76
CHEMISTRY OF NATURAL PRODUCTS DISSERTATION SUBMITTED FOR THE DEGREE OF MASTER OF PHILOSOPHY IN CHEMISTRY APRIL, 1984 NAZMJSEN PARVEEM ALIGARH MUSLIM UNIVERSITY ALIGARH
CHEMISTRY OF NATURAL PRODUCTS
DISSERTATION SUBMITTED FOR THE DEGREE OF MASTER OF PHILOSOPHY
IN CHEMISTRY
APRIL, 1984 NAZMJSEN PARVEEM
ALIGARH MUSLIM UNIVERSITY ALIGARH
DS530
Eef No
DEPARTMENT OF CHEMISTRY M L I G A R H MUSLIM UNIVERSITY
ALIGARH. U P . INDIA
P, 'ones: < Office "^5!' Res. . .
Date V5/I98»f
fills la to certify that the d isser ta t ion
i s the original contribiAion of the csudidate and
i s sui table for subnission for the p a r t i a l fulfilmoit
of the degree of Master of Philosophy,
('Hizan*ud.£&n xOiwi )
Supervisor.
sen.'vs of y r a t i t u t e t o '>»-, M'^-jri. U* r-h^-n^ ^rhjs'? conBis t '^nt
guldan<7e a n i oncouragcsniont hei-pe^l. i n c a r r y i m ou t t h e worTc,
p r e e e n t e d In t h i s d i s s e r t a t i in .
I am h i g h l y g r a t e f v j f' Pr« l e s s o r W. Hahiicu ,0111 Ir"van,
t a-i ^l1n•Ly ins'ibte^! t-j Di , ti..i.Tau3''3e,K.i, Pool Of'- 'ic^r,
Oep'Ttmi^nt of Cneiid.:-!try Cor h i s k i i r i in -ceres t , contlnuOTiP
h e J n ancl encouragc»ii"int. I n t l io l a s t b u t n o t t h e i e s s t v 1
F<ni h i g h l y thanxfu i t o l i r , 5!,K,R )->• •For h i s kiiKl IH^I ^ infi
C O ' < * • -.; ! l | - . - i j - . I h ( - •- " ' , 'W <•.' -'t . <U ' . • • ' r.Tj-
C O N T E N T S
Page No.
INTRODUCTION^
Conat i tu«i te of Taxus Alkaloida and Terpenplds , , , Bcdysonea , , , litgnlne , , , Glycosides . , , Flavanoids , . ,
Chemistry of Bif lavanoids (1) Natural Biflavanoids (A) Biaryl type of Biflavanoida
The Amentoflavone group . , , The Agathleflavone group . , , The Cupreaauf lavone ijroup , , , The Robustaflavone group , . * Tlie succedaneaflavone group «• .
(B) Garcinla and Talwania Biflavanoids The Garcinlaflavanone group . . » The T»lwanlaflavone gro»jp , . «
(c) Biaryi e ther type of Biflavanoids The Hinokiflavone group . . . . The ochnaflavone group ' *.,.
(4 1 )fclKti:action ax;d Pur i f i ca t ion » , . ( i l l )ldGntif j c a t i on and SUtructuce Elucidat ion
W end IR , . , Xroton Magnetic Resonance , , , • C NKR , , »
Fass Spectroa<-:oj)y . « ,
1~4 5
7-8 9
10-13 13-14 15 . 16 l e
17-18 19
19-21 21
2i 23 "J4 35-an 39--45
BlHLlCXU<AItfy
Pin3L-'CATIONS
(1 ) Afostract
(2) ComtTftUilcated, R K p e r e l n t l a .
46
47-51 52-57
58-05
66
^
I N T R O D U C T I O N
- 1 «
liaxua ia a metrber o£ Taxaceae, a family of
3 geoera and about 13 spaciea with two genera (Torrya & Xaxua)
occurr ing only ijn the a o r t h e m henilspher«, and nonotyplc
Auatrotaxua« an endentio of new Calendonla. I t has sevan
species d i s t r i b u t e d in E'jrope, Asia , Worth Africa and North
and Central Anverlca.
The chemistry of Taacua has spec ia l s ign i f i cance 2
because of the occxarrence of antltviinor taxanes , raoltlrig 3 4 5
hormones ' , a lka lo ids and other p t ^ s l o l o g l c a l l y important
coitipoutids in i t s var ious s p e c i e s . Although there a re
sca t t e red r epo r t s about var ious types of na tu ra l organic
conpounde by d i f f e r en t groups/ ye t no attenipt ha« been made
for the extensive study of Taxufl c o n s t i t u e n t s .
1. CONSTITUENTS OF TAXUS ;
Alkaloids , dlterp«molds with taxanes ske le ton ,
ecdyaones, l ign ina , bl£lavanold<i and cyanoglucoaides ar«
cormionly found to occur in Taxua.« Out of the seven Sj ois?8
of Taxua. Tj,xu8 baccata hiia he&n rnoat extensively 8tu<!i <,
AhfJ^LClQ-l /;1D THRPFNOCnSt
A mixture of alicalolds (tdxlns) ^& fouud to occvir in 8 9< lO
ne*HiieB and be r r i e s of Taxus baccata L. and 'raxua cuajgldatg.
Taxin I , an ea ter a lka lo id , la the major const i tueTt and Tax in i t
iB the minor a lkaloid^^. Taxinefi a re believed to be re«|»t^«1bl«
- 7 -
for the poisonous properties of the Yew.
OH OH
'OR
( I ) Toxin I (R « fi - diinethylamino - / 9 - phenyl pacx>polnate}
TtM naturally occurring oKitanea with the taxane
skeleton «r« the typical diterpenold conetituants in the genus
Taxus.
CU) R»H, R '«0H*
( I I I ) « -H, lif=^OAc
CiV) R*H,R'*Cmii<iB»9tt
Cv) R»OH,R'*C'""<''"*'<*
OAc OAc i M«
CVt) RsOH
CVlO''*OAC
«Mr ) «#
12 13
Earlier reports on the etrtxjture of these conpovndM
•re not conclusive. In 1967, Chiang e t a l . reported the
ocourrenoe of -Paxlnine ' A ( I I ) , Texlnlne H ( I I I ) , ( IV) ,
(V), (VI) and (VII) from the learee of Taxua-cuspldftta . iMtac on Mlyasakl and COM'orkera Isolated Taxualn (VXII) froiB
15 the heartwood of Taxus^ouspldata
(6^KH. o ) were a l so Isolated fronn ^jucue-ahlnensle
Taxinlne A and l^xlnlne 16
R0--
CVIlt) R = Ac
OR
OAc
M •o,ci=<^^
H OAc «
ClXa) To;<ogifin»
Ha
OAc
(IX b) Taxagifin* G ' -- "
e x ) Turool
- 4 -
The occxirrence of novel taxanaa such as Taxaglf ine (IX*,
IXb) and tetraol (X) along with 19-hydroocybaccatin In the 17 leave© of Taxua baccata la noteworthy. Actlv* con^Jowida
17 (antltumcar) obtained from trimk bark of Taxug )gaccata
are "Paxol (XI)/ lo-deacetyl taxo l , cephal<»mannine and
10~deacetyl cephalomannlne. Later tMo oooipounde were alao 2 10
obtained from Taxua walllchlana . 'Paxua hrevl fo l ia conatltutea two taxanea XTI and XIII whloh are not reported
from other T^ua apeciea.
' ' p - N H - C H - C H - C O O -
V \ \ r < ^ OH
CXI) Toxol .
Q" 6 OCOMe
O-c-o
AeO
AcO--
OAc
MO
OH i ^*^ OBi
( X M ) ( X I I I )
ECDYS0NE3!
Phytoecdysterolds, well known moltlxig horttnnea,
are another ln¥>ortant claea of naturally occurring organic 3 39 20
conapoxmd iso lated from Taxua spec ies , Taxus baccata ' " ' ,
T, Chinenaisj \ T.ouapidata and T. ^ l l l c h l a n a contain
«cdyaterone (XIV) while Taxlaterone (XV)# Ponaaterone A 24
and Makiaterone were ^Iso i so lated from Taxua cuapidata*
HO-
M [ ^ ^
M«[ 1 1
1 IJ OH
Ho (XIV) Bodyst^qne
Mo ^ O H
Me
Me
Me
Me
H b
- 6 -
LIQNINS t
25
The llgmlna are d i s t r i b u t e d in the r o o t s , h e a r t -
w o o d ^ ' ^ ^ ' ^ ^ , leaves ^^ and needles ^^ o£ Taxua s p e c i e s .
Taxlres inol (XVI ) / . i so t ax i r e s ino l (XVTI) and 3,4-divaniXlyl
tetrateydrofuran (XVIIl) were I so la ted from t h e heart wood o£
Taxus baccata^^ ' while roo t s* etems and needles of Tjaxua -25
wal l ichlana contain a d i f f e r e n t l i g n i n , m i s o l i o v i l (XIX).
HO {XV JO i s o t a x i r e e i n o l
. 9H2OH 0^^
(XVI ) Taxlresinol
- 7 ~
x °V" ::" HO
(xv l l i ) 3,4-dlvanlIlyl tetrahyclrofucan
OH I
ixix) i so l iov l l
GLYCOSIDES I
A novel glycoside^ taxitcatin (XX} wa« jtaolat<a« iBtmS
the l^voa o^ Ta^q baocat^ -. It» starueture as 3 ,S-*i«ei«McIk»rf-«
phenol glucosjde, waa confir|tned by i t s syntheaia from 3^§*^i*>
msthcocypheniS l and iTC^-acetobroHiogiucoae
EpimerJc ryanoqlucosJ 3e^ (R) -Taxiphyxlin (xxib^
and {s)-Dhurrlji (xxia) along with t r i g l o c h l n i n (XXIia) and
ieo- tx lg lcchtnl i j (xx i lb ) were q u a n t i t a t i v e l y determined
from Taxua baccata^^ using high preasure l i q u i d chromato
graphy. StereochenvJcal aspects of the b iosyn thes i s of these
gli¥2osides In Taxua cuspidata^^ have been discussed by
CH2OH
(XXIfc) (s)-Dhurrln
CHjOH H
(XXIb) (R)-TSa;xiphyllin
H H
HO2C/ V - /
HO2C—' 0-gJu, HO2C O-SJu.
(XXIla) •nriglochinln (xx i lb ) i t io t r lg locni i i ln
- 9 "
FIAVANOIDS t
33 Among the flavanoldlv datiatltwsxita of Takus are tha
blflavanoids of amentoflavone s e r i e s , scteteuflaVone (XXZIZ)
ginJogetln (XXV)^*, and sciadopiteln (XXVI)^^ wttre lao lated
from the leaves of Taxus baccat^a '>••>*, an^ Taanttli CHpidata ,
Sequolaflavone (XXXV) was a lso Itolated frcm T. baocata 34
(XXIII) R, R- - R « H; R. - MB 3 4 2
(XXIV) R.
(XXV ) R,
Rj - R^ » H; Rj » MS
R^ - H; Rj - R3 * Me
(XXVI) R- " H; Rj - R3 - R4 Ms
- to-
(1) NATURAL BIgLAVAMOIDS
BlfXavanolda, dimera of mano£lavanolda ncty be
c l a s a i f i e d tn to t h r e e ca t egor i ea t
(A) Bla ry l type
( B ) Garclnla and Taiwania b l f lavanolds
(C) Blaryl e the r typa
The formatIon of a l l t jT^ of n a t u r a l l y occurr ing
blf lavanoid may be eocplalned in terma of aocidative ooi;f;llng 36 of two chalkone uni ta .
<A) BI/ilYL ryPE OF BIFI/.VANOIDS» In t h i s c a t e g o r y Of
bl f lavanolds , two monoflavanold unl ta a r e l inked through
A or B r i ng .
Tha ainentoflavone qioup? This groui) i a repreaanted by
<I - 3 ' , 1 1 - 8 ) ItnRad bif lavonea (XXIII to XXXDC).
flavanone - flavonea (XL t o XLi l ) , biflawanonea (XLIII , XLIV)
a'l'l b J lu teo l ln (XLV).
~ Jti -
R R2 R^ R* R5
H
H H H
(XXVII) Amentoflavone^'^"^^ H H R H H
(xxrv) sequolaflavone*^"^' CSL. H H H N
( X X V I H ) B l l o i b e t i n * ^ * ^ H H H Cli3
(XXIIl) sot«t«ufl«von«**''*^ H CH3
(XXIX} Podocarpusflavone • A H H H H CH-
(XXV) Glnkgetin**'*^'*'' oij H « CH3 H
<XXX) l o o g i n k g e t l n * ^ " * ^ ' * ^ H H H CH, CH3
(XXXI) Podocarpsuf lavone - B* cai, M H H CTL
(XXXII) I - 4% II - 7 - Dl -o - nethylanMttofla- H CH H CH. vcate
H
(XXXIII)I - 7, II - 7 - D± - O -
methylamentoflavone'*'*^ *^3 * ^ H H H
(XXXIV) Heveaflavons"** QHJ CH3 H H CH3
(XXXV) icaya£lavonB*®'^° H CH^ H H CH3
(XXVI) saladopity8i4i*^"^^'*^'*''<M3 H H CH3 CH3
(XXXVI) I - 4 ' , I - 7 , I I - 7 - if l-o-methyl amento- (sn^ cH, H CHJ H flavcne
(XXXVII)I - 4- . It - 4' , I -- 7, " IT - ;-r«^jra-u-TOethyl- CHj ' CHj H. CKj CHj uttiont ofalavoniS^
<XXXVIil) Kttx*-0-i<«thyl-mTWtfito- OH, CH, CHL a i - Cai.
- 1? -
OH O
(XXX23C) 1 - 4 ' , I I - 4 ' , 1 - 5 , I I - S , I I - 7«I^mt«hydrcKy - I * 7-
o-tnethyl, I - 6-C-methyl £1 - 3 ' , I I - 8 ? bl£l«von«^^
R'O
R- R^ R R*
(XL) 2,3-Dihydroamentoflavone^*'^' H H H H
( X L D l I - 4 ' , I I - 7 Dl-oHmethyl-a/
3-<lihyaroani«ito£lavone^*'^' H CKj H CHj
(XLII) 1 - 4 S II - 4 ' , I - 7-^fri-
o-msthyl -t, 3-aibyfiroan«nto- CH H CM- CHj
flavoiM
HjCO
OCH,
- 33 «
R (<HII ) 1 - 7 , 1 - 4 ' , II - 4' --nrl-o-fnethyl
1 - 5 , II - 5, II - a'-Itihydraocy ^ - 3% . 1 1 - 8 7 bifl^vanone*^
(XLTV) 1 - 7 , 1 - 4 ' , 11 - 4 S II - 3 ' -t*tra-o-*wt;hyl I - 5# II - 5-<5ihy- CHj
(Xtv) 8Uut«oli4i
The A.<j thi8 flayong proig)i ititoiw ar* ( 1 - 6 , H-6> linkod
biflavwioldc aotapriBinq blflavonea (XWI to h) flavanonyi «
(IiTI).
m o
li ~
(Xl.'v ' t A ' lAt iUf 1". ,60
( X L V i l ) I - 7 - o - r T e t h y l a q a t h i a
{ X W I I I ) l - 7 , i i - 7 - D i - o - m e t h y l -
a g a t h i s f l a v o n e
(XLIX) I I - 4 * , l - l - D i - o - m e t h y l -
a g a t h i s f l a v o n e ^ ^ ' ^ ^
( L ) I I - 4 ' , 1 - 7 , l l - 7 ~ ' r r l - o -4 4^
n t e t h y l a g a t h i s f i a v o n e
CH3 H
H
H
CH3 CH^ H
CH3 H CH^
CH3 CH3 CH3
( L I ) R h u a f l a v o i . e 63 a
( L I I ) R hu« 11 a v a i w n e 63b
- IS -
T t e C u | y e s s u t l 9 v o T j e u r o u p g i T h i s g r o u p e o m i f i s e s / " ! - « , 1T-B7,
l i n k e d M f l a v o n e s ( L I I T t o L I X ) / f i a v o n o n e - f l a v o n e (U<) a n d
b l f 1 a v a n o n e { L X I ) .
( L i n )
( L l V )
(LV)
( L V I )
( I V I I )
C u p c e s s u t l a v a n o n e
X-4 • -o-Trtethyl c u p r e s s u f l a - H 65
v o n e
I ~ 7 - o - f n e t h y l c i t p r e s s u f l a v o n e ' CHj
I - 7 , I I - 7 ~ D l - o - n i e t h y l - CH ^ , 6 0 , 6 6 ^
I - 4 V l i : - 4 ' , I - 7 / l l - 7 ~ D l - o - CH. 44C
m e t h y l c u p a r e s s u f l a v o n e ^^^ j ,
( L V t l l ) l - 4 ' / l I - 4 ' / 1 - 7 , 1 1 - 7 -
Trl-<s«.niethyl C u p r e s s u -
flavoJW!
(LDC) T e t r a - o - n » e t h y l C u p c e a s u -
o r H
o r CM-,
CM.
M
H
H
CH3
H
CH3
013
H
CH3
H
H
CH3
H
CH3
H
CH.
R
H
H
H
H
U
H
CH3
H
CH3
f l avon t t 66
16 -
OH 0
(IX.) Kesuafierrone'^;^ 63cl OH 0 (DC;) Meauaf errone-A^-*'^
Thfe Robuataflavone giroupt This group la represented by robua-
taflavone (1X7.1) ; a ^1-3', 71-4^^' linked biflavOfte and I t s
methyl ethers which are not coirpletely characterised.
The ^c«<ten«Mi UAvmwia qroMP* A / * l - * * II*^? linked
blfXavanone, aiKoedimeaflavenoi^e (Dc l l l ) lathe only nisjrtoec
o£ th is group laolated ao far.
~ 17 -
(LXIXI) SuQoedaneaflavanone 63c
<B) OhRCmiA AND TAIWANIA BIFIAVAN0ID3«
GARCPJIA BIPIAVOWOipS;
F i r s t i s o l a t e d from o a r c l n i a inorel ls 68 t h i s gro\ip
comprieea / ~ I ~ 3 , I I - « 7 Unload £ lavanc«y l - f l avones ^UKTV,
IXVl, LXVll7, b i f lavanonea / " L X I X t o UQCI^, fXavanone-chromon©
IISOCV) and blf lavariold g l j c o e l d e a (LJCV, DCVIII, u a i v ) .
itJCIV) Volkenelflavon©^^'"'^''^ {» BCH-IIl«tali)Otaflavc3n«)
(LKV) Spicataside^'^
(WCVI) puki^etixi*'®'''*'''^ (»DOH-JCT w«areXlof lavcane J
• H
i-IXjllUl?.
H
H
H
OH
- 10 -
(LSCVII) I I - 3 • -o-methyl fukuge t in
(DCVIII )FuktQ JLside^*
73 H
B-4)-<rluc,
0M«
OH
OH 0
(IX3X) OB-la .75
75
(UCX) GB-1
(IXXl) GB-2a
(LXXII) GB-2^
75
(UCXIII ) I l - 4 • -o-methyl-GB-2
(Kola f l avone)
76
(LXXIV) Xanthochymusside 72
H
OH
H
OH
<M
H
H
H
H
H
H
H
OK
OH
OH
H
H
H
H
CH3
OH ^ - D - g l u c OH H
(tx<V) 1 - 4 ' , T-5 , I I - 5 , 1-7, I I -7-Peaj ta hydroocyflavanone
/ ~ l - 3 , I I -B7 Chroinone'^'''
- 19 -
laiwiJila Bif lavanolds; These a re / " l - S • , 11-37 l inked
bif]avonea (LiOCVI to LXXVIII). Taiwanla flavone (LXXVI) was
f i r s t synthesied by Moljrieux e t e i . ^ ^ * in 1970 and i t a 7flto
occurrence in na ture was reportedj seven years l a t e r .
R1 R2 R3 7fi
(LXXVI) Taiwan la flavone H H H
(L>or"/ll)i-7-oHTvethyl taiwanlaflavoni CH, H
o r
vone
H
ll~7-o~r(\ethyl t a l w a n l a f l a - H CH^ H
(LXXVTll) I I - 4 ' , I -7 - .Dl -o - ine thy l - CH^ H CH-
taiwan i a f l a v o n e
o r
I I - 4 ' , T T - 7 - D l - o - m e t h y l - . H CRj CH^
t a i w a n I a f l a v o n e
(O) Bj>^Vl'_J-'MiL TYrE_ OF BIFIy\VAMOTD3;
Thfse b l f l ava i i o Jds hav^ / " l - 4 * - o - l I - « 7 d i r r y l
eMier l i n k . Sev«»ral bi f l avone (X:<XTK to L;:>1XVT) of tn:.b
- ?0 «
group and one flavanonyl - flavone (DOCXVII) occur In nature.
Hinoklflavone (UCXIX)
(LXXX)
(liOQCl)
(LXXXii) Cryptomerln-A
79
46 Keocr yptoinsr In
80 laocryptomarin
e i a
82 (IJOCX I I I ) Chamaecypar In
(IXXXIV) 1-7. I I - 4 ' - D l - o - i n e t h y l -hlnolclflavcme 81b
8 la (LXXXV) Cryptomerln - B
(UOOCVI) 1-7, I I - 7 , I I - 4 ' - ' m - o -foethyl h inokl f lavone **
H
M
H
H
H
H
H
H
R*
M
Me
H
H
Me
MS
H
HA
R-
H
H
H
H
H
H
H
H
R'
H
H
M»
H
Me
H
Me
fte
R"
H
H
H
M9
H
hte
Me
Me
HCYrYQv-^,^"V«
OH O
OH
( 1 ^ ' . A A V I 1 ) i~2,S-^iit\y\iJ >>i.inv-iK*ti«.' '>»ie'
- 21 -
rpG Oehnaflavone groupt Mentaers of th i s group are
^ 1 - 3 • _o- i i -4_^ linked diaryl ethers and cons t i tu te the
f i r s t eoeatnple of natural ly occurlng blflavanolds In which
neither Of the A - ring I s involved in the IntArf lavonyl liidc.
Only three confounds (LXXXVIII to XC) with t h i s tjrpe l inkage 83
have been i so lated so far .
(UCJOCV^
(IXXXDC)
le (-osi)®^ •
I«-4 • -o-fl»thylochna-flavone ^ ( - OSIl)
R 1
H
H
R 2
H
H
R'
H
m
R*
H
H
1 R
M
H
(XC) 1-7, 1-4 •-Di-o-insthjl-83
ochnaflavone(« OSlIlJ
H Me Me H H
or
1-4 •, n-7-Dl-o-{nBthyl- H ochnafl«vone(- osill)®^
H Me
- 22 -
/ — l i ^ ' / . .
Acetone and tx ich loroe thylene li&ve be«jn used for
the ext rac t ion of the b l a r y l and b l a ry l e ther type, of
biflavanoids'^'*^' ^^, from defa t t ed rnater ia l . Chioroform
has been used for the extracticwi of Garcinla b i f lavanolda '
Crude Crysta ls of b i f iavanolds may be obtained by solvent
treatment or by sKtract ion with d i l u t e a l k a l i and subsequent
a c i d i f i c a t i o n / ^
Fear the p u r i f i c a t i o n of Crude b i f lavanoids Colunn
Chromatography on s i l i c a gel and magnesivini s i l i c a t e have
S4,57b been used with g r e a t e r stKceas . ' Good separat ion on
polyaniides and polyvinyl x^yrrolldone have been repor ted . '
For moni t^ inq the separat ion on coluims of adso rba i t , TIJC
i s eiTfjloye<2» T'- o vcmat widely used developing systems for
TU2 on s i l i o a gel a r e toluene /• e thy l farmato / forntic acid
(5 : 4 : 1 ) and benxen*--' / pyr id ine / formic acid
(36 ! 9 : 5) , Biflavanoids can be sei3=»rated in l a r g e r
amcunt nsix\q p repara t ive Tl.-cp'^'^^' v i s u a l i z a t i o n of
bif lavanoid.^ j s ef£«3ct-ftd e i t he r b-/ spray reaq^»rtt or in UV
l igh t .® ' '
Countercurrent distr ibutica^ has be-sn used for the
separation of isomeric biflavoneo in two phase systet-ne.
( i i i ) ipyynX'Pic^-Z'iou AND STRUCT'-JRE ELUCXDATION
Methyl e thera ^nd a ce t a t e* are usua l ly pc^.p<-u.>sd frort
t h e paro i i t and parf-.i^illy iif-th-flat-^xl n a t t i r a l b l f l a v a n o i d a .
C h a r a c t a c i s t l o s h a d e s In UV l i g h t and R- v a l u e s on 'WC,
p r o v i d e f a i r l y a c c u r a t e method of I d s i t i f l c a t i o n of t h e
t y p e of b i f l a v a n o i d s . fh,, flj-.a! i d e n t - x f i c a t i o n I s dune
by cottari.Bcn of fch>5 comiyinH-yir \j:fh a u t h e n t i c s a n p l e ,
CO"ChrcHi»tograpny, mi>:«3 m.Ps . of thfe s u b s t a n c e aisd
I t s d e r i v a t i v e , UV, m, NMl and mass s p e c t r o n e t r y a r e omploytad
f o r t h e s t r u c t u r e -St l tKidat lon, S t r t x j t u r e s can f i n a l l y b e
suppor t ed by s y n t h e s i s .
U l t r a v i o l e t spoct-Tc -'-"^j---/ has bo&ti, foo/vJ '.o b*.«
e x t r e m e l y ueefu.' £or t h e i-x>A- i<m of p h e n o l i c -^i-.^p-'
v i l s t r i n c t i o n o± b l f l a v a n o l d s it'om t h e i x fSTonosK-Cs and 4?
£--h,;-droxi'bifi,aVjnoi»'?.s show ati^tni^ heiid a round H»t-C cs^"'
v h i c h h e l p s In t l » detor tat i ia t i i '* ' 3f Sh.- ^JXk•cc<yiatior.
43 p a t t e r n .
PROTOn MAGNETIC RESC tAWCE GFECmOSCOPys
Proton magnetic; r e s o n a n c e apectroacc^jy i s one x>£
t h e roost coir«cw\ly used techrii<3(u«s of s t r u c t u r e deters j i lnut ion
of b t f l a v a n o i d s . ^ * P m data, of some r o p r e s e s i t a t i v e b i f l a v a -
24 -
noids of d i f fe ren t groups a re l inked in tabl® - 1. Bensawasi-
ind-uced s h i t t daV.a haa be»n \J3»:M exterts lvely for tii@ d i a t i n c -c, 3 88
t ion of bif lavanoida of cliffertsn-t group® «' ' ' -*• Pararra<|net,ic
induced shift® wifch Eu (FOD)- provides another potent ia l ly
useful technique for the determination of linkages and t l ^
dist incti(»i o£ di f ferent methoxy protons in methylated
bifXavanoids.®^'^^
- 25 -
TABI - la
PHR dat« of amentoflavone hexamethyl ether (XXXVIII); 44b
6 5
HaCO
OCH3O
(XXXVIII)
OCH,
3" r i"
Asalgnadl Position Chemical s h i f t s of Protons ( r-scaX^}
H -
H -
H -
H -
H -
H -
H -
H -
H -
o;i
6*
2 '
2 '" , 6 '"
5«
3 ' " , 5 " '
6"
3 , 3"
e < j
05) - • ? . • «
OM - 4 ' . 4 '
2 . 1 0 ( d , d ; J - - « H a , J j - S H S )
2 . 1 6 ( d ; J
2 . 6 0 ( d , J
2 . 8 8 ( d , J
3 . 2 4 ( d , J
3 . 3 8 ( a ) •
1 . 4 2 ( 9 )
3 . 4 a ( a )
J . 5 2 • » )
i^"h ( < 1 ;
' , ^ ' 1 1 , <?
1 V
n
*
•
a
' ><
3HS)
911s)
9Has)
9Ha)
.- IH3f>
i '.f 3H e-.tfCh*
i 4; 3ii c*K,U>
ll 1 5 / -IH <;<ji,:i/
a e- Kingle t* d w d o u b l e t jsjux^truia run in CJ', i , • ^ *' T, a .-iS A*k jrUf-rnctJ T ^"s<!ur«i
H3CO LYl-}"^ o
-rT OCN.
\ ;~'c 1J
OCH,
<3 ci
!j \ - / OCH,
Assigned P o s i t i o n Chemical s h i f t of ProtonsCV 3 c a l « )
H — 2 / 6 j 2 ,6
H - 3 \ : ' . : / " , 5 ' "
OCIx.,~ £ ' , 5"
OCIU- 7- 7"
2 . 7 0 (d^ J » 9H25)*
' ?3 id} 7 « 9025)
' » 8 5 J a # 6H)
6.3.4 ( s ^ oH) '
1 .23 (fc, 6K)
S « s i ng j l e t , d = ' d o u b l e t . Spectrum run* i n CDci, a t 60 m ; TM9 a s i n t e r n a l s t a n d a r d » r 1 0 , 0 0 .
H3CO
OCH^O
OCH,
(XCII)
Assigned P o s i t i o n
K » 2 ' . 6 '
K ~ 2'" , 6 ' "
H ~ 3 \ 5-
H - 8 tt* ill
H - 3 , 5
H - 3 . 3»'
OCHj- 5 "
CCH3- 7 , 7"
CCHj- 4 ' , 4 " CCH3 5
C h e m i c a l s h i f t o^ P r o t o n s ( f' S e a l s )
7.A2 \A, J ^ 9Hz)
2.63 ( d , ' j » 9H2j)
2.S»9 ( d , J a» 9HS)
3 ,09 ( s )
3 .22 ('&, J « 9H2)
3 . 4 7 , 3.49 (6, IH each )
5 .95 Ca^ 3H)
6 . 1 2 , 6.14 < s , 3H each )
6 . 2 2 , 6.24 ( 8 , 3H each )
6 .41 ( a . 3H)
t « s i n g l e t , d » d o u b l e t * apectr imt run in cr>' l^ a t lOO MCI TMS as zm i n t e r n a l s t a n d a r d « T KX.OO.
P.'R d«3ta of RhusflavoHe hf>pta rmt-hvl cthc-r ( x - I l i )
6 .,
HXO ^ 4'
pCHgO
OCH-
(XCIII)
A s s i g n e d F o s i t i c m •
H . 2 ' " , 6"'
H - ji
H - 2 . 6
H - oC
H - 3 , 5
H - 3 ' , 5
H - 3"
H - 3*
H - 6"
OCH3-. ^"
OCK3- *" '
OCH3- *
OCH3- ^' OCH^- 4 ' , 7"
OCH-- 6*
8 *» a lnglQt; <3 t=
Chemical
2.46 2 .50 2.57 3,03 3 .11
3 .06
3 ,38
3 .42 3 .48 5 , 9 0
6.2:/ 6 . 2 0
6 .15 6.07
6 .60
d o j b l e t .
( d .
( d .
( d .
( d .
(d ,
( d . ( s )
( s )
( s )
( s ;
( s ; ( a ;
( 3 ;
{ 9 ;
S h i f t s of
J = 9H?.) J =16H2)
J = 9Hz) J =16Hz)
J « 9Hz)
3H) 3H) 3H) 3H) 6H)
1H)
Protona( r .;ouie),TMS«lC
- 29 -
TABLE - l e
P^R d a t a of Rhusf lavanone ( L I I ) S3b
(L I I )
Assigned Pos i t l cm Chemical s h i f t Values o£ Rrotana ( T" a o « l « )
C« - 5^ 5"
H - 2*, 6 '
H - 2 ' " . 6 ' "
H - 3 ' , 5*
H - 3 ,5
H - 6 " , 8
H - 2 . 2"
H - 3 , 3"
- 2 . 5 7 , - 2 . 4 3 {a# IH e a c h )
2 .59 (d,- J » 9Hz)
2 .74 ( d , J - 8Hz)
3 .12 ( d , J « 9Hz)
3 .21 <d, J » 8H2)
3i>93. 3 .88 ( s , IH e a c h )
4 .52 ( d , d; J • 12Hz, 4H2)
7 ,20 - 6 ,77 (m, 4H)
s => s i n g l e t , d« d o u b l e t , ro « m u l t l p l e t .
- jr* «
TABIE - If
PW Spectaruro of succedaneaflavwie ( IKI I I ) 63c
6' 5'
5'" 6
(ZXIII)
J^asigned Posi t ion Chanoioal s h i f t of Ptotoaa ( T sca l e )
CH . 5 , 5"
OH - 7 , 7»
GH - 4% 4«"
M - 2 * . f>\ 2'" ,'b'"
H - 3 ' , 5 ' . 3 ' " , 5 "
H *" o# W^
H -• 2, %"
H - i , 3 »
- 2 . 3 5 (8» 2H>
• ^ . 4 7 ( 8 , 2H)
0 . 3 8 ( 8 , 2H)
2 . 6 2 ( d , J - knz, 4H)
3 . 1 4 ( d , a m BHZ^ 4H)
3 . 9 5 ( 8 , 2H)
4 . 2 3 - 4 . 6 7 (m; 2H)
6 . 6 7 - 7 . 3 0 (in, 4H}
8 •» 8 ing l«?t , d - d o t i b l e t , in « i t i u i t i p l e t , DMSO-cl, acJ s o l v e n t .
• 3t •
TABLE " Ig
*H NMR d a t a f o r Ta lwan ia f l avone heocamethyl e t h q r (3CCIV)^^
6 5 H^O
3 - 2"
OCH,
( x c i v )
Aaaigned E^oton Chamical S h i f t valutta of P ro ton ( T S e a l s I
H - 6 '
H - 2 '
M - 2 ' " , 6 ' "
H - 5*
H - 3 " ' , 5 " '
H - 3
H - 8 , 8*
H - 6« 6*
2 .22 ( d d , J « 9Ha, 3HZ>
2.29 ( d )
2.64 ( d , J - 9Ha)
3 .08 (d )
3 .25 (d*,. J • 9He)
3 .47 (« )
3 .46 ( d , J • SKz}
3.49 i d , J • 3H»)
3 .64 (d« J - 3K2}
3 ,69 ( d , J • 3H2)
9 » a i « g l © t , d • d o u b l e t / dd » dowbie dovslJlet.
- 32 «
TABLE - Ih 88
vm. data for hlnokiflavone pentamethyl ether (XCV).
OCH-
(XCV)
ASSIGHMEUT
H - 2 ' , 6'
H - 2 , 6
H - S"
H - 3 % 5 ' ** HI
H - 3 , 5
H - 3 , 3 -
M > 8
H - 6
Olio -
• m mmtimtt
Speatxxm t-^n f lO.CO.
»7-
<I
, 4
• ^oublttt. in c o c i ^ a t
CHEMICAL SHIFT VALUES ^ ^ a l « '
a. 12 ( d , J - 9H»*)
2 . 2 0 ( d , J - 9 H B )
2.95 ( 0 )
2 ,98 ( d , J « 9Ha)
3 . 0 6 ( d , J - 9HZ)
3 . 3 8 , 3 . 4 1 ( s , IH each)
J5,45 <d, J • 2Hz)
3 U 3 ( d / J « 2H2)>
6 .06 » 6 .09
f
100 hHsi TMS a s an i n t e r n a l standard m
- 3 i •
TABLE - l i
PMR data for Penta-o-methvl-ochna£ldvone (XCVI) 83
H3CO
OCH3
.(XCVI)
Assigned Posl t icm Chetnloal S h i f t , T ScaX* • ' " « I I IIr i w n
H - 2"' , 6"
H - 6*
H. - 2*
H - 5 '
H - 3 ' " , 5"'
H - 3 , 3 -
H - 8 , 8«
H - 6 , 6«
OCH3-. 5 , 5"
2 .19 (d* J • 9Ha)
2 .28 (dd, J - 9Ha, 2.S Has)
2 . 3 0 ( d , . J " 2.5 HZ)
2 .89 ( d , J • 9HZ)
2 .99 ( d , J - 9Hz)
3 , 4 1 , . 3 . 4 5 ( s , IH each)
3 . 4 6 , 3 .49 ( s , IH each)
3 .66 ( d , 2H, J « 2.5 HZ)
6 .08 ( 8 , 6H)
6 .12 i.B, 9H)
N
s $ i
H H
H H
I
3
I »-*
H
V M
M H
H H
i
o:
H
" V H H
I V M
m H
H
f M
O H
o
30 •ON • ^
r^ O «^ * I in —•
5o
• » : » in w
S3 r4tft • •
^ f%
OHO H o a o Sorto » ^
i . 5 - i ^ *^.^*:J.% mo'tnoi'
r4?lo X
SB*? S
CM M •
« a O.
33
§••£
m o*
5
U
7 a
K 10
^ «•) in i?
<o O • #
333 lt<0«0O
H r i n j T O r< »-l»>«WJ tH • • • o • • • • f*! ?
333 ;p • «^o 3 • « * « O
^ • ^
^^
0 • i I
?!
«
^4
I
s 1 '8
34
%» «4
- 35 -
^^C-NW SPECTROSCOPY OF BIPIAVANOIDS
A number of papers demonstxating the Utility
o£ C-NMR for the atrusture elucldaticn of the blfleva*
76 91 92 92 nolds have' appeared recently. ' ' * Wagner at al,
13
have dlacuased the c-NMR spectra of various C-C llnJcsd
blflavanolds. The assignments of the slgnaJLs In the
^ C-NMR spectra of blflavanolds have been achieved on
the basis of off-resonance/ proton coi;qpled spectra and
by analogy with published data ot oonpounds with similar
funoticmal groups on the basis of chemical dhlft of the m
Signals and their multiplicities in the offHresonanoe
spectra^ i t was possible to determine the laterflavmoid
linkage in blflavanolds. This method proved equally
inportant for the location of atethcocy substitution directly
in a naturally occurring mathylated blflavanolds. C-NMI
data of some blflavanolds of different C-C lijikage are
given below.
36 «
283 1160
OH
102-8 126-3 H 6 0
(XXVII) Amentoflavone
128-6 u«.2 93-7 i / 3^t57-0 ^ 1641 / \ 161-3
l o S f 102-8 !28-2 116-2
(XLVI) A g a t h i s f l a v o n e
J7 -
9 5-7 '^SS
01
94-6
165 2 - * ! ' ' ' ^ l l
IOI-2-y^N^ Ju96-4
/ O H 0
/ 12 81
^ ° - Y ^ _ J ^ 1 j l29?\
^ - U 9 6 ' 3 J ^ V " "
i02rif^2\ f ^ 0 '2 8.|
1 ^\ 1 1292 \ -
I28'I
H5-3
A '57-3
>^OH
"^ ns-a
y^OH
115-4
(LII^ Rhusflavonon*
12 8-1 114-6
128-1 115-9
(LXiv) -VtoikcwalfUvofs*
- 38 -
94-0
163-7
9A ' '
128 3 1)61
(LXII) R<»k)i*staflavone
(
127-9 U6'l
21 •7-^^
161-3 ^ ^ ' 0
(LII I ) Cijpreasuflavone
94 7
165-3,
42 2 128-3 115-4
(LXII I ) Succ«dan?ia-f3«vanc«e
~ 39
mSB SPECTROSCOPY OF BIFLAVANOIDS
Moat o£ th« reported mass speotral dst« i» on
pormsthylated d«rlvatiyea of 0 - «nd C~ Xinksd b^flava'*
nolda.'*^*'^®*/''®'''^''^'-®^ The two ekeletorte of C-C llnJwwJ
blflavanoids/ generally, j^olloi# the fragrosntatlon pattern
of monoflavanolds giving alndlar A- and B- ring fra^mcnta
(PigtHre 1-5) . . In addition/ molecular ion ( M*'*) and lonS
such aa^ ^ - HT*", ^ - CHaT ' /^ - 2CH3J7 and M** or
j£^37* a»e GoKiTionly bbaerved in 0~ and C- linked' b i f l^ya-
nbida . (lOaJble -2),. The o« linKa<^ biflavanoida tend to
undargo fiaaicm on both aidea of the 0 - linkage to y i e ld
lona which undergo further fragmaitation (retro-DielaWvl^er
reaction} to form uaual A- and B- ring fragmenta ( t iguce - 4) .
- . 4 0 -
Some conmon MS fragnianta from Q~ and C- linlcad
blf lavanolda (S t ruc tu re s of th« fragment* arm glvati in
Figure 1-4);
• • • • . ^ ^ - — I • • » I I I . . , _ , . — - . J , • • • • , , . . . . . ^ ^ . - n - !• , - T -I •••• ' I f f • • I 11 I - I 111 I - II u r n 1 ^'1 • — - i r ••• • . ! • • I n • i l l II t [
FRAGMENT 0 CUPRESSUFLAVONfiO AMI;NTOFLAVONEO AGATHISFLAVONBO HINCKIFlAVONB IONS ^ HEXMEIHYI^. 0 HEXAMETOYL damKAMEWXh .A WSNThy^'THXh KlIflBR
0 ETOER{XCI)'^ ^ ETHEft(XXXVIII)^'E'IHER<iOCIl)**" ETHER(XCVtIir*
M^*
^-ir Z^ T 157^
/M - 307*"'
/S - 467^-^
^1
1
"2-^
A *•
M^Va or
622 ( iqo)
621.(38)
607 (8)
592 (16)
576 (4)
US (1)
132 (14)
135 (26.)
-
311 (14)
622
621
' 607
592
576
245
132
136
. ISO
^ 311
(100)
(31)
(33)
( 8 )
(10)
(5)
(3 )
(16)
(3)
(5) .
622 (90)
621 f l7)
• 607 (54)
S92 (40) . * •
245 '(22)
-
13b (65)
-
311 (100) •
60a ( i 9 )
607 (12)
^93 (3«>
578 (11)
-i
132 (8)
135 (19)
-
3 U (22)
29? {29)
« 41
HgCQ
H3C0 r-III CM
^k 132(3)
H3PO '"/x §76(10)
i ••
fM-30j,B92(8>
M3CO
" A 135(16)
- 42 '
OCH '3
^^^° ^ ' J 245(22) (490 apptort of mft 245)
)CH3 0
M*, 62 2(90)
H3CO OCH
OCH3O
m/2 31 f (100)
OCH,
m/j 135 (65)
r i g . 2» MS fragments from AgathidfXavone pwHhefchyJ
~ < 3
1 OCH,
OCH
H3CO . 0
»f. M . 622 ((00)
J "
1 '" ^
^ ^ 0
rrV - * " > • • •
-0-°°r^
"»/ 24'5ilO
V Q
'f . I
0* • ^ "*/z m 020)
£M-4ea*>76 (4)
f M-i/, 621 C38) rM-r5/.607(8>
rM-30j;S92 08)
- #<
H,CO
fH?'
«lg» 4t M« «» ,^'mtkt^ »•!
^{z 167(7)
M;a i2 C59)
OH
m/z I07CI8)
H3CO
>H 0
">/-• 446(100)
\ r
H3CO
•' /z 2 8 4 ( 5 )
•OH 5
-H
'"/z 2fl3 ( 7 )
ether (XCVIII)^^
4C.
The f l r a t b l f lavanold syn thes i s was performed! by 96 Nakasawa, " who synthes ised gtnkget in (XXV) by the
Condensation of 3 * - iodo - 5 , 7 - 4 ' - trlmethoxyflavone and
8 - iodo - 5 , 7 , 4 ' - trlmethaxyflavone by the Ullroann method*
using triraethyl formamide In the presence of ac t ive aojjpwr
pqwder. Lat ter on, syn thes i s of cupressuflavone ( L l l l ) , O*^ QCs ^f\
cypiTessuflavc^ie hexari^thyl other (XCI)# ' ' agat^il«f le»vo *a
hexamathyl ether <X1CI1) ' , Permthylated d e r i v a t i v e s o*
fuku^etln (LXVl) and Pentamethyl etheor of ochnsflayone (3QCV1J 7 8 a fs "9 *
were repor ted . Mol-ynewt e t e l , obtained / l ^ 3 , I I - 2 / " '
and ^ - 3 , I I - Sjy'- l inked b l f lavanoids by*the ox ida t ive
coupling of aplgenln w^lth the addi t ion bf Potassium ferricy*nidte,
The j / l ~ 3 , I I ~ 3 j7 linked bi f lavones have now been i s o l a t e d 78b , lOOa ,
from na tu ra l source . However,, P e l t e r e t a l and
NatraJan e t al^^^*^ f a i l ed to obta in n a t u r a l l y occurlng b l f i e -
vones by oxidat ive diweiMl8atic«i i reactlons.
D I S C U S S I O N
47 -
Const i tuents o£ Taxus v/.^llichi.ana
Prom the acetone e x t r a c t of the leaves o£ Taxua
vallichi-ana Zucc. (Taxaceae), amantoflavone, .mono-o^nwehyX
amentoflavone, di -o-methyl amantoflavone (two de r iva t i ve* )
and Sciadopltysln have been iBola ted . Araentoflavone and
Scladopltysln were cha rac t e r i s ed by s p e c t r a l s tudied and
other cons t i t uen t s were i d e n t i f i e d by TIC ( i . e . oonparison
o£ parent biflavcxies and t h e i r methylated d e r i v a t i v e s with
the authent ic satniplea).
2 3»4
The occurrence o£ antitumor taxanes , molting honaotiaa ' ^
alhal61ds and other phys io log ica l ly Innoportant compounds * In
T;axus st imulated our i n t e r e s t in Taxua s p e c i e s . A nurrber of
bif lavonea of amentoflavone s e r i e s have been repor ted from 33 • . . - 35 „ > 3
Taxus baccata ^ Taxua Cuspidate a leb & Zucc , Ecdysone #
taxanes^ and l ign ins^^ have been I so la t ed from Taxus wall lohlami
Zucc. (Himalayan Yew) and there i s no r ^ x s r t of b i f lavanoid
c o n s t i t u e n t s . Taxus walllchiarfa leaveac were inves t iga ted for - • • iHBii > » • • • • 1 1 ^ • rmt»
bif lavanolds and amentoflavone, mono«o-methyl and di-o-roethyl
amsitoflavone and Sciadopl tysln were I so la ted and charao te r i««d .
The coarse ly powdered fresh leaves of Taxus wajUUchiana
Zucc. co l lec ted from Royal Botanical Garden, Godawari,
Lall tpur (Nepal) were cxt-ractod with acetonf. The acetone
e x t r a c t s w. -«=. pur l f l e ' i by !jolvf>nt truntnirnt f< 1 5cweti } / coJu'r.
aisrotoatoqr^ >hy (ailica qe l ) and a. ye l lov soJ f'? f rist- wan
obtained wu; 'h responded to the usual flavaiiolds coXcir t e s t .
Thin layer SirotrMtogj a;jruc «?:>?&ri~'i'-«'-A«i Cusir.i a i l l c - ; qeJ ,
BFP, 3 6 J 9 : 5 (levelcping aoivent systeso) " of the tiavonc-idic
mixture revealed the presence of tour bands label led as W - I
(R^ 0.18) , TO-II (R^ 0 .34 ) , W-III (a-R^ 0.55, b-R^ 0.58>
and 1W-IV (Rj 0,73) cocreapondlng to amento£lavone tuanor d l -
and tri.-o-n»thyl anentoflavone, respect ive ly .
The flavanoidic mixture was then subjected to coluim
chromatography using s i l ' c a gel and e lu tad with benzene^
benzpne-ethyiacetate ( 9 : 1 , 8t2, 7»3, I t l ) and then f ina l ly '
with mthylacetate, TW-IV wae obtained ijt pure form with b^neeHei
ethylacetate (9:1) as e l u ^ i t . Rest of the bands TN~I to Tir-IZI
were pres«it in the fractions benaene-ethylSKjetate (8»2# TiS,
I t l ) and ethylacetate . These fractions were contained and each
band was separated in pure form by preparetive TLC ( s i l i c a Qei*
BPF, 36:9:5) . Aft«r establ ishing the homogeneity by chromato
graphy in d i f f ero i t develqpers, such as benzene-pyridine-
formic-acid (36«9i5) , Toluene-ethyl formate-formic acid
(TEP, 5:4:1) , and itoluene-pycidine-acetic-acid (TPA, 10:1:1)«
each fraction was methylated separat^ely. All of them gave
same methyl ether which was identical wiidh authentic sample
- A 9 -
of anxantoflavone l''»xamethyl etfv?.r <R va lues , m.p. , m.n.p
c h a r a c t e r i s t i c fiouresc':-nt in UV l i g h t ) .
W«~l on ace ty l a t i on with ace t i c aiihi'drlde and pyr id ine
gave W lA which wvas cJiaractexieed na arrtentoflavone bfocaacetate
on cotrparlsori! of i t s NMR spectrum with authent ic san;* l e . Tlie
minor fractions TW-li, TW-IIIa and TW-lllb could not be i8oiat«<3
in manageable quantity for their spectral s tud ies . TW-II
corr««pond to nr>no-o-methyl amentoflavone while W - I l l a and
TW i i i b are conparable with di-o-methyl amentoflavpne.
The examination of vn-iv and I t s methyl eth^r indicated
i t to be trl"0~iT»ethyl amerstoflavcaie. I t was then ece ty la t ed
with, pyridine and ace t i c anhydride, on usual work up and
crys ta l l i za t ion d£ acetylated product (TW-rvA), colourleaa
needle l i k e cryetala (m.p. 240-41') were cjbtalned. The
H NM» spectrum of TW-rvA integrated for pct>tan8 of the three
mettKaKy groups and three acetooey groups. l t » MMR data of
W-rvA and sciadopltysin tr iacetate are given in Table - 3 .
- 50 -
TABLE - 3
Chemloai s h i z t o« Protons o£ TM rVA and Sciadopltyirln
t r l a c e t a t e .
Assignment TW rvA
H - i - e
H-I-6
H*-II-6
K - I - 2 ' , 6'
H-I-§ •
H - I I - 2 ' , 6 '
H - I I - 3 ' , 5 '
H - I - 3 , I I - 3
OMe / (Ac
I -A', IX - 4'
1 - 7 , 1 1 - 7
1 - 5 , I t - 5
3 . 1 8 (d ,J«2.5H»)
3 . 3 8 {a ,J«2.5H?)
3 . 0 0 ( a )
1.97 " 2.07 (q)
3.C6 (d,J-9H2)
2.54 (d,J»9Mz)
.3.18 (d,J»9Mz)
3 . 3 7 , 3 . 4 0 (a)
( 6 . 2 5 , 6 . 1 5 )
C6.20) 7 .97
Sc iadopl tygl i i txX ac«tAt«
7 . 5 5 , 7 .59
3.15 (d,<J«2»Sim)
3 .44 (d,J-2.SHis)
3 .04 ia)
2.04 ~ 2*10 icT/^
2»eS (d,J«9H«)"
2 .61 (d ,J-9H«)
3.23 (d»«7-9Hs)r
3*22, 3 .43 (a)
( 6 . 2 7 , 6 . 1 7 )
( 6 . 2 2 ) , 7 . 9 7
7 . 5 4 , 7 .59
s a a i n g l e t , d«doublet , q»»quarJ:ifet speotruip run in CDCX, a t
60MH2, TMS as i n t e r n a l standard!- f 1 0 . 0 0 . Figure m
parenthsses show che«nical s h i f t o f methfoxy pro tons .
The N?i" spectrun) of TW rvA was i d e n t i c a l with
sciadopltysin t r iace ta te . Thus the structure of TW IV
waa assigned as 1-5, I1-5 , I1-7 trihydroxy, 1-4' , 11 -4 ' ,
1-7-trl-o-methyl amentaiflavcme (sc iadopltys ln) .
K3CO
OCH-
soiadopitygln
-E.^\-P-_EJ*-i_H_EJ^-AAJ'-.
•- 52 -
All rn.ps were recorded on Kofler hot j r l rmscopic
3tage and asca uncorrected. Thin layer chron«itocfraphy wei.«
perfortned on s U t c a ge l G(BDH). N^« spectxa w«re recorded
on 60 MHz instrument, chemical s h i f t s a re eKpress*'*^ In Y
soaXe r e l a t i v e to T 5S as i n t e r n a l s tandard . All the reagent*
u«ed were of 'AHAIAR' grade Except formic ac id (E.f-Mrcrk).
KXIRACTION OF BIFLAVC^OIDS FROM I £ A V E 3 or TAXUa WiUjlCHliiim.
ZUOC.(TAXACpAE)t
Dried and Powdered leaves of l ^ t t s v a l l i c h l a n a ^ i cc .
( 1 Kg> were completely exhausted with petroleum e the r
(40-60*). The e x t r a c t s were concentrated f i r s t a t a tmos
pher ic pressure and then under diminished p r e s s u r e . An
o i l y green res idue l e f t behind gave negat ive t e s t for
f lavanolds .
The pe t ro l t r ea t ed leaves were coinpletely exhausted *rlth
bo i l ing acetone t i l l tha e x t r a c t was almost c d l o u r l e a s . The
co[A>ined acetone extrswst were concentra ted , a dark green gunroy
mass obtained was refluoced with petroleinn e the r (40-60*),
benzen, chloroform, success ive ly t i l l the solvent in cash
eacb was almost c o l o u r l e s s . , The res idue l e f t behind was
then t rea ted with bo i l ing water . The inso luble mass was
disaolved in alcohol and d r i ed under reduced p res su re .
-53-
A yellow so l id res idue (2 g) thus obta ined, responded to
usual flavanold coloxjr t e s t .
PxArlflcdtion g£ Blflavanutd Mixture by Colunri Oironigito^rdphyt
A well s t i r r e d suspension of- s i l i c a gel (100 g) In
dry petroleum e ther (40-60") was poured i n to a colufm
(ICX) cm long and 50 imi In d i ame te r ) . When the adsorbent
was well M t t l e d , the excess petroleum e ther was allowed
t o pass thtough the coliuin. The yellow s o l i d r e s i d u e (2fl)
adsorbed on s i l i c a gel was added to the colunn. The coltmn
was eluted with organic so lvents success ively in the order
of increasing order of p o l a r i t y . (Table - 4 ) .
TABLE - 4
Solvent Natute of Product
1. Petzx>leum e ther (40-60*)
2. Benzene
3 . Behzene-Ethylacetate (9 :1)
4 . Benzene-Ethylacetate ( 8 i 2 , 7i3 & I j l )
5 . Ethyl ace ta t e
Oreenlsh gunnroy mass
Green waxy products
Yellow so l id
Yellow- Solid
Brownish yellow so l id
Fract ions 3 - 5 gave p o s i t i v e colour teefc ior
f lavanoids . They were checked by TLC, 3rd was in puae
forro while 4 th area 5 t h were mixtures and they w^re corrbin*^.
Separation of Blflavanolda mixtures by y repara t ivg layec
Chroma tog raph; ''»
Using a th in l aye r spreader (Desaga, Heide lberg} ,
g l a s s p l a t e s (40 x 200 cm) were coated wi th a wel l s t i r r e d
sufl^>en8lan of s i l i c a gel (35 g in 70 ml of water for two
p l a t e s ) to g ive a iayor approocimataly O.S im in thic)cneas*
After drying for 2 hrs a t room temperature« the p l a t e s
weore ac t iva ted a t l i o - 120* for 1 hour.
Thft conp lex l ty of the b i f lavanoid mixture obta ined
a f t e r p u r i f i c a t i o n by colutm chromatograptq' was examined
by TLC using the following solvent systems:
(a ) Ben asene-pyridine-formic acid (BPP,. 3 6:915 )
(b) Toluene-Ethyl for ma te-formie ac id (TEP, 5*4 »1)
(c) 'Toluene-pyrldlne-acetic ac id (TPA, 10»1»1)
In solvent system ' a ' the spo ts were compact and the
d i f fe rence in R# values were so marked a6 to make i t the
developing solvent system of choice for p repa ra t ive TIC.
solut ion of b l f l avonyl •mixture ( f r ac t i ons 4 th & 5 th ) » • *
in pyridine was applied to plates with the help of mechanical
55--
applicatcjc (Desage Heidelberg)/ 2 ctn from the lo*#er edge of
the p la tes . The plates mounted on a stadnless s t e e l £r«si«
ware plated in a Desage g lass charrber (4S x 7'>. x 25 cm)
containing 500 ml of the developing solvent (BPP, 36t9ta)*
When the solvent frorat had travelled 16 cm frora the start l im
l i n e , the plates were taken out and dried at roons fcecnperature.
The position of the bands were inarked in UV l i g h t . The B»i1e«d
pigment 2Bone8 were scraped as separate bands and eXuted Witli
dry acetcme. The eluent in each case was d i s t i l l e d off luqMl
on treatment with^ %raiter yielded ycillow preolpitat* . I t «ms
f l l terecl / washed with water and dried. The homogeneity of
the pigments was again checked by TIC using the dif ferent
solvent systems. The components were labe l led as W-I
{Rj 0.18, 50 n^), TW-ll (R^ 0 .34 ) , W - l I l (a-R^ 0 .55 , b-«^9,89}
and W-IV (RjjO.'ja, 200 Big)
W>l^ Metharlatlont
W - i was methylated with dlRiethylstai^iata and
anhydrous potassium carbonate In dry acetone on water
bath for 6 hrs, Refluxing continued unt i l i t gave a
negative a le . Fecl^ t e s t . I t was f i l t ered and the residue
evaporated to dryness, itie yellow residue l e f t behind wa*
treated with petroleum eth«r and than dissolved In chlocofcc*
- S6 -
and washed wjth water. On TIC ejeamination* the methylated
product was found to be amentofl^vone hexaimsthyl ether.
^•6. 11-^^ I - ? . Jl-'l. 1 - 4 S Il-4*.^:^aaeetcocy
/ f ^ • , l l - | 7 blflavone ( w » ) t -
A mixture o£ I'lf I (30 mg), pyridine (1 qil) and eoetSd
anhydride (2 nd) waa heated on w«t«r bath for 2 hoore to glim
an aoetate which alo^ly oryatal l i svd from CKTl - BtoH as
coloutleaa needles (20 rag) m.p. :M2 - 4^)
2,74 (d^H, H-I-8); 3.16 (d, lH, H-I -«) | 2.99(i , lH# HrXX<4>l
4.02 (q# 1»» H-I-6') l 1.97 (d , IH, H-M* )l ^.Sa (d.lH,H-I-«* h
2.51 (d,2H, H-I1-.2', 6 ' ) ; 2.94 ( 4 , 2H, H-II-3*, 5')> 3 .34 , 3 .3*
( « , IH each, H-I-^, 11-^ )l 7 .72 , 7 .77, 7 .95 , 7 .99 , 7 .55 , 7 .5 t
( a , 3H each, QW-1-4• , 11 -4 ' , 1-7, i i - t , 1-5, II-6 reapectl irely) .
TW-II and W - I H
TW-11 and TW-III (minor firaotiona) ware methylated
separately by lufual method and. id^ i t i f l ed as amantoflAvone
hexamettiyl ether.
This fraction was methylated aa usual a»d TIJC
mam^(0iX<M Kiatisd hexaiaethyl etbar of Msentxtflavone.
S7 t.
I^f 11-^, Il-T-TrlacetOiKv. 1-4'« 11-4'. I~7-tri'Ot«t|HyJ|.
/ I - 3 ' , II - 87blflavon« (TW IVA):
1W iv (50 0)0} wa« ac«tyl«f:«il with pyridln* and «3*«iie
anhydride atKS an acetate (iw xvi } Mie obt«ined whioh
cryatalllBed fr<Mn CHfillj- EtOH a» oXoivleas needle« (40 09}/
in,p. 240 - 41 ' .
um (CD<a.3) I vamea in T acgg
3.18 (d, IH, H«-l-8)l 3.39 (d,lH, H-l-O; 3,00
(a, IH, H-lI-6); 1,97-2.07 (q, 2H, H-I^2S 6*); 3,0S (d, IH/
H-I-5'>; 2.57 (d, 2H, H-II-2S 6'); 3,l6 (d, IH, H-ir-3', .6')l
3.37* 3.40 (i, iK each^ H-l-a» 11-3)i 6.15, 6.25. 6,20 (•« »«
each, OMe -1-4', ll-4', 1-7 respectively)! 7.55, 7.59, 7.97
(a, 3H each, cue - i-S, il-5^ lX-7 reapeotlvely).
a i 1 li i 2 2 B. & S H X
SP -
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P U B 5; X C h T T O -J
1, The Const!tuent»5 of th'S h^'-.la-/an Yew - T - ug gail lcit iang,
Sixxr,, Naaaieesa Parveen, ll.H. Taufeeq, K.u. Khari/ Abstr.jc t
presented In the l 8 t Annual Confcuronce, The Islamic s o c i e t y
for Science and Culture ( India)« Apr i l 28-29, 1984.
2. The C<»istituent« of the Himalayan Yew - yaxus wal l ichiana
Z u c c , Na2aieen Parveen/ H.M. Taufeeq, N,U, Khan, E>cp<r<lufcla,
contnxjnicated.
