NO172856B - PROCEDURE FOR MANUFACTURING TUMOROUSING AGENTS - Google Patents

Description

The present invention relates to the production of 4<1->deoxy-13(S)-dihydro-4<*->iododoxorubicin with formula (II); The microorganism Streptomvces peucetius strain DSM 2444 is used for the stereoselective reduction of the acid chloride of the 13-ketone functional group of 4<1->deoxy-4•-iododoxorubicin (I) ; to give specifically 4<1->deoxy-13-(S)-dihydro-4<1->iododoxorubicin-HCl, which is one of the two possible C-13 stereoisomers 4<1->deoxy-13-dihydro-4<1->iododoxorubicins. The new compound (II) hereinafter referred to as FCE 24883 is useful as a tumor suppressor and exhibits on experimental tumors an activity comparable to that of 4'-deoxy-4<1>;iododoxorubicin (I). The substrate for the microbial stereo-selective reduction is a doxorubicin semi-synthetic analog described in applicant's US patent 4438105 (March 20, 1984). More particularly, the present invention relates to a biosynthetic method by which a mutant of the genus Streptomvses peucetius designated strain M 87 F.I. and deposited at the Deutsche Sammlung von Mikroorganismen where it is registered under accession number DSM 2444 and which is characterized by its ability to stereoselectively reduce the 13-ketone functional group of 4<1->deoxy-4<1->iododoxorubicin (I). Compound FCE 24883 (II) which appears accumulates in the fermentation liquid. FCE 24883 (II) can be recovered from the fermentation medium and impure solutions thereof can be concentrated and purified. The invention thus provides a method for the production of 4'-deoxy-13(S)-dihydro-4<1->iododoxorubicin of formula (II), which method comprises cultivating Streptomyces peucetius strain M 87 F.I. (DSM 2444) in the presence of 4<1->deoxy-4<1->iododoxorubicin and recover the resulting 4<1->deoxy-13(S)-dihydro-4'-iododoxorubicin as such as set forth in claim 1's characterizing share. ;Detailed description of the invention ;The microorganism Stre<p>tom<y>ces peucetius subspecies aureus ATCC 31428 has been mutated using nitrosoguanidine to give a laboratory microorganism designated Streptomyces peucetius strain M 87 F.I. which selectively converts compound (I) to compound (II). S. peucetius strain M 87 F.I. has been given the accession number DSM 2444 by the Deutsche Sammlung von Mikroorganism, West Germany where it has been deposited. ;The morphology of the mutant strain M 87 F.I. cannot be separated from that of the original S. peucetius ATCC 31428, but both cultures are clearly distinguishable in their cultural and biochemical characteristics. Indeed, the mutant strain M 87 forms F.I. on agar not the straw yellow to lemon yellow soluble pigment that characterizes its origin S. peucetius ATCC 31428. Furthermore, the mutant strain M 87 F.I. selectively transform the compound (I) into the compound (II) while the parent S. peucetius ATCC 31428 is not selective in this respect. This characteristic of the mutant M 87 F.I. makes it very useful as described here. ;The transformation process ;The stereoselective biotransformation according to the present invention can be carried out in a growing culture of S. peucetius strain M 87 F.I. by adding compound (I) as substrate to the culture during the incubation period. Compound I to which the hydrochloride can be added after dissolution in sterile distilled water. The preferred but not limiting concentration range of compound (I) in the culture is approx. 50-200 jig per litre. The culture is grown in a nutrient medium, containing carbon sources, e.g. an assimilable carbohydrate and a nitrogen source, e.g. an assimilable nitrogenous compound or proteinaceous material. Preferred carbon sources include glucose, sucleose, glycerol, starch, corn starch, dextrin, syrup and the like. Preferred nitrogen sources include silage juice, yeast extract, dry yeast, soybean meal, cottonseed meal, corn meal, casein, fish meal, solid fermentation material, animal peptone, meat extract, ammonium salts and the like. Combinations of these and carbon sources can be used advantageously. Trace metals e.g. zinc, magnesium, manganese, cobalt, iron etc. does not necessarily need to be added to the fermentation media since tap water and non-purified ingredients are used as components of the medium before sterilization. ;The biotransformation process can take place from approx. 72 hours to 8 days. The incubation temperature during the biotransformation process can range from approx. 25°C to approx. 37°C, where 29°C is preferred. The contents of the transformation chambers are aerated by shaking at approx. 250 r.p.m. or by stirring with sterilized air to facilitate the growth of the microorganisms, and this increases the efficiency of the transformation process. ;Analysis methods ;The development of the microbial transformation reaction is monitored by withdrawing samples of the formulations at different time intervals and extracting at pH 8.0 with a 9:1 dichloromethane:methanol mixture. 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 80:20:7:3 (by volume), it is found that the compound FCE 24883 (II) is present at Rf mean value 0.50 while 4'-deoxy-4'-iododoxorubicin (I) is found at Rf 0.60. A quantitative assessment of the two anthracyclines can be carried out after TLC using the above-mentioned elution systems by scraping off and eluting with methanol the corresponding red colored zones and final spectrophotometric determination at 496 nm. ;Isolation Procedure ;The entire fermentation medium in which compound (I) has been subjected to conversion to FCE 24883 (II) is filtered using diatomaceous earth. The red mycelial cake is extracted with an organic solvent which is miscible with water such as methanol and other lower alcohols, dioxane, acetonitrile, acetone, and preferably acetone is used. The mycelial extracts are collected, concentrated under reduced pressure and combined with the filtered fermentation liquids, adjusted to pH 8.0 and then extracted with an organic solvent immiscible with water such as n-butanol, chloroform, dichloromethane or preferably a dichloromethane: methanol 9:1 mixt. The organic extracts contain FCE 24883 (II) together with compounds (I) and some small degradation products. ;Purification procedure ;The organic extract is concentrated under reduced pressure to dryness and residues dissolved in dichloromethane are chromatographed on a column of silica gel buffered at pH 7 with a gradient of dichloromethane:methanol:water mixture. Compound I is first eluted with a 95:5:0.25 mixture followed by FCE 24883 (II) with a 90:10:0.5 mixture. From the combined fractions after washing with water, concentration to a small volume in the presence of a propanol, addition of an equivalent of hydrochloric acid and of an excess of a hexane, a precipitate of pure FCE 24883 (II) is obtained as the hydrochloride. ;Chemical and physical properties ;FCE 24883 (II) as a free base is soluble in polar organic solvents and aqueous alcohols, while its hydrochloride is soluble in water and lower alcohols but has limited solubility in organic solvents. Hydrochlorides of FCE 24883 have the following physiochemical properties : ;Melting point: 200°C (dec) ;Specific rotation: [cx]23" + 188°(c 0.05,CH3OH) ;U.V. and VIS absorption spectrum: X^^g 232, 254, 290 and 480 nm (E<1>%cm = 492, 370, 127, 163). IR 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, 75, 85, 81 730, 710, 540, 480, 450 and 415 can"1. ;1H-NMR spectrum (DMS0d6, 200 MHz, 22°C): 14.03 (bs, 2H, 0H-6, OH-11), 7.6-7.9 (m, 3H, H-2,H -3), 5.26 (m.1H,H-1'), 4.96 (d, J=5.2 Hz, 1H, OH-13), 4.92 (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, 1H, CH (H) - OH), 3.60 (dq, J= 1.0, 6.0 Hz, H-5<1>), 3.48 (ddd. J=7.2, 6.7, II, 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 1.14S (d, J= 6.0 Hz, 3H, CH3-5<«>) ;Molecular formula: C27<H>30NIO10 • HC1 ;m/z in FD equivalent to free base: 656(MH); ;655(M); and 416(M<+>) corresponding to the aglyzone. ;A selective high pressure liquid chromatography (HPLC) method allows to separate (two peaks with retention times of 18.8 and 19.3 min.) the two C-13 stereoisomeric alcohols present in a sample of synthetic 41-deoxy-13 -dihydro-41-iododoxorubicin prepared by a NaBH4 reduction of I. ;Using the same HPLC method, FCE 24883 (II) appears as a single peak with a retention time of 19.3 min, corresponding to that of the slower moving component of the synthetic 13-dehydroderivative. ;HPLC method ;Column: 2 RP Spherisorb S30DS2 (C18 3p, Phase Seperation England) 150 x 4.5 mm connected in series. ;Temperature: 45°C ;Mobile phase A: 0.05 M aqueous KH2P04, adjusted to pH 3.0 with ;1 M H3PO4/CH3OH = 80/20 (by volume) ;Mobile phase B: CH3OH ;Elution: Isocratic in 30 minutes (42%a + 58%B) Flow rate: 0.6 ml/min. ;Detection: 254 nm. ;Structure analysis ;Acid hydrolysis of II (0.2 N aqueous HCl, 80°C, 30 minutes) gives a red precipitate of the corresponding aglyzone (III), while the sugar component, namely 3-amino-2,3,4,6- tetradeoxy-4-iodo-L-lyxohexohexose (IV) present in the aqueous phase has not been identified after comparison with an authentic sample obtained by acid hydrolysis of compound I. ;The absolute (S) configuration at C-13 of III has been 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 authentic samples of 13-(S)-dihydroadriamycin ion obtained as described by S. Penco et al. in Gazzetta Chimica Italiana, 115, 195, 1985. ;Biological activity ;The cytotoxic activity of FCE 24883 (II) was investigated "in vitro" on Hela and P 388 cell colony formation in comparison with compound I and doxorubicin. As indicated in Table I, compound II was as active as 4'-deoxy-4'-iododoxorubicin (I) and doxorubicin. ;The "in vivo" antitumoral activity was investigated for FCE 24883 (II) against disseminated Gross leukaemia. C3H mice were injected intravenously with 2*10<6> cells/mouse and treated with compounds under study 24 hours after the tumor injection.

Table II shows the results of both experiments. At the optimal dose, FCE 24883 (II) was found to be more active than doxorubicin and as active as 4'-deoxy-4<1->iododoxorubicin (I) with little toxicity shown at the active doses. The antitumor activity of compound II assessed as mean survival time of treated versus control mice is comparable to that of I and doxorubicin.

Description of preferred embodiments

The following non-limiting examples are given to describe in greater detail the method according to the present invention.

Example 1

A culture of Stre<p>tomyces peucetius strain M 87 F.I (DSM 2444) has been grown for 14 days at 28"C on agar slants in the following maintenance medium (medium SA): glucose 3%, dry yeast 1.2% , NaCl 0.1%, KH2P04 0.05%, CaC03 0.1%, MgS04 0.005%, FeS04.7H20 0.0005%, ZnS04.7H20 0.0005%, CuS04.5H20 0.0005%, agar 2% , tap water up to 100 ml, pH 6.7 where sterilization is carried out by heating in an autoclave to 115°C for 20 minutes.

The spores of the thus grown culture are collected and suspended in 3 ml of sterile distilled water and the suspension is inoculated into 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: dry yeast 0.3%, peptone 0.5%, Ca (N03)2.4H20 0.05%, tap water up to 100 ml. Sterilization by heating in an autoclave to 120°C for 20 minutes. The pH of this medium after sterilization is between 6.8 and 7.0. The inoculated bottles are shaken for two days at a temperature of 28°C on a rotary shaker operated at 250 r.p.m. and describes a circle of 7 cm in diameter. 1.5 ml of the culture, grown as described above, is inoculated into 300 ml Erlenmeyer flasks containing 50 ml of the following biotransformation medium: yeast extract 1.5%, KH2PO4 0.25%, glucose 1.5%, tap water up to 100 ml , pH 6.9 sterilization by heating in an autoclave to 115°C for 20 minutes. The glucose solution is sterilized separately and added to each sterilized bottle at the appropriate concentration.

The flasks are then incubated at 28°C under conditions described for the growth phase for 24 hours. At this time, 1.0 ml of a solution of compound I in sterilized distilled water at a concentration of 5 mg/ml was added to each bottle. The shaken bottles are incubated for an additional 2 days to obtain a 70% conversion of compound I to compound

II.

Example 2

A culture of S. peucetius strain M 87 F.I. grown on solid medium as described in example 1. The spores of three slant cultures are combined and collected in 10 ml of sterile distilled water, where the suspension thus obtained is inoculated into a 2 liter round-bottomed bottle with barrier plates containing 500 ml of the growth medium described in example 1. The bottle is incubated for 48 hours on a rotary shaker operated at 120 r.p.m. and describes a circle with a diameter of 7 cm at a temperature of 28°C. The entire growth is inoculated into a 10 1 stainless steel fermenter containing 7.5 1 of the biotransformation medium described in Example 1, and sterilized with steam at 120°C for 30 minutes where the glucose solution is sterilized separately and added at an appropriate concentration to the sterile -certe fermenter. The culture is allowed to grow at 28°C with agitation at 230 r.p.m. and aerated with an air flow of 0.7 1/1 medium/min. After 48 hours, the substrate compound is added at the concentration described for example 1 and the culture is incubated for 3 further days to obtain a 60% conversion of compound I to compound II.

Example 3

The culture/medium (5 L) from a fermentation obtained according to Example 1 was filtered using 2% diatomaceous earth as a filter. The wet filter cake was extracted with acetone (3 L). After filtration, 2 further extractions with acetone were carried out to ensure a complete recovery of the red pigments. The combined acetone extracts were concentrated under reduced pressure and the concentrate (1 L) was combined with the filtered medium and extensively extracted at pH 8 with a dichloromethane:methanol 9:1 mixture. The organic extract containing compounds I and II with some decomposition 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 (w/15 phosphate buffers) 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 combined fractions after washing with water concentration to a small volume in the presence of n-propanol, addition of an equivalent amount of hydrochloric acid and an excess of n-hexane, pure FCE 24883 (II 0.30g, 60%) was obtained as the hydrochloride (melting point 200°C). By following the same procedure, untransformed compound I (0.13 g, 26%) was also obtained as hydrochloride.

Example 4

A sample of FCE 24833 (I) (200 mg) was dissolved in 0.2 N aqueous hydrochloric acid (15 ml) and heated for 30 min. to 100 °C. A crystalline red precipitate (0.12 g) of aglysone (III) was collected by filtration, washed with water and dried. Mass spectrum: m/e 416 (M<+>). The aglyzone (III) was identified as 13-(S)-dihydroadriamycinone by comparison with an authentic sample.

Claims (2)

1. Process for the preparation of 4<1->deoxy-13(S)-dihydro-4<1->iodo-doxorubicin of formula II characterized in that the method comprises growing Streptomyces peucetius strain DSM 2444 under aerobic conditions in a culture medium containing sources of nitrogen, carbon and mineral salts in the presence of 4<1->deoxy-4<1->iodo-doxorubicin of formula (I) in the form of its hydrochloride at a final concentration of 0.1 mg/ml of the medium, and at a temperature from 27 to 30°C, separate the resulting culture liquid, extract the separated mycelium with acetone and the liquid phase with a mixture of dichloromethane:methanol (9:1 v/v) at pH 8.0, combine the resulting extracts and concentrate them to dryness under reduced pressure, then dissolve the concentrate in dichloromethane and chromatograph this on a column of silica gel buffered to pH 7.0 and successively elute with a mixture of dichloromethane:methanol:water (95:5:0.25 v/v) and (90:10:0.5 v/v) respectively, concentrate the resulting second eluate in the presence of n-propanol, recover at known way the desired product (II) as its hydrochloride in pure form by adding an equivalent part of hydrochloric acid and an excess of n-hexane. 2. Method according to claim 1, characterized in that the cultivation is carried out from 3 to 5 days.