R. Pinchin, and A.C. ]=5.0 and 12.0 Hz, 1, H-3), 2.5 (m, 3, H-4a Pinto, Citncia e Cultura, Suplemento, 125 and H-5), 2.2-1.8 (m, 2, H-4), 2.08 (s, 3, CH3(1978). C=O), 1.43 (s, 3, CH,), 1.36 (s, 3, CH,); 13C 9. F.W. Wehrli and T . Nishida, “Progress in nrnr (25.2 MHz, CDCI,) 6 195.9(s, C-6), 170.2 the Chemistry of Organic Natural Prod(5, CH,-C=O), 133.8 (d, C-9), 130.8 (s, C-7a), ucts.” Ed. by L. Zechmeister, Springer 127.4 (d, C-7), 126.9 (d, C-8), 124.7 (d, C-lo), Verlag, Vienna, Vol. 36, 1979, p. 125. 74.6 (s, C-2), 74.5 (d, C-la), 70.7 (d, C-3). 42.7 10. C.H. Brieskokn and W . Biechele, Tet(t, C-5), 39.0 (d, C-4a), 31.4 (t, C-4), 27.9 (9, rahedron Letterr, 31,2603 (1969). CH, ax), 2 1.1 (9, CH,-C=O), 17.6 (q, CH, eq.). 11. J . W . Wallace, Phytochemistry, 10, 452 (1971). SYNTHESISOF l.-Isocatalponol(O.304 g) in C.H. Brieskorn and R. Pohlmann, Arch. 12. CHCI, (5.0 ml) was treated with m-chloroperPharm., 309,829 (1976). benzoic acid (0.226 g) in CHCI,. Treatment of Rereived 22 May I985 the reaction mixture with BF3*Et20(traces) fol- zyxw zyxwvut zyxwvuts zyx zy zyx