alkaloides-_vincristine_camptothecin

Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 11

A SEMINAR ON

CLASSIFICATION, ISOLATION, STRUCTURE DETERMINATION, STEREOCHEMISTRY, BIOLOGICAL ACTIVITY OF

ALKALOIDES:- VINCRISTINE,CAMPTOTHECINGLYCOSIDES:- CALANOLIDES, GLYCERRHITINIC ACID

Guided By:Dr. N. M. PATELPrincipal & H.O.D

B. M . SHAH COLLEGE OF PHRM. EDU. AND RESEARCH, MODASA

Given By:KINJAL H. SHAHM. Pharm.- III


VINCRISTINEVINCRISTINE Vincristine is an indole alkaloid Vincristine is an indole alkaloid

obtained along with vinblastine from obtained along with vinblastine from the common periwinkle, the common periwinkle, Cathranthus Cathranthus roseus. roseus. G.DonG.Don

This plant was previously known as This plant was previously known as VVinca rosea Linn.inca rosea Linn., belonging to the , belonging to the natural order Apocynaceae.natural order Apocynaceae.

Synonyms:- Leurocristine; VCR; LCR.Synonyms:- Leurocristine; VCR; LCR.

The structure of a vincristine The structure of a vincristine methiodide, was then determined by methiodide, was then determined by the combination of two the combination of two crystallographic methods based on crystallographic methods based on the anomalous scattering of X-rays.the anomalous scattering of X-rays.

Vincristine is bisindole alkaloids, Vincristine is bisindole alkaloids, containing vindoline attached to a containing vindoline attached to a

cathranthincathranthin Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 33

VINCRISTINEVINCRISTINE Physical properties:-Physical properties:-

Melting rangeMelting range 218-220218-22000

SolubilitySolubility Practically insoluble in water; soluble in Practically insoluble in water; soluble in alcohol and chloroformalcohol and chloroform

Specific optical Specific optical rotationrotation

DD25 25 + 26.2 + 26.200 (ethylene chloride); [ (ethylene chloride); []D]D23 23 + + 171700

pH RangepH Range pH of a 0.1% solution is 3.5 to 5.0pH of a 0.1% solution is 3.5 to 5.0

Loss on DryingLoss on Drying Dried at 40Dried at 400 0 ; pressure not exceeding 0.7 ; pressure not exceeding 0.7 kPa for 16 hours, loses not more than kPa for 16 hours, loses not more than 12.0% of its weight.12.0% of its weight.

Dissociation Dissociation constantconstant

pKa 5.0, 7.4 (in 33% DMF)pKa 5.0, 7.4 (in 33% DMF)Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 44

PHENOLICSPHENOLICS• Mode of action:-Mode of action:-• Vincristine's anti-cancer properties result from its ability to Vincristine's anti-cancer properties result from its ability to

inhibit cell division during early mitosis. inhibit cell division during early mitosis. • Vincristine binds to the tubulin monomers preventing the Vincristine binds to the tubulin monomers preventing the

formation of spindle microtubules. By binding to the building formation of spindle microtubules. By binding to the building blocks of microtubules, vincristine disables the cell's blocks of microtubules, vincristine disables the cell's mechanism for aligning and moving the chromosomes.mechanism for aligning and moving the chromosomes.

• Vincristine stops the separation of the duplicated Vincristine stops the separation of the duplicated chromosomes and prevents cell division. chromosomes and prevents cell division.

• While vincristine works to keep the cancer cells from dividing, While vincristine works to keep the cancer cells from dividing, it does not selectively inhibit cancer cell division. It can also it does not selectively inhibit cancer cell division. It can also halt the division of some healthy cells, leading to some of the halt the division of some healthy cells, leading to some of the common side effects. common side effects.

VINCRISTINEVINCRISTINE


VINCRISTINEVINCRISTINE Common side effects include:-Common side effects include:-• Hair loss Hair loss • Pain/redness at location of injection Pain/redness at location of injection • Nausea/stomach pain/vomiting Nausea/stomach pain/vomiting • Lowered blood cell count Lowered blood cell count • Numbness Numbness • Headache Headache • Constipation Constipation • Nervous system problems such as neuropathy or sensory impairment Nervous system problems such as neuropathy or sensory impairment • Blurred or double vision Blurred or double vision • Back pain Back pain • Overall weakness Overall weakness Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 66

VINCRISTINEVINCRISTINE Ultra violet spectrum:-Ultra violet spectrum:-

The Uv absorbance The Uv absorbance spectrum of vincristine sulfate in spectrum of vincristine sulfate in methanol was scanned from 200 methanol was scanned from 200 to 400 nm using a Pye-Unicum SP to 400 nm using a Pye-Unicum SP 8-100 spectrophotometer.8-100 spectrophotometer.

maxmax nm nm loglog AA1111

%%cmcm

218218 4.724.72 568.75568.75

252252 4.244.24 188.75188.75

285285 4.184.18 165.60165.60

293293 4.234.23 185.6185.6Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 77

VINCRISTINEVINCRISTINE Infrared Spectrum (IR) :-Infrared Spectrum (IR) :-

The IR absorption spectrum of The IR absorption spectrum of vincristine sulfate as a KBr disc (1%) vincristine sulfate as a KBr disc (1%) was recorded over a Pye-Unicum SP was recorded over a Pye-Unicum SP 3-300 Infrared Spectrophotometer.3-300 Infrared Spectrophotometer.

Frequency CmFrequency Cm-1-1 Functional Functional GroupGroup

34503450 Free OHFree OH

29202920 -CH stretch-CH stretch

17251725 Ester C=OEster C=O

16801680 Lactam C=OLactam C=O

12251225 C-O-CC-O-CDr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 88

PHENOLICSPHENOLICSVINCRISTINEVINCRISTINE 11H-NMR Spectrum:-H-NMR Spectrum:-

obtained by Varian XL 200 NMR Spectrophotometer for a solution in Dobtained by Varian XL 200 NMR Spectrophotometer for a solution in D22O.O.

Chemical shiftChemical shift AssignmentAssignment

7.18 – 7.49 (m)7.18 – 7.49 (m) 4H, aromatic protons of catharanthine. ( C 9’, 10’, 4H, aromatic protons of catharanthine. ( C 9’, 10’, 11’, 12,)11’, 12,)

6.66 (s)6.66 (s) H, aromatic proton of vindoline (CH, aromatic proton of vindoline (C99).).

6.42 (s)6.42 (s) H, aromatic proton of vindoline (CH, aromatic proton of vindoline (C1212).).

3.913 (s)3.913 (s) 3H, methoxy protons (C3H, methoxy protons (C1111).).

3.757 (s)3.757 (s) 3H, ester protons of catharanthine (C3H, ester protons of catharanthine (C16’16’).).

3.650 (s)3.650 (s) 3H, ester protons of vindoline (C3H, ester protons of vindoline (C1616).).

2.068 (s)2.068 (s) 3H, acetate protons of vindoline (C3H, acetate protons of vindoline (C1717).).Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 99

VINCRISTINEVINCRISTINE


VINCRISTINEVINCRISTINE Vincristine chemically is 22-oxo-Vincristine chemically is 22-oxo-

vinblastine, it can therefore be vinblastine, it can therefore be prepared from vinblastine by prepared from vinblastine by specific N-oxidation.specific N-oxidation.

Vinblastine is first synthesized from Vinblastine is first synthesized from catharanthine and vindoline catharanthine and vindoline followed by conversion into followed by conversion into vincristine.vincristine.

Total synthesis of vinblastine:-Total synthesis of vinblastine:-

1. Catharanthine1. Catharanthine

2. Cathranthine N-oxide2. Cathranthine N-oxide

3. Trifluoroacetate derivative3. Trifluoroacetate derivative

4. Immonium ion.4. Immonium ion.Dr. Harish & Purvi Dr. Harish & Purvi kakranikakrani 1111

VINCRISTINEVINCRISTINE Total synthesis of vinblastine:-Total synthesis of vinblastine:-

5. Anhydro vinblastine5. Anhydro vinblastine

6. Vinblastine.6. Vinblastine.

Synthesis of Vincristine:-Synthesis of Vincristine:-

Two methods are available.Two methods are available.

First is to oxidize the N- methyl First is to oxidize the N- methyl group of vinblastine with chromium group of vinblastine with chromium trioxide in acetone at low trioxide in acetone at low temperature (-60temperature (-6000c) to give c) to give vincristine directly (7).vincristine directly (7).


VINCRISTINEVINCRISTINE Synthesis of vincristine:-Synthesis of vincristine:-

The other method involved The other method involved microbial N- demethylation microbial N- demethylation followed by N-formylation followed by N-formylation procedure as follows:-procedure as follows:-

Vinblastine (1) was Vinblastine (1) was incubated with the incubated with the microorganism Streptomyces microorganism Streptomyces albogriseolus to afford N- albogriseolus to afford N- demethylvinblastine (2), which demethylvinblastine (2), which was N-formylated to produce was N-formylated to produce vincristine (3).vincristine (3).


VINCRISTINEVINCRISTINE Biosynthesis of vincristine:-Biosynthesis of vincristine:-

When radioactive vindoline(1) and When radioactive vindoline(1) and labelled cathranthine (2) were fed into 6-labelled cathranthine (2) were fed into 6-week old differentiated Cathranthus week old differentiated Cathranthus roseusplants,labelled anhydrovinblastine roseusplants,labelled anhydrovinblastine (3)has been isolated.(3)has been isolated.

Administration of labelled (3) to cell-free Administration of labelled (3) to cell-free extracts of C.roseus afforded radioactive extracts of C.roseus afforded radioactive vinblastine. It has also be shown that vinblastine. It has also be shown that when (3) was incubated at room when (3) was incubated at room temperature in solutions of the cell-free temperature in solutions of the cell-free extracts from C. roseus leaves at pH 6.3, extracts from C. roseus leaves at pH 6.3, both radioactive vinblastine(4) and both radioactive vinblastine(4) and vincristine(5) were obtained after 3 and vincristine(5) were obtained after 3 and 50 hours respectively.50 hours respectively.


VINCRISTINEVINCRISTINE Isolation of vincristine:_

Svoboda’s Method:-

Svoboda et al. devised an extraction scheme by which numerous individual vinca alkaloids have been isolated.

The ground whole plant material is extracted with skelly B(n- hexane) for de fatting followed by extraction with tartaric acid and org solvents such as benzene and ethyl chloride.

Separation is achived by chromatography and isolation is done by gradient pH technique.


VINCRISTINEVINCRISTINE Supercritical fluid extraction:-

Previously developed extraction methods involve aqueous or alcohol extraction followed by pH control and re- extraction with org. solvent, which is very long and tedious process and also use large quantity of toxic org. solvent.

The aerial proportions and roots (200mg) were extracted with methanol (50*3) & evaporated in vaccum. Each extract was dissolved in 1 ml methanol and filtered through a membrane of 0.45mm pore size These experiments were conducted in triplicate.

SFE was performed using an ISCO Supercritical Fluid Extractor model SFX 3560 equipped with two ISCO 260D syringe pumps using CO2 in the pressure range of 10.2-34.0 MPa at 40,60 & 800c.


VINCRISTINEVINCRISTINE Supercritical fluid extraction:-

All extracted analytes were collected in 20 ml vials containing 10 ml of methanol.

After evaporation, each extract was re-dissolved in 1 ml methanol and filtered through a membrane of 0.45m pore size. These experiment conducted in triplicate.

HPLC – ESI/MS Analysis:-

An aglient 1100 series HPLC system equipped with an autosampler a photodiode array detector, a column over, a binary pump & a degrasser was used.

Separation of vinblastine and vincristine were perfomed using a Zorbax Bonus RP-18 at 400c.Dr. Harish & Purvi Dr. Harish & Purvi

kakranikakrani 1717

CAMPTOTHECINCAMPTOTHECIN 20(S)-Camptothecin (CPT), a

pentacyclicalkaloid, was first isolated by Wall and co-workers in 1966from extracts of Camptotheca acuminata, a tree native toChina.

CPT has a planar pentacyclic ring structure, that includes a pyrrolo[3,4-β]-quinoline moiety (rings A, B and C), conjugated pyridone moiety (ring D) and one chiral center at position 20 within the alpha-hydroxy lactone ring with (S) configuration (the E-ring). Its planar structure is thought to be one of the most important factors in topoisomerase inhibition.Dr. Harish & Purvi Dr. Harish & Purvi

kakranikakrani 1818

CAMPTOTHECINCAMPTOTHECIN

Physical properties:-Physical properties:-Molecular weightMolecular weight 348.111348.111

SolubilitySolubility The parent compound is extremely water The parent compound is extremely water insoluble, insoluble in all organic insoluble, insoluble in all organic compounds except dimethyl sulfoxide in compounds except dimethyl sulfoxide in which it exhibits moderate solubility.which it exhibits moderate solubility.

Specific optical Specific optical rotationrotation

[[]D]D25 25 + 31.3 + 31.300

Melting pointMelting point 264-267264-26700cc

DescriptionDescription Brown crystalline powder, easy to loss Brown crystalline powder, easy to loss activity under light.activity under light.



Mode of Action:-• Camptothecin act by binding to the topoisomerase I-DNA

complex, leading to an accumulation of DNA strand breaks upon replication ultimately causing cell death during the S-phase of the cell cycle.

• The complex is normally a transient intermediate which involved in DNA relaxation during a number of critical cellular processes, including replication transcription, recombination, repair, chromatin assembly and chromosome segregation, and generally rapidly reversible without CPT.


CAMPTOTHECINCAMPTOTHECIN Topoisomerase are essential nuclear

enzymes that modify the topological state of DNA through the introduction of transient breaks in the phosphodiester backbone of DNA

Type I topoisomerases make single strand DNA cuts and type II topoisomerases make double strand DNA cuts. Thetopoisomerization reactions carried out by either the type I or type II enzyme involve a Y transient DNA break. During this reaction, the enzyme becomes covalently linked to the break site via a phosphotyrosine bond. Dr. Harish & Purvi Dr. Harish & Purvi

kakranikakrani 2121


Camptothecin affects the activity of the enzyme topoisomerase I, whose normal action is to cleave, unwind, and religate DNA. When camptothecin binds to topoisomerase I, it will be able to cleave but not to religate DNA. Thereby, camptothecin causes single strand breaks in DNADr. Harish & Purvi Dr. Harish & Purvi

kakranikakrani 2222

PHENOLICSPHENOLICSCAMPTOTHECINCAMPTOTHECIN Isolation:-

Sucessive extraction of plant material is done by org. solvent in order of heptane, ethanol, chloroform and water. The aqueous fraction partially concentrated; a yellow precipitate was formed and collected by filtrate. This material was subsequently further purified by chromatography on a silica gel column and crystalisation.


CAMPTOTHECINCAMPTOTHECIN Derivatives of Camptothecin:-Derivatives of Camptothecin:-


CAMPTOTHECINCAMPTOTHECIN Biosynthesis :-

Hairy root culture of Ophiorrhiza pumila with tracer technique using C13 glucose.

Breakdown of glucose provide acetyl CoA which is precursor for MVA Pathway and also provides glyceraldehyde 3- phosphate and pyruvate which are precursors of MEP Pathway.


CALANOLIDESCALANOLIDES The calanolides and

inophyllums represent novel HIV inhibitory coumarin derivatives, isolated from the tropical rainforest tree, Calophyllum lonigerum and Calophyllum inophyllum.

Both compounds were found to inhibit HIV 1 These compounds can be considered as NNRTIs.


CALANOLIDESCALANOLIDES Racemic synthesis:-

Chenara et al.was the first research group to establish the racemic calanolide A in 1993 and the related calanolide C and D via a five-step synthesis with 15% overall yield starting from phlorglucinol and then constructed the coumarin (A, B rings) followed by the chromanone ring (C) using a Lewis acid-promoted Claisen rearrangement. The chromene ring was built last. Finally,Luche reduction of the chromanone provided racemic calanolide A


CALANOLIDESCALANOLIDES PHARMACOLOGY OF CALANOLIDES:-

(+) Calanolide A, (-) Calanolide B are the most potent candidates of anti-HIV-1agents among Calophyllum coumarins.

CalanolideA is the only naturally occurring anti-HIV agent to be at an advanced stage of a phase II clinical trial, but it has been obtained in relatively low yield isolated from Calophyllum lanigerum.

Some structural modifications of (+)-calanolideA or (-)-calanolideB have been carried out. However, no compounds showed anti-HIV-1 activity superior to the two lead.


GLYCYRRHETINIC ACIDGLYCYRRHETINIC ACID Glycyrrhetinic acid is a pentacyclic

triterpenoid derivative of the beta-amyrin type obtained from the hydrolysis of glycyrrhizic acid, which was obtained from the herb liquorice.

Research in 2005 demonstrated that with a proper functional group a very effective glycyrrhetinic artificial sweetener can be obtained.

The structure of glycyrrhetinic acid is similar to that of cortisone. Both molecules are flat and similar at position 3 and 11.

This might be the basis for licorice's anti-inflammatory action.Dr. Harish & Purvi Dr. Harish & Purvi

kakranikakrani 2929

GLYCYRRHETINIC ACIDGLYCYRRHETINIC ACID Stereochemistry:-

Two isomers:-

1. 18 acid

2. 18 acid.

It is more stable in 18 configuration because the carboxylic group in 18 acid is axial while in 18 acid is equatorial.


GLYCYRRHETINIC ACIDGLYCYRRHETINIC ACID Mecanism of action:-

• Glycyrrhetinic acid inhibits the enzymes (15-hydroxyprostaglandin dehydrogenase and delta-13-prostaglandin) that metabolise the prostaglandins PGE-2 and PGF-2α to their respective 15 keto-13,14-dihydro metabolites which are inactive.

• This causes an increased level of prostaglandins in the digestive system. Prostaglandins inhibit gastric secretion but stimulate pancreatic secretion and mucous secretion in the intestines and markedly increase intestinal motility.


GLYCYRRHETINIC ACIDGLYCYRRHETINIC ACID Mecanism of action:-

• They also cause cell proliferation in the stomach. The effect on gastric acid secretion, promotion of mucous secretion and cell proliferation shows why licorice has potential in treating peptic ulcer.

• PGF-2α stimulates activity of the uterus during pregnancy and can cause abortion, therefore, licorice should not be taken during pregnancy.


GLYCYRRHETINIC ACIDGLYCYRRHETINIC ACID Isolation of glycyrrhetinic acid:-Isolation of glycyrrhetinic acid:-

Crude drug

Extract with chloroform; filter

Marc extracted with 0.5 M H2SO4

Cool and shake with chloroform, conc. And dry

Glycyrrhetinic acid


GLYCYRRHETINIC ACIDGLYCYRRHETINIC ACID Therapeutic uses:-

Traditionally it has been used as an expectorant and demulcent in cough mixtures.

Also used as a flavouring agent.

Due to mineralocorticoid like activity it is employed in place of corticosteroids in Rheumatoid Arthritis.

Also used in peptic ulcer in the form of deglycyrrhizinated liquorice.


REFERENCESREFERENCES1.1. J. B. Harbone & H. Baxter J. B. Harbone & H. Baxter phytochemical dictionary; A Handbook Of Bioactive phytochemical dictionary; A Handbook Of Bioactive

Compounds From Plants; Taylor and Francis; Revised Reprint:1995, 185, 271,678.Compounds From Plants; Taylor and Francis; Revised Reprint:1995, 185, 271,678.

2.2. Manuchair Ebadi Manuchair Ebadi Pharmacodynamic Basis Of Herbal Medicine; Taylor & Francis; Pharmacodynamic Basis Of Herbal Medicine; Taylor & Francis; second Edition; 195.second Edition; 195.

3.3. Wilson & Gisvoid’s Wilson & Gisvoid’s Textbook of organic and pharmaceutical chemistry; Lippion Textbook of organic and pharmaceutical chemistry; Lippion cott Williams & Wilkins; tenth Edition; 379,381-382.cott Williams & Wilkins; tenth Edition; 379,381-382.

4.4. Svoboda G., : Svoboda G., : Lloydia Isolation of various Vinca Alkaloides; 24:173, 1961.Lloydia Isolation of various Vinca Alkaloides; 24:173, 1961.

5.5. Paul M. Dewick,Paul M. Dewick, Medicinal Natural Products; A Biosynthetic Approach; Wiley Medicinal Natural Products; A Biosynthetic Approach; Wiley Science Publications; Science Publications; Third Edition; 383-384.Third Edition; 383-384.

6.6. Analytical Profiles Of Drug Substances Edited By: Analytical Profiles Of Drug Substances Edited By: Klaus FloryKlaus Flory; Harry G. Brittain ; Harry G. Brittain Academic Press, Vol-I: 463-480; Vol-22: 517-553; Vol-23: 611-658.Academic Press, Vol-I: 463-480; Vol-22: 517-553; Vol-23: 611-658.

7.7. Burger’s Burger’s Medicinal chemistry & Drug Discovery; A Wiley Interscience Publication; Medicinal chemistry & Drug Discovery; A Wiley Interscience Publication; Volume 1; 995.Volume 1; 995.


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alkaloides-_vincristine_camptothecin

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