IL87347A - Process for the synthesis of vinblastine and vincristine - Google Patents

Description

PROCESS FOR THE SYNTHESIS OF VINBLASTINE AND VINCRISTINE The Applicant ;- THE UNIVERSITY φ BRITISH COLUMBIA 2194 Health Sciences Mall VANCOUVER, B.C.

V6T 1W5 - CANADA.

The Inventors 1. James P. Kutney 2327 McMullen Avenue VAncouver, B.C. , CAN V6L 2E2. 2. Lewis S.L. Choi 7672 Kerrywood Cres.

Burnaby, B.C., CAN V5A 2G1. 3. Jun Nakano U80-5, Kagtsube-cho Moriyama-shi, Shiga, 524 Japan. 4. Hiroki Tsukamoto 122, Oyama-7 Kariya, Aichi, 448 Japan. 5. Camille A. Boulet c/o G.C. Boulet 265 Heath Rd.

Edmonton, Alta., CAN T6R iG5. 6. Michael McHugh c/o 1019 Sauchiehall St.

Glasgow, G3 7T2 Scotland, U.K.

Process for the Synthesis of Vinblastine and Vincristine Field of the Invention The present application relates to a further improvement of a new and improved method for producing dimer alkaloid compounds especially of the Catharanthus (Vinca) alkaloid groups and, in particular, is a further improved method for producing the anti-viral, anti- leukemic (antineoplastic) compounds, vincristine and vinblastine of Formula I.

I The above compound, when R is COOCH3, and is OCH3, is vinblastine (NSC 49482) and when R is COOCH3, and ^ is OCH3 and ^ 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 aliphatic center C^g in tne indole unit and an aromatic carbon C15 in the vindoline portion.

II % III Summary of the Invention The specific improvements in the present application are set out below.

Firstly, improvement of the novel reduction method, in which conversion of the iminium intermediate (Formula VI) to the enamine (Formula VIII) viii R«C02CH3 as described in step (e), post, is achieved by the use of various new 1 ,4-dihydronicotinamides (Formula IX, B 12 = R 14= F 15 = Ri6 = H; R13 = CONH2 and may contain different functional groups ;such as aryl, cajrboxylic esters, sugars, carboxylic acid, and carboxylate salt, as represented by Formulae XXVII to XXXII listed in Table 1) and 1,4-dihydropyridine compounds such as Formulae XXXIII and XXXIV. Of special importance is t e presence of electron rich functional groups (e.g., carboxylic esters and carboxylate salts in Rj_| of Formula IX, capable of coordination with the positively charged iminium intermediate (Formula VI).. Such coordination increases both the regioselectivity (1,4-reduction over 1,2-reduction ) and the rate of the reduction of iminium intermediate (Formula VI), leading to an improved yield of the enamine (Formula VIII). Further improvement in the formation of the latter compound is achieved by performing the reduction of iminium intermedia e (Formula VI) and maintaining the reaction mixture before any subsequent manipulation at a low temperature (0 to -70 °C) preferably below -40°C. ft IX Specific examples related to the above improvements for the synthesis of enamine (Formula VIII) by the 1 , 4-reduction of the iminium intermediate (Formula VI) are given in the Examples 1-9 (Procedures C to K). Results of these examples are summarized in Tables 2 and 3. Table 2 indicates reduction procedures (Procedures I and J as in Examples 7 and 8) , employing reducing agents Forrula XXXI and Formula XXXII, respectively, afford the best yields of the enamine (Formula VIII). Table 3 shows the effect of temperature in the reduction of iminium intermediate (Formula VI) by the reducing agent Formula XXXII. Lowering the reduction temperature to -40 °C (Procedure K(ii) Example 9) resulted in an increase in both the ratio of 1,4-reduction production, enamine (VIII), versus 1 , 2-reduction product, 3 , 4-dehydrovinblastine (VII), (4.2:1), as well as the overall yield of the reaction (85%).

The second area of improvement relates to the oxidative transformation of the enamine '(Formula Vlit) to the iminium intermediate (Formula XVI) which was described in the parent application. Various parameters for this oxidative transformation have been studied to optimize the yield of vinblastine (Formula I) production.

Table 4 indicates the effect of ferric chloride concentration. Two equivalents of ferric chloride provide the highest yield of vinblastine (I).

In a similar manner, other metal ions may be utilized, being selected from cupric, cuprous, mercuric and silver in open air or with a stream of air/oxygen bubbled through the solution.

Table 1. KADH Models for 1,2-Versus 1,4-Reduction of Iminiun Intermediate R«C02CH3 Table 2. Effect of Reducing Agent on 1,4- vs. 1,2'Reduction of Iminium VI 1.4 Reduction Procedure l,4:l,2-Reduction Products4 Yield (%) 1:1 75 B" 1:1 60 C 0.9 60 D 1 40 £ 1.1 60 F 2 65 G .5 70 H .1 65 I .3 70 J 2 70 Typical Procedure: 100 n Iminiun) VI in 6 ml methanol fco which reducing agents C-J (1-6 eq.) dissolved in 6 ml methanol were added. Full details in Experimental Section.

By reverse phase HPLC quantitation.

Combined 1,2-reduction (3,4-dehydroviriblastine, VII) + 1,4-reduction (enamine VIII) products.

All of these reactions were conducted at 20° C.

Procedures A and B are presented in the parent application and are cited here for comparison.

Table 3. Effect of Teaperature on 1,4· vs. 1,2-Reduction of lainium VI. 1 Λ Reduction Procedure Temp. ( C) 1,4:1,2-Reduction Products Yield D 20 2.2:1 70 K (i) -20 3.2:1 80 K (ii) -40 4.2:1 85 1 Typical procedure as in Table 2. 2 Quantitation by HPLC. 3 Combined 1,2-reduction (3,4-dehydrovinblastlne, VII) + 1,4-reduction (enamine VIII) products.

Table 4. Effect of Ferric Chloride on Production of Vinblastine (I) from Enamine VIII3 Amount of FeCl. Oxidation Conditions % Yield of (Equivalents) (Temp., tine) Vinblastine2 1 At a rate of 60 ml/min. 2 fiy reverse-phase HPLC quantitation, after reductive work-up with NaBH Enamine VIII generated at -40° C (Procedure (ii)) Table 5. Effect of Time of Oxidation on Production of Vinblastine (I) from Enanine VIZI3 (min) % Yield of , Vinblastine 1 6.2 5 15.4 10 15.5 15 15.7 45 6.5 Reaction conditions: - 2 eq. ferric chloride added, air bubbled through the solution at 60 ml/mln at 0° C.

By reverse-phase HFLC quantitation after reductive vork-up with NaBH Enamine VIII generated at -40° C (Procedure K (11)) Table 6. Effect of Oxidation Temperature on Production of Vinblastine (I) 3 from Enanine VIII Temp., ° C1 % Yield of 2 vinblastine -40 3.7 -23 6.2 0 19.6 20 20.6 45 16.0 Reaction conditions: 2 eq. ferric chloride added, air bubbled through the solution at 60 ml/min for 15 min.

By reverse-phase HFLC quantitation after reductive work-up with NaBH Enamine generated at -40° C (Procedure (ii)).

Table 7. Effect of Dilution on Production of Vinblastine (I) from Enamine VIII3.

Dilution Factor1,4 % Yield of , Vinblastine 1 19.6 5 25.2 10 * 30.1 20 29.6 50 24.7 Reaction conditions: 2 eq. ferric chloride added,oair bubbled through the solution at 60 al/min for 15 ain. at 0 C.

By reverse-phase HPIX quantitation after reductive work-up with MaBH Enamine generated at -40° C (Procedure K (11)).

Dilution Factor 1 - 100 ag Iminium VI in 6 nl Methanol to which reducing agent Formula ZXXII (6 eq.) in 6 ml methanol was added.

(Total volume - 12 ml) Dilution Factor 5 - Total volume of 60 ml, etc.

Table 5 indicates results relating to yield of vinblastine versus time of oxidation. The maximum yield of vinblastine (I) is reached after 5 to 20 minutes of aeration in the presence of 2 equivalents of ferric chloride.

Table 6 shows the results of various oxidation temperatures, in the presence of two equivalents of ferric chloride., on the yield of vinblastine (I). The temperature range of 0°C to 20°C provides the highest yield of vinblastine (I) after a reductive work-up with NaBH^ , as quantified by reverse-phase HPLC.

The effect of dilution of the enamine (VIII) solution on the production of vinblastine (I), is indicated in Table 7. A dilution factor of 10 to 20 on the enamine (VIII) solution before aeration in the presence of ferric chloride (2 equivalents) at 0°C, affords the best yield of vinblastine (I) as quantified by reverse-phase HPLC after reductive work-up with NaBH^ . % In summary, the specific improvements in the present application as they relate to the oxidative transformation of the enamine (VIII), involve the dilution (5 to 50 folds) of the enamine (Formula VIII) solution, obtained in the above reduction of iminium intermediate (Formula VI), by the same solvent used in the reduction. For practical purposes, the preferred dilution factor is usually in the range of 8 to 12 folds. The above procedure is conducted at a low temperature (0°C to -70°C), preferably below -40 °C and under cover with inert conditions such as argon or an inert gas of Group Zero of the Periodic Table (nitrogen, helium, neon, etc.). After this dilution process, the oxidative transformation of the enamine (Formula VIII) can be carried out as described above (aeration at 60 ml/min for 15 min at 0°C in the presence of ferric chloride (2 eq. ) ) as well as those in the parent application, to afford the corresponding iminium intermediates (Formula XVI).

Formula XVI . =C02CHj The third area of improvement relates to the reduction of iminium intermediates (Formula XVI) by alkali metal borohydride (NaBH4, KBH4, LibH4# etc.) to vinblastine (Formula I) and/or leurosidine (Formula XXXV). The iminium intermediates (Formula XVI and Formula XVIa) produced from the oxidative transformation of enamine (Formula VIII) are reduced by the addition of alkali metal borohydride at low temperature (4°C to -20°C), preferably at 0°C. The reduction is conducted at pH lower than 8.5 and preferably at 7.5 to 8. The total reaction mixture is then concentrated in vacuo at low temperature (0°C to 10 °C) before extraction and isolation of alkaloid products as described in the parent application.

Formula XXXV ( urosidine) R-CO2CH3 XVla For practical purposes, all the above improvements can be incorporated, as indicated, directly into the overall process conducted in a one-pot operation from the indole unit (Formulas II, XXII or XXIII) and the dihydroindole unit (Formula XXI, R = H) to the final products of Formula XXI. Isolation of the various intermediates (Formulas XXIV, VI, VIII and XVI) is omitted as summarized in Scheme 1.

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: Scheme 1. Optimuxt procedure for one-pot process - vinblastine fron. catharanthine and vlndoline.

Enamine Anhydrovinblastine Ratio 4.2: 1 Overall Yield: Vinblastine (42%); Lcurosidine (17%); Anbydrovinbtastine ( Formula XXI i*- as pictured and in that formula alk represents a lower alkyl group of C^-Cg and preferably C^- C3 aryl is mono-aryl such as benyzl, styryl, and xylyl; R^ is a member of the group consisting of hydrogen, alk, CHO and CORg where Rg is alkyl o aryl; R2 and R3 are members of the group consisting of hydrogen and -CO-alk> 4 is a member of the group consisting of COO-alk, CONH-NH2, CONH2, CONHR6, and CON(R6)2 where R6 is alkyl; Z is a member of the" group consisting of -CH2-CH2- end -CH¾CH- and R. is a member of the indole family represented by Formula XXII where R7 is a member of the group consisting of hydrogen; or COO-alk; R8 is a member of the group consisting of hydrogen, OH, O-alk, OCO-alk or alkyl Rg is a member of the group consisting of hydrogen, OH, O-alk, OCO-alk, or alk; is a member of the group consisting of hydrogen, OH, O-alk, OCO-alk, or Formula XXIII where Rj^ is a member of the group consisting of hydrogen or COO-alk; R12 is a member consisting of alkyl.

Specific Description of the Invention This invention is specifically a process for the production of compounds represented by the following formulas: XXI wherein in Pormula XXI ■ , Alkyl « CH3 or (CH2)n6H3 where n=l-5 Rl ■ CH3 or CHO R2 β H or CO-alk R3 ■ H R4 « COO-alk or CONRj^Rj^" where R13 and R14 can be any member of the group consisting of hydrogen, alkyl, substituted alkyl, aryl or substituted aryl functions Z ' - -CH=CH- or -CH2-CH2- R = Formula XXII or XXIII R7 «= H or COO-alk R8 β H, OH, O-alk, OCO-alk or alkyl R9 , s H, OH, O-alk, OCO-alk or alkyl R10 β H, OH, O-alk, OCO-alk R - H or COO-alk , B H or alkyl wherein the process is for the synthesis of a dimer derived from an indole unit of the natural Iboga alkaloid family containing an aza bicyclo-octane portion and a dihydroindole unit of the natural Aspidosperma and Vinca alkaloid families, the stereochemistry of the carbon-carbon linkage between these two units being identical with that of vinblastine which consists of (a) forming an N-oxide intermediate in the cold at a temperature of -70 °C to +40 °C from said indole unit by oxidizing the bridge nitrogen and without isolating said intermediate; (b) treating said -oxide indole intermediate in the presence of one member of 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 lb), coupling said reaction product with a dihydroindole unit in the presence of acetic anhydride, halogenated acetic anhydride, and acetyl chloride at a low temperature of about -70 °C to +40 °C under inert conditions; (d) the product of step (c) is solated by solvent evaporation preferably at. low temperature in the range of -20°C to 0°C; (e) the product of step (d) is reduced by 1,4-dihydropyridine compounds represented by Formula IX where R3 and 5 in Formula IX are carboxylic esters (C00-alk) and R R2, R and R6 are members of the group consisting of H, alkyl, aryl (Hantzch ester series) or N-substituted 1 , 4-dihydronicotinamides where R ± is a substituted alkyl or substituted aryl function, for example, benzyl, and R3 is CONR Rg where R?. and Rg is one member of the group consisting of hydrogen, alkyl or aryl function; (f) the product of step (e) , an enamine, Formula VIII, is isolated by solvent evaporation, preferably at low temperature in the range of. -20 °C to 0°C; (g) the product of step (e), a solution of enamine VIII, is diluted (5 to 50 folds) by the same solvent employed, preferably in the range of 8 to 12 folds at a low temperature (0°C to -70°C), preferably below -40°C; (h) the enamine obtained in step (f) or the enamine solution obtained in step (g) is used to prepare iminium intermediates, Formula XVI or XVIa, by a number of oxidative processes including: (1) controlled aeration/oxygenation in which a solution of the enamine is stirred in open air or with a stream of air/oxygen bubbled through the solution; (2) as in (1) but with the addition of a metal ion; (3) as in (1) but with the addition of a flavin coenzyme, as represented by Formula XII, to generate, in situ, the corresponding 1 , 5-dihydroflavin coenzyme, as represented by Formula XIII; (4) as in (1) but with the addition of hydrogen peroxide and/or hydroperoxides as represented by the Formula R-OOH where R is alkyl or aryl (i) the product, an iminium intermediate or iminium intermediates obtained in step (h) is isolated by sovlent evaporation, preferably at low temperature in the range of -20 °C to 0°C; (j) the product obtained in step (i) or the iminium intermediate solution obtained in step (h) is converted to the target compounds of Formula XXI, for which vinblastine and vincristine are examples and leurosidine (Formula XXXV) by reduction with alkali metal borohydride (NaBH^ , KBH4, LiBH4) in suitable solvents (organic and/or aqueous) as used in the oxidative processes of step (h).

Of special importance is the presence of electron rich functional groups (e.g., caqrboxylic esters and carboxylate salts) in of Formula IX, capable of coordination with the positively charged iminium intermediate (Formula VI). Some examples of these 1,4-dihydropyridine compounds are given in Table 1 (e.g., Formulas XXXI and XXXII). Such coordination increases both the regioselectivity (1 , 4-reduction over 1,2-reduction) and the rate of the reduction of iminium intermediate (Formula VI), leading to an improved yield of the enamine (Formula VIII).

The reduction is conducted under an inert atmosphere at -60°C to +60°C but preferably in the temperature range -20 °C to -40 °C. The solvents employed are alcohols, acetonitrile or higher members of this series, dimetheyl sulfoxide, dimethylformamide, various ethers such as dioxane and tetrahydrof uran , and chlorinated hydrocarbons.

The oxidative processes in step (h) (l)-(4) are conducted 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. An aqueous buffer (e.g., phosphate, Tris-HCl, MES buffers) at pH 5-9, but preferably in the range of 6-8, can be used as co-solvent. The reaction temperature may vary from -60°C to +60°C.

Of special importance is the presence of electron rich functional groups (e.g., caqrboxylic esters and carboxylate salts) in R-^ of Formula IX, capable of coordination with the positively charged iminium intermediate (Formula VI). Some examples of these 1,4-dihydropyridine compounds are given in Table 1 (e.g.. Formulas XXXI and XXXII). Such coordination increases both the regioselectivity (1 , 4-reduction over 1 , 2-reduction ) and the rate of the reduction of iminium intermediate (Formula VI), leading to an improved yield of the enamine (Formula VIII).

The reduction is conducted under an inert atmosphere at -60°C to +60°C, but preferably in the temperture range of -20 °C to -40 °C. The solvents employed are alcohols, acetonitrile or higher members of' this series, dimethyl sulfoxide, dimethylformamide, various ethers such as dioxane and tetrahydrofuran and chlorinated hydrocarbons.

The product of step (e) above, an enamine, Formula VIII, is isolated by solvent evaporation, preferably at low temperature in the range of -20 °C to 0°C.

The process of this invention also prepares an enamine and oxidizes said enamine to the iminium intermediate (Formula XVI and XVIa) by a number of oxidative processes including: (a) controlled aeration/oxygenation in which a solution of the enamine is stirred in open air with a stream of air/oxygen bubbled through the solution; (b) as in step (h)(1) but with the addition of a e (c) as in step (h)(1) but with the addition flavin coenzyme, as represented by Formula XII: n R' is H or kyl to generate, in situ, the corresponding 1 , 5-dihydroflavin coenzyme, as represented by Formula XIII s H or* The oxidative processes are conducted in organic solvents such as alcohols, acetonitrile or higher members of this series, dimethyl sulfoxide, dimethylformamide, ethers such as dioxane, tetrahydrofuran, aromatic hydrocarbons such as benzene, toluene, etc.

EXAMPLES The following examples (Examples 1 - 8) were conducted at 20 °C.

Example 1 Reduction of Iminium Intermediate (Formula VI) with 1-Diphenylmethyl ( -1 , 4-dihydronicotinamide (Formula IX, R± -diphenyi methyl; R2 = R4 = R5 = R6 = H ; R3 = CONH2) (Formula XXVII) - Synthesis of Enamine (Formula VIII) - Procedure C) To a stirred solution of iminium intermediate (VI, 100 mg ) in degassed ethanol (6 ml) was added 1-diphenylmethy1-1 , 4-dihydronicotinamide (Formula XXVII) (76 mg, 2.5 equivalents) in methanol (6 ml) under a positive atmosphere of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 60 min. After this, reverse phase HPLC analysis (Waters Radial-Pak C-^g or CN cartridge, methanol-H 20-E t 3 as solvent system) indicated, among other products, formation of enamine (VIII) and 3 ' ,4 · -dehydrovinblastine (VII) in a ratio of 0.9:1 (60% yield).

Example 2 Reduction of Iminium Intermediate (Formula VI) with 1-Benzyl-3-cyano-l,4-dihydropyridine (Formula IX, R = benzyl; R2 · R4 = R5 = R6 = H; 3 = CN) (Formula XXXIII) - Synthesis of Enamine (Formula VIII) - (Procedure D) .

To a stirred solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 ml) was added l-benzyl-3-cyano-1 , 4-dihydropyridine (Formula XXXIII) (206 mg, 10 equivalents) in methanol (10 ml) under a positive atmosphere of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 60 min. After this, 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 (40% yield).

Example 3 Reduction of Iminium Intermediate (Formula VI) with 1-Benzyl-1 , -dihydronicotinyl- ( 2 ' -carbamoyl-pyrrolidinyl ) -amide (Formula IX, R^ = benzyl; R2 = R = R5 = Rg = H; R3 = (2' carbamoyl-pyrrolidinyl) carbonyl) (Formula XXXIV) -Synthesis of Enamine VIII) - (Procedure E).

To a stirred solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 ml) was added l-benzyl-1,4-dihydronicotinyl- ( 2 ' -carbamoyl-pyrrolidinyl ) -amide ( Formula XXXIV) (163 mg, 5 equivalents) in methanol (5 ml) under a positive pressure of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 30 min. After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and- 3 ' ,4 ' -dehydrovinblastine (VII) in a ratio of 1.1:1 (60% yield).

Example 4 Reduction of Iminium Intermediate (Formula VI) with 1,4-Dihydro-1- (l-methoxycarbonyl isobutyl) -nicotinamide (Formula IX, R^ = l-methoxycarbonyl isobutyl; R2 = 4 = R5 = Rg = H; 3 = CONH2) (Formula XXVIII) - Synthesis of Enamine (Formula VIII) - (Procedure F) To a stirred solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 ml) was added 1, 4-dihydro-l-(1-methoxy carbonylisobutyl) -nicotinamide (Formula XXVIII) (150 mg, 6 equivalents) in methanol (6 ml) under a positive pressure of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 30 min. After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 31 ,4 ' -dehydrovinblastine (VII) in a ratio of 2:1 (65% yield) . , Example 5 Reduction of Iminium Intermediate (Formula VI) with 1-(2,,3',4,,6' -Tetraacetyl- ( beta ) -D-glucopyranosidy1 ) -1 , 4r dihydrdnicotinamide (Formula IX, = (2 ', 3 ', 4 ' , 6 ' -Tetraacetyl- (beta )-D-glucopyranosidyl; R2 = R4 = R5 = Rg - H; R3 = CONH2) (Formula XXIX) - Synthesis of Enamine (Formula VIII) - (Procedure G) To a stirred solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 ml) ws added Ι-^'^'^',β1-tetra-acety1- ( beta ) -D-glycopyranosidy1 ) -1 , 4-dihydronicotin-amide (Formula XXIX) (238 mg, 5 equivalents) in methanol (10 ml) under a positive atmosphere of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 60 min. After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 31 ,4 * -dehydrovinblastine (VII) in a ratio of 1.5:1 (70% yield).

Example 6 Reductio of Iminium Intermediate (Formula VI) with 1,4-Di-hydro-l-(2'-methoxycarbonyl isopropyl) -nicotinamide (Formula IX, R^ = 2'-methoxy carbonylisopropyl; R2 = 4 = 5 = g = H; R3 = CONH2) (Formula XXX) - Synthesis of Enamine (Formula VIII) - (Procedure H) To a stirred solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 ml) was added 1 , 4-dihydro-l- ( 2* -methoxy-carbonylisopropyl) -nicotinamide (Formula XXX) (82 mg, 3.5 equivalents) in methanol (7 ml) under a positive atmosphere of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 30 min. After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 3 ' , ' -dehydrovinblastine (VII) in a ratio of 1.1:1 (65% yield).

Example 7 Reduction of Iminium Intermediate (Formula VI) with 1,4- Dihydro-l-(l ' ,2 6 = H; R3 = CONH2) (Formula XXXI) - Synthesis of Enamine (Formula VIII) - Procedure I) To a solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 ml) was added 1 , 4-dihydro-l- (1 · ,2 ' -fdi^nethoxy carbonyl) ethyl'-nicotinamide (Formula XXXI) (148 mg, 5 equivalents) in methanol (10 ml) under a positive atmosphere of argon, the reducing agent being added portionwise at the rate of 1 equivalent each 30 min. After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 3 · ,4 · -dehydrovinblastine (VII) in a ratio of 1.1:1 (70% yield).

Example 8 Reduction of Iminium Intermediate (Formula VI) with 1,4-Dihydro-l-( sodium-isobutyl-l-carboxylate) -nicotinamide (Formula IV, R^ = sodium-isobutyl-l-carboxylate; R2 = R4 = R5 = R6 = H R3 = CONH2) (Formula XXXII) - Synthesis of Enamine (Formula VIII) - (Procedure J) To a solution of iminium intermediaqte (VI, 100 mg) in degassed methanol (6 ml) wqs added 1 , 4-dihydro-l-( sodium-isobutyl-l-carboxylate) -nicotinamide ( Formula XXXII ) (130 mg, 5 equivalents) in methanol (6 ml) under a positive atmosphere of argon, the reducing agent being a'dded portionwise at the rate of 1 equivalent each 30 min. After this, reverse-phase HPLC analysis (as 'described above) indicated, among other products, formation of enamine (VIII) and 3 ' , 41 -dehydrovinblastine (VII ) in a ratio of 2.2:1 (70% yield) .

Example 9 Reduction of Iminium Intermediate (Formula VI) with 1,4-Dihydro-1- ( sodium-isobutyl-l-carboxylate) -nicotinamide (Formula IX, ^ = sodium-isobutyl-l-carboxylate; 2 = 4 = R5 = R6 = H; R3 = CONH2 ) (Formula XXXII) at low temperature - Synthesis of Enamine (Formula VIII) (Procedure K) (i) To a solution of iminium intermediate (VI, 100 mg) in degassed methanol ( 6 ml ) at -20 °C was added 1,4-dihydro-1- ( sodium-isobutyl-l-carboxylate) -nicotinamide (Formula XXXII) (155 mg, 6 equivalents) in methanol (6 ml) under a positive atmosphere of argon, the reducing agent being added in one portion. After 45 min. at this temperature reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 3',4' -dehydrovinblastine (VII) in a ratio of 3.2:1 (80% yield). (ii) Carrying out the reaction above at -40 °C gave, after 60 minutes, enamine (VIII) and 3',4'-dehydrovinblastine (VII) in a ratio of 4.2:1 {85% yield).

Example 10 Synthesis of Vinblastine (Formula I) by Oxidation of Enamine (Formula VIII) to Iminium Intermediate (Formula XVI) with Air in the Presence of Ferric Chloride at High Dilution. (Method 5) The solution containing the enamine (VIII), (Procedure K (ii)) obtained from the iminium intermediate (VI, 200 mg) was diluted five-fold with methanol before oxidation (total vol.: 120 ml). Ferric chloride (75 mg, 2 equivalents) was then added, and air bubbled through the solution, at 0°C, for 20 min. Sodium borohydride (200 mg) was added, and the solution concentration in vacuo before adding water (100 ml) and extracting with ethyl acetate (3 x 200 ml). The combined organic extract was dried over Na2S04 and the solvent evaporated in vacuo. The crude product was purified by column chromatography (silica gel, TLC grade, 15 g). Elution with ether: chloroform (10:7) gave 3',4'-de-hydrovinblastine (VII, 18 mg, 11%). Further elution with ether: chloroform: methanol (10:7:0.5) gave vinblastine (I, 62 mg, 37%) .

Example 11 One-Pot Conversion of Catharanthine (Formula II) and Vindoline (Formula III) to Vinblastine (Formula I) and Leurosidine (Formula XXXV) - Overall Procedure To a solution of catharanthine 500 mg, 1.5 mmol) in dry dichloromethane (4.5 ml) at -15 °C under a positive atmosphere of argon was added m-chloroperbenzoic acid (330 mg, 1.9 mmol) in one portion, and the mixture stirred at -10 to -15 °C for 5 minutes. After this time the reaction mixture was cooled to -40 °C and a solution of vindoline (III, 450 mg, 1 mmol) in dry dichloromethane (1 ml) was added, followed immediately by trif luoroacetic anhydride (1 ml, 7.1 mmol). After 2 h at -60°C volatiles were removed in vacuo (high vacuum pump) and dry, degassed methanol (12 ml) added after flushing the system with argon. The resulting orange solution was cooled to -40 °C and a solution of l,4-dihydro-l-(sodium-isobutyl-l-carboxylate)-nicotinamide (Formula XXXII) (1.5 g, 6 mmol) in dry degassed methanol (12 ml) was added under a positive atmosphere of argon. After reduction was complete (by reverse-phase HPLC monitoring), cold methanol (about 300 ml) was added, keeping the temperature of the solutionn between -5 and 0°C. Ferric chloride (330 mg, 2 mmol) was then added and dry air bubbled through the solution at a rate of 60 ml/min for a period of 20 min. Sodium borohydride (1 g) was added and the solution concentrated in vacuo (water aspirator) before adding water (100 ml) and extracting with ethyl acetate (3 X 150 ml). The combined organic extract was dried over Na2S04 and the solvent evaporated in vacuo to give the crude product which was purified by chromatography as previously described to give 3 · , 4 » -dehydrovinblastine (VIII, 95 mg, 12%), vinblastine (I, 315 mg, 39%) and leurosidine (XXXV, 130 mg, 16%).

Claims (11)

1. - 34 - 87347/3 C L A I M S : - 1. A process for the production of dimer alkaloid compounds comprising the steps of: (a) oxidizing an indole unit having a bridge nitrogen and being represented by the following formula: wherein l< represents hydrogen or COO-alkyl and , n^, ^, Rg and independently represent H; said oxidation being carried out in the cold, at a temperature from about -70°C to about +40°C, thereby oxidizing the bridge nitrogon of said indole; unit and forming an N-nxide derivati fcrrrula: wherein: e as above, and without iso ating sai erivative; (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; 87347/2 (c) without isolating the product of step (b), stereospecifically coupling said product of step (b) with a dihydroindole unit represented by the formula: said coupling being conducted 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, represented by the formula: wherein R=C00CH3 and R1f R2, R3, and R4 are the same as defined above; - 36 - 8734*7/4 (d) reducing said first ίminium intermediate by reaction with 1 ;4-dihydropyridine compound as described in the specification said reduction being conducted unde an inert atmosphere at a temperature in the range of -60°C to +60°C; - 37 07347/3 (e) diluting said enamine obtained in step (d) by a factor of 5 to 50 fold with a solvent at 0°C to 7QUC and transforming said enamine intermediate b} ixidation unde controlled aeration conditions at a temperature in the range of -60°c to +60°c to a second iminium intermediate, as represented by the following formula: wherein R'=COOCH, 1 j and R^ are the same as defined in step (c), g is : «0H or C2Hg and R6 is OH or C2H5» with the proviso that Rg and R6 cannot be the same; and 38 - 87347/3 (f) reducing the second iminium intermediate obtained in step (e) to form said dimer alkaloid compounds, represented by the formula: wherein R, R1 , R2, R3 , R4» " are the same as defined in step (e) and R'5 and R'6 are OH or C2H-., with the proviso that R1^ and R'g cannot be the same, using an alkali metal borohydride, at a low temperature in the range of 4°C to -20°C and a pH below 8.5.

2. The process according to Claim 1 , wherein 13 and R15 are COOZ, wherein Z is alkyl or a cation and aryl .

3. The process according to Claim 1 , wherein R1 ¾ is C0NR1 7R1 8 , wherein R^ 7 and R1 FT independently may be H, 7 alkyl, aryl , or taken together, R and R^ Q can form a ring structure containing up to four carbon atoms, said ring structure may further be substituted by C0NR1 9R20 , wherein .q and R?n are H or alkyl . 87347/2

4. The process according to Claim 1 wherein the oxidative transformation step (e) is selected from the group consisting of: (1) 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; (2) 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), cuprous 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; (3) 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; (4) controlled aeration/oxygenation in which a solution of said enamine and a flavin coenzyme, represented by the following formula: 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-dihyroflavin coenzyme, represented by the following formulas 87347/2 (5) 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 (6) 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. 87347/3

5. The process according to Claim 1, wherein R, is an electron-donating substituent as described in the specification.

6. The process according to Claim 1, wherein R¾1 is selected from carboxylic esters and carboxylate salts.

7. The process according to Claim 1 , wherein the reducing agent used in step (f) comprises contacting the reaction product from step (e) with an alkali metal borohydrate selected from the group consisting of NaBH^, KBH4 and LiBH^.

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

9. A process according to Claim 1, wherein the dimer alkaloid compound is vinblastine.

10. A process according to Claim 1, further comprising the step of oxidizing said vinblastine to obtain the dimer alkaloid compound vincristine.

11. A process according to Claim 1, wherein the dimer alkaloid compound is leurosidine. For the Applicant, Patent Attorney