HRP921220A2 - New antitumor agent obtained by microbial stereoselective reduction of 4'-deoxy-4'-iodoxorubicin - Google Patents

Description

The proposed invention relates to a derivative of doxorubicin, to its preparation and to pharmaceutical compositions containing it.

The proposed invention is 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin of formula (II):

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and pharmaceutically acceptable salts thereof.

For the stereoselective reduction of the 13-ketone functional group of 4'-deoxy-4'-iododoscorubicin (I)

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used the microorganism of the genus Streptomyces to specifically obtain 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin, one of two possible C-13 stereoisomers of 4'-deoxy-13-dihydro-4'-iododoxorubicin. The new compound (II), further designated as FCE 24883, can be used as an antitumor agent and shows activity on experimental tumors comparable to that of 4'-deoxy-4'-iododoxorubicin (I). The substrate for the microbial stereoselective reduction is a doxorubicin semisynthetic analog, described in our US-A-4,438,105 (March 20, 1984).

In more detail, the proposed invention relates to a biosynthetic process, in which a mutant of the Streptomyces peucetius species, designated as species M 87 F.I., is deposited in the Deutsche Sammlung von Mikrooganismen, where it is registered under serial number DSM 2444. It is characterized by the ability stereoselective reduction of the 13-ketone functional group of 4'-deoxy-4'-iododoxorubicin (I). The species FCE 24883 (II) can be easily isolated from the fermentation broth, and its crude solutions can be concentrated and purified.

The invention also provides a process for the preparation of 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin of formula (II), or its pharmaceutically suitable salts, and is characterized in that the strain Streptomyces peucetius M87 F.I. (DSM 2444) in a solution of 4-deoxy-4'-iododoxorubicin, and separates the resulting 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin as such, or in the form of a pharmaceutically acceptable salt.

The invention includes within its framework the new antitumor anthracycline FCE 24883 (II) in its pure form as hydrochloride.

The invention also provides a pharmaceutical composition comprising compound (II) or a pharmaceutically acceptable salt thereof in admixture with a diluent or carrier.

Mutation of Streptomyces peucetius, subspecies aureus ATCC 32428, was performed using nitrosoguanidine, in order to obtain a laboratory microorganism designated as Streptomyces peucetius, species M 87 F.I., which selectively converts compound (I) into compound (II). Species S. peucetius M 87 F.I. has received the application serial number DSM 2444 in the Deutsche Sammlung von Mikroorganismen, SR Germany, where it is deposited in the permanent collection.

Morphology of the mutant species M 87 F.I. it does not differ from the morphology of the parent S.peucetius ATCC 31428, although both cultures clearly differ in terms of cultivation conditions and their biochemical characteristics. In fact, the mutant species M 87 F.I. does not produce the straw-yellow to lemon-colored soluble segment on agar, which is characteristic of its parent species S. peucetius ATCC 31428.

In addition, the mutant strain M 87 F.I. can selectively transform compound (I) into compound (II), while parent S. peucetius ATCC 31428 is not selective in this regard. That feature of the mutant M 87 F.I. makes it very useful, as demonstrated here.

Transformation process

The stereoselective biotransformation according to the present invention can be carried out on a growth culture, species S. peucetius M 87 F.I., by adding compound (I) as a substrate to the culture during the incubation period.

Compound (I) in the form of hydrochloride can be added after dissolution in sterilized distilled water. The preferred, but not limiting, mass concentration range of compound (I) in the culture is 50-200 μg/l. The culture is grown in a nutrient medium containing a carbon source, eg, an assimilable hydrocarbon, and a nitrogen source, eg, an assimilable nitrogen compound, or a proteinaceous material. Preference is given to carbon sources that include glucose, sucrose, glycerol, starch, corn starch, dextrin, molasses, etc. Suitable sources of nitrogen are: corn steep liquor, yeast extract, dry brewer's yeast, soy flour , cotton seed flour, corn flour, casein, fish meal, solid waste from distillation, animal peptone, meat extract, ammonium salts, etc. A combination of the above sources of carbon and nitrogen is suitable. It is not necessary to add trace metals such as zinc, magnesium, manganese, cobalt, iron, etc. to the fermentation medium, because tap water and unpurified ingredients are used as components of the medium before sterilization.

The biotransformation process can last approximately 72 hours to 8 days. The incubation temperature during biotransformation ranges from 25°C to 37°C, and the optimum is 29°C. The content of the container in which the transformation is carried out is aerated by shaking at about 250 rpm, or by mixing with sterile air, in order to facilitate the growth of microorganisms and improve the efficiency of the transformation process.

Analytical methods

The course of the microbial transformation reaction was monitored by taking a fermentation sample at different time intervals and extracting it at pH 8.0 with a mixture of dichloromethane: methanol (9:1). When a sample of the organic extract is subjected to thin layer chromatography (TLC), using as eluent a mixture of chloroform : methanol : acetic acid : water, in a volume ratio of 80 : 20 : 7 : 3, the compound FCE 24883 (II) appears at a mean Rf value of 0 .50 while 4'-deoxy-4'-iododoxorubicin (I) occurs at an Rf of 0.60. Quantitative determination of both anthracyclines can be carried out by TLC, using the above-mentioned elution systems, by scraping the corresponding intestinally stained zones and eluting with methanol, and finally determining spectrophotometrically at 496 μm.

Isolation procedure

The entire amount of fermentation broth, in which compound (I) has undergone conversion to FCE 24883 (II), is filtered using diatomaceous earth. The red mycelial cake is extracted with a water-miscible organic solvent, such as methanol and other lower alcohols, dioxane acetonitrile, and acetone. The use of acetone is preferred. The mycelial extracts are collected, concentrated under reduced pressure, combined with the filtered fermentation liquid, adjusted to pH 8.0, and then re-extracted with an organic solvent that is immiscible with water, such as n-butanol, chloroform, dichloromethane or preferably a mixture of dichloromethane : methanol : (9 : l). Organic extracts contain FCE 24883 (II), together with compound (I) and smaller amounts of degradation products.

Purification process

The organic extract is evaporated to dryness under reduced pressure, and the residue dissolved in dichloromethane is chromatographed on a column with silica gel, buffered to pH 7, with a gradient mixture of dichloromethane:methanol:water. First, compound (I) is eluted with a mixture of 95 : 5 : 0.25 and then FCE 24883 (II) with a mixture of 90 : 10 : 0.5. From the collected fractions, after washing with water, concentrating to a small volume, in the presence of n-propanol, adding an equivalent amount of hydrochloric acid and n-hexane, a precipitate of pure FCE 24883 (II) is obtained, as a hydrochloride, that is, pure FCE 24883 (II) is precipitated ) binding a molecule of hydrochloric acid to itself.

Chemical and physical properties

FCE 24883 (II) as a free base dissolves in weak organic solvents and aqueous alcohol solutions, while its hydrochloride dissolves in water and lower alcohols, and poorly in organic solvents. The hydrochloride compound FCE 24883 has the following physical and chemical properties:

melting point: 200°C (decomposes)

specific rotation: [α]23°D+188 ̊ (c 0.05, CH3OH)

U.V. and VIS absorption spectrum: [λ]H2 max 232, 254, 290 and 480 μm

(E1% 1cm = 492, 370, 127, 163)

I.R. spectrum (KBr): peaks at the following frequencies:

3400, 2970, 2920, 1610, 1580, 1472, 1440, 1410, 1380, 1355, 1320, 1280, 1235, 1210, 1110, 1080, 1060, 1030, 1010, 985, 90, 90, 90, 90, 90, 90, 94. 860, 830, 810, 785, 755, 730, 710, 540, 480, 450 and 415 cm-1

1H-NMR spectrum (DMSOd6, 200 MHz, 220°C): 14.03 (b. 2H, OH-6. OH-11), 7.6-79 (m, 3H, H-2, H-3) , 5.26 (m, 1H, H-1'), 4.96 (d, J=5.2 Hz, 1H, OH-13), 492 (m, 1H, H-7), 4.55 ( m, 1H, H-4'), 4.51 (t, J=6.7 Hz, 1H, OH-14), 4.20 (s, 1H, OH-9), 3.97 (s, 3H , 4.OCH3), 3.76 (ddd, J=3.5, 6.7, 11.0 Hz, CH(H)-OH), 360 (dq, J=1.0, 6.0 Hz, H-5'), 3.48 (ddd, J=7.2, 6.7, 11.0 Hz, 1H, CH-(H)-OH), 3.37 (ddd, J=5.2, 3.5, 7.2 Hz 1H, H-13), 3.02 (m, 1H, H-3'), 2.81 (m, 2H, CH2-10), 2.15 (dd, J= 2.0, 15.3 Hz, 1H, H-8e). 1.97 (dd,J=6.0, 15.3 Hz, 1H.H-8ax). 1.7-1.9 (m, 2H, CH2-2') and 1.14?(d, J=6.0 Hz, 3H, CH3,-5').

Molecular formula: C27H30NIO10 ∙ HCl

m/z in FD equivalent to free base: 656 [MH]+;

655 [M]+ corresponds to the aglycone.

The method of high-performance liquid chromatography (HPCL) enables the separation (two peaks with retention times of 18.8 and 19.3 min) of two C-13 stereoisomers of alcohol, present in the sample of synthetic 4'-deoxy-13-dihydro-4'-iododoxorubicin, prepared by means of NaBH4, by reduction of compound (I).

Applying the HPCL method, the compound FCE 24883 (II) appeared as a single peak, with a retention time of 19.3 min, which corresponds to the value of the component that moves more slowly in the synthetic 13-dihydro derivative.

* HPLC method

Column: two RP Speherisorb S30DS2 (C18 3 μ, Phase Separation U.K.) 150x4.5 mm, connected in a row

Temperature: 45 °C

Mobile phase A: 0.05 M aqueous KH2PO4, adjusted to pH 3.0 with 1 M H3PO4/CH3OH = 80/20 (vol.)

Mobile phase B: CH3OH

Elution: isocratic 30 min (42%A + 58%B)

Flow: 0.6 ml/min

Detection: 254 µm.

Explanation of the structure

Acid hydrolysis of compound (II) (0.2 N aqueous HCl solution, 80°C, 30 min) gives a red precipitate of the corresponding aglycone (III), while the sugar component is 3-amino-2,3,4,6-tetradeoxy-4 -iodo-L-lyxohexohexose (IV), present in the aqueous phase, identified after comparison with an authentic sample obtained by acid hydrolysis of compound (I).

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The absolute (S)-configuration at C-13 of compound (III) was determined by direct comparison (1H-NMR and mass spectrum, TLC) of its 9,13-0-isopropylidene-14-0-t-butyldiphenylsilyl derivative with the corresponding derivative of an authentic sample of 13-(S)-dihydroadriamycinone, obtained as described by S. Penco et. al. Gazetta Chimica Italiana, 115 195 (1985).

Biological activity

The cytotoxic activity of FCE (II) was tested "in vitro" on a colony of HeLa and P 388 cells, by comparison with compound (I) and doxorubicin. Compound (II) is as effective as 4'-deoxy-4'-iododoxorubicin (I) and doxorubicin (Table 1).

The "in vivo" antitumor activity of the compound FCE 24883 (II) was tested on disseminated Gross leukemia. C3H mice were intravenously injected with 2-10 6 tumor cells/mouse, and 24 hours later were administered test compounds.

Table 2 shows the results of the experiment. It can be seen that at the optimal dose, FCE 24883 (II) is more effective than doxorubicin, and equally as 4'-deoxy-4'-iododoxorubicin (I), with less pronounced toxicity as a means of survival time of treated mice compared to control mice and can be compare with that for compound (I) and doxorubicin.

Table 1

"In vitro" activity of 13-(S)-dihydro-4'-iodo-doxorubicin (H, FCE 24883) compared to 4'-deoxy-4'-iodo-doxorubicin (I, FCE 21954) and doxorubicin (Dx)

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a) Dose that reduces the number of cells by 50% compared to untreated controls.

b) Human neck epithelioid carcinoma cells

c) P388 leukemic cells

Table 2

Activity of 13-(S)-dihydro-4'-iodo-doxorubicin (H, FCE 24883) compared with 4'-deoxy-4'-iododoxorubicm (I, FCE 21945) and doxorubicin (Dx) against disseminated Gross leukemia

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a) C3H mice were given i.v. per 2∙106 leukemic cells, so after that they were given i.v. given a suitable match

b) (average survival time of treated mice/average survival time of control mice) x 100

c) concluded on the basis of the autopsy findings of a dead mouse

The following non-limiting examples are provided for a more detailed description of the processes and products of the subject invention.

Example 1

Culture of Streptomyces peucetius, species M 87 F.I. (DSM 2444) was grown for 14 days at 28°C on an agar medium with the following composition maintained (medium SA): glucose 3%, dry brewer's yeast 1.2%, NaCl 0.1%, KH2PO4 0.05% . sterilization was carried out by heating for 20 min in an autoclave at 115°C.

The spores of the cultures thus grown were collected and suspended in 3 ml of sterilized water; this suspension was introduced (inoculated) into a 300 ml Erlenmeyer flask containing 60 ml of liquid nutrient medium with the following composition: dry brewer's yeast 0.3%, peptone 0.5%, Ca(NO3)2 • 4H2O 0.05% , tap water up to 100 ml.

Sterilization was carried out in an autoclave for 20 min at 120°C. After sterilization, the pH of this medium is 6.8 to 7.0. The inoculated dishes were shaken for 2 days on a rotary shaker at 250 rpm turning in a circle with a diameter of 7 cm at a temperature of 28°C. In Erlenmeyer flasks with a volume of 300 ml, 1.5 ml of the culture, grown as described above, was placed in 50 ml of the following biotransformation medium: yeast extract 1.5%, KH2PO4 0.25%, glucose 1.5% tap water to 100 ml; pH 6.9; sterilized by heating for 20 min in an autoclave at 115°C. The glucose solution was sterilized separately, so it was added to each of the sterilized containers as much as needed.

The flasks were then incubated for 24 h, at 28°C, under the conditions described for the seeded phase. Then, 1.0 ml of a solution of compound (I) in sterilized distilled water, with a mass concentration of 5 mg/ml, was added to each flask. The flasks were shaken and incubated for 2 days longer, during which a 70% conversion of compound (I) to compound (II) was achieved.

Example 2

Culture of S. peucetius, species M 87 F.I. was grown on solid medium as described in example 1. Spores from three cultures were collected and placed in 10 ml of sterilized distilled water. The thus obtained suspension was introduced (inoculated) into a 2 1 collection flask with a round bottom, in which there is 500 ml of the seeded phase, described in example 1. The vessel was incubated for 48 hours on a rotary shaker at 120 rpm and rotating in a circle of diameter 7 cm at a temperature of 28°C.

The entire content of the culture was transferred to a stainless steel fermenter with a volume of 10 liters, which contains 7.5 l of the biotransformation medium described in example 1, and was sterilized for 30 minutes with steam at 120°C. The glucose solution was sterilized separately and the required amount added to the sterilized fermenter. The culture is grown at 28°C with agitation at 230 rpm, aerated with air at a flow rate of 0.7 liters/liter of medium/min. After 48 days, the substrate compound was added in the concentration as in example 1, and the culture was incubated for another three days, after which a 60% conversion of compound (I) into compound (II) was achieved.

Example 3

The entire amount of liquid (5 L) from the fermentation obtained according to example 2 was filtered using 2% diatomaceous earth. The wet extraction cake was extracted with acetone (3 1). After filtration, two additional acetone extractions were carried out, in order to ensure the extraction of red pigments. The collected acetone extracts were concentrated under reduced pressure, and the concentrate (1 liter) was combined with the filtered broth and thoroughly extracted, at pH 8, with a mixture of dichloromethane:methanol (9:1). The organic extract containing compounds (I) and (II) and some degradation products was evaporated to dryness under reduced pressure. The residue was dissolved in dichloromethane and chromatographed on a column with silica gel, buffered to pH 7 (M/15 phosphate buffer), with a gradient mixture of dichloromethane:methanol:water. After some degradation products, compound (I) was eluted with a 95 : 5 : 0.25 mixture, and then FCE 24883 (II) was isolated with a 90 : 10 : 0.5 mixture.

From the collected fractions, after washing with water, concentrating to a small volume in the presence of n-propanol, adding an equivalent amount of hydrochloric acid and an excess of n-hexane, pure FCE 24883 (II, 0.30 g, 60%) was obtained in the form of hydrochloride (m.p. 200°C, with decomposition). Following the same procedure, the unchanged compound (I) (0.13 g, 26%) was also recovered as the hydrochloride.

Example 4

The sample GCE 24883 (I) (200 mg) was dissolved in a 0.2 N aqueous solution of hydrochloric acid (50 ml) and heated for 30 min at 100°C. A red crystalline precipitate (0.12 g) of aglycone (III) was separated by filtration, washed with water and dried. Mass spectrum: m/e 416 (M+). The aglycone (III) was identified as 13-(S)-dihydroadriamycinone by comparison with an authentic sample.

Claims (5)

1. Process for preparing 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin of formula (II) [image] indicated by the fact that under aerobic conditions, in an aqueous culture medium containing an assimilable carbon source, an assimilable nitrogen source and mineral salts, a mutant of the Streptomyces peucetius species, designated as the M 87 F.I. species, is grown. (DSM 2444), in the presence of 4'-deoxy-4'-iododoxorubicin (I) [image] in the form of its hydrochloride and that the obtained 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin (II) is recovered in the form of hydrochloride. 2. The process according to claim 1, characterized in that it is carried out from 72 hours to 8 days, at a temperature of 25 °C to 37 °C, primarily at 29 °C. 3. The method according to claim 1 or 2, indicated by the fact that the anthracycline glycosamide of formula (I) is then extracted, according to claim 1, from the micellar cake with acetone, and from the filtered fermentation liquors with a mixture of dichloromethane:methanol, 9:1 (v/ v), at pH 8, and the purchased extracts are evaporated to dryness under reduced pressure. 4. The process, characterized by the fact that the crude product obtained by the process according to claim 3 is dissolved in dichloromethane and then purified by chromatography on a silica gel column, buffered at pH 7, whereby the mixture of dichloromethane : methanol : water is first used as the elution system . in the presence of n-propanol evaporates to a small volume, so the desired 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin (II) is recovered in its pure form as a hydrochloride, after adding an equivalent amount of hydrochloric acid and an excess of n-hexane. 5. A compound characterized by the following chemical structure: 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin hydrochloride, formula (II) [image]