Cardiac glycosides Flavonoids 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 Apocyanaceae Liliceae 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 StrophanidiolStrophanthus Scillarenin (squill) sugars: 1-4 β-linked at C3 in various combinations glucose, rhamnose, deoxy-sugars eg digitoxose, digitalose

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 digitoxigenin A digitoxin OH gitoxigenin B gitoxin digoxigenin C digoxin diginatigenin D diginatin formylester gitaloxigenin 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 Two ways: 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 Advantages 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 flavonol isoflavone flavone

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) (b) Hesperidin (‘citrin’) 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