FI63945C - FOERFARANDE FOER FRAMSTAELLNING AV NYA TUMOERER MOTVERKANDE ANRACYKLINGLYKOSIDANTIBIOTIKA - Google Patents

Description

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Patent-och ragistaratyralMn 'AmMcm uti ^ d och νϋ. · Μ * · η puMicmrf 31.05.83 (32) (33) (31) l * yr «l *« r • wolk · *' * - t * j * rd pilots * 05.10.76 2i4.05.77 Japan-Japan (JP) Sho51-120237 Sho52-60908 (Tl) Zaidanhojin Biseibutsu Kagaku Kenkyukai, lU-23, Kamiosaki 3-chome, Shinagawa-ku, Tokyo, Japan-Japan (JP) ( (72) Hamao Umezava, Tokyo, Tornio Takeuchi, Tokyo, Toshikazu Oki,

Kanagawa-ken, Taiji Inui, Kanagawa-ken, Japan-Japan (JP) (7 * 0 0y Boreni'is & C: o Ab (514) Method for the preparation of anthracycline-glucoside antibiotics against the growth of new tumors - Förfarande för framställning av nya tumörer motverkande anthracyclinglycosidantibiotics (62) Separated from application 772915 (patent 57 ^ * 0 -Avdelad frän trap 772915 (patent 57UI4I4)

The invention relates to a process for the preparation of antitumor anthracycline glycoside antibiotics MA144 having the formula R10C00CH3

Wv®2®3 OH D (H Ö (II)

/ "Oj

iCchJ

N "« 3

f OH H

wherein R1 is a hydrogen atom or a hydroxyl group.

In particular, the invention relates to a process for the preparation of anthracycline glycoside antibiotics by simple acid hydrolysis from the corresponding anthracycline glycosides having the same backbone scheme, i.e. aclavinone, L-rhodosamine and 2-deoxy-L-fucose.

_ · I.

2 63945

Various types of anthracycline glycosides have been previously found in culture broths of microorganisms belonging to the genus Streptomyces and have also been described in the literature. Of these, daunomycin (U.S. Pat. No. 3,616,242) and adriamycin (U.S. Pat. No. 3,590,028) have already been used clinically against human cancer, and aclasino-mycin A, carminomycin and rubidazone are being tested clinically with great interest in cancer chemotherapy.

When studying Streptomyces cultures for metabolites with antitumor activity, the inventors have discovered new compounds which, after being purified and classified on the basis of their physicochemical properties, have confirmed that the antibiotics now known as MA144-G1, G2, -L, -N1, -S1, -S2, -U1, -U2 and -Y are novel compounds with antitumor activity and low toxicity in animals.

The parent application FI-772.915 of this application discloses the preparation of these antibiotics by culturing MA144-developing strains belonging to the genus Streptomyces. At the same time, the inventors have found that these MA144 antibiotics can be prepared enzymatically as well as by a chemical conversion method involving reduction and hydrolysis. In particular, compounds of the MA144 series having the formula a hydrogen atom, i.e. MA144-S1, and a hydroxyl group, i.e. MA144-S2, can be prepared by simple acid hydrolysis. Accordingly, the invention relates to a chemical conversion process for the preparation of novel antibiotics MA144-S1 and -S2, wherein the invention is characterized in that the hydrolysis of the general formula R o cooch3 OH 0 OH 0

OiST ^ «3

I S

An anthracycline glycoside compound according to R3, wherein R1 and R2 represent the following groups: 3 63945 R1 R3 - cfT ^ j

MA1HU-G1 0 / ° \ J

G2 OH \ / MA1 * + * + - N1 H "j HO ^ - 'MA144-U1 H A — 0., U2 OH) HO 1-'

OH

MA144-Y H

Aclasinomycin A H _ _ MA1MM-M1 H "V7 ° \ j

M2 OH \ __ J

Rhodorubin A OH / C7 ^ \ l Fey HO - '

Sinerubin A OH A 0. i 63945 4

The process according to the invention is suitably carried out with a dilute inorganic acid such as hydrochloric acid or sulfuric acid using similar anthracycline glycosides as starting materials, such as, for example, aclasinomycin A, MA144-N1, -G1, -U1, -Y, -M1, -G2, -U2, rhodirubin A, synerubin A and -M2 having the same framework pattern, aclavinone, L-rhodosamine and 2-deoxy-L-fucose. The formed MA144-S1 and -S2 can be recovered, separated from the hydrolyzate and purified by conventional methods for the separation and purification of water-insoluble antibiotics. These methods include solvent extraction, precipitation, concentration, gel filtration, countercurrent partitioning, chelating and column chromatography.

The novel anthracycline antibiotics prepared according to the invention have been found to have antimicrobial and antitumor activity. In particular, the compounds have an effect on gram-positive bacteria, in addition to inhibiting the growth of various mammalian tumors, such as L-1210 and P-388 leukemia in mice. Besides, their toxicity is low. Thus, the compounds of the invention can be used as antibacterial and antitumor drugs.

The chemical structure of the novel compounds MA-144-S1 and -S2 according to the invention corresponds to the 2-deoxy-L-fucosyl-L-rhodosaminyl-aclavinone or -e-pyrromycinone of the formula II. The physicochemical properties of the compounds are shown in Table 1.

5 63945 MA mu_SI_S2_

Appearance Slightly basic amorphous Slightly basic amorphous ___yellow powder_red powder

Elemental analysis C H NOCHNO

found 61, 37 6.45 1, 97 29.36 60.09 6.13 1, 88 30.94 calculated 61, 79 6.84 2.00 29.72 60.41 6.34 1, 96 31, 13

The general formula ^ 36 ^ 45 ^ 13 ^ C36 ^ 45 ^ 14 ^

Molecular weight 699.8 715.8

Melting point (° C) 144 ... 147 154 ... 158

Specific rotation + 77 ° ΓαJl ° (C = 1.0, CHCl 3)

Solubility Soluble in acidic water, methanol, ethanol, n-butanol, acetone, ethyl acetate, chloroform, benzene, toluene, dimethylsulfoxide, methylcellosolve, dimethylformamide.

Slightly soluble in water, n-hexane, cyclohexane, diethyl ether and petroleum. The hydrochloride salt is soluble in water, methanol, ethanol and slightly soluble in chloroform, acetone and ethyl acetate.

Rf values ** * C: M = 20: 1 0.14 0.14

Reaction Acidic aqueous and methanolic solution The acidic aqueous solution is red to yellow and turns and turns purple-blue to purple as a preposition and turns purple and turns reddish-brown in a concentrated brown concentrated solution.

^ SO ^ solution at.

Ultraviolet light Fig. 5 Fig. 6

IbsS ^ thioSpStri 230 (638)> 258'5 <371> '234.5 (607) »258.5 <306), (E1%) 289.5 (160), 432 (177) 293 (110), 491 (189) 1 an max (full line) ______________ 0.1 N HCl-MeOH 229.5 (652), 258.5 (380), 234.5 (629), 258.5 (318), (dashed line) _289 , 5 (163, 431 (192) - 193 (114), 491 (197) - 0.1 N NaOH-MeOH 237.5 (553), 286 (141), 242 (606), 566 (244), (-. -.- line) _320 (90), 524 (161) _606 (210) _ 1nfrared absorption spectrum (KBr) _Figure 1_Figure 2_

Nuclear Magnetic Resonance Spectrum (PMR) Fig. 3_ Fig. 4_______ 6 6394 5

The antimicrobial activity of the MA144 components MA144-S1 and -S2 against various microorganisms is examined below. Table 2 shows the minimum inhibitory concentration of the antibiotics according to the invention determined by the broth dilution method.

Table 2 Antimicrobial activity of the MA144 component

Tissue microorganism ^ 1 C (wg / ml) ^

Staph. aureus FDA 209P 6.25 6.25

Staph. aureus, Smith 3.1 0.78

Bac. subtilis ATCC 6633 3.1 3.1

Bac. cereus ATCC 9634 1.56 0.78

Bac. megaterium NRRL B-938 3.1 3.1

Sar. lutea ATCC 9341 1, 56 1, 56

Mic. flavus 1.56 0.78

Cory. bovis 1 81 0 0.78 0, 78

Ps. Fluorescent NIHJB-254> 100> 100

PR. morganii> 100> 100

Mycobact. ashtray ATCC 607> 100> 100

Can. albicans IAM 4905> 100> 100

Can, tropicalis IAM 4942> 100> 100

As can be seen from the table, the MA144 components of the invention have antimicrobial activity, especially against gram-positive bacteria, and can therefore be used to treat mammals against diphtheria, tuberculosis, pneumonia, tetanus and other infectious diseases caused by gram-positive bacteria.

The antitumor effect and acute toxicity of the MA144 components are examined below. The MA144 components of the invention have been shown in animal experiments to have a pronounced antitumor effect and low toxicity, so that they can be used to inhibit the growth of mammalian tumors. In particular, the compounds of the invention have been found to have a pronounced inhibitory effect on murine leukemia L1210. Thus, BDF 2 mice weighing 19-22 g were implanted intraperitoneally with 1 x 10 6 L1210 cells / mouse, and 24 hours after implantation, the test compound was injected intraperitoneally once a day for nine consecutive days. The following table shows the percentage prolongation of survival of animals surviving on the thirtieth day relative to control animals, and the corresponding LD5Q values after once intraperitoneal injection of the compound into mice.

Therapeutic efficacy and toxicity of MA 144 components against murine leukemia L 1210

Prolongation of survival time (% experimental / control animals)

Compounds MA 144- S1 S2 Adriamycin _doxorubicin_

Anti-leukemic effect L1210, dose (mg / day) 20 - 1 0 98 - 5 140 85 toxic 2.5 168 110 219 1.25 133 145 236 0.6 114 130 176 0.3 96 118 157 0.15 - 97 143

Toxicity (mouse) LD ^ q

Intraperitoneal dose (mg / kg) 24.4 · 12.5 12 ... 14

The cytotoxicity of MA144 components to cultured L1210 cells is examined below. The MA 144 components of the invention inhibited the growth of mammalian cultured tumor cells, especially at low concentrations, and completely inhibited RNA synthesis. In this experiment, L1210 cells were seeded in RPMI medium (Nissui, Rosewell Park Memorial Institute 1640) containing 20% calf serum, then cultured at 37 ° C for 3 days in a CCl 2 incubator, and 0.1 μg / ml was added on the first day. a compound of the invention. Included-14 ...

In this C test compound, the compounds of the invention were used at a concentration of 0.5 μg / ml for RNA synthesis and 1.0 μg / ml for DNA synthesis, and C-thymidine or uridine was added to the medium for 60 minutes at 37 ° C. The effect of the compounds on DNA and RNA synthesis and growth was demonstrated as a percentage of inhibition compared to the control experiments, as shown in the following table. The results show that even at low concentrations, MA1HH components significantly inhibited the growth and RNA synthesis of cultured L1210 cells. These results support the therapeutic effect on tumors in animal experiments.

Composition of RPMI medium L-echinin hydrochloride 240.0 mg L-aspartic (monohydrate) 56.8 mg L-aspartic acid 20.0 mg L-cystine dihydrochloride 65.0 mg L-glutamic acid 20.0 mg L-glutamine 300.0 mg glutathione 1.0 mg glycine 10.0 mg L-histidine hydrochloride (monohydrate) 20.3 mg L-hydroxyproline 20.0 mg L-isoleucine 50.0 mg L-leucine 50.0 mg L-lysine hydrochloride 40.0 mg L- methionine 15.0 mg L-phenylalanine 15.0 mg L-proline 20.0 mg L-serine 30.0 mg L-threonine 20.0 mg L-tryptophan 5.0 mg L-tyrosine 20.0 mg L-valine 20.0 mg biotin 0.2 mg

Ca-pantothenate 0.25 mg choline chloride 3.0 mg folic acid 1.0 mg I-inositol 35.0 mg nicotinamide 1> 0 mg para-aminobenzoic acid 1 50 mg pridoxin hydrochloride 1> 0 mg riboflavin 0.2 mg thiamine hydrochloride 1.0 mg cyanocobalamin 0.005 mg sodium chloride 6000.0 mg potassium chloride 400.0 mg calcium nitrate (anhydrous) 69.5 mg 9,63945 disodium hydrogen phosphate (anhydrous) 801.0 mg magnesium sulfate 48.8 mg glucose 2000.0 mg phenol red 5.0 mg sodium bicarbonate

The above substances are dissolved in water to form 1 liter of aqueous solution.

Effect of MA144 components on growth and synthesis of cultured L1210 cells.

Estymis-%

Growth of RNA in DNA

Compound (in 2 days) _synthesis_

Aclasinomycin 89.7 81.6 69.6 MA144-S1 86.0 74.1 76.5 -S2 88.1 65.6 48.8

The process of the invention can be carried out as a simple hydrolysis using dilute inorganic acids such as hydrochloric acid or sulfuric acid. The anthracycline glycosides used as starting materials can be used either in purified form, mixtures, salts or in impure form, for example by using substances containing these anthracycline glycosides, such as culture broth or crude broth extracts.

The chemical process is illustrated by the following reaction scheme: 63945

TO

R, O OOOCHj COOCH, ΦΟΟ> “· 0H 0 / r-Q? hydrolysis OH 0 OH 0 IPy ->

/ G; 0v? r ~ ^ CH CH

Iyy ^ s o (^ n <«3 1 PH" oh R3

where H 1 = H or OH R 3 = H

RV

CH3 »<Si) (¾ 0 Jj

HW3 ° N

hoV / ° \ L / or V7

OH

63945

The reactions can be represented as follows:

Aclasinomycin A MA144-N1 MA144-G1 MA144U1 MA11 + 14-Y Hydrolysis MA144-S1 MA144-M1 ^ (R1 = H »R3 = H) MA144-G2 MA144-U2

Rhodinorubin A Hydrolysis ^ MA144-S2

Sinervin A <R1 * 0H · VH) MA144-M2

The starting materials used in the process according to the invention are described in the literature as follows: MA144-G1, -G2, -N1, -U1, -U2, -Y from the parent application of this application FI-772915; aclasinomycin A from FI patent publication 54,496; MA144-M1 and -M2 from DE-A-2,715,255; rhodirubin A from U.S. Patent 4,127,714 and synerubin A from U.S. Patent 3,864,480.

The reaction conditions for applying the process of the invention are conventional. Depending on the inorganic acid used, the dilution may be, for example, 0.1-pair% in the case of hydrochloric acid or 0.5-pair% in the case of sulfuric acid. The hydrolysis is usually carried out at room temperature and the reaction time varies from 10 minutes to 1 hour depending on the acid concentration.

The hydrolytic formation of MA144-S1 and -S2 is identified using thin layer chromatography on silica (Merck Co 6 ° F254 silica plate, n-butanol: acetic acid: water 4: 1: 1 or chloroform: methanol 20: 1) after neutralization and concentration. R7 ~ values are compared to authentic sugars and the chemical structure of the starting glycoside.

MA144-S1 or -S2 can be isolated and purified by conventional methods used to purify water-immiscible antibiotics. These include solvent extraction, solvent precipitation, concentration, gel filtration, countercurrent partitioning, chelation with metal ions, and column chromatography.

An organic solvent such as chloroform, acetone, n-butanol, methanol, ethanol and ethyl acetate may be used for the extraction, or they may be present in the hydrolyzate as an acidic aqueous solution. Alternatively, they can be extracted as a water-insoluble organic solvent at a pH of 6 to 9, concentrated under reduced pressure, mixed with an acidic aqueous solution having a pH below 4, and re-extracted as a weakly basic solution as an organic solvent. By repeating this method, the product is obtained in very pure form.

Alternatively, or in connection with an extraction process, the products may be recovered by column chromatography using adsorbents such as activated carbon, silica gel or modified dextran.

Countercurrent partitioning or liquid chromatography using suitable organic solvents may also be applied. The active extracts obtained by these methods are concentrated under reduced pressure to give a yellow (-S1) or red (-S2) powder.

The solution containing MA144-S1 and -S2 may also be lyophilized alone or with at least one substance which may be serum, serum protein, globulin, gelatin, glycerol, sugar, amino acid, deoxyribonucleic acid or an inorganic or organic acid such as hydrochloric acid, phosphoric acid , acetic acid, succinic acid or pantothenic acid.

If necessary, the above-mentioned purification and separation methods can be repeated several times to obtain a cleaner product.

The following examples illustrate the invention.

Example 1 400 mg of MA144-M1 was dissolved in 100 ml of 0.5% hydrochloric acid and hydrolyzed at 20 ° C for 15 min. The reaction mixture was neutralized with a dilute basic solution to pH 7.0, after which MA144-S1 was extracted twice with 200 ml of chloroform. The chloroform extracts were combined and evaporated under reduced pressure. Chromatography on a 13,63945 silica gel column (3 x 25 cm column) using a 5: 100 mixture of methanol and toluene afforded the active fractions containing MA 144-N1, which were combined, evaporated and added to n-hexane. 237 mg of MA144-S1 were obtained as a yellow powder.

Example 2

In the same manner as in Example 1, but starting from 500 mg of the following compounds: aclasinomycin A, MA144-N1, -G1, U1 and -Y, after hydrolysis, neutralization, separation and purification, a yellow powder of MA144-S1 was obtained, the yield of which was as follows. / mg

Aclasinomycin A 302 MA14I + -N1 286 -G1 290 -U1 273 -Y 289

Example 3

In the same manner as in Example 1, but starting from 500 mg of the following compounds: MA144-G2, -U2, rhodorubin A, sinerubin A and MA144-M2, was obtained by hydrolysis with dilute hydrochloric acid in aqueous solution and purification of red MA144-S2 powder to give the following yield: Yield / mg MA144-G2 293 -U2 278 -M1 310 rhodorubin A 287 synerubin A 265

Claims (1)

A process for the preparation of anti-tumor anthracycline glycoside antibiotics MA1UW of the formula R10COOCH3OH0OH? MyIOH d H having a hydrogen atom or a hydroxyl group, characterized in that the hydrolysis of the general formula R10COOCH3 CH 9 CH o KX 3 <«» OHO OH 0 / CHo 7 CH0 door 3 k OH An anthracycline glycoside compound according to R3, wherein in the formula and R3 denote the following groups: 15 6 3945 __ MA144-G1 H G2 OH o MA144-N1 II <& hon— 'MA144-U1 H / -0., U2 OH | iH3 y HO) - 'OH MA144-Y H _ ° €) Aclasinomycin AH 0-0 MA 144-M1 H HO r-0 M2 OH KCH3 N \ _! / Rodorubin A OH n Θ HO x- / Sinerubiini A OH —O 16 63945 For the preparation of anthracycline-glycosidantibiotics based on the formula MA1UU with the formula: R- 0 COOCH3 -Ä, M '«3 I OH H for R the hydrolyser of the anthracyclyl glycosides with all the compounds of formula R1 o cooch3 OCOC® "'" · OH 0 0 0 i @ 7 ° nC®3 ° 0H ®3 R3 i vilken formula R1 och R ^ betecknar feljande grupper: