AU601857B2 - A new antitumor agent obtained by microbial stereoselective reduction of 4'-deoxy-4'-iododoxorubicin - Google Patents

Description

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i s x v AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION Form

(ORIGINAL)

FOR OFFICE USE 6 0 B 01857 Short Title: Int. Cl: Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: I 7 -i Priority: Related Art:

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TO BE COMPLETED BY APPLICANT Name of Applicant: Address of Applicant: FARMITALIA CAtLO ERBA s.r.l.

VIA CARLO IMBONATI, 24 20159 MILAN

ITALY

CLEMENT HACK CO., 601 St. Kilda Road, Melbourne, Victoria 3J04, Australia.

Actual Inventor: Address for Service: SComplete Specification for the invention entitled: A NEW ANTITUMOR AGENT OBTAINED BY MICROBIAL STEREOSELECTIVE REDUCTION OF 4'-DEOXY-4'-IODODOXORUBICIN The following statement is a full description of this invention including the best method of performing it known to me:f*l!;'Si l: f- A A_e,

DESRIPIO

AIE NIUO GN BANDB IRBA STROEETV EUCINO 'DOY4I"DDXRUII STERESELECIVE RDUCTIN Oydr-DEOY-4'-ododoxorubicin hvn h formula (11) 3 He 7 O_ 4 dP and pharmaceutically acceptable salts thereof.

A microorganism belonging to the genera Streptomyces is employed for a stereoselective reduction of the 13-ketone functional group of 4'-deoxy-4'iododoxorubicin (I) 0 OH 0 to give specifically 4'-deoxy-13-(S)-dihydro-4'iododoxorubicin, one of the two possible C-13 stereoisomeric 4'-deoxy-13-dihydro-4'-iododoxorubicins.

The new compound hereinafter designated FCE 24883, is useful as an anti-tumor agent and displays on experimental tumors an activity comparable with that of 4 4'-deoxy-4'-iododoxorubicin The substrate for the microbial stereoselective reduction is a doxorubicin semi-synthetic analogue disclosed in our US-A-44381)5 (March 20, 1984).

More particularly the present invention relates to a biosynthetic process by which a mutant of the species Streptomyces peucetius, designated strain M 87 F.I. and deposited at the Deutsche Sammlung von Mikroorganismen where it is registered under the accession number DSM 2444, is characterised by its ability to stereoselectively reduce the 13-ketone functional group of 4'-deoxy-4'-iododoxorubicin Compound FCE 24883 (II) which results accumulates in the fermentation broths. The FCE 24883 (II) can be recovered from fermentation broths and crude solutions of it concentrated and purified.

The inve*tion therefore also provides a process for the preparation of 4'-deoxy'13(S)-dihydro-4'iododoxorubicin of formula (II) or a pharmaceutically acceptable salt thereof, which process comprises culturing Streptomyces peucetius strain M 87 F.I. (DSM 2444) in the presence of 4'-deoxy-4'-iododoxorubicin and recovering the resultant 4'-deoxy-13(S)-dihydro-4'- 2 5 iododoxorubicin as such or in the form of a pharmaceutically acceptable salt.

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

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

i i-ip 5 Detailed Description of the Invention; the microorganism Streptomyces geucetius subsp. aureus ATCC 31428 has been mutated, using nitrosoguanidine, to give a laboratory microorganism designated Streptomyces peucetius strain M 87 F.I. which selectively transforms compound into compound S. peucetiusstrain M 87 F.I. has been given the accession number DSM 2444 by Deutsche Sammlung von Mikroorganism, West Germany, where it has been deposited in the permanent collection.

The morphology of the mutant strain M 87 F.I.

is indistinguishable from that of the parent S. peucetius ATCC 31428, whereas both cultures are clearly distinguishable in their cultural and biochemical characters. In fact mutant strain M 87 F.I. does not produce an agar media the straw yellow to lemon yellow soluble pigment which characterises its parent S.

peucetius ATCC 31428.

Moreover mutant strain M 87 F.I. can selectively transform compound into compound (II) whereas the parent S. peucetius ATCC 31428 is not selective in this respect. This property of mutant M 87 F.I. makes it highly useful, as herein disclosed.

The transformation process The stereoselective biotransformation of the present invention can be effected in a growing culture of S. _eucetius strain M 87 F.I. by adding compound I as substrate to the culture during the incubation period.

Compound I, as the hydrochloride, can be added after solubilization in sterile distilled water. The preferred, but not limiting, range of concentration of compound I in the culture is about 50-200 micrograms per liter. The culture is grown in a nutrient medium containing a carbon sources, for example, an assimilable carbohydrate and a nitrogen source, for example an assimilable nitrogen compound or proteinaceouF material.

i :r r~-llm~.

6 Preferred carbon sources include glucose, sucrose, glycerol, starch, corn starch, dextrin, molasses and the like. Preferred nitrogen sources include corn steep liquor, yeast extract, brewer's dry yeast, soy bean meal, cotton seed meal, corn meal, casein, fish meal, distiller's solids, animal peptone, meat extract, ammonium salts and the like. Combination of these carbon and nitrogen sources can be used advantageously, Trace metals, for example, zinc, magnesium, manganese, cobalt, iron and the like, need not necessarily be added to the fermentation media, since tap water and unpurified ingredients are used as components of the medium prior to sterilization.

The biotransformation process can range from about 72 hours to 8 days. The incubation temperature 2 during the biotransformation process can range from about C to about 37°C, with 29°C being preferr.ed. The content of the transformation vessels are aerated by shaking at about 250 r.p.m. or by agitating with sterilized air, to facilitate growth of the microorganism, and this enhance the effectiveness of the transformation process.

a Analytical Methods The progress of the microbial transformation 25 reaction is monitored by withdrawing samples of fermentations at various time intervals, and extracting at pH 8.0 with a 9:1 dichlorpoTcthane:methano'L mixture.

When a sample of the orgar.c extract is subjected to thin layer chromatography (TLC), using as eluent a mixture of chloroform:methanol:acetic acid: water 80:20:7:3 (by volume), compound FCE 24883 (II) is found to occur at Rf medium value of 0.50, while 4'-deoxy-4'-iododoxorubicin is found at Rf 0.60, A quantitative estimation of the two anthracyclines can be performed after TLC using the above mentioned eluting systems, by scraping off and 7 7 eluting with methanol the corresponding red coloured zones and finally spectrophotometric determination at 496 nm.

Isolation procedure The whole fermentation broths, in which compound I has been subjected to conversion into FCE 24883 are filtered with the aid of diatomaceous earth. The red mycelial cake is extracted with a water miscible organic solvent, such as methanol and other lower alcohols, dioxane, acetonitrile, acetone, preferentially acetone is employed. The mycelial extracts are collected, concentrated under reduced pressure and combined with the filtered fermentation liquors, adjusted at pH 8.0 then extracted with a water-immiscible organic solvent such as n-butanol, chloroform, dichloromethane, or preferentially a dichloromethane:methanol 9:1 mixture. The organic extracts contain FCE 24883 (II) along with compound I and some minor degradation products.

Purification Procedure The organic extract is concentrated under reduced pressure to dryness and the residue, dissolved in dichloromethane, is chromatographed on a column of silica gel, buffered at pH 7, with a gradient of dichloromethane:methanol:water mixture. Compound I is eluted first with a 95:5:0.25 mixture, followed by FCE 24883 (II) with a 90:10:0.5 mixture. From the pooled fractions, after washing with water, concentration to a small volume in the presence of n-propanol, addition of an equivalent of hydrochloric acid and of an excess of n-hexane, a precipitate of pure FCE 24883 as the hydrochloride is obtained.

t j ie ~ii' -uL-rruru-ul '-ilYW 8 Chemical and Physical Properties FCE 24883 (II) as free base is soluble in polar organic solvents and aqueous alcohols, while its hydrochloride is soluble in water and lower alcohols but slightly soluble in organic solvents. The hyJrochloride of FCE 24883 has the following physicochemical properties: melting point: 200°C (dec.) specific rotation: 23 188° (c 0.05, CH OH) U.V. and VIS absorption spectrum: h 2 0 232, 254, 290 max and 480 nm (E 492, 370, 127, 163).

1 cm 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, 965, 940, 920, 900, 890, 870, 860, 830, 810, -1 785, 755, 730, 710, 540, 480, 450 and 415 cm lH-NMR Spectrum (DMSOd6, 200 MHz, 22 0 14.03 (bs, 2H, OH-60H-11), 7.6-7.9 3H, H-l, H-2, 5.26 1H, 4.96 J=5.2 Hz, 1H, OH-13), 4.92 1H, H-7), 4.55 1H, 4.51 J=6.7 Hz, 1H OH-14), 4.20 1H, OH-9), 3.97 3H, 4-OCH 3 3.76 (ddd, 6.7, 11.0 1H, CH OH), 3.60 (dq, J=1.0, 6.0 Hz, 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 1H, 2.81 2H, CH 2 10), 2.15 (dd. J=2.0, 15.3 Hz, 2 1H, H-8e), 1.97 (dd, J=6.0, 15.3 Hz, 1H, H-8ax), 1.7-1.9 2H, CH 2 and 1.14 J=6.0 Hz, 3H, CH z2 -3 Molecuar Formula: C 7

H

3 0 NIO1 0 HC1 m/z in FD equivalent to the free base: 6561MHI; 6551M1; and 416(M corresponding to the aglycone.

A selective high pressure liquid chromatography (HiLC) method* allows to separate (two peaks with retention times of 18.8 and 19.3 minutes) the two C-13 .i

I

i' i 9 stereoisomeric alcohols, present in a sample of synthetic 4 '-deoxy' 3-dihydro-4'-iododoxorubicin, prepared by NaBH 4 reduction of I.

Using *-ie same HPLC method, FCE 24883 (II) appears as a single p2ak with a retention time of 19.3 minutes corresponding to that of the slower moving constituent of the synthetic 13-dihydroderivative.

*HPLC method Column: two RP Spherisorb S30DS2 (C18 3 A. Phase Separation 150x4.5 mm connected in series.

Temperature: 45 0

C

Mobile Phase: A: 0.05 M aqueous of KH 2

PO

4 made to pH with 1M H PO 4

/CH

3 OH 80/20 (by vol.) Mobile Phase: B: CH OH Elution: isocratic for 30 minutes (42%A 58%B) Flow rate: 0.6 ml/min.

Detection: 254 nm.

Structure elucidation Acid hydrolysis of II (0.2N aqueous HC1, 80 0

C,

30 minutes) gives a red precipitate of the corresponding aglycone (III), while the sugar constituent, namely 3-amino-2,3,4,6-tetradeoxy-4-iodo-L-lyxohexohexose, (IV), present in the aqueous phase, has been identified after comparison with comparison with an authentic sample obtained upon acid hydrolysis compound I.

1 .L4j i_ 10 FCE 24883 (11) Acid hydrolysis (HCfl OH L OH I OH0 K IH /3O oci 0 OH H OH K-H.C 3 III: l3-(S)-dihydroadriamycinone

IV

The absolutc; (S)-configuration at C-13 off III has been determined by direct comparison (lH-NMR and mass spectra, TLC) of its 9 ,lJ-0-iopropylidene-14-O-tbutyldiphenysilyl derivative with the corresponding derivative of an authentic samples of l 3 -(S)-dihydroadrianycinone, obtained as described by S.

Penco et al. in Gazzetta Chemica Italiana, 115, 195, 1985.

Biologicjal activity The cytotoxic activity of FCE 24883 (11) was tested "in vitro" on HeLa and P 388 cells colony formation in comparison with compound I and doxorubicin.

As reported in Table 1, compound II resulted as potent as 4 '-deoxy--4'-iododoxorubicin and doxorubicin.

The "in vivo"l antituimor activity, FCE 24883 (II) was tested against disseminr.-d Gr'o~s leukemia. C38mice were injected intravenously wic.h 2.19 6 cell/mouse and treated with compounds under study 24 hours after the tumor injection.

11 Table II shows the results of two experiments.

At the optimal dose, FCE 24883 was found more potent than doxorubicin and as potent as 4'-deoxy-4'iododoxorubicin with a minor toxicity displayed at the active doses. The antitumor activity of compound II, evaluated as median survival time of treated over control mice, can be compared with those of I and doxorubicin.

TABLE 1 In vitro activity of 13-[S]-diidro-4'-Iodo-Doxorubicin (II, FCE 24883) in comparison with 4'-deoxy-4'-lodo- Doxorubicin FCE 21954) and Doxorubicin (Dx).

I

~lC-1-- Compound ID50 (n/ml) a) HeLa b

P

388 c DX 10.8 8 FCE 21954 10.4 2.1 FCE 24883 (II) 8 a) Dose giving 50% reduction of cell number in comparison with untreated controls.

b) Human cervic epithelioid carcinoma cells.

c) P388 leukemia cells.

1 12 TABLE 2 Activity of 13-[S]-diidro-4'-Iodo-Do. i~ in (II, FCE 24883) in comparison with 41-deoxy-i Doxorubicin FCE 21954) and Doxorubicin (DX) against disseminated Gross leukemia.

Compound Dose a) T/C% b) Toxic deaths C) (mng/Kg)

DX

FCE 21954 (1) 10 13 1G .9 4 5.2 6.8 4 5.2 6.8 8.8 200 225 250 240 260 145 180 220 220 140 (200, 200) (230, 220) (,260, 240) (240, 240) (260, 260) (160, 1210) (180) (220, 220) (220, 220) (140) 0/20 0/20 2/20 0/20 4/20 19/2 0 0/10 0/20 0/20 5/9 FCE 24883 (11) a) C3H mice were injeQted iLv. with 2x10 6 let~kemna cells and treated with the compounds the dlay after the tumor injection.

b) (Median survival time of treatHd mice/median survival time of controls) x 100.

c) Evaluated on the basi of autopsy findings on dead mice.

DEjscripation of th peerred qm'bodiments The following non-limitative examples arc- given in order to~ describe in more detail the processee and the product of the subject invention.

i 1 13 Example 1 A culture of Streptomyces peucetius, strain M 87 F.I. (DSM 2444) has been grown for 14 days at 28 0 C on agar slants of the following maintenance medium (medium SA): glucose, brewer's dry yeast, NaCI, 0.1%,

FH

2

PO

4 0.05%; CaCO 3 MgSO 4 0.005%; FeSO 4 .7H 2 0, 0.0005%; ZnSO 4 .7H 2 0, 0.0' CuSO 4 .5H 2 0, 0.0005%; agar, tap water up to 100 ml; pH 6.7; sterilization is carried out by heating in an autoclave at 115 0 C for minutes.

The spores of the so grown culture are collected and suspended in 3 ml of steriledistilled water; this suspension is inoculated in 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: brewer's dry yeas., peptone, Ca(NO 3 2 .4H 2 0, 0.05%; tap water up to 100 ml.

Sterilization by heating in autoclave at 120 0 C for minutes. The pH of this medium after sterilization is between 6.8 and 7.0. The inoculated flasks are shaken for 2 days at a temperature of 28 C on a rotary shaker running at 250 r,p.m. and describing a circle of 7 -m in diametr. 1.5 ml of the culture grown as described above a.e inoculated in 300 ml Erlenmeyer flasks containing ml of the following biotransformation medium: yeast extract, KH 2

PO

4 0.25% glucose, tap water up to 100 ml, pH 6.9; sterilization by heating in autoclave at 11 5C for 20 minutes. The glucose solution is sterilized separately and added to each sterilized flask at the proper concentration.

The flasks are then incubated at 28 0 C under the conditions described for the seed phase, for 24 hours.

At this time 1.0 ml of a solution of compound I in sterile distilled water at a concentration of 5 mg/ml are added to each flasks. The shaken flas k s are incubated for 2 days more obtaining a 70% conversion of compound I into compound II.

I I -14- Example 2 A culture of S._peucetius strain M 87 F.I. is grown on solid medium as described in example 1, The spores of three slants are pooled and collected in 10 ml of sterile distilled water; the suspension so obtained is inoculated in a 2 1 baffled round-bottomed flask containing 500 ml of the seed mediun described for example 1. The flask is incubated for 48 hours on a rotary shaker running at 120 r.p.m. and d- 3cribing a circle of 7 cm in diameter at a temperature of 28 C. The whole seed is inoculed in a 10 1 stainless-steel fermenter containing 7.5 1 of the biotransformation medium described in example 1 and sterilized by vapour at 120 0 C for 30 minutes, the glucose solution being sterilized separately and added at the proper concentration to the sterilized fermenter. The culture is allowed to grow at 28 0 C under stirring at 230 r.p.m.

and aerated with an air flow of 0.7 lite!1iter of the medium/minute.

After 48 hours the substrate compound is added at the concentration described for example 1, and the culture incubated for 3 more days obtaining a conversion of compound I into compound II.

iExample 3 4 25 The whole beer (5 1) from a fermentation obtained according to example 2, was filtered using 2% diatomaceous earth as filter aid. The wet filter cake was extracted with acetone (3 After filtration two additional extractions with acetone were effected to ensure a complete recovery of the red pigments. The combined acetone extreots were concentrated under reduced pressure and the concentrate (1 1) was combined with the filtered broth and exhaustively extracted at pH 8 with a dichloromethane:methanol 9:1 mixture. The organic extract, containing compounds I and II with some l 15 degradation products, was concentrated under reduced pressure to dryness. The residue, dissolved in dichloromethane, was chromatographed on a column of silica gel, buffered at pH 7, (M/15 phosphate buffer) with a gradient of dichloromethane:methanol:water mixture. After some degradation products compound I was eluted with a 95:5:0.25 mixture followed by FCE 24883 (II) with a 90.10:0.5 mixture.

From the pooled fractions, after washing the water, concentration to a small volume in the presence of n-propanol, addition of an equivalent of hydrochloric acid and of an excess of n-hexane pure FCE 24883 (II, 0.30g, as the hyrochloride 200 0 C, dec,) was obtained. Following the same procedure, untransformed compound I (0.13g, as the hydrochloride was also recovered.

Example 4 A sample of FCE 24833 (200 mg) was dissolved in 0.2 N aqueous hydrochloric acid (50 ml) and heated for 30' at 100°C. A crystalline red precipitate (0.12 g) of aglycone (III) was collected by filtration, washed with water and dried.

Mass spectrum: m/e 416 The aglycone (III) was identified as 13-(S)-dihydroadriamycinone by comparison with an a" hentic sample.

41

Claims (6)

1. -4'-Deoxy-13(S)--dihydro-4'-iododoxorlubicin having the formula (II) 0 OH 4K NH, 00 0 o 0 0 o oo0 0 09 00 0 a 0 9 00o a0 0 0000 o oo 0 0 0 0 00 0 000000 0 0 and pharmaceutically acceptable salts thereof.

2. A salt according to Claim 1 which is the hydrochloride salt.

3. A microbiological process of pre~paring 4'-deoxy-13(S)-dihydro-4'-iodorubiai-,having the fotmula (II)0 OH OH 'O 0 00 0 0 0 0 00 0 a 0 00 0 0 00 0 00 0 0 0 0 00 H 3C 4 0 0 00 a0 0 00 0 009000 a 0 the process comprising cultivati ng, under aerobic conditions in an aqueous cultural medium containing an assimilable source of carbon, an assimilable source of nitrogen and mineral salts, a mutant of the species V 17 Streptomyces peucetius, designated as strain M 87 F.I. (DSM 2444), in the presence of 4'-deoxy-4'-iodo- doxorubicin (I) 0 OH 0 Sits hydrochloide. I o°°c in the form of its hydrochloride and recovering the rasultant 4'-deoxy-13(S)-dihydro-4'-iododoxorubicin (II) as its hydrochlok'ide.

4. A pruiess according to Claim 3, which is S' carried out at a t nperature from 250 to 37°C (preferably 290) for a period of from 72 hours to 8 days.

A process according to Claim 3, further comprising extracting the anthracycline glycoside of formula of Claim 1 from the mycelial cake with acetone and from the filtered fermentation liquors with a mixture of dichloromethane: methanol (9:1 v/v) at pH 8, and concentrating to dryness the combined extracts under reduced pressure.

6. A process which comprises dissolving the raw product, as obtained following the procedure of Claim 3, c)iCh\or mer-0 k ne- Sin dichlorC~~tane nd submitting the product to a chromatographic purification on a column of liggel, buffered at pH 7, using as eliting system first a mixture of dichloromethane:methanol:water (95:5:0,25 v/v) for eliminating the unreacted starting material followed by a mixture of dichlorometane:methanol:water (90:10:0.5 concentrating to a small volume the eluted fractions in the presence of n-propanol, and recovering 18a the wanted 4'-deoxy-l 3(S)-dihydro-4'-i.ododoxorubicin (II) as its hydrochloride in pure form by addition of~ one equivalent of hydrochloric acid and an excessofRe4ftp- DATED this 12th day of August 1988 FARMITALIA CARLO ERBA S.R.L. Dy its Patent Attorneys GRIFFITH HACK CO. Fellows Institute of Patent Attorneys of Australia 00 0 0 O00 a 00 00 000 00 0 000 QoO00 00 0 0 00 0 0 0 00 0 0 0 0 0 00 a 00 00 0 C C -I>