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TERPENOIDS
3.1.1 Introduction
Terpenes are the most widespread and chemically interesting group o f natural
products, possessing a carbon frame work comprised o f five carbon isoprene unit
arrangements. The monomeric unit is called isoprene as one o f the Nature’ s favorite
Q-1 Q Q
building block in terpene biosynthesis ' . For centuries, human beings have known that
volatile oils with a variety o f fragrances and flavors could be isolated from plants. They
occur in every part o f the plant and are called essential oils. They are o f great commercial
importance in perfume and flavoring industries. Many essential oils have been used
medicinally 99' 104. The chemical constituents o f essential oils have a large variety o f
structures. Such substances are o f interest because as was pointed out by Wallach in 1887
and reemphasized by Ruzika in 1935, the components o f the essential oils can be
regarded as derived from isoprene (2-methyl-1,3-butadiene). The ensuing theory that
these compounds (terpenoids) are biosynthesized from branched five carbon modules was
fist stated by Ruzicka and Stook in 1922. Ruzicka’ s theory has been one o f the most
fruitful in organic chemistry. It has inspired and guided incredible amount o f research in
structure determination and organic synthesis in vivo and in vitro. Beyond that much
research in physical chemistry in the areas o f molecular rearrangements particular has
been sparked and sustained by the monoterpene o f camphene series 105' 109. in their own
light, the terpenoids have long mirrored the development o f organic chemistry. For many
years they were an interesting subject for academic research. More recently with the
advent o f sophisticated methods o f isolation, separation and structure elucidation, terpene
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research has expanded greatly and has established these compounds as one o f the most
diverse and intriguing group o f natural products.
3.1. 2 Classification of Terpenoids
Terpenes often posses a carbon frame work consisting o f five carbon units. This
particular five carbon unit, known as isoprene unit is commonly presented by a symbol
C5. During the formation o f terpenes the isoprene units are linked together in head to tail
fashion and the number o f units incorporated in a particular terpene serves as a basis for
the classification o f compounds (Table-1). Thus monoterpenes are composed o f two
isoprene units and have the molecular formula C 10H 16, contains three isoprene units.
Diterpenes, C20H 3 2 , have four isoprene units and triterpenes, C 3 0H48 are composed o f six
•• 110-117isoprene units
Table-1:c . Name Parent Sub-classes Occurrence
10 Monoterpenoids G PP Iridiods Oils
15 Sesquiterpenoids FPP Abscesic acid Oils, Resins
20 Diterpenoids G G PP Gibberllins Resin, Bitters
25 Sesterterpenoids GFPP Resin, Bitters Heartwood
30 Triterpenoids Squalene Phytosterols, Cardenoloids, Saponins
Resin, Bitters, Heartwood, Latex
40 Carotenoids Phytones Green tissues Root, Fruit
O1
O
Rubbers G G PP Latex, Root
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3.1. 3 Nomenclature
Terpenes are named according to their biological source o f material. The names
may be derived on the basis o f plant genus, plant family, plant species, the local plant
name or some trivial name o f author’ s choosing. In the earlier literature trivial names
have been given to many natural products including terpenes because they were isolated
and described in the literature long before their structure were known. The following
operators designate the structural modification to the parent structure. A prefix indicates
to which enantiomeric series as a compound belongs e.g. cyclo, nor, homo, seco, friedo,
abeo, ent etc. I f the pefix are more than one, they are arranged in alphabetical order to
define the location and stereochemistry o f substituents. Some o f the most commonly used
operators to modify these skeletal are as follows: “Nor” is used to describe the
substraction o f methylene and the numbering preceding the prefix, nor is the number o f
atoms or groups eliminated. The term “Seco” is used to designate the cleavage o f a bond
between two atoms o f parent structure, while “cylco” refers to the formation o f single
bond between two atoms o f parent which results in the formation o f additional ring.
“Homo” is used to show the addition o f methylene to the parent structure. The operator
“Friedo” is used to designate the migration o f groups or atoms around a ring system
while “Abeo” refers to modification in ring size. Another term “Ent” is used to indicate
the inversion at all chiral centres implied in the parent structure 118"127.
3.1. 4 Biosynthesis
Isoprenoids the largest family o f naturally and commercially important
products, are synthesized ubiquitously among eubacteria, archaebacteria and eukaryotes
through
35
Table -2: Name and structure of parent compounds of central terpenoid pathway
condensation o f isopentenyl diphosphate (IPP) and its isomer dimethyl allyl diphosphate
(DMAPP). Two distinct and independent biosynthetic routes for the formation o f IPP
exist: the mevalonate pathway and non-mevalonate (deoxyxylulose 5-phosphate (D O X Y)
pathway). The evolutionary history o f the enzymes involved in both routes and the
phylogenetic distribution o f their genes across genomes suggest that the mevalonate
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pathway is connected with archaebacteria and the D O X Y pathway is connected to
eubacteria. Consequently eukaryotes have inherited these genes for IPP biosynthesis from
prokaryotes. In lower photosynthetic organisms like Chlamydomonas reinhardii and
Chloretla jusco use exclusively the D O XY pathway whereas rhodophyte Cyanidium
caldraum posses both the D O X Y and M V A pathway.
Mevalonate pathway
Mevalonate pathway is an important metabolic pathway that provides cells
essential bioactive molecules, vital in multiple cellular processes. This pathway converts
mevalonate into sterol isoprenoids, such as cholesterol, indispensable precursor o f bile
acids, lipoproteins and steroid hormones and into a number o f hydrophobic molecules,
nonsterol isoprenoids. These intermediates o f the mevalonate pathway play important
role in post-translational modification o f a multitude o f proteins involved in intracellular
signaling and are essential in cell growth/differentiation, gene expression, protein
1 11Qglycosylation and cytoskeletal assebbly ’ .
Mevalonate pathway starts from two molecules from acetyl CoA by Claisen
condensation to give acetoacetyl CoA. An aldol addition o f third unit o f acetyl CoA
yields 3-hydroxy 3-methyl glutaryl CoA (HM G-CoA) which is reduced by an enzyme
HMG-CoA reductase to mevalonic acid. Mevalonate kinase which catalyses the
phosphorylation o f mevalonate at C 5 and loss o f carbon dioxide and inorganic phosphate
gives IPP, which is subsequently converted to DMAPP. IPP and DM APP combine to
yield geranyl diphosphate (GPP). Further condensation o f GPP with DM APP gives
famesyl diphosphate (FPP). Further condensation with prenyl units gives geranylgeranyl
(GGPP). FPP can dimerises to form squalene (C 3 0), the precursor o f triterpenes.
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DOXY Pathway
The reassessment o f the isoprenoid biosynthesis in plants and microorganisms
was initiated in the late 1980s by studies performed independently by Rohmer, Sahm,
Arigoni and their respective coworkers. Experiments by Flesch and Rohmer 109,”
incorporating 13C-labelled acetate into bacterial hopanoids, revealed unexpected labelling
patterns in the terpenoid moiety o f the molecules under study. Originally, the existence o f
distinct acetyl CoA pools was proposed to explain how the anomalous labelling patterns
could arise via the mevalonate pathway 130, but other possibilities were also considered.
Later, the data were definitely reinterpreted in terms o f a novel pathway, the
deoxyxylulose pathway discussed below 111.
Independently, Arigoni and his research group studied the incorporation o f
various 13C-labelled glucose samples into the isoprenoid side chain o f ubiquinone by the
bacterium Escherichia coli ljl and into ginkgolides in seedlings o f the tree Ginkgo bilolia
m . The labelling pattern o f ginkgolide A observed after feeding with [ 1 3C] glucose (as a
mixture with unlabelled glucose). Five blocks o f two contiguous 13C atoms and one block
i t
of three contiguous C atoms were shown to be transferred together from the C-
labelled carbohydrate to the ginkgolide. The formation o f the hexacyclic diterpene from
the linear precursors involves a sequence o f skeletal rearrangements corresponding to
reshuffling o f the original Cs-moieties 133134. As a result o f one o f these rearrangement
38
39
processes, C-4 o f IPP. The observed block o f three contiguous 13 C atoms therefore
indicated that carbon atoms 1, 2 and 4 o f the corresponding IPP molecule stem from a
single, universally 1 3C-labelled glucose molecule. One o f the original direct linkages
between the carbon atoms is disrupted during the formation o f IPP, and subsequently
reestablished in the rearrangement process.
Intermediates and mechanism of the novel pathway
From the glucose-labelling data, it was concluded that the diversion o f 13C to
isoprenoids had occurred via intermediates in the triose phosphate pool. Initially, the
135groups o f Rohmer and Sahm proposed that dihydroxyacetonephosphate could undergo
an acyloin-type condensation with ‘ activated acetaldehyde’ . The resulting branched chain
carbohydrate was assumed to undergo a rearrangement resulting in the observed
reshuffling o f the three-carbon moiety.
Arigoni’ s group l3 6 l 3 7 5 however, suggested a head-to-head condensation o f
glyceraldehyde 3-phosphate and ‘activated acetaldehyde’ generated from pyruvate by
thiamine-pyrophosphate-dependent decarboxylation (Scheme-4). The decisive evidence
in favor o f this hypothesis was the incorporation o f the putative condensation product 1 -
deoxy-D-xylulose (13, R = H), which was shown to occur into the isoprenoid sidechain o f
ubiquinone in E. coli with exceptionally high efficiency. The carbohydrate was also
incorporated into ginkgolides o f G. biloba and into diterpenes o f Salvia miltzorrhiza
1W 1 'IQ
’ . The head-to-head condensation o f the two building blocks is now a universally
accepted mechanism, and Rohmer, Sahm and their coworkers l 4 0 ' 142 have reinterpreted
tiieir earlier isotope incorporation data accordingly.
40
11 COOH
H OPPOPP
F A) ?HOH
CH2OPTPP
CH,OP
OH13
OPP
Scheme-4: The proposed mechanism for the formation o f 1-deoxyxylulose 5-phosphate formation
[13, R = P 0 3H2] catalysed by 1-deoxyxylulose 5-phosphate synthase
Subsequent studies in different research groups confirmed the incorporation o f
isotope-labelled deoxyxylulose into terpenoids in several experimental systems. Thus,
incorporated into isoprene emitted by the higher plants Populus nigra, Chelidonium
majus and Salix viminalis, and into the phytol moiety o f chlorophylls in the red alga,
Cyanidium caldarium, in the green algae, Scenedesmus obliquus and Chlamydomonas
reinhardii and in the higher plant Lemna gibba. It is important to note that 1-deoxy-D-
xylulose (13, R = H ) had been shown previously to serve as a precursor in the
biosynthesis o f thiamin (15) 123' 125 and pyridoxol (1 4 )146’147.
13C-labelled deoxyxylulose was incorporated into carotene and the phytol moiety o f
chlorophyll in cell cultures o f Catharanthus roseus 143 into menthone by Mentha x
piperita l44, and into ubiquinone by E. coli 145. ‘H-labelled deoxyxylulose was
Scheme -5: The biosynthesis o f pyridoxol (14) and thiamine phosphate (15) from 1-deoxyxylulose [3, R =
H or l-deoxy-D-xylulose 5-phosphate [3, R = P 0 3H2]
O P P
13 2 1
Scheme- 6: Skeletal rearrangement o f 1-deoxyxyxylulose conducive to IPP formation
OPP
OPP
Terpenoids
R = P or PP
Scheme -7: 1,2-ketol intermediates in the biosynthesis o f terpenoids via the deoxyxylulose pathway
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Crosstalk between the deoxyxylulose and mevalonate pathways
Arigoni and coworkers had already noted in 1968 148 that 14C-mevalonate is
efficiently used as a precursor o f triterpenes and sterols in the plant Menyanthes
trifooliuta. Mevalonate was also incorporated into the monoterpene alkaloid loganin,
albeit with much lower efficiency. Similarly, 14C mevalonate labelled sterols from
various plants at high rates, whereas (3-carotene and the isoprenoid sidechains o f
chlorophyll and plastoquinone acquired only low levels o f the radiolabel I49. Four
decades later, these confusing data found a conclusive explanation when Arigoni’ s group
150 was able to show that both terpenoid pathways, the mevalonate and the deoxyxylulose
pathways, operate in higher plants. In G. biloba seedlings, sterols were found to be
formed preferentially via the mevalonate pathway, whereas ginkgolides are formed via
the deoxyxylulose pathway, as described in detail above. More specifically, Arigoni and
coworkers found evidence for compartmental separation o f the two terpenoid
machineries: the mevalonate pathway appears to operate in the cytoplasm, whereas the
deoxyxylulose pathway appears to be located in plastids.
The compartmental separation o f the two pathways is not absolute. Schwarz and
Arigoni 131 found that approximately 1-2% o f ginkgolides derive their isoprenoid
precursors from the mevalonate pathway. More recently, experiments using [1-13C]- and
[2,3,4,5 13C4]-l-deoxy-D-xylulose (13, R=H) showed that C. roseus cells predominantly
derive the monomers for formation o f phytol and carotenoids from the deoxyxylulose
pathway 151. On the other hand, 6 % o f the building blocks used for sitosterol formation
152are derived in the same system from the deoxyxylulose pathway . Generally, it appears
that in higher plants, steroids are preferentially formed via the mevalonate pathway,
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whereas as monoterpenes, diterpenes, carotenoids, phytol and plastoquinone are formed
predominantly via the deoxyxylulose pathway. The ‘ crosstalk’ between the two
metabolic pathways is best explained by the exchange o f metabolic intermediates
between the cytoplasm and the chloroplasts. The specific intermediate involved in the
intercompartmental exchange and the regulation o f the process is unknown.
Carotenoid Chlorophylls
HOT
Scheme-8: Crosstalk o f mevalonate and deoxyxylulose pathway in cell cultures o f Catharanthus roseus
Biosynthesis of Monoterpenoids
A ll the experimental evidences supports the view that isopentenylpyrophosphate
( 1 ) combines with 3,3-dimethyallylpyrophosphate (2 ) to give geranyl pyrophosphate. IPP
and geranylpyrophosphate (3) now serves as the precursor for monocyclic monoterpenes.
The mechanisms involved in ring closure are not certain but a favored one is via ionic
intermediate 153-155.
Biosynthesis of Sesquiterpenoids
Sesquiterpenoids are formed by condensation o f IPP (1) with D M APP (2) to give
geranylpyrophosphate (3). Further addition o f a molecule o f IPP ( 1 ) form 2E, 6E -
farnesylpyrophosphate (4), which has fifteen carbons atoms characteristic o f
IPP+
DMAPPTriterpenes
44
sesquiterpenes. These two steps are catalysed by the same enzyme, famesyl
pyrophosphate synthetase 156 which has been isolated from different sources.
The biosynthesis o f cylic sesquiterpenoids has been explained using hypothetical
routes with formal cationic intermediate, and is based on the cyclisation o f
famesylpyrophosphate or nerolidylpyrophosphate. Intra-molecular attack o f the cationic
centre formed in the solvolysis o f pyrophosphate one double bond that is allylic to this
group, followed by further cyclisation, rearrangements, deprotonation, could lead to the
formation o f a large variety o f compounds characteristic o f this group. There exists a
group o f sesquiterpenes formed by cyclization without the intervention o f
pyrophosphate group. There are mono- and bicyclic famesenes, which are formed in
analogues way to the cyclization o f diterpenoids and tritepenoids by attack o f an
electrophile on gemdimethyl double bond. One example is the formation o f albicanol
from 2E, 6 £’-farnesylpyrophosphate.
Biosynthesis of Bicyclic Diterpenoids
Bicyclic diterpenes also formed from C20 units derived from the cyclization o f the
GGPP or sometimes from its isomers. Cyclization o f GGPP is initiated by the protonation
o f double bond o f the starting o f isopropylidine unit. The type o f cyclisation occurs in
I C O
several steps. GGPP (5) first cyclises to give rise a bicyclic labdadienol pyrophostate
One o f the first characteristic features o f the diterpenoid substance appeared at this stage
i.e. the formation o f normal (21) and antipodal (22) A/B ring junction.
This may rise through different modes o f coiling o f the open-chain precursor on
the cyclase enzyme surface. Examples o f both series are quite widespread and there are
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even reports o f both series occurring along side each other in the same plant.
Furthermore, the vast majority o f the diterpenoids possess a trans relation between H-19
and C-10 methyl groups. Subsequent modification o f the labdadinol may lead one hand to
compounds related to manool and on the other hand to labdanolic acid and agathic acid
159,160 and its derivatives. In both series a new chiral centre is created at C-13.
Biosynthesis of Triterpenoids
Most o f the triterpenoids fall into one o f two broad classes: tetracyclic series,
structurally related to steroids and a very large pentacyclic class, including a number o f
skeletal types.
The cyclization o f squalene (7) can be promoted either by oxidative and non-
oxidative agents.
3.1.4.1 Oxidative cyclisation
Oxidative cyclization o f squalene give rise to tetracycles, which differ slightly
from each other in their cyclisation mechanisms. The damarenediols, euphol and
tirucallol are derived from 2 , 3-epoxy squalene molecule, held in active site o f the
enzyme in “ chair-chair-chair-boat” conformation, while lanosterol and cycloartenol are
derived from “ chair-boat-chair-boat” conformation.
3.1.4.2 Non-Oxidative cyclisation
The non-oxidative cyclisations are rare and occur from a proton attack at the 2, 3-
double bond o f squalene. In the non-oxidation cyclisation, the formation o f the various
skeletons could be derived from the “ chair-chair-chair-chair” squalene conformation.
46
3.1.4.3 Oxidative cyclisation and Non-Oxidative cyclisation
Both terminals o f the squalene molecule are involved in these cyclizations. The
biosynthesis o f some terpenes is explained by independent electrophilic attacks at both
terminals o f the squalene molecule 161. These two attacks are both oxidative and non-
oxidative.
47
Coumarins
3. 2.1 Introduction
The isolation o f coumarins was first reported by Vogel 162 in Munich in 1820. He
associated the pleasant odor o f tonka bean from Guiana with that o f colver, Melilotus
officinallis. The name coumarin originates from Caribbean word ‘ coumarou’ for the
tonka tree, which was known botanically at one time as Coumarouna odorata. Coumarin
is accepted trivial name for the compound whose structure ( 1 ). Although coumarin ( 1 )
was the first coumarin to have its structure elucidated, it was not the earliest to be
isolated. Vauquelin 163 in 1812 extracted glycoside from Daphne alpine. This was later
named as daphnin.
Neilson remarks the excellent review o f coumarins o f the Umblliferae. The
activities o f some o f the compounds as a fish poison and in increasing the fermentation o f
yeast l64. During the past two decades there has been marked reawaking o f interest in
natural coumarins. This has led to discovery o f these natural compounds in hundreds o f
plant species. However, much interest has also emanated from the wide range o f
physiological activity many display and from the characteristic fluorescence o f most
coumarins on ultraviolet (U V ) irradiation.
Structural types
A feature common to many coumarins is the presence o f isoprenoid chains,
frequently o f one, but often o f two or three units, attached to carbon atom o f the nucleus
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or to the oxygen atom or to both. The prenyl group may be found to be simple but it is
often encountered as the corresponding epoxide or vicinal glycols or in a variety o f
oxidized or skeletally rearranged forms (Scheme-7). Indeed in the field o f natural product
chemistry, coumarins offer the best example o f the class o f compound exhibiting the
greatest number o f biogenetic modification o f the simple isoprenoid unit. The structural
variations o f this type encountered in the natural coumarins are oxidative interactions can
lead to the hydroxyisopropyldihydrofuran moiety, which by dehydrogenative loss o f the
hydroxyisopropyl group, furnishes the furan ring. When nuclear prenylation o f the
umblliferone occurs at C - 6 this leads to demethylsuberosin, marmesin and finally to the
linear furanocoumarin psolarin. On the other hand alkylation at C - 8 affords the angular
furanocoumarin angelicin, via osthenol and columbianetin. Any oxidative cyclisation
may prevail forming hydroxydihydropyrans, which on dehydration produce the linear and
angular pyranocoumarins, xanthyletin and seselin, respectively.
3. 2. 2 Classification
1. Simple, which has implied coumarin ( 1 ) and its hydroxylated, alkoxylated and
alkylated derivatives and their glycosides.
2. Furanocoumarins, o f linear and angular types with substituents at one or both o f the
remaining benzoid positions and including dihydrofuranocoumarins.
3. Pyranocoumarins, the six membered ring analogues.
4. Coumarins substituted in the pyrone ring, such as 4-hydroxycoumarins and 3-
phenylcoumarins.
49
There after within each section entries are presented in following order:
a) Coumarins with one C-alkyl or C-aryl substituent, the order depending on the
position o f substituent on the nucleus and the chain length and oxidation level within
each substituent.
b) Dihydrofuranocoumarins
c) Furanocoumarins
d) Dihydropyranocoumarins
e) Pyranocoumarins
f) Coumarins with two or more C-alkyl or C-alkyl/C-aryl substituents.
3. 2. 3 Nomenclature
Most coumarins have been assigned trivial names, principally to make
reference to them easier while structural studies are in progress. Common suffix found in
trivial names such as ‘o f and ‘ one’ are quite inconsistent with structure in many cases.
The prefix ‘ iso’ is found to have such diversity o f structural meaning when used to
differentiate an isomeric coumarin from one o f the known structure that no clear
guidelines can be given enable one to deduce the structure o f the ‘ iso’ named coumarin
from a knowledge o f the structure o f the named coumarin. It is uncommon to find the
prefix ‘ oxy’ used when reference is made to the epoxide o f the naturally occurring
coumarin. A different approach was initially adopted in naming the aflatoxins, a group o f
highly toxic and exceedingly carcinogenic coumarins isolated from strains o f Aspergillus
flavus. Some trivial names have originated from the local names for the plant rather than
from the Latin name o f botanical source. Others have been derived from the geographical
location in which the coumarin sources were found growing.
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leading to the name 5, 6 -benzo-a-pyrone. With the passage o f time, the accepted correct
name has been variously, 5,6-benzo-2-pyrone, 2/7-1 -benzopyran-2-one, 2H-
benzo[b]pyran-2 -one and 2 //-chromen-2 -one.
52
53
Occurrence
Almost all the natural coumarins except few have been isolated from botanical
sources. Coumarins are distributed in all parts o f the plant; fraxoxide and isofraxoside
were isolated from most organs o f two Campanula spp. I65. Psoralen was located in the
seed coat o f Psolarea subacaulis seeds. Extensive study showed the coumarins are found
54
in endosperm and embryo o f the seeds. In contrast to these discrepancies, it is well
established that fruits are parts richest in coumarins. Fruits o f Angelica contain about 3%
o f coumarin compounds, their concentration increases during the course o f followed by
roots as for as high coumarin content is concerned. And then by stems and roots 166' 171.
The roots o f Astrodaucus orientalis and Daucus carota were the richest organs in
coumarins with contents o f 0 .8 % and 2 % respectively 172.
Coumarins were mainly present in the bark o f Periploca gracea and in the stalks
o f Conium maculatum (0.5%) and Malabaila dasynytha (1% ) 173. Heracleum
lehmannianum the amount o f coumarins was more than twice that o f stem, in
Peucedanum longifolium the reverse was true for its furanocoumarin content (1.35% in
stems) and (0.7% in leaves) l74. Roots o f Ruta graveolens contained relatively high
levels o f rutaretin and isorutaretin. Furanocoumarins isolated from leaves o f Angelica
archangelica differed in kind from those o f the roots 175. Six coumarins were obtained
from the roots o f Angelica dahurica. Moreover the leaves in contrast to the bark
contained traces o f phenolic compounds 176. Unripe fruits o f Poncirus trifoliata were
found to be rich in simple coumarins as opposed to furacoumarins, which were
predominant in ripe fruits ,77.
3. 2. 4 Biosynthesis
The benzopyran nucleus can be regarded as a derivative o f 2'-hydroxycinnamic
acid, formed by lactonization o f the carboxyl 2'-hydroxy functions. As such, the simple
coumarin nucleus is phenyl propanoid, having a C6 benzene ring linked to a C3 aliphatic
178side chain. It has been recognized that phenylpropanoids are most common derivatives
via the shikimate-chorismate biosynthetic pathway.
55
HO
M arm esin
HO
PsoralenO 'O
H O ' ~ O Dem ethylsuberosin
H O ' v O
U m belliferone
HO
O O o
Xanthyletin
HO
Osthenol
OH
O o
C olum bianetin
A ngelicin
Seselin
Scheme-13: Umbelliferone derived coumarins
56
Simple coumarins: Coumarins formed by plants originate via shikimic and chorismic
acids as well as phenylalanine and cinnamic acids with carbon dioxide being the ultimate
179 181source ' . The formation o f the phenylpropanoid amino acids phenylalanine and
tyrosine via this pathway and the conversion o f former to trans-cinnamic acid through the
action o f phenylalanine ammonia-lyase. The first step in the biosynthesis o f the coumarin
nuleus involves ortho-hydroxylation o f cinnamic acid. Brown 182-184 proposed the basis o f
tracer investigation that trans-cinnamic acid is the common precursor o f all coumarins
and that the ortho or para hydroxylation leads to the elaboration o f coumarin or the 7-
hydroxycoumarin.
Very early in studies on coumarin biosynthesis the trans isomer o f cinnamic acid,
trans-2'-glucosyloxycinnamic acid and its aglycone were implicated. Kinetic studies after
administration o f 14C 0 2 provided definite choice that trans-2'-glucosyloxycinnamic acid
• 1 is an intermediate in the formation o f coumarin . Glucosylation o f trans-2'-
hydroxycinnamic acid has been demonstrated in cell free extracts o f Morus alba.
Cinnamic acid to 7-oxygenated coumarins
The 2'-hydroxylation o f the branch point intermediate, trans-cinnamic acid, leads
to coumarin, so 4'-hydroxylation engenders the 7-oxygenated coumarins. The latter is
common to the biosynthesis o f lignin and numerous other phenolic products o f the
secondary plant metabolism. A significant role is played by glycosides in the formation
o f 7-oxygenated simple coumarins. Bound forms have been found to predominate in the
cases o f umbelliferones in H. macrophylla 186 and Pimpinella magna 187 and herniarin in
57
1 o o
Lavandulla officinalis . In the former case the glycoside is mostly 7-p-D-
glycosyloxycoumarin. In Daphne, 7, 8 -dihydroxy coumarin exists as both the 7 and 8 -
58
glycosides. In Cichorium intybus act in an analogue way on aesculetin 7-glycoside,
converting it via hydrolysis and trans glycosylation to the 6 -glycoside 189.
The conversion o f cinnamic acid via 4'-hydroxycinnamic acid has been shown by
tracer technique 190191. Furanocoumarins which are mostly umblliferrone derived were
also synthesized from both 4' and 2'-hydroxycinnamic acid in P. magna 192. Herniarin is
likewise formed via 4'-hydroxycinnamic acid 1 9 3 19 4 by a pathway in which trans-2 '-
glycosyloxy 4'-methoxycinnamic acid appears to participate.
Furanocoumarins: The origin o f the two non-benzoid carbons o f the furan ring from
mevalonate is now well documented. Such an origin was first indicated by, co-occurrence
o f simple coumarins, prenylated and the isopropyldihydrofuranocoumarins marmesin and
columbianetin l95. It is possible that the two carbons derive from C -l and C-2 o f an
isoprenoid precursor originating respectively from C-5 and C-4 o f mevalonate. Ruta
graveolens was shown to contain a particular transferase able to mediate the prenylation
o f position- 6 o f umblliferone by dimethylallylpyrophosphate, an intermediate well
known to be mevalonate derived, to form the naturally occurring demethylsuberosin
(DMS), 6 -dimethylallylumblliferone ,96. Requiring a divalent cation, this prenylase
(dimethylallylpyrophosphate: umblliferone dimethylallyltransferase) is highly specific as
to the length o f the isoprenoid chain, being inactive against geranyl and farnesyl 197
pyrophosphate and requires a free hydroxyl group at coumarin position-7. No prenylation
at position-8 , which would lead to angular furacoumarin. Prenylation probably involves
the formation o f a resonance stabilized phenolate anion with contributing structures.
59
Pyranocoumarin: Coumarins o f the xanthyletin, linear coumarins and seselin, angular
pyranocoumarin types share with psolaren and angelecin, respectively, a common
biosynthetic pathway except for the final cyclisation step. A non-enzymatic
transformation, DMS-dihydroxyxanthyletin, cannot be ruled out. Mode o f cyclisation
depends upon the pH at the synthetic site, explicable on the basis o f the formation o f the
carbocation, followed by loss o f proton.
Phenylcoumarins: The 3-phenylcoumarins or coumestans may be regarded as substituted
as 4-hydroxycoumarins. Coumestrol. 4', 4'-trihydroxychalcone-4'-glycoside, a known
60
precursor o f isoflavone, also was coumestrol precursor. Berlin studies on cell suspension
cultures o f P. aureus roots 198 studied dihydroxylisoflavone (daidzein), 2', 4', 7-
trihydroxyisoflavone and 4', 7- dihydroxyisoflavanone (dihydrodaidzein) as possible
coumestrol precursors. Scheme-16 entails the alternative route 2'-hydroxylation at the
isoflavanone (dihydrodaidzein). Scheme-17 provides the way o f coumestan synthesis.
The formation o f 9-O-methylcoumestrol from the pterocarpan could also
be justified.
61
62
Flavonoids
3. 3. 1 Introduction
Flavonoids are the diverse and widespread group o f natural products, which have
prominent position among the natural phenols. The name flavonoid is derived from
Greek word ‘ flavus’ , which means ‘ yellow ’ 199’200. Flavonoids are united by their
derivation from the aromatic heterocycle, flavone, which itself occurs naturally as a
farina in Primula plants. Some flavonoids are intensely coloured, e.g. the anthocyanins
and provide a wide range o f red to blue colours to flowers, fruits and leaves. Flavonoids
in general are universally distributed in higher plants. They also occur in mosses and
liverworts and rarely in fungi.
Besides colour, flvonoids have a variety o f roles in the growth and development
201o f plants. The biological importance o f flavonoids has been reviewd by Williaman ,
who listed thirty three different manifestations o f activity under the heading
‘bioflavonoids’ .
3. 3. 2 Classification
Flavonoids can be classified according to their biosynthetic origin. Some
flavonoid classes are both intermediates in biosynthesis as well as end products; these
include chalcones (the first C 15 structure to be formed from melonyl coenzyme A and p-
coumaryl coenzyme A ), flavanones, flavanon-3-ols and flavan-3, 4-diols. Other classes
are known as only known as end products o f biosynthesis, e.g. anthocyanins,
proanthocyanidins, flavones and flavonols. Two further classes o f flavonoids are those in
which the 2-phenyl sidechain o f flavanone isomerizes to 3-position, giving rise to
63
isoflavones and related isoflavonoids. Further isomerization to the 4-position gives rise to
neoflavonoids.
Flavonoids may also be classified according to molecular size. While majority o f
the flavonoids are monomeric, a significant are dimeric, trimeric, tetrameric and
polymeric structures have been described. Most biflavonoids are based on carbon-carbon
linking o f two similar units. The highest molecular weight flvanoids are the oligomeric
and polymeric proanthocyanidins, derived biosynthetically from flavan-3-ols.
Most flavonoids occur naturally associated with sugars in conjugated form within
any class may be characterized as a monoglycoside, diglycosidic, etc. Glycosidic
complexity is considerable and monosaccharides associated with flavonoids include
glucose, galactose, arabinose, rhamnose, xylose, apiose, allose, mannose, galacturonic
acid and glucuronic acid. Mono-, di- and tri-saccharides may be linked through a single
phenolic hydroxyl or may be variously linked to two or more phenolic groups. A special
group o f mainly flavone based C-glycosides occur in plants and those may additionally
be present as O-glycosides as well. Sulphated conjugates are common in the flavone and
flavonol series, where the sulphation may be on a phenolic hydroxyl or on an aliphatic
hydroxyl o f a glycosidic moiety.
3. 3. 3 Nomenclature
There are two parallel systems o f nomeclature, one based on trivial names such as
flavan and chalcone and the other based on systematic chemical names such as 3, 4-
Dihydro-2-phenyl-2//-l-benzopyran or flavan. Trivial names are employed extensively in
the flavonoid literature. Some names indicate the class o f compound e.g. ending ‘ inidin’
denotes anthocyanidin (pelargonidin), while the ending ‘ etin’ denotes a flavonol
64
(quercetin). Certain glycosides o f quercetin have related names quercitrin, the
rhamnoside; isoquercitrin, the 3-glycoside; quercimeritrin, the 7-glycoside. There is little
consistency in the naming o f flavonoids and many names derived from the generic or
specific name o f the plant source (e.g. tricin from Triticum, hypolaetin from Hypolaena
etc).
3. 3. 4 Biosynthesis
The main biosynthetic units o f flavonoids are the phenyl propanoid derived from
shikimate pathway; they are common elements o f other classes o f phenylpropanoids,
such as lignin, stilbenes and cinnamate esters. The sequence o f reactions converting
phenylalanine into the CoA ester derivatives o f substituted cinnamic acids was therefore
termed ‘ general phenylpropanoid metabolisn’ 202. The enzyme catalyzing the individual
steps are phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate CoA
ligase.
Phenylalanine ammonia-lyase catalyses the anti-elimination o f ammonia and the
(pro-35)-proton from I-phenylalanine to yield trans-cinnamic acid. The enzymes from
many plants also convert Z-tyrosine into trans-4-coumarate. The second enzyme o f the
general phenylpropanoid metabolism, cinnamate 4-hydroxylase (trans-cinnamate 4-
mono-oxygenase), catalyzing the 4-hydroxylation o f cinnamate to yield 4-coumarate.
Cinnamate 4-hydroxylase was first detected in microsomal enzymes o f pea 203.
Cinnamate 4-hydroxylase is a mono-oxygenase which catalyses the NAD PH and 0 2 -
dependent hydroxylation o f trans-cinnamate to give trans-4-coumarate 204’205. The
enzyme is highly substrate specific and is inhibited by the reaction product, trans-4-
coumarate 206’207.
65
Table-3: Different flavonoid structures
The CoA esters o f 4-coumarate and o f other substituted cinnamic acids play a
central role as a intermediates at the branch point between general phenylpropanoid
metabolism and the various subsequent specific pathways. The enzyme responsible for
CoA ester synthesis from various cinnamic acids is 4-coumarate CoA ligase. The basic
properties o f 4-coumarate CoA ligase from various sources are fairly uniform. 4-
coumarate CoA ligase is specific for the activation o f substituted cinnamic acids in the
presence o f ATP, M g and CoASH, with the respective cinnamoyl-AMP being an
intermediate in this reaction 208-214.
Some o f the specific pathways into general phenylpropanoid metabolism require,
in addition to 4-hydroxylation, further substitution o f the aromatic ring. The most
prominent additional reactions are hydroxylation and subsequent methylation in the 3 and
66
5 positions. Flavonoid specific methyl transferases have been found in various plants it
appears that general phenyl propanoid metabolism includes a ‘ general’ methyltransferase.
67
68
Aurone Isoflavone
A A
B D
AChalcone » Flavanone > Flavone
C F
y y
Dihydrochalcone Flavanon-3-ol ^ Flavonol
c
y
Flavanon-3,4-diol > Anthocyanidins
G
y
Flavan-3-ol|_|
->- Protoanthocyanidin
G
y
Flavan
Scheme -22: Biosynthetic pathways of flavonoids: A: Cyclisation, B: Oxidative
cyclisation, C: Bioreduction, D: Aryl migration, E: Dehydrogenation, F: Didroxylation, G:
Dehydroxylation, H: Polymerization
69
70
LIGNANS
3. 4. 1 Introduction
The lignans comprise a class o f natural products which are derived from
cinnamic acid derivatives and which are related biochemically to phenylalanine
metabolism. They fall into five major subgroups, and the nomenclature in use is neither
consistent between the groups nor even within the subgroup.
Many lignans show physiological activity, such as tumor inhibiting
podophyllotoxin. This specific activity leads to interference with cell division by two
different mechanisms in animals including humans. Some are active in suppressing the
central nervous system and inhibiting cyclic-AMP phosphodiesters, while others act as a
fish poison or germination inhibitors. In Chinese traditional medicine lignans are used for
the treatment o f viral hepatitis and protection o f liver.
The growing interest in the lignans and neolignans and increasing number o f
variations o f their skeleton make a rational for naming them a necessity. Robinson 215
recognized in 1927 a common feature o f many natural products was a C 6C 3 unit (i.e., a
propylbenzene skeleton) derived from cinnamyl units. In a review o f natural resins
91 f\Haworth proposed that the class o f compounds derived from two C 6C3 units with P,
P'-linked should be called lignans. The nomenclature o f the diverse range o f structures
classified as lignans depending largely on trivial names and i f necessary the appropriate
numbering derived from the systematic name. This resulted sometimes in alternative
numbering systems for closely related compounds with potential ambiguity in the naming
o f analogues. For example, in the arylnaphthalenes, the aryl group might be attached to
the naphthalene at C-l or C-4 depending on the location o f functional groups.
71
9 1 7In an extensive review by Hearon and MacGregor the different skeletal types
were consistently numbered although there was no correlation between them.
218 219Freudenberg and Weinges ’ proposed in 1961 that the C 6C3 unit be numbered from 1
to 9 and the second unit from 1' to 9' to provide a consistent numbering system. Thus a
lignan all had an 8 -8 ' linkage. In addition, they proposed the term cycloneolignans for
lignans with an additional ring.
The term neolignan was defined by Gottlieb 220 as including the lignans and also
related compounds where the two C 6C3 units are joined by other bonds (e.g, 3-3' instead
o f 8 -8 '). With the aryl naphthalenes the additional ring was formed between 7 and 2'
rather than 7 and 6 ' as proposed by Weinges 22'.
3. 4. 2 Classification
Lignans are classified into following groups
1. Lignan: I f the two units (I) are linked by a (3,p'-bond the parent structure lignan (II) is
used as the for naming the lignan.
r\ 7
3 2
(I) (II)
2. Neolignan: I f the two C 6C3 units (I ) are linked by a bond other than a p,P'-bond the
parent structure, neolignan, is used as the basis for naming the neolignan. The locants o f
72
the bond linking the two units are cited in front o f the name and with the second number
primed. Where there is a choice o f locants, preference is made in the order:
(a) position 8
(b) lower unprimed numbers
(c) lower primed numbers
3. Oxyneolignans:
I f the two units (I ) are linked by an ether oxygen atom and not directly bonded
together the parent structure, oxylignan, is used as the basis for naming the neolignan.
The locants o f two positions linked by the either oxygen are cited in front o f the name
with the second number primed. Where there is a choice o f locants, preference is made in
order to:
(a) position 8
(b) lower unprimed numbers
(c) lower primed numbers
7 8
3,3'-neolignane 8,3'-neolignane
73
8,3'-oxyneolignane 3,4'-neolignane
4. Cyclolignans: I f the carbon skeleton o f a lignan has an additional carbocyclic ring
which is formed by direct bonding between two atoms o f the lignan skeleton this is
indicated by the prefix cyclo- and the two locants which identify the relevant bonds. I f
there is a choice o f locants, lower numbers are preferred in the order:
(a) position 8
(b) lower unprimed numbers
2,7'-cyclolignane 2 ,2 '-cyclolignane
5. Cycloneolignans: I f the carbon skeleton o f the neolignan with the two C 6C3 units (I)
directly bonded has an additional carbocyclic ring which is formed by direct bonding
between two atoms o f the neolignan skeleton this is indicated by the prefix cyclo- and the
74
two locants which identify the relevant bond. I f there is a choice, lower numbers are
preferred in the order:
(a) position 8
(b) lower unprimed numbers
(c) indicated hydrogen
5',7-cyclo-8,3'-neolignane 6//-2,6'-cyclo-l ,3'-neolignane
3. 4. 3 Nomenclature
Lignans are a large group o f natural products characterized by the coupling o f
two C 6C3 units. For nomenclature purposes the C6C3 unit is treated a propylbenzene and
numbered from 1 to 6 in the ring, starting from the propyl group and with the propyl
group numbered from 7 to 9, starting from the benzene ring. With the second C 6C 3 unit
the numbers are primed. When the two C6C3 units are linked by a bond between 8 and 8 '
the compound is referred to and named as a lignan. In the absence o f the C - 8 and C-8 '
bond, and where the two C 6C3 are linked by a bond between positions 8 and 8 ' the
compound is referred to and named as a lignan. In the absence o f C - 8 and C-8 ' bond, and
where the two C 6C 3 units are linked by a carbon-carbon bond it is referred to and named
as neolignan. The linkage with neolignans may include C - 8 or C-8 '. Where there are no
direct carbon-carbon bonds between the C6C3 units and they are linked by an ether
75
oxygen atom the compound is named as oxyneolignan. The nomenclature provides for
the naming o f additional rings and other modifications following standard organic
nomenclature procedures for naming natural products. Provision included naming the
higher homologues. The sesquineolignans have three C 6C 3 units and dineolignans have
3. 4. 4 Biosynthesis
The lignans comprise a group o f natural compounds with their carbon skeletons
derived from two phenylpropane units together by at least one C-C bond between the two
central positions (P and P ') o f the two C3 chains. Both phenyl propane units are almost
The most popular explanation for the origin o f the P, P’ bond in an oxidative
coupling between two C6-C3 units. For this explanation the /?-hydroxystyrene radicals
form quinimethide (quinine methide) dimmers which, either directly or after
rearrangement to more stable structures, can be used by plants for synthesizing lignan as
well as lignins. This idea is in contrast with the following argument:
four C6C3 units 222-224
7 8
2
9
3 5 6
(I) (II)
22^certainly derived from shikimic acid '.
76
(a) One electron oxidation
x = ch3, ch2, och 3
R = H, OH, OCH3
III
II III
h i
IV
(b) Two electron oxidation
IV
Scheme-25: Dehydrodimerisation of P-hydroxystyrenes by either 1- or 2-electron
mechanisms. The quinomethide structures react further
77
(a) Some lignans are formed in nature with the oxidation level o f the p and (3' carbon
atom in a lower state than expected from such styrene precursors and from the oxidative
dimerization and polymerization process which usually give lignins.
(b) A ll known lignans show optical activity (sometimes this optical activity is masked
by internal compensation) so differing from the lignins and their degradation products.
(c) Trimeric or tetrameric lignans have not been found in nature; such polymers
should be expected as intermediates i f conversion o f dimeric lignans into polymeric
lignins occurs.
Scheme-25: Probable biosynthesis of some lignans
For these reasons Neish’ s hypothesis appears to be more likely, namely, that the
lignans are formed through initial stereospecific oxidation process o f /?-hydroxy styrenes
units by different enzymes to those active in the biosynthesis o f lignins; after such
dehydrodimerisations stereospecific reductive processes can occur. The sites for lignan
biosynthesis (oxidation-reduction sites) are probably separate from those involved in
lignification.
Podophyllotoxin isolated from Podophyllum hexandrum is a lignan o f therapeutic
importance, for its anti-tumour activity. Most lignans have been isolated from plants o f
the Magnoliaceae and Piperaceae families.
78
Gottlieb proposed the name neolignans for those bis-arylpropanoids in which two
C6-C3 units o f 4-allyl or 4-propenylphenols are joined together by bonds other than the (3,
P' carbon-carbon bond typical o f lignans. According to Gottlieb the neolignans are
formed through oxidative coupling o f the type shown, followed by typical degradative
processes o f the intermediate quinomethides (Michael addition, aromatisation), as
observed for the lignans. In the synthesis o f neolignans atleast one position o f coupling o f
two C6-C3 units is different from the P one. The units involved need not be o f the styrene
type, i.e. allyl phenols can be incorporated.
The neolignans probably arise at sites similar to those producing lignins. Enzymes
o f the same kind (laccases and peroxidases) act either on pairs o f molecules with a 4-
propenyl and/or 4-allylphenol structure (to give the neolignans) or on /?-hydroxycinnamyl
(to give products called lignols).
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