DK146541B - METHOD OF ANALOGUE FOR THE PREPARATION OF 4'-EPI-DAUNOMYCINE OR ITS INDIVIDUAL ALFA OR BETA ANOMERS THEREOF - Google Patents

Description

U6541

The invention relates to an analogous process for the preparation of 4'-epi-daunomycin or the individual le oC or β-anomers thereof. Daunomycin and daunomycinone are described in British Patent No. 1 o33,383.

The complete chemical name of the 41-epi-daunomycin-oC anomer is 7-O- (3'-amino-2 ', 31,61-tridesoxy-o (-L-arabino-hexopyranosyl) -aunomycinone of formula I:

0 OH

C0CH3

I I x OH

H3C0 o OH o K \ nh2

The complete chemical name of the 4'-epi-daunomycin / 3-anomer is 7-O- (31-amino-21,3 ', 61-tridesoxy- ^ - L-arabino-hexopyranosyl) -daunomycinone of formula II:

0 OH

^^ 000H3 H3CO 0 OH! (II) nh2 H0 \ l f / U6541 2

The process according to the invention is characterized by the characterizing part of the claim and comprises condensation of daunomycinone of formula III:

0 OH

COCH3 | | '' 'OH (III) H, C0 0 OH i ύ i

OH

with a derivative of 4'-epi-daunosamine (3-amino-2,3,6-tri-deoxy-L-arabino-hexose) of Formula IV: HO a-O to CH3 Vv OH (IV) nh2 to prepare the pharmacologically active glycosides of formulas (I) and (II). In practice, hexose IV must be protected and converted into a 1-derivative such as, for example, a halide suitable for condensation with daunomycinone. After the condensation, the protecting groups are removed.

The 3-amino group in hexose IV must be protected by groups that can be removed without further degradation of the products containing various chemically sensitive groups. The N-trifluoroacetyl group can be satisfactorily removed by weak alkaline treatment.

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The introduction of trifluoroacetyl groups is described in Med.

Chem., 1974, 17, 659.

The hexose IV must be converted into derivatives that are of sufficient stability to be used in the synthesis. The instability of 1-halogen derivatives of 2-deoxy carbohydrates is well documented (W. W. Zorbach et al., Advances in Carbohydrate Chemistry, 1966, 21, 273).

The tri-trifluoroacetyl derivatives of hexose IV are reacted in the usual manner with dry hydrogen chloride; hereby the corresponding 1-chlorohexoses are obtained. These are solids that can be stored for several days under anhydrous conditions.

With regard to suitable reaction conditions for the synthesis of the glycoside bond, the well-known Koenigs-Knorr reaction (J. Conchi, GA Lewy and CA Marsh, Advances in Carbohydrate Chemistry, 1957, J_2, 157) indicates a variety of conditions which include solvent modifications, temperature. , catalyst and hydrogen chloride or hydrogen bromide acceptor. The significance of the modifications is that in order to obtain a reasonable reaction rate, a number of optimal conditions are necessary.

The reaction of daunomycinone with hexoses is known from J.

With. Chem., 1974, Γ7, 659-660.

The process of the invention comprises a mild treatment of daunomycinone (III) with I-chloro-N, O-di-trifluoroacetyl derivative of hexose IV in an organic solvent such as chloroform or dichloromethane in the presence of a catalyst consisting of a mercuric halide (e.g. mercuribromide) and a hydrogen chloride acceptor (e.g. mercuric oxide), and by removal of N-trifluoroacetyl with dilute alkali, the subject 4'-epi-daunomycin is obtained.

The process according to the invention is illustrated by the following example, where the temperatures are given in ° C.

Example 1 g of 2,3,6-tridesoxy-3-trifluoroacetamido-L-arabinohexose is slurried in 20 ml of anhydrous diethyl ether and treated at 0 ° C with trifluoroacetic anhydride. After standing for two hours at 0 ° C and one hour at room temperature, the solvent is removed at reduced pressure and the residue is recrystallized from dichloromethane. The product obtained is treated in anhydrous diethyl ether at 0 ° C with anhydrous gaseous hydrogen chloride. After standing at + 5 ° C overnight, the solvent is removed in vacuo to give a quantitative yield of 2,3,6-tridesoxy-N, O-di-trifluoroacetyl-L-arabinohexopyranosyl chloride.

NMR (CDCl1): 1.30 δ (d, J = 6.0 Hz, 3H, CHg) 2.25-2.806 (m, 2H, C (2) H₂) 4.20-4.656 (m, 1H, C (5) H) 4.65-5.156 (m, 2H, C (3) H and C (4) H) 6.256 (m, WH = 6.0 Hz, 1H, C (1) H) 6.445 (wide s, 1H, NH)

A solution of 0.5 g of daunomycinone in anhydrous chloroform is treated with 1.0 g of mercuric oxide, 0.25 g of mercuribromide, 10 g of molecular sieve (3 Å, Merck) and 0.5 g of 2,3,6-tridesoxy-N, 0 di-trifluoroacetyl-L-arabinohexopyranosylchlorid. The mixture is stirred for 24 hours, freed from solids by filtration and evaporated under vacuum. The residue is dissolved in methanol, refluxed for 15 minutes, evaporated to dryness and chromatographed on a silica column with a mixture of chloroform: benzene: methanol 10: 20: 3 as eluent. The main product is a 70:30 ratio of <A- and / 3-7-0- (N-trifluoroacetyl-4'-epi-daunosaminyl) -aunomycinone (yield after recrystallization from chloroform 0.3 g).

This material is converted quantitatively by treatment with 0.1 N sodium hydroxide as described above into a mixture of the corresponding <x- and / <3-glycosides as free bases.

The product is separated into <x and / a 3 anomers by silica gel chromatography using a chloroform: methanol: water solution system 135: 20: 2 (volume ratio) as the eluent. One obtains <* - anomer (4'-epi-daunomycin, I), [oc] + 320 ° (c = 0, o45, methanol), m.p. 199-201 ° C, yield 0.16 g and / 3 anomer (II), m.p. 182-184 ° C, yield 0.06 g.

Biological activity of compounds I (41-epi-daunomycin, c <-anomer) and II (41-epi-daunomycin, β-anomer)

Compounds I and II exhibit excellent biological properties as potent inhibitors of cell mitosis and proliferative activity in cultured cells in vitro. They have also shown significant activity on cell transformations induced by oncogenic viruses. They have antitumor activity in mice as shown by an increase in mean survival time at nontoxic doses in animals having a number of experimental tumors (Tables 1-4).

The cardiotoxic "in vitro" activity of compound I is very low (lower than that of daunomycin), as shown in the following experiments.

6 146541

The method consists in growing single cardiac cells isolated by tripinisation from the heart of newborn mice. After 3-4 days, the cultures showing clusters of pulsating cells can be studied both in terms of frequency and rhythm (A. Necco, T. Dasdia, IRCS, 2, 1293, 1974).

Table 1

Effect of Compounds I and II on mitotic index and proliferative activity of cultured HeLa cells at different exposure times.

Results are expressed as% of untreated control samples.

Compound Dose Mitotic Index Colony Number (gig / ml) 2 t 4 t 8 t 2 t 4 t 24 t 0.025 226 * 100 117 113 88 48 I 0.05 I89 * 103 122 115 56 23 0.1 79 140 O 77 23 3 DI50 0.16 0.056 0.027 0.25 137 103 85 108 86 70 yr 0.5 95 67 88 101 37 18 1 52 40 O 98 17 6

The «> 1 0.47 °> 33 __J? 0__

Daunomycin DI ^ 0.098 0j036 0> 021 * Metaphasic Blockage 146541 7

Table 2

Effect of compounds I and II on foci formation and on cell proliferation in cultured mouse fibroblasts infected with Moloney Sarcoma virus (3 days treatment).

Compound Dose Fooi - formation Cell proliferation B / A

__ (^ / ml) kW- Lnt, ol »r. '' tø ™ _ 0.0062 51 75 j 0.025 O 0.006 23 0.013 0.1 O 14 0.4 o 1 0.0062 48 87 n 0.025 44 80 0.1 5 ° '01 28 °> 064 6 · 4 0 , 4 O 14

Daunomycin 0.006 0.0086 1.4

Table 3

Effect of compounds I and II on mean survival time and on the number of female female mice (Swiss CDI) intraperitoneally inoculated with Sarcoma 180 ascites (1 x 10 ** cells / mouse).

The compounds were used the day after the tumor transplant.

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Compound Dose Mean survival time Number of survivors (mg / kg)% of control samples on day 60 0.22 111.1 1/10 τ 1.1 120.8 1/10 5.7 174.5 0/10 0.26 96.6 0 / 10 II 1.3 114.8 1/10 6.7 118.6 1/10

Table 4

Effect of compounds I and II on Gross transplant-borne leukemia. Mean survival time of C 1 H female mice intravenously inoculated with a suspension of leukemic lymph nodes.

C

glands and spleen (2.5 x 10 10 cells / mouse). The compounds were used once daily for five days, the first time the day after the tumor transplant.

Compound Daily dose Mean survival time (mg / kg) <f0 of control samples 1.5 115 I 2.25 143 3 162 3.75 122 4.5 120 1.5 106 II 2.25 102 3 121