Anthracycline glycosides

Details Anthracycline glycosides Anthracycline glycosides

1999-08-27

2001-02-13

Peselev, Elli (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C536S006400, C435S078000

Reexamination Certificate

active

06187758

ABSTRACT:

The present invention relates to anthracycline glycosides, their isolation and pharmaceutical compositions containing them.
The present invention provides anthracycline glycosides of formula I:
wherein R represents one of the two following residues:
or a pharmaceutically acceptable salt thereof. The compounds of the invention are anti-tumour agents. The new anthracyclines are useful as remedies for gram positive and gram negative bacterial infections and they inhibit growth of tumor cells.
The preferred compounds according to the present invention are in particular:
compound A:
4′-O-[3″-hydroxy-1″-(2′″-hydroxy-1′″-methyl-4′″-oxo-pentyloxy)-butyl]-daunorubicin of formula:
and compound B:
4′-O-[4″-hydroxy-3″-(1′″-hydroxy-ethyl)-5″-methyl-tetrahydrofuran-2-yl]-daunorubicin of formula:
The present invention further provides a process for the preparation of an anthracycline glycoside of formula I or a pharmaceutically acceptable salt thereof, which comprises culturing
Streptomyces peucetius
, in particular
Strepromyces peucetius
ATCC 27952, and isolating a resultant anthracycline glycoside of formula I as such or in the form of a pharmaceutically acceptable salt thereof. The anthracycline glycosides of this invention may be obtained therefore by culturing
Streptomyces peucetius
in a culture medium containing a carbon source, nitrogen source, inorganic components and, optionally micro-components under aerobic conditions. They may be isolated by conventional procedures such as solvent extraction, column chromatography, salt formation, preparative thin layer chromatography (TLC) and/or crystallization.
Thus, the present invention also provides a process for preparing a compound of the formula I as above defined, or a pharmaceutically acceptable salt thereof by means of fermentation, extraction and purification.
The pharmaceutically acceptable salts of the compounds of formula (I) include salts with pharmaceutically acceptable acids, either inorganic acids, for example, hydrochloric, hydrobromic, nitric or sulphuric acid; or organic acids, for example, citric, tartaric, maleic, fumaric, methanesulfonic or ethanesulfonic acid.
Preferred salts according to the invention are hydrochlorides.
The new anthracycline derivatives of the present invention are endowed with useful antitumoral activity.
The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and, as an active principle, an anthracycline analogue of formula I or a pharmaceutically acceptable salt thereof.
Fermentation process.
The production was carried out by well known methods. The micro-organism was cultured in a previously sterilized liquid culture medium under aerobic conditions at a temperature ranging from 25 to 35° C. (preferably at 30° C.) over a period of time varying from 5 to 8 days (preferably 7 days) and a pH value which initially was from 6.5 to 7.0 and at the end of the fermentation process from 5 to 7.0.
The culture medium consisted of a carbon and a nitrogen source as well as of mineral salts.
As carbon source starch, dextrin, glucose, maltose can be used.
As nitrogen source sorbean meal, dry yeast, meat, peptone or casein can be used.
All ammonium salts such as ammonium nitrate, ammonium sulphates, ammonium phosphates may be used.
The mineral salts useful for the production may vary depending on the medium employed.
The fermentation may be carried out in flasks or in pilot scale fermenters of various capacities.
Extraction Procedure.
At the end of the fermentation process the new anthracycline glycosides were principally found in the mycelium which was separated from the fermentation supernatant by filtration at pH 7.5.
The mycelium was extracted with water-miscible organic solvents such as acetone and methanol and then the mycelial extracts were concentrated in vacuo to obtain an aqueous solution. After adjusting to pH 7.2 the aqueous solution was loaded on Amberlite column. The selected fractions obtained were concentrated in vacuo and then extracted in alkaline conditions with dichloromethane. The organic extracts were concentrated to a small volume and then precipitated by addition of petroleum ether to obtain a crude extract.
Purification Procedure
The purification procedure consists of conventional methods like column chromatography, preparative TLC and crystallization.
The crude extract obtained from the Extraction Procedure was chromatographed on a silica gel column eluted by step elution with methanol. The fractions obtained were monitored by TLC and the selected fractions were concentrated in vacuo to give a red powder. This powder may be purified by flash chromatography on silica gel or by preparative TLC in order to obtain sufficiently pure anthracycline glycosides to determine their structure by mass spectrometry and NMR analysis and to study their biological activity. The resultant compound may be then converted into a pharmaceutically-acceptable salt.
Biological Activity
A) Antibacterical activity
The “in vitro” minimun inhibitory concentration (MIC) of the novel anthracycline glycosides in comparison with daunorubicin was determined for some microoganisms using the standard tube dilution procedure and is reported in Table 1.
TABLE 1
Antibacterial activity
MIC in &mgr;g/ml
Microorganism
Daunorubicin
Compound A
Staphylococcus
25
6.25
aureus
ATCC 14154
Staphylococcus
6.25
3.12
aureus
ATCC 65389
Bacillus subtilis
3.12
1.56
ATCC 6633
Bacillus cereus
6.25
1.56
ATCC 9634
Sarcina lutea
3.12
0.78
ATCC 9341
Micrococcus
6.25
1.56
flavus
ATCC 10240
Candida albicans
>100
>100
ATCC 10231
Escherichia coli
>100
>100
K12
Klebsiella
50
25
pneumoniae
ATCC
10031
B) Cytotoxic activity
The cytotoxic activity of the novel anthracycline glycosides was tested “in vitro” in comparison with daunorubicin on cultured L1210 cells as shown in Table 2.
TABLE 2
Cytotoxic activity of the novel anthracycline
glycosides on the growth of L1210 cells
Product
IC
50
(&mgr;g/ml)
Daunorubicin
0.259
Compound A
0.085
Compound B
0.222
The cytotoxic activity of compound A and compound B has also been evaluated in comparison to Doxorubicin on a panel of solid human tumor cell lines: 4 human mammary adenocarcinoma cell lines (MCF-7, MCF-7/Dx, SKBR-3 and MDA-MB-231, 2 human ovarian carcinoma cell lines (A2780 and OVCAR3) and 2 human colon adenocarcinoma cell lines (HCT116 and, HT29). Cytotoxicity was evaluated by the SRB assay (J.N.C.I.: 82, 1107-1112 and 1113-1118, 1990) in 96 well plates at 72 h after treatment with different concentrations of the compounds. Concentrations inhibiting the growth by 50% (IC50 values) were calculated. The results are reported in Table 3. Both compunds are more potent than Doxorubicin on all the tested cell lines. In particular compound A is active also on the MCF7 clone resistant to Doxorubicin and is particularly potent (IC
50
<10 nM) on 2 breast carcinoma cell lines (SKBR-3 and MDA-MB-23) and on both tested colon and ovarian carcinoma cell lines.
TABLE 3
Cytotoxic activity of the novel anthracycline glycosides on
solid human tumor cell lines
DOXORUBICIN
COMPOUND A
COMPOUND B
(IC
50
nM)
(IC
50
nM)
(IC
50
nM)
mammary carcinoma cell lines:
MCF7
940 ± 400
15
100
MCF7/DX
>10000
100
>200
SKBR-3
110 ± 20 
1.4
8.9
MDA-MB-231
540 ± 270
0.03
4.67
colon adenocarcinoma cell lines:
HCT 116
80 ± 20
0.3
4.8
HT29
160 ± 50 
3.2
11.2
ovarian carcinoma cell lines:
A2780
250 ± 140
0.4
7.5
OVCAR-3
140 ± 10 
0.08
20
Suitable routes of administration include parenteral administration, for example intravenous administration. For parenzeral administration a liquid formulation may be prepared using the anthracycline glycoside of formula I or salt thereof and a sterile diluent or carrier which may either dissolve the active compound or provide a suspension for it. The parenteral formulation may be prepared in the form of a sterile solid for reconstitution prior

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