CS272792B2 - Method of 4-deoxy-13/s/-dihydro-4-iodofuricine production - Google Patents

Abstract

The invention concerns the microbiological method of producing 4'-deoxy-13(S)-dihydro-4'-idarubicin of formula II, where, in aerobic conditions and in an aqueous culture medium containing a source of carbon, nitrogen and inorganic salts, a mutant of the family Streptomyces peucetius, strain M 87 F.I. (DSM 2444) is cultivated in the presence of 4'-deoxy-4'-iododoxorubicin of formula I, in the form of hydrochloride, and the resulting 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin of formula II is isolated in the form of hydrochloride. The product has anti-tumour effect.<IMAGE>

Description

[57] The invention relates to a microbiological process for the production of 4'-deoxy-13 (S) -dihydro-4-iodo-dicricin of formula II by culturing under aerobic conditions in an aqueous nutrient medium containing a usable carbon, nitrogen and inorganic salt of a Streptomyces peucetius mutant. strain M 87 FI (OSM 2444) in the presence of 4'-deoxy-4'-iodo doxorubicin of formula I, in the form of hydrochlorides, and the resulting 4-deoxy-13 (S) -dihydro-4'-iodo-doxorubicin of formula II is isolated as hydrochlorides. The product has an antitumor effect.

Italy (11) (13) B 2 (51) Int. Cl. ⁵ C 07 H 15/24 A 61 K 31/71

The present invention relates to a microbiological process for the preparation of 4'-deoxy-13 (S) -dihydro-4'-iodoic acid. It is an effective new anticancer drug.

The present invention therefore a method of producing 4'-deoxy-13 (S) ^-dihydro-4, Formula II -joddoxorubicinu

II and the pharmaceutically acceptable salts thereof.

This is done by cultivating under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts, a mutant of the species Streptomyces peucetius strain M 87 FI (DSM 2444) in the presence of ^{4-deoxy-4'-iododoxorubicin} of formula AND

whereby a stereoselective reduction of the ketone group at the 13-position of the compound is obtained, so that only one of the two possible 4-deoxy-13-dihydro-4-iodo-oxorubicin stereoisomers is formed.

The novel compound of formula II, designated FCE 24883, is useful as an antitumor agent and has, in experimental tumors, an effect comparable to that of 4'-deoxy-4-iodo-doxorubicin of formula I. The substrate for stereoselective reduction is a semisynthetic analogue of doxorubicin.

The method according to the invention is a biosynthetic process in which a mutant of the family Streptomyces peucetius, strain M 87 FI, deposited in the collection of Deutsche Sammling vom Microorganism under number DSM 2444, is capable of stereoselectively reducing the ketone functional group at position 13 of the starting The compound FCE 24883 of formula II resulting from this process accumulates in the culture medium. The substance can then be isolated from the fermentation broth, concentrated and further purified.

Accordingly, the pharmaceutically acceptable 4'-Deoxy-13 (S) -dihydro-4-iodo-xorubicin of formula II or a pharmaceutically acceptable salt thereof can be obtained by growing Streptomyces peucetius strain M 87 F.I. (DSM 2444) in the presence of 4'-deoxy-4'-iodo-oxorubicin and then recovering the resulting 4'-deoxy-13 (S) -dihydro-4'-iodo-oxorubicin in free form or in the form of a pharmaceutically acceptable salt thereof.

Particularly preferred is the resulting compound in its pure form or its hydrochloride.

The compound may be formulated into a pharmaceutical composition comprising a compound of formula II or a pharmaceutically acceptable salt thereof as an active ingredient in admixture with a pharmaceutically acceptable diluent or carrier.

... Mutation of Strepromyces peucetius subsp. aureus ATCC 31428 was achieved by treatment with nitrosoguanidine to form a laboratory microorganism, which was designated Streptomyces peucetius strain M 87 F.I. and which is capable of selectively transforming a compound of formula I to a compound of formula II. S. peucetius strain M 87 F.I. was deposited under DSN 2444 in the collection of Deutsche Sammlung von Microorganism, Germany, and forms a permanent part of this collection.

Morphology of the M 87 Mutant F.I. It is indistinguishable from the morphology of the original S. peucetius strain ATCC 31428, but culture media are clearly discernible by their growth and biochemical properties. Mutant M 87 F.I. does not form a straw yellow to lemon yellow soluble pigment on agar media, which is a hallmark of the original S. peucetius strain ATCC 31428.

In addition, the mutant is M 87 F.I. capable of selectively transforming a compound of formula I to a compound of formula II, whereas the original S. peucetius strain ATCC 31428 is non-selective in this sense. This property of the mutant makes the mutant highly useful in the above sense.

Transformation

The stereoselective biological transformation underlying the process of the invention can be carried out in a growing culture of S. peucetius M87 F.I. by adding a compound of formula I to the culture as a substrate during incubation.

The compound of formula (I) may be added as the hydrochloride after dissolution in sterile distilled water. The preferred concentration range of the compound of formula I in the nutrient medium is 50 to 200 µg / L, but the method is not to be limited to this concentration range. Said microorganism is grown in a culture medium containing a carbon source, for example, useful carbohydrates, a nitrogen source, for example, a useful nitrogen compound or proteinaceous material. Useful preferred carbon sources include glucose, sucrose, glycerol, starch, corn starch, dextrin, molasses, and the like. A preferred nitrogen source is, for example, corn steep liquor, yeast extract, dried brewer's yeast, soy flour, cottonseed flour, corn flour, casein, fish flour, distillers, animal peptone, meat extract, ammonium salts and the like. Preferably, a combination of carbon and nitrogen sources may be used. It is not necessary to add trace elements such as zinc, magnesium, manganese, cobalt, iron and the like to the fermentation broth, since the tap water and the unpurified components contain the trace elements mentioned as nutrient media.

The biological transformation can be performed for 72 hours to 8 days. The temperature during the biological transformation may range from 25 to 37 ° C, preferably at 29 ° C. The contents of the transformation vessels are aerated by shaking at 250 rpm or by stirring using sterilized air, thereby promoting the growth of the microorganism used, thereby increasing the efficiency of the transformation process.

Analytical methods

The microbiological transformation process can be monitored by taking samples of the fermentation broth at various intervals and extracting them at pH 8.0 with a 9: 1 mixture of dichloromethane and methanol. thin layer, eluting with a mixture of chloroform, methanol, acetic acid and water in an 80: 20: 7: 3 by volume ratio, the FCE 24883 compounds of formula II have mean Rj values of 0.50, while 4-deoxy-4'- ioddoxorubicin of formula I has an R _{f of} 0.60. The quantitative determination of both anthracycline derivatives can be carried out after thin layer chromatography using the above-mentioned elution system by removing the corresponding red-colored portions and eluting with methanol, followed by spectrophotometric determination at 496 nm.

Isolation

The resulting broth in which the compound of formula I was converted to the compound of formula II is filtered using diatomaceous earth. The red mycelial cake is extracted with an organic solvent miscible with water, for example methanol or other lower alcohol, dioxane, acetonitrile, acetone, preferably acetone. The mycelial extracts are combined, evaporated under reduced pressure and combined with the filtered fermentation broth, adjusted to pH 8.0 and then extracted with a water-immiscible organic solvent such as π-butanol, chloroform, dichloromethane, preferably a mixture of dichloromethane and The organic extracts contain the compound FCE 24883 of formula II together with the compound of formula I and, to a lesser extent, some degradation products.

Cleaning

The organic extract was evaporated to dryness under reduced pressure, the residue was dissolved in dichloromethane and then chromatographed on a silica gel column with pH 7 using an increasing gradient of dichloromethane in methanol / water. Compound I elutes first at a 95: 5: 0.25 volume ratio and then elutes FCE 24383 of Formula II. In a mixture of the above components in a volume ratio of 90: 10: 0.5. The fractions containing this material were combined, washed with water, evaporated to a small volume in the presence of n-propanol, treated with an equivalent amount of hydrochloric acid and an excess of π-hexane to give a precipitate of pure hydrochloride of compound FCE 24883 of formula II.

Chemical and physical properties

FCE 24883 of formula II in free form is soluble in polar organic solvents and in a mixture of water and alcohols, whereas its hydrochloride is soluble in water and lower alcohols, but only slightly soluble in organic solvents. The hydrochloride of FCE 24 883 has the following physical and chemical properties:

Melting point: 200 DEG C. with decomposition

Specific rotation M + 188 ⁰ (c 0.05, CH, OH) ⁰ , η 0

Absorption spectrum in ultraviolet light: 1/2 max

232, 254, 290 and 480 nm (? ^1% = 492, 370, 127, 163).

cm

The infrared spectrum in potassium bromide has peaks at: 3,400, 2970, 2920, 1610, 1580,

472, 1 440, 1 410, 1 380, 1 355, 1 320, 1 280, 1 235, 1 210, 1 110, 1 080, 1 060, 1 030, 1 O10,. 985, 965, 940, 920, 900, 890, 870, 860, 830, 810, 785, 755, 730, 710, 540, 480, 450, and 415 cm ^-1 .

¹ H-NMR spectrum (DMSOd 6, 200 MHz, 22 ° C):

14.03 (broad (hereinafter b, s, 2H, OH-6, OH-11, 7.6-7.9 (m, 3H, H1, H.-2, H-3)), 5.26 (m , 1H,

Δ-Γ), 4.96 (d, O = 5.2 Hz, 1H, OH-13), 4.92 (m, 1H, H-7) _; 4.55 (m, 1H, H-4) 4.51 (t, = 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, 1H, CH (H) -OH), 3.60 (dq, 3 = 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, D = 5.2, 3.5, 7.2 Hz, 1H, H-13), 3.02 (tn, 1 H, H-3 '), 2.81 (m, 2H, CH ₂ -10), 2.15 (dd, 3 = 2.0, 15.3 Hz, 1H, H-Be), 1 , 97 (dd, J = 6.0, 15.3 Hz, 1H,

H-Bax), 1.7 to 1.9 (m, 2H, CH ₂ -2 '), and cf 1.14 (d, 3 = 6.0 Hz, 3H, CH-5').

Molecular formula: C ^ HH gNNlO. ^. HCl mass spectrum in free base equivalent: 656 (MH) ⁺ , 655 (M) ⁺ and 416 (M) ⁺ , corresponding to aglycone.

In HPLC, two peaks with a retention time of 18.8 and 19.3 minutes can be separated, the two peaks corresponding to the two stereoisomeric alcohols present in the synthetic 4-deoxy-13-dihydro-4'-iodo-oxorubicin sample, which was prepared by reducing the compound of formula I with sodium borohydride.

When using high pressure liquid chromatography, FCE 24883 of formula II appears to be a single peak with a retention time of 19.3 minutes, corresponding to a slower moving

EN 272792 02 component of the synthetic 13-dihydroderivative.

High pressure liquid chromatography

Columns: two columns of RP Spherisorb inhaler resin. S30DS2 (C18, 3 (rm, Phase Separation U.K) 150 x 4.5 mm, columns were sequentially in series.

Temperature: 45 ° C

Mobile phase A: 0.05 M aqueous potassium dihydrogen phosphate solution, adjusted to pH 3.0 by the addition of an 80:20 mixture of 1M phosphoric acid and methanol.

Mobile phase B: methanol

Elution: Isocratic for 30 minutes (42 MA + 58 MB)

Flow rate: 0.6 ml / min.

Detection: at 254 nm.

Lighting structure

Hydrolysis of the compound of formula II under acidic conditions for 30 minutes at 80 ° C in a 0.2 N aqueous hydrochloric acid solution yields a red precipitate of the corresponding aglycone of formula III, while the sugar component, J-amino-2,3. The 4,6-tetra-deoxy-4-iodo-L-lyxohexohexose of formula IV present in the aqueous phase was identified by comparison with an authentic sample obtained by hydrolyzing the compound of formula I in an acidic medium.

The absolute configuration (S) - at position 13 of compound III was determined by direct comparison of 1 H-NMR spectrum, mass spectrum and thin layer chromatography 9,13-0-isopropylidene CS 272792 B2

The 14-O-tert-butyldiphenylsilyl derivative with the corresponding authentic sample of 13- (S) -dihydroadriamycinone, obtained by the method of S. Penoo et al., Gazzet ta Chimica Italiana, 115, 195 (1985).

Biological efficacy

The cytotoxic activity of FCE 24883 of Formula II was investigated in vitro for colony formation of HeLa cells and P 388 'cells' compared to that of Formula I and doxorubicin. As further evident from the data in Table I, the compound of formula II appears to be as active as 4'-deoxy-4'-iodo-doxorubicin of formula I and doxorubicin.

The anti-tumor activity of FCE 24883 of formula II in vivo was investigated against multiple Gross leukemia. C3H mice were infected intravenously with 2 x ^{10 6} cells / mouse and then treated with the compounds 24 hours after tumor infection.

Table II shows the results of two experiments. At the optimal dose, FCE 24883 of Formula II was more potent than doxorubicin and as effective as 4'-deoxy-4'-iodo doxorubicin of Formula I at lower toxicity at effective doses. The antitumor activity of the compound of formula II, evaluated as the median survival of treated animals versus control, can be compared to that of the compound of formula I and doxorubicin.

Table I

In vitro activity of 13- (S) -dihydro-4'-iodo doxorubicin FCE24883 of formula II compared to the activity of 4'-deoxy-4'-iodo doxorubicin FCE 21954 of formula I and doxorubicin (Dx)

(a) Compound Ιϋ ng / ml

Hela ⁰ · ¹ P388 ^(c)

Dx

FCE 21954 (I)

10,8 8

10.4 2.1

3.5 dose providing a 50% reduction in cell counts compared to the control of b-cell epithelioid carcinoma of the human cervix c ^ leukemia cell P388

Table II

The efficacy of 13- (S) -dihydro-4'-ioddoxorubicin FCE24883 of formula IIf in comparison with the activity of 4'-deoxy-4'-ioddoxorubicin FCE 21954 of formula I and doxorubicin (Dx) against disseminated Ross leukemia

CS 272792 Β2

Compound- dose ^a ) (mg / kg) T / CX ^(b) - death due to toxicity Dx 10 200 (200) 0/20 13 225 (230) 0/20 16.9 250 (260, 240) 2/20 FCE 21954 '4 240 (240) 0/20 5.2 260 (260) 4/20 6.8 145 (159, 130) 19/20 FCE 24883 4 180 (180) 0/10 5.2 220 (220) 0/20 6.8 220 (220) 0/20 8.8 140 (140) 5/9

C3H mice were administered intravenously 2. 10 µl of leukemia cells and then mice were treated intravenously the day after tumor infection, the mean survival time of the treated mice / mean survival time of the control x 100 was evaluated based on the autopsy findings in the mice.

The following examples illustrate the process of the present invention.

He did

Culture Streptomyces peucetius strain M 87 F.I. OSM 2444 is grown for 14 days at 28 ° C on sloping agar on the following maintenance medium (SA medium): glucose 3%, dried brewer's yeast 1.2%, sodium chloride 0.1%, potassium dihydrogen phosphate 0.05X, calcium carbonate 0.1 X, magnesium sulfate .0.005 X, ferrous sulfate.

. 7Η ₂ 0 0.0005 X, zinc sulphate. 7H ₂ 0 0.0005 X, copper sulphate. 5H ₂ 0. 0.005 X, agar 2X tap water to 100 mL, pH 6.7. Sterilization is performed by heating in an autoclave for 20 minutes at room temperature. 115 ° C.

The spores of the culture thus grown are isolated and suspended in 3 ml of sterile distilled water. The suspension is then used to inoculate 300 ml Erlenmeyer flasks containing 60 ml of the following liquid broth: dried brewers yeast 0.3X, peptone 0.5X, calcium nitrate. 4H ₂ 0 0.05 X, tap water up to 100 ml. Sterilization is performed by heating in an autoclave at 120 ° C for 20 minutes. After sterilization, the pH of this medium is in the range of 6.8 to 7.0. The inoculated flasks are shaken for 2 days at 28 ° C on a rotary shaker at 250 rpm. The shaker swing has a diameter of 7 cm.

Then, 1.5 ml of this culture is used to inoculate 300 ml Erlanmayer flasks containing 50 ml of the following biotransformation, nutrient medium: yeast extract 1.5X, potassium dihydrogen phosphate 0.25 X, glucose 1.5 X, water from water to 100 ml. The medium is pH 6.9: This medium is then sterilized by heating in an autoclave at 115 ° C for 20 minutes. The glucose solution is sterilized separately and added to each sterilized flask to the appropriate concentration.

The flasks were then incubated as above for 24 hours at 28 ° C. 1.0 ml of a solution of compound I in sterile distilled water is then added to each flask to a concentration of 5 mg / ml. The flasks are then incubated for a further 2 days with shaking to give 70% conversion of compound I to compound II.

Example 2

Culture of S. peucetius strain M 87 F.I. The spores from three cultures on slanted agar are collected and collected in 10 ml of sterile distilled water and the suspension thus obtained is inoculated into a 2-liter round-bottomed flask containing 500 ml of growth medium. The flask was incubated for 48 hours on a rotary shaker at 120 rpm with a 7 cm swing at 28 ° C. The entire contents of the flask are used to inoculate a 10-liter stainless steel fermenter containing 7.5 liters of the biotransformation medium of Example 1, steam sterilized at 120 ° C for 30 minutes, the glucose solution is sterilized separately and added at the appropriate concentration. to the sterilized fermenter. Thereafter, the culture is grown under stirring at 28 ° C at 230 rpm and aerated with air passing through at a rate of 0.7 liters per liter of culture medium per minute.

After 48 hours, the substrate was added as in Example 1 and the culture was incubated for a further 3 days to obtain a 60% conversion of Compound I to Compound II.

Example 3 liters of the fermentation broth obtained in Example 2 are filtered using 2% diatomaceous earth as a filter aid. The wet cake was extracted with 3 L of acetone. After filtration, two further extractions of acetone are carried out to achieve complete extraction of the red pigments. The acetone extracts are combined, evaporated under reduced pressure and 1 liter of the concentrate is mixed with the filtered broth and extracted thoroughly at pH 8 with a 9: 1 mixture of dichloromethane and methanol. Organic extract containing compounds I and II and some degradation products Evaporate to dryness under reduced pressure. The residue was dissolved in dichloromethane and chromatographed on a silica gel column in M / 15 phosphate buffer □ pH 7 using a gradient of a mixture of dichloromethane, methanol and water. Some degradation products leave first, then compound I is eluted with a 95: 5: 0.25 by volume mixture and then FCE 24883 Formula II with a 90: 10: 0.5 mixture.

From the pooled fractions, after washing with water, concentrating to a small volume in the presence of n-propanol and adding an equivalent of hydrochloric acid and excess n-hexane, a total of 0.30 g of pure FCE 24883 of formula II is obtained as the hydrochloride, m.p. C with decomposition. In the same way, a total of 0.13 g of unconverted compound I is obtained in the form of the hydrochloride in a yield of 26%.

Example4

A sample of 200 mg of FCE 24883 of Formula II was dissolved in 50 mL of a 0.2 N aqueous hydrochloric acid solution and heated at 100 ° C for 30 min. 0.12 g of a crystalline red precipitate of the aglycone of formula III is then collected by filtration, washed with water and dried.

Mass spectrum: m / e 416 (M ^& lt ^{; +} & gt ^; ). The aglycone of formula III was identified as 13- (S) -dihydroadriamycinone by comparison with an authentic sample.

Claims (4)

SUBJECT OF THE INVENTION 1. A process for producing ^4> deoxy-13- (S) -dihydro-4'-jodrubícínu II microbiological process, characterized in that it is grown under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts a mutant of the family Streptomyces peucetius, strain M 87 FI (DSM 2444) in the presence of 4'-deoxy-4'-iodo-xorhicin of formula I I. HCl as the hydrochloride and the resulting 4'-deoxy-13- (S) -dihydro-4-iodo-oxorubicin of formula II is isolated as the hydrochloride. 2. The method according to claim 1, wherein the cultivation is carried out at a temperature of 25 to 25 ° C. ; 37 ° C, preferably at 29 ° C for 72 hours to 8 days. 3. A method according to claim 1 or 2, characterized in that s? The anthracycline glycoside of formula H according to item 1 is extracted from the mycelial cake with acetone and the filtered fermentation broth with a 9: 1 by volume mixture of dichloromethane and methanol at pH 8, the extracts are combined and evaporated to dryness under reduced pressure. 4. The process of claim 3 wherein the crude product obtained is dissolved in dichloromethane and the solution is purified by chromatography on a silica gel buffer, pH 7, eluting first with a 95: 5 mixture of dichloromethane, methanol and water. 0.25 to remove unreacted starting material of formula I and then a 90: 10: 0.5 mixture of dichloromethane, methanol and water, the fractions obtained are evaporated in the presence of n-propanol and the desired 4'-deoxy-13- (S). 1-Dihydro-4'-iodo-oxorubicin of formula II is isolated as its hydrochloride in pure form by adding an equivalent amount of hydrochloric acid and an excess of n-hexane.