AU617342B2 - Process for the synthesis of dimer alkaloid compounds - Google Patents

Description

ir Y--"C S F Ref: 66995 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION 617342

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Spocificatioi Lodged: Accepted: Published: Priority: Related Art: 0* 0

U

0* 0 0* 0 00 0 00 0 P 0 Go 0 00 0 00 0b 00 0' 0O *0 00 *0 0: 000I 0 Name and Address of Applicant: Address for Service: The University of British Columbia 2194 Health Sciences Mall Room 331, I,R.C. Building Vancouver B.C. V6T

CANADA

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Males, 2000, Australia Complete Specification for the invention entitled: Process for the Synthesis of Dimer Alkaloid Compounds The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 CII(Il~~ LIL--- I a t' r .1 Abstract The present invention relates to the synthesis of dimer alkaloid compounds, particularly those of the Catharantus (Vinca) family, from an indole unit, such as cantharanthine, and a dihydroindole unit, such as vindoline. A multi-step process is disclosed including the steps of a 1,4-reduction of a first dimeric iminium intermediate to an enamine compound by reaction with a 1,4-dihydropyridine compound; oxidative transformation of the resulting enamine to a second iminium intermediate under controlled aeration; (3) reduction of the second iminium intermediate to form the target dimer alkaloid compounds. The entire process can be conducted in a one-pot operation to obtair the target compounds without isolation of the intermediates.

o 0 o oo o 0 6 a 1A BACKGROUND OF THE INVENTION The present invention relates to the synthesis of dimer alkaloid compounds, particularly those,of the Catharantus (Vinca) family, such as vinblastine, vincristine and leurosidine, sometimes referred to S hereinafter as the target compounds or the tarqet dimer lkaloid compounds.

The dimeric alkaloid,; of present interest have .remarkable anti-viral, anti-tumor and anti-leukemic """properties. In the past, these alkaloids were isolated o from the Catharantus species, particularly C. roseus, in small quantities. More recently, progress has been made in the synthesis of these compounds patents "4,144,237 and 4,279,817 to James P. Kutney, and parent S.application U.S. serial number 07/011,810).

S'Unfortunately, these prior art methods also yielded o"mall quantities of the target compounds. The present invention overcomes the difficulties encountered in the ;prior art insofar as it yields dramatically increased a osquantities of the target dimer alkaloid compounds.

SUMMARY OF THE INVENTION The present invention relates to the multi-step process for preparing dimer alkaloid compounds,, particularly those of the Catharantus (Vinca) family, 1 2 such as Vinblastine, vincristine and leurosidine, as described in parent application U.S. serial number 07/011,810, filed February 6, 1987, the disclosure of which is incorporated herein by reference.

Briefly, the present process comprises the following steps: forming an N-oxide derivative in the cold at a temperature of from about -770 to about +40 0 from an indole unit having a bridge nitrogen, by oxidizing the bridge nitrogen and without isolating said derivative; treating said N-oxide derivative in the presence of at least one member selected from the group consisting of acetic anhydride, halogenated acetic anhydride, and acetyl chloride, to effect a Polonovskitype fragmentation reaction; without isolating the product of step stereospecifically coupling said product of step (b) q.«oso with a dihydroindole unit in the presence of at least or one member selected from the group consisting of acetic .o anhydride, halogenated acetic anhydride, and acetyl chloride at a low temperature of about -70 0 C to about 6 +40 0 C, under inert conditions, to form a first iminium intermediate; oo reducing said first iminium intermediate by reaction with a 1,4-dihydropyridine compound, thereby t* fforming an enamine; preparing a second iminium intermediate by oxidative transformation of the enamine obtained in step under controlled aeration conditions; and reducing the product obtained in step to form the target dimer alkaloid compounds.

All of the above steps can be conducted in a onepot operation from the reaction of the indole unit and the dihydroindole unit to the final products without isolation of the intermadiater.

nr 4 0 0 oc a 04 44 4 4 I 4 44 3 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the synthesis of dimer alkaloid compounds, particularly of the Catharantus (Vinca) family, such as vincristine, vinblastine and leurosidine, as represented by the following formula I: 8,

N

17 RI R 4

O-R

2 O-R3 wherein in Formula I, alk CH 3 or (CH2)n CH 3 where n

R

1

CH

3 or CHO;

R

2 H or CO-alk;

R

3

H;

R4 COO-alk or CONR 1 3

R

1 4 wherein R 13 and R 1 4 are selected frofi the group consisting of hydrogen, alkyl, substituted alkylr aryl or substituted aryl -CH=CH- or -CH2-CH2- II or IIa; pt 0 00 04 4 4I 4 aC p *4 #0 IIa wherein,

R

7 H or COO-alk; .1 -2 -r t ii I -CLLIII -LIIIII--_-i__l 111111113- 1_ 00 01 0 D 00 0R 0R 04 09 0 -4-

R

8 H, OH, 0-alk, OCO-alk or alkyl;

R

9 H, OH, 0-alk, OCO-alk or alkyl; R0 H, OH, 0-alk, OCO-alk; R1 H or COO-alk; and

R

12 H or alkyl.

According to a broad form of the invention there is provided a process for the production of dimer alkaloid compounds represented by Formula 1, which comprises the steps of: forming an N-oxide derivative in the cold, at a temperature from about -77°C to about +40 0 C from an indole unit having a bridge nitrogen, &s herein defined, by oxidizing the bridge nitrogen and wthout isolating said derivative; '0 treating said N-oxide derivative in the presence of at least one member selected from the gro.p consisting of acetic anhydride, halogenated acetic anhydride, and acetyl chloride, to effect a Polonovski- type fragmentation reaction; 'o without isolating the product of step stereospecifically coupling said product of step b) with a dihydroindole unit in the presence i of at least one member selec.ed from the group consisting of acetic 20 anhydride, halogenated acetic anhydride, and acetyl chloride at a low temperature of about -70°C to about under inert conuitions, to form 0o0 a first iminium intermediate; reducing said first iminium intermediate by reaction with a S 1,4-dihydropyridine compound, thereby forming an enamine intermediate; -25: preparing a second iminlum intermediate by oxidative Stransfermation of said enamine intermediate obtained in step under controlled aeration conditions; and oi f) reducing the product obtained in step to form the target dimer alkaloid compounds.

When, in Formula I, alk=CH 3

R

1

=CH

3

R

2

=CO-CH

3

R

3

=H,

R

4

=COOCH

3 Z -CH=CH-, R Formula II, R 7

COOCH

3

R

8

OH,

R

9 C2H 5 and R 10 H, the product is vinblastine; when alk CH 3 R1 CH 3

R

2

CO-CH

3

R

3 H, R 4

COOCH

3 Z -CH=CH-, R Formula II, R 7

COOCH

3

R

8

C

2

H

5

R

9 OH and R 1 H, the product is leurosidine; and when alk CH 3

R

1 CHO, R 2

CO-CH

3

R

3 H, R 4

COOCH

3 Z -CH-CH-, R Formula II, R 7

COOCH

3 R8 OH, R 9 g C 2

H

5 and R 10 H, the product is vincristine.

0r 0, 0.

001 0( 01 0 01 0 0

AIM

r 5 Throughout specification and claims, alk and alkyl represent a CI-C6 alkyl, and preferably C 1

-C

3 alkyl, and aryl represents a mono-aryl such a benzyl, xylyl, etc. All percentages are percentages by weight, all time periods are in minutes, and all temperatures are in unless otherwise specifically noted.

Throughout the specification and claims the term "substituted" is meant to include, for example, the following substituents: alkyl, carboxy, alkoxy, aryl, aryloxy, amino, carboxyamino, sugar units, carboxyalkyl and salts thereof.

The present process of preparing the target dimeric alkaloids comprises the initial formation of an N-oxide derivative from an indole unit having a bridge nitrogen as represented by Formula III, #0 0 0 9 99 9 0 99 0 *a 9 90'0 9 0900 0 0 *09 00 9 09 0 0 III 4 9 a 0 4 0 0 «0 a a 09 o a 4 04 a 004 4 9 wherein R represents hydrogen or COO-alk and R1, R 2

R

3 and R 4 independently, represent H, OH, 0-alk, OCO=alk, alkyl or aryl. Throughout the specification and claims the term "indole unit having a bridge nitrogen" means a compound as definec hy Formula III. The indole unit is oxidized by oxidizing the bridge nitrogen to form an N-oxide derivative as represented by Formula III(a) IIIa ~Vl R and related analogues as represented by Formula I11(b) 0li wheei R 1 2,R 3 an R ae he am a Fomua IN alkyli grup repRsend byar the saes Formula III, ~jwhr t:n 0-10. Preferably, the indole unit u-Eilized in step is ccitharanthine '(Formula III when R =COOCH- 3 and S:R11 R 2 and R 4 are6 H).

::The N-oxide deri ative is oxidized at the bridge nitrogen at a temperature in the range of about -77 0

C.

Z :to about -400C. by reaction with a peracid such as m- :4:chloroperbenzoic 'acid or p-nitroperbenzoic acid ,in an inert organic solvent such a methylene chloride or other polyhalo organic solvents (step The Noxiaie intermediate thu formed is used In the next step S(step without is'olation. The thus-treated N-oxide derivative obtained instep undergoes a 0 fragmitnea~lti reaction (ste~p and the iminium S:intermediate thus formedi is coupled (step with a Sdihy&ioindole unit, such as vindoline; as represented byoo** .IV 0 ~14 OH 4 4

A

H3CO 1717v in whi~h tlie two respective compounds (Formula Illa and 6 Formula IV) are linked via a carbon-carbon bond involving the aliphatic center C8 in the N-oxide derivative and an aromatic carbon C 15 in the dihydroindole unit.

The fragmentation reaction which fragments the

C

5

-C

18 bond of the indole N-oxide derivative is carried out in the presence of a reagent such as tri u9oroacetic anhydride. To maximize the subsequent coupling reaction which promotes the formation of a natural dimer bonded at C18 (indole unit) and C 1 (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 trichloroacetic anhydride, acetic anhydride, acetyl halides and tosyl anhydride. These reagents bring about a Polonovski-type fragmentation of the C 5

-C

18 a« bond in the compounds shown in Formulae IIIa and IIIb.

«o The reaction temperature, time and pressure S 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 o" temperature of the fragmentation and cloupling steps may S* vary from about -70 0 C to about 40 0 C; preferably from about -70C to about -30 0 and most preferably from about -600 to about -40 0 C, The formation of the Noxide derivative, the fragmentation step and the coupling step, may be conducted in the open or under S cover in an inert gas atmosphere such as argon or any .other inert gas of Group Zero of the Periodic Table such as helium, neon, etc. or nitrogen. Due to the low temperature necessary for the later stage reactions, the reaction time for each of steps may vary from several minutes to several days. Typically, step 7 wou'ld take from about 5 min. to about several hours, step would take from about 5 min. to about 1 hour, and step would take from about min. to about several hours.

The above-described conditions for the coupling reaction in the present process represent an important improvement over the prior art (as described, for example, in U.S. Pat. No. 4,279,817; Helv. Chim. Acta, 52, 2858 (1976) and in Reaction Scheme In particular, the present coupling (step allows for the preparation and isolation of a relatively unstable dihydropyridinium intermediate, formed in the coupling of the S 10 N-oxide derivative and the dihydroindole unit.

Reaction Scheme I 00 H e a 0 0 0 0 00 a 0 indne a o4 a C O (R R2, R3, R4=H RCOOCH3 a

HOT

R CMeCOOCH I4.o~H 8 The relatively unstable indole-dihydroindole dimer intermiediate formed by the stereospec if ically coupling step is characterized by an irniniun salt functioh at -Ehe Nb atom Of the indble moiety. The unistable adimeji is heiinafter referred to as an inmini.un intermediAtdc The iminium ixtermedi,ate is represe- tuci by Formulas V or VI

N~N

*1 00 0 41 Swhejein R 1

R

2 1 R 3

R

4 1 R 5

R

6 R7~, and R 8 are as Sopreviousl r discussed.

The prior art method, as described in U.S. patent 4,279,817, reduces this uns-table iminium intermediate by reaction with alkali metal borohydride (NaB~i 4 KBH4, ~LiBl 4 to obtain 'certain stable dirneric alkaloids. For #.instance, reduction of the itninium intermediate of *Formula V1 by reaction with alkali metal borohydride, ~gives the 3' ,41-dehydrovinblastine compound as 0009representea by Formula VII and shown in reaction scheme 0 1Reaction 'Scheme 1I 04 0 Nalkali* iihetal N H borohVcdhie CH3O 1 R o-CH ,41-deliydtovinblastine) CH3 0i R=C02CH3 Myr.

In the present ihvention, however, the relatively unstable iminium intermediate (Formula V or VI) need not be reduced and, in fact, may-be isolated by various chromatographic techniques. For example, said iminium intermediate may be isolated by applying the reaction mixture of step directly onto 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 about 4 0 C to about ro¢m 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 -100 0 and below about 5 mm, Hg. The resulting solid (Formula V or VI) is then dissolved in a suitable organic solvent, such as halogenated hydrocarbons, ethers, alcohols, acetonAtrile, or the like or in various aqueous buffer solutions. The pH of the buffer solution may vary from about 2 to about Suitable aqueous buffer solutions include, for example, phosphate, Tris HC1, and MES buffers. The solution of iminium intermediate (Formula V or VI) can then be purifiea by the chromatographic methods described above. Alternatively, the iminium intermediate solution can be used directly for subsequent reactions.

Example 1 shows the preparation of the iminium intermediate by reaction of the catharanthine, (the indole unit of Formula III when R=COOCH 3 and RI, R 2

R

3 and R 4 H) with vindoline (the dihydroindole unit of Fornma a IV).

9r 9' eO 9 98 4 9 9+ 9 9 a 8 9 0 0* 69 9 9 19a Example 1 *0 Rb 0 19e* Pkeparation of the Iminium Intermediate (Formula VI) via Modified Polonovski Reaction.

'he reaction was performed under anIydrous r H~~Lr~ 7 49 9 9 9 00- 0 6 0 0 0 4 9 9 0 CH9 cofiditiohs. All glcissware ras oven-dried a 120 0

C.

'lie solvent, zethylefie 'chloride, and coupling rEeagent, 'luoroacetic anhyd. ide, were di~ttilled fizbni P 2 0 5 pr.±jr to use.

To a olution 6f 6atharan-Ehine (Fo3imu'la 111, mgi 0;6 6iuol) in dry methy'len~a chloride (2 ml) at -2 0 C. undpr a positEive atmosphere of argon was added m.:d6ilor6perbbnzoic a6id (132 mg, 0.8 rnmol) and the mixture was stiried for 5 minutes. To the cathiar*ntfine N-oxide (Ila; R COOCH 3

R

1

R

2

R

3 and R 4 thus fbrmed was added a solution of vindoline (IiI, 270 mig; 0*.6 inxnol) in methylene chloride (1 iil) and the mix:Eure was cQoled to -60 0 C. Trifluoro-, acetic anhydride (0.2 ihl, 1.5 mzimnol) was added to the stiired rea~tion mixture maintained at -SOOC for 2 hou.rs. After thi time, the solvent an d excess reagents w6i:e reinoved in. .vacuo at -2d 0 C. to leave a reddish-brown residue containing the iminium intermediate. The inteimediate was characterized by reverse phase high peizformance liquid chiomatbgraphy (HPLC) (Waters Radial-Pak C 1 8 or CN cartridge, niethanol-H O-Et 3 N as solvrent system). It was shown that -Ehe yield of the iminium intermr-diate of Formula VI in this reactEion exceeded The iminiun intermediate (Formulae V or VI). was then converted to an enamine compound by reducing the irninium inltermediate with a 1,4-dihydropyridine as illustrated, for example, in reaction schemG' III.

Reaction SchemeIII 130' 1,4 dihydropyridine OC0-CH 3 11 0 1,4 (Encmiie) Vill R= C0 2 CH3 As indicated above., the conversion may take place either OLter isolating the iminiuMh intermediate or by treatinq, the reaction mass obtainied in step Producti36h of the target compound iiB dependent upon which imfihuin intermjediate is emplbyed. In other words, the iminium intermediate V leads to, analogs of It vihblastihe, and iihiuni intermediate VI leaLds to the 4 target compoufids; Vinblastine and leurosic'Lne.

Viiicristine, in turn, is produced by the oxidation of' vinbiastine, specifically, the oxidation of the R, substituent methyl group. dr example, one method for the o. iaatibn of said mdthyl group by reactihng the vinblastihb with Jones reagent (Cr:03) in acetone andi acetib anihydfide at very low temperature, such as Kutney et al, Heterocycle Vol. 9, p.

201, 1978). In order to limit donfusion, howevor, the 0following discussion will be directed to the iminium, aintermediate of Formula VI. Note however, analog6us steps can be conducted with the imininn intermediate of ~Formula V.

When the starting indole unit has a C3-C4 double bond catharanthine (Formvla III when R=COOCH-3 and

R

1

R

2 0 R 3 and R4 H) the resultavit coupling intearmediate contains an alpha, beta-unsaturated Sixniniun functional group as represented by Formula VI.

This iminium intermediate can be converted in step via a 1,4-reduction to tlbw enamine represented by SFormula VIII: o R4 Reagents used for this reduction include .1,4dihydropyridine dompounds (the so-w.alled NADEI models) 12 as represented by Formula IX: 91 0 0 4 041 0 t 0 4,4 04 p 4 #4 0 0 4 wherein Rl 5

R

16 R 17 RIB, R 19 and R 20 1 independently, can be H, alkyl, substituted alkyl, aryl and substituted aryl. Two series of -'ch compounds are readily available [Chem. Rev. 82, 232 (1982); Chem. Rev. 72, 1 (1972)], The first series is known as Hantzch esters wherein

R

1 and 0: R 19 in Formula IX are lower alkyl carboxylic esters, e.g. COOCH H 17 0. Th se o d s r e5st e N s b t t t d i y r n c t n m d s o m l Th eodsre steNsbttte ,-iyrnctnmds(oml 15 I) i whch 15 is a substituted alkyl or substituted aryl function, IX nwhc to e~g. benzyl, and R 17 is CONR 21 R 22 wherein R 2 and R 2 2, ~*independently, can be hydrogan, alkyl, substituted alkyl, aryl and substituted aryl. The term "substituted'! is as herein~pfore defined. The ~jterm aryl includes N-containing heterocyclic ring structures, for exam'ple, pyrrolidine, Which may further be substituted by C0NR 23

R

2 hri R3and

R

24 are H or alkyl, 2 4 hri *ao One preferred class of 1,4-dihydropyridUites which may be employed in "he 1,4-reduction are selected from 1,4-dihydropyridine compounds of t Formula IX wherein R 16

R

18 R 19 and R 20 are hydrogen, is alkaryl and s-No 00 o 4 4,4444 0 4 p 4 40 4* 4 4 *4 *0 30' CO

CONH

2 Another preferred class of 1,4-dihydropyridines are the 1,4-dihydronicotinami~e, wherein R 16 RIB, R 19 and R 0are hydrogen, R 17 is CONH 2 and R 15 is a functional group such as alkaryl, lower alkyl carboXylic esters, sugars, carboxylic acids and carboxylate salts.

Most preferably, 1,4-dihydron-Icotinamides are employed in the reduction, wherein R 15 is 'selected from the electron rich functional group consisting of lower alkyl carboxylic esters and carboxylatm. salts, Extensive research has shown the~t these 1,4-dihydronlcotinamides, provided I 13 with such electron rich functional groups, are particularly capable of coordination with the positively charged iminium intermediate Formula VI), increasing both regioselectivity 1,4-reduction over 1,2-reduction) and the rate of the reduction of the iminium intermediate (Formula VI), thus leading to an improved yield of the enamine (Formula VIII). Specific examples of these preferred and most preferred 1,4-dihydropyridine compounds are provided in Table 1 (Formulae IX-A to

IX-J).

0 f S tt 0 0 0 II 4 0 I 00 e *0 0 -K41ii 0 0 4 0 OtYS 0 0 00 0900 00090 00 00 0 0 0 004 Oa 0 0 00 0000 0 00 .0 0 0 00 0000 4 @000 0 0 4 0 000 0 0 000 040 0 Tab le I Formula Reducing Agents Procedure Reducing Agents Procedure

NH

2 N)0

IX--:A-

Ix-C

IX-F

IX-G

R'1 CH 2

CBH

5

-&CH(C

6

H

5 2 C! OR"

R"O

OR"

R' Aic jOMe EtO I QI O Me Me

CN

CH

2

C

6

H

5

CO-

CH

2

C

6

H

B-

D

Formul a

IX-B

IX-D

I-.

IX-E

H

IX-B1 Ix-I Ix-J M eO2C ItC0 2 Me OpeNall Ku The above reductants can be used alone or in 'combination. The reduction is conducted under an inert atmosphere such as argon or an inert gas of Group Zero of the Periodic Table (helium, neon, etc.) or nitrogen.

Various solvents may be employed during the reduction step Suitable solvents include, for example, alcohols, acetonitrile or higher members of this series, dimethyl sulfoxide, dimethylformamide, various ethers such as dioxane and 'tetrahydrofuran and chlorinated hydrocarbons, normally without an aqueous buffer.

The progress of the reduction step is monitored by direct analysis of the reaction mixture on an appropriate chromatographic system, preferably 'reverse phase high performance liquid chromatography.

his method is used to optimize the reaction S,"temperature, time, pressure and concentration of °°reactants. The reaction temperature may vary from Sabout -600 to about +60 0 and preferably from about to about +20 0 C. and most preferably from about -60° to about -20 0 C. The reaction time may vary from, several minutes to several days depending on other parameters.

0404. The following experimental examples (Examples 2- 12, procedures A to K) were conducted for the synthesis "of the enamine from the iminium intermediate by .ro reaction with the reducing agents of Table I, in accordance with reaction scheme III.

0 t r f S,,Example 2 :Reduction of Iminlum Intermediate (Formula VI) with J1 l-Benzyl-l,4-dihydronicotinamide [Formula IX, R 15 benzyl,

R

1 6' R 18

R

19 and R 20 H; R 17

CONH

2 (Formula IX-A) Procedure A] To a stirred solution of iminium intermediate (VI, 100 mg) in degassed acetonitrile (5 ml) was added r29 i l-benzyl--1,4-dihydronirotinami'de (135 mrg, 0.63 nunol, 6 equivalents) under a positive atmosphere of argon (greater than 760 mm Hg) at room temperature (20 0

C.)

over a period of 5 hours. After this time, the reaction mixture, as monitorod by reverse phase HPLC (Waters Racdipl-Pak C 1 8 or CN cartridge, methano~l /1 2 0/Et 3 N soi:Vent sYstem), indicated complete cohivetsibn of VI to a mixture of enamine VIII and 3',41-dehydrovinblastine (VII) ih a ratio of 1:1 yield).

Example 3 Reduction of Iminium Intermediate (Formula VI) with 1-Benzyl-1,4-dihydronicotlnamide [Formula IX, R.

1 benzyl; 0 *R, 6 R 18 R 19 and R 20 4;H; R 17

CONH;

049 (Formula IX-A) Procedure A] 0 ~To a stirred solut 3n of iminium ifttermediate (VI, 100 mg) in methanol (5 ml) kept initially at 0 0

C

0for 0.5 hours was added dropwise or in portions, a solution of l-benzyl-.l,4,-dihydronicotinamide (56 mg, 0.26 mmol, 2.5 equivalents) in methanol (2 under a Spositive atmosphere of argon (greater than 760 mmn Hg) Sover a period of 5 hours. During this time thG solution was allowed to warm up to room temperature.

OHPLC monitoring, as in Examplel, indicated complete Sconversation of VI to a Mixture of enamine VIII and 3',41-dohydrovinblastine (VII) in a ratio of 1:1 4 yield).

00 0 0d Example .4 The mixture of enamine (VIII) and 31 r4 1 dehydrovinblas tine (VII) obtained as described in Example 2 abc-ve, was treated with excess sodium borohydride (500 mg) at 0 0 C. The reaction mixture, was then naade basic with NH 4 0H and extracted with ethyl acetate (3 x 200 The combined organic phase was dried over magnesiumn sulphate. The product obtained, aftel: removal of or Iganic sol-ent, was subjected to preparative thin layer chromatography on silica gel (meth anbl/et'hyl acetate as: 6luti-ng system). The produc,-,t *As showii to b'e mixEitu' of unreacted. 3 ',41d6h ?Abvinblastin6 (VIi), an'd the known coampounds 4'deo xov inbias tine R COOCH3) and 4 1-deoxo-4epiviiiblastine (XI, R ;-COOCH 3 The presence of the latter compounds provided unambiguous evidence for the striu~re 'of enainine VIII,.

Exam The6 procedure 6f Ek~.xple 4 was repeated, except th4 tEhe "nxtu~e obt ained in Example 3 was treated with A t 0 excess sodium borohydride instead of the mixture from Exarip;le 2. The pr~auct" was again shown to be a mixturea too of utireacted. 31,4 1-dehydrovinblastine (VII) and the o known compounds 4 1-deoxovinblastina R COOCH 3 and 41-deoxo-41-epivinblastine (XI, R= COOCH 3 The presence of the latter compounds provided unamLiguous evidence for t~Ie structure of enarnine VIII.

Example.6 4 Reduction of Iminium Intermediate (Formula VID with 4 carbonyl-2,6-DlmethyI-4-Phenyl-1 ,4 Dihydropyridine [Formula IX, R 15 R 17 1 and R 19 'COOCH 2 CH 3 R 16 and J 20 CH 3

R

18 =phenyl; (Formula IX-B) Procedure B1.

To a stirred solution o3f irninium intermediate (Vi 100 m4) in degass~d acetonitrile (3 ml) was added 3; 5-diethO'xycarI38nyl-2 6-dimethyl-.4-phenyl-1, 4aihjd:ropyridirie (264 mg, 8 equivalents) in ethanol (12 ml) under a positive atiosphere o argon (greater than 760 mW 11g) aind at atemperature of abotit -20 to aboutr N

I.'

The reaction mixture was ref luxed for 3 hours.

After this time, reverse phase HPLC analysis (as described above) indicated, -among other products, formation of enamine VIII and 3',41-dehydrovinblastine (VII) in a ratio of 1:1 (60% yield).

Example 7 Reduction of Iminium Intermediate (Formula VI) with l-Dipnenylmethyl-1,4-.dihydronicotinamide [Formula IX, diheylmehy; 16 1 R 18

R

19 and R 20

=H;

R 17

CONH

2 (Formula IX-C) Procedure CL.

To a stirred solution of iminium intermediate 100 mg) in degassed ethanol (6 ml) was, added 1d;Lphenylmethyl- 1, 4-dihydronicotinanide (Formula IX-C) S (7J mg, 2.5 equivalents) in methanol (6 ml) under a Spositive atmosphere of argon (greater than 760 mm Hg) and at a temperature of 20 0 C. the reducing agent being Sadded portionwise at zhe rate of 1 equivalent each mi.n. After this, reverse phase HFLC analysis (Waters Radial,-Pak C 1 8 or CN cartridge, methanol-1i 2 0-Et 3 N as Qasolvent system) indicated, among other products, Sformation o'f enamnine (VI II) and 3, 4' S:dehydrovinbl as tine (VII) in a ratio of 0.9:1 Syield) Example 8 Reduction of Iminium Intermediate (Formula VI) with l.-BenzY1 -3-cyano-l,4-dihydropyridine [Formula IX, R 1 benzyl; I15 jR 16 1 R 18

R

19 t and R 2 o H; R 17

-CN;

(Formula IX-D) Procedure 0).

To a ptirred solution of iminium intermediate (VX, 3,00 mg) in degassed methanol (6 ml) was added Ibenzy>-3-cyano-l, 4-dibiydropyridine (Formula XX-D) ,(206 mg, 10 equivalents) in methanol (10 ml) under a r 4 19 positv atmosphere of (greater than 760 mmn Hg) argon and at a temperature of 20 0 C. the reducing agent being added portionwise at the rate of, 1 equivalent each min. After this, Zeverse phase HPLC analysis (as described above) indicated, among other products, formation of enamine (V III) and 3',4' dehydrovinblastine (VII) in a ratio of 1: 1 (40% yield).

Example 9 Reduction of Iminium Intermediate (Formula VI) with 1-ez l14dhdoio iy -21c raolyr ldnl -md ,Formula IX, R 1 benzyl; R 16 R 18 R, 9 and *kO2 H; (2 carbamoyl pyrro i d ifnl carbonyl; Coo "Formula IX-E) Procedure El.

To a stirred solution of iminium intermediate 100 mg) in degassed methanol (6 ml) was added 1- Ienzyl<, .4-dihydronicotinyl 2 1carbam'ylyrrolindinyl)-amide (Formnula IX-E) (163 equivalents) in methanol (5 ml) under a positive pressure of argon (greater than 760 mm Hg) and at a ,,,,temperature of 200C., the reducing agent being added OOportionwise at Lt e rate of I. equivalent each 30 min.

*.After this, reverse-phase EPLC analysis (as described ~above) indicatedf among other products, formation' of 0 -6namnine (VIII) and 41-dehydrovinbJ.astiae (VII) in a 00 00Yatio of 1.1:1 (60% yield).

2xample 1leduction of Iminium Intermediate (Formula VID with 1 ,4-Dhydro-1-(1--metho) ycarbonyI 1 sobutyl)-nicotinamide [Formula IX, R 1 1methoxycarbonyl isobutyl; R1,and R 20 H; R 17 ICONH 2 (Formula IX-F -Procedure F).

Mz7

IL

.L -i (L a. a a a.a a aa a 9 a a a a.

a, a a: a aaaa a a a aaaa aa a a a a a ao aaaa) a a a a. a ao a a a aa Qa a a a a a a aI To a stirred s6liutioh of iminium intermediate (VI, 100ob mg) in degassed methanol (6 ml) was added 1,4dihydro-1- 1-methoxy carbonylisobityl) -nicotinamide (Formula IX-F) (150 ing, 6 equivalents) in methanol (6 ml) undbr a positiVe Pressure of argon (greater than 760 mm Hg) and at a temperature 6f 20 0 the reducing ageit beixig added portionwise at the rate of 1 equivaleiit each 30 min. After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 31,4'dehydrovin-blastine (VII) in a ratio of 2:1 yield); Examle -i Reduction of Iminium Intermediate (Formula VI) with 61-Tetraacetyl-(beta)-D-glUtopyranosidyl)- 1,4-dihydronicotinamilde EFormula IX, R 15 (2,,46 Tetraacetyl-(beta)-D-glucopyranosidyl; R 16

R

18

R

19 and R 20 H; R 17

CON

2 (Formula IX-G) Procedure GL To a stirred solution of iminium intermediate (VI, 100 mg) in degassed methanol (6 mi) was Adaded 1- (2 4' 6 '-Tetra-acetyl- (beta) -D-glycopyranosidyl) 1,4-dihydronicotinamide (Formula XXX) (238 Mg, equivalents in methanol (10 ml) under a pot tive atmosphere 6f argon (greater than 160 mm Hg) ad at a temperature of 20 0 the reducing agent being added portioinwise at the rate of 1 equivalent each 60 min.

After t1is, reverse-phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 4' -dehydrovinblastine (VIt) in a ratio of 1.5:1 (70% yidld).

21 Example 12 Reduction of Iminium Intermediate (Formula VI) with 1 ,4-D ihyd ro-lI-(2'me thoxy car bonyl IIsopropylI) -n Icot in am id e [Formula IX, R 15 21-methoxy carbonyl-isopropyl;

R

16

R

18

R

19 an 20 H; R 17 CONH 2 (Formula IX-H Procedure HJ.

To a sti-rred solution, of iininiumi ntermnediate 100 mg) in degassed me~thanol (6 ml) was added 1,4dihydro- I- (P '-mphoxy-ca,bony~isQpropy.) -nicotinamide (;Formu~l~a IX-H) (82 mg, 3.5 eg- iivalents) in mnethanol (7 ml) under a posit.,ve atmosphere of argon (greater th4,n 760 mmn Hg) acrdi at a temperature of 20 0 the reducing agent being added portionwise at the rate of ,,:one equivalent each 30 inm, After this, reverse-phase HPLC analysis (as described above) indicated, among other products, formatlon of enamine (VII11) and 3 4- .**dehy4i;oinblas tine (VII) in a ratio of *yield) Example 13 Reduction of Iminlum Intermediate (Formula VI) with 1 'l4-Dihydro-lWl I 21-dimethoxy carbonyl ethyl )-nicotinamide 4 Formula IX, R 1 l t ,1 -dimothoxy carbonyl ethyl; .0R 16 R 8

R

1 20 H RI 7

"CONH

2 4 4 :(Formula IX-I ?rocedure 2.

To a solutio4 of iminium intermed.Vate (VI, 100 m, 4 4 ig) 4~n degassed miethanol (6 mil) wks added 1,4-dihydro- 1-dimethoxy carbonyl ethyl)-nicotinamide (2Formnula IX-I) (148 mg, 5 equivalents) in methanol ml) under a positive atmosphexe of argon, (greater than 760 mm Hg) and at a temperature of 20OCi, the reducing agent bei~ng added portionwise at the rate of I.

equivalent each 30 min. After this, reverse-phase HPILC analysis (as described. above) indicated, among other, 22 pro'diicts, f ormation of ehamine (V'III) and 3 ,4' dehydroviilblastifie (V.11) in a ratio of 1.1:1 yield).

Example. 1.4 Reduction of Iminium Intermediate (Formula VI) with 1,47pihydro -1(oimiouy abxlt)nctnmd [Formula IX, R 15 =sodium-isobutyl-l-carboxylate; R1 6

R

18 1 R 19 and R H; R CONH 2 (Formula IXJ-3 Procedure J1.

To a solution of iminium intermediate (VII 100 mg) in degassed methanol (6 ml) was added 1,4-dihydro- 1 (sodium-isobutjl-l-carboxylate) -nicotinamide (Formula IX-J) (1Omg, 5 equivalents) in methanol (6 iTl) under a positive atmosphere of arqr ,n (greater than 760 mm Hg) ~~and at a temiperature ot 20 0 the reducing agent being Sadade~ poitionwi.&e at the rate of I equivalent each :min. After this, reve:ise. -phase HPLQ analysis (as ,described above) indicated, among' other products, ~formation o f enamine (I II) and 3',4dehydrovinblastine (VII) in a ratio of 2.2:1 yield).

Reduction Qf Iminium Intermediate (Formula VI) with 1 ,4-Dihydro -1-(.sodiUm-isobuty1-carboxyate)-nicotinamide tFormula IX, R 15 sodium-isobuty!-l-.carboxylate; R 16 0* 20 H RCN J R 18

R,

9 and R 2

;R

17

-CN

2 S(Formula IX-J at loW temperature Procedure K), To a solution 6f iininiwn, intermediate (VII 100 mg) in degassed metfiano2. (6 mlJ.) at -20 0 C *~as added 1,4dihydro-l- s odium- isobutyl- 1-carboxylate) -nIcotinaxnide (Fomu2la IX-JT) (155 mg, 6 equivalents) in methanol (6 ml) under a positive atmosphere ot argon, (greater than 36 carried ouit Within the fO11o'wind 4 2.3 mm Hg) the reducing agent being ad1ded in one p O:tion. After 45 min. at t1~s temperature, reverse- 1phase HPLC analysis (as described above) indicated, among other products, formation of enamine (VIII) and 3 1,4 1-dehycdrovinb~astine (VIIX) in a ratio of 3.2;1 yield).

Example 16 The procedure of Example 15 was repeated, except that the reaction was carried out at -40 0 C. After minutes, enam 'ine (V 'III) and 31,41-dehydrovinb],astine (VII) were obtained in a. ratio of 4.2:1 (85% yield).

The prgcess of this example is designated, as Procedure Results 94 Examples 2-1,6 are summarized in Tables 2 and 3.

4 "0 41 a0 0V 0 to 4 a 4 S S 0 0 4

S

o 40s a 4 a a 0 4 a a p *00 S Table 2 Effect of Reducing Agent on 1,4-vs. 1,-2-Reduction of Iminium vi Examiple Reduction 1,4-1,'2-Reduction Yield?(%) itumbers Procedure Productsi 2,3 6 7 8 9 11 12 13 14 16 0.:1 -1:1 2.3:1 2.2:1 2.2:1 3.2:1 1. By reverse phase HPJIC quantitatton.

2. Combined 1, 2-reduction 4-dehydrovinbiastine, 1, 4-reduction. (enamine VIII) products.

VI) Efec of Teprtr on l,'-vs 1,-euto of .,V vs a. ato a Effect o Tepaueductions Tep,2-R. 1 :,-eduction Yiel (1)numV Numbers Procedure (OC) Products 2.2:1 3.2:1 -4.2:1 1. Quantitation by HPLC.

2. Combined 1,2-reduction (3,4-dehydrovinblastine.,VII) 1,4-reduction'(enamine VIII) products.

MMIMMO -AM 26 Table 2 indicates that the reduction procedures (Procedures I and J as in Examples 13 and 14, respectively) employing reducing'agents of Formula IX-I and Formula IX-J afforded the best yields of the enamine (Formula VIII) at 20 0 C. Table 3 shows the effect of temperature in the reduction of the iminium intermediate (Formula VI) using the reducing agent of Formula IX-J. From Table 3, it is apparent that lowering the reduction temperature to -40 0 C (Procedure L, Example 16) from -20 0 C. (Procedure K, Example resulted in an increase in the ratio of the 1,4reduction product, versus the 1,2-reduction product, as well as an increase in the overall yield. The results for procedure J (lesser yield and lesser 1,4-reduction product) are consistent with the results for procedures K and L even though procedure J used 5 equivalents of reductant as opposed to 6 equivalents of reductant as used in procedures K and L.

In summary, increased yields of the enamine (Formula VIII) are afforded by employing the 1,4dihydropyridines of Formula IX wherein Rij is'an Selectron-rich substituent, such as carboxylic esters and carboxylic salts in the 1,4-reduction of the S iminium intermediate (Formula VI). The reduction is best conducted under cover with inert conditions such as argon, at a low temperature in the range from about -60 0 to about +60 0 preferably in the temperature range of from about -600 to +20 0 and most preferably to about -20 0

C.

The enamine (Formula VIII) formed in the above e, reduction step may be used directly for subsequent reaction or may be isolated by various chromatographic l, 0* techniques, for example, the enamine may be isolated by applying the reaction mixture from step directly onto an appropriate chromatographic system such as a I column, thin layer or high performance liquid chro.satographic system. Preferably reverse phase and/or gel-permeation chromatographic separation 27 inethods dr employed. Thei~ emperatuire bf the' isolation procedure may vary from about 4 0 C to room t6mperature.

Alternatively, volat ile reagents and solveHE's present in the reaction mixture are removed under reduced pressure and temperature, preferably blelow -10 0 C. The resultant r esidue ciifi be purified by the chromnatographic meth6as de c'ibed abo-vd befor further characterization or tranisformat idn.

Treatment of the enamiine VIII with alkali metal borohydride (NaBH 4

KBH

4 LiBH 4 in accordance with known 'procedures produices the 4 '-deoxoxvinl&lastine compounds (Fbrniula X, R=COOCH 3 and 4'deoxo-4'-epivinblastine compounds (Formula XI, R=COOCH 3 p pp p i p ~p p p p Q a .4 0 o 0 p pp p 0 However, under: oxidative conditions of the ,,,,resent robess step the enamine VIII can be ,o,,transformed to a second iminium ihtermediate as represeneda by Formalae XVI ana XVIa.

0 0* V4Y 28 R 1 OO R 1 I N R N N CH0 OC-CH3 CH0 CH3 CHO H3O II II I C P 0 CHR 0

R-CO

2 CH3 xv R=CO 2 CH3 Oxidative procedures that are useful for converting the enamine (Formula VIII) to the second iminium intermediate (Formulae XVI and XVIa) include, for example: controlled aeration/oxygenation; addition of flavin coenzymes (Formula

XII):

#0 b oo 000100 S o* [riboflavin, Formula XII, R H; flavin mononucleotide (FMN) Formula XII, R P032- flavin adenine Sdinucleotide (FAD), Formula XII, R (P03)2 2 adenosine] followed by controlled aeration/oxygenation; addition of the reduced form of the flavin coenzymes (Formula XIII): 0r0 I i i 29 N

XIII

0 [dihiydrori'boflavin, Formula XIII, R dihydroflavin, mononucleotide (FMNH2), Fbrmula XIII, R=P02dihydroflavin adenine dinucleotide (FADH 2 Formula XIII, R (P0 3 2 -adenosine],, followed by controlled aeration/oxygenation; 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, followed by controlled aeration/oxygenation; additI'Sn of flavin coenzyme afialogues having the isoalloxazine structure as represented by Formula XIV; wa aV 0 6 6 rersne Foml XV 1 where RI, R 2 and R 3 independently, can be alkyl, substituted alkyl, aryl and substituted aryl functions, followed by controlled aeration/oxygenation; 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; addition of peracids as represented by the Formula R-C03H, where R can be an alkyl, substituted alkyl, aryl or substituted aryl functions; addition of superoxides; addition of a hydroxyl radical (OH) generated in a variety of ways, for example, by the use Sof hydrogen peroxide in the presence of ferrous ion; or (11) addition of a metal ion which is a good electron acceptor, for example, ferric ion (Fe+ 3 cupric ion (Cu+ 1 1 mercuric ion (Hg 2 2 and silver ion o followed by controlled aeration/oxygenation.

0 The oxidative procedures and described above, are conducted in organic solvents such D* as alcohols, acetonitrile or higher members of this series; dimethyl sulfoxide; dimethylformamide: 'rarious ethers such as dioxane, tetrahydrofuran; and aromatic ao hydrocarbons such as benzene, toluene, etc.

The oxidative procedures involving flavin coenzymes (conditions and require an aqueous buffer (for example, phosphate, Tris HC1, MES buffers) at pH 5-9, but preferably in the range 6-8, as solvent. An organic co-solvent, e.g. alcohols; 31 acetonitrile or higher members of this series; dioxane; tetrahydro-furan; dimethyl sulfoxide; or dimethylformamide can be used.

The progress of the oxidative procedure 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 -600 to about +60 0 C. and preferably from about 4 0 C. to room temperature. The reaction time may vary 'from several minutes to several days depending on, for example, the temperature and the particular oxidative conditions. The reaction is generally conducted at atmospheric pressure.

In an alternate embodiment, the enamine solution obtained from the 1,4-reduction of the iminium intermediate (Formula VI) is initially diluted 5-50 fold by the same solvent used in the reduction, at a low temperature (0OC to -7 0 The diluted enamine solution is then oxidized (step to the second iminium intermediates (Formulas XVI and XVIa) by one of ~the oxidative procedures described hereinabove.

g eo Among the prefered oxidative procedures employed Sin the present process are: t 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; as in step but with addition of ferric S chloride; 0 C as in step but with the addition of a flavin coenzyme, as represented by Formula XII, to generate, in situ, the corresponding dihydroflavin coenzyme, as represented by Formula XIII; or S(d) as in step but with the addition.of hydroge peroxide and/or hydroperoxides as represented 32 by the Formula R-OOH where R is alkyl or a'ryl.

The oxidative procedures ai 6niicted in organic solvents such as alcohiols; acetonitrie or higher members of this series; dinietehl sulfoxide; dimethylformamide; ethers such as dioxane; tetxahydrofuran; aromatic hydrocarbons such as benzene, toluene, etc. An aqueous buffer phosphate, Tris-HCi, ME'S 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 about -600 to about +60 0

C.

Various parameters for the oxidative trans formation step have been stUdied to optimize the yield of the target compounds, particularly vinblastine (Formula The results of these studies have been illustrated in Tables 4-7, as follows: V 4 0 14 to 4 li-i 33 Table 4 Effect of Ferric Chloride on Production from Enamine V111 3 of Vinbiastine Am~ount of FeC13 Oxidation Conditions Yield of (9quivalents) (Temp., time) Vinblastinp 2 0 0 C, 5 mein 0 1Airl, 0 0 CI 5 min 13.3 2 Air 1 0 0 C, 5 mein 19.0 3 Air', OOC, 5 min 10.4 1. At a rate of 60 mi/rein.

2. By reverse-phase HPLC quantitation, after reductive work-up with NaBH 4 3. Enamine VIII generated at -40 0

C

Procedure L) Table Effect of Time of Oxidation on Production of Vinbiastine from Enamine V111 3 Time' (mein) Yield of Vinblastine 2 *4 9 k 4 94 4 9 9, 4 444 0@49 9 .4,5, p. a a.

*4 @9 .9 '5 4 *4 pp 55 1 94 @9 4 4 4* 44 44* 4 8.2 15.4 15.5 15.7 Reaction conditions: -2eq. ferric chloride addod, air bubbled through the solution. at 60 rel/rin at OOC.

2. By reverse-phase TIVLC quantitation after reductive with NaBH 4 3. Enamine VIII genevated at -40 0

C

(Procedure L).

34 Table 6 Effect of Oxidation Temperature of Producti~ft of Vinbiastine from Enazuine VIII-3 Temp., OCi Yield of ',.r:nblas tine~ 3.7 -23 6.2 0 19.6 20.6 16.0 1. Reaction conditions: 2eq. ferric chloride added, air bubbled through the solution at 60 m..I/nun for min.

2. By reverse-phase HPLC quantitation after reductive work-up with NaBH 4 3. Enamine generated-at -40 0

C

(Procedure L).

Table 7 Effect of Dilution on Production of Vinbiastine from Enamine V111 3 4. 00 .0 000*0 0 0 0 00 000 0 4 Dilution Factdr 1 4 %Yield of Vinblastine 2 1 19.6 5 25.2 30.1 29.6 50 24.7 1. Reaction conditions: 2 eq. ferric chloride added, air bubbled through the solution at 60 ml/min for 15 min. at

QOC.

2. By reverse-phase HPLC quantitation after reductive work-up with NaBH4.

3. Enamine generated at -40 0

C

I ProcedurecL).

41. D lution Factor 1-100 mg Iminium VI in 6 ml methanol to which reducing agent Formula IX-J (6 eq.) in 6 methanol was added. (Total volume 12 ml) Dilutionl Factor 5 Tot~l volume of ml; Dilution Factor 10 Total volume' of 120 ml; Dilution Factor 20 aTotal volume 240 ml; Dilution Factor Total volume of 600 ml..

Table 4, which indicates the effect of ferric ch.soride concentration on the yield of vinblastine shows that the yield first increases and then decreases with increasing ferric chloride concentration. Maximum vinblastire yield is seen to occur at a ferric chloride concentration of about two equivalents, Table 5, which sets forth the results relating to the yield of vinblastine versus the time of oxidation, indicates that the maximum yield of vinblastine is reached after about 5 to about minutes of aeration in the presence of 2 equivalents of ferric chloride and that the yield significantly decreases with an oxidation time of 45 minutes.

Table 6, which sets forth the results of various oxidation temperatures on the yield of vinblastine showo that a temperature in the range of from about 00 to about 20 0 C. provides the highest yield of vinblastine after a reductive work-up with NaBH 4 as quantified by reverse-phase HPLC.

Table 7, which sets forth the effect of dilution of the enamine (VIII) solution on the production of vinblastine shows that a dilution factor of about S 5 to about 20 on the enamine (VIII) solution before aeration in the presence of ferric chloride (2 equivalents) at 0oc., affords the best yield of vinblastine as quantified by reverse-phase HPLC after reductive work-up with NaBH 4 Summarizing the results set forth in Tables 4-7, in tho oxidative transformation (step of the 99 o enamine, the dilution factor of the enamine solution obtained in the 1,4-reduction (step of iminium intermediate (rormula VI), by the same solvent used in the reduction, is preferably in the range of 5 to fold and most preferably about 10 fold (8 to 12 fold).

The dilution procedure is conducted at a low t* temperature (from about 0O et about -70 0 preferably below about -40 0 C aAd under inert cover such as argon.

The oxidative transformation of the enamine is best 36 carried out within the following parameters: (1) aeration at about 60 mi/mmn for 5 to 20 min~utes, and preferably about 15 minutes; at a 'temperature in the range from about 00 to about 20 0 C. and preferably about 20 0 C, in the presence of about 2 equivalents of ferric chloride, to afford the corrQeyconcU second iminium intermediates (Formulae XVT and XVlt), Reduction of these second iminium intermediates (Formulae XVI find XVIa) in step by reaction with an alkali metal borohydride (NaBH 4

X(BH

4 1 IiBH 7 etc.) lead to the target compounds vinblastine and leurosidine, and the by-products 31,41-dehydrovinbiastine (Fo;rmula VII) leurosine (Formula XVII), catharine (Formula XVIII), vinamidine (Formula XIX) and the reduction product of vi'namidine (Formula XXC).

NN

C~aCO NW Mcc Oft 6N g 00 *d O4#@ H A aI

ACMO

00 9 0 4 37 The reduction is carried out in suitable solvents (organic and inorganic), such as those described above in the oxidative transformation step The reduction is conducted at low temperature, in the range from about -20 0 C to about 4 0 and preferably at about 000. and at a pH lower than 8.5, and preferably between about 7.5 and about 8. T, total reaction mixture may then be concentrated in vacuo at a low temperature in the range from about 00 to about 10 0 C. before extraction and isolation of the target compound.

The following examples (Examples 17-21. methods describe the synthesis o vinblastine by oxidation of the -,'amine (Formula VIII).

Example 17 Synthesis of Vinblastine (Formula I) by Oxidation of Enamine (Formula VIII) to Iminium Intermediate (Formula XVI) with Flavin Mononucleotide (FMN Formula XII R PO3 2 Method 1.

To a stirred reaction mixture containing 100 mg °o of the enamine (VIII) obtained as described above (Example 2, Procedure from the iminium intermediate S" VI there was added FMN (80 mg, 1 equivalent) dissolved in Tris HCL buffer (2 mi) under a positive atmosphere igreater than 760 rn Hg) of argon. The solution was kept in the dark at room temperature (20 0 for 16 hours. After this time, the inert atmosphere of argon was replaced by air and the reaction mixture was j stirred for another 2.5 hours. Reverse phase HPLC S analyses indicated transformation of the enamine VIII to the iminium intermediate XVI as well was to other by-products. Sodium borohydride (500 mg) was added at OOC. and the r'sction mixture was made basic with S* and extracted with ethyl acetate (3 x 200 ml). The combined organic extract was dried over magnesium sulphate and the solvent was removed in vacuo to 38 provide a crude product (85 mg). Purif ication of the crude product by thick layer chromatography (silica gel, methanol; ethyl acetate 1:5) allowed the separation of the following dimeric products: viixblastine (Formula I, 22 mg, 30,41dehydrovint~lastine (Formula VII, 16 mg, leurosine (Formula XVII, 8 mg, catharine (Formula XVIII, 7 mg, vinamidine (Formula XIX, 5 mg, and the redu(..,-ion product of vinarnidin~e (Formula XX, 19 mg, Example 18 Synthesis of Vinblastine (Formula I) by oxidation of the Enamine (Formula VIII) with Hydrogen Peroxide to the Iminium Intermediate (formula XVI) Method 2.

To a solution containing 100 mg of the enamine (VIII) obtained from iminium intermediate VI (4xample 2, Procedure A) there was added hydrogen peroxide 1.2 ml, 95 equivalents under an inert atmnosphere-of :argon. The reaction mixture was stlirred at room 2temperature for 5.5 hours when reverse phase HPIJC analyses indicated complete conversion of enamine VIII.

a 0 S Sodium borohydride (500 mg) was added at 0 0 C. and the w~4' u-Ing solution was extracted with ethyl acetate (3 x 200 ml). The combined organic extract was dried over magnesiumn sulfate and removed in vacuo. The resulting product mJixtnure was separatect by thxipk layer chromatography (silica gel, methanol /ethvyl acetate) to give the following dimeric alk~aloids: vinblastine (I, Q q* 4 mg, 31,41-dehydrovinblastine (VII, 5 mg, Ot leurosine (XVII, 13 mg, catharine (XVIII, 5 mg, the rzduced form of. vInamidine (XX, 30 mg, 27.6%) %0 00 39 Example 19 Synthesis of Vinblastine by Oxidation of the Enamine (VIII) with Air to the Iminium Intermediate (Formula XVI) Method 3.

A Zolution containing 100 mg of the enamine (VIII) obtained from the iminium intermediate VI, (Example 2, Procedure A) was stirred in open air at room temperature for 3 hours. After this time, sodium borohydride (500 mg) was added at OOC. and the reaction mixture was made basic with NH40H and extracted'with ethyl acetate (3 x 200 ml). The combined organic extract was d,'ied over MgSO 4 and removed in vacuo. The resulting crude product was separated by thick layer chromatography (silica gel, methanol/ethyl acetate) to give vinblastine 4 mg, Example Synthesis of Vinblastine by xidation of the Enamine (Formula VIII) with Air in the Presence of Ferric Chloride, to the Iminium Intermediate (XVI)- Method 4.

o 0 *00o" To a stirred solution containing 100 mg the 00 enamine (VIII) obtained from the iminium intermediate VI (Example 2, Procedure A) there was added ferric chloride (1 equivalent) and air bubbled through the °solution at OOC for a period of 0.5 hours. Sodium 0 a, borohydride (500 mg) was added at OOC. and the reaction S mixture was made basic with NH 4 0H before extraction with ethyl acetate (3 x 100 ml). The combined organic extract was dried over Na2SO 4 and the solvent was removed in vacuo. The crude product was purified by S thick layer chromatography (silica gel, methanol/ethyl acetate) to give vinblastine 37 mg). Basedon enamine (50 mg) present in the mixture, the yield of i

L

vinblastine was Example 21 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 A solution containing 200 mg the enamine (VIII) obtained ,rom the iminium intermediate VI (Example 16, Procedure L) was diluted five-fold with methanol before oxidation (total vol.: 120 ml). Ferric chloride mg, 2 equivalents) was then added, and air was bubbled through the solution, at UdC., for 20 min. Sodium borohydride (200 mg) was then added, and the solution was concentrated in vacuo before adding water i100 ml) and extracting with ethyl acetate (3 x 200 ml). The combined organic extract was dried over Na2SO4 and the solvent was evaporated in vacuo. The crude product was 'purified by column chromatography (silica gel, TLC grade, 15 Elution with ether: chloroform (10:7) Sgave 3',4'-dehydrovinblastine (VII, 18 mg, 11%).

Further elution with ether:chloroform:methanol oo o (10:7:0.5) gave vinblastine 62 mg, 37%).

o ;For practical purposes, isolation of the intermediates VI, VIII, XVI, XVIa) is not required and the entire process for the indole unit (Formula III) and the dihydroindole unit (Formula IV) is preferably conducted in a one-pot operation as Sillustrated in Example 22.

44 *4 994 4 f 41 Example 2.2 One-Pot Conversion. of Catharanthine (Formula III, R1, R2, R3t and R 4 H, R COOCH 3 and Vindoline (Formula III) to Vinblastine (Formula I) and LeuroEidine- Overall Procedure.

To a solution of catharanthine (500 mg, 1.5 mmol) in dry dichloromethane (4.5 ml) at -15 0 C under a positive atmosphere (greater than 760 mm Hg) of argon there was added m-chl6roperbenzoic acid (330 mg, 1.9 mmol) in one portion, and the mixture was stirred at- 100 to -15 0 C. fo 5 minutes. After this time, the reaction mixture was cooled to -40 0 C. and a solution of vindoline (IV, 450 mg, 1 mmol) in dry dichloromethane (1 ml) was added, followed immediately by trifluoroacetic anhydride (1 ml, 7.1 mmol). After 2 hours at -60 0 C. volatiles were removed in vacuo (high vacuum pump) and dry, degassed methanol (12 ml) was added after flushing the system with argon. The resulting orange solution was cooled to -40 0 C and-a solution of 1,4-dihydro-l-(sodium-isobutylcarboxylate)-nicotinamide (Formula IX-J) (1.5 g, 6 mmol) in dry degassed methanol (12 ml) was added under S0" 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 solution between about -50 to about 0 0 C. Ferric chloride (330 mg, 2 mmol) was then added 1 o and dry air was bubbled through the solution at a rate of about 60 ml/min for a period of 20 min. Sodium borohydride (1 g) was added and the solution 0. 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 S Na 2

SO

4 and the solvent was evaporated in vacuo to give S the crude product which was purified by chromatography as previously described to give 3',4'-dehydrovin- 42 'blastine (VII, 95 mg, vinbiastine (315 mg, 39%) and leurosidine (130 mg, 16%).

In summary, the present 'invention significantly differs form the prior art in several important steps, specifically the characterization of the unstable, intermediates V, VI; VIII, XVI and XVIa. Theseintermedialtes can be isolated, but isolation is not essential- and the entire process, monitored carefully for said intermediates, can be conducted in a one-pot operation, as illustrated in reaction scheme IV.

Reaction Scheme IV 0 o 04 9 t9 o 9

(CF

3

CO)

2 0 -1500 C CH3

CH

3 R C Cz CH 3

O~(

3

CONH

2

N

1,2-Reduction Ratio 4.2: 1 Vill Enamine Anhyd rovin bla stine A 9- 43 Reaction Scheme IV continued IMPROVEMENTS TO PATENT: C0 2

CH.

'a 0 0 a 0*0 FeC19 ml/min, '15 min Dilution Factor I CH3

X'VI

XXXV

Vinbiastine Leurosidine Overall Yield: Vinblastine Leurosidine Anhydro~vinblastine (18 A47S 44 The principles, preferred embodiments, and modes of operation of the present invention have been described in the foregoing' specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded ,as illustrative rather than restrictive.

Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

4 o S9 4 e *14 MMWMM.W

Claims (17)

1. A process for the production of dimer alkaloid compounds represented by the following formula: I CO-R2 RI R 4 O-R 3 wherein: o 4r t 0 4 4 0O o 0) 0 0 4 4 Ct C9 4 .4 00 ,44 4 4 04 4 44* 4 04. CH 3 or (CH 2 )n CH 3 where, n CH 3 or CHO; H or CO-alk; H; COO-alk or CONR 13 R 14 wherein R 13 and R14 are selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl or subsztitu-ted aryl;-j or- otr'/ -CH=CH- or -CH 2 -CH 2 SII or IIa; Ia. and wherein, R, R7 R 9 R 10 R 1 1 R12 H or COO-alk; H, OH, 0-alk, H, OH, 0-alk, H, OH, 0-alk, H or COO-alk; H or alkyl, OCO-alk or alkyll OCO-alk or alkyl; Oco-alk; and which comprises the steps of: 4 46 forming an N-oxide derivative represented by the following formula R, 3 N Rq H R in the cold, at a temperature from about -77°C to about +40 0 C from an indole unit having a bridge nitrogen, by oxidizing the bridge nitrogen and without isolating said derivative; op treating said N-oxide derivative in the presence of at least one member selected from the group consisting of acetic anhydride, halogenated 0 acetic anhydride, aid acetyl chloride, to effect a Polonovskt- type fragmentation reaction; without isolating the product of step stereospecifically coupling said product of step 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*C to about +40 0 under Inert conditions, to form a first Imlnlum intermediate; reducing said first iminium Intermediate by reaction with a 1,4-dlhydropyridine compound, thereby forming an enamine Intermediate; S0 preparing a second iminlum intermediate by oxidative transformation of said enamine intermediate obtained in step under controlled aeration conditions; and reducing the product obtained in step to form the target dimer alkaloid compounds.

2. The process according to claim 1, further Including the step of diluting said enamine obtained in step by a factor of 5 to 50 with a solvent fold prior to performing step KEH/173f I"' Fv\ A^C* C^?VL 1 1 46A

3. The process according to claim 1 or claim 2, wherein the 1,4-dihydropyridine compound that is used in step to reduce said first iminium intermediate is represented by the following formula: H R1 8 R19 Ri17 N R 16 RIS wherein R 1 5 is H, alkyl, arylalkyl, diarylalkyl, alkoxy, alkoxycarbonyl, alkoxycarbonylalkyl, dialkoxycarbonylalkyl, alkali metal salts thereof, Saryl, lower alkyl carbonylic esters or salts thereof, carbonylic acids, and sugar units; E0 aa ao a K 3 u Ut KEH/173f lu-- r 47 R 16 R 18 and R 20 independently, are H, alkyl or aryl; R 7 is H, alkyl, carboxylate and salts thereof, aryl, cyano, CONR 21 R 22 wherein R21 and R 22 independently are H, alkyl, aryl, or taken together, R 21 and R22 form a ring structure containing up to four carbon atoms, said ring structure further substituted by CONR 23 R 24 wherein R3 and R24 are H or alkyl; and R 19 is H, alkyl, carboxylate and salts thereof, and aryl.

4. The process according to claim 3, wherein R17 and R19 are carboxylate. The process according to claim 3, wherein R1 is CONR 21 R 22 and wherein R 21 and R 22 independently is H, alkyl, aryl, or taken together, R 21 and R 22 form a ring structure containing up to four carbon atoms, said ring structure further substituted by CONR 23 R 24 wherein R 23 and R24 are H or alkyl, o o 6. The process according to claim 3, wherein R 15 is an 0 0 15 electron-donating substituent selected from the group consisting of alkyl, aryl, carboxy, carboxylate and salts thereof, and sugar units.

7. The process according to claim 6, wherein R is selected from o o. 15 the group consisting of Ic er alkyl carboxylic esters and carboxylate salts,

8. The process according to claim 7, wherein R 15 Is 1',2 1 dimethoxy carbonyl ethyl.

9. The process according to claim 7, wherein R 15 is ad. sodium-isobutyl-l-carboxylate. The process of any one of claims 1 to 9, wherein the reduction of step is conducted in an inert atmosphere at a temperature in the aoo* range from about -60 0 C to about +60*C. in the presence of at least one solvent selected from the group consisting of lower alkyl alkanols, acetonitrile, dimethyl sulfoXide, dimethylformamlde, dioane, 0* oo tetrahydrofuran, and chlorinated lower hydrocarbons,

11. The process according to claim 10, wherein the reduction of step S(d) is conducted at a temperature in the range of about -20° to about -60 0 C.

12. The process according to any one of claims 1 to 10 wherein the oxidative transformation step is selected from the group consisting of: 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; r, r Of 0? t~ 0 0 000001 0 0 0 0* o 0 0 000 0 4*04 o 0 0 0*40 I 4 00 40 *0 Vt 0 >0 4 Vt U 0 1 Vt Vt Li Vt Vt Vt Vt Vt *0 Vt Vt 04 0 4 Vt 0 4 i~4 Vt VtVtVt 00* 4 4 I Vt 44 4 Vt 4 0 ~Vt 44 4 0 Vt 4 Vt 40 48 controlled aeration/oxygenation in which a solution of said enamine and a metal ion, selected from the group consisting of ferric ion cupric ion (Cu+ cuprous Ion mercuric ion (Hg 2 and silver Io (Ag is stirred in open air or with a stream of air/oxygen bubbled through the solution; 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; 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, wherein the flavin coenzymes generates, in situ, the corresponding 1,5-dlhyroflavlng coenzyme; 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 aeratlon/oxidation being conducted in an organic solvent at a pH of 5-9 and a reaction temperature of about -60" to about

13. The process according to claim 12, wherein the oxidative transformation step is conducted at a pH in the range of 6-8, 14, The process according to claim 12 or claim 13, wherein about two equivalents of ferric chloride are employed in step

15. The process according to any one of claims 12 to 14, wherein the time of aeration in the oxidative transformation step is from about five to about twenty minutes,

16. The process according to any one of claims 12 to 15, wherein the oxidative transformation step Is conducted at a temperature in the range from about 0 to about

17. The process according to any one of claims 12 to 16, wherein said enamine obtained in step is diluted from about 5 to about 20 fold with a solvent, prior to performing step 18, The process according to claim 17, wherein said enamine Is diluted 8 to 12 fold, 19, The process according to any one of claims 1 to 18, wherein said reduction step Is conducted at a temperature In the range from about to about and at a pH between about 7,5 and about 8.5, and wherein r 49 the reaction mixture in step is concentrated in vacuo at a temperature between about 00 and about 10'C. before extraction and isolation of the target compound. The process according to any one of claims 1 to 19, ,,herein the reducing used in step comprises contacting the reaction product from step with an alkali metal borohydride selected from the group consisting of NaBH 4 KBH 4 and LiBH 4

21. The process according to any one of claims 1 to 20, wherein steps are conducted in a one-pot operation without isolation of any intermediate products.

22. The process according to any one of claims 1 to 22, wherein at least one of the intermediates formed in steps and is isolated S prior to being further reacted. ,23. The process according to claim 22, wherein all of said S ntermediates are isolated prior to being further reacted.

24. A process according to any one of claims 1 to 23, wherein the target compound is vinblastine. 444* A process according to claim 24, further comprising the step of oxidizing said vinblastine to obtain the target cceipound vincristine.

26. A process according to any one of claims 1 to 23, wherein the target compound is leurosidine. 0. 27. A process for the production of dimer alkaloid compounds, which process is substantially as herein described with reference to Example 2 a and Example 17, Example 2 and Example 18, Example 2 and Example 19, Example 2 and Example 20, Example 16 and Example 21, or Example 22, 28, A dlmer alkaloid compound of Formula I as defined in claim 1 S whenever prepared by p process according to any one of claims 1 to 27. 0 .O t DATED this TNENTY-SEVENTH day of DECEMBER 1990 The University of British Columbia Patent Attorneys for the Applicant SPRUSON FERGUSON