GB2034707A

GB2034707A - Anthracycline glycosides

Info

Publication number: GB2034707A
Application number: GB7936407A
Authority: GB
Original assignee: Farmitalia Carlo Erba SRL
Current assignee: Pfizer Italia SRL
Priority date: 1978-10-25
Filing date: 1979-10-19
Publication date: 1980-06-11
Also published as: GB2034707B

Abstract

<IMAGE> Anthracycline glycosides, useful as antitumour antibiotics, of the above formula are provided. The daunorubicin derivatives (R = H) are prepared by condensation of daunomycinone with the appropriate (previously unknown) protected amino sugar, followed by removal of the protecting group, following techniques known per se. The doxorubicin derivatives (R = OH) are prepared from the daunorubicin derivatives by 14-bromination and hydrolysis, also following techniques known per se. The amino sugars are prepared from methyl 2,3,6-trideoxy-3-trifluoroacetamidoc-???-L-threo-hexopyranoside-4-ulos e.

Description

SPECIFICATION Anthracycline Glycosides DESCRIPTION The invention relates to anthracycline glycosides, to processes for their preparation and to pharmaceutical compositions containing them.

The invention provides anthracycline glycosides of the general formula I

in which R represents a hydrogen atom or a hydroxy group, one of R, and R2 represents a methyl group, and the other of R1 and R2 represents a methoxy group, and their hydrochlorides.

The anthracycline glycosides I may be named as follows: R = H, R1 = cm3, R2 = CH3O:4'-C-methyl-4'-O-methyl-daunowbicin R = H, R, = cm30, R2 = CH3: 4'-epi-4'-C-methyl-4'-O-methyl-daunorubicin R = OH, R1 = CH3, R2 = C H30: 4'-C-methyl-4'-O-methyI7doxornbicin R = OH, R1 =CH30, R2 = CH3:4'-epi-4'-C-methyl-4'-O-methyl-dioxorubicin They are referred to hereinafter, for convenience, as compounds IA, IB, IC and ID respectively.

Compounds IA and IB may be prepared by a process according to the invention, which process comprises condensing dau nomycinone with 2,3,6-trideoxy-3-trifluoroaceta mido-4-C-methyl-4-O- methyl-L-lyxo-hexopyranosyl chloride or 2,3,6-trideoxy-3-trifluoroacetamido-4-C-methyl-4-O-methyl- L-arabino-hexopyranosyl chloride (hereinafter, for convenience, referred to as compounds IIA and 118 respectively in an inert organic solvent in the presence of a silver salt catalyst and of a molecular sieve as a dehydrating agent, and removing the trifluoroacetyl protecting group from the resultant anthracycline glycoside by mild alkaline hydrolysis.

The inert organic solvent is suitably chloroform or methylene dichloride. Silver trifluoromethanesulphonate is the preferred silver salt. Compounds IA and IB may be isolated as their crystalline hydrochlorides.

Compounds IC and ID may be prepared from compounds IA and IB respectively by a further process within the scope of the invention. The said further process comprises brominating the compound IA or IB at the 1 4-position and hydrolysing the resultant 1 4-bromo derivative to give the compound IC or ID. The bromination and hydrolysis conditions are suitably those described in our Patent Specification No. 1 21 71 33 or those described in United States Patent Specification No. 38031 24.

Compounds IIA and IIB are themselves novel, and may be prepared from methyl 2,3,6-trideoxy-3 trifluoroaceta mido-a-L-threo-hexopyra noside-4-u lose, a known compound described in United States Patent Specification No. 4039663. The first step of the preparative process comprises reacting the methyl 2,3 ,6-trideoxy-3-trifluoroaceta mido-ex-L-th reo-hexopyranoside-4-u lose with methylmagnesiu m iodide either in diethyl ether solution at - 780C or in tetrahydrofuran solution under reflux.The reaction is highly stereospecific, yielding in the case of diethyl ether solution at -780C methyl 2,3,6-trideoxy-3 trifluoroacetamido-4-C-methyl-cr-L-lyxohexopyranoside and in the case of tetrahydrofuran solution under reflux methyl 2,3,6-trideoxy-3-trifluoroacetam ido-4-C-methyl-a-L-a rabinohexopyranoside.

The remaining steps of the preparative process comprise treating the methyl 2,3,6-trideoxy-3 trifluoroacetamido-4-C-methyl--L-lyxo (or arabino)-hexopyranoside with diazomethaneborontrifluoride etherate reagent in methylene dichloride at - 700 C, hydrolysing under acid conditions the resultant methyl 2,3,6-trideoxy-3-trifIuoracetamido-4-C-methyl-4-O-methyL-L-lyxo (or arabino)hexopyranoside, reacting the resultant 2,3,6-trideoxy-3-trifluoroacetamido-4-C-methyl-4-0-methyl-cr- L-lyxo (or arabino)-hexopyranose with p.nitrobenzoyl chloride in pyridine solution, and treating the resultant 1-O-p.nitrobenzoyl-2,3,6-trideoxy-3-trifluoroacetamido-4-C-methyl-4-O-methyl-L-lyxo (or arabino)-hexopyranose with dry hydrogen chloride in anhydrous methylene chloride to obtain the compound IIA or IIB.

Reaction conditions for the reaction between the 2,3,6-trideoxy-3-trifluoroacetamido-4-C-methyl- a-L-Iyxo (or arabino)-hexopyranoside and the diazomethane-borontrifluoride etherate reagent are described by J.O. Deferrari et. al. in Methods in Carbohydrate Chemistry, Vol. VI,p. 365, 1972, Academic Press, New York and London, although the temperature given above (-70 C) is lower than that described there.

Daunomycinone is described and claimed in our Patent Specification No. 1003383.

The invention further provides a pharmaceutical composition comprising an anthracycline glycoside according to the invention in admixture with a therapeutically acceptable diluent or carrier.

The invention is illustrated by the following Examples, in which degrees of temperature are degrees Celsius.

EXAMPLE 1 2,3,6-Trideoxy-4-C-methyl-4-O-methyl-3-trifluoroacetamido-L-lyxo-hexopyranosyl chloride (II A) To 100 ml of an ethereal solution of methylmagnesium iodide (2.06 g of Mg + 7 ml of CH3l) cooled to - 780C there was added under stirring 20 ml of an ethereal solution of methyl 2,3,6-trideoxy 3-trifluoroacetamido-(x-L-threo-hexopyranoside-4-ulose (1.170 g, 4.6 mmol).

After 3 hours at --78 OC, water was added, the ethereal layer was separated off and the aqueous layer was extracted with diethyl ether. The combined ethereal extracts were washed with water and dried over anhydrous sodium sulphate. The diethyl ether was evaporated off and the residue was crystallized from a mixture of diethyl ether and hexane to give methyl 2,3,6-trideoxy-4-C-methyl-3 trifluoroacetamido-α-L-lyxo-hexopyranoside (1.180 g, 95%). m.p. 133--134 ; [a] = - 1280 (e = 1 in chloroform).

The carbon-i 3 Nuclear Magnetic Resonance Spectrum (DMSOd6) of the compound shows the C-4 methyl group signal at 21.3 p.p.m. (down field from tetramethylsilane, used as internal reference), a characteristic chemical shift value for equatorial methyl groups in branched-chain sugars.

A solution of 1 g (3.7 mmol) of methyl 2,3,6-trideoxy-4-C-methyl-3-trifluoroacetamido--L-lyXo- hexopyranoside, prepared as above described, in 10 ml of dry methylene dichloride was treated at - 700 with 0.05 ml of borontrifluoride etherate. While maintaining the temperature at - 700, an excess of diazomethane dissolved in methylene dichloride was added until a faint yellow colour persisted. After 60 minutes at - 700, a white solid (polymethylene) was removed by filtration, and the filtrate was washed successively with 10% sodium bicarbonate solution and with water, after which it was dried with anhydrous magnesium sulphate. The residue, which was obtained by evaporation of the solvent, was chromatographed on a silica gel column.Elution with a 95:5 chloroform: acetone mixture gave pure methyl 2,3,6-trideoxy-4-C-methyl-4-O-methyl-3-trifluoroacetamido-α-L-lyxo-hexopyranoside (0.68 g, 65%) as a syrup. [a], = -- 1290 (c = 1.1 in chloroform); mass spectrum m/e 285 (M+). The pmr spectrum (CDCI3) showed absorptions at 1.22 (s, CH3-C-4), 1.28 (d, CH3-C-5), 3.33 and 3.48 (two s,-OCH3) and 4.77 s (broad sC-i-H).

A solution of 0.60 g (2.1 mmol) of the compound last prepared in 1 2 ml of acetic acid was added to 48 ml of water and heated at 1 000 for 2 hours. The solvent was evaporated off to give 2,3,6 trideoxy-4-C-methyl-4-O-methyl-3-trifluoroacetamido-α-L-lyxo-hexopyranose (2 9,97%) as a syrup.

[D - 950 (c = 1.1 in chloroform). The pmr spectrum (CDCI3) showed absorptions at 1.22 (s, CH3-C-4), 1.26 (d, CH3-C-5), 3.46 (s, CH3O) and 5.37 S (broad s, C-i-H).

A solution of 0.50 g (1.85 mmol) of the compound last prepared in 12 ml of dry pyridine was treated at O 0 with 0.68 g of p.nitrobenzoyl chloride under stirring. After 10 hours at room temperature he reaction mixture was poured into iced water and the resulting precipitate was filtered off and washed with water to neutrality. The precipitated 1 -p-nitrobenzoate (mixture of a and ss anomers) was dissolved in chloroform and dried over magnesium sulphate. The residue obtained by evaporation off of the solvent gave 0.735 g o 2,3,6-trideoxy-4-C-methyl-4-O-p.nitrobenzoyl-3-trifluoroacetamido-L-lyxo- hexopyranose (90%) m.p. 180-i ; [aC]D = - 420 (c = 1 in chloroform).

A solution of 0.70 g (1.66 mmol) of the compound last prepared in 1 5 ml of dry methylene chloride was saturated at 0 with anhydrous hydrogen chloride. The resulting precipitate of p.nitrobenzoic acid was filtered off under anhydrous conditions and the filtrate was evaporated to a residue which was 2,3,6-trideoxy-4-C-methyl-4-0-methyl-3-trifluoroacetamido-L-lyxo-hexopyranosyl chloride (II A, 0.45 g). This material was suitable for condensation with daunomycinone without further purification.

EXAMPLE 2 2,3,6-Trideoxy-4-C-methyl-4-O-methyl-3-trifluoroacetamido-L-arabino-hexopyranosylyl chloride (11 B) To a refluxing solution of methylmagnesium iodide (3.1 g of Mg + 10.5 ml of CH3l) in dry tetrahydrofuran (150 ml) was added methyl 2,3,6-trideoxy-3-trifluorQacetamido-a-L-threo- hexopyranoside-4-ulose (1.76 g, 6.9 mmol) in dry tetrahydrofuran (35 ml). After refluxing for 3 hours the reaction mixture was diluted with chloroform and water, the phases were separated and the aqueous layer was then extracted with chloroform. The combined chloroform extracts were washed with water and dried over anhydrous sodium sulphate. The residue (1.85 g), obtained by evaporation off of the chloroform, was chromatographed on a silica gel column. Elution with a 97.3 chloroform: acetone mixture afforded two compounds.The minor compound was identified as methyl 2,3,6-trideoxy-4-C- methyi-3-trifluoroacetamido-a-L-lyxo-hexopyranoside (0.28 g,15%), whereas the major compound was crystallized from diisopropyl ether and identified as methyl 2,3,6-trideoxy-4-C-methyl-3trifluoroacetamido-a-L-arabino-hexopyranoside (1.40 g, 75%). m.p. 140-141 , [a]23 = - 1 55 (c = 1.1 in chloroform), mass spectrum m/e 271 (M+). The carbon-i 3 Nuclear Magnetic Resonance spectrum (DMSOd6) of the compound showed the C-4 methyl group signal at 13.6 p.p.m. (down field from tetramethylsilane, used as internal reference), a characteristic chemical shift value for axial methyl groups in branched-chain sugars.

Treatment of methyl 2,3,6-trideoxy-4-C-methyl-3-trifluoroacetamido--L-arabino- hexopyranoside (1.30 g, 4.8 mmol), prepared as above described, in methylene dichloride (10 ml) with diazomethane/borontrifluoride, as described in Example 1, gave the corresponding 4-0-methyl derivative (1.03 g, 75%) as a syrup. [a]o23 = - 1180 (c = 1 in chloroform); mass spectrum m/e 285 (M+); pmr spectrum (CDCI3): 1.12 (s, CH3-C-4), 1.1 7 (d, CH3 -C-S), 3.22 and 3.33 (two s, OCH3) and 4.66 S (m, C-i-H). Acid hydrolysis of the 4-0-methyl derivative (1 g, 3.5 mmol) as described in Example 1 gave 2,3,6-trideoxy-4-C-methyl-4-O-methyl-3-trifluoroacetamido-L-arabino-hexopyranose (0.94 g. 95%) as a syrup. [a] 23 = - 700 (c = 1 in chloroform); p,r spectrum (CDCI3) 1.12 (s, CH3 -C-4), 1.16 (d, CH3 -C-S), 3.24 (s, OCH3) and 5.28 S (broad s, C-i-H).

Treatment of the compound last prepared (0.90 g, 3.3 mmol) with p.nitrobenzoyl chloride in pyridine as described in Example 1 gave the corresponding 1 -O-p.nitrobenzoyl derivative (1.26 g, 90%).

m.p. i7Si760;[a]23=4O0 (c= 1.1 inchlorform).

A solution of the 1-O-p.nitrobenzoyl derivative (1 g, 2.4 mmol) in dry methylene dichloride was saturated at 0 with anhydrous hydrogen chloride. After filtering off the precipitated p.nitrobenzoic acid, the solution was evaporated to dryness to give 2 ,3,6-trideoxy-4-C-methyl-4-0-methyl-3- trifluoroacetamido-L-arabino-hexopyranosyl chloride (II B, 0.65 g).

EXAMPLE 3 4'-C-Methyl-4'-O-methyl-daunorubicin (I A) (IMI 96) To a solution of daunomycinone (0.816 g, 2.05 mmol) in dry methylene dichloride (80 ml) was added 2,3 ,6-trideoxy-4-C-methyl-4-O-methyl-3-trifluoroacetam ido-L-lyxo-hexopyranosyl chloride (I I A, 0.45 g), prepared as described in Example 1, in 10 ml of methylene dichloride and molecular seive (4 A Merck, 5 g). The mixture was then treated with 0.40 g of silver trifluoromethanesulphonate in anhydrous diethyl ether (10 ml) under vigorous stirring.After 1 hour at room temperature, the reaction mixture was neutralized with a saturated aqueous solution of sodium bicarbonate, and the organic phase was separated off and evaporated under vacuum Chromatographic purification of the crude residue on a column of silicic acid, using 95:5 chloroform: acetone as the eluent, gave 0.72 g of 4' C)methyl-4'-O-methyl-N-trifluoroacetyl-daunorubicin. m.p. 9i930; [a] o' = - 2220 (c = 0.05 in chloroform).The pmr spectrum (CDCI3) showed absorption at 1.1 9 (s, CH3-C), 1.32 (d, CH3 --CC-5'), 239 (s, CH3-CO), 3.45 (s, C-4'-O-CH3), 4.04 (s, C-4-O-CH3), 5.25 (broad s, C-7-H), 5.47 (broad s, '-H), 13.17 and 13.86 S (two s, phenolic OH).

A solution of 0.6 g of the compound prepared above in 10 ml of acetone was treated with 45 ml of 0.2 N aqueous sodium hydroxide and stirred under nitrogen at room temperature. After 2 hours the reaction mixture was adjusted to pH 3.5 with 1 N aqueous hydrogen chloride and then extracted with chloroform to eliminate impurities. The aqueous phase, adjusted to pH 8.2, was extracted twice with chloroform (50 and 30 ml portions). The combined organic extracts were dried over sodium sulphate, concentrated to a small volume and acidified to pH 4.5 with 0.5 N methanolic hydrogen chloride.

Addition of excess diethyl ether gave 4'-C-methyl-4'-0-methyidaunorubicin (IA) as the hydrochloride (0.38 g, 68%): m.p. 178-1 800 (with decomposition); [aJ = + 2850 (e = 0.05 in methanol).

EXAMPLE 4 4 '-C-Methyl-4 '-0-methyl-doxorubicin (IC) (IMI 97) A solution of 4'-methyl-4'-0-methyl-daunorubicin hydrochloride (0.3 g, 0.5 mmol), prepared as described in Example 3, in a mixture of 4 ml of anhydrous methanol, 11.5 ml of dioxan and 0.3 ml of ethyl orthoformate was treated with 1.2 ml of a solution containing 0.93 g of bromine in 10 ml of chloroform. After 2 hours at 100 the reaction mixture was poured into a mixture of 60 ml of diethyl ether and 30 ml of petroleum ether. The resultant red precipitate, after being filtered off and washed with diethyl ether several times completely to remove the acidity, was dissolved in a mixture of 10 ml of acetone and 10 ml of 0.25 N aqueous hydrogen bromide.After 20 hours at 300 the reaction mixture was treated with 0.45 g of sodium formate in 5 ml of water and stirred at 300 for 48 hours. The resultant mixture was extracted with chloroform in order to remove some lipophilic impurities. The aqueous phase, after being adjusted to pH 7.6 with aqueous sodium bicarbonate, was repeatedly extracted with chloroform until the extracts were colourless. The combined chloroform extracts were dried with sodium sulphate and evaporated to a small voiume (about 1 5 ml) under vacuum. To the resulting red solution, adjusted to pH 3.5 with anhydrous methanolic hydrogen chloride, was added an excess of diethyl ether to give 4'-C-methyl-4'-O-methyl-doxorubicin (IC, 0.21 g) as the hydrochloride. m.p.

185-186 (with decomposition); [a]23"= + 2910 (c=0005'in methanol).

EXAMPLE 5 4'-Epi-4 '-C-methyl-4'-O-methyl-daunoruhicin (IB) (IMI 98) The synthesis of the compound IB, starting from daunomycinone and 2,3,6-trideoxy-4-C-methyl4-O-methyl-3-trifluoroaceta mido-L-arabi no-hexopyranosyl chloride llb) prepared as described in Example 2, was carried out according to the procedure described in Example 3.

4'-Epi-4'-C-methyl-4'-0-methyl-daunorubicin (IB) was obtained as the hydrochloride in the form of red crystals. m.p. 187-i 880 (with decomposition); [aS]23 = + 251 0 (c = 0.05 in methanol).

EXAMPLE 6 4 '-Epi-4 '-C-methyl-4'-O-methyl-doxorubicin (ID) (IN11101) The 14-bromo-derivative of compound IB, prepared as described in Example 5, was obtained and successively hydroxylated at the 14-position according to the procedure described in Example 4. 4'-epi 4'-C-methyi-4'-0-methyl-doxorubicin (ID) was obtained as the hydrochloride in the form of red crystals.

m.p. 190-i 91 0 (with decomposition); [a]23' = + 2720 (c = 0.025 in methanol).

BIOLOGICAL ACTIVITY The anthracycline glycosides according to the invention display antitumour activity. The compounds have been tested against HeLa cels "in vitro" (time of exposure to the drugs: 24 hours) and on P 388 leukemia in mice in comparison with daunorubicin and doxorubicin. The results of the "in vitro" tests, shown in Tabie 1 are reported as ID50 (Inhibiting Dose 50% in ng/ml). It can be observed that all the compounds inhibited the cell viability of HeLa cells in vitro. The antitumour activity was tested "in vivo" on P 388 leukemia in comparison with daunorubicin and doxorubicin.

The date reported in Table 2 show that all the new anthracycline glycosides display antitumour activity at non-toxic doses.

TABLE 1 Activity on Hela cells viability in vitro

Compound ID > o ng/ml Daunorubicin 10 4 '-C-methyl-4 '-O-methyl-daunoru bi cin (I-A) 34 4'-epi-4'-C-methyl-4'-O-methyl-daunorubicin (1-8) 2.5-3,4 Doxorubicin 10 4'-C-methyl-4'-0-methyl-doxorubicin (I-C) 5 4'-epi-4'-C-methyl-4'-O-methyl-doxorubicin (I-D) 27 TABLE 2 Activity against P 388 leukemia

Dose(a) 7 No. of toxic Compound mg/kg deaths/total Daunorubicin 2.9 163 0/48 4.4 161 3/48 6.6 156 28/48 4-C-methyl-4 '-O-methyl- 6.6 140 0/18 daunorubicin (I-A) 22,5 155 0/20 33.7 160 0/26 50.5 137 1/5 4'-epi-4'-C-methyl-4'-O-methyl- 6.6 140 0/8 daunorubicin (I-B) 10.0 147 0/8 22.5 150 0/8 Doxorubi ci n 4,4 190 0/28 6.6 201 0/28 10.0 241 5/28 4'-Cmethyl-4-OmethyI-doxorubi cin 4.4 165 0/10 (I-C) 6.6 172 0/10 10.0 202 2/17 4 '-epi-4'-C-methyl-4 '-O-methyl- 4.4 165 0/8 doxorubicin (I-D) 6.6 185 1 0/8 10.0 205 0/8 (a) Mice were treated i.p. on day 1 after tumour cell inoculation.

(b) Median survival time of treated mice, over median survival time of control x 100.

Claims

1. An anthracycline glycoside of the general formula I

in which R represents a hydrogen atom or a hydroxy group, one of R, and R2 represents a methyl group and the other of R, and R2 represents a methoxy group, or a hydrochloride thereof.

2. 4' -C-methyl-4'-0-methyl-daunorubicin or its hydrochloride.

3. 4'-epi-4'-C-methyl-4'-O-methyl-daunorubicin or its hydrochloride.

4. 4'-C-methyl-4'-0-methyl-doxorubicin or its hydrochloride.

5. 4'-epi-4'-C-methyl-4'-0-methyldoxorubicin or its hydrochloride.

6. A process for the preparation of 4'-C-methyl-4'-O-methyl-daunorubicin or 4'-epi-4'-C-methyl 4'-O-methyl-daunorubicin, the process comprising condensing daunomycinone with 2,3,6-trideoxy-3 trifluoroaceta mido-4-C-methyl-4-0-methyl-L-Iyzo-hexopyranosyl chloride or 2,3,6-trideoxy-3trifluoroacetamido-4-C-methyl-4-O-methyl-L-arabino-hexopyranosyl chloride in an inert organic solvent in the presence of a silver salt catalyst and of a molecular sieve as a dehydrating agent, and removing the trifluoroacetyl protecting group from the resultant anthracycline glycoside by mild alkaline hydrolysis.

7. A process according to claim 6 wherein the inert organic solvent is chloroform or methylene dichloride.

8. A process according to claim 6 or claim 7 wherein the silver salt catalyst is silver trifluoromethanesulphonate.

9. A process for the preparation of 4'-C-methyl-4'-O-methyl-daunorubicin or 4'-epi-4'-C-methyl 4'-0-methyl-daunorubicin, the process being substantially as described herein with reference to Example 3 or Example 5.

1 0. A process for the preparation of 4'-C-methyl-4'-0-methyl-doxorubicin or 4'-epi-4'-C-methyl4'-0-methyl-daunorubicin, the process comprising brominating 4'-C-methyl-4'-O-methyl-daunorubi,oin or 4'-epi-4'-C-methyl-4'-O-methyl-daunorubicin at the 14-position and hydrolysing the resultant 1 4- bromo derivative.

ii. A process for the preparation of 4'-C-methyl-4'-O-methyl-doxorubicin or 4'-epi-4'-C-methyl4'-O-methyl-doxorubicin, the process being substantially as described herein with reference to Example 4 or Example 6.

12. A pharmaceutical composition comprising an anthracycline glycoside according to any of claims 1 to 5 in admixture with a therapeuticaly acceptable diluent or carrier.