1 134126 1
The present invention relates to a new and improved method particularly
for producing diner alkaloid compounds especially of the Catharanthus (Vinca)
alkaloid group and, in particular, is an improved method for producing the
antiviral, antileukemic (antineoplastic) compounds, vincristine and
vinblastine,
of Formula I.
OH
Indok Uoit
(Catharanthine) V
N
H u'
H
N
14
Is
na,Ydrudl off ~ I
(Vmdoline) Ii~O I 1 % ~lOAe
HOC 1
The above compound, when R is COOCH3, and Rl is OCH3, is vinblastine
(NSC 49482) and when R is COOCH3, and R1 is OCH3 and N1 is N-CHO (N-formyl),
vincristine (NSC 67574).
The present series of dimeric alkaloids, including important antitumor
agents, are formed from an indole, such as catharanthine (Formula II, R -
COOCH3),
and a dihydroindole unit, e.g. vindoline (Formula III), in which the halves
are
linked via a carbon-carbon bond involving an aliphatic center C18 in the
indole
unit and an aromatic carbon C15 in the vindoline portion,
N
N N
1!
OH
H H,~ N OAe
COOC
H~ H
II III
13~r1 Z6 1
2
More particularly, according to one aspect of the
present invention there is provided a process for the
production of dimer alkaloid compounds represented by the
following formula:
io N/~''~,
m
s ,'
1 2
3
R1 R,~ O-R3
wherein:
alk = CH3 or (CH2)n CH3 where n = 1-5;
R1 = CH3 or CHO;
R2 = H or CO-alk;
R3 = Hs
R4 = COO-alk or CONR13R14 wherein R13 and R14 are
selected from the group consisting of hydrogen, alkyl,
substituted alkyl, aryl or substituted aryl;
Z = -CH=CH- or -CH2-CH2-;
R = XXII or XXXIII
RR
R9
X~I
72936-2
- 2a _ ~ J 41 2 6 1
Ria
J
XXIII
and wherein,
R7 = H or COO-alk;
R8 = H, OH, 0-alk, OCO-alk or alkyl;
R9 = H, OH, O-alk, OCO-alk or alkyl;
R10 = H, OH, O-alk, OCO-alk;
R11 = H or COO-alk; and
R12 = H or alkyl,
which comprises the steps of:
(a) forming an N-oxide derivative in the cold, at a tem-
perature from about -77° to about +40°C from an indole unit
having a bridge nitrogen by oxidizing the bridge nitrogen and
without isolating said derivative;.
(b) treating said N-oxide derivative in the presence at
least of one member selected from the group consisting of
acetic anhydride, halogenated acetic anhydride, and acetyl chloride,.
to effect a Polonovski-type fragmentation reaction;
(c) without isolating the product of step (b), stereo-
specifically coupling said product of step (b) with a dihydroindole
unit in the presence of at least.one member selected from the
group consisting of acetic anhydride, halogenated acetic anhydride,
and acetyl chloride at a low temperature of about -7Q° to about
+40°C., under inert conditions, to form a first iminium inter-
mediate;
~~
134126 1
- 2b -
(d) reducing said first iminium intermediate by reaction
with a 1,4-dihydropyridine compound, thereby forming an enamine
intermediate;
(e) preparing a second iminium intermediate be oxidative
transformation of said enamine intermediate obtained in step (d)
under controlled aeration conditions; and
(f) reducing the product obtained in step (e) to form
the target dimer alkaloid compounds.
The conditions for the coupling reaction, as described
in the present invention, relate to an important modification of
the method developed in these laboratories [United States Pat. No.
4,279,817; Helv. Chim. Acta, 59, 2858 (1976)] [Scheme 1). In
particular, the present modification allows the preparation and
isolation of the highly unstable dihydropyridinium intermediate
VI (R = COOCH3), being formed in the coupling of catharanthine N-
oxide (Formula IIa) with vindoline (Formula III).
In general, the Nb-oxide derivative of the indole unit
(Formula IIa) or related analogues (Formula IV, wherein R1 = R2 =
R3 = R4 = H or R2 = R3 = R4 = H and R1 = alkyl group of structure,
(CH2)nCH3 where n = 0-10) prepared with a peracid such as m-chloro-
perbenzoic acid or p-nitroperbenzoic acid in an inert organic
solvent such as methylene chloride or other polyhalo organic sol-
vents, is achieved at various temperatures; for example, -77°C,
0°C, room temperature and above. The N-oxide intermediate thus
formed is used for the next step without isolation. The fragmen-
tation reaction which fragments the C5-C18 bond of the indole Nb-
C
~3~~z~ ~
- 2c -
oxide intermediate is carried out in the presence of a reagent
such as trifluoroacetic anhydride. To maximize the subsequent
coupling reaction which promotes the formation of a natural dimer
bonded at C1$ (indole unit) and C15 (dihydroindole unit), the
dihydroindole unit may be added to the reaction mixture prior to
the fragmentation reaction. As alternative reagents for the
trifluoroacetic anhydride component used in fragmentation and
coupling, there may be utilized tr.ichloroacetic anhydride, acetic
anhydride, acetyl halides and tosyl anhydride. These reagents
bring about a Polonovski-type fragmentation of the C5-C1$ bond
in the compounds shown in Formulas IIa and IV.
<IMG>
4
131261
The reaction temperature, time and pressure conditions in general are
similar to those employed in the Polonovski reaction which, in its original
application, involved the dealkylation of tertiary and heterocyclic amines by
acylation of the corresponding N-oxides with acetic anhydride or acetyl
chloride
(cf. Merck Index, 8th ed., 1968, page 1203). The temperature of the
fragmentation
and coupling reaction may vary from -70° C to 40° C and
preferably at the low
temperature range. The portions of the reaction relating to the formation of
the
N-oxide compound may be conducted in the open or under inert gas atmosphere
such
as argon or other inert gas of Group Zero of the Periodic Table such as
helium,
nitrogen, neon, etc. The same inert atmosphere conditions are employed in the
fragmentation and coupling portions of the reaction and under a positive
temperature control preferably in the range of -40° C to -60° C.
N
a4
N ,
H ,
i
IV ~~ndolih~
V
H
N I
H ~~ r
virWo~i~e
VI whdolsne
VII
co,~e
-H
I
it
N
v~ndcline
VIII
~ 1 341 26 1
Due to the low temperature necessary for the later stage reactions, the
reaction time may vary from several hours to several days.
The first formed indole-dihydroindole dimer intermediate, after the
(stereospecific) coupling reaction, possesses an iminium salt function at the
Nb atom
of the indole moiety as represented by Formulas V or VI. Reduction of this
iminium
intermediate by reacting with alkali metal borohydride (NaBH4, KBH4, LiBH4)
produces
the dimeric alkaloids) as described in U.S. Pat. No. 4,279,817.
In the present invention, the iminium intermediate (Formula V or VI) may
be isolated as such by careful manipulation. After the coupling reaction is
completed, the reaction mixture is applied directly on an appropriate
chromatographic system such as column, thin layer or high performance liquid
chromatography, preferably reverse phase and/or size-exclusion separation
methods.
The temperature of the operation may vary from 4° C to room
temperature.
Alternatively, volatile reagents and solvents which are present together with
the
iminium intermediate in the reaction mixture may be removed under reduced
pressure
and temperature, preferably below -loo C. The resulting solid (Formula V or
VI) is
then dissolved in various organic solvents, such as halogenated hydrocarbons,
ethers, alcohols, acetonitrile, etc., and/or various aqueous buffers. The pH
of
the buffer may vary from 2 to 10. The solution of iminium intermediate
(Formula V
or VI) can then be purified by the chromatographic methods described above
before
further characterizations. Alternatively, the iminium intermediate solution
can be
used directly for subsequent reactions. The use of inert atmosphere conditions
may
or may not be necessary.
When the starting indole unit has a C3-C4 double bond (e. g. catharanthine
(II)), the resultant coupling intermediate contains an a,B-unsaturated iminium
functional group as represented by Formula VI. Reduction of VI with alkali
metal
borohydride (NaBH4, KBH4, LiBH4), gives 3',4'-dehydrovinblastine compounds
(Formula
1341261
VII). The present process utilizes a novel reduction method in which
conversion of
the iminium intermediate (Formula VI) to the enamine (Formula VIII) can be
achieved. Reagents used for this reduction include 1,4-dihydropyridine
compounds (the so-called NADH models) as represented by Formula IX, where Rl,
H R4
Rs R3
R' ( ~ Ix
N Rz
R,
R2, R3, R4, RS and R6 can be any member of the group consisting of H, alkyl,
substituted alkyl, aryl and substituted aryl. Two series of such compounds are
readily available [Chew. Rev. 82, 232 (1982); Chem. Rev. J,~, 1 (1972)]. The
first is known as Hantzch esters where R3 and R5 in Formula IX are carboxylic
esters, e.g. carboethoxy (COOC2H5). The second series is the N-substituted 1,4-
dihydronicotinamides (Formula IX) in which R1 is a substituted alkyl or
substituted aryl function, e.g. benzyl, and R3 is CONR7R8 where R.~ and R8 can
be
any member of the group consisting of hydrogen, alkyl, substituted alkyl, aryl
and substituted aryl functions.
The above reductants can be used singly or in combination. When 1,4-
dihydropyridines are used to reduce iminium VI, organic solvents, such as
alcohols, acetonitrile or higher members of this series, dimethyl sulfoxide,
dimethylformamide, various ethers such as dioxane, tetrahydrofuran, etc.,
chlorinated hydrocarbons, etc., are employed, normally without an aqueous
buffer
as co-solvent. The progress of the reduction is monitored by direct analysis
of
the reaction mixture on an appropriate chromatographic system, preferably
reverse phase high performance liquid chromatography. This method is used to
optimize the reaction temperature, time, pressure and concentration of
1341261
reactants. The reaction temperature may vary from -60° C to 60°
C, and
preferably from 4° C to room temperature. The reaction time may vary
from
several minutes to several days depending on other parameters. The reduction
is
conducted under cover with inert conditions such as argon or an inert gas of
Group Zero of the Periodic Table (nitrogen, helium, neon, etc.).
The enamine (Formula VIII) formed in the above reduction may be used
directly for subsequent reaction or isolated by careful manipulation. The
reaction
mixture is applied directly on an appropriate chromatographic system such as
column, thin layer or high performance liquid chromatography, preferably
reverse
phase and/or gel-permeation separation methods. The temperature of the
operation
may vary from 4° C to room temperature. Alternatively, volatile
reagents and
solvents present in the reaction mixture are removed under reduced pressure
and
temperature, preferably below -10° C. The resultant residue can be
purified by the
chromatographic methods described above before further characterization or
transformation.
Treatment of the enamine VIII with alkali metal borohydride (NaBH4, KBH4,
LiBH4) produces the 4'-deoxovinblastine compounds (Formula X, R - COOCH3) and
4'-
deoxo-4'-epi-vinblastine compounds (Formula XI, R - COOCH3). Whereas, under
oxidative conditions, the enamine VIII can be transformed to the vinblastine/
vincristine series via the iminium intermediate (Formula XVI).
X XI
8 _
1 341 26 1
Oxidative conditions that are used for converting enamine (Formula VIII)
to an iminium intermediate (Formula XVI) include:
(1) controlled aeration/oxygenation;
(2) addition of flavin coenzymes [riboflavin, Formula XII, R - H; flavin
mononucleotide (FMN), Formula XII, R - P032 ; flavin adenine dinucleotide
(FAD),
Formula XII, R - (P03)22 -adenosine) followed by controlled
aeration/oxygenation;
XII
(3) addition of the reduced form of the flavin coenzymes [dihydro-
riboflavin, Formula XIII, R ~ H; dihydroflavin mononucleotide (FirINH2),
Formula
XIII, R - P032 ; dihydroflavin adenine dinucleotide (FADH2); Formula XIII, R -
(P03)2 -adenosine] followed by controlled seration/oxygenation;
' ~OH
H
N
[
~N
H I
0
XIII
9
~341~61
(4) addition of flavin coenzyme analogues having the isoalloxazine
structure as represented by Formula XIV, where R1, R2 and R3 can be a member
of the
group consisting of alkyl, substituted alkyl, aryl and substituted aryl
functions,
and followed by controlled aeration/oxygenation;
R,
/ N ~N~O
~~~i H~'H
R
j
0
XIV
(5) addition of the reduced form (1,5-dihydro) of the above flavin coenzyme
analogues as represented by Formula XV, where Rl, R2 and R3 can be a member of
the
group consisting of alkyl, substituted alkyl, aryl and substituted aryl
functions,
and followed by controlled aeration/oxygenation;
R
H
N
~1
N ~ NH xv
3 H I
0
(6) addition of hydrogen peroxide and/or hydroperoxides as represented by
the Formula R-OOH, where R can be an alkyl, substituted alkyl, aryl or
substituted
aryl function;
(7) addition of peracids as represented by the Formula R-C03H, where R can
be an alkyl, substituted alkyl, aryl or substituted aryl functions;
(8) addition of superoxides;
to - 1 3412fi 1
(9) addition of a hydroxyl radical (OH) generated in a variety of
ways, for example, by the use of hydrogen peroxide in the presence of ferrous
ion.
(10) addition of a metal ion which is a good electron acceptor, for
example, ferric ion (Fe+3); cupric ion (Cu+2); cuprous ion (Cu 1), mercuric
ion
(Hg2+2) and silver ion (Ag+1) followed by controlled aeration/oxygenation.
The oxidative processes involving controlled aeration/oxygenation
(condition (1)), the flavin coenzyme analogues (conditions (4), (5)),
peroxides
(condition (6)), peracids (condition (7)), superoxides (condition (8)),
hydroxyl
radical (OH) (condition (9)) and metal ions capable of electron transfer
(condition (10)) can be carried out in organic solvents such as alcohols,
acetonitrile or higher members of this series, dimethyl sulfoxide,
dimethylformamide, various ethers such as dioxane, tetrahydrofuran, aromatic
hydrocarbons such as benzene, toluene, etc.
The oxidative processes involving flavin coenzymes (conditions (2),
(3)), require an aqueous buffer (for example, phosphate, Tris HC1, MES
buffers)
at pH S-9, but preferably in the range 6-8, as solvent. An organic co-solvent,
e.g. elcohols, acetonitrile or higher member of this series, dioxane,
tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, can be used.
The progress of the oxidative process is monitored by direct analysis of the
reaction mixture on an appropriate chromatographic system, preferably reverse
phase
high performance liquid chromatography. This method is used to optimize the
reaction
temperature, time, pressure and concentration of reactants. The reaction
temperature
may vary from -60° C to 60° C and preferably from 4o C to room
temperature. The
reaction time may vary from several minutes to several days depending on other
parameters. The reaction is conducted at atmospheric pressure.
11 1341261
The conversion of intermediate (Formula XVI) to vinblastine (Formula I) may
be achieved by reacting XVI with alkali metal borohydride (NaBH4, KBH4, LiBH4)
in
suitable solvents (organic or aqueous) as used in the oxidative process
(conditions
(1)-(10)).
For practical purposes, isolation of intermediates (Formulas V, VI, VIII,
XVI) is not required and the entire process from the indole unit (Formula II)
and
the dihydroindole unit (Formula III) to the end product vinblastine (Formula
I) may
be preferably conducted in an one-pot operation.
DO ~1
co~~ +le~
I
r,
N
N
vindoline
XVI
0
N.~ , ,.
N
..
H Me,
XIX
xx
12 1341261
In summary, the present method is applicable to the production of dimer
products from catharanthine and dihydrocatharanthine with vindoline as
starting
materials and phenyl, alkyl and amide derivatives embraced by the following
formulas: to a
n N v
a z
R s;
z ~ ~ " XXI
dt-0 j~ O-R~
ti
R ~ 14 O-R~
Ra
wRv
~N
R' XXI I
N
H Rio
R~~
~N
Rte XXIII
p V
H
Formula XXI is as pictured and in that formula alk represents a lower
alkyl group of Cl-C6 and preferably Cl-C3; aryl is mono-aryl such as benzyl,
styryl, and xylyl; R1 is a member of the group consisting of hydrogen, alk,
CHO
and CORS where R5 is alkyl or aryl; R2 and R3 are members of the group
consisting of hydrogen and -CO-alk; R4 is a member of the group consisting of
C00-alk, CONH-NH2, CONH2, CONHR6, and CON(R6)2 where R6 is alkyl; Z is a
member of the group consisting of -CH2-CH2- and -CH-CH- and R is a member of
the
indole family represented by Formula XXII where R~ is a member of the group
consisting of hydrogen, or C00-alk; R8 is a member of the group consisting of
hydrogen, OH, 0-alk, OCO-alk or alkyl; R9 is a member of the group consisting
of
hydrogen, OH, 0-alk, OCO-alk, or elk; R10 is a member of the group consisting
of
hydrogen, OH, 0-alk, OCO-alk, or Formula XXIII where R11 is a member of the
group consisting of hydrogen or C00-alk; R12 is a member consisting of alkyl.
r
13 1 34~ ~6 1
The present invention differs from the prior art [U.S. Pat. No.
4,279,817] in several important stages specifically the isolation and
characterization of unstable intermediates V, VI, VIII and XVI, and their
subsequent
conversion to the clinical drugs vinblastine and vincristine. The prior art
describes a method for producing dimer alkaloids which are analogues of the
clinical
drugs, for example, 3',4'-dehydrovinblastine of Formula VII.
Intermediates V and VI are prepared by contacting vindoline or a vindoline
derivative, [Formula XXI when R is H) with an indole derivative represented by
a
compound of Formula XXIV where R13 is a member of the group consisting of
hydrogen or
C00-alk or by a compound of Formula XXV where R14 is a member of the group
consisting
of H or C00-alk and R15 is a member of the group consisting of hydrogen or
alkyl.
0 0
N~ N
t4 ~ N
ie ,e
H RU ~ H Rn ~s
XXIV XXV
In addition to catharanthine, any indole unit represented by Formula XXVI may
be
employed. In the Formula XXVI, R, Rl, R2 and R3 are members of the group
consisting of hydrogen, OH, 0-alk, OCO-alk, alkyl or aryl. In the Formula
XXVI,
as previously stated, alk is lower alkyl C1-C6 and preferably C1-C3, and aryl
is
mono-aryl such as benzyl, xylyl, etc.
R
R~
N
R N
H
XXVI
14 X341261
In Formulas XXI - XXVI and generally in this application and claims,
alk and alkyl mean lower alkyl as defined in Formula XXI and aryl means mono-
aryl as similarly defined in Formula XXI.
The intermediate VI, thus obtained, is utilized in a highly specific and
novel process involving 1,4-reduction, to afford the next important
intermediate
VIII. Intermediate VIII is further converted by a novel process to the novel
intermediate XVI and the latter is utilized in another new reductive process
to
afford the compounds of general structure XXI, when R is a compound of Formula
XXII.
In a similar series of reactions, intermediate V, leads to compounds of
general structure XXI when R is a compound of Formula XXIII.
Another highly significant and novel feature of this invention is that the
isolation of intermediates VI, VIII and XVI is not essential and the entire
process, monitored carefully for VI, VIII and XVI by HPLC, can be conducted to
the dimeric products of Formula XXI from the starting indole (Formulas II,
XXII
and XXIII) and dihydroindole (Formula XXI, R - H) units in a one-pot
operation.
15 1341261
EXPERIMENTAL PART
Preparation of the Iminium Intermediate (Formula VI) via Modified Polonovski
Reaction
The reaction was performed under anhydrous conditions. All glassware
was oven-dried at 120° C. The solvent, methylene chloride, and coupling
reagent, trifluoroacetic anhydride, were distilled from P205 prior to use.
To a solution of catharanthine (II, 200 mg, 0.6 mmol) in dry methylene
chloride (2 ml) at -20° C under a positive atmosphere of argon was
added m-
chloroperbenzoic acid (132 mg, 0.8 mmol), and the mixture stirred for 5 min.
To
the catharanthine N-oxide (IIa), thus formed, was added a solution of
vindoline
(III, 270 mg, 0.6 mmol) in methylene chloride (1 ml) and the mixture cooled to
-
60° C. Trifluoroacetic anhydride (0.2 ml, 1.5 mmol) was added to the
stirred
reaction mixture maintained at -60° C for 2 hours. After this time, the
solvent
and excess reagents were removed ~ vacuo at -20° C to leave a reddish-
brown
residue containing the iminium intermediate. The latter was characterized by
reverse phase high performance liquid chromatography (HPLC) (Waters Radial-Pak
C18
or CN cartridge, methanol-H20-Et3N as solvent system). It was shown that the
yield of VI in this reaction exceeded 80% by reduction of VI (NaBH4, methanol,
0°
C) to the known 3',4'-dehydrovinblastine (Formula VII).
Reduction of Iminium Intermediate (Formula VI) with 1-Benzvl-1.4-
dih~dronicotinamide (Formula IX. R1 ~ benzyl. R2 - R4 ~ R5=
R6 - H: R3 - CONH2~ - SXnthesis of Enamine (Formula VIII) - (Procedure A)
To a stirred solution of iminium intermediate (VI, 100 mg) in degassed
acetonitrile (5 ml) was added 1-benzyl-1,4-dihydronicotinamide (135 mg, 0.63
mmol, 6 equivalents) under a positive atmosphere of argon at room temperature
(20° C) over a period of 5 hours. After this time, the reaction
mixture, as
16 13412fi 1
monitored by reverse phase HPLC (Waters Radial-Pak C18 or CN cartridge,
methanol/H20/Et3N solvent system), indicated complete conversion of VI to a
mixture of enamine VIII and 3',4'-dehydrovinblastine (VII) in a ratio of 1:1
(75$ yield).
Alternatively, to a stirred solution of iminium intermediate (VI, 100 mg)
in methanol (5 ml) kept initially at 0oC for 0.5 hours was added dropwise or
in
portions, a solution of 1-benzyl-1,4-dihydronicotinamide (56 mg, 0.26 mmol,
2.5
equivalents) in methanol (2 ml) under a positive atmosphere of argon over a
period
of 5 hours. During this time the solution was allowed to warm up to room
temperature. HPLC monitoring, as above, indicated complete conversion of VI to
a mixture of enamine VIII and 3',4'-dehydrovinblastine (VII) in a ratio of 1:1
(75$ yield).
In one experiment, the mixture of enamine (VIII) and 3',4'-
dehydrovinblastine (VII) obtained as described above, was treated with excess
sodium borohydride (500 mg) at 0° C. The reaction mixture was then made
basic
with NH40H and extracted with ethyl acetate (3 x 200 ml). The combined organic
phase was dried over magnesium sulphate. The product obtained, after removal
of
organic solvent, was subjected to preparative thin layer chromatography on
silica gel (methanol/ethyl acetate as eluting system). The product was shown
to
be a mixture of unreacted 3',4'-dehydrovinblastine (VII), and the known
compounds 4'-deoxovinblastine (X, R - COOCH3) and 4'-deoxo-4'-epivinblastine
(XI, R - COOCH3). The presence of the latter compounds provided unambiguous
evidence for the structure of enamine VIII.
%1~ ,~
~l ~~ ~C f~~~; Y ~~
l~ 1341261
Reduction of Iminium Intermediate (Formula VI) with 3 5-Diethoxycarbonvl-
2 ~-Dimethvl-4-Pheny~]L-1 4-Dihvdrowridine (Hantzch ester analogue Formula
I~1~3=~5 - COOCH2~1 3~2=~6~3~4 - .phenyl ) -
Alternative Synthesis of Enamine (Formula VIII) - (Procedure B)
To a stirred solution of iminium intermediate (VI, 100 mg) in degassed
acetonitrile (3 ml) was added 3,5-diethoxycarbonyl-2,6-dimethyl-4-phenyl-1,4-
dihydropyridine (264 mg, 8 equivalents) in ethanol (12 ml) under a positive
atmosphere of argon. The reaction mixture was refluxed for 3 hours. After this
time, reverse phase HPLC analysis (as described above) indicated, among other
products, formation of enamine VIII and 3',4'-dehydrovinblastine (VII) in a
ratio of 1:1 (608 yield).
Synthesis of Vinblastine (Formula I) by Oxidation of Enamine (Formula VIII) to
Iminium Intermediate (Formula XVI) with Flavin Mononucleotide (FMN
Formula XII. R - P032 ) - Method 1
To a stirred reaction mixture containing the enamine (VIII) obtained as
described above (Procedure A) from iminium VI (100 mg) was added FMN (80 mg, 1
equivalent) dissolved in Tris HC1 buffer (2 ml) under a positive atmosphere of
argon. The solution was kept in the dark at room temperature (20° C)
for 16
hours. After this time, the inert atmosphere of argon was replaced by sir and
the rection mixture stirred for another 2.5 hours. Reverse phase HPLC analyses
indicated transformation of enamine VIII to the iminium intermediate XVI as
well
as to other products (see later). Sodium borohydride (500 mg) was added at
0° C
and the reaction mixture made basic with NH40H and extracted with ethyl
acetate
(3 x 200 ml). The combined organic extract was dried over magnesium sulphate
and the solvent removed ~n vacuo to provide a crude product (85 mg).
18 1341261
irification of the latter by thick layer chromatography (silica gel,
methanol: ethyl acetate 1:5) allowed the separation of the following dimeric
products: vinblastine (Formula I, 22 mg, 23%); 3',4'-dehydrovinblastine
(Formula VII, 16 mg, 17%); leurosine (Formula XVII, 8 mg, 9%), catharine
(Formula XVIII, 7 mg, 7.5%); vinamidine (Formula XIX, 5 mg, 5.6%) and
the reduction product of vinamidine (Formula XX, 19 mg, 20%).
Synthesis of Vinblastine (Formula I) b~~Oxidation of the Enamine (Formula VIII
with Hydrogen Peroxide to the Iminium Intermediate (Formula XVI) - Method 2
To a solution containing the enamine (VIII) obtained from iminium
intermediate VI (100 mg, Procedure A) was added hydrogen peroxide (30%, 1.2
ml,
95 equivalents) under an inert atmosphere of argon. The reaction mixture was
stirred at room temperature for 5.5 hours when reverse phase HPLC analyses
indicated complete conversion of enamine VIII. Sodium borohydride (500 mg) was
added at 0° C and extracted with ethyl acetate (3 x 200 ml). The
combined
organic extract was dried over magnesium sulfate and removed j_n vacuo. The
resulting product mixture was separated by thick layer chromatography (silica
gel, methanol/ethyl acetate) to give the following dimeric alkaloids:
vinblastine (I, 4 mg, 4%), 3',4'-dehydrovinblastine (VII, 5 mg, 4.8%),
leurosine
(XVII, 13 mg, 12.5%), catharine (XVIII, 5 mg, 4.8%), the reduced form of
vinamidine (XX, 30 mg, 27.6%).
Svmthesis of Vinblastine (I) by. Oxidation of the Enamine (VIII) with Air to
the
Iminium Intermediate (Formula XVI) - Method 3
A solution containing the enamine (VIII) obtained from the iminium
intermediate (VI, 100 mg) by Procedure A was stirred in open air at room
temperature for 3 h. After this time, sodium borohydride (500 mg) was added at
19 13412fi1 ..
0° C and the reaction mixture made basic with NH40H and extracted with
ethyl
acetate (3 x 200 ml). The combined organic extract was dried over MgS04 and
removed in vacuo. The resulting crude product was separated by preparative
thick layer chromatography (silica gel, methanol/ethyl acetate) to give
vinblastine (I, 4 mg, 4%).
Synthesis of Vinblastine (I) bx Oxidation of the Enamine (Formula VIII) with
Air
in the Presence of Ferric Chloride to the Iminium Intermediate (XVI) - Method
4
To a stirred solution containing the enamine (VIII) obtained from the
iminium intermediate (VI, 100 mg, Procedure A) was added ferric chloride (1
equi-
valent) and air bubbled through the solution at 0° C for a period of
0.5 hour.
Sodium borohydride (500 mg) was added at Oo C and the reaction mixture made
basic
with NH40H before extraction with ethyl acetate (3 x 100 ml). The combined
organic
extract was dried over Na2S04 and the solvent removed in vacuo. The crude
product
was purified by thick layer chromatography (silica gel, methanol/ethyl
acetate) to
give vinblastine (I, 37 mg). Based on enamine (50 mg) present in the mixture,
the
yield in this step is 70%.
