CATHARANTHUS

ALKALOIDS

DEEKSHA PAHWA

B.PHARM
PUNJAB UNIVERSITY
CHANDIGARH
 CONTENTS

 INTRODUCTION

 CATHARANTHUS ALKALOIDS

1. Introduction
2. Extraction procedures of Vinca alkaloids
3. Biosynthesis of Catharanthus alkaloids
4. Cell and organ cultures
5. Semi-synthetic procedures of production of vinblastine

and vincristine
6. Mechanism of action
7. Clinical indications of the Vinca alkaloids
8. Toxicity
9. Effect of weather on the production of alkaloids in C.

roseus
10.Chemical Modifications of the Vinca alkaloids leading to
the synthesis of novel alkaloids
11. Other novel cytotoxic alkaloids isolated from
Catharanthus roseus.
12. Vinca alkaloids in clinical trials.

 TABLE : Vinca Alkaloids: Comparative Pharmacokinetic and

Toxicologic Characteristics

 REFERENCES
 INTRODUCTION

The genus Catharanthus (Family- Apocynaceae) is comprised of eight species of

mostly perennial herbs. The single species Catharanthus pusillus is native to
India. All other species, are native to Madagascar. The chromosome number for
all Catharanthus species is 2n = 16.

The different species are :

• Catharanthus coriaceus Markgr. Madagascar.

• Catharanthus lanceus (Bojer ex A.DC.) Pichon. Madagascar.
• Catharanthus longifolius (Pichon) Pichon. Madagascar.
• Catharanthus ovalis Markgr. Madagascar.
• Catharanthus pusillus (Murray) G.Don. Indian subcontinent.
• Catharanthus roseus (L.) G.Don. Madagascar.
• Catharanthus scitulus (Pichon) Pichon. Madagascar.
• Catharanthus trichophyllus (Baker) Pichon. Madagascar

Ornamental cultivars of Catharanthus are all derived from species native to

Madagascar. Germplasm of C. roseus and C. trichophyllus has provided the
primary material for development of ornamental cultivars. As an ornamental,
Catharanthus, more commonly known as Madagascar Periwinkle or Vinca is
valued for its drought and heat tolerance.

In addition to its value as an herbaceous ornamental, alkaloid extracts of

Catharanthus roseus have been used in folk medicine as an antidiabetic, diuretic,
and antidysenteric, an anti haemorrhagic and for wound healing. In Europe, it was
mainly used as an antidiabetic, for easing lung congestion and throat
inflammation, in south and central Africa; as a poultice to stop bleeding in the US;
in India in case of insect stings; against eye inflammation in the Caribbean and as
an astringent, diuretic and cough remedy in China.
Research on this plant was stimulated by its mention in folklore and investigations
started in 1950 for antidiabetic activities. Antidiabetic activity could not be
confirmed. However, the test animals became susceptible to bacterial infections
on account of transient depression of the bone marrow leading to a net decrease in
the number of circulating leukocytes. This led the researchers to undertake
extensive examination for the possible immunosuppressive principles causing
these effects. A number of dimeric indole alkaloids showing antileukaemic
activity have subsequently been isolated and two of these, vincaleukoblastine
(vinblastine) and leurocristine (vincristine), are now extracted commercially from
C.roseus and used, either alone, or in combination with other forms of therapy for
cancer treatment.
Catharanthus roseus G. Don., has been variously designated Vinca rosea L. and
Lochnera rosea L. It is a rich source of alkaloids belonging to the category of
terpenoid indole alkaloids which are isolated from the three varieties of the plant:

(i) ‘roseus’ with violet or rose coloured flowers

(ii) ‘albus’ with white flowers and
(iii) ‘ocellatus’ with white flowers having red eye.
Of these, the red purple variety contains the highest amount of vincristine and
vinblastine.

Catharanthus roseus var. roseus Catharanthus roseus var. ocellatus

Catharanthus roseus var. albus

 CATHARANTHUS ALKALOIDS

1. Introduction

The aerial parts of the plant contain from 0.2 to 1% alkaloids. [1] About 150
alkaloids have been isolated from Catharanthus roseus.[2]
Of particular interest is a group of 20 dimeric alkaloids which contains those
with antineoplastic activity, including vincristine and vinblastine. These alkaloids
are formed by the coupling of two moieties: an indole moiety and a dihydroindole
moiety. Thus, this led to referring them as “dimer alkaloids” or “bisindole
alkaloids. [1]
Vinblastine is produced by coupling of two monomer alkaloids catharanthine
(indole) and vindoline (dihydroindole), both of which occur free in the plant.
Vincristine is structurally similar to vinblastine, but has a formyl group rather than
a methyl on the indole nitrogen in the vindoline derived portion.
Because these alkaloids are only minor constituents of the plant (vincristine is
obtained in about 0.0002% yield from the crude drug), large quantities of raw
materials are employed in the extraction procedures.
Also there is a growing demand for vincristine rather than vinblastine, but the
plant produces a much higher proportion of vinblastine. Fortunately, it is now
possible to convert vinblastine into vincristine either chemically or via a
microbiological N-demethylation using Streptomyces albogriseolus. [2]
Other binary alkaloids which are active are leurosidine (20’-epivinblastine),
leurosine (15’, 20’-epoxy vinblastine). [1]
Some alkaloids, for example, ajmalicine, lochnerine, serpentine and
tetrahydroalstonine, also occur in other genera of the family. [2]

2. Extraction procedures of Vinca alkaloids

Various extraction procedures employed are:

a. Supercritical fluid extraction (SFE)

b. Soxhlet extraction
c. Solid-liquid extraction
d. Hot water extraction
Supercritical fluid extraction
Supercritical temperature of a substance is one above which the substance can no
longer exist as a liquid, no matter how much pressure is applied. Similarly,
supercritical pressure is a pressure above which a substance can no longer exist as
a gas, no matter how high the temperature is raised. Under these conditions, the
gas and liquid phases both possess the same density and no division exists
between the two phases. This is the “critical state”. In practice, conditions
somewhat above the critical temperature and pressure for a particular substance
are usually used and these “supercritical fluids” exhibit properties intermediate
between those of the liquid and gaseous phases. Most commonly used is
supercritical fluid carbon dioxide (tc = 31oC and pc = 74atm). To render it more
polar, a small amount of modifier, e.g. methanol, may be added to the carbon
dioxide.[6] Other substances under development for use as supercritical fluids in
SFE are: ethane, ethane, propane and nitrous oxide.
In case of vinca alkaloids, vindoline is extracted by SFE. According to an
optimization study conducted for the process, a remarkably high content of
vindoline, 58 wt% was extracted from the leaves of Catharanthus roseus using
SFE under conditions of temperature as 35oC and the pressure being 300 bars. The
addition of 3 wt% ethanol as a cosolvent, only slightly improved the extraction
yields.[3]

Solid-liquid extraction
The plant extract from C. roseus is a complex mixture of alkaloids with a wide
range of polarities. The traditional solid-liquid extraction procedure for
Catharanthus alkaloids from an aqueous acidic medium is based on their general
basic properties. The alkaloids form salts in aqueous acidic media, showing
improved solubility and enhanced stability at low pH values. In addition, protons
in the aqueous acidic media assist in breaking the sample matrix to release the
analytes more easily.
As a further modification of the above process, water insoluble embonic acid
complexes of catharanthine and vindoline were prepared by adding an aqueous
alkaline (pH 10.5) solution of embonic acid (4,4’-methylene-bis-3-
hydroxynaphtalenecarboxylic acid) to the aqueous acidic solution (pH 1.5) of the
plant extract containing the alkaloids as their soluble hydrochloride salts. The
alkaloids were exhaustively precipitated when pH 5 was reached during this
process. The precipitate mostly consisted of stable embonate complexes of
catharanthine and vindoline, which were useful starting materials for vinblastine
synthesis.[4]
According to a recent study conducted, SFE method of extraction from dried
leaves of C.roseus was optimized to give higher yields of the pharmacologically
important indole alkaloids. Quantification of the alkaloid concentration was in the
range of 0.18 - 31 microg/ml. The yields obtained for catharanthine, vindoline,
vinblastine and vincristine were 2.7, 2.0, 1.3and 1.1 microg/g.[5]
Also different methods of extraction were compared for various indole alkaloids
and best recoveries for catharanthine (100%) were obtained using SFE at 250 bar
and 80oC, using 6.6vol% methanol as modifier for 40 minutes; for vindoline by
Soxhlet extraction using dichloromethane in a reflux for16 hours; and for
3’,4’-anhydrovinblastine by solid-liquid extraction using a solution of 0.5M
H2SO4 and methanol (3:1v/v) in an ultrasonic bath for 3hours.[5]

3. Biosynthesis of Catharanthus alkaloids

The various Catharanthus alkaloids belong to the class of terpenoid indole

alkaloids, that is, they consist of two moieties derived from two separate
metabolic pathways- The Mevalonate pathway which gives the non tryptophan
moiety; and the tryptophan moiety is obtained from tryptophan. The complex
structure of these alkaloids usually contains two nitrogen atoms; one is the indole
nitrogen (in the tryptophan- derived moiety) and the second is generally two
carbons removed from the Beta- position of the indole ring. The non- tryptophan
moiety is derived from mevalonic acid and it is a C10-geraniol (monoterpenoid)
contribution in the case of these alkaloids. This portion with suitable
rearrangements leads to formation of three types of alkaloids-
(i) Coryanthe- type alkaloids
(ii) Iboga- type alkaloids
(iii) Aspidosperma- type alkaloids
it is believed that the coryanthe- type monoterpenoid moiety is metabolically most
primitive. The reactive form of terpene involves an aldehyde group. The loss of
one carbon atom during biogenesis, to give C9 unit is largely common.
Geraniol by a series of conversions forms loganin and then secologanin (a
monoterpenoid glucoside). The molecular characterization of CYP72A1 from
Catharanthus roseus was described nearly a decade ago, but the enzyme function
remained unknown. However in a recent study conducted, it was shown that
CYP72A1 converts loganin into secologanin.[56]
A key intermediate in the biogenesis of the monoterpene indole alkaloids is
3alpha (S)- strictosidine, formed by the enzymatic condensation of tryptamine and
secologanin. The enzyme responsible for this important reaction, strictosidine
synthase, has been isolated and characterized from cell cultures of a number of
species including Catharanthus roseus.
Strictosidine then leads to formation of cathenamine (a coryanthe- type of
alkaloid); enzyme involved is cathenamine synthase. Cathenamine further gives
rise to ajmalicine (enzyme- ajmalicine synthase) and serpentine. Both ajmalicine
and serpentine are also coryanthe- type alkaloids. Cathenamine through a series of
reactions also leads to formation of catharanthine (iboga- type) and vindoline
(aspidosperma- type). Catharanthine and vindoline are monomeric indole
alkaloids and occur free in the plant. 3’,4’-Anhydrovinblastine is a key
intermediate from the coupling of catharanthine and vindoline and the enzymes
involved are peroxidases. It is further converted to vinblastine.

4. Cell and organ cultures

In efforts to improve the production of alkaloids, cell cultures of C.roseus have

received considerable attention. Earlier attempts at production in cell cultures
failed because a part of the complex pathway was not active, i.e. from tabersonine
to vindoline. The enzyme responsible for the conversion is tabersonine 16-
hydroxylase (T16H), a cytochrome P450-dependent enzyme. However, later it
was found that T16H is induced in the suspension culture by light. [51]
To date success has been achieved in obtaining total alkaloid yields corresponding
to 0.1 – 1.5% (dry weight, cultivated cells). Alkaloids including catharanthine,
vindoline and ajmalicine have been identified and isolated from these cell lines;
however, the more useful dimeric alkaloids have only been isolated in traces from
the cell cultures. [2]
It has been observed that leaf organ cultures of C.roseus synthesize a variety of
alkaloids. Also dimeric alkaloids have been detected in organ cultures of C.roseus,
suggesting the possibility of an efficient production system for these valuable
alkaloids. The dimers occurred only in those cultures which also contained
vindoline and catharanthine. [9]

However in a research conducted recently, the final dimerization step of the

terpinoid indole pathway, leading to the synthesis of the dimeric alkaloid,
vinblastine, was demonstrated to be catalyzed by a basic peroxidase and for the
first time the cloning, characterization and localization of a novel basic
peroxidase, CrPrx (Catharanthus roseus peroxidase), from C. roseus was done.
The CrPrx nucleotide sequence encodes a translation product of 330 amino acids
with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature.
The molecular mass of this unprocessed and unmodified protein is estimated to be
37.43 kDa. CrPrx was found to belong to a 'three intron' category of gene that
encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were
found to be present in specific organs and were regulated by different stress
treatments.
It is proposed that CrPrx is involved in cell wall synthesis, and also that the gene
is induced under methyl jasmonate treatment indicating its potential involvement
in the terpenoid indole alkaloid biosynthetic pathway. [55]

In one of the experiments conducted, a two-liquid-phase bioreactor was designed

to extract indole alkaloids from C.roseus ‘hairy root cultures’, using silicon oil.
Partition studies between silicon oil and culture medium showed that the affinity
of tabersonine and löchnericine for silicon oil is nine times higher than for the
aqueous phase.
Also, all measured alkaloids' specific yields were higher using silicon oil and
elicitation, suggesting that the silicon oil, by acting as a metabolic sink for
tabersonine and löchnericine, decreased their negative feedback effect on the
production of the other useful alkaloids in the aqueous medium and so was
efficient in increasing metabolic fluxes of the secondary metabolism pathways. [10]

The transformed root culture seems to be the most promising for alkaloid
production. The genetically transformed roots, obtained by the infection with
Agrobacterium rhizogenes, produce higher levels of secondary metabolites than
intact plants. Also, whole plants can be regenerated from hairy roots. The content
of indole alkaloids in the transformed roots was similar or even higher when
compared to the amounts measured in studies of natural roots. The predominant
alkaloids in transformed roots are ajmalicine, serpentine, vindoline and
catharanthine, found in higher amounts than in untransformed roots. Transformed
hairy roots have been also used for encapsulation in calcium alginate to form
artificial seeds.[57]

• Role of the Non-Mevalonate Pathway in Indole Alkaloid Production by

Catharanthus roseus Hairy Roots :

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway)

leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur
in most bacteria and in all higher plants. Treatment with the antibiotic
fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably
inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and
lochnericine by Catharanthus roseus hairy root cultures in the exponential growth
phase.
These results suggest that the DXP pathway is a major provider of carbon for the
monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus
hairy roots in the exponential phase.[58]
5. Semi-synthetic procedures of production of vinblastine and
vincristine
Vindoline and catharanthine are the major monomer alkaloids as well as
biosynthetic precursors for the "dimeric" alkaloids, vinblastine and vincristine.
Low "dimeric" alkaloid contents in the plant have encouraged intense research for
alternative production methods involving cell cultures,[18,19] metabolic
engineering,[20] semi-synthesis,[21,22] or even total chemical synthesis.[23] Total
synthesis has proved difficult due to structural complexity of the molecules and
complicated reaction steps involving stereochemical constraints. Various semi-
synthetic procedures have been developed for these alkaloids on the basis of
chemical[21,22] or enzymatic[24] coupling of commercially available catharanthine
and vindoline.
The present semi-synthetic process is partially based on an earlier method
involving photoactivation of catharanthine, as well as photolytic singlet oxygen
production in an aqueous reaction solution.[9] In that method the high energy
needed for singlet oxygen production from water, however, results in excessive
catharanthine consumption by many side reactions.
As an improvement to the above photochemical method, singlet oxygen (1O2) is
here produced in situ from hydrogen peroxide and sodium hypochlorite [19]. An
allylic hydrogen in the protonated catharanthine moiety is presumably attacked by
singlet oxygen to yield a hydroperoxide of catharanthine which then possibly
undergoes reduction in the presence of sodium borohydride. Then a final aromatic
substitution reaction yields vinblastine through the crucial C18'-C15 bond linking
the top indole half of the catharanthine moiety (C18') and the bottom
dihydroindole half of the vindoline moiety (C15).

6. Mechanism of action

Vinblastine and vincristine are antimitotics. They bind to tubulin and prevent the
formation of the microtubules which help in the formation of the mitotic spindle.
Thus, these compounds block mitosis and cause an accumulation of cells in the
metaphase (“metaphase arrest”).[11-16] This contributes to their major
pharmacological action.[1]
The microtubule assembly also plays a role at other levels, particularly in
neurotransmission (axon microtubules). Hence blockade of this activity is
responsible for the neurotoxicity caused as a side effect by these alkaloids. [1]
They are generally in vitro inhibitors of the biosynthesis of protein and nucleic
acid, elevate oxidized glutathione, alter lipid metabolism and membrane lipids,
elevate cyclic AMP and inhibit calcium-calmodulin regulated cyclic AMP
phosphodiesterase.[17]
The treatment of cell population with vincristine or vinblastine, leads to an
accumulation of cells in the M and G2 phase and the effect is lethal in the S
phase.[1]

7. Clinical indications of the Vinca alkaloids

Vincristine is used more commonly to treat pediatric malignancies, which likely

reflects a combination of the higher level of sensitivity of pediatric malignancies
to vincristine and to the better tolerance of higher vincristine doses in children. In
both children and adults, however, vincristine is an essential component of the
chemotherapy regimens used to treat acute lymphocytic leukemia, lymphoid blast
crisis of chronic myeloid leukemia, and both Hodgkin and non-Hodgkin
lymphomas. It also plays a role in some multimodality therapies of Wilms tumor,
Ewing sarcoma, neuroblastoma, and rhabdomyosarcoma, as well as in the
treatment of multiple myeloma and small-cell lung cancer in adults.[34]

Vincristine

On the other hand, vinblastine has been a mainstay component of chemotherapy

regimens for germ cell malignancies and some types of advanced lymphomas. On
a historical note, vinblastine has also been used alone or in combination with
other agents to treat Kaposi sarcoma and bladder, breast, and some types of brain
malignancies.[34] Vincristine has a superior antitumour activity as compared with
vinblastine, but the former is more neurotoxic.[2]
 Vinblastine

Desacetyl vinblastine (vindesine), initially identified as a metabolite of

vinblastine, was introduced in the 1970s.[25-29] Vindesine is available only for
investigational purposes in the United States, but is registered elsewhere. The
agent was principally evaluated in combination with other agents, particularly
cisplatin and/or mitomycin C, in treating non-small-cell lung cancer, but it has
also demonstrated consistently favorable results in several hematologic and solid
malignancies.[28,29]

V inde
sine
The newer, orally active,[2] vinblastine derivative semi-synthesized by Potier et al.
Vinorelbine (5’-norhydro Vinblastine), has broader anticancer activity and lower
neurotoxic side-effects than the other Catharanthus alkaloids.[8] It is structurally
modified on its catharanthine nucleus, resulting in substantially greater
lipophilicity as compared to the other Vinca alkaloids. The potent antitumor effect
of Vinorelbine with minor neurotoxicity was explained by Vinorelbine having
stronger activity on mitotic microtubules than axonal microtubules.[53]
It is effective in combination with chemotherapeutic agents such as anthracycline,
fluorouracil and Taxol. It is approved in the United States for treating non-small-
cell lung cancer as either a single agent or in combination with cisplatin, and has
been registered to treat patients with advanced breast cancer elsewhere. [30-33]
Vinorelbine has also demonstrated anticancer activity in advanced ovarian
carcinoma and lymphoma; however, a unique role in the therapy of these
malignancies has not been defined.
 Vinorelbine Tartrate

8. Toxicity

Although the Vinca alkaloids are quite similar from a structural standpoint, their
toxicologic profiles differ significantly. All of the Vinca alkaloids induce a
characteristic peripheral neurotoxicity, but VCR is most potent in this regard. The
neurotoxicity is principally characterized by a peripheral, symmetric mixed
sensory-motor, and autonomic polyneuropathy.[32,35,36,37-41] The primary pathologic
effect is axonal degeneration and decreased axonal transport, most likely caused
by a drug-induced perturbation of microtubule function.
Toxic manifestations include constipation, abdominal cramps, paralytic ileus,
urinary retention, orthostatic hypotension, and hypertension. severe neurotoxicity
is observed less frequently with VBL, VDS, and VRL, as compared to VCR.[42,43]
VRL has a lower affinity for axonal microtubules than either VCR or VBL, which
seems to be confirmed by clinical observations.[30-32,43,44]
VCR treatment in patients with hepatic dysfunction or obstructive liver disease is
associated with an increased risk of developing neuropathy because of impaired
drug metabolism and delayed biliary excretion. Neutropenia is the principal dose-
limiting toxicity of VBL, VDS, and VRL. Thrombocytopenia and anemia are
usually less common and less severe.
Gastrointestinal toxicities, aside from those caused by autonomic dysfunction,
may be caused by all the Vinca alkaloids. [26,28,37,38,41,46]
Mucositis occurs more frequently with VBL than VRL or VDS, and is least
common with VCR. Nausea, vomiting, and diarrhea may also occur to a lesser
extent. Pancreatitis has also been reported with VRL. [47]

The Vinca alkaloids are potent vesicants and may cause significant tissue damage
if extravasation occurs. If extravasation occurs or is suspected, treatment should
be discontinued immediately and aspiration of any residual drug remaining in the
tissues should be attempted. [48] The application of local heat and injection of
hyaluronidase, 150 mg subcutaneously, in a circumferential manner around the
needle site are thought to minimize both discomfort and latent cellulitis, perhaps
by facilitating drug dispersion.
Because of their remarkable vesicant properties, the Vinca alkaloids should not be
administered intramuscularly, subcutaneously, intravesically, or intraperitoneally.
Direct intrathecal injection of VCR and other Vinca alkaloids, which has occurred
as inadvertent clinical mishaps, induces a severe myeloencephalopathy
characterized by ascending motor and sensory neuropathies, encephalopathy, and
rapid death.[26,28,49,50]

Mild and reversible alopecia occurs in approximately 10% and 20% of patients
treated with VRL and VCR, respectively.

The only known effective intervention for Vinca alkaloid neurotoxicity is

discontinuing treatment or reduction of the dose or frequency of drug
administration. Although a number of antidotes, including thiamine, vitamin B12,
folinic acid, pyridoxine, and neuroactive agents (eg, sedatives, tranquilizers,
anticonvulsants), have been used, these treatments have not been clearly shown to
be effective.[26,28] Folinic acid protects mice against otherwise lethal doses of the
Vinca alkaloids, and there are anecdotal reports of its successful use following
VCR overdosage in man; however, prospective studies have never been
performed.

9. Effect of weather on the production of alkaloids in C. roseus

In a study one set of plants were grown in rainy season from March-April to Sept-
Oct. Another set was grown in winter season from Sept-Oct to March-Apr. [62]
Vincristine was totally absent from root material. It is also reproted that bisindole
alkaloids and vindoline accumulate only in green tissue and are not found in root
or cell suspension cultures. Also full sunshine is reported to give a higher sontent
of alkaloids than shade.

Table-1

Amount of different alkaloid (mg/gm) under difference season in leaf and

root material of two varieties of c-roseus

Type of Variation Variety Ajmaline Vincristine Ajmalicine

Tissue in Season
Rainy roseus 1.09 1.39 0.4
alba N 3.83 N
Leaf
Winter roseus 3.38 8.75 0.91
alba N N N
Rainy roseus 4.52 N 2.1
alba N N N
Root
Winter roseus 3.0 N N
alba 0.85 N N

Table -2

Season Variety Total Alkaloid

Leaves (mg/kg)a Roots (mg/kg)a
roseus 45 24
Rainy
alba 40 28
roseus 48 26
Winter
alba 39 20
LSD at 5% 3.5 2.8
a = on dry weight basis
Total alkaloid content was highest in ‘roseus’ variety during winter. Highest
amount of root alkaloid was noted in ‘alba’ variety when grown in rainy season
and in ‘roseus’ in winter season. Minimum alkaloid content was noted in ‘alba’
variety during the winter months.

Present study showed that seasons have impact on the biomass and alkaloid
production, both qualitatively and quantitatively on genotypes of C. roseus and
variety ‘roseus’ was found to be superior to variety ‘alba’.

10. Chemical Modifications of the Vinca alkaloids leading to the

synthesis of novel alkaloids

Several hundred derivatives have been synthesized and evaluated for their
pharmacological activities, the majority being modified in the vindoline moiety,
bearing several reactive centers. These efforts led to the identification of the
amido derivative vindesine, registered in Europe in 1980 and now available in
several countries. Then novel chemistry permitted the semisynthesis of
derivatives modified in the velbenamine "upper" part of the molecule, creating a
new potential in the Vinca alkaloids medicinal chemistry: as a result, vinorelbine,
obtained by C' ring contraction of anhydrovinblastine, and is now marketed
worldwide. Several strategies aimed at the total synthesis of vinblastine
derivatives have been investigated, giving the opportunity to design rationally
certain compounds. Modifications in the D' ring appeared to induce dramatic
changes in the tubulin interactions. These observations have been confirmed
recently by the identification of unprecedented pharmacological properties
exerted by the novel fluorinated Vinca alkaloid, vinflunine.[52]

The two second-generation Vinca alkaloids, vinorelbine and vinflunine, affect

microtubule dynamics very differently from vinblastine, a first generation Vinca
alkaloid. For example, vinblastine strongly suppresses the rate and extent of
microtubule shortening in vitro, whereas vinorelbine and vinflunine suppress the
rate and extent of microtubule growing events.
There was doubt whether these differences result in differences in mitotic spindle
organization that might be responsible for the superior antitumor activities of the
two second-generation Vinca alkaloids.
However,Despite differences in their actions on individual dynamic instability
parameters, morphologically detectable differences in spindle effects among the
three drugs were minimal, indicating that overall suppression of dynamics may be
more important in blocking mitosis than specific effects on growth or shortening.
the peak intracellular drug concentration at the mitotic IC(50) value was highest
for vinflunine (4.2 +/- 0.2 microM), intermediate for vinorelbine (1.3 +/- 0.1
microM), and more than 10-fold lower for vinblastine (130 +/- 7 nM), suggesting
that intracellular binding reservoir(s) may be partially responsible for vinflunine's
high efficacy and minimal side effects. [54]

11. Other novel cytotoxic alkaloids isolated from Catharanthus

roseus
(i) According to a recent study, BM6, a new semi-synthetic vinca alkaloid,
exhibits its potent in vivo anti-tumor activities via its high binding affinity for
tubulin and improved pharmacokinetic profiles.
The aim of this study was to evaluate the anti-tumor activities and to establish the
mechanism of the action of 3-decarboxyl-acetyloxylmethyl-anhydrovinblastine
(BM6).
BM6 was characterized by its superior in vivo activity to vinorelbine in
preclinical tumor models, though BM6 exerted in vitro cytotoxic activity against a
wide spectrum of tumor cell lines with IC(50) values generally 10-fold higher
than the classic Vinca alkaloids. Of note, BM6 displayed more potent cytotoxic
activity against multidrug-resistant sublines.

BM6 also induced significant cell cycle arrested in mitosis and cytoskeleton
disruption via interacting with the Vinca binding site on tubulin. Encouragingly,
the features in term of its higher tubulin binding affinities and better
pharmacokinetic profiles highlight BM6 distinct from other Vinca alkaloids. [59]

(ii) Two New bisindole alkaloids, Vingramine 1 and Methylvingramine 2, were

isolated from the Seeds of Catharanthus roseus. Their structures were determined
by one- and two-dimensional NMR experiments. They possess a new bisindole
skeleton involving an indole alkaloid part B with loss of 5',6'-ethylene, a C7'-C16'
linkage, a 14'-O-19'-tetrahydrofuran, and a N-4'-isobutyramide group. The 12-
methyl vincorine part A and part B are connected via an 11,10'-biphenyl linkage.
These alkaloids display, in vitro, cytotoxic activity against nasopharynx
carcinoma KB cells. [61]

(iii) 16-Epi-Z-isositsirikine, a monomeric indole alkaloid with antineoplastic

activity from Catharanthus roseus.
The compound displayed antineoplastic activity in the KB test system in vitro and
the P-388 test system in vivo. [60]
12. Vinca alkaloids in clinical trials

Vinflunine : Vinflunine is a new Vinca alkaloid uniquely fluorinated, by the use

of superacid chemistry, in a little exploited region of the catharanthine moiety. In
vitro investigations have confirmed the mitotic-arresting and tubulin-interacting
properties of vinflunine shared by other Vinca alkaloids. However, differences in
terms of the inhibitory effects of vinflunine on microtubules dynamics and its
tubulin binding affinities have been identified which appear to distinguish it from
the other Vinca alkaloids.
Studies investigating the in vitro cytotoxicity of vinflunine in combination therapy
have revealed a high level of synergy when vinflunine was combined with either
cisplatin, mitomycin C, doxorubicin or 5-fluorouracil. Furthermore, although
 vinflunine appears to participate in P-glycoprotein-mediated drug resistance
mechanisms, it has proved only a weak substrate for this protein and a far less
potent inducer of resistance than vinorelbine. Vinflunine was identified in
preclinical studies as having marked antitumour activity in vivo against a large
panel of experimental tumour models, with tumour regressions being recorded in
human renal and small cell lung cancer tumour xenografts. Overall its level of
activity was superior to that of vinorelbine in many of the experimental models
used. Interestingly, an in vivo study using a well vascularised adenocarcinoma of
the colon has suggested that vinflunine mediates its antitumour activity at least in
part via an antivascular mechanism, even at sub-cytotoxic doses. Therefore, these
data provide a favourable preclinical profile for vinflunine, supporting its
promising candidacy for clinical development. Phase I and Phase II evaluations of
vinflunine have been completed in Europe and phase III clinical trials are now
ongoing. [62-63]

vinflunine

Table-3

Vinca Alkaloids: Comparative Pharmacokinetic and Toxicologic

Characteristics

Vincristine Vinblastine Vindesine Vinorelbine

Formula C46H56N4O10 C46H58N4O9 C43H55N5O7 C45H54N4O8

Standard adult 1–2 6–8 3–4 15–30

dose range
(mg/m2/wk)
Route i.v. i.v. i.v. i.v., oral

Bioavailability n.a. n.a. n.a. 79-91%

Protein Binding ~75% ~75% 65-75% 43% (oral)

Pharmacokinetic Triphasic Triphasic Triphasic Triphasic

behavior

Plasma half-lives
α (min) <5 <5 <5 <5
β (min) 50–155 53–99 55–99 49–168
γ (h) 23–85 20–64 20–24 18–49

Clearance 0.16 0.74 0.25 0.4–1.29

(L/h/kg)

Primary route Hepatic Hepatic Hepatic Hepatic

metabolism and metabolism and metabolism and metabolism and
biliary biliary biliary biliary
elimination elimination elimination elimination
Principal toxicity Neurotoxicity Neutropenia Neutropenia Neutropenia

Other toxicities Constipation, Alopecia, Alopecia, Neurotoxicity,

SIADH neurotoxicity, neurotoxicityvomiting,
mucositis constipation,
mucositis
SIADH = syndrome of inappropriate secretion of antidiuretic hormone.
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