FI80462C - FOERFARANDE FROSTAELLNING AV DIMERA CATHARANTHUS-ALKALOIDER. - Google Patents

Description

1 ε O 4 6 2

Method for the preparation of dimeric Catharanthus alkaloids Förfarande för framställning av Dimera Catharanthus alkaloider

The present invention relates to a process for the preparation of dimeric Catharanthus alkaloids of formula I

»· £ ** OH

a A 'V / \ (' I 18 / <n

I *, * \ J] iiH

Η, οοΛτ ^ Ν ^^ ΟΑο R is COOCH3 wherein R is CH3 or CHO, or derivatives thereof.

The dimeric alkaloids of the formula I described above, vincristine (R = CHO) and vinblastine (R = CH3>) are used in the treatment of cancer as such or in the form of various derivatives.

These compounds have not been economically synthesized but have been produced by extraction from plants of the genus Catharanthus. However, the concentration of the compound in the plants is low, so when recovered in this way the price of the final product is very high.

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The dimeric alkaloids of the formula I, i.e. the bis-indole alkaloids, consist of monomeric alkaloid units, catharanthine (III) and vindoline (II). Both monomers are the main alkaloids (up to 1% by dry weight) of Catharanthus roseus (red winter) and easily isolated.

Ko. the plant is cultivated e.g. In southern Europe, Japan and as an ornamental plant in Finland in greenhouses.

However, the synthetic combination of these starting materials into the final product of formula I has been difficult in the past [(Lounasmaa, M and Nemes, A. (1982) Tetrahedron, 38, p. 223, (1982)].

It has now surprisingly been found that these monomeric units can be simply joined together to form a final product of formula I having the desired stereochemical configuration at the 16 'position. The process according to the invention is characterized in that vindoline of formula II. (¾ frns / CHj H3CO3 ^ 'N ^> 0Ac H5 OH ui) h3c cooch3 and catharanthine of formula III H COOCH3 ^ (III) 38C462 are irradiated with UV light in aqueous solution. Simultaneous photolytic oxidation can also be coupled to the dimerization, whereby vincristine (I, R = CHO) is formed as the main product.

The reaction mechanism is assumed to be as follows: 1. Cataranthine (III):

Under acidic conditions, photolysis converts catharanthine (III) to an excited state in equilibrium with the surrounding acidic solvent.

hl / = ctcv * h “° ch3 h / Mr a m jh *

In reality, the rules of photochemistry are a radical reaction, but electron transfers are shown in the diagram as electron pair transfers for clarity.

In its excited state, III 'tends to return to its aromatic form, in which case the appropriate nucleophile, here vindoline (II), has the opportunity to participate in the reaction (at the position of the molecule indicated by the <-nm arrow). If the excited III 'state is considered, then the conjugated double bond systems 4 80462 form a plane at which the N-bridge structure in the drawing rises above the drawing. This means for the further reaction that the approaching nucleophile can approach the molecule only below the level of the drawing. This results in the native C-16 'S configuration in vinblastine (I, R = -CH3). The enamine formed in intermediate III 'is normally hydrolyzed by an acidic aqueous solution. Due to the structure of the molecule, the water-OH group of the double bond can attack C-20 'only from the other side of the molecule and the unambiguous enantiomer is formed.

2. Vindoline (II)

Vindoline (II) has two factors that make it a 10-carbon quite efficient nucleophile for the dimerization of two alkaloids (II & III). The molecule has an aromatic moiety attached to it with both a methoxy group and an nitrogen having an odd pair of electrons. Both cause an "electron pressure" in the molecule precisely to C-10. The matter can be sketched, for example, by resonance forms II 'and II "® ch3COOCH3 CH3cooch3 H3CO CH3COOCH3 I * I I"

Resonance forms II * and II "explain a possible further reaction; e.g. nucleophilic substitution with excited catharanthine (III) to give vinblastine (I, R = -CH3) as a product.

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Surprisingly, the present reaction can be carried out by very simple methods by mixing the reaction components together in an aqueous solution and irradiating the mixture with UV light.

The reaction can be carried out at various temperatures ranging from about -10 to + 60 ° C. Normally normal pressure is used, but even increased pressure can be used in conjunction with reduced temperature.

The reaction is carried out in an aqueous solution the pH of which is adjusted to between about 1.5 and about 7. Inorganic acids such as hydrochloric or hydrobromic acids or organic acids such as formic acid can be used to adjust the pH.

The invention can advantageously be carried out in the "Hanovia" photoreactor shown in Figure I. This reactor consists of a 1 liter, three neck "Pyrex" bottle 1. An arc element 2 placed in a glass tube is inserted into the bottle, which has a cooling medium inlet connection 3 and an outlet connection 4. The bottle is equipped with a magnetic stirrer. The general course of the method is as follows. The flask is filled with one liter of deionized water, 1-2 mg of starting material is added to an aqueous solution, the pH of which is adjusted to 3-4 with acid (preferably HCl, HBr or HCOOH). The mixture is stirred with a magnetic stirrer and a UV light lamp placed in a quartz jacket in the middle of the pyrex bottle is turned on. The wavelength of light is between 180 and 400 nm (mainly 254, 265, 297 and 366 nm). The reaction time is about 15 minutes to 8 hours, but may also be longer depending on the acid used and the concentrations of the starting materials.

Upon completion of the reaction, the reaction products are separated and analyzed by conventional analytical methods.

The present invention is explained in more detail in the following examples. It should be noted, however, that the invention is not limited to these exemplary embodiments of the invention, but the details of the invention may vary within the scope of the claims set forth below.

Example 1 The "Hanovia" reactor was filled with one liter of deionized water and the pH of the solution was adjusted to 3.5 with hydrobromic acid. To the acidic aqueous solution was added 1.8 mg (4.4 μταοί) of vindoline (II) and 1.7 mg (4.4 pmol) of catharanthine (III). The reaction mixture was irradiated for 0.5 hours at about 8 ° C. The water was evaporated under reduced pressure to give 3.4 mg of a yellowish viscous oil as an evaporation residue.

Analysis

The reference substances in all analytical steps were dimeric Catharanthus alkaloids; vinblastine (I, R = -CH3) and vincristine (I, R = -CHO).

HPLC

Reaction samples run on three different columns with acetate buffer in LC give a completely identical signal to vinblastine (I, R = -CH 3) with a retention time of up to more than 40 minutes. About 15 to 20% reaction yield can be deduced from the LC.

HPTLC

Upon elution of the basic (NH 4 OH), concentrated reaction mixture with methanol, a slower moving spot corresponding to the vinblastine standard appeared on the TLC plate. The spot is visible on the TLC plate in the UV range of 366 nm. In contrast, the starting materials are visible in the UV range at 254 nm (some of the starting material was vindoline).

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MS

The mass spectrum shows a strong peak m / z 809 (= M + -1), which is typical for such organic compounds. However, the signal clearly indicates that dimerization between vindoline (II, MW = 455) and catharanthine (III, MW = 336) has occurred.

UV

The UV spectra of the synthesis product separated by LC and the standard substance (I, R = -CH 3) measured in methanol were completely identical.

UV: λ max (δ MeOH): 223 (4.76), 258 (4.32) nm.

RIA

The reaction product separated by liquid chromatography gave a positive response in the RIA analysis of vinblastine (I, R = -CH3).

1): Huhtikangas, A., Lehtola, T., Lapinjoki, S. and Lounasmaa, M., Planta Medica, 1987, 85? 2): Lapinjoki, S.,

Veräjänkorva, H., Huhtikangas, A., Lehtola, T. and Lounasmaa, M., Journal of Immunoassay, 7, 113 (1986).

In Examples 2-7, the procedure described in Example 1 above and the analysis of the final product were repeated, but using the parameters shown in Table I.

8 80462 0

4-) Γ> H N OJ

C r — t iH

ctj <#> i i i i i id m ro σ \ r-4 a t — i U) l: θ (ö - oooooo

Oj O <—I '—t i — I t — I i — H

e · ή O I I I I I

: (0 4 -) ^ - αο oo oo oo co 0 4-) - * M (0 u o r- ~ in in (C M JO »

O) .H - 'CN (N <N O O <N

01 (0 rH i — I

—T> 1 (0 4-) Φ 4-) Φ <d o a aa

4-) 4-) Q. r-H —I rH V4 O O

c> iauuuoaoo

> 1: <0 ιβ 3 S K S U U

O tn i * <jo K 03 Äö iti c__ n -h J c 03 m m in o o o £ 3 - »H Qj <C * h ro ro oo - * r E-i 4 -) __ ω (0 -h

rH C

JO -H

C: (0 • h m r-nn-invor ^ CTio>: <ö O ·> «* - ·> - -

IfO 03 rH rH r-H CO r-H O

6 C

•B

c> Q) r-, Ό tr__ • ή ε

(1) * - * rH

c c • H · Η

(0 -H

: 0 -U

4-> C

jo O in H1 Φ h eo: (Ö> h * · * · “·» * · *

1-3 (0 rH rH rH CO rH rH

4-) tO M

*B

1 M

• H M

to in cn ro m m r ~ W Φ e

II

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Example 8 The "Hanovia" reaction was filled with a liter of deionized water which was saturated with oxygen by introducing O 2 gas into it. The pH of the solution was adjusted to 2.5 with hydrochloric acid. To the acidic aqueous solution were added 1.8 mg (4.4 pnol) of vindoline (II) and 1.7 mg (4.4 pmol) of catharanthine (III). The reaction mixture was irradiated for 0.5 hours at about 8 ° C. The water was evaporated under reduced pressure to give 3.4 mg of a yellowish viscous oil as an evaporation residue.

Analysis

Catharanthus alkaloid: vincristine (I, R = -CHO) was used as a control in all assay steps.

HPLC

According to the standard substance (vincristine, R = -CHO), the reaction mixture run in acetate and phosphate buffer showed a yield of about 30% vincristine.

HPTLC

Upon elution of the basic (NH 4 OH), concentrated reaction mixture with methanol, a slower moving spot appeared on the TLC plate, corresponding to the vincristine standard.

MS

The reaction product isolated by HPLC gave a typical m / z = 825 (M + + 1) peak in chemical (NH 3) ionization.

UV

The isolated reaction product gave an identical UV spectrum with the standard substance (I, R = -CHO).

Λmax (log ε, eluent): 222 (4.63, 253 (4.21), 293 (4.18) nm).

Claims (6)

A process for the preparation of dimeric Catharanthus alkaloids of the formula I r U OH w., JH is CH3 or CHO, characterized in that vindolin, of formula II ^ c® ff Tji / 13 80 462 is irradiated with UV light in an aqueous solution either in non-oxidizing or oxidizing conditions. Process according to Claim 1, characterized in that the pH of the aqueous solution is adjusted to between 1.5 - 7. 3. A process according to claim 2, characterized in that the pH is controlled with acid, preferably hydrochloric or hydrobromic acid or an organic acid. 4. A process according to claim 1 or 2, wherein the reaction temperature is -10 ° - + 60 ° C. Process according to claim 2, characterized in that the reaction is carried out at normal pressure. Process according to Claim 1, characterized in that the reaction is carried out at elevated pressure, preferably at a lowered temperature.