Bisht, K S

Gupta, Suman

Jain, Rajni

Jain, S C

Malhotra, Sanjay

Olsen, C E

Parmar, V S

Singh, Rominder

NOPR

Indian Journal of ChemistryVol. 37B, August 1998, pp. 828 - 831Chemical constituents of Taxus canadensisC E Olsen", Rominder Singh", Suman Gupta, K S Bisht,Sanjay Malhotra, Rajni Jain, S C Jain & V S Parmar*Department of Chemistry, University of Delhi,Delhi 110007, India'Chemistry Department, Royal Veterinary and AgriculturalUniversity, DK-!87! Frederiksberg C, Copenhagen, Denmark'Research Laboratories, Merck & Co. Inc., West Point,Pennsy!vania 19486, USAReceived 6 May 1998; accepted 22 June 1998Phytochemical investigation of Taxus canadensisleads to the identification of four new esters, namely 3-(4-hydroxyphenyl)propyl pentacosanoate 1, 3-(4-hydr-oxyphenyl)propyl hexacosanoate 2, 3-(4-hydroxyphe-ny\)propy\ dotriacontanoate 3 and 3-(4-hydroxyphenyl)-propyl tetratriacontanoate 4 along with one known ester,3-(4-hydroxypheny\)propy\ tetracosanoate 5. Six addi-tiona\ compounds, sciadopitysin 6, ginkgetin 7, rhodo-dendrin 8, taxicatin 9, taxinine 10 and ~-sitosterol havealso been isolated and identified.The genus Taxus belongs to the family Taxaceaeand its various species are of significance as theyhave been found to possess a number of biologicalactivities such as cytotoxic, antileukemic, sedative,antiseptic, tranquilising and antimitotic. Earlierphytochemical work on Taxus canadensis hasresulted in the isolation of ten different taxanes'".In the present investigation of the petroleum etherand ethanol extracts of the leaves and twigs ofTaxus canadensis, four new fatty acid esters,namely 3-(4-hydroxyphenyl)propyl pentacosanoate1, 3-(4-hydroxyphenyl)propyl hexacosanoate 2, 3-(4-hydroxyphenyl)propyl dotriacontanoate 3 and 3-(4-hydroxyphenyl)propyl tetratriacontanoate 4have been isolated and characterized, in addition to3-(4-hydroxyphenyl)propyl tetracosanoate 5(earlier known only once, i.e., from Piper clarkii'),sciadopitysin 6, ginkgetin 7, rhododendrin 8,taxicatin 9, taxinine 10 and ~-sitosterol. Of thesecompounds only taxinine has been reported earlierfrom this plant.The dried leaves and twigs of Taxus canadensiswere exhaustively extracted with hot petroleumNote ..Iether and ethanol in succession. The concentrate ofthe petroleum ether extract was columnchromatographed over silica gel, and elution withpetroleum ether-chloroform gave ~-sitosterol.Further elution with petroleum ether-chloroformfurnished two waxy solids, named mixture 'A' andmixture 'B'. The IR and IH NMR spectra of thesesolids were quite similar. The IR spectra of bothsubstrances showed the presence of a phenolic OH(3300 cm') and an ester moiety (1720 and 1142cm-I). The IH NMR spectra exhibited signals foran aliphatic methyl (80.88), a -CH2CH2CH2-moiety (84.08, 2.61 and 1.91) attached to electron-withdrawing groups on both sides, a methylene ato a carbonyl group (82.30), another methylene ~-to a carbonyl group (81.62) and a broad singlet at8 1.26. The presence of a -CH2CH2CHZ- moietywas further confirmed by the expected cross peaksin the IH_IH COSY NMR spectra of 'A' and 'B'.The peaks in the aromatic region showed a patterntypical for a para-disubstituted benzene ring.The CI mass spectra of mixture 'A' exhibited, asonly significant ions, pseudo molecular ions[M+Ht corresponding to three major components5, 1 and 2 with M, values of 502, 516 and 530.Integration of ion traces mlz 503, 517 and 531 overthe evaporation profile indicated that the threecompounds were present in an approximate ratio of5:1:2. Trace amounts of homologs (M, 474, 488,544, 558, 586, 614) were also present. In the EImass spectra, intense signals common to allcompounds were present at mlz 107 ([HO-Ph-CH2tt and 134 ([HO-Ph-CH2CH=CH2t),the latter being formed by ~-cIeavage at the esterbond. In addition, peaks due to a- and ~-cIeavage(with McLafferty rearrangement) at the ester bondand charge retention by the carboxylic acid moietywere observed for all the three major components.For 5, these peaks were observed at mlz 351 and368, for 1at mlz 365 and 382 and for 2 at mlz 379and 396. The presence of 5 and 1was confirmedby GC/MS of the silylated mixture. The samplewas first derivatized (as TMS derivative) using bissilyltrifluoroacetamide and pyridine in acetonitrile.Total ion current (TIC) extraction of TMSJ3"Hon--.....:::::r• \ ,. 2 05· : ~~CH3o1 n=232 n=243 n=304 n=325 n=22 ~0Uf\I HOOH 0HO (OHO 0H~OH8oH10derivatived mixture-A suggested that scan numbers245 and 427 contain appreciable ion current. Themass spectrum of the ions extracted at around scannumbers 245 and 427 showed molecular ion peaksat mlz 574 and 588, respectively. In addition bothshowed an intense ion at mlz 206([TMC-O-Ph-CH2CH=CH2"t) due to ~-cleavageat the ester bond. The solid 'A' was thus identifiedas an inseparable mixture of 3-(4-hydroxyphenyl)propyl pentacosanoate 1, 3-(4-hy-droxyphenyl)propyl hexacosanoate 2 and 3-(4-hy-droxyphenyl)propyl tetracosanoate 5 in the ratio1:2:5.NOTES 829OROH 06 R=CH37 R=HOH~deH'OH~9 OCH3···""IOCOCH=CHPhThe IR, IH and l3C NMR spectra of mixture-Bwere very similar to those of mixture-A suggestingthe same basic structure for their components. TheCI mass spectra showed major [M+Ht peakscorresponding to compounds 3 and 4 with M,values of 614 and 642, respectively, in anapproximate ratio of 2: 1. The EI mass spectraconfirmed the molecular weights. Other homologs(M, 558, 586, 600, 628) were also present in traceamounts. The solid 'B' was thus identified as aninseparable mixture of 3-(4-hydroxyphenyl)propyldotriacontanoate 3 and 3-(4-hydroxyphenyl)propyltetratriacontanoate 4 in the ratio of 2: 1.830 INDIAN J CHEM, SEC.B, AUGUST 1998The ethanolic extract of Taxus canadensis oncolumn chromatography yielded five crystallinecompounds 6-10. Their physical constants andspectral data were in good agreement with thosereported for sciadopitysin 67,8, ginkgetin 77,8,. 9 . . 910 d .. 1011-14rhododendnn 8 , taxicatm an taxinme ,respecti vely,Experimental SectionGeneral. All melting points are uncorrected. IHand I3C NMR spectra were recorded on a BrukerAC 2S0P spectrometer at 2S0 and 62.9MHz,respectively (chemical shifts in 8, ppm relative toTMS). ElMS and ClMS were recorded on a JeolAXS05W mass spectrometer at 70 eV. GC-MSwas performed on a VG Trio I using a 30m DB-1capillary column at 2S0°C.Extraction and isolation. The leaves and twigsof Taxus canadensis were procurred from OdenseUniversity Campus, Denmark, in January 1992.The dried plant material (1 kg) was extractedexhaustively with petroleum ether and ethanol insuccession in a soxhlet apparatus. Both extractswere concentrated below SO°(;:- under reducedpressure.Column chromatography of the petroleum etherextract (2Sg) was carried out on a silica gelcolumn. The fractions eluted with petrol-chloroform (1:1, 1:4 and 3:7) mixtures yielded ~-sitosterol, an inseparable mixture of three esters 1,2 and 5 (mixture-A) and another mixture of twoesters 3 and 4 (mixture-B), respectively.Column chromatography of the ethanol extract(l50g) on elution with chloroform-methanol (9: 1)gave sciadopitysin 6, ginkgetin 7 and taxinine 10and the chloroform-methanol (7:3) eluates yieldedtaxicatin 9. Rhododendrin 8 was obtained as amajor component (4% by weight of the ethanolicextract) from the chloroform-methanol (3:2)eluates.Mixture-A (compounds 1, 2 and 5). Whitewaxy solid (l5mg); UV (MeOH): 226, 280nm; UV(MeOH+NaOMe): 226, 243 (sh), 281, 302 (sh)nm; IR (nujol): 3300, 1720, 1240, 1142 cm':ElMS (relative intensity): mlz 107 [HO-Ph-CH2t(24), 134 [HO-Ph-CH2CH=CH2t (l00), 351, 365,368, 379, 382, 396, S02, 516, S30, 544, 558, 586,614 (all less than 3% along evaporation profile);CIMS (relative intensity): mlz 475, 489, S03, 517,531, 545, 559, 587, 615 (intensities vary alongevaporation profile); IH NMR (CDCh): 80.88 (t,J=7Hz, 3H, -CH3), 1.26 (brs, C-4''H to C-23"H/C-24''H/C-25''H), 1.62 (brm, 2H,-OCOCH2CH2), 1.91 (m, 2H, -COOCH2CH2),2.30 (t, J=7.5 Hz, 2H, -OCOCH2-), 2.61 (t,J=7.6Hz, 2H, ArCH2-), 4.08 (t, J=7Hz, 2H,-COOCH2- 6.75 (d, J=10Hz, 2H, C-3'H and C-5 'H), 7.03 (d, J=10Hz, 2H, C-2'H and C-6'H); 13CNMR (CDCI3): 814.11 (C-24"/C-25 "/C-26"),22.68 (C-23 "/C-24"/C-25"), 25.03 (C-3"),29.18-29.69 (C-5" to C-22 "/C-23 "/C-24"), 30.46(C-4"), 31.25 (C-2), 31.92 (C-2"), 34.39 (C-3),63.54 (C-1), 115.25 (C-3' and C-5'), 129.38 (C-2'and C-6'), 133.34 (C-1 '), 153.84 (C-4'), 174.01(C-1 ").Mixture-B (compounds 3 and 4). White solid(20 mg), mp 80-84°C; UV (MeOH): 224, 276nm;UV (MeOH+NaOMe): 216,240 (sh), 280, 296 (sh)nm; lR (nujol): 3300, 2850, 1725, 1505, 1240,1160, 1000 cm'; ElMS (relative intensity): mlz107 [HO-Ph-CH2t (21), 134 [HO-Ph-CH2CH=CH2t (100), 463, 480, 491, 508, 586, 614, 628,642 (all less than 3% along evaporation profile);ClMS (relative intensity): mlz 531, 545. 559, 573,587, 601, 615, 629, 643, 657 (intensities varyalong evaporation profile); IH NMR (CDCI3):80.88 (t, J=7Hz, 3H, -CH3), 1.26 (brs, C-4"H toC-31 "HC-33 "H), 1.62 (brm, 2H, -OCOCH1-CH2-) , 1.91 (m, 2H, -COOCH1CH2-), 2.30 (t,J=7.5 Hz, 2H, -COCH2-), 2.61 (t, J=7.6Hz, 2H,ArCH2-), 4.08 (t, J=7Hz, 2H, -COOCH2-), 4.70(l H, brs, -OH), 6.75 (d, J=10Hz, 2H, C-3'H andC-5'H), 7.03 (d, J=10Hz, 2H, C-2'H and C-6'H);13CNMR (CDCI3): 814.11 (C-32"/C-34"), 22.68(C-31 "/ C-33 "), 25.03 (C-3 "), 29.18-29.70 (C-5"to C-30" /C-32 "). 30.46 (C-4 "). 31.2·5 (C-2),31.93 (C-2"), 34.39 (C-3), 63.54 (C-l), 115.23 (C-3' and C-5'), 129.47 (C-2' and C-6'), 133.40 (C-1'), 153.78 (C-4'), 174.03 (C-l ").Compound 6. Greenish yellow powder (25 mg),mp 297-300°C (lit.7 mp 315°C, lit.8 mp 290°C). Allthe spectral data were identical with those ofsciadopitysin 7,8.Compound 7. greenish yellow powder (20mg),mp >300°C (lit.7(,mp 320°C, lit.8 mp 327°C). Allthe spectral data were in good agreement with thevalues reported for ginkgetin 7,8.Compound 8. Shiny colourless cubes (6.5g),mp 188-190°C (lit.9 mp 189-191°C). All thespectral data tallied well with those reported forrhododendrin in literature".Compound 9. Cream coloured crystalline solid(200 mg), mp 173-175°C (lit.1Omp 170-71 0C). Thespectral data matched well with the data reportedfor taxicatin 10.Compound 10. Light green crystalline solid(60mg), mp 261-262°C [lit.11 mp 264-265°C];[a]D24+126.8° (c 0.78, MeOH), lit.12 [a]D18+137°(CHCh). All these spectral data are identicalwith those for taxinine 1011-14•Compound 11. Colourless needles (80mg), mp136°C. The mixed melting point with an authenticsample of ~-sitosterol remained underpressed. Co-TLC with an authentic sample of ~-sitosterol andsuperimposable IR spectra confirmed its identity.AcknowledgementThe authors thank the Council of Scientific andIndustrial Research (CSIR), New Delhi, and theDanish International Development Agency(DANIDA), Denmark for financial support. Theyare also grateful to Prof. Dr Per M Boll forencouragement and suggestions during the courseof this work.NOTES 831References1 Parmar V S, Jha A, Bisht K S, Taneja P, Singh S K,Kumar A, Poonam & Olsen C E, Phytochemistry, (inpress).2 Zamir L 0, Nedea M E, Belair S, Sauriol F, Marner 0,Jacqmain E, Jean F I & Garneau F X, Tetrahedron Left,33,1992,5173.3 Bourbeau G, Chem Abstr, 48, 1954, 12371.4 Zamir L 0, Nedea M E, Belair S, Sauriol F, Marner 0,Jacqmain E, Jean F I & Garneau F X, Tetrahedron Left,33, 1992, 6548.5 Gunawardana G P, Premachandran V, Burres N S,Whittern D N, Henry R, Spanton S & McAlpine J B, J NatProd,55, 1992,1686.6 Boll P M, Hald M, Parmar V S, Tyagi 0 D, Bisht K S,Sharma N K & Hansen S, Phytochemistry, 31, 1992, 1035.7 Khan M S Y, Kumar I, Prasad J S, Nagarajan G R,Parthasarathy M R & Krishnamurthy H G, Planta Med,30, 1976, 82,8 Reddy B P& Krupadanam G L D, Indian J Chem, 35B,1996,283.9 Parmar V S, Vardhan A, Taneja P, Sinha R, Patnaik G K,Tripathi S C, Boll P M & Larsen S, J Chem Soc, PerkinTrans I, 1991,2687.10 Ushiyama M & Furuya T, Phytochemistry, 28, 1989, 3009.11 Kurono M, Nakadaira Y, Onuma S, Sasaki K & NakanishiK, Tetrahedron Lett, 1963, 2153.12 Dictionary of organic compounds, s" Edn (Chapman &Hall, New York) 1982,5109.13 Appendino G, Gariboldi P, Pisetta A, Bombardelli E &Gebetta B, Phytochemistry, 31, 1992,4253.14 Kurono M, Maki Y, Nakanishi K, Ohashi M 0, Veda K,Uyeo S, Woods M C & Yamamoto Y, Tetrahedron Left,1965,1917.

Chemical constituents of Taxus Canadensis

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Chemical constituents of Taxus Canadensis

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