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Harish & Purvi

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 A SEMINAR ON

CLASSIFICATION, ISOLATION, STRUCTURE DETERMINATION,

STEREOCHEMISTRY, BIOLOGICAL ACTIVITY OF

ALKALOIDES:- VINCRISTINE,CAMPTOTHECIN
GLYCOSIDES:- CALANOLIDES, GLYCERRHITINIC ACID

Given By: Guided By:

KINJAL H. SHAH Dr. N. M. PATEL
M. Pharm.- III Principal & H.O.D

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B. M . SHAH COLLEGE OF PHRM. EDU. AND RESEARCH, MODASA 2
 VINCRISTINE
 Vincristine is an indole alkaloid
obtained along with vinblastine from
the common periwinkle, Cathranthus
roseus. G.Don
 This plant was previously known as
Vinca rosea Linn., belonging to the
natural order Apocynaceae.
 Synonyms:- Leurocristine; VCR; LCR.
 The structure of a vincristine
methiodide, was then determined by
the combination of two
crystallographic methods based on
the anomalous scattering of X-rays.

 Vincristine is bisindole alkaloids,

containing vindoline attached to a
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cathranthin
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 VINCRISTINE
 Physical properties:-
Melting range 218-2200
Solubility Practically insoluble in water; soluble in
alcohol and chloroform
Specific optical D25 + 26.20 (ethylene chloride); []D23 +
rotation 170
pH Range pH of a 0.1% solution is 3.5 to 5.0
Loss on Drying Dried at 400 ; pressure not exceeding 0.7
kPa for 16 hours, loses not more than
12.0% of its weight.
Dissociation pKa 5.0, 7.4 (in 33% DMF)
constant
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 PHENOLICS
VINCRISTINE
• Mode of action:-
• Vincristine's anti-cancer properties result from its ability to
inhibit cell division during early mitosis.
• Vincristine binds to the tubulin monomers preventing the
formation of spindle microtubules. By binding to the building
blocks of microtubules, vincristine disables the cell's
mechanism for aligning and moving the chromosomes.
• Vincristine stops the separation of the duplicated
chromosomes and prevents cell division.
• While vincristine works to keep the cancer cells from dividing,
it does not selectively inhibit cancer cell division. It can also
halt the division of some healthy cells, leading to some of the
common side effects.
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 VINCRISTINE
 Common side effects include:-
• Hair loss
• Pain/redness at location of injection
• Nausea/stomach pain/vomiting
• Lowered blood cell count
• Numbness
• Headache
• Constipation
• Nervous system problems such as neuropathy or sensory impairment
• Blurred or double vision
• Back pain
• Overall weakness
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 VINCRISTINE
 Ultra violet spectrum:-
The Uv absorbance spectrum
of vincristine sulfate in methanol
was scanned from 200 to 400 nm
using a Pye-Unicum SP 8-100
spectrophotometer.

 max nm log A11%cm

218 4.72 568.75

252 4.24 188.75

285 4.18 165.60

293 4.23
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185.6
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 VINCRISTINE
Infrared Spectrum (IR) :-
The IR absorption spectrum of
vincristine sulfate as a KBr disc (1%)
was recorded over a Pye-Unicum SP
3-300 Infrared Spectrophotometer.

Frequency Cm-1 Functional

Group
3450 Free OH
2920 -CH stretch
1725 Ester C=O
1680 Lactam C=O
1225 C-O-C
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 PHENOLICS
VINCRISTINE
 H-NMR Spectrum:-
1

obtained by Varian XL 200 NMR Spectrophotometer for a solution in D 2O.

Chemical shift Assignment

7.18 – 7.49 (m) 4H, aromatic protons of catharanthine. ( C 9’, 10’,
11’, 12,)
6.66 (s) H, aromatic proton of vindoline (C9).
6.42 (s) H, aromatic proton of vindoline (C12).
3.913 (s) 3H, methoxy protons (C11).
3.757 (s) 3H, ester protons of catharanthine (C16’).
3.650 (s) 3H, ester protons of vindoline (C16).
2.068& (s)
Dr. Harish Purvi 3H, acetate protons of vindoline (C17).
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 VINCRISTINE

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 VINCRISTINE
 Vincristine chemically is 22-oxo-
vinblastine, it can therefore be
prepared from vinblastine by
specific N-oxidation.
 Vinblastine is first synthesized from
catharanthine and vindoline
followed by conversion into
vincristine.
 Total synthesis of vinblastine:-
1. Catharanthine
2. Cathranthine N-oxide
3. Trifluoroacetate derivative
4. Immonium ion.
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 VINCRISTINE
 Total synthesis of vinblastine:-
5. Anhydro vinblastine
6. Vinblastine.
 Synthesis of Vincristine:-
Two methods are available.
First is to oxidize the N- methyl
group of vinblastine with chromium
trioxide in acetone at low
temperature (-600c) to give
vincristine directly (7).

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 VINCRISTINE
 Synthesis of vincristine:-
The other method involved
microbial N- demethylation
followed by N-formylation
procedure as follows:-
Vinblastine (1) was
incubated with the
microorganism Streptomyces
albogriseolus to afford N-
demethylvinblastine (2), which
was N-formylated to produce
vincristine (3).

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 VINCRISTINE
 Biosynthesis of vincristine:-
When radioactive vindoline(1) and
labelled cathranthine (2) were fed into 6-
week old differentiated Cathranthus
roseusplants,labelled anhydrovinblastine
(3)has been isolated.
Administration of labelled (3) to cell-free
extracts of C.roseus afforded radioactive
vinblastine. It has also be shown that
when (3) was incubated at room
temperature in solutions of the cell-free
extracts from C. roseus leaves at pH 6.3,
both radioactive vinblastine(4) and
vincristine(5) were obtained after 3 and
50 hours respectively.
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 VINCRISTINE
 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.
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 VINCRISTINE
 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.

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 VINCRISTINE
 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 0.45m 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
Dr. Harish
Zorbax & Purvi
Bonus RP-18 at 400c.
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 CAMPTOTHECIN
 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
Dr. topoisomerase
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 CAMPTOTHECIN

 Physical properties:-
Molecular weight 348.111

Solubility The parent compound is extremely water

insoluble, insoluble in all organic
compounds except dimethyl sulfoxide in
which it exhibits moderate solubility.
[]D25 + 31.30
25 0
Specific optical
rotation
Melting point 264-2670c

Description Brown crystalline powder, easy to loss

activity under light.
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 CAMPTOTHECIN
 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.

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 CAMPTOTHECIN
 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
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 CAMPTOTHECIN

 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
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DNA
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 PHENOLICS
CAMPTOTHECIN
 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.

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 CAMPTOTHECIN
 Derivatives of Camptothecin:-

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 CAMPTOTHECIN
 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.

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 CALANOLIDES
 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.

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 CALANOLIDES
 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
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 CALANOLIDES
 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.
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 GLYCYRRHETINIC 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
Dr. anti-inflammatory
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 GLYCYRRHETINIC 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.

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 GLYCYRRHETINIC 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.

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 GLYCYRRHETINIC 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.

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 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
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 GLYCYRRHETINIC 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.

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 REFERENCES
1. J. B. Harbone & H. Baxter phytochemical dictionary; A Handbook Of Bioactive
Compounds From Plants; Taylor and Francis; Revised Reprint:1995, 185, 271,678.
2. Manuchair Ebadi Pharmacodynamic Basis Of Herbal Medicine; Taylor & Francis;
second Edition; 195.
3. Wilson & Gisvoid’s Textbook of organic and pharmaceutical chemistry; Lippion
cott Williams & Wilkins; tenth Edition; 379,381-382.
4. Svoboda G., : Lloydia Isolation of various Vinca Alkaloides; 24:173, 1961.
5. Paul M. Dewick, Medicinal Natural Products; A Biosynthetic Approach; Wiley
Science Publications; Third Edition; 383-384.
6. Analytical Profiles Of Drug Substances Edited By: Klaus Flory; Harry G. Brittain
Academic Press, Vol-I: 463-480; Vol-22: 517-553; Vol-23: 611-658.
7. Burger’s Medicinal chemistry & Drug Discovery; A Wiley Interscience Publication;
Volume 1; 995.

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 THANK YOU

Dr. Harish & Purvi

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