A Tetracyclic diterpene and triterpenes from euphorbia segetalis

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Pergamon PII ] S9920Ð8311"86#90900ÐX

Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 068Ð072\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9920Ð8311:87:,Ðsee front matter

A TETRACYCLIC DITERPENE AND TRITERPENES FROM EUPHORBIA SEGETALIS MARIA!JOSEł U[ FERREIRA\ ANA MARGARIDA MADUREIRA and JOSEł R[ ASCENSO$ Faculdade de Farmacia\ Centro de Estudos e de Cie¼ncias Farmace¼uticas\ Universidade de Lisboa\ Av[ das Forcžas Armadas\ 0588 Lisbon Codex\ Portugal ^ $ Instituto Superior Tecnico\ Centro de Qu(mica Estrutural\ Av[ Rovisco Pais\ 0985 Lisbon Codex\ Portugal "Received in revised form 18 September 0886#

Key Word Index*Euphorbia se`etalis ^ Euphorbiaceae ^ tetracyclic diterpene ^ segetalol ^ penta! cyclic triterpenes ^ tetracyclic triterpenes[

Abstract*A new tetracyclic diterpene\ with a novel carbon skeleton\ has been isolated from the acetone extract of the whole plant of Euphorbia se`etalis[ Seven known compounds were also isolated ] the pentacyclic triterpenes friedeline\ lupenone\ and glutinol\ the tetracyclic triterpenes dammaradienol\ cycloartenol and 13! methylenecycloartanol and b!sitosterol[ The structure of the new compound and its derivatives have been extensively characterised by high!_eld NMR spectroscopic methods including 1D NMR techniques[ Þ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION

Euphorbia se`etalis L[ "Euphorbiaceae# is a herb com! monly encountered in Portugal[ It produces a large amount of latex which causes severe irritation of the skin and eyes during collection[ Euphorbia species have a}orded a large number of polyfunctional diter! penoids with the tigliane\ ingenane and daphnane skeletons ð0\ 1Ł[ Most of them are skin irritants and many of them are skin tumour promoters[ Non!irri! tant polyfunctional macrocyclic diterpenoids\ with the lathyrane and jatrophane skeletons\ have also been isolated from Euphorbia species[ Some of these com! pounds have shown antitumor activity ð2\ 3Ł[ They are considered biogenetic precursors of the irritants ð4Ł[ In continuation of our research on Euphorbia species ð5Ð03Ł we have isolated\ from the non!saponi_able part of the acetone extract of the whole plant\ a hith! erto unknown polyfunctional tetracyclic diterpene parent alcohol "0b#[ The known compounds 1Ð7 were also isolated[ The chemical constituents of the latex of Euphorbia se`etalis have been investigated and the presence of the compounds 5Ð7 have also been reported ð04Ł[ RESULTS AND DISCUSSION

Acetone extract of Euphorbia se`etalis L[ "whole plant# was saponi_ed and the non!saponi_able part

Author to whom correspondence should be addressed[ 068

was chromatographed on a silica gel column[ A fur! ther fractionation of the less polar fractions yielded the known pentacyclic triterpenes friedeline "1#\ lup! enone "2#\ and glutinol "3#\ and the tetracyclic tri! terpenes dammaradienol "4#\ cycloartenol "5# and 13! methylenecycloartanol "6# and b!sitosterol "7# ident! i_ed by their physical and spectroscopic data[ Acety! lation of one of the more polar fractions a}orded a tetraester derivative "0# of a polyfunctional diterpene alcohol with a novel tetracyclic diterpene skeleton\ named as segetalol "0b#\ which seems to be derived from rearrangement of the bicyclic jatrophane skel! eton since the cyclopropane ring\ present in the lathy! rane skeleton\ is absent[ The tetraester derivative of segetalol "0 ^ 0\4\03!tri! acetate!2!benzoate# was obtained as crystals "EtOAcÐ

079

M[!J[ U[ FERREIRA et al[ Table 0[ 0H NMR spectral data of compounds 0\ 0a and 0b "in CDCl2\ 299 MHz#

H

0

0a

0b

0a 1a 2a 3a 4b 6a 6b 7b 00a 00b 01b 03b 05b!CH2 06a!CH2 07a!CH2 08b!CH2 19a!CH2 04!OH$ OH OH Benzoyl moiety 2? 3? 4? 5? 6? Acetyl moieties R0 R2 R3

4[93 d "09[1# 1[77 m "W0:1 14[9# 4[79 dd "4[3\ 5[8# 1[39 dd "4[0\ 01[9# 4[57 d "01[9# 0[37 dd "5[8\ 03[0# 0[71 dd "09[4\ 03[0# 2[12 m "W0:1 18[4# 0[67 dd "3[4\ 03[9# 0[85 dd "09[1\ 03[9# 3[11 m "W0:1 16[9# 3[72 s 9[73 d "6[4# 0[97 s 0[95 s 0[04 s 9[59 s 1[79 s * *

3[42 m "W0:1 19[9# 1[54 m "W0:1 14[9# 4[70 dd "3[4\ 5[2# 1[37 dd "3[4\ 01[9# 4[46 d "00[6# 0[36 dd "5[8\ 03[0# 0[71 dd "09[4\ 03[0# 2[06 m "W0:1 18[4# 0[61 dd "3[4\ 03[9# 0[78 dd "09[1\ 03[9# 3[00 m "W0:1 16[9# 2[20 s 9[86 d "6[4# 0[09 s 0[96 s 0[02 s 9[68 s 2[36 s 1[17 brs 1[79 brs

7[91 brd "6[1# 6[35 brt "6[7# 6[47 brt "6[4# 6[35 brt "6[7# 7[91 brd "6[1#

6[83 brd "6[1# 6[28 brt "6[7# 6[59 brt "6[4# 6[28 brt "6[7# 6[83 brd "6[1#

1[03 s 0[83 s 1[09 s

* 1[90 s *

3[22 d "09[1# 1[24 m "W0:1 14[9# 3[10 dd "3[1\ 5[9# 0[76 dd "3[9\ 00[4# 3[96 d "00[6# 0[38 dd "5[5\ 02[7# 1[16 dd "09[4\ 03[9# 2[96 m "W0:1 18[4# 0[61 dd "3[4\ 03[9# 0[71 dd "09[1\ 03[9# 3[91 m "W0:1 16[9# 2[06 s 0[91 d "6[4# 0[97 s 0[90 s 0[00 s 9[63 s * * * * * * * * * * * * *

 In CDCl2:CD2OD\ 8 ] 0[ $ Hydroxyl protons were not observed for 0b ^ Exchangeable with the solvent[

n!hexane#[ Its molecular formula was assigned as C22H31O09 by LD!FTICR mass spectrometry with the ion at m:z 510[15534 ðM¦NaŁ¦[ The IR spectrum of compound 0 exhibited the characteristic absorptions of an hydroxyl group "2344 cm−0#\ carbonyl groups "0632 ester carbonyl ^ 0695 cm−0 ketone# and an aro! matic ring "0358\ 667 and 603 cm−0#[ The EI mass spectrum of 0\ with a molecular ion peak at m:z 487\ showed a base peak at m:z 245 ðMÐ1×HOAcÐ C5H4CO1HŁ¦ and fragment peaks at m:z 185 ðMÐ 2×HOAcÐC5H4CO1HŁ¦\ 094 ðC5H4COŁ¦ and 66 ðC5H4Ł¦ indicated the presence of the benzoyl moiety and the three acetoxyl groups which was supported by its 0H NMR and 02C NMR spectra "see Tables 0 and 1#[ Apart from the signals of the ester groups\ the 0 H NMR spectrum of 0 showed signals for a sec! ondary methyl group "d 9[73\ d\ J  6[4 Hz# and four tertiary methyl groups "d 9[59\ 0[04\ 0[95 and 0[97# as well as four protons geminal to ester functions "d 4[93\ d ^ 4[79\ dd ^ 4[57 d ^ 3[72\ s#[ A singlet at d 1[79\ which disappeared with D1O\ revealed an hydroxyl group which was not acetylated indicating its tertiary nature[ In addition to the signals of the ester groups\ the 02C and DEPT NMR spectra of 0 showed signals of 19

carbon atoms corresponding to _ve CH2\ two CH1\ eight CH "four oxymethines#\ and _ve quaternary car! bons "a carbonyl group at d 113[6 and a C0OH at d 71[5#[ Among the 19 carbons\ there were no sp1 carbon atoms indicating the saturated nature of 0[ Based on the thirteen degrees of unsaturation given by the molecular formula "C22H31O09#\ a tetracyclic diterpenoid skeleton "C19H21O5# was proposed for 0[ The COSY "0HÐ0H correlation# and HETCOR "0HÐ 02 C correlation# experiments of 0 led to the estab! lishment of the structure of three main fragments\ separated by quaternary carbons ] CH"OR0#0CH "CH2#0CH"OR1#0CH0CH"OR2#\ CH10CH0CH 0CH1 and CH"OR3#[ The quaternary carbons bridg! ing these fragments were assigned by analysis of the 0 HÐ02C two and three bond correlations of the HMBC spectrum of 0[ The hydroxylic carbon C!04 is cor! related with the oxymethine protons H!2 "d 4[79# and H!03 "d 3[72# and with H!1 "d 1[77# and H!3 "d 1[39#[ The down_eld shift of the carbonyl resonance at d 113[6 suggested the presence of a methyl!substituted _ve membered ring ketone ð05Ł which agrees with the correlations of this carbon with the protons H!7 "d 2[12# and H!01 "d 3[11# and with the geminal methyl

Terpenes from Euphorbia se`etalis Table 1[ 02C NMR spectral data of compounds 0\ 0a\ 0b "in CDCl2\ 64[3 MHz# C

0

0a

0b

DEPT

0 1 2 3 4 5 6 7 8 09 00 01 02 03 04 05 06 07 08 19

63[5 26[7 62[9 32[1 57[5 42[1 24[3 35[3 113[6 36[9 24[5 39[6 40[8 61[1 71[5 09[1 05[3 11[7 18[3 04[2

61[7 28[3 64[6 31[2 58[0 42[1 24[5 35[4 114[9 36[0 24[6 39[5 40[2 60[1 74[9 8[5 05[6 11[8 18[3 05[1

61[5 28[4 63[1 32[5 56[4 42[9 24[6 35[7 115[7 36[0 24[4 39[4 40[7 69[8 74[9 8[2 05[2 11[6 18[6 04[4

CH CH CH CH CH C CH1 CH C C CH1 CH C CH C CH2 CH2 CH2 CH2 CH2

Benzoyl moiety$ 0? 1? 2?:6? 3?:5? 4?

055[0 018[6 017[4 018[6 022[1

054[5 018[3 017[5 018[5 022[2

* * * * *

CO C CH CH CH

069[8 10[9 * *

* * * * * *

CO CH2 CO CH2 CO CH2

Acetyl moieties$ 058[8 R0 19[5 R2 069[5 19[8 R3 058[6 19[7 In CDCl2:CD2OD\ 8 ] 0[ $See structures 0Ð0b

groups at C!09 "d 0[95 and 0[04#[ Similarly\ cor! relations of the quaternary carbon C!02 with H!01\ H!7 and Me!06 "d 0[97#\ and of C!5 with the protons geminal to the ester functions H!03 and H!4 "d 4[57# and Me!19 "d 9[59# indicated the presence of the inner _ve membered ring[ The after mentioned 1D long range0HÐ02C chemical shift correlation also led to the location of the four ester functions[ The ester carbonyl carbon at d 055[0 correlates with the aromatic protons at d 7[91 and with H!2 indicating that it is attached to C!2 and to the aromatic ring[ The ester carbonyl at d 058[6 is bound to C!03 since it correlates with H!03 and the methyl at d 1[09[ Similarly\ the carbonyl at d 058[8 correlates with H!0 and the methyl at d 1[03 and is attached to C!0[ The remaining ester carbonyl carbon resonance at d 069[5 correlates with H!4 and with the methyl at d 0[83 and therefore is bound to C!4[

070

The coupling constants of the protons H!0\ H!2\ H! 3 and H!4\ in compound 0\ were similar to those reported for euphoratines A\ B and C ð06Ł and euphac! tins A and C ð07Ł[ Thus\ the con_guration at C!1 to C!4 and C!04 must be identical to that of these model compounds[ However\ it di}ered from that of eupho! ractines D and E ð06Ł and from euphactins B and D ð07Ł with an a!methyl group at C!1[ The relative con_guration of these chiral centres of 0 was con! _rmed by a NOESY spectrum[ The enhancements observed in this spectrum also led to the ster! eochemistry of the remaining carbons[ The strong NOE enhancements of H!3 at H!2 and H!0 con_rm that they are located on the same side of the molecule[ Furthermore\ the enhancement of H!3 at Me!06 indi! cates that both lies also on the same face[ The hydroxyl group at C!04 has also the same b!orientation as the functional group at C!2 since the hydroxylic proton is correlated with Me!05[ The strong NOE cross peaks between the vicinal methine protons H!7 and H!01 show that they have the same b!con_guration as opposed to the methyl groups at C!02 and C!5 which show the a!con_guration[ This relationship is derived from the absence of NOE correlations between these two methines and Me!06 and Me!19[ In addition\ H! 7 is also correlated with the oxygen!bearing methines H!4\ H!03 and Me!08\ which allocates these groups on the b!face of 0[ Figure 0 illustrates the most relevant NOE correlations used to establish the stereo! chemistry of 0[ Alkaline hydrolysis of 0\ with 9[0 M potassium hydroxide!methanol\ at room temperature\ yielded the parent alcohol 0b and the partially hydrolysed diester derivative 0a "4!acetate!2!benzoate#[ The molecular formula of 0a was assigned as C18H27O7 by LD!FTICR mass spectrometry with the ion at m:z 442[07761 ðM¦KŁ¦[ The EI mass spectrum of 0b\ the parent alcohol\ showed a molecular ion peak at m:z 257 indicating a molecular formula of C19H21O5[ Com! pounds 0a and 0b gave essentially the same 0H NMR and 02C NMR spectra as 0 except for the chemical shifts of carbons and protons which are dependent of the functional groups[ The 0H NMR and 02C NMR spectra of 0a showed the removal of two acetoxyl groups from 0[ The doublet at d 4[93 "d 63[5# and the singlet at d 3[72 "d 61[1# were diamagnetically shifted to d 3[42 "d 61[7# and to d 2[20 "d 60[1#\ respectively\ and the acetyl methyl singlets at d 1[03 "d 058[8 and

Fig[ 0[

071

M[!J[ U[ FERREIRA et al[

19[5# and d 1[09 "058[6 and 19[7# disappeared[ In addition\ the signals of Me!05 and Me!19 were para! magnetically shifted to d 9[86 and to d 9[68 ^ this was due to the deshielding e}ect of the hydroxyl groups at C!0 and at C!03 on Me!05 and Me!19\ respectively[ The 0H NMR and 02C NMR spectra of 0b\ the parent alcohol\ revealed the hydrolysis of the remaining ester groups of 0\ the benzoyl group at C!2 and the acetoxyl group at C!4[ The signal of H!3 was diamagentically shifted to d 0[76 "Dd  −9[48 ppm# owing to the deshielding e}ect of the benzene ring in H!3 of 0 and 0a[ The signal of H!6b appeared shifted down_eld[ The other proton resonances of 0b remained prac! tically unchanged[ EXPERIMENTAL

General Mps uncorr[ ^ IR ] KBr or _lm ^ 0H NMR "299 MHz# and 02C NMR "64[3 MHz#\ Varian Unity!299 NMR spectrometer\ CDCl2\ TMS as int[ standard ^ MS ] Kratos MS14RF "69 eV# and Finnigan!FT!1990 for LD!FTICR!MS[ Plant material The plant material was collected at Leiria\ Portugal\ and identi_ed by Dr[ Teresa Vasconcelos from the Department of Botany and Biologic Engineering of Instituto Superior de Agronomia\ University of Lisbon[ A voucher specimen has been deposited at the Herbarium "LISI# of Instituto Superior de Agron! omia[ Extraction and isolation The air dried whole plant "1[0 kg# was extracted with Me1CO "3×09 l# at room temp[ for 3 days[ Each extract was _ltered on a Buchner funnel and evapor! ated under red[ pres[ at low temp[ "39>#[ The combined extracts gave a residue of 079 g[ Saponi_cation A 09) KOH soln[ in MeOH "0 l# was added to the total extract[ The mixture was left at room temp[ for 25 h[ After concentration of the MeOH\ at red[ pres[ the residue was suspended in 0 l of H1O and extracted several times with Et1O[ The combined extracts\ con! taining the non!saponi_able part\ were dried "Na1SO3# and evaporated\ yielding a residue of 009 g[ The non! saponi_able part was then dissolved in hot Me1CO and cooled[ The ppt[ was _ltered o} "5 g of Me1CO insoluble part#[ The _ltrate was evaporated giving 093 g of Me1CO soluble part[ Separation of the above!mentioned extract "093 g\ Me1CO soluble part# was performed by CC on silica gel "0 kg# with n!hexaneÐEtOAc mixtures of increasing polarity[ Repeated chromatography of the less polar

frs on silica gel or AgNO2Ðsilica gel "0 ] 8 ^ 1 ] 7# and crystallisation "M1COÐMeOH# a}orded 1 "3 mg ^ n! hexaneÐEtOAc\ 08 ] 0#\ 2 "59 mg ^ n!hexaneÐEtOAc\ 08 ] 0#\ 3 "8 mg ^ n!hexaneÐEtOAc\ 8 ] 0#\ 4 "69 mg ^ n! hexaneÐEtOAc\ 6 ] 0# 5 "599 mg ^ n!hexaneÐEtOAc\ 6 ] 0#\ 6 "019 mg ^ n!hexaneÐEtOAc\ 6 ] 0#\ 7 "199 mg ^ n!hexaneÐEtOAc\ 2 ] 0#[ The fraction eluted with n!hexaneÐEtOAc "0 ] 0# was acetylated with Ac1OÐpyridine "0 ] 0# at room temp[ overnight[ The usual workup gave a residue which was chromatographed twice on silica gel col! umns with n!hexaneÐEtOAc and CH1Cl1ÐEtOAc mix! tures yielding 199 mg of 0 "CH1Cl1ÐEtOAc\ 8 ] 0#[ Se`etalol!0\4\03!triacetate!2!benzoate "0#[ Mp 040Ð 042> "EtOAc!n!hexane# ^ ðaŁ19 D −48[55> "CHCl2 ^ c 9[29# ^ LD!FTICR!MS m:z 510[15534 ðM¦NaŁ¦ KBr "C22H31O09Na requires 510[15691# ^ IR nmax cm−0 2344\ 1876\ 1784\ 0632\ 0695\ 0358\ 0272\ 0172\ 0121\ 0012\ 0920\ 667\ 603[ 0H NMR and 02C NMR ] see Tables 0 and 1 ^ EIMS "probe# 69 eV\ m:z "rel[ int# ] 487 ðMŁ¦ "0#\ 479 ðMÐH1OŁ¦ "9[4#\ 427 ðMÐHOAcŁ¦ "07#\ 412 ðMÐHOAcÐCH2Ł¦ "01#\ 367 ðMÐ1×HOAcŁ¦ "22#\ 359 ðMÐ1×HOAcÐH1OŁ¦ "4#\ 307 "02#\ 304 "20#\ 262 "04#\ 245 ðMÐ1×HOAcÐC5H4CO1HŁ¦ "099#\ 230 ðMÐ 1×HOAcÐC5H4CO1HÐCH2Ł¦ "00#\ 227 ðMÐ 1×HOAcÐC5H4CO1HÐH1OŁ¦ "08#\ 220 "00#\ 203 "44#\ 293 "03#\ 188 "0#\ "09#\ 185 ðMÐ2×HOAcÐ C5H4CO1HŁ¦ "56#\ 170 "31#\ 167 "16#\ 127 "57#\ 109 "29#\ 078 "14#\ 047 "14#\ 023 "44#\ 094 ðC5H4COŁ¦ "62#\ 80 "67#\ 66 "52#[ Alkaline hydrolysis of compound 0 Compound 0 "14 mg# was treated with 9[0 M KOH in MeOH "3 ml# at room temp[ for 2 h[ After con! centration\ the residue was suspended in 1 ml of H1O and extracted with EtOAc "3×4 ml#[ The combined extracts were dried "Na1SO3# and evaporated\ yielding a residue of 04 mg[ After prep[ TLC "CH1Cl1ÐEtOAc\ 2 ] 0# 09 mg of 0a and 3 mg of 0b were obtained[ Se`etalol!4!acetate!2!benzoate "0a#[ Gum ðaŁ19 D film −26[49> "CHCl2 ^ c 9[08# ^ IR nmax cm−0 ] 2326\ 1862\ 1818\ 0606\ 0369\ 0271\ 0170\ 0121\ 0005\ 0915\ 603[ 0 H NMR and 02C NMR ] see Tables 0 and 1[ LD! FTICR!MS m:z "rel[ int# ] 442[07761 ðM¦kŁ¦ "099# "C18H27O7K requires 442[24338# 426 ðM¦NaŁ¦ "8#\ 338 ðM¦NaÐHOAcÐH1OŁ¦ "6#\ 318 "5#[ Se`etalol "0b#[ Mp 183Ð185> "EtOAcÐn!hexane# ^ film −0 ðaŁ19 2238\ D −07[85> "MeOH c 9[02# ^ IR nmax cm 1847\ 1804\ 0619\ 0323\ 0272\ 0927[ 0H NMR and 02C NMR ] see Tables 0 and 1[ EIMS "probe# 69 eV ] m:z "rel[ int# ] 257 ðMŁ¦ "0#\ 249 ðMÐH1OŁ¦ "7#\ 221 ðMÐ 1×H1OŁ¦ "17#\ 206 ðMÐ1×H1OÐCH2Ł¦ "4#\ 203 ðMÐ 2×H1OŁ¦ "00#\ 188 ðMÐ2×H1OÐCH2Ł¦ "4#\ 185 ðMÐ 3×H1OŁ¦ "2#\ 180 "52#\ 174 "09#\ 170 ðMÐ3×H1OÐ CH2Ł¦ "2#\ 152 "00#\ 134 "02#\ 115 "8#\ 119 "8#\ 089 "27#\ 061 "099#\ 043 "56#\ 014 "29#\ 091 "56#\ 84 "45#\ 44 "41#[ Acknowled`ements*We are grateful to Junta Nacional de Investigacža½o Cient(_ca and PRAXIS

Terpenes from Euphorbia se`etalis

XXI for _nancial support[ We also thank Dr[ Teresa Vasconcelos of the Instituto Superior de Agronomica for the identi_cation of the plant[ We are also grateful to Dr[ Joaquim Marcžalo for high resolution mass spectrum[ REFERENCES

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