CA1341261C

CA1341261C - Process for the synthesis of 3',4'-anhydrovinblastine, vinblastine and vincristine

Info

Publication number: CA1341261C
Application number: CA000527897A
Authority: CA
Inventors: James P. Kutney; Lewis S.L. Choi; Michael Mchugh; Camille A. Boulet; Hiroki Tsukamoto; Jun Nakano
Original assignee: University of British Columbia
Current assignee: University of British Columbia
Priority date: 1987-01-22
Filing date: 1987-01-22
Publication date: 2001-06-26
Anticipated expiration: 2018-06-26
Also published as: DE3801450C2; DE3801450A1; GB2204036A; SE8800170D0; JPH01131187A; GB2204036B; FR2611202A1; ZA88408B; GB8801296D0; IT1215751B; IL85154A; JPH0613531B2; SE467874B; IL85154A0; SE8800170L; IT8819156A0; FR2611202B1; CH675724A5; NL8800134A

Abstract

A process for the synthesis of bisindole alkaloids of the vinblastine and vincristine family from an indole unit, such as catharanthine, and a dihydroindole unit, such as vindoline, wherein conversion of an initially-formed dimeric iminium intermediate to an enamine is achieved by a novel 1,4-reduction method employing various 1,4-dihydropyridine compounds. The resulting enamine can be transformed to a second iminium intermediate by controlled aeration and oxidation in the presence of various oxidizing reagents. The second iminium intermediate is then reduced with alkali metal boro-hydride to produce the target compound vinblastine or vincristine.
The entire process can be conducted in a one-pot operation to obtain the target product without isolation of the intermediates.

Description

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

Is na,Ydrudl off ~ I
(Vmdoline) Ii~O I 1 % ~lOAe 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 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 ,' 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

X~I

- 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;
~~

- 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.

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 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, 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 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 _ 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

XIII

~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 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 (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.

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

N.~ , ,.
N
..
H Me, XIX
xx 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.

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

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.

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 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).

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 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%.

Claims

1. A process for the production of dimer alkaloid compounds represented by the following formula:
wherein:
alk = CH3 or (CH2)n CH3 where n = 1-5;
R1 = CH3 or CHO;
R2 = H or CO-alk;
R3 = H;
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 XXIII

and wherein, R7 = H or COO-alk;
R8 = H, OH, O-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 temperature 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 Polonovski-type fragmentation reaction;

(c) without isolating the product of step (b), stereospecifically 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 -70° to about +40°C, under inert conditions, to form a first iminium intermediate:
(d) reducing said first iminium intermediate by reaction with a 1,4-dihydropyridine compound, represented by the following formula:
wherein:
R1, R2, R4, and R6 are selected, independently, from the group consisting of H, alkyl, substituted alkyl, aryl and substituted aryl;
R3 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, carboxylic esters, and -CONH2;
R5 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl and carboxylic esters, thereby forming an enamine intermediate;
with the proviso that, when R3 is -CONH2 then, R2, R4, R5, and R6 are H and R, is CH2Ph;
(e) preparing a second iminium intermediate by 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,

2. The process according to claim 1 wherein in the compound of formula IX, R1 is H or alkyl, and R2, R4 and R6 are each, independently, hydrogen, alkyl or aryl and R3 and R5 are each carboxylic esters.

3. The process according to claim 1 or 2, wherein the 1,4-dihydropyridine reducing agent used in step (d) is selected from the group consisting of:
1-benzyl-1,4-dihydronicotinamide; and 3,5-diethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine.

4. The process of any one of claims 1, 2 or 3, wherein the reduction of step (d) is conducted in an inert atmosphere at a temperature in the range from about -60° to about +60°C. in the presence of at least one solvent selected from the group consisting of lower alkyl alkanols, acetonitrile, dimethyl sulfoxide, dimethylforamide, dioxane, tetrahydrofuran, and chlorinated lower hydrocarbons.

5. The process according to claim 4, wherein the reduction of step (d) is conducted at a temperature in the range of about -20° to about -60°C.

6. The process according to any one of claims 1 to 5, wherein the oxidative transformation step (e) is selected from the group consisting of:
(i) controlled aeration/oxygenation in which a solution of said enamine is stirred in open air or with a stream of air/oxygen bubbled through the solution;
(ii) controlled aeration/oxygenation in which a solution of said enamine and a metal ion, selected from the group consisting of ferric ion (Fe+3), cupric ion (Cu+2), curpous ion (Cu+1), mercuric ion (Hg2+2) and silver ion (Ag+1) is stirred in open air or with a stream of air/oxygen bubbled through the solution;
(iii) controlled aeration/oxygenation in which a solution of said enamine and a flavin coenzyme is stirred in open air or with a stream of air/oxygen bubbled through the solution;
(iv) controlled aeration/oxygenation in which a solution of said enamime and a flavin coenzyme is stirred in open air or with a stream of air/oxygen bubbled through the solution, wherein the flavin coenzyme generates, in situ, the corresponding 1,5-dihydroflavin coenzyme;
(v) controlled aeration/oxygenation in which a solution of said enamine and a member selected from the group consisting of hydrogen peroxide and hydroperoxides represented by the Formula R-OOH, where R is alkyl or aryl and mixtures thereof is stirred in open air or with a stream of air/oxygen bubbled through the solution said aeration/oxidation being conducted in an organic solvent at a pH of 5-9 and a reaction temperature of about -60° to about +60°C.

7. The process according to claim 6, wherein the oxidative transformation step (e) is conducted at a pH in the range of 6-8.

8. The process according to any one of claims 1 to 7, wherein the reducing used in step (f) comprises contacting the reaction product from step (e) with an alkali metal borohydride selected from the group consisting of NaHB4, KBH4 and LiBH4.

9. The process according to any one of claims 1 to 8, wherein steps (a) - (f) are conducted in a one-pot operation without isolation of any intermediate products.

10. The process according to to any one of claims 1 to 9, wherein at least one of the intermediates formed in steps (c), (d) and (e) is isolated prior to being further reacted.

11. The process according to any one of claims 1 to 10, wherein all of said intermediates are isolated prior to being further reacted.

12. A process according to any one of claims 1 to 11 wherein, in the starting materials, alk is -CH3, R2 is -COCH3, R3 is hydrogen, R4 is -CO2CH3, Z is -CH=CH- and R is a moiety of formula XXII as defined in claim 1 in which R7 is -CO2CH3, R8 is -OH, R9 is -C2H5 and R10 is hydrogen to obtain vinblastine and recovering the vinblastine so obtained.

13. A process according to Claim 12, further comprising the step of oxidizing said vinblastine so obtained to obtain the compound vincristine.

14. A process for the production of 3',4'-anhydrovinblastine which comprises the steps of:
(a) forming an N-oxide derivative of catharanthine in the cold, at a temperature 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), stereospecifically coupling said product of step (b) with vindoline 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 -70° to about +40°C, under inert conditions, to form an iminium intermediate:
(d) reducing said first iminium intermediate by reaction with a 1,4-dihydropyridine compound, represented by the following formula:

wherein:
R1, R2, R4, and R6 are selected, independently, from the group consisting of H, alkyl, substituted alkyl, aryl and substituted aryl; and R3 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, carboxylic esters, and -CONH2; and R5 is selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl and carboxylic esters;
with the proviso that, when R3 is -CONH2 then, R2, R4, R5, and R6 are H and R1 is CH2Ph.