Quinoline alkaloids

N Quinoline nucleus (liquid, b.p. 238)

-Quinoline is a colorless liquid with an odour similar to that of pyridine, miscible with most organic solvents and with water to the extent of 0.7%.

A-Major example for quinoline alkaloids is Cinchona alkaloids (Rubiaceae) which represent pairs of diastereoisomeric alkaloids: quinine, cinchonine, quinidine and cinchonidine and their dihydroderivatives. Alkaloids

1-Source. 2-Biosynthesis. 3-Structures and chemical classes. 4-Preparation in brief. 5-Chemical testing and assay in brief. 6-Applications in therapy. 7-Related drugs. -Cinchona contains 6-7% total alkaloids, 1/2 - 2/3 of which should be quinine and cinchonidine.

-Cinchona also contains minor alkaloids of quinoline and indole types such as cinchonamine and quinamine .

B-Rutaceae quinoline alkaloids are derived from anthranilic acid e.g. skimianine and platydesmine. Structure of Cinchna alkaloids -The structure of Cinchona alkaloids can be dissected to two ring systems: quinoline and quinuclidine.

CH 2 N

N N Quinuclidine nucleus Ruban ring system (8,9`-quinuclidyl-methylquinoline)

H HO N H R

N Quinine, R=OCH3 Cinchonidine, R=H H H HO N HO N H H R R

N N

Quinine, R = OCH3: 8S,9’R Quinidine, R = OCH3: 8R,9’S Cinchonidine, R = H Cinchonine, R = H

*Cinchona alkaloids occur in salt form with organic acids e.g. quinic acid (tetrahydroxyhexahydrobenzoic acid) and cinchotannic acid. HO COOH

HO OH OH Quinic acid Biosynthesis of Cinchona alkaloids 1-biosynthesized from amino acid tryptophan and secologanin to give strictosidine thus called terpenoid indole alkaloids (Mannich like reaction).

2-Rearrangement of indole skeleton of strictosidine into quinoline skeleton. 1-Quinine -represents 1/2 - 2/3 of the total alkaloids of Cinchona -colorless needles, m.p 177 oC.

-Insoluble in water but soluble in alcohol, ether, CHCl3 and boiling benzene. -Quinine sulfate is the most frequently used salt. -Quinine forms double compounds with some solvents such as benzene, toluene and phenol.

-Solutions of quinine in oxygenated acids e.g. H2SO4 & H3PO4, but not halogenated acids have blue fluorescence. -It also forms insoluble tartarates. -On heating with KOH/amyl alcohol, quinine isomerizes into quinidine (quinidine is semi synthesized from quinine). Tests for identity 1-Quinine + Oxygenated acids  blue flourescence. 2-Thalleioquine test. 3-Herpathite test. 4-Rosquin test (Erythoquinine test). Physical and chemical characters of Cinchona alkaloids Alkaloid test Quinine Quinidine Cinchonine Cinchonidin

1-Solubility Soluble in Soluble in. Soluble in Soluble in alc.,

alc., ether & alc., benzene alc., benzene, CHCl3 CHCl3 & ether benzene, moderately sparingly sol. insol. in soluble in

in H2O ether ether, insol. in H2O

2- Tartarates Insolube Soluble Soluble Insoluble 3- Optical rotation Levo Dextro Dextro Levo

4-Reactions A-Fluorescence + + - - B-Thalleioquin + + - - c-Rosequin + + - - d- Herpathite + - + - e- Mono-sulfate Insolube Soluble Soluble Soluble Tests for identity 1-Quinine + Oxygenated acids  blue flourescence.

2-Thalleioquine test:

Solution in water + 2-5 drops Br2 water + 1 ml NH4OH  green color, extractable with CHCl3.

3-Herpathite test: Boiling mixture of quinine + glacial acetic acid + alcohol + drops of conc. H2SO4 is treated with 3.5 ml of 1% iodine solution. On cooling green crystals of iodosulfate of quinine are obtained.

4-Rosquin test (Erythoquinine test): Dil. soln. of quinine + bromine water gives faint yellow color. Addition of 2-3 drops 10% Potassium ferricyanide + 2 drops of dil. NH4OH produces red color. Therapeutic uses of Cinchona alkaloids 1-Quinine: -It is an anti-malarial drug (Malaria is caused by protozoon Plasmodium falciparum). -It acts by intercalation with DNA double helix and complexes with toxic hemoglobin breakdown products formed by the parasite. -Quinine is now replaced by other synthetic anti- malarial agents e.g. chloroquin, but still used in case of drug resistance. -Quinine has also a mild curare- like skeletal muscle relaxant effect and thus used to treat nocturnal recumbency leg cramps especially in the elderly. -Other Cinchona alkaloids have anti-malarial effect also, but less effective than quinine. 2-Quinidine sulfate: -is the major alkaloid used from Cinchona.

-It is used in treatment of different cardiac arrhythmias by membrane stabilization as it combines with Na+ channels in the membrane.

-It inhibits fibrillation (uncoordinated contraction of the heart muscle - Quinidine is rapidly absorbed by GIT and overdose may lead to diastolic arrest, which makes it unsuitable as anti-malarial agent.

-Quinidine gluconate is used in treatment of severe malaria caused by P. falciparum, but it's use is limited due to its effect on the heart.

3-Totaquin (Mixture of Cinchona alkaloids) has been used as substitute for quinine in time of shortages. Quinine and related alkaloids, especially quinidine were also isolated from the bark of Remijia pedunculata (Rubiaceae).

Gravimetric estimation of total alkaloids 1-Known amount of Powdered Cinchona is treated with lead subacetate.

2-The mixture is extracted with ammoniacal alcohol in a soxhlet till exhaustion.

3-The extract is concentrated by removing most of the alcohol and treated with dil. H2SO4 to form alkaloidal bisulfate.

4-Acid solution is washed with CHCl3 and chloroformic extract is washed with H2SO4.

5-Combined acid solution is alkalinized with NH3 and extracted several times with CHCl3 till exhaustion.

6-Chloroformic extract is washed with little water and distilled in a tared dry flask. Alcohol (5 ml) is added and residue dried to constant weight at 100oC. Assay of quinine and cinchonidine 1-The residue containing total alkaloids, from the last experiment is converted to neutral sulfates, then to tartarates by treatment with sodium potassium tartarate.

2-The precipitated tartarate is decomposed by NaOH to give free bases which are extracted with CHCl3.

3-The chloroform extract is evaporated and dried at 100 oC. The amount of quinine and cinchonidine is weighed. An HPLC assay and separation of cinchona alkaloids has been published, J. chromatography 295, 276-281 (1984).

Isoquinoline alkaloids *Many alkaloids contain the aromatic or reduced isoquinoline ring system. *Isoquinoline was first isolated and characterized from coal tar first and crude petroleum and occurs as a colorless liquid with benzaldhyde- like odour.

N

Isoquinoline

*Isoquinoline alkaloids are widely-distributed in nature e.g. in families Rubiaceae (Ipecacuanha), Papaveraceae (Opium) and Menispermaceae (Curare). More than 700 isoquinoline alkaloids of Pharmaceutical interest have been characterized. *Isoquinoline alkaloids are generally biosynthesized from aromatic amino acids: phenylalanine and tyrosine. 1-Ipecacuanha alkaloids (Terpenoid tetrahydroisoqunoline alkaloids)

-Source: alkaloids are localized in the cortex of the roots and rhizomes of Cephalis ipecacuanha and C. accuminata (Rubiaceae), which contain 2-2.5% total alkaloids, 70% of which is emetine.

-Major alkaloids are emetine and cephaline, while psychotrine and its O-methyl ether occur in smaller amounts. MeO MeO MeO N MeO N

NH H N

OMe OR OMe OR R=Me, emetine R=H, cephaline R=Me, O-methylpsychotrine R=H, psychotrine

*Psychotrine and cephaline are phenolic, while emetine and O-methylpsychotrine are non phenolic. -These benzyltetrahydroisoquinoline alkaloids occur naturally mainly in Rubiaceae genera such as Cephalis and Pogonopus. They also exist in families Alangiaceae and Icacinaceae.

Biosynthesis:

Ipecac. alkaloids are biosynthesized from two dopamine and a secologanin molecule.

Mannich reaction of one molecule of dopamine and one molecule of secologanin gives 3a- desacetylipecoside, which on reaction with a second molecule of dopamine produces emetine and related alkaloids. Isolation of Ipecac. Alkaloids

Alcoholic extract of the rhizome or root

1-concentrate 2-add lead acetate to precipitate tannins and coloring matter

Filtrate tannins + coloring matter evaporate to dryness

Residue of crude alkaloid mixture + HCl Hydrochlorides, filter Mixture of alkaloid

Filtrate of alkaloid reject insoluble material Hydrochlorides +

NaOH + ether

aqueous contains Ether Contains the non cephaline + psychotrine Phenolic emetine + HCl + NH4OH + ether

ether aqueous cephaline psychotrine ext with CHCl3

Chromatographic isolation was reported in phytochemistry 52, 1169-1176 (1999). Pharmacological actions and uses of Ipecac. Alkaloids 1-Emetine and cephaline are emetic alkaloids in case of drug overdose. They act by irritation of the stomach. Emetine also causes emesis by stimulation of chemoreceptor trigger zone

2-Cephaline is more toxic than emetine and in small doses, these alkaloids have expectorant effect (in this case, crude extract is used not pure alkaloid).

3-Emetine has been used to treat amoebic dysentery with no effect on bacillary infection. However, it is toxic and cumulative and the treatment should not be continue for more than 10 days. 4-Both emetine and cephaline are potent protein synthesis inhibitors acting at the stage of translocation. They have anti- tumor and antiviral effects but too toxic to be used therapeutically.

5-O-methyl psychotrine possesses fairly low effect on protein synthesis but showed promising ability to curb viral replication by inhibition of HIV-reverse transcriptase (potential to be used in treatment of AIDS infection).

6-Emetine inhibits protein synthesis in the animal and protozoon. It inhibits DNA synthesis and kills viruses but is not anti-bacterial. Emetine is extremely toxic causing cardiac toxicity, hypotension and muscular weakness, tremors and pain in the extremities. It is slowly excreted from the body in 60 days. It is no longer used in therapeutics. 8-Dover`s powder: powdered opium is combined with powdered Ipecacuanha to give a popular sedative and diaphoretic (promotes perspiration to take place at the onset of colds and influenza). (-)2,3 dehydroemetine is equal to emetine in activity and less toxic (being readily excreted). A-Assay of Ipecac. alkaloids by acid/base titration

10 grams of Ipecacuanha powder

is treated with CHCl3-ether mixture (3:1) + NH4OH concentrate the extract

add 20 ml N/1 H2SO4 shake and separate the layers

upper layer lower layer contains alkaloid sulfates (organic solvent layer )

extracted with 10 ml

N/10 H2SO4 and combined

Combined acid layer

alkalinize with NH4OH and extract with CHCl3 till exhaustion CHCl3 ic extract washed with water and dehydrated over anhydrous

Na2SO4, solvent evaporated

-Residue treated with 2 ml neutral alcohol and heated at 100oC to remove volatile bases

-Dissolve in 15 ml N/10 H2SO4 and back titrate excess acid using N/10 NaOH using methyl red as indicator

Estimation of total alkaloids B- Non-phenolic alkaloids (emetine) a-The above titrated liquid is mixed with 5 ml NaOH and enough ether to extract the non phenolic alkaloids (emetine). b-Ethereal extract is washed with NaOH and then with water. c-Evaporate the ether, dissolve the residue in 10 ml

N/10 H2SO4 and back titrate excess acid with N/10 NaOH using Me red as indicator.

% phenolic alkaloids = % total alkaloids- % of non phenolic alkaloids. Chemical testing for Ipecacuanha alkaloids

1-Alkaloid in dil. HCl + Ca hypochlorite or H2O2 gives orange color due to hexacyclic iminium salt.

2-Mandalin`s reagent produces green color changing to yellow in the presence of HCl in case of emetine only.

3-Froehd`s reagent results in dirty green color changing to grass green after addition of HCl (emetine). Cephaline gives dirty green color which changes to green-blue in presence of HCl.

4-Coupling with P-nitrodiazobenzene gives a dye soluble in NaOH to give purple color in case of the phenolic alkaloids only (cephaline and psychotrine).

5-Erdmann’s reagent gives cherry red in case of psychotrine. Isoquinoline alkaloids *Many alkaloids contain the aromatic or reduced isoquinoline ring system. *Isoquinoline was first isolated and characterized from coal tar first and crude petroleum and occurs as a colorless liquid with benzaldhyde- like odour.

N

Isoquinoline

*Isoquinoline alkaloids are widely-distributed in nature e.g. in families Rubiaceae (Ipecacuanha), Papaveraceae (Opium) and Menispermaceae (Curare). More than 700 isoquinoline alkaloids of Pharmaceutical interest have been characterized. *Isoquinoline alkaloids are generally biosynthesized from aromatic amino acids: phenylalanine and tyrosine. Opium alkaloids Opium is the air-dried milky exudate (latex), obtained by incision of the unripe capsule of Papaver somniferum family papaveraceae.

Opium contains over 40 alkaloids belonging to four groups: 1-Benzylisoquinoline e.g. papaverine, noscapine (narcotine).

2-Phenylethylamine alkaloids e.g. narceine.

3-Phenanthrene group e.g. morphine, codeine and thebaine (modified benzyltetrahydroisoquinoline alkaloids).

4-Diisoquinoline alkaloids (cryptopine and protopine). It was found that opium from different geographical sources has different content of alkaloids. Meconic acid O OH

O HOOC COOH b-Hydroxy-g-pyrone-a,a-dicarboxylic acid

-Powdered opium contains 3-5% of meconic acid. This is an organic acid characteristic of opium and is important only because of forensic reasons. Chemical test for meconic acid

-Aqueous extract of opium with CaCl2 to give a precipitate of Ca meconate.

-Ca meconate is decomposed by repeated treatment with warm HCl to give meconic acid, which is tested by adding FeCl3 to it’s aqueous solution to give deep purplish red coloration.

-The produced color is not affected by adding dil. HCl or boiling in difference from Ac/ Form. The color is also not affected by HgCl2, SnCl2 (not thiocyanat).

-This test was used to check the urine of opium smokers. 1-Benzylisoquinoline alkaloids -These alkaloids are weakly basic. -They can be extracted from their salt solution with chloroform.

a-Papaverine (1% of opium):

MeO

MeO N

MeO

MeO Biological activities for papaverine

1-t is a smooth muscle relaxant on pulmonary, cerebral and peripheral blood vessels, used in conjunction with codeine.

2-It also relaxes smooth muscles of bronchi, intestine, ureter and biliary ducts.

3-It can be used in treatment of cramps and to relieve cerebral and peripheral ischemia.

4-It inhibits cAMP phosphodiesterase and decreases intracellular Calcium leading to decrease of excitability and conductibility of the cardiac muscle.

5-Papaverine is sometimes used in treatment of GIT spasms and in expectorant preparations. 6-It was used as an effective treatment of male impotence by direct injection and vasodilator.

7-It is devoid of any CNS effects and is widely used as vasodilator and anti-ischemic in cerebral circulatory insufficiency and improves symptoms of senility, loss of attention and memory. Test (Warren`s test)

0.5 mg papaverine + crystal of KMnO4 + 0.2 ml of Marqui`s reagent, stirring  green color. b- Narcotine (noscapine) (2-10%)

The action of this alkaloid is devoid of any narcotic effect thus, it is also called anarcotine.

It can be dissolved in hot alkali because of opening of the lactone ring system to give alkali narcotinate salt.

It is not soluble in cold alkali or ammonia and used as an anti-tussive agent. O

O NMe

OMe O OMe O OMe -It has teratogenic effect and may deform a fetus.

-Recent studies showed that noscapine possesses an anti-tumor action similar to that of podophyllotoxin and colchicine by binding to tubulin arresting cells in mitosis. 2-Phenylethylamine alkaloids (narceine) -Narceine can be chemically prepared from narcotine.

-Narceine is a weakly basic alkaloid which forms well crystallized hydrochlorides, picrates and Aurichloride.

-It is an example of amphoteric alkaloids, which is not used medicinally. 2-Phenanthrene alkaloids (morphinans) *Morphine, codeine and thebaine are representative examples of this class. *They are biosynthetically related to the benzyl isoquinoline alkaloids such as papaverine as all of them are derived from the same precursor molecule. Tyrosine

HO MeO

NH HO MeO N Aromatisation

Methylation MeO HO MeO

Papaverine (S)-norcoclaurine Several steps HO

MeO MeO O O O NMe NMe NMe HO HO MeO Thebaine Codeine Morphine

Thus, the phenanhrene alkaloids can be classified as modified benzyl tetrahydroisoquinoline compounds as well.

O NH

Phenanthrene nucleus morphinan (A)Morphine (8-15%)

*Morphine is the most important opium alkaloid, produced only by Papaver somniferum, while thebaine is produced by ten species e.g. P. bracteatum. HO

O NMe

HO Morphine is classified as narcotic analgesic agent, characterized by:

1-A phenolic and alcoholic hydroxyl groups charactrised by acidic characters.

2-It tends to induce sleep and causes nausea, vomiting, hypotension, constipation, urine retention by an action on smooth muscle of sphincters.

3-It is a habit forming CNS depressant.

4-The major use of morphine is in the relief of terminal persistent pain in cancer also in kidney and gall stones.

5-It is orally active but used by injection to obtain rapid relief of acute pain. *Morphine is hexahydrophenanthrene-4,5-oxide-9,13-N methylethylamine.

*It has 5 chiral centers and the active form is (-) 5R, 6S, 9R, 13S, 14R. Inversion of configuration at positions 9, 13 causes disappearance of activity.

*Morphine can be assayed by acid/base titration.

*It also dissolves in caustic alkalies and alkaline earth hydroxides because of phenolic nature and can be precipitated by addition of (NH4)Cl or (NH4)2SO4 or HCl/NH4OH.

*Important salts are the sulfate, hydrochloride and acetate. In contrary to codeine, morphine is sparingly soluble in organic solvents such as CHCl3, ether and benzene. Reactions of morphine

1-Iodic acid test: Solution of morphine in dil. H2SO4 reduces KIO3 solution.

2-Brouadrel-Boutomy test.

3-Sodium nitrite test: gives red-orange color (due to the formation of nitrosomorphine). This reaction is used in colorimeteric assay.

4-Morphine + conc. HNO3  blood red color, which does not react with SnCl2 in difference from brucine.

5-Addition of FeCl3 gives greenish blue color because of phenolic hydroxyl group at C-3. Structural requirements for narcotic analgesic activity

1-Quaternary central carbon (No. 13).

2-Phenyl group attached to C- 13,(carbons 1, 2, 3, 4, 11, 12)

3-Tertiary nitrogen atom.

4-Two-carbon bridge (C15-C16) attaching the central carbon and tertiary nitrogen. *Analgesics of morphine series (SAR) 1- Activity is increased by: -Any modification of C-6 alcoholic OH group. - Introduction of C-14 OH or C-5 methyl. - Replacement of N-methyl by another group e.g. N-CH2-CH2-.

2- Activity of morphine is decreased by: - Methylation of phenolic OH group. - Quaternization of nitrogen. - Fission of N containing ring or opening of 4,5 oxide.

3- Condensation of thebaine and acetylenic dienophils. HO

O NMe

HO Mechanism of tolerance to morphine -Increased biotransformation of the drug and adaptation to its actions. Morphine congeners 1-Hydromorphone HCl (Dihydromorphinone = Dilaudid) -More powerful narcotic analgesic and tends to depress respiration. -It causes less nausea and vomiting than morphine. 2-Apomorphine HCl - Formed by acid catalyzed rearrangement and dehydration of morphine. -Apomorphine is an emetic drug, which can be administered subcutaneously. It is a potent anti-parkinsonism agent, but not used because of emesis.

3-Diacetylmorphine (Heroin) -Prepared from morphine by acetylation. -Actions of heroin are more pronounced than morphine (not used therapeutically) OAc

O NMe

OAc 4- Opoids

-Totally synthetic or semi-synthetic morphine like compounds.

-Prepared by replacement of N-methyl by a higher alkyl e.g.nalorphine.

-They have pain relieving and anti-tussive properties.

-They are not habit forming. MeO

O NMe

HO Codeine B-Codeine -The most widely used opium alkaloid (0.2-0.7%), used as anti-tussive.

-It is semi-synthesized by: a-Methylation of morphine using methyliodide, dimethylsulfate or diazomethane. b-Demethylation and reduction of thebaine.

-Codeine is less potent, less toxic and less habit forming than morphine. -It's analgesic action is due to due partial demethylation in the liver.

-It is contraindicated in case of respiratory insufficiency and asthmatic cough.

-Toxic signs include meiosis, dizziness, constipation, allergic skin reactions, histamine release and puffy face.

-Codeine is soluble in organic solvents such as ether (c. f. morphine). Codeine congeners 1-Hydrocodone bitartarate: -Used as anti-tussive, but it is more addictive than codeine.

MeO

O NMe

O

2-Ethylmorphine (codethylene)

H5C2O

O NMe

HO

-Used as anti-tussive, similar to codeine. 3-Oxycodone: MeO

O NMe HO O -More potent anti-tussive and more addictive than codeine.

4-Pholcodine (3-morpholinylethyl morphine): -Specific anti-tussive, but not strong analgesic with prolonged action. -It does not induce addiction. O N O O NMe

HO Reaction of codeine:

Acid solution of codeine (in HCl) + drops of dil. FeCl3 + dil. solution of K ferricyanide --- dull green, but not blue as in the case of morphine.

MeO

O NMe

HO C-Thebaine MeO

O NMe

MeO

-Can be separated as thebaine salicylates.

Pharmacologically: 1-Thebaine produces excitement, which progresses to strychnine like seizures.

2-Small doses show narcotic analgesic action. -Thebaine can be used in: 1-It can be efficiently (75% yield) converted to codeine by acid catalyzed hydrolysis, then selective borohydride reduction.

-The advantage of this reaction is that the conversion of thebaine to morphine and then to heroin is much more difficult and low yielding. There is also interest in cultivating of Papaver bracteatum.

2-Synthesis of oxycodone, oxymorphone (potent analgesics).

3-Diels Alder reaction of diene ring system to give adducts with very potent analgesic activity. Papaveretum is a mixture of purified opium alkaloids, as their hydrochlorides and is now formulated to contain only morphine (85.5%), codeine (7.8%) and papaverine (6.7%). It is used for pain relieving during operations. Methods of assaying opium A-Determination of morphine in opium can be done as follows:

1-Morphine can be soluble as phenate, using Ca(OH)2.

2-Morphine is precipitated as free base, using NH4Cl which is insoluble in water, then acid-base titration.

B-Gravimetrically Using 1-chloro-2,4-dinitrobenzene to give insoluble dinitrophenyl ether.

C-Colorimetric assay Using nitrite or iodic acid methods.

3- Curare alkaloids (Bisbenzyltetrahydroisoquinoline alkaloids) -Source: 1-Dried plant extract of the bark and stem of Chondodendron (or Chondrodendron) tomentosum family Menispermaceae (isoquinoline type). 2-stem and bark is of Strychnos toxifera family Loganiaceae (indole type). -Biosynthesis: Dimeric alkaloids, biosynthesized by oxidative coupling of R and S N- methyl coclaurine: MeO MeO

NMe NMe HO HO H H

HO HO

( R)-N-methylcoclaurine S)-N-methylcoclaurine MeO

NMe HO O H

O H MeN OH

OMe (+)-(R,R) isochondrodendrine d-Tubocurarine chloride is soluble in water, alcohol and alkali and is not soluble in organic solvents such as CHCl3 and ether. d- Tubocurarine chloride -This is the only useful alkaloid of curare. - It is phenolic, quaternary bisbenzyl tetrahydroisoquinoline. MeO MeO NMe NMe O OH O OH H H

O O H H Cl- + OH MeN OH Me2N OMe OMe d-tubocurarine chloride curine (l bebeerine)

Dimeric non-quaternary alkaloids such as curine & isochond rodendrine are derived from two R-N-methyl coclaurine. Separation of mixture of curine and d- tubocurarine

1-Aqueous solution of curare is treated with

NH4OH and kept until all curine precipitates and filtered.

2-Filtrate + alcoholic HgCl2 double salt of d-tubcurarine and HgCl2 precipitates.

3-Precipitate is suspended in alcohol and decomposed by H2S gas. The filtrate from H2S is diluted with ether, when d- tubocurarine is crystallized out. Pharmacological activities of d-tubocurarine -Competitive neuromuscular blocking agent, used as muscle relaxant before surgery without deep anesthesia.

-It controls convulsions of strychnine and tetanus as well as in Parkinson's disease to relax rigid muscles.

-Curare kills by producing paralysis of respiratory muscles (artificial respiration).

-Curare is used in abdominal surgery and tonsillectomy to relax tense muscles.

-Anti-choline esterase drugs e.g neostigmine and physostigmine are effective antidotes for moderate curare poisoning. Structure activity relationship

1-Quaternary nitrogen.

2-Two quaternaries are more effective than one quaternary 10 carbons or 14A apart.

3-Di-O-methyl d-tubocurarine iodide (prepared by reaction of d-tubocurarine with methyl iodide in ethanolic KOH) is more effective than d- tubocurarine.

Estimation

1-Biological assay (Head-Drop-test).

2-Spectrophotometrically & spectrofluoremetrically Color reactions of d-tubocurarine

1-Aqueous solution + FeCl3  slight green color.

2-d-tubocurarine solution + solution of Na2SO4 gives yellow-brown precipitate.

3-Powder + Hg(NO3)2 solution cherry red color gradually appearing.

4-Aqueous solution of tubocurarine + conc. H2SO4 + 1% KIO3 soln. , 30` pink color water bath 5-tubocurarine gives pink precipitate with ammonium reineckate and picric acid solutions.

6-Microchemical: drop + ammonium molybodate (Froehd`s) solution  residue

+ H2SO4 (moisten) deep blue  green yellow color.

7-Microchemical: drop + ammonium vanadate (mandalin`s reagent)  residue +

H2SO4 (moisten)  deep blue green  yellow.

Indole alkaloid

-Biosynthetically derived from amino acid tryptophan. -Indole nucleus consists of a benzene ring fused with a pyrrole ring.

NH -Indole is a colorless solid, boiling with decomposition at 245 oC, extremely volatile and miscible with most organic solvents. It has a pleasant smell and may be used as perfume base.

-There are over 1400 alkaloids (the largest group of alkaloids) containing indole nucleus, including many pharmacologically active alkaloids such as vinblastine, ergotamine and reserpine. -Indole nucleus may be present in one of the following forms:

O NH NH N

Dihydroindole Oxindole Indolenine

N N NH NH NH

Carbazole b-carboline g-carboline Alkaloids derived from amino acid tryptophan: 1-Simple tryptamines e.g. gramine & b-carboline e.g. Harmine in Peganum harmala.

2-Indolenes formed by cyclization of tryptamine e.g. physostigmine.

3-Ergolines e.g. Ergot alkaloids (isoprenoid indole).

4-Monoterpenoid indole alkaloids are from tryptophan + monoterpene (secologanin). More than 3000 alkaloids belong to this class, usually encountered in three plant families : Rubiaceae, Loganiaceae and Apocynaceae. The largest number is present in Apocynaceae 1-Simple tryptamines and b-carbolines

These indole derivatives are hallucinogens with structural homology to neurotransmitters: serotonin, adrenalin and nor adrenalin. Their use is rooted in the social life of most primitive civilizations.

Examples: a-Agaricaceae (central America) e.g. mushroom Psilocybe semilanceolata CH CH3 3

OH OP3H2 N N CH CH3 3

NH NH Psilocine psilocybine b-Myresticaceae (south American): e.g. Virola elongata

R1

NH2 N HO R2

NH NH Tryptamine, Mono N-methyl, N, N dimethyl tryptamine Sertonin c-Mimosaceae (south America) e.g Seeds of Anadenan thera peregrina CH3

N CH HO 3 Bufotenin

NH d-Malpighaceae (south America) e.g. Hallucinogenic plants. Banistreopsis caapi ( Ayahuasica). They contain b-carboline alkaloids harmine, harmol, harmaline, harmalol

e-Zygophyllaceae (serian Rue) Seeds of Peganum harmala contain alkaloids identical to those of Ayahuasica f-Convolvulaceae Alkaloids are hallucinogens, but they are not tryptamines (they are ergoline). These alkaloids are found in ololiuqui e.g. Rivea carymbosa & Ipomea violaceae and are semilar to those of ergot e.g. ergine. Ergot alkaloids (Ergoline = )octahydroindoloquinoline) (Isoprenoid indole)

-Ergot is the dried sclerotium of the fungus Claviceps purpurea family Claviciptaceae arising in the ovary of rye plant, Secale cereale family Poaceae.

-Ergotism (st.Antony`s fire) ?.

-The sclerotia contain 0.15-0.5% alkaloids (50).

-The medicinally useful compounds are derivatives of (+) lysergic acid, which are levorotatory and can be separated into two groups: 1-Water soluble amino alcohol derivatives, which represent 20% of total alkaloids e.g. ergometrine.

2-Water insoluble peptide derivatives representing 80% of total alkaloids e.g. ergotamine further classified to ergotamine, ergoxine and ergotoxine groups (ergopeptines) according to the amino acid. (+) lysergic acid (derivatives are levorotatory) = ergolines Features of ergot posioning: -Alimentary upsets. -Circulatory changes e.g. coldness of hands & feet. -Neurological changes. -Ergot alkaloids are also produced by other fungi e.g. Aspergillus, Rhizopus & Penicillium also by some convolvulaceous plants e.g. Rivea & Ipomoea. -Derivatives of lysergic acid can easily isomerize to the inactive derivatives of isolysergic acid in presence of base/heat (enhanced in polar solvents and goes through enol intermediate).

The reverse effect is achieved by refluxing with HAc or treatment with alcoholic phosphoric acid.

-Nomenclature of isolysergic acid derivatives is by addition of “in” syllable.

-Assurance the stability of levorotatory active alkaloids in medicinal preparations should be insured.

-Action of Ergot alkaloids proceeds via interaction with a-adrenergic, dopaminergic & serotonergic receptors.

Structural types of ergot alkaloids Group A: Acid-amide derivatives of lysergic acid or isolysergic acid:

1-R= NH2 ------ergine, isoergine (erginine).

2-R= 2-aminopropanol (alkanolamide type) e.g. Ergometrine, ergometrinine (ergonovine, ergonovinine).

3-R=cyclic tripeptide, peptide type, further classifiable to: a-Ergotamine group, which yields pyruvic acid + lysergic acid +proline + phenylalanine by hydrolysis e.g. ergotamine. b-Ergotoxine group, which yields dimethylpyruvic acid by hydrolysis due to terminal valine. Group B: clavine series, their names end in clavine e.g. ergoclavine, elymoclavine. These are biosynthetic intermediates of lysergic acid. A-Ergometrine (ergonovine, ergobasine) -Derivative of (+) lysergic acid and 2-aminopropanol. -Exerts its oxytocic effect by an action on adrenergic and serotonergic receptors.

-It has oxytocic effect, injected in the final stages of labor and immediately after childbirth to reduce postpartum hemorrhage. -It is also valuable after caesarian operations. -It is orally active and used as maleate salt. -Ergometrine stimulates a- adrenergic receptors in the myometrium and causes uterine hypertonicity.

-Methylergonovine maleate (semi-synthetic homologue) is slightly more active and longer acting than ergonovine.

-Ergometrine occurs as crystalline needles, soluble in alcohol, sparingly soluble in benzene and CHCl3, insoluble in ether, CCl4.

-Ergometrine maleate is readily soluble in water, less soluble in alcohol and insoluble in CHCl3 and ether. Isolation a-Powdered ergot is defatted with petroleum ether, moistened with absolute ethanol containing 5% methanol. b-The mixture is rendered alkaline with ammonia and percolated with same solvent. c-Extract is concentrated + N/4 H2SO4 till acidic to congo red. Alcohol is distilled, cooled and solidified fat is removed. d-The mixture is alkalinized with Na2CO3, extracted with CHCl3, concentrated and cool to 2 oC ergometrine, recrystallised from benzene. Chemical tests of ergometrine:

1-It gives the general tests of ergot alkaloids (Van Urk test, keller`s reaction).

2-It gives negative reaction with Mayer`s reagent (no precipitate).

3-It's aqueous soln. has blue fluorescence. B-Ergotamine tartarate -Actions 1-A vasoconstrictor peptide alkaloid. 2-Agonist of a-adreno and serotonin receptors. 3-Inhibitor of reuptake of nor epinephrine. 4-A serotonin antagonist. -Uses -Used together with caffeine as specific analgesic in migraine -Caffeine increases absorption of ergome trine by 45%. -Dihydroergotamine methanesulfonate is more effective, better tolerated and produces less digestive upsets. -Physical properties of ergotamine Hygroscopic prisms, which darkens and decomposes on exposure to air, heat or light, -Insoluble in water, slightly soluble in alcohol, freely soluble in CHCl3 and ether. Isolation a-Powdered ergot + Aluminum sulfate solution (moistening) double compounds (fix alkaloids). b-Extract with benzene or ether to remove different extractive matter e.g. fats, sterols, and coloring matter. c-Powdered ergot is then moistened with ammonia, extracted with ether. d- Ergotamine is obtained by evaporation of ether and recrystallization from methanol.

Reaction of ergotamine 1-Marqui`s reagent  faint brown.

2-Froehd`s test  grey blue.

3-Mandalin`s test  purple-brown.

4-Vitali`s test  dark orange  purple  brown. Synthetic analogues

1-Methysergide maleate (N-methylmethyl ergonovine salt).

-is a serotonin antagonist used in treatment of vascular headache (prophylaxis of sever migraine).

-Inhibits increase of permeability caused by serotonin and decreases the release of histamine by mastocytes.

2-The ergotoxine group (Ergoloid mesylates)

-used to relieve symptoms of idiopathic decline of mental capacity in geriatric (old-aged individuals). They produce vaso-relaxation, increased cerebral blood flow, lowering of systemic blood pressure and bradicardia. Ergotoxine alkaloid

-Mixture has oxytocic and vasoconstrictor effect.

-In dihydroergotoxine=codergocrine, the reduction of the double bond reverses the vasoconstrictor effect leading to cerebral vasodilatation and increase of blood flow and an improve in mental functions and mental performance.

OH O N NH H N O O O NMe R H

NH

1-Ergocristine, R=

2-Ergocornine, R=

3-Ergocryptine, R= 3-Lysergic acid diethylamide (LSD)lysergide -It is the most notorious of lysergic acid derivatives, widely abused hallucinogen and most active pyschotomimetic agent known.

-It behaves as agonist/antagonist at 5-HT-receptors.

-It is non addictive, but may lead to schizophrenia being used in experimental psychiatry.

-LSD is a central sympathetic stimulant with two fold action producing central sympathetic stimulation that parallels a slight depression. . Occurrence in higher plants

-Ergot alkaloids occur also in higher plants such as seeds of Rivea corymbosa and Ipomoea violacea morning glory (Fam. Convolvulaceae), which contain ergine (lysergic acid amide) as major component.

-These plants are still used for magico religious purposes and also abused as hallucinogens (ololique). Minor constituents are ergometrine, erginine and other clavine alkaloids. Specific reactions of ergot alkaloids

1-Aqueous solution of alkaloidal salts gives blue fluorescence.

2-Van Urk test.

3-Conc H2SO4 ----- blue color.

4-Keller`s reaction: Conc H2SO4 + glacial acetic acid containing a trace of FeCl3---- blue color. Assay of ergot alkaloids

1-Colorimetric assay using P-dimethylaminobenzaldehyde, which gives blue color measured after 5 minutes at 590 nm. This is used for determining the total alkaloid content (the color is due to reaction of indole nucleus). % Water soluble alkaloids =total-%water insoluble alkaloids.

2-Fluorimetric assay for ergometrine depends on quantitative separation by paper chromatography and fluorescence of separated spots on the chromatogram is compared with those of standard spots, prepared at the same time (under UV light). Terguride is a synthetic ergot indole alkaloid analogue, which acts as agonist at dopamine D2 receptor and used in case of parkinsonism and in schizophrenia.

O N NH

H NMe

NH

T erguride Ellepticine (Pyridocarbazole alkaloid)

-Ellepticine and 9-methoxyellepticine are both naturally occurring alkaloids. Side effects include haemolysis and cardivascular effects.

-Ellepticine is oxidized in vivo to 9-hydroxyellepticine, which is thought to be the active form.

-Poor water solubility gave problems in formulation and derivatives with 9-hydroxy and quaternized nitrogen (e.g. elleptinium acetate =9-hydroxy N-methylellepticinium acetate) showed high activity.

-The mechanism of action is via intercalation between base pairs of DNA causing partial unwinding of the helical array

2-The calabar bean alkaloids (ordeal bean) Me Me HO MeHN O NMe NMe NMe O NMe Pyrrolydine indole Physostigmine ( indolene) Eseroline Source: 1-seeds of Physostigma venenosum (Fabaceae) containing 0.15% alkaloids (0.3 % is physostig mine). 2-Eserine can also be prepared by fermentation using Streptomyces griseofuscus in 0.88 g / l yield.

-Physostigmine is a reversible choline esterase inhibitor enhancing the endogenous activities of acetylcholine. -Physical properties: colorless prisms, m.p. (106 oC), which may acquire a reddish tint, soluble in alcohol, benzene, insoluble in pet. ether, hydrolyzed by KOH to methylamine, CO2 and eseroline (oxidized to rubreserine).

-Biosynthesis from tryptophan+SAM (pyrrolidine indole=indoline)

-Why called calabar bean and ordeal bean?. Pharmacological actions -Physostigmine is a reversible choline esterase inhibitor enhancing the endogenous activities of acetylcholine.

-It is a parasympathomimetic drug, which causes: 1- meiosis, sialrrhia, bradicardia, hypotension and bronchiospasms.

2-As eye drops, it produces meiosis, contraction of ciliary muscle and a decrease in intraocular pressure, thus used to treat glaucoma.

3-Salts include: a-eserine salicylate injection is used as an antidote due to intoxication with hyoscyamine or atropine. b-Eserine SO4is ointment applied to conjunctiva. Other actions of physostigmine include:

(1)Eserine also used also in intoxication with curare and in treatment of Parkinson disease.

(2)Neostigmine is used in myasthenia gravis.

(3)carbamate insecticides e.g. carbaryl.

(4)Physostigmine (synapton) was found useful in treatment of Alzheimer. Physostigmine analogues are used for that purpose e.g. rivastigmine and eptastigmine.

(5)In therapeutics, synthetic drugs e. g. neostigmine and pyridostigmine are indicated in myasthenia gravis and postoperative intestinal and bladder atony and in stubborn constipation. Synthetic analogues should retain: 1-Carbamate (methylurethane) functionality.

2- Aromatic ring.

3- Quaternary ammonium system to ensure water solubility.

O

O NHMe O + + Me2N NMe O Me2N NMe3 O O

carbaryl neostigmine pyridostigmine Synthetic analogues Color tests

1-Solution + traces of H2SO4 + few drops ammonia  bluish colored residue, dissolving in alcohol to give blue color.

2-Alkaloid salt solution + KOH, in contact with air  rubreserine (red needles) further oxidation eserine blue.

3- Solution + K ferricyanide + FeCl3 blue color. Isolation 1-extracted with alcohol in a soxhlet, alcohol is distilled and residue washed with water, rendered alkaline with Na2CO3, extracted with ether.

2-Ether extract is concentrated + 5% H2SO4  physostigmine sulfate + Na salicylate precipitate of physostigmine salicylate.

NH4OH, KOH, NaOH are not used as alkalinizers because they catalyze oxidation to rubereserine 3-Nux vomica alkaloids (Terpenoid indole) -Source: The seeds of Strychnos nux vomica (Loganiaceae) contain 5% total alkaloids mainly strychnine and brucine (dimethoxystrychnine). The alkaloids are present in the large thick-walled endosperm cells.

N N CH3O CH O 3 N N

O O O O

Strychnine brucine -Strychnine is used as tool in physiology & neuroanatomy research (fatal dose 60-90 mg in humans). -It's only medicinal use is in very small doses as appetite stimulant and general tonic, sometimes with iron salts in case of anemic patients. -Both brucine and strychnine are also used in synthetic chemistry to resolve mixtures of racemic acids acting as optically active bases. A-Strychnine -A CNS stimulant toxin causing tonic convulsions.

-Strychnine binds to its sites in spinal cord leading to contraction of the diaphragm and asphyxia.

-It has two nitrogen atoms, but the amide nitrogen is not basic.

-It is used as vermin (mice) killer, formerly used as bitter tonic, circulatory stimulant & appetite stimulant in very small doses.

-Biosynthetic origin: terpenoid indole alkaloid derived from strictosidine.

-Strychnine and brucine occur as salts with malic & chlorogenic acid (caffoyl derivative of quinic acid). OH OH OH HOOC O C HC CH OH OH O

Chlorogenic acid

-Physical properties: Strychnine (strychnidine-23-one) is very bitter prisms (alcohol, m.p. 268oC), levorotatory, soluble in water, ethanol, CHCl3 and gives many crystalline salts with acids.

-Color reactions: 1-Mandalin`s  blue  purple  red 2-Vitali`s test  orange

3-+K2CrO7 + conc. H2SO4  streaks of blue -violet color Isolation

1-Powdered Nux vomica seeds are mixed with lime, water and dried, extracted with CHCl3 in a soxhlet.

2-CHCl3 extract is shaken with successive portions of dil. H2SO4.

3-Combined acid extracts are filtered, treated with excess ammonia….alkaloids.

4-Precipitate is extracted with 25% ethyl alcohol to dissolve brucine.

5-Alcohol extract is filtered and the residue (strychnine) is purified by repeated crystallization from ethyl alcohol. Brucine is crystallized from filtrate as oxalate. B-Brucine -Physical properties: prisms (from alcohol), m.p 178, sparingly soluble in cold water more soluble in boiling water, ethyl alcohol, CHCl3, acetone. It is monoacidic base, forming salts with HCl, H2SO4.

-It is much less poisonous than strychnine.

-Brucine is purified from the alcoholic filtrate, after separation of strychnine as oxalate.

-Uses of brucine: alcohol denaturant (makes alcohol unfit for drinking). . Assay of Nux vomica alkaloid a-Total alkaloids of Nux vomica are assayed by acid/ base titration. In order to determine strychnine, brucine is decomposed using 15 ml of a

3% w/v solution of H2SO4/H2O and 2 ml conc. HNO3. b-UV spectrophotometric determination depends on the fact that max for strychnine occurs at 254 nm, while that for brucine is at 264 nm. Thus strychnine is dissolved in absolute ethanol and absorbance measured at 254 nm. If brucine is present, it should be destroyed by potassium persulfate and sulfuric acid. 4- Rauwolfia alkaloids (Yohimbine alkaloids)

-Source: Rauwolfia (snake root) dried root of R. serpentina Family Apocynaceae, containing 0.8-2.4 %of 60 b-carboline alkaloids (15% of which is reserpine and rescinamine). -In India & Africa, the drug is used as antidote to treat snake bites, stomach pains, fever, vomiting and insanity. Deserpidine and reserpine are major in R. canescenss

-Biosynthetic origin: terpenoid indole -Pharmacological actions: 1-The drug possesses a lowering effect on blood pressure in hypertensive states.

2-The drug causes slow down of pulse and a general sense of euphoria and a tranquilizer effect.

3-Higher doses of reserpine may lead to mental depression, which is not prevalent when powdered root is used.

4-In severe hypertension, Rauwolfia acts in synergism with thiazide diuretics.

5-Reserpine was claimed to cause promotion of breast tumor.

6-Reserpine is of secondary interest now, being marketed with thiazide diuretics in USA and France.

7-It produces lasting drop in blood pressure and heart rate by peripheral depletion of catecholamines, while the depletion of central neurotransmitters explains it's sedative activity. Rescinamine and deserpidine possess the same action. OMe O C N 11 A B D OMe MeO NH E O OMe

MeO2C OCH3 Reserpine

-Deserpidine=11-demethoxyreserpine used in a similar way like reserpine.

OMe O C N 11 A B D CH CH OMe MeO NH E O OMe

MeO2C OCH3

Rescinamine Reserpine Physical properties: -White to pale buff to slightly yellow crystalline powder which darkens slowly and rapidly in solution after exposure to light.

-It dissolves in CHCl3, acetic acid, but not in water, ether, benzene and alcohol.

Isolation -As a weak base, it is purified by extraction from dil.

HCl solution, by CHCl3.

-CHCl3ic extract is then washed with 10% Na2CO3 and evaporated to dryness.

-The residue is crystallized from MeOH. Color tests

1-+ Froehd`s reagent  yellow  blue

2-+ vanillin/H2SO4  rose pink color

3-+P-dimethylaminobenzaldehyde + glacial acetic acidred excess glacial green color. HAc

Quantitative determination

Reserpine and rescinamine are isolated and determined by:

1-Non- aqueous titration.

2-Fluorimetrically after treatment with NaNO2/ H2SO4 reagent, which produces 3,4-dehydro derivative (oxidation).

3-Biologically: by an effect on blood pressure. Yohimbine

C N A B D NH E

OH MeO2C -Yohimbine is b-carboline indole alkaloid prepared from the bark of Pausinystalia yohimbe (Rubiaceae), alkaloid content is 6%.

-Yohimbine has been successfully used to treat impotence in patients with diabetic or vascular problems. It acts by increase cholinergic and decrease adrenergic activity (aphrodisiac).

-It is contraindicated in hepatic and renal insufficiency. 5- Catharanthus alkaloids (Vinca alkaloids) Madagascar periwinkle -Source: Dried whole plant (aerial parts) of Catharanthus roseus (Vinca rosea) family Apocynaceae containing 90-150 alkaloids the major of which are vinblastine, vincristine with anticancer properties.

-Biosynthetic origin: the alkaloids are bisindole binary alkaloids which are also terpenoid indole.

N N OAc OH

NMe CO2Me MeO NH CO2Me

Vindoline Catharanthine N

NH OH

CO2Me

N OAc OH

CO2Me MeO NR

R=CH3 in vinblastine, R=CHO in vincristine -Mechanism of action: The alkaloids act by arrest of cell division in metaphase.

-Twenty binary cytostatic alkaloids were isolated from the plant, characterized by having indole and dihydroindole component.

1-Vinblastine is used to treat: -Generalized Hodgkin's disease, a cancer affecting lymph glands, spleen and liver (IV or orally). -Advanced testicular cancer, kaposi sarcoma and breast cancer unresponsive to other therapies.

2-Vincristine is used to treat acute leukemia and in combination therapy of Hodgkin's disease.

3-Vindesine has been introduced to treatment of acute lymphoid leukemia in children. Activities of different bisindole alkaloids 1-Vincristine has a superior anti-tumor activity compared to vinblastine but is more neurotoxic.

2-It is clinically more important especially in the treatment of childhood leukemia also in lung and breast cancer.

3-The plant contains mainly vinblastine with far less amounts of vincristine.

4-Vindesine is a semi-synthetic derivative of vinblastine, which has anti-tumor activity similar to that of vincristine and is effective in refractory cancer types. It has no cross resistance with vincristine or vinblastine. 5-It is now possible to convert vinblastine to vincristine by: a-Controlled chromic acid oxidation at very low temperature. b-Microbiological N-demethylation using Streptomyces albogriseolus.

-It is also possible now to synthesize the binary alkaloid vinblastine from monomeric units such as catharanthine and vindoline, present in C. roseus in much higher amounts via stereospecific coupling in 40% yield by reacting catharanthine-N-oxide + trifluoroacetic anhydride + vindoline (Polonovski reaction or biomimetic synthesis).The reaction mimicsbiosynthesis. N

NH OH

CO2Me

N OH OH

NMe CONH2 MeO Vindesine A side effect of therapy with this drug is severe constipation, reminiscent of paralytic ileus, that may lead to occlusion.

Polonoviski reaction mimics biosynthesis of the alkaloids Color test:

1-Alkaloids + vanillin HCl orange color.

2-Alkaloid+ Marqui`s reagent  red color.

Assay methods:

Spectrophotometeric.

Chromatography.

Radioimmuno assay. Vincamine N N -Source: common periwinkle (Vinca minor). HO -Uses: MeOOC a-In geriatrics to increase cerebral blood flow by vasodilatation. b-Vincamine is employed in psychological and behavioral problems of cerebral senility (attention deficit, memory loss and dizziness) in a dose of 40-60 mg/day. c-The alkaloid is sometimes combined with rutin in cerebrospinal accidents and cranial trauma. d-It is contraindicated in cerebral tumor with intracranial hypertension. e-Continuous release forms are used because of the low bioavailability of the drug. Indolzidine alkaloids

-Structure: consist of fused five and six-membered rings, with nitrogen atom at the ring fusion.

-Biosynthesis: derived from lysine via pipecolic acid + acetate, which provides carbons of the pyrrolidine ring.

8 1 7 2 6 N 3 N 5 4

Indolizine Indolizidine

-Source: Indolizidine alkaloids were obtained from fungal and animal sources, rare in plants.

-Swainsonine and castanospermine are the only used alkaloids because they inhibit glycosidase enzyme due to an analogy with the saccharides as in case of deoxynojirimycin (from Morus species).

-The high bioactivity of these alkaloids motivates research in the areas of their detection, extraction, isolation, structural determination, synthesis and biosynthesis.

-Polyhydroxylated indolizidines are extracted by alcohol/water, being highly soluble in water, purified by ion exchange chromatography and separated by preparative TLC method.

-Quantitative estimation of these alkaloids is by GC/MS of the TMS derivative. OH OH OH OH OH HO H OH HO O OH HO N NH HO OH Glucose Swainsonine Deoxynojirimycin

Swainsonine Source: 1-It is the first water-soluble, polyhydroxylated indolizidine obtained from toxic Australian legume Swainsona canescens

2-Swainsonine was also obtained from other plant sources such as Astragalus lentiginsus and several other Swainsona, Astragalus, Oxytropus and Ipomoea species. Swainsonine has also been obtained from fungi Rhizoctonia leguminicola and Metarhizium anisopliae. Therapeutic applications of swainsonine

1-Swainsonine acts as a modulator of the immune system as it stimulates IL-2 production by T-helper cells leading to proliferation of T-lymphocytes.

2-It is also effective as anti-metastasis agent, inhibiting the spread of some tumors, which can be due to altering oligosaccharides processing by inhibiting complex glycosidation of glycoproteins.

3-It was successful in clinical trials with terminally ill patients suffering advanced malignancies.

4-It has glycomimetic activity (inhibits oligosaccharide processing enzymes e.g. lysosymal glycosidase (a- mannosidase) leading to wasting, locomotor syndrome (mannosidosis) in grazing animals (live stock). Castanospermine

-It is also a polyhydroxylated indolizidine from the Australian Moreton Bay chestnut (Castanospermum australe), toxic in animals causing gastroenteritis.

-Therapeutic applications:

1-It has in vitro anti-human Immunodeficiency virus by inhibition of glycosidase enzymes involved in glycoprotein biosynthesis (the glycoprotein coating is essential for proliferation of AIDS virus).

2-The ester 6-O-butanoylcastanospermine is now in clinical trials as anti- AIDS agent. It was also tested in animals as an inhibitor of transplant rejection. OH OH H OH H OH HO HO O N O HO N Purine alkaloids (xanthine alkaloids)

-Structure: Purine ring system is a heterocyclic nucleus consisting of 6- membered pyrimidine ring fused to 5-membered ring of imidazole type.

-Natural occurrence: as xanthine (2,6-oxypurine) and Purine alkaloids are methylated derivatives of xanthine.

-Characters of Purine bases: 1-Weakly basic and their salts are hydrolyzed by water and may form salts with alkali due to acidic nature of amides. 2-They give negative test with Mayer`s, but give brown precipitates with tannic acid. 3-Murexide test is positive with all of purine alkaloids and may be performed by treating the residue of C2H2 extract with H2O2 and dil. HCl, which on evaporation gives a bright red color turning to purple on exposure to ammonia vapors. 4-Quantitative estimation: performed by reversed phase HPLC of methyl alcohol extract using methanol/water (35:65) as mobile phase. 5-Solubility: of the purine alkaloids in water is increased by organic acids e.g. citric acid and organic acid salts e.g. Na benzoate or acetate. Biological activity 1-They competitively inhibit phosphodiesterase enzyme and increase the level of cAMP and release endogenous epinephrine leading to relaxation of pulmonary blood vessels and bronchi.

2-They also inhibit uterine contraction and possess weak positive chronotropic and inotropic effects on the heart.

3-They also stimulate CNS, diuresis and increase gastric acid secretion.

4-Caffeine is the best CNS stimulant and has weak diuretic action, while theobromine has little stimulant action and is an effective diuretic, which relaxes smooth muscle. On the other hand, theophylline has low stimulant action and is an effective diuretic and smooth muscle relaxant. 1-Caffeine CH O 3 CH 3 N N

O N N

CH3 -Biosynthesis of purine alkaloids seem to derive from amino acids: aspartic acid, glycine, glutamine and methionine.

-Natural occurrce of caffeine: a-Kola nuts: nuts of Cola nitida family Sterculaceae. In fresh nuts, it is present as tannates, which release free caffeine by drying. Kola contains 3.5% caffeine and 1% theobromine. b-Coffee beans: Beans of Coffee arabica family Rubiaceae which contain 1-2% caffeine. c-Tea: leaves of Kamellia sinensis (Theaceae), which contains 1-4% caffeine (Theine) d-Mate (Paraguay tea): leaves of Ilex paraguariensis family Aquifoliaceae which contains up to 2% caffeine. Commercial preparation of caffeine

1-From tea, tea dust or tea sweepings.

2-It can be prepared synthetically from theobromine by methylation, using MeI.

3-It is also obtained as bi product in the manufacture of caffeine-free coffee (decaffeinated coffee), which is subjected to aqueous percolation prior to roasting.

4-Most of caffeine of commerce is collected in condensers attached to coffee roasters (caffeine is librated from association with chlorogenic acid and partially sublimes). properties of Caffeine Physical 1-Prisms, m.p. 238 oC, sublimes without decomp. at 178 oC.

2-Water solubility increases by heating with citric (citrated caffeine) also benzoic acid, benzoate, salicylate and bromide.

3-It gives crystalline salts with several acids.

4-It is soluble in CHCl3, sparingly soluble in ether, ethanol. Uses of Caffeine 1-Central stimulant, which is used with other agents e.g. cafergot to treat migraine.

2-Caffeine + Na benzoate is used intramuscularly as analeptic in treatment of poisoning, diuretic and circulatory stimulant.

3-It has also stimulatory effects on heart and respiration. Chemical testing

1-Murexide test: Caffeine in conc. HCl + KClO3  yellow residue which gives purple color on exposure to ammonia vapors, destroyed on treatment with fixed alkali. 2-Solution of caffeine + Tannic acid  white precipitate, dissolving in excess of tannic acid.

3-Solution of caffeine +I2 + HCl red brown precipitate dissolving in NaOH. 4-Solution of caffeine + Mayer`s reagent does not give precipitate 5-Solution of caffeine+ Wagner`s precipitate soluble in NaOH.

6-Drop of caffeine solution + drop of 5% KI+ drop of BiCl3 solution gives orange precipitate dissolving by heating and deposits on cooling.

Isolation of caffeine from tea 1-Powdered tea leaves refluxed with water and filtered. 2-Filtrate + lead acetate to precipitate tannins filtered.

3-Filtrate is concentrated and excess lead acetate is removed by Na2HPO4 and filtered. 4-Filtrate is concentrated, caffeine is extracted with chloroform and residue, crystallized from water. 2-Theobromine (3,7-dimethylxanthine)

O CH3

N NH

O N N

CH3

-Source: ripe seeds of Theobroma cacao family Sterculaceae (Cacao beans). The content is 1.5-3% (beans), 0.7-1.2% (husk shell) as it passes from the beans to the husk during fermentation and roasting.

Uses: as diuretic and smooth muscle relaxant with little action on CNS and preferred over caffeine in cardiac edema. also has vasodilator effect. -Physical properties: White crystalline powder, which sublimes at 260oC sparingly soluble in water, solubility in water is less than that of caffeine. It's water solubility is increased in presence of Na acetate and Na salicylate. It is not soluble in organic solvents such as ether, pet. ether and benzene.

-Chemical properties: Theobromine is amphoteric in nature, behaving as weak acid and weak base (dissolves in NaOH to give sodium salts and in mineral acids to give hydrochloride salt). It also forms insoluble salts with AgNO3. Chemical tests 1-Murexide test gives positive reaction with theobromine. 2-Theobromine+ conc. Hl + bromine solution, heat to expel excess bromine, cool and add drops of ferrous sulfate + dilute ammonia give blue color. 3-Aqueous solution of theobromine + Draggendorff's reagent orange red precipitate + drops of iodine tincture (1/10) or HCl 1/100  light brown brown precipitate.

Isolation of theobromine 1-Powdered seeds defatted with pet. ether and made into a paste with Ca(OH)2 and refluxed with 80% alcohol. 2-Concentration and cooling provide some theobromine as a precipitate. 3-The filtrate is concentrated, acidified with HCl, alcohol is almost evaporated and ammonia added, which gives crude theobromine. 4-Crude theobromine is collected and crystallized from boiling water or boiling dilute alcohol. 3-Theophylline O CH 3 NH N

O N N

CH3 -Source: a minor alkaloid in tea and in cola.

-Physical properties: 1-Occurs as tablet form, which melts at 270-274 oC. 2-It's water solubility is (1:120) and solubility increases in presence basic compounds such as ethylene diamine e.g in Aminophylline. 3-It is freely soluble in ammonium hydroxide and alkali hydroxides.

4-It is slightly soluble in organic solvents as alcohol, CHCl3 and ether. 5-Weak base, extracted from aqueous solution with organic solvents

Chemical tests : 1-Murexide test +ve.

2-+AgNO3 precipitate of silver theophyllinate. Isolation 1-Alcoholic extract is concentrated to a syrup, kept till all caffeine is precipitated and filtered.

2-Filtrate acidified with dil H2SO4 and diluted with water and filtered from precipitated resinous matter and other impurities.

3- Filtrate is rendered alkaline with ammonia, AgNO3 solution is added and left for 24 hr to give precipitates of silver salts of xanthine, adenine and theophylline.

4-The precipitate is collected and dissolved in dilute HNO3. On cooling silver salts of adenine and hypoxanthine are precipitated leaving silver theophyllinate in solution, which is alkalinized with ammonia and precipitate decomposed using

H2S to precipitate Ag2S, filtrate is concentrated to produce crystals of theophylline. Uses 1-As smooth muscle relaxant, diuretic and causes sympathomimetic relief of bronchial asthma.

2-It is used as micro granules for sustained release in acute attacks of chronic asthma, bronchiospasms with obstructive pulmonary disease.

3-Water soluble derivatives are used e.g. monoethanolamine, ethylene diamine and piperazine. It is a cardiac stimulant acting by lowering the venous pressure.

4-Theophylline preparation (aminophylline) can be used in Angina and hypertension. Separating a mixture of caffeine, theobromine & theophylline 1-Caffeine is extracted from the mixture with benzene.

2-Remaining theobromine and theophylline are treated with NH4OH which dissolves theophylline and leaves theobromine insoluble.

Quantitative determination of caffeine 1-Gravimetrically e.g. in caffeine sodium benzoate solution: NaOH added and solution extracted with CHCl3. CHCl3ic extract is evaporated to dryness and residue weighed.

2-Non-aqueous titration.

3-Colorimetrically: depends on the blue color that caffeine produces with glacial acetic acid and phosphomolybdic and absorbance is measured at 670 nm.

4-GLC using N-methyl -2- pyrrolidone as an internal standard.

Quantitative determination of theobromine Theobromine can be estimated iodometrically as it gives with excess iodine/HCl an addition compound. Excess iodine is back titrated with

Na2S2O3. Imidazole (=glyoxaline) group (Pilocarpine)

H H C H 2 5 CH2 CH N 3

O O N -Source: leaves of Jaborandi, a small shrub native to Brazil & Paraguay (Pilocarpus Microphyllus Family Rutaceae) contain 0.5-1% pilocarpine. Other main sources of pilocarpine are P. jaborandi and P. pinnatifolius.

-Physical properties: It is a syrupy liquid easily soluble in water, ethanol, and chloroform and insoluble in ether and light petroleum.

-Structurally: pilocarpine contains imidazole ring system and most important pilocarpine salts are nitrate and hydrochloride.

-Biosynthesis :from amino acid histidine and acetate, but this was not proved experimentally. Although histidine is ubiquitous, imidazole alkaloids are very rare and restricted to some plants of Rutaceae, Cactaceae and Fabaceae. 1-Action of caustic alkalies: Caustic alkalies open the lactone ring and form salts of pilocarpic acid with subsequent loss of its physiological activity: H H C H 2 5 CH2 CH N 3

O CH2 N OH OH

2-Effect of KMnO4 Imidazole ring is destroyed yielding NH3+ CH3NH2 + pilopic acid + homopilopic acid. H H H H C H C H 2 5 COOH 2 5 CH2COOH

+ O O O O Pilopic Acid homopilopic acid

3-Aqueous dilute acid Causes demethylation of pilocarpine  pilocarpidine. H H C H 2 5 CH2 Pilocarpidine NH

O O N 4-Effect of base and heat is isomerzation to isopilocarpine, which lacks the biological activity. H H C H 2 5 CH2 CH N 3

O O N Isopilocarpine 5-Precipitating agents: Pilocarpine produces precipitates with Mayer`s, wagner`s, gold chloride and silicotungestic acid reagents.

6-Specific color tests a-Helch`s color test: solution in dil H2SO4 + H2O2 + K2Cr2O7 (1:300) + benzene  violet in benzene layer (pilocarpine perchromate). This test was used as basis for colorimetric assay of pilocarpine. b-Ekker`s color test: Pilocarpine nitrate or HCl + sodium nitroprusside + NaOH solution + HCl  red color + Na2S2O3  green color. Uses of pilocarpine 1-Is a cholinergic drug, used to treat glaucoma (increased intraocular tension). Pilocarpine has a meiotic action, causes stimulation of muscarinic receptors of the eye leading to contraction of ciliary muscle. It also enhances outflow of aqueous humor. The actions are similar to those of physostigmine and the two agents are sometimes combined.

2-Pilocarpine also increases salivary secretion gives also relief of mouth dryness for patients undergoing radiotherapy in mouth and throat cancer.

3-The analogues of pilocarpine were investigated for use in Alzheimer as muscarinic agonists.

4-As a parasympathomimetic drug, pilocarpine causes increase in gastric secretion, intestinal motility, bronchiospasms and bradicardia. Isolation of pilocarpine 1-Leaves extracted with alcohol containing 1% HCl. Extract is distilled off under vacuum. The residue is diluted with water and filtered from the separated resinous matter.

2-Filtrate is alkalinized with NH3 and extracted with CHCl3.

3-Chloroform is evaporated and the residue treated with

NaOH solution and shaken with CHCl3, which removes isopilocarpine and pilosine leaving pilocarpine and pilocarpidine in aqueous solution.

4-Aqueous solution is neutralized with dil. acid, rendered alkaline with NH3 and extracted with CHCl3.

5-Evaporation of CHCl3 gives pilocarpine and pilocarpidine. Pilocarpine nitrate is crystallized from dilute alcohol. Assay 1- Volumetrically. 2-Acid dye technique Terpenoid alkaloids This group is classified into: 1-Alkaloids having mixed biogenetic origin: 1.Monoterpenoid indole e.g. Cinchona, Nux vomica 2.Monoterpenoid tetrahydroisoquinoline e.g. Emetine in ipecacuanha 3.Isoprenoid indole – Ergoline in ergot 4.Furanoquinoline alkaloids of anthranilic acid and isopentenyl pyrophosphate (in plants from Rutaceae e.g. skimmianine)

2-Alkaloids totally biosynthesized from isoprene units: These alkaloids incorporate nitrogen late in the biosynthesis. Some authors call them pseudoalkaloids:

a- Monoterpenoid alkaloids e.g. Skytanthine in valerian root and Actinidine . They have no biological importance.

b- Sesquiterpenoid alkaloids e.g. from water Lilies Nuphar luteum and Nymphea alba (Nymphaeaceae) used as itch-relieving treatment of skin disorders, cracks, bruises, insect bites, burns, sunburns and diaper rashes. H H

NH OH N

O O

Deoxynupharidine Nuphamine c- Diterpenoid alkaloids

-Highly toxic alkaloids with very complex structures of C20 or C19 (norditerpenoid)

-They are obtained from genera Aconitium, Consolida and Delphinium family Ranunculaceae e.g. Aconitine:

-Thy are ether soluble ester alkaloid used as arrow poison in China and India. It is a neurotoxin acting on sodium channels and used in murders (25 µg/kg → 90 % mortality). d- Steroidal and triterpenoidal alkaloids:

- C21 steroidal alkaloids from Hollarhenna pubescens (Apocynaceae) e.g. holaphylline. They arise from pregnenolone by amination at C-3 or C-20 and used to make aldosterone. O

H2N Holaphylamine

- C24 in Buxaceae (derived from cycloarternol)

- C27 occur in Solanum and Veratrum. - Tritepene alkaloids the only known examples are yuzurimine and daphniphylline isolated from (Daphniphyllaceae or Euphorbiaceae) Steroidal alkaloids -Source: Families Liliaceae and Solanaceae contain steroidal alkaloids based on C27 cholestane. Examples include Veratrum and Solanum alkaloids.

-Steroidal alkaloids resemble saponins existing as glycosides with surface activity and hemolytic properties being toxic if ingested.

CH3

CH3

Steroidal nucleus

-Biosynthesis: they derived from isoprene units, and can be classified as steroids or triterpenes (mevalonate pathway). 1-Solanum alkaloids

-Many plants in Solanaceae accumulate steroidal alkaloids e.g. solasodine and tomatidine, which are usually present as glycosides e.g. solasonine, tomatine. Solanidine (glycoside is solanine) is present in potato (Solanum tuberosum).

-These compounds are employed for commercial steroid production by degradation of side chain in a similar way to steroidal sapogenins (via Marker degradation) to provide progesterone, testosterone, estradiol or corticosteroids. -These steroids are closely related to steroidal saponins (structurally and biogenetically) and may be viewed as nitrogen-containing saponins.

-They share saponins in the following: 1-They cause membrane alteration by interaction with membrane steroids in analogous manner to saponins. 2-They have choline esterase inhibitory activity and possess molluscicide, insecticide and cytotoxic effects. However, they are not used therapeutically because of toxicity. H H

H N H H H 1,3 b-DGlc b-D-Gal O 1,2 Solanidine a-L-Rha

a-Solanine -Physical properties: needles (from alcohol), mp = 285 oC, bitter taste, sparingly water soluble, readily soluble in alcohol, insoluble in CHCl3 and ether.

-Chemically, Solanine is a very weak base, which is not affected by alkali hydrolysis. Acid hydrolysis produces solanidine (aglycone) + galactose + glucose + L-Rhamnose. Isolation 1-Powdered drug is extracted with alcohol containing 1% acetic acid. The alcohol is evaporated under vacuum and residue extracted with acetic acid.

2-Acetic acid extract is alkalinized with NH3 and heated to precipitate solanine, recrystallized from alcohol.

Chemical testing

1-+SbCl3/ CHCl3 give red color.

2-+ H2SO4 + formaldehyde produce violet color, used in colorimetric assay.

3-Few mg heated with alcohol and cooled results in a jelly.

Assay 1-Colorimetric  based on violet color with HCHO / sulfuric. 2-Acid dye technique 2- Veratrum alkaloids -Veratrum has been used in the same way as Rauwolfia and sometimes in combination with it.

-Veratrum alkaloids are biosynthesized from cholestrol.

-Veratrum alkaloids have teratogenic effect on animals grazing on Veratrum californicum due to compounds: cyclopamine, jervine and cyclopamine glucoside (cycloposine).

1-Varatrum viridi (green or American Hellebore):

-Dried rhizomes and roots of Veratrum viridi (Liliaceae). The drug contains a unstable complex mixture of alkaloids that account for problems encountered in the biologic standardization of the drug.

-V. viridi possesses hypotensive, cardiac depressant and sedative properties. It has been used in the treatment of hypertension in small doses without affecting cardiac or respiratory rate. 2-Veratrum album (white or European Hellebore):

-Dried rhizome of V. album which contains a complex mixture of alkaloids e.g. ester alkaloids protoveratrine A and B (C nor D homo steroids) are the most active.

-White hellebore possesses hypotensive properties but is not used therapeutically. Both white and green Hellebore are currently employed as insecticides. Tropolone alkaloids (Pseudo-aromatic seven membered unsaturated ring)

O OH

Colchicum alkaloids

-Source: 1-Seeds and corms of Autumn crocus C. autumnale (Liliaceae), which contain 0.8% and 0.6% of colchicine, respectively. It is native to the black sea region (Georgia). 2-Cholchicine is extracted from Gloriosa superba in 0.9% yield by several European companies. 3-Colchicine is also present in other liliaceous plants e.g. Bulbocodium, Gloriosa, Merendera, Sandersonia (family Colchicaceae). -Chemically, the compound has only a weekly basic amidonitrogen. It is considered as an alkaloid as it is precipitated by many alkaloid reagents.

-Physical properties pale yellow, amorphous scales or powder that gradually darkens on exposure to light. It is soluble in water and ether, freely soluble in alcohol and CHCl3, neutral to litmus.

O MeO NH H MeO OMe O

OCH3 Application in therapy and uses 1-Colchicine is used as gout suppressant by inhibition of leukocyte migration and reduction of lactic acid production by leukocytes leading to decreased deposition of uric acid. It also decreases phagocytosis, which reduces inflammation. 2-It has antimitotic activity and interfere with the formation of mitotic spindle, which may lead to change in chromosome number (polyploidy). New varieties of plants of pharma cognostic and economic value could thus be obtained. 3-Injection of colchicine in animals can affect the dispersal of tumors. Thus it has been employed in treatment of various neoplastic diseases.

Mechanism of action Colchicine binds to tubulin and prevents polymerization to microtubules leading to multiplication of chromosomes without cell division, which starts on cessation of treatment leading to polyploidy. Tests of identity 1-Cochicine + HCl  intense yellow color.

2-Alcoholic solution + FeCl3  red color. 3- Colchicine + conc. HNO3  dirty violet  brown  yellow. 4-Strong bases change ring C to benzenoid ring (allocolchicine or colchicic acid methyl ester)

5- Mineral acids lead to demethylation of OCH3 in ring C with the formation of methyl alcohol.

Isolation 1-Powder is extracted by percolation with hot 90% alcohol. Alcoholic extract is concentrated to a syrupy consistency + water  precipitate of resins + fats, filter.

2-Extraction with CHCl3, CHCl3ic extract is concentrated to syrup and treated with small amount of alcohol, while warm; on cooling  additive compound of colchicine and CHCl3. 3-Additive compound is decomposed by suspending in H2O, heating and then evaporation. The residue is crystallized from ethyl acetate. Tests of identity

O O MeO NH MeO NH H MeO base H MeO OMe OMe O COOCH3 OCH3 Colchicine allocolchicine

-Assay

1-Colorimetric with FeCl3. 2-Gravimetrically. -The biosynthesis of cholchicine is from phenylalanine and tyrosine and intermediate is autumnaline.