cardiac glycosides flavonoids anna drew with grateful acknowledgement for inspirational teaching...

Cardiac glycosidesFlavonoids

Anna Drew

with grateful acknowledgement for inspirational teaching received at

The School of Pharmacy, University of London

Cardiac glycosides

• Plant glycosides with specific action on heart

• Historical use:– to assasinate people, arrow poisons

• Historical sources:– South American toad skins, African plant extracts

• Modern use:– to treat congestive heart failure (dropsy)– aglycone structure important for activity

Sources

• Scrophulariaceae• Digitalis purpurea leaves (foxglove)• Digitalis lanata leaves – white flowers

• Apocyanaceae• Strophanthus vine seeds – Africa

• Liliceae• Urginea bulbs (squill) – Europe, India• Convallaria leaves (lily of the valley) –

also produces a volatile oil perfume

Active compounds

• steroid nucleus• AB cis-junction• CD cis-junction

– not planar

• C14 = 3y -OH

• C3 = 2y –OH– sugars attached

• C17 = lactone ring– classified into 2 groups

Cardenolides more common

opens in alkali

Bufanolides

• sugars:– 1-4 β-linked at C3 in various combinations– glucose, rhamnose, deoxy-sugars

• eg digitoxose, digitalose

StrophanidiolStrophanthus

Scillarenin (squill)

Extraction• large molecular weight molecules with sugars -> polar

– soluble in water and alcohol

• expensive – long process, solvents

Digitalis

• Scrophulariaceae family– foxglove - biennial flowering plants

• cases of poisoning rare• natural emetic if eaten in excess

– Digitalis purpurea leaf – purple, British• -> Digitalis Tablets B.P.• -> Tincture of Digitalis B.P.• commercially grown Holland, E. Europe• NB no extraction for these products

– Digitalis lanata leaf – white, Mediterranean

• used for manufacture of pure glycosides• ie digoxin, lanatoside C• commericially produced Holland, Equador,

USA

Chemistry of D.lanata• compounds belong to

cardenolide series– 5 membered lactone ring– approx 96 compounds

• [1930-1950 Stroll worked on structures]

R1 R2 Names 1y 2y

H H digitoxigenin A A digitoxin

H OH gitoxigenin B B gitoxin

OH H digoxigenin C C digoxin

OH OH diginatigenin D D diginatin

H formylester gitaloxigenin E E gitaloxin

*

* Acetyl group confers crystalline properties - makes compounds more easily isolated

[i] Digitoxose• sugar found on primary glycosides of D.lanata• glucose on the end of a chain of O-linked digitoxose sugars at C3

• during harvesting and drying enzymes can remove acetyl groups and the end glucose

– hence drying method needs to be followed or glycosides degrade further– after collection dried as rapidly as possible at 60oC, stored in airtight containers

protected from light (contain no more than 6% moisture)

• expect about 10 compounds from D.lanata– important ones:

• Digoxin “Lanoxin” – Wellcome – 0.25 μg white tablet• Digitoxin “Digitalin” 0.25 μg small pink tablet• Lanatoside “Cedilanid” 0.10 μg – less well absorbed but used for rapid digitalisation• Others not marketed, used experimentally

Some cardioactive glycosides from D.lanata:

Ref: Trease & Evans

Chemistry of D.purpurea

• Steroid cardenolides– contains 30 glycosides, 6 main ones– only has 3 aglycones

• Purpurea 1y glycosides– do NOT have acetylated digitoxose third sugar

• but these are found in smaller quantities

– called ABE series

Aglycones 1y 2y

digitoxigenin A digitoxin

gitoxigenin B gitoxin

gitaloxigenin E gitaloxin

[ii] Digitalose

• found in both species

• only strospeside important as emergency injection for heart attacks – quickest acting cardiac glycoside

Assay of Digitalis B.P.

• required to contain not less than 0.3% total cardenolides calculated as digitoxin

• important to guarantee reproducibility of products (drug dosage)

• narrow therapeutic index• can cause cardiac arrest• slowly excreted, bound to serum proteins• long term therapy for patients• patients tend to be older, weak

• Digitalis B.P. tablets

– crushed dried leaves -> green tablet– contain 30 glycosides each with different

onset, action and excretion profiles– in different amounts

• influenced by growing conditions• (temp, water, sun, drying process)

– assay for each glycoside as accurately as possible – dilute effects by adding grass

• Two ways:

[1] Biological assay

• British method - inaccurate but safer

• Tincture of extract of leaves or tablets• diluted with saline so alcohol <6%v/v• guinea pigs (6 test, 6 control) x 3 =36

– expensive but can average results• measure volume injected into vein of leg/foot before

heart stops beating• monitor heart rate via ECG

– or open chest wall and watch inserted needle with flag on move– Better to watch ECG – have to differentiate from death from too

large an injected volume• trained staff required, can calculate potency• assay acceptable within 80-120% error margin (not that

accurate)

• Disadvantages– inaccurate, expensive– injecting material IV (avoiding absorption,

excretion)– end point is death– toxicity test not therapeutic assessment

• Advantages– assessing some biological activity– safety mechanism

[2] Chemical assay

• Problem: 30 different glycosides – can measure them accurately but may not correlate with therapeutic activity of drug

• Make a tincture (with alcohol)• decolourise with lead subacetate• extract glycosides by partition with CHCl3• evaporate to give residue (containing cardiac glycosides)• hydrolyse with HCl to remove sugars leaving aglycone

– residue contains gitoxigenin and digitoxigenin (AB series)– gitaloxigenin -> gitoxigenin when acid hydrolysed

• Colourimetric assay to separately determine material amounts(i) total aglycone

• purple colour with dinitrobenzoic acid and alkali

(ii) digitoxigenin only• green colour with FeCl3 + acetic acid

– can substract answers to work out• Digitoxigenin (A series) content• Gitoxigenin (B series) content

• Advantages:– precise method (reproducible 2%, standard error 5%)– unqualified staff, quicker

• Disadvantages:– doesn’t correlate with biological activity– only estimating approx 60% therapeutic material

• BOTH methods used in industry

Flavonoids

• mainly O-linked glycosides• occur in plants, lichens, moss• those in free state and glycosides largest

naturally occurring group of phenols• aromatic, based on γ-pyrone moiety• can get several forms of flavonoids depending

on nuclei

flavone isoflavone flavonol

• often yellow (flavus Latin – yellow)

• known for a long time

• interest in them for– anti-inflammatory (and analgesic) properties– anti-allergic effects– antithrombotic, vasoprotective properties

• decrease capillary fragility• phlebitis – changes in vessel walls in extremities

-> plasma leakage -> oedema– mainly due to high oestrogen, sometimes in males

– tumour inhibition promotion– protective for gastric mucosa

• sugars:– glucose, rhamnose, arabinose, xylose– 2-3 attached to phenolic groups in middle of

structures

Examples

(a) Rutin (Vitamin P)– from Fagopyrum esculentum

(buckwheat)= rhamnoglucoside of quercetin

(b) Hesperidin (‘citrin’)– from citrus industry= hesperetin (methyl eriodictyol),

rhamnose, glucose

Isolation

• easy• water and alcohol soluble• give brightly coloured

solutions• crystallise easily

• may give colour reactions eg– MgCl2 -> violet -> orange– alkali KOH -> orange

• easy to detect

Coumarins

– aromatics based on α-pyrone

– widely distributed in plants• Leguminosae• Rubiaceae• Umbelliferae• Solanaceae

– first medicinal compounds from clover

• certain types toxic to animals in summer

• anticoagulant activity found• dicoumarols produced clinically