FI90566B - Microbiological method for production of 4'-13(S)-dihydro- 4'-iododoxorubicin - Google Patents

Description

1 90566

The present invention relates to a doxorubicin derivative, its preparation and pharmaceutical compositions containing it. This invention relates to a microbiological process for the preparation of 41-13 (S) -dihydro-4'-iodidoxo-rubicin.

Accordingly, the invention relates to 4'-deoxy-13 (S) -dihydro-41-iodo-doxorubicin of formula (II):

j ° k H ^ 0H

Uf 'OH

1 II f / C

OCH, o OH H 'o H3CTTH! - / (Π) and pharmaceutically acceptable salts thereof.

A microorganism belonging to the genus Streptomyces is used for the reduction of the functional 13-ketone group of 4'-deoxy-4'-iodidoxorubicin according to formula (I) 2 90566 o oh o H I n

^ 3 ^ · 0H

1 V

!% Α ν ^ · χ / 0A

i Ίι i /.

0CH3 0 ° H / i, ** T ^ Λ * / * ''

4 f 'NH2 I

to give specifically 41-deoxy-13 (S) -dihydro-4 '- iodidoxorubicin, one of the two possible C-13 isomeric 4'-deoxy-13-dihydro-4'-iodidoxorubicin. The novel compound of formula (II), hereinafter referred to as FCE 24883, is useful as an antitumor agent and has been shown in experimental tumors to have activity comparable to that of 41-deoxy-4'-iodidoxorubicin of formula (I). The substrate for microbial stereoselective reduction is a semi-synthetic analog of doxorubicin disclosed in our publication US-A-4438105 (March 20, 1984).

More particularly, this invention relates to a biosynthetic method characterized in that a mutant of the species Streptomyces peucetius, designated strain M 87 F.I. and stored in 1 ai-15 The Deutsche Sammlung von Microorganismen, in which it is registered or assigned the number DMS 2444, is capable of stereo-selectively reducing the 13-keto functional group of 4'-deoxy-4'-iodidoxorubicin (I). The compound FCE 24883 (II) obtained is precipitated in the fermentation broth. FCE 24883 (II) can be recovered from the fermentation broth and its crude solutions are concentrated and purified.

The invention thus also relates to a process for the preparation of 3,90566 4 1 -deoxy-13 (S) -dihydro-4'-iodidoxorubicin of the formula (II) or a pharmaceutically acceptable salt thereof, which process comprises culturing Streptomyces peucetius strain M 87 F.I. (DMS 2444) in the presence of 4'-deoxy-4'-iodidoxorubicin and the resulting 4'-deoxy-13 (S) -dihydro-4'-iododoxorubicin is recovered as such or as a pharmaceutically acceptable salt.

The invention includes a new antitumor anthracycline of formula (II) FCE 24883 in pure form and as hydrochloride.

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

15 Streptomyces peucetius subsp. aureus ATCC 31428 has been mutated using nitrosoguanide to give a laboratory microorganism, designated Streptomyces peucetius strain M 87 F.I., which selectively converts a compound of formula (I) to a compound of formula (II). S. peucetius 20 to strain M 87 F.I. has been issued or assigned number DSM 2444 at the Deutsche Samlung von Mikroorganism, West Germany, where it is stored in a permanent collection.

Mutant strain M 87 F.I. the morphology does not differ from the morphology of the parent microorganism S. peucetius ATCC 31428, whereas the two cultures are clearly different in their culturing and biochemical properties. Mutant strain M 87 F.I. does not produce an agar medium soluble pigment from oyster yellow to lemon yellow, which is characteristic of its parent microorganism 11e S. peucetius ATCC 30 31428.

In addition, mutant strain M 87 F.I. can selectively convert a compound of formula (I) to a compound of formula (II), while the parent organism S. peucetius ATCC 31428 is not selective in this respect. This mutant M 87 F.I. the feature makes it very useful as shown above.

The stereoselective biotransformation of this invention can be performed on S. peucetius strain M 87 F.I. in a growth culture by adding a compound of formula (I) to the culture as a substrate during the incubation period.

The compound of formula (I) as well as the hydrochloride may be added after dissolving in sterile distilled water. However, a preferred, although not limiting, concentration of a compound of formula (I) in culture is about 50-200 ug / liter. The culture is grown in a medium containing a carbon source, for example an assimilating carbohydrate and a nitrogen source, for example an assimilable nitrogen compound or a proteinaceous substance. Preferred carbon sources include glucose, sucrose, glycerol, starch, corn starch, dextrin, molasses and the like. Preferred nitrogen sources are corn steep liquor, yeast extract, dry brewer's yeast, soybean meal, cotton soybean meal, cornmeal, casein, fish meat, distiller's solids, animal peptide, meat extract, meat extract, meat extract. A combination of these carbon and nitrogen sources can be used advantageously. Trace elements such as zinc, magnesium, manganese, cobalt, iron, and the like may not need to be added to the fermentation broth prior to sterilization because tap water and unpurified ingredients are used as components of the medium.

The biotransformation process can take from about 72 hours to 8 days. Incubation during the biotransformation process may range from about 25 ° C to about 37 ° C, with a temperature of 29 ° C being preferred. The contents of the transformation vessels are aerated by shaking at about 250 Merr./min or mixed with sterilized air to accelerate the growth of microorganisms and this increases the efficiency of the transformation process.

The progress of the microbial transformation reaction is monitored by sampling the fermentation broth at various intervals and extracting at pH 8.0 with dichloromethane: methanol (9: 1).

When a sample of the organic extract is placed on a thin layer chromatography plate (TLC) and a mixture of chloroform, methanol, acetic acid and water (80: 20: 7: 3, by volume) is used as the eluent, FCE 24883 (II) is found to have a R-chimer of from 0.50 while 4'-deoxy-4'-iodidoxorubicin of formula (I) is found at 0.60. Quantitation of the two anthracyclines can be performed using the above elution systems by TLC, after which the corresponding red-stained bands are scraped and eluted with methanol, and finally a spectrophotometric determination at 496 nm.

All fermentation broths in which the compound of formula (I) has been subjected to the conversion of the compound of formula (II) to FCE 24883 are filtered with the aid of silicon.

The red mycelial cake is extracted with a water-miscible organic solvent such as methanol or other lower alcohols, dioxane 11a, acetone triil, acetone, preferably acetone. The mycelial extracts are collected, concentrated under reduced pressure and combined with filtered fermentation broths, the pH is adjusted to 8.0, followed by extraction with a water-immiscible organic solvent such as n-butanol, chloroform, dichloromethane or preferably a mixture of dichloromethane and methanol 1 (9: methanol). ). The organic extracts contain FCE 24883 (II) together with the compound of formula (I) and some minor degradation products.

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

The compound FCE 24883 (II) as a free base is soluble in polar organic solvents and aqueous alcohols, while its hydrochloride is soluble in water and more soluble in 1-alcohol but is sparingly soluble in organic solvents. FCE 24883 hydrochloride11a has the following physicochemical properties: melting point: 200 ° C (dec.) Specific rotation: ^] p3 + 188 ° (c 0.05; CH2OH) \ H2 ° 20 UV and VIS absorption spectra: Λ max 232, 254, 290 and 480 nm (E1% 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, 25 965, 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 (bs, 2H, OH-6, OH-11), 7.6-7.9 (m, 3H, H-1, H -2, H-3), 5.26 (m, 1H, H-Γ), 4.96 (d, J = 5.2 Hz, 1H, OH-13), 492 (m, 1H, H-7) ), 4.55 (m, 1H, 30H-4 '), 4.51 (t, J = 6.7 Hz, 1H, OH-14), 4.20 (s, 1H, OH-9), 7,90566 3.97 (s, 3H, 4-OCH 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-1 3), 3.02 (m, 1H, H-3 '), 2.81 (m , 2H, CH 2-10), δ 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: C H NIO .HCl 27 30 10 10 m / z in FD equivalent to the free base: 656 (MH +); 655 (M +) and 416 (M +) corresponding to aglycone.

Selective high performance liquid chromatography (HPLC) can separate (two peaks with retention times of 18.8 min and 19.3 min) the two C-13 stereoisomeric alcohols present in the synthetic 4'-deoxy-13-dihydro-4 in a sample of i-iodo-doxorubicin prepared by reduction of a compound of formula (I) with NaBH 4.

Using the same HPLC method, FCE 24883 (II) 20 appears to have only a single peak with a retention time of 19.3 minutes corresponding to the slower moving component of the synthetic 13-dihydro derivative.

* HPLC method column: two RP Spherisorb S30DS2 (C18, 3 y, Phase 25 Separation U.K.) 150x4.5 mm, combined in series,

temperature: 45 ° C

mobile phase A: 0.05 M aqueous ΚΗ3Ρ04, prepared at pH

to 3.0 1 M H 3 PO 4 / CH 3 OH: 11 a (80:20, vol.) mobile phase B: CH 2 OH

30 elution: isocratic 30 minutes (42¾ A + 58% B) flow rate: 0.6 ml / min detection: 254 nm.

8 9 O 566

Acid hydrolysis of a compound of formula (II) (0.2 N aqueous HCl, 80 ° C, 30 min) gives a red precipitate of the corresponding aglycone of formula (III), whereby the sugar moiety, i. 3-Amino-2,3,4,6-tetradeoxy-4-iodo-L-xylohexohexose-5 si (IV) in the aqueous phase has been identified by comparison with a reliable sample obtained after acid hydrolysis of a compound of formula (I).

FCE 24883 (II) acid hydrolysis (HCl)

/.8 T

| Λ / γγ 0H ^ _o Ί I 11 P [- t4, HjC-7 0 / och3 s; f \ r nv «c '

III: 1 3- (S) -di Hydroadri Amys i noni I_V

The absolute (S) configuration of the compound of formula (III) at the C-13 position has been determined by direct comparison (1H-NMR-10 and mass spectra, TLC) of its 9,13-O-isopropylidene-14-O-tert-one. butyldiphenylsilyl derivative to the corresponding derivative of an authentic sample of 13- (S) -dihydroadriamycinone obtained as described by S. Penco et al. have described in Gazzetta Chimica Italiana, 115, 195, 1985.

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Biological activity

The cytotoxic activity of FCE 24883 (II) was tested in vitro on HeLa and P 388 cells for colony formation, compared to the compound of formula (I) and doxo-5 rubicin. As shown in Table 1, the results of the compound of formula (II) are as effective as those of 4'-deoxy-4'-iodidoxorubicin (I) and doxorubicin.

The in vivo antitumor activity of FCE 24883 (II) was tested against diffuse Gross-1 leukemia.

£ 10 C3H mice were injected intravenously with 2.10 cells / mouse and treated with compounds 24 hours after tumor injection.

Table II shows the results of two experiments. At the optimum dose, FCE 24883 (II) was found to be more effective than doxorubicin and as effective as 4'-deoxy-4'-iodidoxorubicin (I), with lower toxicity at the active dose11a. The antitumor activity of the compound of formula (II) is estimated over the average lifespan of the treated mice compared to control mice, can be compared to the activity of the compound of formula (I) and doxorubicin.

10 90 566

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12 9 0 566

The following examples are provided to illustrate the method and product of this invention in more detail.

EXAMPLE 1 5 Streptomyces peucetius strain M 87 F.I. (DMS 2444) culture was grown at 28 ° C on an agar surface with the following medium (medium SA): glucose 3%, dry brewer's yeast 1.2%, NaCl 0.1%, ΚΗ ,, ΡΟ ^ 0.05 %, CaCO 3 0.1%, MgSO 4 0.005%, FeSO 4 .7H 2 O 0.0005%, ZnSO 4 .7H 2 O 0.0005%, 10 CuSO 4 .5H 2 O 0.0005%, agar 2%, tap water to 100 ml, pH 6 7; sterilization is performed by heating in an autoclave at 115 ° C for 20 minutes.

The spores of the culture thus grown are collected and suspended in 3 ml of sterile distilled water, with this suspension, etc., 300 ml batch flasks containing 60 ml of the following liquid medium are dried: dry brewer's yeast 0.3%, peptone 0.5%, Ca ( NO 2) 2-4H 2 O 0.05%, tap water to 100 ml. Sterilize by heating in an autoclave at 120 ° C for 20 minutes. After sterilization of this medium, the pH of 20 is 6.8-7.0. The inoculated flasks are shaken for 2 days at 28 ° C in a 250 rpm rotary shaker with a circular diameter of 7 cm. 1.5 ml of the culture grown as described above are inoculated into 300 ml conical flasks containing 50 ml of the following bio-transformation growth medium: yeast extract 1.5%, KH 2 PO 4 0.25%, glucose 1.5%, tap water to 100 ml, pH 6.9; sterilization by heating in an autoclave at 115 ° C for 20 minutes.

The glucose solution is sterilized separately and added to each sterilized bottle at the correct concentration.

The flasks are then incubated for 24 hours at 28 ° C under the conditions described above for the inoculum solution. During this time, 1.0 ml of a solution of the compound of formula (I) in sterile 13 90566 distilled water at a concentration of 5 mg / ml is added to each flask. The shake flasks are incubated for a further 2 days to convert more than 70% of the compound of formula (I) to the compound of formula (II).

EXAMPLE 2 S.pecetius strain M 87 F.I. the culture is grown on a solid medium as described in Example 1. The spores of the three oblique surfaces are combined and collected in 10 ml of sterile-10 distilled water, and the suspension thus obtained is inoculated into a 2-liter round-bottomed flask containing 500 ml of the inoculum solution described in Example 1. The flask is incubated at 28 ° C for 48 hours in a rotary shaker at 120 rpm with a circular diameter of 7 cm. The entire inoculum is transferred to a 10 liter stainless steel fermentor containing 7.5 liters of the biotransformation medium described in Example 1 and steam sterilized at 120 ° C for 30 minutes, the glucose solution is sterilized separately and added to the sterilized fermentor at the appropriate concentration. The culture is allowed to grow at 28 ° C with stirring at 230 rpm and aeration with an air flow of 0.7 l / medium 1 / min.

After 48 hours, the substrate is added at the concentration shown in Example 1 and the culture is further incubated for 3 to 3 days to give a 60% conversion of the compound of formula (I) to the compound of formula (II).

EXAMPLE 3

From the fermentation obtained according to Example 2, the whole broth (5 L) was filtered, using 2% silicon-30 earth as a filter aid. The wet filter cake was extracted with acetone (3 L). After filtration, it was extracted twice more with acetone, 14,90566, to ensure complete recovery of the red pigment. The combined acetone extracts were concentrated under reduced pressure and the concentrate (1 L) was combined with the filtered broth and extracted under vacuum at pH 8 with a 9: 1 mixture of dichloro-5 methane and methanol. An organic extract containing the compounds of formulas (I) and (II) after a few with decomposition products, was concentrated to dryness under reduced pressure. The residue, dissolved in dichloromethane, was chromatographed on a silica gel column buffered to pH 7 (M / 15 phosphate buffer), eluting with a gradient of dichloromethane, methanol and water. After some decomposition products, the compound of formula (I) eluted with 95: 5: 0.25, followed by FCE 24883 (II) with 90: 10: 0.5. The combined fractions, after washing with water, concentrated to a small volume in the presence of n-propanol, added an equivalent amount of hydrochloric acid and excess n-hexane, gave pure FCE 24883 (II, 0.30 g, 60%) in water. as the chloride (m.p. 200 ° C, dec.). Following the same procedure, the unchanged compound of formula (I) (0.13 g, 26%) was also recovered as the hydrochloride.

EXAMPLE 4 A sample of FCE 24883 (II) was dissolved in 0.2 N aqueous hydrochloric acid (50 ml) and heated at 100 ° C for 30 min. The crystalline red precipitate of Aglycon (III) (0.12 g) was collected by filtration, washed with water and dried. Mass spectrum: m / e 416 (M +). Aglycone (111) was identified by comparing 13- (S) -dihydroadriamycinone to an authentic sample.

Claims (4)

A microbiological process for the preparation of 4'-13 (S) -dihydro-41-iodidoxorubicin of formula (II) 5. OH Πμ OCHj o1 OH / \ J H3cy-0 / (n) fNH2. HC1 15 I characterized in that Streptomyces peucetius strain M 87 F.I. is cultured under aerobic conditions in an aqueous culture medium containing an assimilable carbon source, an assimilable nitrogen source 20 and mineral salts. (DSM 2444), in the form of its hydrochloride, in the presence of 4'-deoxy-4 · -iododoxorubicin of formula (I) .25 ° OH 0 'Oh OCH. o OH Process according to Claim 1, characterized in that the process is carried out at a temperature of from 25 ° C to 37 ° C (preferably 29 ° C) with a reaction time of from 72 hours to 8 days. Process according to Claim 1 or 2, characterized in that the anthracycline glycoside of the formula (II) according to Claim 1 is extracted from the mycelium with acetone and the filtered fermentation broth with a mixture of dichloromethane and methanol (9: 1, v / v) at a pH of 8. and the combined extracts are concentrated to dryness under reduced pressure. 15 3 H 0: ö ”m2. hci (i) 35 and the resulting 4'-deoxy-13 (S) -dihydro-4 1-iodidoxorubicin of formula (II) are recovered as the hydrochloride. Process according to Claim 1, characterized in that the crude product obtained by the process according to Claim 1 is dissolved in dichloromethane and the product is chromatographed on a silica gel column buffered to pH 7, eluting first with dichloromethane. a mixture of methanol and water (95: 5: 0.25, v / v / v) to remove the unreacted starting material of formula (I) followed by elution with a mixture of dichloromethane, methanol-water (90: 10: 0, 5, v / v / v), the eluted fractions are concentrated to a small volume in the presence of n-propanol and the desired 4'-deoxy-13 (S) -dihydro-41-iodidoxorubicin (II) is recovered as the hydrochloride in pure form. by adding an equivalent amount of chloro-30 hydrochloric acid and an excess of n-hexane. 17 90566