Acylphenols from Myristica crassa as New Acetylcholinesterase Inhibitors

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I, CNRS,Gif-sur-Yvette CedexFrance Vallqy,'ltualaL!mpuI Malaysia

Keywords O Myristica crassa O Myristicaceae O Malabaricone o Acylphenol O Acetylcholinesterase o Lc.Ms

Abstract V A significantacerylcholinesterase inhibitory activity was observedfor the ethyl acetateand methanolextractsfrom the leavesand the fruits of Myistica crossa.Threenew dimeric acylphenols, giganteoneC (5), maingayones B and C (6

and 7) were isolatedtogetherwith the known malabaricones B and C (2 and 3) and giganteone A (4).Compounds 2 and3 possess significant inhibitory acdviry on acetylcholinesterase. LC/MS study was particularly useful to discriminate structuresof compounds6 and 7.

Introduction

received April 4, 2008 r e v i s e d J u n e1 8 , 2 0 0 8 a c c e P t e dJ u n e2 9 , 2 0 0 8 Bibliography OOI 10 1055/s-2008-1081336 P l a n t aM e d 2 0 0 9 ;7 4 : l - 6 @ Ceorg ThiemeVerlagKC Stuttgart New York P u b l i s h eo d n l i n eI t 5 5 N0 0 3 2 - 0 9 4 3 Corespondence Dr. Mort Utoudon Institut de Chimiedes 5 u b s t a n c eN saturelles U P R - 2 110 CNRS 'l avenuede la Terrasse 9l 198 Cif-surYvetteCedex France Tel: +13-l-6982-3085 F a x :+ 3 3 - 1 - 6 9 0 7 - 7427 marc litaudon@icsn,cnrs-9if fr

compoundsisolatedalongwith malabaricone A V (l ) andpromalabaricones B and C (8 and9),pregenus The Myristica(Myristicaceae)is wideviouslyisolatedfromM maingayt[6] (O Fig.r ). spreadin the tropicsofAsia,Australia andOceania, of which l0 speciesare found in peninsular Malaysia [1j. tn this genus,Myrisrica fragrans Materials and Methods and other Myrisficospeciesareusedin folk med- V icine in Asia[2]. Theyare known ro produceliP l a n tm a t e r i a l gnansand acylphenols mainlyof the 2,6-dilry- Ripefruitsof M. uassaKingwere collecredarJerdroxyphenyltype suchas malabaricones A- D antur, Pahang(Malaysia)in March 1996 and t3l, I4l, t5l, [6],[7].Theseacytphenots showedinidentifiedby rhebotanistT.LeongEng.d voucher terestinganrimicrobial adivity [5], cytotoxicity specimen (KL-4564)is deposited ar the Herbarion variouscell lines[6], [7],antinematocidal ac_ um of the ForestResearch lnstitute,Kepong,Mativity [8], antipromasrigote acriviry[9] and a laysia, healingpropertyagainstindomethacin-induced gastriculceration[10]. In addition,organicex- Extraction and isolation tractsof M.Jragronsseedswerefoundto possess Thedriedpowderedfruits (50 g) weremacerared in virro and in vivoacerylcholinesterase-inhibitat roomtemperature with EtOAc(3 x 200mL.t h i n ga c t i v i t i elsl l l , [ 1 2 ] each)and percolated. Thefiltrarewasevaporated In continuationof our searchfor novelbioactive undervacuumto yielda gummy residue(2.03g). compoundsfrom the Malayanflora,we have Theextractresidue(50 mg)was filteredon a polylauncheda bioassay-guided study on an ethyl amidecartridge(Chromabond pAG,1 g; Macheracetateextract of the fruits of M. crassoKing, ey-Nagel)using an EtOAc-MeOH I :l mixrure. which exhibitedpotentacetylcholinesterase in- The filteredextractwas then solvedin DMSO hibitoryactiviry.This srudy,which was accom- (15mg in 300pL)andinjected at onceon a semiplishedwith the aid of HPLC, HPLC-ESI-MS and preparativeKromasilo C-18 column (5,r.rm, NMRanalysis, Iedto the isolarion and identifica- 2 5 0 x 1 0 m m ;T h e r m o e l e c t r o A n ) g. r a d i e n m t o_ rion of threenew arylphenoldimers,giganteone b i l ep h a s ec o n s i s t i nogf A - M e C N , B H 2 O i;s o C ( 5 ) a n d m a i n g a y o n eBsa n dC ( 6 a n d 7 ) a l o n g c f a t i c2 0 %A f o r 2 m i n , 2 0 - 1 O 0 % A o v e r3 2m i n w i t h t h e k n o w nm a l a b a r i c o nB e s( 2 ) a n d C ( 3 ) followedby isocraticl00Z A for 3l min; flow andgiganteoneA (4) Herein,we reportrhesrruc- rate:3 ml/min was usedjn orderto obtainnine turalcharacterization of thenewcompounds and fractions(24m1 each).The HpLCseparations the acerylcholine'irerase inhibitoryacriviryof the wereperformedusinga Watersautopurificarron MaiaAet al Acylphenolsfrcm Myristtco plantaMed 200g: 74: I _ 6

o

OH

OH

o

o

1 :Malabaricone A : R1= R2= H 2 : Malabaricone B : R1= OH, Re= H 3 :Malabaricone C : R1= Re= OH OH

o

OH

C OH 5 : Giganteone OH OH

o

OH R2

Giganteone A

o

OH

6 : Maingayone B : R1= OH, R2= H 7 : Maingayone C : R1= H, Re=OH

8 : P r o m a l a b a r i c oBn: e R= H 9 : Promalabaricone C : R = OH F l g , 1 C h e m i c a l s t r u c t u r eosf m a l a b a r i c o n e s AB , a n d : C( l - 3 ) , g i g a n t e o n e s A a n d C ( 4 a n d 5 ) , m a i n g a y o n eB s a n d C ( 6 a n d 7 ) , a n d p r o m a l a b a r i c o n eBsa n d C (8 and 9)

systemequippedwirh a UV-Visdiode array derector(lg0600nm) anda PI.ELS 1000ELSDdetecor(polymerlabofarory). Thefilteredextractand the fractionswere evaporatedand dissolvedin DMSOat a concentration of l0 mg/mL,anddistributed into a 96-well mother plate. The remaining EtOAc extracr (1.989)was submittedto flash chromatography on reversed phase(Versapack, C-18Carrridge 40x75 mm) usinga srepgradie n t m o b i l ep h a s ec o n s i s t i nogf A - M e C NB , - H 2 O2 : 0 - 1 0 0 %A for 70min; flow rare:10ml/min ro give j0 fractions(fA ro fl).A partof fractionc (300mg) wassubmittedro HPLC(6 injections of 50mg in 1.5mL)of an MeCN-HrO 60:40 mrxture,on a preparativeSymmetry@ RP-18column (7 ym,250x20 mm; Waters) usinga gradientmobilephasestartingfromA - MeCN,B - H2O; isocratic40X A for 6 min, 40-80% for 12 min; flow rate: 22 mL/ min,to givemalabaricone C (3) (tR12 min) and malabaricone B (2) (tp 14min).A secondpartof fr G (146mg),wassubmirred ro H P L C (i3n j e a i o n os f 4 9 m g i n 1 m L )o f a M e C N - H r 6 O0 : 4 0m i x ture, on a preparativeSymmetry@RP-18 column (7 iim, 2 5 0 x 2 0 m m ;W a t e r s u ) s i n ga g r a d i e n m t o b i l ep h a s es r a r t i n g f r o mA - M e C NB, - H 2 O6; 0 : 4 0 r o 8 0 : 2 0 f o r 3 5 m i n ; f l o w r a r e : 2 2m l / m i nt o g i v eg i g a n t e o nAe( 4 )( r R5 . 5m i n ) .g i g a n r e o nCe( 5 ) ( 1 0 1 m g ,r * 7 . 9 m i n )a n d t h e m i x t u r eo f m a i n g a y o n eBs+ C ,( 6 a n d7 ) ( 8 . 3m g ,t p9 . 5m r n ) .

lvlaaa Aet al Acylphenolsfrom /ttyrsfico

PlantaMed 2008; 74: I - 6

G e n e r a el x p e r i m e n t a lp r o c e d u r e s lR spectra wereobtainedon a NicolletFTIR205 spectrophotometer TheUV spectrawererecordedon a perkin-Elmer Lamba5 spectrophotometer. NMR spectrawere recordedwith a Bruker (500 and 600MHz for rH, and 'l25 and l50MHz spectrometer forr3Cin CD3OD). I C - D A D - M Sa n a l y s i s HPLCof the activefractionwas performedusinga Waters2295 Alliance system, with a DADfrom 210to 450nm Theseparation was achievedon a Symmetryo Rp-l8 column (3 5 pm, '150mmx2.1 mm; Waters) equippedwith a guard column, mainrained at 30 'C.Themobilephasegradientconsisted of A H2O,B - MeOH-0.1 %formicacid,isocraric 80%A for 6 min.8025%over'12min, 25-0% for 27 min; flow rate:200rl/min; lnjectionvolume:10gL;sampleconcenlration I mg/ml in MeOH, andall of theeluentwasallowedinto the MS.Atmospheric pressure ionization-high resolutionmass spectrometry was performedon a time of flightmassspectrometef (LCT;Micromass) with an electrospray ionizationsource(Lockspray) The source parameters were as follows:sourceand desolvation temperat u r ew e r e1 2 0" Ca n d3 5 0" C ;n e b u l i z earn dd e s o l v a t i ogna sf l o w s were20 and 520 L/h, respectively: capillaryvoltageand cone voltagewereset ar 3000V and 40 V in rhe ESI.and -2500 V and-40 V in the ESI-mode Lockmass wasa polyalanine solution in ESI-anda leucineenkephalin solutronin ESI-in orderto allow

for internalmasscalibration.ComplementaryLC-MS/MS experimentswererealizedusinga U3000LCsystem(Dionex)coupled to an orbitrap mass spectrometer(lJlQ-orbitrap:Thermoelectron) equippedwith an electrosprayion source.Sourceparameterswere as follows:sourcevoltage:3.2kV, capillarytemperature: 300'C, capillaryvoltage:-40 V, tube lens voltage:-80V, sheathgas,capillary gas and sweep gas flows were respectively fixed at 40, 10 and 7. MSnspectrawere acquiredin the negative ionizationmode,with detectionon either the linear ion trap (low resolution)or orbitrap(high resolution).

( 1 2 s M H zC , D 3 o D )6: = 2 s . 8( c - 3 ) , 3 0 . s( c - 4 ) , 3 0 . 6( C - 5a n dC 6),30.3(C-7),32.s(C-8),36.3 (C-9),4s.8(C-2),108.4(C-18and c-20),r11.s(cl6), 116.2(C-14),116.6(C-l1), 120.7(C-ls),13s.S (c-r0),136.8(C-19),1,t4.0(C-13),146.0(C-12),163.4(C-17and (C-l ); HR-ESI-IS seeo Table1. C-21),209.7 Giganteone C (5): Colorlessamorphoussolid; W (MeOH):i-* ( l o ge ) = 2 1 2( 3 . 9 \ . 2 7 2 ( 3 . 53)5, 0( 3 . 2 )n m ; l R :v - * = 3 4 3 9 ,2 9 2 t , 2857,1681,1620,1597,1454,1135,1015cm-l:rH- and |3C-NMR (CDsOD): seeo Table2;HR-ESI-MS seeo Tablet. Maingayones B+C (6 and 7): Colorless amorphoussolid; UV ( M e o H ) :1 - * ( l o g e ) = 2 0 7( 4 . 1 ) ,2 7 5 ( 4 . 1 ) , 3 5 3( 3 . 3 )n m ; t R : v*=3380, 2930, 2857, 1701,16'12,1525, 1470,1361.1015, 993cm-t;1H-and |3C-NMR (CD3OD): seeo Table2;HR-ESI-MS seeo Tablel.

Biological activity Acetylcholinesterase(AChE) ftom Electrophoruselectric.$ (C 2888) was purchasedfrom Sigma.Inhibition of AChEactivity wasdeterminedby the spectroscopic methodof Ellman[13],using acetylthiocholine iodide as substrate,in 96-well microtiter plates.All solutionswere broughtto room temperatureprior to Results and Discussion use.Aliquotsof 200p1 of a solutioncontaining640;iL of 10mM v of 5,5-dithio-bis-2-nitrobenzoic acid (DTNB)in 0.1M sodium Groundfruitsof M. crassawereextractedby ethylacetateto give phosphate, pH 8.0,19.2mL of the samebuffer,and '13pL of a sol- a crudeextract.which exhibiteda significantacerylcholinesterution of AChE(100U/mL)in water,were addedto eachwell, folaseinhibitoryactivity(97%t 1 at 100pg/ml ).A smallamounrof lowedby2gLof a DMSOsolurionof the inhibitor(0.9% finalvol- the extractwas submittedto HPLCfractionationas it was done ume).The reactionwas initiated by adding20 1L of acetylthio- in a previous study[14].Fraction 4 (26-34 min)showeda srrong cholineiodide(7.5mM) to eachwell and wasfollowedby moni- biological activity(80%r 5 at 10pglml). lt wasanalyzedby LCtoringtheappearance of thethiolaredianionproduced by reduc- HRMS(LCl)and LC-MS/MS. Resulrs of rheLC-HRMS analysis of tion ofDTNBat 412nm every13s for l20s at 25'C in a Molecu- the activefraction,which contained6major compounds, are lar DevicesspectraMax 384 Plusplatereader.Eachinhibitorwas presentedin O 1.A largeamountof the crudeextractwas then (from 1 mg/ml to 0.05pg/mLby evaluatedat ten concentrations submittedto C-18flashchromatographyfollowed by preparadilutingby a factor3 ). Percentage inhibitionwascalculatedrela- tive HPLC, allowingus to isolafecompounds 2-7 (O Fig.1). tivetoa controlsampleof DMSO(% inh.=[l - (slopecpd/slope Compounds2-4 were identified basedon their massspectra DMSO)lxlag) with SoftMax@ Prosoftware.IC56 valuesdisplayed and NMRcharacteristics as malabaricones B,C and giganreone representthe meantstandarddeviationfor six assays(SD= A, respectively [3],[7]. V{x - x)'z/n).Tacrinehydrochloride(Sigma,purity > 99%)was Compound5 was isolatedas a colorless solid.lts lR spectrum = 3430cm-r and a usedasreference compound. displayeda broadhydroxyabsorptionat vmax Malabaricone B (2): Colorlessamorphous solid; 1H NMR conjugatedcarbonylbandat -l681cm-r.Thetandemmassspec(500MHz,CD3OD): 6= 1.35(8H,m, CH2-4,5, 6, 7),1.55(2H,m, tra in the negativeionizationmode of the parent ion m/z=697 C H 2 - 8 )1, . 6 7( 2 H ,m , C H 2 - 3 ) 2, . 4 9( 2 H ,t , J = 8 H z ,C H 2 - 9 ) , 3 . 1 1 showeda basepeak at mlz=587, resultingfrom a cleavageto (2H, t, .1=3 Hz, CH2-2),6.35 (2H, d, J = AUz, H-18 and H-20), the carbonylgroupat C-l and leadingto the ion rn/z= 569 by fur6.69(2H,d,J=8 Hz,H-12andH-14),6.97(2H,J=8Hz,H-l1 and ther lossof H2O,On the otherhand,the cleavage d to the carbonH - 1 5 ) ,7 . 1 9( l H , t , 8 H z , H - 1 9 ) ;r 3 CN M R ( 1 2 5 M H 2 C , D 3 O D ) : yl groupat C-1'leadsto the ion m/z=465,which undergoes a 6=2s.8(c-3),30.3(c-4),30.s( c-s and C-6),30.6(c-7),33.0(ccleavage o to the carbonylgroup at C-l to givethe ion m/z= 355 '108.4 (c-18andC-20),111.s (C16).116.0 (O Fig,2).TherH-,_r3C-, 8),36.1(c-9),4s.8(c-2), and2 D NMRspectraof compound4 de(C-12andC-14),130.3 (C-11andC-ls),131.0(c-l0),136.9(c-19), picted four benzenerings a, a', b, b': 1,2,3-trisubstituted a (C-l7 andC-21), 209.7(C-l); HR-ESI-MS 156.2(C-13),163.4 see [ f i - 6 . 3 2 ( 2 H , H - 1 8 a n d 2 0 , d , J = 8 H z ) , 7 . 1 7( 1 H , H - 1 9 ,t , o Tablel. / = 8 H z ) ] 1, , 2 , 4 , 5 - t e t r a s u b s t i tbu[t&e =d 6 . 5 3 ( 1 H ,H - l 4 ,s ) , 6 . 6 5 MalabaiconeC (3): Colorlessamorphoussolid; 1H NMR ( l H , H - I 1 ,s ) 11, , 2 , 3 , 4 - t e t r a s u b s t i tau' [t fei = d 6 . a 0 ( 1 H ,H - 2 0 'd, , ( 5 0 0M H z ,C D 3 O D6) = : 1 . 3 6( 8 H ,m , C H 2 - 4 ,5 , 6 , 7 ) ,1 . 5 6( 2 H ,m , / = 8 . 5 H 2 ) ,7 . 0 4( 1 H , H - 1 9 ' ,d , J = 8 . 5 H 2 ) 1l , 4 - d i s u b s t i t u t eb d' C H 2 - 8 )1, . 6 7( 2 H ,m , C H 2 - 3 ) , 2 . 4 (52 H , t , J = 8 H 2 C , H 2 - 9 ) , 3 . 1 2 [ q j = 6 . 9 3( 2 H , H - 1 1 ' a n d1 5 ' ,m ) , 6 . 9 4 ( 2 H , H - 1 2 'a n d l 4 ' , d , ( 2 H ,t , J = 8 H 2 ,C H 2 - 2 ) .6 . 3 5( 2 H , d , l = 8 H 2 , H - 1 8a n d H - 2 0 ) , / = 8 Hz)1. Analysis of thealiphaticregionindicated16methylene 6 . 4 8( 1 H ,d d , J = 8 H z , 2 H zH, - ] s ) , 6 6 2( 1 H ,d , J = 2 H z . H - 1 1 ) ,groupsdistributed intorwo n-ocrylchains(c andd) (o Fig.2) In 6 . 6 7( l H , d , J = 8 H 2 ,H - 1 4 ) , 7 . 2(01 H ,t , / = 8 H 2 , H - ] 9 ) ;' 3 CN M R the HMBCspectrum,the quaternarysp2carbonat 6= 1350 (C-

Tabh1 [M - H]- and[M + Hl' ions(ESl)andrawformulaaregivenfor compounds 2-7

.1748 3 5 7 . 1 7I1 713.3342 6 9 7 . 3 4I0 6 8 33 5 8 6

€21H7sOa

c 2 rH ? s o s L42' '49v10

c42H1eos o8 c42H5r

3 5 03

143;l906 3 5 9r 8 7 2 1 1 53 4 1 7 6 9 93 5 2 0 7 0 73 5 6 1

rIl,,27v4

c { 2 H s r o lo c . 2H 5 1o s Cr2HstO8Na

'I 3 0 1 0

8 7 9 2

MaiaAet al Acylphenolsfrom Myristico PlantaN4ed2008: 74: 1 -6

t:: l-O.€c,*+) Ealcil;€?:ft} :1.IlF+'+.'isEirri),:

z l 4-7

-RG{.i)

2F.1 30;!:30.7 lz:2' 33r8, 135.0 'l'193

-..

145J 'l:43:9

1;iFlrdr 2,-2++8,4:.

8 9 10 1..:l

I :'

1A T3 +4

..

16 1:7' 18 19 20 2l 1', t' J -

8' t

10f '| 1', .13: 11

6,53(Fl:

1at

t

,ir5*i 1.61{++l} 1,36:=t 35 frn) t,53 + l ,54:(n)'

1n*en

135:1

i5.:32r(d,8)

&L f,=1.4..(r,

'6i:93:(lt)i.,_: 'l6aAid-;i8)

tJo.I

6.6014A

I lOi/

14 6 . 1 144.2 116.4 120.8 1 11 . 4 160,6. .7 10-1 131.6 124.0 I 60.7. 35.1 326

t9] I to. I

130.3

6.6s(dd,8,ra) 6 . 4 6( d .8 )

6 ; 3 1( d ,8 . 5 ) 7 . 5 9( d ; 8 ' s )

4.s5.{t,,8} 2is7-2 39,(m) r.23'(m) 1.06- 1.35('r:!) 1,5a({n) 2-49{t.r7}

zt,o

3 0 . 3- 3 0 . 6 36;2 1 3 51 130.3 1',16.2 '156.4

6,96(m) 6:6E,{ri)

130i3 't161

5 . 6 8( m ) 6 . 9 6( m )

I to.l

1 l 1 3 1 6 13

l8'

zo!

13.0 36.4

I fo.5

6,94r$;8) 6.93(m)

l6'

,19.

+5,5 26,0 3 0 r l- 3 0 . 6

:

129.5 ill.5 I 6l:6 r 08,5 .136,9 10 8 . 5 t6316 2lo;2 46.0 25.9 3n.2:30;7 33:.2

,68",.(d;'1:8': '.7,:$711,'81

,?,061-.0'glTr;!7 3) r'.igf=r,oztqr) 'r1-':it6:1;}5:.tm) i.r5i(m): 2.1i2(t, &)

Table2'H-(500and 600MHz)andtsC(l25and 15 0 M H z )N M Rd a t ao f g i g a n Bs t e o n eC ( 5 )a n dm a i n g a y o n e ( 5 a n d7 ) a n dC i n C D 3 O D

7-04..(!;.8:5) 6.40(d;:E.5)

Itt.) 't39.1 107.6

',] 5 . 3 0( d ,8 ) 1) 6.79(t,8 6 . 3 0( d ,8 . 1)

'

130.3 'r 1 85 1 5 77 108,9 1 2 81 108.9 157.7

tbt.b 21! t ForcomDound7, the NMRdata tH- (600 MHz)and trc- (150 M Hz) from c-8to C-15 hasto be switchedwith thosefrom C-8'to c'1 5 b ' A s 5 i g n m e n( at n b e i n t e r c h a n g e d .

to the protonat 6=6.53(H-14)of ring b and 10)wascorrelated at 6= 1.35and 2.29 (CHzI and 9) of chainc to the methylenes thus linking C-10 to the n-ocryl c chain. The carbonyl at 6=209.8(C-1) was correlatedto the methyleneat 6=3.08

355 +

465 (-H)'

Frg.2 MaihE5FM5fragmentation(m/z values)for compound5'

a 4 e2d0 0 87; 4 :I - 6 M a i aA e ta l A c y l p h e n of rlos mM y t i s t i c oP l a n f N

(CHr-2) and to the aromaticprotonsat 6=6.32(H-18and H20,JW-coupling) of ring a endingthe n-ocrylc chainandthus, -15-substituted malabaricone I was definedas a the substructure concorrelations long-range in the HMBCspectrum, C.Similarly, nectedthe n-octyld chainto the b'ring and to the carbonylat 6=210.2(c-1J that was linked to the ring a'. Substructurell B.Thequaterto an 18'-substituted malabaricone wasassigned to the protonat narycarbonat 6= 129.5(C-15)was correlated 6= 6.65(H-]1) of the nng b, and at = 6 7.04(H-l9J of the ringa', I and ll, showingthe linkageat C-'15and C-18'ofsubstructures (o Fig.2).Compound5, namedgiganteone C,is a respectively B,IinkedthroughCdimerof malabaricone C and malabaricone 15andC-18',respectively, powder, amorphous 6 and7,obtainedasa colorless Compounds w e r ea n a l y s ebdy N M R s p e c t r o s c o pays a l : 2 n o n - s e p a r a b l e absorption bandsofa conjumixtufeTheIRspectrumrevealed gatedcarbonylar 1702cm-rtogetherwith hydroxybandsat foursubstispectradepicred andHSQC 3385cm-rThetH-,COSY tetrarings:a, a',bl or b2,b'l or b2; one 1,2,3,4 tutedbenzene a', one1,2,4trisubstituted a, one 1,2,3trisubstituted substituted bI or b2,The 1H-and r3Cb1 or b2 and one.1.4disubstituted

T a b l e3 P r o d u cito n so b exPeritainedfrom LC-MS" mentsin the ESt-modefor c o m D o u n d6sa n d7 341 3 4 1 ,2 9 9 2 , 31 299,273:231 -

281,?71' '

n -'s:,D/t:r 283: .SupelcosiltRp-18column(5!m,250mmx46mm).mobilephasegradient;A-HrO.B-ACN-Ollformicacid,50%Bfor1min.50-65%Bforl0min,65XBfor 25 min.flow rate: I mLimin

NMRspectraof both compoundswere very similarto the extent perfectlyoverlappedwith one that almostall of the resonances another.The presenceof these isomerswas subtly detectedby a slight differencein the chemical shifts of the C-2 protonswhich at 6 3.06and 3.09 in compounds6 and7.Theconnecresonated the four rings a, a', b1 or b2 and bl or b2 were rivities benareen by thoroughanalysisof the HMBCspectrumand Lcestablished The tetrasubstitutedring a is adjacentto the CMS/MSanalysis. 1 carbonylwhich in turn is linked to ring bl or b2 by an n-octyl were Proventhroughthe conelation chain.Theseconnectivities of C-l andC-3with Hz-2, C-1with H-18,andC-9with Hsignals Ringa is 15 (6.46or 6,96 in compounds5 and 7, respectively). '(C-20, C16'-r H-1') while ring bl linkedto ring a'through C-l or b2 is connectedto both rings a and a'by an n-octylchain which is attachedto C-l'(C-10' --+H2-9', C-3' -+ H-l'). The of compounds6 and 7 and theircommainHMBCcorrelations pleteassignments are depictedin o F19.3and o Table2.Compounds6 and 7 coutdbe differentiatedthroughLC-MS.experimentsusingthe negativeionization mode (o Table3)'LC-MS2 experimentsof [M - Hl- at mlz'683leads to a basepeakat m/ ofthe C-l'/C-16'bond' z = 573resultingmainlyfrom the cleavage commonto both structures.The presenceof two otherionsat m/

Eg,3

MainHMB€{onelitionsfot compounds6 and7.

1

z 3 4 5 8 9 Tacrina

11 . 0 +2 . 1 9.4+1.6 I t-t t t.)

> 7-0 >70 >70 > t40 >,140, 0 : 115 1 0 . 0 2 0

T a b f e 4 l n h i b i t i o no f E l e c t r o ' AChEactiviphoruselectricus t i e sf o rc o m P o u n dI s- 9

z= 341 and 357 alloweddistinctionof compounds6 and 7. The extractionof these ions from the total ion current showed that a differenceof 0.4min separatesthe retentiontimes of the two Forcomisomericcompoundsdisplayedon the chromatogram. pound6, the ruptureof the C-lJC-16'bondled to fragmentsat m/z-325 and 357 consistentwith the presenceof two hydroxy groupson ring bl and one hydroxylon ring b1. For compound 'bond bewvEenthe phenolicring 7, the cleavage of the C-20/C-l a and the carbonchain led to a signalat m/z=347,which conof wo hydroxygroupson ring b2 and only firmedthe presence onehydroxyon ring b2. In the lattercase,the ion m/z= 341could comefrom the two fragmentsI and ll (o Fig.3).Bothions at m/ z=357 (compound5) and 341 (compound7) undergoa rearrangementof the phenylring a leadingto the ions at mlz'289 after the lossof a neutral fragment andm/z=273,respectively, ln addition,we noticedthe presenceof the ionsat m7 C4H4O. to the carz=247 andmlz=231,resultingfrom the a cleavage bonylgroupat C-l ofthe parentionsmlz=357and341'respectively.Finally,from the ionsmlz = 325 and 341 (fragmentI in 6 and 7 respectively),the loss of C2H2Oresultedfrom the rearrangementof ring a', led to the ions mlz-283 and 299; both lossof COand H2O,Thesedata ionsundergoinga subsequent confirmedthe structures6 and 7 depictedin o Flg' 1 for maingayonesB and C,respectively. inhibitory activity was assayedby the The acerylcholinesterase elecfrirus methodof EllmanI13l usingAChEfrom Electrophorus compound(o Table4)' Onlythe monowith tacrineasreference moiety,suchas malabarhavingan acylphenol mercompounds with iconesA, B and C (1-3) exhibitedsignificantinteraction aromatic of rwo the that be deduced AChE.Thus,it can Presence contribringsis essential for the activiryand that dimerization utesto a greatdecreasein activiry Moreoverthe presenceof a sincecompound1 is asacphenolring(ring B) is not necessary 2 and 3. Theseresultscouldexplainthe in tive as compounds inhibitory activitiesobvifro and in vivo acerylcholinesterase seeds[11], [12]. of M.fragrans servedfor the extracts

Acknowledgements V Thanksaredue to O.Thoison,ICSN,for her help in rhe LC-DADC. LeCallonecwho performedthe acerylcholinesMS analysis, Pr.B Bodowho hasprovidedsamples teraseinhibitionassayand B and C. Dr. A. Maia A and promalabaricones of malabaricone from the Institutde Chimiedes wassupportedby a scholarship (ICSN-CNRS) Naturelles Substances

Maia Aet al Acylphenolsftom Myristi.o PlantaMed 2008: 74: I 6

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9 Ruposhree t Ajay BK SubrataC Mitali C.Antipromastigoteactivity of 7 WhitmoreTC.Myristicaceae.ln : Whitmore TC,editor. Treefl ora of Ma_ the malabaricones of Myristicamalabarica(rampatri).phytotherRes laya:A manualfor foresters,volume 1 HongKong:Longman;'l972: 2007:21:592-5 3 1 5- 4 5 l0 DebaslirhB,Ajay BK RonjitGK SanitipBK,SubrataC.Healingproper2 layaweera DMA Medicinalplantsusedin Sri Lanka;part lV. Colombo: tiesofmalabariconeB and malabaricone C.againstindomethacin_in.l NationalScience Council:l9g2: - 107 ducedgastriculcerationand mechanismofiction. EurJ pharmacol 3 Purushothuman KK, Saradah ConnollyJD. MalabariconesA _ D, novel 2 0 0 8 ;5 7 8 :3 0 0 - j 2 diarylnona-noids from Myristica malabaricaLam (Myrisricaceae;. 11 M.ukherjee PK,KumarV, Houghtonp Screeningof lndian medicinal J ChemSocIPerkinIl; 1977:587- A plants for acerylcholinesterase activiry. phytother Res 2007: Zl: 4 CoorayNF,JanszER,WimalasenaS, WueselkeraTp,Noir gM. Acylresor_ 1142-5 cinolsfrom seedkemels of Myristica dacryloides.phyrochemistry I 9 8 7 :2 6 :3 3 6 9 -7 . t 5 OrabiKY.Mosso./S, El-FeratyFS.lsolarion and characterizariono[ rwo antimicrobial agentsfrom mace(Myristicafragrans).J Nar prodlggl; 54:856-9 6 VanCuongP, Jossang A, SdvenetI, BodoB Cytotoxicarylphenolsfrom Myristicamaingayi.Tetrahedron 74 Bousserouel 2000; 56: 1707_ 13 H, LitaudonM, Morleo B, MartinM_T,7loisonO, Nosjean O 7 U.anCuongP, lossang4 Sevenet er al. New biologigallyactivelineartriterpenesfrom the barkof three T, Bodog. Novel rytotoxjc acylphenol dimersof Mynsticagiganrea;enzymaticsynrheslsof giganieones n e w - c a l e d o n iC a nu p a n i o p ssi sp e c i e sT.e t r a h e d r o2n0 0 5 :6 j : g 4 5_ 5 l A andB.Tetrahedron 15 Mukherjee 2002;58: 5709- 14 PK,Kumat V,Mdt M, Houghtonp,f.Acerycholinesterase in_ 8 HosoiS, Kiuchi F, Nokamuro N, /mosho M, Ahad Ali M, Sasakiy et al. h i b i t o r sf r o r np l a n r sp. h y t o r n e d i c i n2e0 0 7 :f 4 : 2 8 9 _ 3 0 0 Synthesis and nematocidal activityof diarylnonanoids relatedto malabaricones. ChempharmBull 1999:47:37-43

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