PT87972B - A process for the preparation of 4'-deoxy-13 (S) -DIHYDRO-4'-IODODOXORUBYCIN WITH ANTI-TUMOR ACTIVITY BY MICROBIAL STEREO-SELECTIVE REDUCTION OF 4'-DEOXY-4'-IODODOXORUBICIN - Google Patents

Abstract

4'-iododoxrubicin of formula (I) and its salts are new. (I) is prepd. by culturing Streptomyces peucetius strain M87 F.I. (DSM2444) in the presence of (II) and recovering (I) as such or as a salt. The strain M87 F.I. is obtd. by mutating S.peucetius subsp. aureus ATCC 31428 using nitrosoguanidine.

Description

The present invention concerns a derivative of dodoxy rubicin, its preparation and pharmaceutical compositions containing it.

O _

According to the present invention, if the post 4 with ¹ deoxy-13 (S) -dihydro-4 -iododoxorubicina ^1, with trutu es ra formula (11)

and their pharmaceutically acceptable salts.

A microorganism belonging to the streptomyces genus is used for the stereoselective reduction of the functional ketone group linked to carbon 13 of the 4'-deoxy-4 ¹ -iodo-doxorubicin

-2Cs structure formula (I).

to specifically provide 4'-deoxy-13- (S) -dihydro-4'-1ododoxorubicin, one of two possible stereoisomers of carbon 13 of 4'-deoxy-13-dihydro-4'-iododoxorubicins. The new composition of formula (II), hereinafter referred to as FCE 24883, is useful as an anti-tumor agent and exhibits an activity comparable to that of 4'-deoxy-4 ¹ -iododoxorubicin (of structural formula) I). The substrate for microbial selective stereo reduction is a semi-synthetic analogue of doxorubicin described in our U.S. Patent A-4438105 of March 20, 1984.

More particularly, the present invention relates to a bio-synthetic process by which a mutant of the species of

Streptomyces pencetius, known as strain M87 F. I. and dep £

-3sited at Deutsche Sammlung von Mi kroorgani sm and which was registered with the deposit number DSM 2444, and which is characterized by its ability to stereoselectively reduce the functional group ketone-13 of the 4 '-deoxy-4 ¹ -iododoxorubicin (I). The resulting FCE 24883 (II) compound accumulates in the fermentation broths. FCE 24883 (II) can be collected from the fermentation broths and the purified crude solutions of its concentrate.

The present invention also provides the process for the preparation of 4 ¹ -deoxy-13 (S) -di-hydroxy-4 ¹ -iododoxirubicin of structure (II) formula or its pharmaceutically acceptable salts, which contain -system in the culture of the strain M 87 FI Streptomyces peuceti us (DSM 2444) in the presence of 4 ¹ -deoxy-4 '-iododoxorubicin and recovery of 4 ¹ - deoxy-1 3 (S) -di-hi dro-4'- i ododoxorubi ci_ na resulting or in the form of a pharmaceutically acceptable salt.

The present invention encompasses within its scope the new anthracycline anti-tumor agent FCE 24883 (11) in pure hydrochloride form.

The present invention also provides pharmaceutical compositions that incorporate the compound of formula (II) or a pharmaceutically acceptable salt thereof, together with a mixture of a pharmaceutically acceptable diluent or carrier.

Detailed Description of the Present Invention

The Streptomyces peucetius microorganism subspecies aureus ATCC 31428 is mutated using nitrosoguanidine to provide a laboratory microorganism called Streptomyces peucetius strain M 87 F. I. that selectively transforms compound (I) into compound (II). The strain M 87 F. I.

-4of _S. peuceti us was assigned the deposit number DSM 2444 by Deutsche Sammlung von Mikroorganism, West Germany, which was deposited in its permanent collection.

The morphology of the mutant strain M 87 F. I. is indistinguishable from the mother strain S_. peuceti us ATCC 31 428, although the cultures of both are clearly distinguishable in their cultural and biochemical characteristics.

In fact, the mutant strain M 87 F. I. does not produce in soluble medium agar the yellow-to-lemon yellow soluble pigment that characterizes the parent strain of Y peuceti us ATCC 31 428.

In addition, the mutant strain M. 87 F. I, can selectively transform the compound of formula (I) into the compound of formula (II), whereas the parent strain S. peuceti us ATCC 31428 is not selective regarding to this feature. This property of the M 87 F. I. mutant makes it highly useful, as described herein.

Transformation process

The selective stereo biotransformation of the present invention can be carried out in a S_ development culture. peucetius strain M 87 F. I. by adding the compound of formula I as a substrate, the culture, during the incubation period.

compound of formula I in the form of hydrochloride, can be added after dissolution in sterile distilled water. The preferred, but not limiting, concentration of the compound of formula I in the culture is between about 50-200 micrograms per liter. The culture is grown in a nutrient medium containing carbon sources, e.g. , a carbohydrate assimilable to a nitrogen bridge, e.g. eg an assimilable nitrogen compound or t

protein material. Preferred carbon sources are glucose, sucrose, glycerol, starch, corn starch, dextrin, molasses and the like. Preferred nitrogen sources are corn infusion liquor, yeast extract, dry beer yeast, soy protein, cottonseed protein, corn protein, casein, fish protein, distillation solids, animal peptide , meat extract, ammonium salts, etc. The combination of these sources of carbon and nitrogen can be utilized in an advantageous way. Necessarily, it will be necessary to add the trace foods to the fermentation medium, e.g. eg, zinc, magnesium, sodium manganese, cobalt, iron and others that are added with common water and in the form of unpurified ingredients prior to sterilization of the medium.

The biotransformation process can take between 72 hours and 8 days. The incubation temperature during the biotransformation process can vary between about 25 ° C and 37 ° C, with a temperature of 29 ° C being the most appropriate. The contents of the transformation vessels are aerated by stirring at about 250 rpm or by stirring with sterile air, to favor the development of the microorganism and to enhance the efficiency of the transformation process.

Analytical methods The microbial transformation reaction process is controlled by taking fermentation samples at different time intervals and extracting with a 9: 1 mixture of dichloromethane: methanol with a pH of 8.0. When a sample of the organic extracts is subjected to thin layer chromatography (TLC) using a mixture of chloroform, methanol, acetic acid, water 80: 20: 7: 3 (by volume) as an element, it appears that O

-6 / ^{ν -χ} 'FCE 24883 compound of formula II appears with an average value of ί Rf 0.50, whereas 4 ^{1 1} -deoxy-4 -iododoxorubicina of formula I having an Rf of 0.60. The quantitative evaluation of the two anthracyclines can be carried out after thin layer chromatography, using the system of elements mentioned above, by scraping and eluting with methanol from the corresponding red stained zones and finally by determination with spectrophotometry at 496 nm.

Insulation process

The total fermentation broths in which the compound of formula I was subjected to conversion into FCE 24883 of formula II, are filtered with the aid of diatomasia soil. The red mycelium cake is extracted with a water-miscible organic solvent, such as methanol or other lower alcohol, dioxane, nitrile acetate, acetone, preferably using acetone. The mycelium extracts are collected, concentrated under reduced pressure and added to the filtered fermentation liquids; its pH is adjusted to 8.0, after which it is extracted with an organic solvent not miscible with water, such as n-butanol, chloroform, dichloromethane or, preferably, a mixture of 9: 1 dichloromethane: methanol . Organic extracts contain FCE 24883 of formula II, together with the compound of formula I and some minor degradation products.

Purification process organic extract Ó concentrated under reduced pressure to dryness and the residue is dissolved in dichloromethane and chromatographed on a column of silica gel, buffered at pH 7, with a gradient consisting of a mixture of dichloromethane, methanol, water . The compound of formula I is first eluted with a mixture at 95, 5, 0.25, followed by FCE 24883 of formula II with a mixture

-7 to 90, 10, 0.5.

The combined fractions, after washing with water, are concentrated to a small volume in the presence of n-propanol, added with hydrochloric acid in the proportion of an equivalent and an excess of n-hexane, obtaining a precipitate of FCE 24883 of formula II in the pure form of hydrochloride.

Physical and chemical properties

FCE 24883 of formula II is a free base soluble in polar organic solvents and in aqueous alcohols, whereas calcium hydrate is soluble in water and lower alcohols, but little soluble in organic solvents. FCE 24883 hydrochloride has the following physicochemical properties:

Melting point: 200 ° C (with decomposition) Specific rotation: + 188 ° θ ³ ° (c 0.05, CH ₃ 0H) UV absorption spectrum and H VIS absorption esoectro _to 0 232, 254, and 290 480 nm cm α X (E] ^% Cm ^{= 492} > ³⁷⁰ ' ¹²⁷ ' ¹⁶³ ) ·

I.R. (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, 965, 940, 920, 900, 890, 870, 860, 830, 810, 785, 755, 730, 710, 540, 480, 450 and 415 cm ^-1 .

1H - NMR spectrum (DMSOd _g , 200 MHz, 22 ° C): 14.03 (bs, 2H, OH-6, 0H-11), 7.6-7.9 (m, 3H, Hl, H-2, H-3 ), 5.26 (m, ΙΗ, Η -1 ¹ ),

4.96 (D, J = 5.2 Hz, ΙΗ, 0H-13), 4.92 (m, 1H, H-7), 4.55 (m, 1H,

H-4 '), 4.51 (t, J = 6.7 Hz, ΙΗ, 0H-14), 4.20 (s, ΙΗ, OH-9), 3.97 (s, 3H, 4-0CH ₃ ), 3.76 (ddd, J = 3.5, 6.7, 11.0 Hz, 1H, CH (H) -OH),

3.60 (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

-8-.

(m, IH, H-3 '), 2.81 (m, 2H, CH ₂ -10), 2.15 (dd, J = 2.0, 15.3 Hz,

IH, H-8e), 1.97 (dd, J = 6.0, 15.3 Hz, IH, H-8ax), 1.7-1.9 (m, 2H, CH ₂ -2 ') and 1 .1 4 (d, J = 6.0 Hz, 3H, ÇH_ ₃ -5 *).

Molecular formula: Ο ^ Η ^ θΝΧ ^ θ.ΗΠ m / 2 in FC equivalent to free base: 656 MH 655 M; and 416 (M) corresponding to the aglycone.

Selective high pressure liquid chromatography (HPLC) allows the separation of the two stereoisomeric alcohols at C-13 (two peaks with retention buffer 18.8 and 19.3 minutes) present in a sample of 4 ¹ -deoxy-13-di- synthetic hydro-4 ¹ -iododoxorubicin prepared by NaBH4 reduction of the compound of formula I.

Using the same method as (HPLC), FCE 24883 of formula II is presented as a single peak with a retention time of 19.3 minutes, which corresponds to the least mobile constituent of the 13-dihydro derivative synthetic.

HPLC process

Column: two 150 x 4.5 mm Spherisorb S30DS2 (C18 S ^ j, Phase Separation U.K) columns connected in series.

Temperature: 45 ° C.

Mobile phase A: 0.05M aqueous KH ₂ P0 ^, with pH 3.0 with H ₃ P0 ₄ lM / CH ₃ 0H = 80/20, by volume

Mobile phase B: CH ₃ 0H

Elution: isocratic for 30 minutes (42% A + 58% B) Flow rate: 0.6 ml / minute Detection: 254 nm.

ί Structure clarification

Acid hydrolysis of the compound of formula II (0.2 N aqueous HCl, 80 ° C, 30 minutes) provided a red precipitate of the corresponding agglomerate of formula III, while the constituting sugar, namely 3-amino-2, 3,4,6-tetradioxy-4-iodo-L-1ixo-hexohexose of formula IV, present in the aqueous phase, was identified after comparison with an authentic sample obtained after acid hydrolysis of the compound of formula I.

III: 13-‘5) - IV

The absolute configuration (S) at Carbon 13 of the compound of formula III was determined by direct comparison (proton magnetic-nuclear resonance and the mass spectrum and thin layer chromatography) of its derivative 9,13-0-isopropylidene-14-0 -t-butyl diphenyl as the corresponding derivative of an authentic sample of 13-S-dihydroadriamycinone, obtained according to metho-10 / /

as described by S. Penco et al. in Gazzetta Chimica Italiana, 115, 195, 1985.

Biological activity

The cytotoxic activity of FCE 24883 of formula II was evaluated in in vitro assays on colony formation of Hela cells and 388 cells compared to the compounds of formula I and with doxorubicin. As indicated in Table I, compound II resulted Fo £ mule as present as deoxy-4 ¹ 4 ¹ -iododoxorubicina of formula I and doxorubicin.

The anti-tumor activity in vivo of FCE 24883 of formula II was tested against total disseminated leukemia. C3H mice were injected intravenously with 2.10θ cells / mice treated with the compounds under study 24 hours after tumor injection.

Table II shows the results of the two experiments.

At the optimum dose, FCE 24883 II was found to be more potent than doxorubicin and as potent as 4 ¹ -deoxy-4 ¹ -i ododoxorubin (I), but with less toxicity exhibited at the active doses.

The anti-tumor activity of the compound of formula II evaluated as an average survival time of control mice under treatment could be compared with that of the compounds of formula I and xorubin.

In vitro activity of 1 3- / s7'di hyd ro-4 ¹ - i ododoxorubicin (II, FCE 24883) compared to 4'-deoxy-4'-iododoxorubicin (I, FCE 21954) and doxorubicin (Dx) .

c) P388 leukemia cells /

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The following non-limiting examples are provided in order to describe in detail the processes and products covered by the present invention.

Description of preferred aspects

Example 1

A culture _of Sjtreptomyces peuceti us, strain M 87 FI (DSM 2444) was grown for 14 days at 28 ° C on agar plates followed by maintenance in medium (SA medium), constituted as follows: glucose 3 %; dry beer yeast 1.2%; Sodium Chloride 0.1%; KH ₂ P0 ₄ 0.05%; CaCOg, 0.1%; MgSo ₄ , 0.005%; FeS0 ₄ 7K ₂ 0, 0.0005%; ZnS0 ₄ · 7H ₂ 0, 0.0005%; CuSO. .5H ₂ 0, 0.0005%; 2% agar; common water 1000 ml; pH 6.7; sterilization was performed by heating in an autoclave at 115 ° C for 20 minutes.

The spores obtained from this culture were collected and suspended in 3 ml of sterile distilled water; this suspension was inoculated in 300 ml Erlenmeyer flasks, containing 60 ml of the following liquid medium; dry beer yeast, 0.3%; 0.5% peptone; Ca (NOg) ₂ .4H ₂ 0, 0.05%; ordinary water 100 ml. Sterilization by heating in an autoclave at 120 ° C for 20 minutes. The pH of this medium, after sterilization, was between 6.8 and 7.0. The inoculated flasks were shaken for two days at 28 ° C on a rotary shaker at 250 rpm and a 7 cm diameter circle. 1.5 ml of the growth culture, described above, was inoculated into 300 ml Erlenmeyer flasks containing 50 ml of a biotransformation medium, as follows: yeast extract 1.5 '; KH _{2 0> 25%} . _{glucose 1>5%;}

-14 common water up to 100 ml pH 6.9; sterilization was performed by heating in an autoclave at 115 ° C for 20 minutes. The gose solution was sterilized separately and added to each sterile vial with the concentration

Then the vials, 28 ° C under the conditions described for 24 hours. At this point, I added a solution containing the tilted compound, with a concentration of ram incubated for two more than 70% of the appropriate formula I compound.

were incubated at the temperature for the sowing phase, 1 ml of formula I was ionized in each vial in sterile water of 5 mg / ml. The flasks were shaken slightly, obtaining a conversion to the compound of formula II.

Example 2

A culture of (3. peuce ti us of the strain M 87 FI developed in solid medium as described in Example I. The spores from three planes were removed and placed in 10 ml of sterile distilled water; the suspension obtained by this method was inoculated into an airy round-bottom flask containing 500 ml of the sowing medium described in Example 1. The flask was incubated for hours on a rotary shaker at 120 rpm, describing a 7 cm diameter circle at a temperature of 28 ° C. The total seed was inoculated in a 10 l stainless steel fermenter containing 7 and a half liters of the biotransformation medium described in example 1 and steam sterilized at 120 ° C for 30 minutes, having sterilized separately the glucose solution and added in the appropriate concentration to the sterilized fermenter. The culture was developed at a temperature of 28 ° C with agitation of 230 rpm and aeration with an air flow of 0.7 liters

medium / minute. After 48 hours the substrate compound was added at a concentration as described in example 1, and the culture was incubated for an additional 3 days, obtaining 60% conversion of compound 1 to the compound of formula II.

Example 3 Complete broth, 5 1, from a fermentation obtained according to the description in example 2, was filtered using a 2% diatomaceous earth filter. 0 wet filter

-it was extracted with 3 liters of acetone. After filtration, two more extractions were made with acetone to ensure complete collection of the red pigments. The combined acetone extracts were concentrated under reduced pressure to a volume of one liter, combined with the filtered broth and exhaustively extracted at pH 8 with dichloromethane-methanol in a 9: 1 mixture. The organic extract, containing the compounds of formula 1 and 2, with some degradation products, was concentrated under reduced pressure to dryness. The residue dissolved in dichloromethane was chromatographed on a column of silica gel, buffered to pH 7 (phosphate buffer M / 15) with a gradient consisting of a mixture of dichloromethane, methanol, water. Then some degradation products of the compound of formula 1 were eluted with a 95: 5: 0.25 mixture and then eluted the compound FCE 24883 of formula II with a 90: 10: 0.5 mixture.

From the combined fractions, after washing with water, concentrating to a small volume in the presence of n-propanol, adding the hydrochloric acid in the proportion of an equivalent with an excess of n-hexane, the compound FCE 24883 of formula was obtained

-16II, in pure form (0.30 g, yield 60%), in the form of hydrochloride with a melting point of 200 ° C, with decomposition. Then, to this process, the compound of formula I, untransformed (0.13 g, 26% yield) was also collected in the form of hydrochloride.

Example 4

A sample of FCE 2483 of 200 mg (I) was dissolved in 0.2 N aqueous hydrochloric acid (50 ml) and heated for 30 minutes at 100 ° C. Filtration collected a crystalline medium (0.12 g) of formula III aglycon which was washed with water and dried.

Mass spectrum: m / e 416 M ⁺ . The formula III aglycon was identified as 13- (S) -dihydroadriamycininone by comparison with an authentic sample.

Claims (5)

Claims 1.- Process for the preparation of 4'-deoxy-13 (S) -dihydro-4'-iododoxorubicin of formula and its pharmaceutically acceptable salts, characterized by the fact that a mutant strain, called strain M 87 FI (DSM 2444), of the species Streptomyces peucetius, under aerobic conditions in an aqueous culture medium _ Ί η J_O containing an assimilable carbon source, an assimilable nitrogen source and mineral salts, in the presence of 4'-deoxy-4'-iododoxoru bicina de formula / f as its hydrochloride, and to isolate the resulting 4'-deoxy-13 (S) -dihydro-4'-iododoxorubicin of formula II as its hydrochloride. 2. Process according to claim 1, characterized in that the compound of formula II is obtained in the form of its hydrochloride. 3. A process according to claim 1, characterized in that it is carried out at a temperature between 25 ° C and 37 ° C, preferably at 29 ° C, for a period from 72 hours to eight days. 4.- Process according to claim 1, characterized by 19 It is used by extracting the anthracycline glycoside from. formula II of the mycelium mass with acetone and the fermentation liquids filtered with a mixture of dichloromethane / methanol 9: 1 v / v at a pH equal to 8, the extracts are combined and the combined extracts are evaporated to dryness under reduced pressure. 5. A process according to claim 1, characterized in that the crude product obtained is dissolved in dichloromethane, the product is subjected to purification by chromatography on a silica gel column buffered to pH7, and the initial compound of formula I that did not react by means of an eluent system consisting first of a mixture of dichloromethane: methanol: water 95: 5: 0.25 v / ve in sequence, by a mixture of dichloromethane: methanol: water 90 : 10: 0.5 v / v of concentrating the eluted fractions to a small volume in the presence of n-propar.ol, and of isolating 4 '-deoxy-13 (S) -dihydro-4'-iododoxorubicin from formula II in pure hydrochloride by adding an equivalent of hydrochloric acid and an excess of n-hexane, Lisbon, July 12, 1988 The Otic Hot Property