FI59101C - FOERFARANDE FOER FRAMSTAELLNING AV ANTIBAKTERIELLA 3- (SUBSTITUERAD AMINO) TOLYPOMYCINONER 3- (SUBSTITUERAD AMINO) -16,17-DIHYDROTOLYPOMYCINONER OCH 3,16-DI (SUBSTITUERAD AMINO) -D,17,17 - Google Patents

Description

R55F1 M (11) NOTICE OF PUBLICATION Q 1 WA 1 j 1 'UTLÄGGN I NGSSKRIFT 0 y 1 u 1 C (45) Patent rV-- Uy 36 1321 ^ T ^ (51) Kv.lk?/lnt.CI.3 C O? D 4-98 / 08 FINLAND — FINLAND (21) Patanttihakamui - PKantamBknlng 750743 (22) H * k * ml * plhf · - Ansdknlngidftg l4.03.75 '' (23) The beginning of the cloud — Glltlghttsdtg l4.03.75 (41) Has become swept— Bllvlt offentlig l6.09.75

Patent and Registration Office .... .......

. ,, (44) Date of NlhUvUulptnon et al. - ratent · och register Styrelsen Ansakin utitgd och uti.skrtftsn pubitcend 27.02.81 (32) (33) (31) Privilege requested — Bigird prlorltit 15.03.74 01.11.74 England-England (GB) 11674/74, 47398/74 ( 71) Alfa Farmaceutici SpA, Via Ragazzi del 99 »No. 5, 40133 Eologna, Italy-Italy (lT) (72) Mario Brufani, Roma, Egidio Marchi, Bologna, Giuseppe Mascellani, Bologna, Italy-Italy (IT) (74) Oy Kolster Ab (54) Method of antibacterial 3 ~ (substituted amino) tolypomycinones, 3- (substituted amino) -1,6,17-dihydrotolypomycinones and 3,16-di (substituted amino) -16,17-dihydrotolypomycinones 3 - (substituted amino) -1,6,17-dihydrotolypomycinone and 3,16-di (substituted amino) -1,6,17-dihydrotolypomycinone

The invention relates to a process for the preparation of new antibiotic compounds and in particular new derivatives of certain tolypomycinones.

The antibiotic Tolypomycin Y is prepared as described in U.S. Patent 3,627,881 by culturing the microorganism Streptomyces Tolypophorus (ATCC 21177) in a suitable nutrient medium. JA Patent 72,37199 discloses a process in which Tolypomycin Y is hydrolyzed to give tolypomycinone of the formula: 59101 CH-, CH 2 λ

CH 3 CO-V

OH OH

'1 17

OH C | B

V "

ΠΜ I

o o________ o CH3

Tolypomycin Y is a very potent antibiotic, but it has the disadvantage that it is very unstable under acidic conditions; on the other hand, tolypomycinone, which is very similar in structure to Tolypomycin Y, has better stability even in the presence of acids but a strongly reduced antibiotic effect.

New derivatives of tolypomycinone have now been discovered which have an unexpectedly good antibiotic effect combined with excellent acid resistance. These derivatives are 3- (substituted amino) tolpomycinones, 3- (substituted amino) -16,17-dihydrotolypomycinones, 3,16-di (substituted amino) -1,6,17-dihydrotolypolysinones.

The invention relates to a process for the preparation of compounds of the general formula: I 59101? 3 Γ3 CH COO Jl OH OH (11)

OH O I

\ x ^ “3 ^ 0 \

OH O H Q

1 ^ 0 3j

^ ^ 2 * nR

0

0- I

0.3 wherein R is H, OH, mono- or dialkylamino, phenyl which may be substituted by hydroxy, lower alkoxy or lower alkyl with an amino group, or a 5- or 6-membered heterocyclic group which may be substituted by lower alkyl -, a nitro or hydroxy group; n is an integer 1-Uj and.Q is one of the groups ch3 ch3 ch3 - CH = C -, - CH2 - CH -, - CHg - C -

NH (CH 2) n

(17) (16) (17) (16) (17) (16) (R means the same as above)

Examples of the group R when it is an alkyl, amino or dialkylamino group include: methyl, amino, dimethylamino, ethylamino, diethylamino, propylamino and dipropylamino.

Examples of the substituent R when it is a heterocyclic group include: pyridyl, piperidyl, morpholinyl, piperazyl, thienyl, furyl »tetrahydrofuryl, imidazolyl, oxazolyl, pyrazolyl, pyryl, pyrrolidyl and triazolyl.

Specific examples are dimethylaminophenyl, N-methylpiperidyl, nitropyridyl, N-methylpyrrolyl and N-methylpyrrolidinyl.

4,5991

Compounds of general formula (II) are prepared by adding an amine? (Cri) li to polypomycinol (formula I) and then preferably adding an oxidizing agent, such as potassium ferricyanide or manganese oxide. The process is generally carried out in the presence of a solvent, for example chloroform, benzene, toluene or dioxane. The temperature at which the reaction is carried out is not critical and is therefore preferably carried out at ambient temperature. The reactive amine and tolpomycinone compounds are preferably used in stoichiometric amounts or the amine is used in excess.

Depending on the reaction sensitivity of the amine, it is possible to either attach the amino substituent solely to the 3 'position, or to both the 3' and 16 'positions, to form 3,16-di- (substituted amino) -1,1,17-dihydrotolypomycinone. Normally, large amounts of amine lead result in substitution at both the 3 'and 16' positions, while equimolar amounts or a reasonable excess of amine cause substitution predominantly at the 3 'position. Small amounts of the 3,16-di- (substituted amino) derivative, which are occasionally formed in the preparation of the 3-derivatives, can be separated by known methods, e.g. from chromatography.

3- (Substituted amino) -1,16,17-dihydrotolypomycinone can be prepared as described above from 16,17-dihydrotolypoinysinone; lo, 17-dihydrotolypomysinonista.

The compounds according to the invention (i.e. the compounds of formula (II)) have an important antibiotic effect, as indicated by the minimum concentration value, and are potent antibacterial agents against both Gran-positive and gram-negative bacteria. Their effect is bactericidal. The compounds of the invention can therefore be used to control infection in both animals and Snow. In addition, the compounds of the invention are stable under the pH conditions common in biological fluids in the gastrointestinal tract; in particular, they are persistent in the ph conditions prevailing in the area of the racket of animals and humans. As an account, Tolyponycin Y and tolypomycinone are the opposite, because the former, despite being a very potent antibiotic, is unstable in the presence of acids, while the latter, despite its better stability under acidic conditions, has a substantially reduced antibiotic effect.

The stability of the compounds of the invention under acidic conditions, such as in human and animal molluscs, makes it possible to administer the compounds orally, which is a substantial advantage because oral administration is generally the most preferred form of administration for antibiotics.

5 59101

The invention is further illustrated by reference to the following examples. The Rf values given in the examples belong to the Rf of tolypomycinone and are calculated from the formula: Rf (one) = Rf for compound / Rf for tolypomycinone. The UV spectrum was determined in methanol and make is given in nm in the examples, log is given in parentheses. Percentages are by weight.

The tolyponysirone derivatives of this invention can also be used in the form of acid addition salts, e.g. with HCl, citric acid, oxalic acid, which are pharmaceutically active. Furthermore, the compounds of general formula (IV) can be used as basic addition salts, e.g. salts of alkali metals or organic bases such as triethylamine.

Example 1 Preparation of 3-benzylaminootolypomyBinone (Compound 1) 50 U mg of benzyl amine a dissolved in 5 ml of chloroform was slowly added over 1 hour to 1 g of tolypomycinone dissolved in 30 ml of chloroform. 1.25 g of manganese dioxide were then added. The mixture was stirred mechanically for 20 hours and filtered, after which 10 ml of a 5% aqueous solution on lemon day was added. The organic phase was separated, extracted with three 10 ml portions of water, dried over anhydrous sodium sulfate and then the solvent was evaporated.

The resulting solid was dissolved in 2 ml of ethyl acetate and passed through a chromatography bead having an inner diameter of k, 6 cm and a height of 46 cm (volume 770 ml) filled with silica gel 60 Merck. The column was eluted with OO / 20 (v / v) ethyl acetate / benzene. The fractions, each 10 ml, which were collected by thin-layer chromatography, TLC, (on silica gel 60 Merck F2) to give a purple component with Rf (one) = 1.36 (mobile phase ethyl acetate), combined, dried, dissolved in 1.5 ml of ethyl acetate. and passed through a second column (inner diameter 2, h cm, height 37 cm, volume 105 ml) packed with silica gel 60 Merck. The column was eluted with 90/10 (v / v) ethyl acetate / methanol containing 1% oxalic acid. Fractions with a purple component with Rf (ykei) = 36 by TLC as above were collected and combined, the solvent was evaporated and the residue was dissolved in 100 ml of chloroform.

The organic solution was then washed with three 20 ml portions of water, dried and the solvent was evaporated. It was 68% in the hall.

The solid product was then recrystallized three times from chloroform with the addition of 15 volumes. part of n-hexane. The analysis showed that the nitrogen content of the product

i-v - K

is 6 591 01 den to be 3.1 ** '! <>> compared to the calculated value 3.¾¾% · UV spectrum: 23h, ^ 3) i 26U (U, 33) i 319, 13) i 362 (3, 37); 535 (3.28).

the manganese dioxide used was prepared as follows: 10 g of MaSO 2 - H 0 were dissolved in 200 ml of water, followed by the addition of 6.2 g of KMnO 2. A solution of 3 G of sodium hydroxide in 20 ml of water was added and the mixture was heated at 30-90 ° C for 3 hours. The solid was collected, washed with water, filtered and dried at 120 ° C for 1+ hours.

Example 2 Preparation of 3,1b-di- (benzylamino) -1,6,17-dihydrotolypomycinone (Compound 2) 300 mg of tolyporaysinone was dissolved in 6 ml of dioxane, and while the solution was stirred and cooled with ice water, 0.5 ml of benzylamine was added. The mixture was stirred for another 6 hours at room temperature. The purple solution was then poured into 60 ml of chloroform and mechanically stirred with 60 ml of a 2.5% aqueous solution of potassium ferricyanide. The pH of the aqueous solution was then raised to 7 by the addition of excess hydrochloric acid with mechanical stirring.

After separating the oceanic layer, the aqueous layer was extracted with 20 ml of chloroform. The chloroform layers were combined, washed with 20 ml of water, dried and the chloroform evaporated in vacuo.

TLC (mobile phase ethyl acetate, silica gel 60 Merck F25 * 0) showed a single spot of Rf (one) = 1.73.

The material was chronographed on a column containing 25 g of silica gel 60 Merck and eluted with ethyl acetate to give 280 mg of product. The analysis showed a nitrogen content of 1.77% * compared to the calculated value of H, 55 UV spectrum: 233 (U, 37h 262 (ä, 3l); 315 (1 *, 19) i 377 (3.87); 522 (3, The nuclear magnetic resonance spectrum (ΛΜΗ) in CDCl 3 (60 Mc) for the product showed the absence of the signal H (3) and the presence of the signal at ^ = 7 → ^ 2 ppm, which is related to the five aromatic protons in the benzyl group.

Example 3 Preparation of 3-methylaminotolypomycinone (Compound 3) and 3,16-di- (methylamino) -16,17-dihydrotolypomycinone (Compound lp) In 500 mg of tolypomycirone dissolved in 5 ml of dioxane was added 0.12 ml of methylamine. A solution of 17.5 l in water / dioxane (1: 1, v / v) was stirred for 30 minutes with mechanical stirring and at a temperature below 10. The mixture was stirred for a further 1 591 01, after which 600 ml of manganese dioxide were added. The mixture was then stirred for 1 hour, after which it was filtered and 1U ml of chloroform and 8 ml of 1N aqueous citric acid solution were added.

The organic layer was then separated, washed with three 10 ml portions of water and dried, after which the solvent was evaporated. The resulting solid was dissolved in a mixture of benzene / ethyl acetate / methanol (10: 90: 1 by volume) and chromatographed through 100 volumes of silica gel 60 Merck saturated with 1) oxalic acid. Elution was performed with benzene / ethyl acetate / methanol (10: 90: 1) with ethyl acetate containing 1% oxalic acid. Each 10 mL fraction was chromatographed using TLC with benzene / ethyl acetate / methanol (10: 90: 1) as the mobile phase. The fractions giving the purple component at Rf (one) = 0.86 were pooled and dried to give 110 mg of 3-methylaminotolypomycinone. Nitrogen content of the product: found 3.87 calculated: 3.79% · UV spectrum: 23k (btk2) ‘t 26 ·» (U, 31 *) »319 (U, 08) i 362 (3.80); 533 (3.17).

Elution was continued using 80/20 methanol / acetone as eluent and fractions giving the purple-brown component at Rf (yk3i) = 0.5U by TLC (same conditions as above) were pooled, combined and dried to give 120 mg of 3.16. di- (methylamino) -l6,17-dihydrotolypomysinonia.

UV spectrum: 23U (ä, 30); 262 (U, 26) ·, 318 (1.03); 373 <3.97) and 530 (3.39).

The experiment was repeated using the same amounts of reactants and under the same conditions, except that chloroform was used as the reaction solvent.

Example h Preparation of 3- (2-morpholinoethylamino) -tolypomycinone (Compound 5) and 3,16-bis- (2-morpholinoethylamino) -1,6-17-dihydrotolyponyoninone (Compound 6) In 200 mg of tolyponysinone was dissolved in 5 ml of chloroform and 1 ml of an 8E solution of 1- (2-aminoethyl) morpholine in chloroform was added over 1 hour, the solution was stirred mechanically for 30 minutes, then 200 ml of manganese dioxide was added. The solution was stirred for another 5 minutes and filtered, after which 30 ml of chloroform was added. The organic solution was washed with U ml of 2: 3 aqueous citric acid solution and then washed with three 10 ml portions of water. The organic phase was dried and the solvent was evaporated.

The product was dissolved in a 60: 37: 5 v / v mixture of ethyl acetate / chloroform / methanol and chromatographed through 100 parts of silica gel 60 through Merck and then eluted with the same solvent mixture. Fractions giving the purple component Rf (one) = 0.58 by TLC (silica gel 60 Merck F25U) using ethyl acetate / chloroform / methanol, 60: 37: 5 »eluent a, were combined and the solvent evaporated to give 0.120 g. compound 5 · Nitrogen content: calculated 5.01 found U, 9U%. UV spectrum: 230 (U, Ul); 26U (J », 27); 59101 8 313 (U.10); 302 (3.33); 533 (3.23).

The column was then further eluted with ethyl acetate / methanol 50:50 (v / v) and fractions giving Rf (one) = 0.08 by TLC (same conditions as above) were combined, dried and the solvents evaporated to give 50 mg of compound o.

Example 5 Preparation of 3- (2-hydroxyethylamino) -tolypomycinone (Compound 7)

To a solution of 130 mg of tolypomycinone in 5 ml of chloroform was added 15 mg of ethanolamine in 0.075 ml of chloroform. After stirring for 1 hour, an additional 1 U mg of amine was added. The mixture was then stirred for 30 minutes, after which it was washed with 5 g; 11a of a 0.5% aqueous solution of citric acid. Then 5 ml of a 5 .mu.l aqueous solution of potassium ferricyanide was added.

The organic layer was dried and the solvent was evaporated in vacuo. The solid product was chromatographed on a 60 Merck silica gel column and eluted with 1% ethyl acetate. of oxalic acid. The fractions which, by TLC (silica gel 60 Merck F25 * 0, gave the purple component Rf (one) = 0.6 U (ethyl acetate as mobile phase) were combined, concentrated, washed with water, dried and the solvent evaporated in vacuo.

Example 6 Preparation of 3- [3- (N, N-dimethylamino) -propylamino] -tholypolylysine (Compound 8)

To a solution of 318 mg of tolypomycinone in 5 ral of chloroform was gradually added over 1 hour, 53 mg of N, N-dimethylpropylenediamine dissolved in 0.5 ml of chloroform, keeping the temperature below 10 ° C and stirring the mixture. Stirring was continued for a further 1 hour, 310 mg of manganese dioxide were added and after stirring for a further 30 minutes the mixture was filtered. 50 ml of chloroform and 5 ml of a 1 # aqueous solution of citric acid were added. The organic layer was separated, washed three times with 10 ml portions of water each time, dried and the solvent was evaporated. The solid was dissolved in 1 mL of methanol and passed through a column of silica gel 60 Merck using methanol / water (v / v Sunde 80:20) as eluent.

Fractions by TLC (silica gel 60 Merck F251 *) gave the purple component Rf (one) = 0.16 (ethyl acetate / pyridine as mobile phase, 70:30 by volume). The methanol was evaporated in vacuo and the remaining aqueous phase was extracted several times with chloroform. The organic extracts were combined, dried and then the solvent was evaporated to give 80 ml of compound 3.

f 9 59101 UV spectrum: 2314 2 Sk (U, 37) i 319 (U, 17); 362 (3, Ö9) V 535 (3.25).

Example 7 Preparation of 3-benzylamino-16,17-dihydrotolypomycinone (Compound ') 300 mg of tolypomycinone was dissolved in 25 ml of ethanol and hydrogenated at room temperature and atmospheric pressure in the presence of 50 mg of palladium / charcoal catalyst. 35 ml of hydrogen at 27 ° C were used over 1 hour.

the catalyst was then filtered off, the filtrate was concentrated to 1/6 of its original volume and 100 ml of ethyl acetate was added. The solution was then shaken for a long time with a 3 / * solution of ferric chloride (1 * portion, 30 ml each). The organic phase was then washed twice with water, dried and the solvent was evaporated. Virtually pure 16,17-dihydrotolypomycinone with Rf (one) = 0.98 (eluent ethyl acetate) was obtained by TLC (silicon gel 60 Merck F25 * 0 · 1 * 00 mg of 16,17-dihydrotolypomycinone & dissolved in 8 ml of dioxane and 0.1 ml of benzylamine was gradually added under ice-water with mechanical stirring, the solution was stirred for a further 1 * 0 minutes with cooling, then 75 ml of chloroform was added, followed by 75 ml of a 3% aqueous solution of potassium ferricyanide. 1 hydrochloric acid.

The organic layer was separated and the aqueous layer was extracted with 30 ml of chloroform.

The organic extracts were combined, washed with 20 ml of water, dried and the solvent evaporated in vacuo.

TLC on silica gel Merc0 Merck F25I + »using ethyl acetate / methanol (90: 1 * v / v) as mobile phase gave a red spot Hf (ykei) = 1.32: 38a. The product was chromatographed on 30 g of silica gel using ethyl acetate / methanol (98: 2 by volume) as eluent.

100 mg of 3-benzylamino-1b, 17-dihydrotolyponysinone were obtained.

Example 8 Preparation of 3- [4- (dimethylamino) -ethylamino] -tholypomycinone (Compound 10)

To a solution of 100 mg of tolypomycinone in 2.5 ml of chloroform was gradually added over 1 hour 12 mg of N, 11-dimethylethylenediamine dissolved in 0.170 ml of chloroform while maintaining the temperature below 10 ° C and continuing to stir. Stirring was continued for a further 1 hour, 110 mg of manganese dioxide were added and after stirring for a further 30 minutes the mixture was filtered.

10 59101 Then 50 ml of chloroform and 5 ml of a 1 liter aqueous solution of citric acid were added. The organic layer was separated, washed with three 10 ml portions of water, needed and the solvent was evaporated. The resulting solid was dissolved in 1 mL of methanol and passed through a column of silica gel b0 Merck using ethyl acetate / methanol (60: v / v) as eluent, with ethyl acetate containing $ 1 w / v. of oxalic acid.

Fractions by TLC (silica gel 60 Merck F25M to give the purple component Rf (one) = 0.5 ° (ethyl acetate / methanol as mobile phase) were combined, the methanol was then evaporated in vacuo and the remaining aqueous phase was extracted several times with chloroform. The residue was recrystallized from chloroform / n-hexane to give 80 mg of 3- [2- (dimethylamino) ethylamino] tolyl pomycinone.

UV spectrum: 23k (U, 1 * 0) i 26¾ (U, 3l) i 319 (M3); 362 (3.35); 530 (3.20).

Example 9 Preparation of (1-methylpiperidyl) methylamino-4-tolpomycinone (Compound 11)

To a solution of 300 ml of tolypomycinone in k ml of chloroform was gradually added 200 mg of h-aminomethyl-ylmethylpiperidine dissolved in 2 ml of chloroform. The solution was stirred for 12 hours at room temperature. With continuous stirring, 1 ml of chloroform was then added, followed by 20 ml of an aqueous solution of potassium ferricyanide. The silicon in the solution was adjusted to Y by the addition of 0.5 L of hydrochloric acid a.

The organic layer was then separated, dried and evaporated in vacuo. TLC (silica gel 60 Merck F25 2) using methanol / acetic acid (9: 1 v / v) as eluent gave a residue with a simple purple spot at Rf (one) = 0.31. The product was chromatographed on a column of silica gel όΰ Merck, eluted with methanol / acetic acid 9: 1 · The fractions containing the purple product were combined and the solvent was evaporated in vacuo. The residue was dissolved in chloroform, and the solution was washed twice with water, dried, and then evaporated in vacuo.

Example 10 Preparation of 3- (3-hydroxypropylamino) -tolypomycinone (Compound 12)

To a solution of 200 mg of tolypomycinone in 5 ml of chloroform was added, with mechanical stirring, a solution of 70 mg of 3-aminopropanol in 1 ml of chloroform. After stirring for 1 hour, an additional 30 mL of chloroform was added, followed by 15 of an aqueous solution of potassium ferricyanide in 3 .mu.l. The pH of the aqueous solution was then adjusted to 7 by the addition of 0.5 hydrochloric acid.

The organic layer was separated, dried and the solvent was evaporated in vacuo. The product gave a spot on TLC (silica gel 60 Merck F25U) at Hf (one) = 0.33 (ethyl acetate as eluent).

The residue was chromatographed on silica gel 60 ° Merck using ethyl acetate as eluent.

Example 11 Preparation of S-ethylamino-1 H -dihydrotolypoinysinone (Compound 13) 300 mg of methylamine were added under reduced temperature and mechanical stirring to 250 mg of 16.17 "dihydrotolypomycinone dissolved in 5 ml of dioxane. Stirring was continued at room temperature for 10 minutes. 50 ml of chloroform were then added, followed by 50 ml of an aqueous solution of potassium ferricyanide 2, 2. The pH of the solution was carefully adjusted to 7 »by adding 0. 5Π hydrochloric acid.

The organic phase was then separated and the aqueous phase was extracted with 20 ml of chloroform. The organic extract was combined with the organic phase and the mixture was dried and the solvent was evaporated in vacuo. TLC gave the purple product at Rf (one) = 1.0q (eluent ethyl acetate / methanol 2: 1) and unreacted tert -17.17 dihydrotolpomycinone. The product was chromatographed on 25 g of silica gel 00 Merck and eluted with ethyl acetate. 1 ^ 5 mg of product was obtained.

The JMR spectrum (Ui) Cl: 33a, όο Mc) indicated the absence of signal il (3).

Example 12 Preparation of? and then filtered, 70 ml of chloroform was added and the solution was washed with 5 ml of 5/2: 8 aqueous citric acid solution and three 20 ml portions of water.

The organic phase was dried and the solvent was evaporated. The residue gave a spot in TLC (silica gel 00 Merck ^ 25 ^) eluting with ethyl acetate / benzene / methanol (70:25:15 v / v) at Rf (one) = 0.77.

The product was chromatographed on a column of silica gel 60 Merck, eluting first with ethyl acetate / benzene / methanol, 88: 10: 2 by volume, (ethyl acetate containing 1% w / v oxalic acid) to separate the unreacted starting material and then ethyl acetate. benzene / methanol, 70:25:15 v / v (ethyl acetate containing 1 w / v oxalic acid) to elute the desired product. The product-containing fractions were combined, concentrated to 1/3 of their original volume, mixed with 70 ml of chloroform and washed several times with 20 ml portions of water.

The organic phase was then dried and the solvent was evaporated. The residue was dissolved in 1 ml of chloroform and stirred by gradually adding, while cooling, 20 ml of n-hexane. 170 ms of 3 "(N-pyridyl) methylamino-tolpomycinone were left.

Example 13 Preparation of 3- (2-Tetrahydrofuryl) -methylamino-tolonopyronylone (Compound 15) 2b0 mg of tetrahydrofurfurylamine dissolved in 2 ml of chloroform was gradually added, with mechanical stirring, to 300 mg of tolpomysirone dissolved in 10 ml of chloroform. After 1 hour, 150 πΐβ of mancen dioxide was added and the suspension was stirred for a further 30 minutes, after which it was filtered. Ml0 ml of chloroform was then added and the solution was washed with 5 / β: 11 & aqueous citric acid solution and then twice with water. The organic phase was dried and the solvent was evaporated. TLC on silica gel 60 Merck F25 ** using ethyl acetate / benzene / methanol (70: 25: 5 v / v) as eluent gave a spot at Rf (one) = 0.37.

The product was dissolved in 2 ml of ethyl acetate and chromatographed on a column containing 35 g of silica gel υϋ Merck, using ethyl acetate containing 1% oxalic acid as solvent. The fractions which gave Rf (one) = 0.07 by TLC were combined, washed several times with water, dried and the solvent was removed. the solid was further purified by chromatography through 1 * 0 g of silica gel 60 Merck using ethyl acetate / benzene / acetone (v / v ^ 9: ^ 9 = 5) as eluent. The product-containing fractions were combined, washed with water, dried and the solvent was evaporated. The solid was dissolved in 1.5 ml of chloroform and precipitated with 20 ml of n-hexane. The product was obtained as 11 * 5 mg, giving a nitrogen content of 3.61% compared to the calculated value of 3Λο / ..

UV spectrum: 23 ^ (U.U3); 261 * (U, 33) and 319 (U.10) ·, 362 (3.30) ·, 535 (3.19).

Example 1 Preparation of β- (piperinylamino) -tolypomyainone (Compound 16) 150 mg of piperinylamine dissolved in 1.5 ml of chloroform was added slowly with mechanical stirring over 2 hours to 300 mg of tolypomycirone dissolved in 10 ml of chloroform. chromatographed on silica gel 60 F25I + Merck using ethyl acetate / bercene / methanol (70: 25: 5 v / v) as eluent. The Rf (one) of the product was 1.20.

59101 After 3 hours of reaction, tolyporaysinone had disappeared from the reaction mixture. 100 ml of chloroform and 5 2/8 aqueous citric acid solution were added, and then the organic phase was washed several times with water and dried, after which the solvent was dried and evaporated. The residual solid was then collected in 2 ml of ethyl acetate (containing 1% oxalic acid) / benzene / methanol (70: 25: 5 by volume) and chromatographed using the same solvent through a column of silica gel bO Merck (inner diameter 2, U cm, height 9.5 cm).

Fractions containing the desired product were combined and washed several times with water. After drying, the solvent was evaporated from the organic phase to give 115 mg of crude 3 '(piperinylamino) tolpomycinone.

The solid was then taken up in ethyl acetate and rechromatographed through a column of silica gel bO Merck (inner diameter lo mm, height 50 mm) using 1% oxalic acid ethyl acetate as eluent. The fractions containing the desired compound Ιό were washed with water and then dried, after which the residue was taken up in 1 ml of chloroform and mixed with n-hexane. 5 * + mg of pure product were obtained.

UV spectrum: 235 (M9); 205 (M2); 317 (U, 20); 370 (3.83); 530 (3.23).

Example 15 Preparation of 3- (p-dimethylamino-benzylamino) -to-polynyninone (Compound 17) 210 mg of p-dimethylaminobenzylamine in 1.5 ml of chloroform was slowly added over 2 hours to 300 mg of tolyponysinone in 5 ml of chloroform.

100 mg of manganese dioxide was then added and the mixture was stirred for 30 minutes. The solid was then filtered off and 5% aqueous solution was added to the filtrate on citrate day. The mixture was washed with water and dried, after which the solvent was evaporated.

The residual solid was taken up in 1 ml of ethyl acetate (containing 1% oxalic acid) / methanol (97: 3 by volume) and chromatographed, using the word solution, through a column of silica gel 60 Merck (internal diameter 3 μm, height 9 cm). Fractions determined by thin layer chromatography (TLC) on silica gel 60 F25 ^ Merck using chloroform / methanol as eluent (volume ratio 95 = 5 containing 3-p-dimethylaminobenzylamino) -tolypomycinone ^ Tif (one) = 0.0g7> were combined, was made somewhat alkaline by the addition of sodium bicarbonate and then washed with water. The organic phase was dried and the solvent was evaporated to give 90 mg of pure product.

UV spectrum: 200 (U, 6oh 320 (U, 22); 362 (3.98); 5? 0 (3.33).

Example 16 Preparation of 3 - [(N-imidiazolyl) ethylamino] n-butyl pomycinone (Compound 18) 190 mg of histamine in 1.6 ml of chloroform / methanol (2: 1 by volume) 1.1 59101 was added slowly over 1 hour. mg of tolyponysinone in 10 ral of chloroform.

220 mg of manganese dioxide, followed after 15 minutes by a further 70 mg of histamine in 0.6 ml of chloroform / methanol (2: 1 by volume), was added. The reaction mixture was chromatographed on silica gel 60 F25I4 Merck using ethyl acetate / methanol (75:25 v / v) as eluent; the Rf (one) of the product was = 0.06.

The reaction was allowed to proceed for k hours, after which the manganese dioxide was filtered off. To the solution were added 100 ml of chloroform and 5 ml of 5,7 aqueous citric acid solution, after which the mixture was washed with water and dried, and the solvent was evaporated. The residue was taken up in 2 ml of ethyl acetate with 1% oxalic acid / methanol (75:25) and chromatographed, using the same solvent, through a column of silica gel 60 Merck (inner diameter 2.7 cm, height 6.5 cm). ).

fractions; containing 3- [2- (k-imidiazolyl) -ethylamino] -tholypomycinone were combined, evaporated to 1 / k of its original volume, taken up in 100 ml of chloroform, washed with water containing a few drops of 1N sodium hydroxide and then with water alone. and dried; the solvent was then evaporated. The residue was dissolved in 1.5 ml of chloroform and then remixed with 30 ml of n-hexane. 205 mg of pure product remained.

UV spectrum: 233 (k, 51); 261 * (it, 36); 319 (k, 16); 36k (3.39); 530 (3.2k).

Example 17 Preparation of 3 - [(β- (methyl-piperazin-1-yl) -propylamino] -7-tolyporjiginone (Compound ^ 9) k28 mg 1 - (3-aminopropyl) -1 + - methylpiperazine in 3 ml of chloroform was added with mechanical stirring to 90 mg of tolpomycinone in 3 ml of chloroform as a 90 minute pig. After that, 200 mg of manganese dioxide were added and the reaction was allowed to continue for 30 minutes. After this time, the solution was filtered, 100 ml of chloroform was added, and the mixture was stirred with 2 ml of 3 aqueous citric acid solution. A small amount of sodium acetate was added to the aqueous phase to facilitate extraction of the desired compound 19 from chloroform. The organic phase was washed three times with water, dried and the solvent was evaporated. The residual solid was collected in 1 ml of acetone / methanol (25:75 by volume) containing 1 oxalic acid and chromatographed through a column of silica gel 60 Merck (inner diameter 2.1 * cm, height 6.2 cm). using the same solvent as eluent. Fractions found by TLC on silica gel 60 F25k Merck (eluent: chloroform / methanol, 90:10 by volume) to contain the desired compound 19 ^ Rf (one) = 0.8? 2 were combined, dried and taken up in chloroform and washed. with water. The organic phase was then dried and the solvent was evaporated in vacuo. The residue was taken up in 2 ml of toluene and precipitated with 30 ml of n-hexane. 65 mg of S- (trans-methylpiperazir-1-yl) propylamino] tolpomycinone were obtained.

UV spectrum: 228 (, 33); 267 (k, 2k); 316 (k, 07); 365 (3.79); 535 (3.15).

59101

Example 18 Preparation of 3- (2 * phenylamino) -tolypomycinone (Compound 20) 260 mg of 2-1-phenylamine in 1.3 ml of chloroform were added with mechanical stirring over 2 hours to 100 mg of tolpomysirone in> * ml of chloroform.

150 mg of manganese dioxide were then added, and after the mixture had reacted for 30 minutes, the manganese dioxide was filtered off.

The filtrate was added with 100 ml of chloroform, and the solution was washed with a mixture of 20 ml of water and 5 ml of 5% aqueous citric acid solution, and then washed three times with water alone. The solution was dried and the solvent was evaporated. The solid thus obtained was chromatographed on a column of silica gel bO Merk (inner diameter 2.2 cm, height 10 cm) using ethyl acetate containing 1% oxalic acid as eluent. Fractions found to contain 3 '(2-phenylamino) -tolyponysinone / Rf (one) -1.0 to 7 r.uC (eluted with ethyl acetate) were washed with water, dried and the solvent was evaporated.

The solid obtained on ice was collected in 2 ml of a 30:30 (v / v) mixture of acetone and water and chromatographed through a column of silanized silica gel όk Merck (inner diameter 3 cm, height 5 cm) using the same solution as eluent. The fractions containing the 3 ′ derivative were combined and the acetone was evaporated. The solution was then collected in chloroform, washed with water, dried and the solvent was evaporated. The solid obtained on ice was collected in 1 ml of chloroform, from which it was reprecipitated by adding n-hexane. 120 mg of pure compound 20 were obtained.

UV spectrum: 233 (> *, 52); 263 (> *, 35); 317 (U.lö); 370 (3.88); 530 (3.23).

Example 19 Preparation of 3- (N-imidiazolid-2-on-1-yl-ethylamino) -tolypomycinone (Compound 21) 10 mg of 1 * (γ- (β-aminoethyl) -2-imidiazolidone in 3 ml of chloroform was added mechanically with stirring over 1.5 hours to 300 mg of tolypomysirone in 5 ml of chloroform, then 120 mg of manganese dioxide was added to the solution, after stirring for 30 minutes, 100 ml of chloroform was added to the mixture, and the manganese dioxide was filtered off, 2 ml of 5% aqueous citric acid solution was added. the organic phase was washed with water and dried over anhydrous sodium sulfate, the solvent was then evaporated in vacuo, the resulting solid was collected in 1.5 ml of chloroform and the solution was applied to a 20 x 20 cm plate on a medium 2 Miru wool silica gel, 60 F25> * Merck, layer and chromatographed using chloroform / methanol (90:10 by volume) as eluent, the fractions were then separated and, after scraping off the silica gel, the fractions with Rf (one) = 0.55, corresponding to 3- (N-imidiazolid-2-on-1-yl-ethylamino) -polypolysinone.

16 59101

The triturated silica gel was then treated twice with 20 ml of methanol / water (9: 1 by volume), after which the desired compound 21 was dissolved in aqueous methanol. This solution was concentrated to a volume of J-8 ml, collected in 100 ml of chloroform and washed with water. The chloroform phase was dried over anhydrous sodium sulfate, followed by evaporation of the chloroform. The remaining solid was dissolved in 1.5 ml of chloroform, from which it was remixed with 30 ml of n-hexane. 150 mg of 3- (1H-imidazolid-2-orryl-ethylamino) -polypomycinone were obtained.

UV spectrum: 23b (U, 35) and 262 (h, 26); 316 (U, 1H); 370 (3.81+); 531 (3.19).

Example 20 Preparation of 3 - [(β-hydroxyphenyl) -ethylamino] -tolypomyelinone (Compound 22) 200 mg of tyramine in 2.5 ml of ethanol was added over 1 hour to 1 + 00 mg of tolyponysynin in 5 ml of chloroform. After the mixture was stirred for 30 minutes, 100 ml of chloroform was added. The solution was then washed several times with water containing 0.5% citric acid and then treated exclusively with water until a silicon value of 7 was reached.

The chloroform phase was then dried and the chloroform was evaporated. The solid was collected in 2 ml of a mixture of benzene / methanol / ethyl acetate (+ 0.5% oxalic acid) (10: 1: 90 by volume) and chromatographed on a column of silica gel bO Merck (internal diameter 2.8 cm, height 10 cm) using the same mixture as eluent. Fractions which showed a spot on Hf (one) = 0.9 by TLC on silica gel 60 F25} i Merck (eluted with benzene / methanol / ethyl acetate, 10: 1: 90 by volume) were mixed with 200 ml of benzene and the solution. washed with water and dried. The benzene was then evaporated. The remaining solid was dissolved in 1.2 ml of acetone and 0.7 ml of water was added. This solution was chromatographed through a column (inner diameter 2.8 cm, height λ cm) packed with silanized silica gel 60 Merck using acetone / water (v0: Uu) as eluent. Virtually all of the desired compound 22 remained at the top of the column, while impurities eluted. After removal of impurities, 3- [3- (p-hydroxyphenyl) -ethylamino] -tolyporaysinone was eluted with a mixture of acetone and ethyl acetate (molar ratio 0:20).

Fractions containing the desired compound were combined and then the solvent was evaporated to substantially dryness; the residue was collected in chloroform and washed with water. The chloroform phase was then dried and the chloroform was evaporated. The remaining solid was collected in 1 ml of chloroform and remixed with r'59101 in n-hexane. 35 mg of pure 3 '(p-hydroxyphenyl) -ethylamine-9-tolypory synone were obtained.

UV spectrum: 225 (fc, 52) and 266 (U, 37); 318 (U, 15) i 305 (3 »night) i 535 (3.2lt).

Example 21 Preparation of 3 - ((4-hydroxy-3-methoxybenzylamino) -tolypomycinone (Compound 23) 2.5 ml of a 2 solution of hydroxy-3-methoxybenzylamine 30 was gradually added to 360 mg of tolpomycinone 3 over 1.5 hours. ml: obtain dioxane. The reaction mixture was chromatographed (layer: silica gel 60 [mu] l Merck, eluent: chloroform / methanol / ethyl acetate, 20: 2.5: 2.5 by volume). The desired compound 23 had an Rf (one) = 0.75 · After 2 hours of reaction, 100 ml of chloroform, 2 ml of 5% aqueous citric acid solution and 20 ml of water were added. The chloroform phase was then washed with water and dried, after which the chloroform was evaporated. The solid was collected in 2 ml of chloroform, chromatographed on a 2 mm thick silica gel layer βθ Merck and eluted with chloroform / methanol (2: 1 / v / v).

The purple spot corresponding to the desired compound 23 was scraped off, suspended in 20 ml of a mixture of methanol and water (90:10 by volume) and stirred for 15 minutes. After filtration, the silica gel was washed again with 20 ml of the same mixture.

The combined filtrates were evaporated to a small volume and then extracted with 100 ml of chloroform. The chloroform phase was washed with water, dried and concentrated to a volume of 2 ml. Thereafter, 3- (U-hydroxy-3-methoxybenzylminino) -tolypomycidone was mixed with 30 ml of n-hexane; 90 mg of this compound was obtained.

UV spectrum: 230 (U, 5Uh 271 (* », 37) i 318 (U, 23) i 522 (3.27).

Example 22 Preparation of 3- [pr (2-pyridyl) -ethylamino] -tolypomycinone (Compound 2U) 190 mg of 2- (2-aminoethyl) pyridine in 2 ml of chloroform were added with mechanical stirring at room temperature over 1 hour to 220 mg of tolpolyysinone. In 7 ml of chloroform. After stirring for 30 minutes, 100 mg of manganese dioxide was added. The mixture was allowed to react for 20 minutes, after which the manganese dioxide was filtered off and 100 ml of chloroform was added to the filtrate. The solution was then washed with 20 ml of water containing 0.5 g of citric acid. The organic phase was dried and the solvent was then evaporated.

The solid obtained from 591 01 18 animals was chromatographed on silica gel 60 F25U Merck (layer: 20 x 20 cm, 2 mm thick) using chloroform / methanol (90:10 by volume) as eluent.

The desired product was scraped off the plate for 2 h and the silica gel containing this was washed a few times with a mixture of methanol and water (90:10 by volume).

.biuotir was then evaporated and the residue was taken up in 1U0 ml of chloroform and washed with water and dried ·, the chloroform was then evaporated.

The residual solid was collected in 1 ml of a water / acetone / ethyl acetate mixture (volume ratio 1: 1: 1) and chromatographed on a column (internal diameter 2.8 cm, height 5 cm) with 3-silanized silica gel 60 Merck using water. / acetone / ethyl acetate as eluent.

Fractions found by TLC on silica gel 60 F25 ^ Merck (eluent: chloroform / acetone, 20:10 by volume) to contain the desired compound 2h./ftf(one) = 0.1 * 6 ^ were partially evaporated and then collected in chloroform. washed with water, dried, evaporated to a volume of 1-2 ml and then the gelatinized product precipitated for 2 h by adding 30 ml of n-hexane.

50 mg of 3 "φ- (2-pyridyl) -ethylamino-7-tolpomycinone were obtained.

UV spectrum: 233 (**, 50); 261 (U, Ul); 317 (htl6) \ 370 (3.38) and 533 (3.22).

Example 23 Preparation of 3- (3-pyridylmethylamino) -tolyproflycinone (Compound 25) 270 mg of 3-picolylamine in 2 ml of chloroform were added with mechanical stirring at room temperature over 1.5 hours to 300 mg of tolpomycinone in 10 ml of chloroform. After the mixture was reacted for 2 hours, 1.50 mg of manganese dioxide was added and the solution was stirred for another 30 minutes. The progress of the reaction was monitored by TLC on silica gel 60 F25 ^ Merck, eluent: chloroform / methanol (25: 5 by volume); Rf (one) of the called compound 25 = 0.79.

The manganese dioxide was then filtered off and the solution was washed with 5 .mu.l of an aqueous solution of citric acid and water and then dried; then the solvent was evaporated. The residual solid was collected in 2 ml of chloroform / ethyl acetate (containing 0.5% oxalic acid) / methanol (15: 13: 2 by volume) and chromatographed through a column of silica gel 60 Merck (inner diameter 2.8 cm, height 7.5 cm), in which case the original solvent mixture was used as eluent. The 3 "(3-pyridylmethylamino) -tolypornysinone was absorbed at the top of the column. it was reprecipitated with n-hexane to give 90 mg of 3 * (3-pyridylmethylamino) -tolypomycinone &.

UV spectrum: 261 (U, t? 5) i 317 (U, 23) i 360 (3.90) ·, 52.5 (3.23).

Example 2k Preparation of 3 - ((2'-piperidinylethylamino) -tolypomycinone (Compound 26) 15 mg of N- (2-aminoethyl) -piperidine was added slowly with mechanical stirring over 1.25 hours to 300 mg of tolpomycinone in 13 ml of chloroform. After the addition of the amine, the mixture was stirred for a further 30 minutes, after which 160 mg of manganese dioxide were added and stirring was continued for a further 20 minutes. The reaction mixture was then chromatographed on silica gel 60 F25l Merck, eluting with ethyl acetate / acetone (15:15 by volume); 26 Hf (one) of the desired compound = 0.36.

The manganese dioxide was then filtered off and 100 ml of chloroform was added to the filtrate. The solution was then washed once with 20 ml of water containing 3 ml of a $ 5 aqueous solution of citric acid and then with water alone a few times. The organic phase was dried over anhydrous sodium sulfate, and then the solvent was evaporated.

The solid residue was taken up in 2 ml of ethyl acetate / acetone / methanol (v / v 200: 175: 25) and chromatographed on a column of silica gel 60 Merck using ethyl acetate (containing 1% pair '/ v / v oxalic acid as eluent). ) / netanoli-3eosta. Fractions containing compound 26 having an Rf (one) of 0.36 were combined, the solvent was partially evaporated in vacuo and the organic phase was then washed with water after the addition of chloroform until the pH was 7. The organic phase was then dried and the solvent was evaporated. The residue was taken up in 1 ml of chloroform, and 35 mg of 3- (γ-piperidineethylamino) -tolypomycinone was precipitated by adding 30 ml of n-hexane.

Example 25 Preparation of β- (5-nitro-pyrid-2-yl) -aminoethylamino-4-tolypoinysinone (Compound 27) h2k mg of 2- (2-aminoethylamino) -5-nitropyridine in 3 ml of a mixture of methanol and chloroform (v / v 1 : 2) was added with mechanical stirring over 1.5 hours to 300 mg of tolypomycinone in a mixture of 5 ml of chloroform and 1 ml of methanol. Then 100 mg of manganese dioxide was added and stirring was continued for another 20 minutes. The reaction mixture was chromatographed on silica gel 6j F25 ^ Merck (eluent ethyl acetate / methanol, 2U: 1 by volume).

20 59101

Rf (one) pressure of the desired compound 27 = 1.27 · The manganese dioxide was then filtered off and 100 ml of chloroform was added to the filtrate. The orgasmic phase was then washed, first with 2 ml of 5% aqueous citric acid solution and 20 ml of water, and then with water alone.

The chloroform was dried and the chloroform was evaporated in vacuo. The solid residue was chromatographed on a column of silica gel 60 Merck (internal diameter 2.8 cm, height 7 cm) using ethyl acetate (containing 1% oxalic acid) / methanol (13: 6: 1 by volume) as eluent.

The fractions containing the desired product were found by TLC Rf (one) = washed with water, dried and the solvent was evaporated. The solid residue was chromatographed through a further column of silica gel using a mixture of water and acetone (6θ: 8θ by volume) as eluent.

Fractions containing the desired compound 27 were concentrated to a small volume and 100 ml of chloroform was added; the solution was washed with water and then dried. The chloroform was evaporated. The resulting solid was collected in 1 ml of chloroform, which was mixed with 120 mg of 3- [3- (5-nitropyrid-2-yl) -aminoethylamino] -β-tolpomycinorne with n-necane.

UV spectrum: 267 (MI); 357 (**, 37); 529 (3.27).

The compound was virtually insoluble in water.

Example 26 Dissolution of 3- (H-methylthyrrol-2-yl, methylamino) -tolypyrrolidone (Compound 28} in 30 ml of thionyl chloride was added to 10 g of II-methyl-pyrrole-2-carboxylic acid. at reflux for 2 hours, after which the thioryl chloride was evaporated in vacuo and the residual oil was distilled under a nitrogen atmosphere at 20 mm Hg to give 8.5 g of N-methylpyrrole-2-carboxylic acid chloride at 105-106 ° C. ethyl ether and a stream of dry gaseous ammonia was allowed to pass through the solution until precipitation of ammonium chloride ceased, the solid was filtered off and the solvent was evaporated in vacuo from the filtrate.

6.3 g of 11-methyl-pyrrole-2-carboxylic acid amide were obtained. This was mixed with an equal amount of phosphorus pentoxide and the mixture was heated in a cooking flask under vacuum. 5 G of pure nitrile were obtained (IR band 2210 cm *). This was dissolved in 20 ml of anhydrous ether and the solution was added dropwise over 1 hour. at 0 ° C with mechanical stirring to a suspension of 2.5 g of lithium aluminum hydride in 100 ml of anhydrous ether. The mixture was then kept at room temperature for 18 hours, after which it was heated under reflux for 2 hours. The reaction was then quenched and 2.2 ml of water was added to the mixture, followed by 1.6 ml of 20% aqueous sodium hydroxide solution and finally 7 ml of water. The solid was filtered off and the solvent was evaporated from ether. The residual oil was distilled to give 3.8 G of 2-aminomethyl-N-methylpyrrole, boiling point 95 ° C / 13 m long.

10 mg of 2-aminomethyl-N-methylpyrrole in 1.5 ml of chloroform was added to 300 mg of tolpomycinone in 3 ml of chloroform. After 1 hour, 150 mg of manganese dioxide was added to the reaction mixture. The progress of the reaction was monitored by thin layer chromatography on silica gel 60 F25 ^ Merck using ethyl acetate / compound 28 Rf (one) = 1.2 / as eluent. After about 2 hours, the reaction was complete and the manganese dioxide was filtered off. 100 ml of chloroform were then added and the solution was washed with water containing citric acid until it reached pH 7 *. The solvent was then evaporated in vacuo and the solid residue was passed through a column (inner diameter 2 cm, height d, 5 cm) containing silanized silica gel using water / acetone / ethyl acetate as eluent (v / v from 16: 1). The fractions containing 28 were combined, twice the volume of chloroform was added, and the solution was then washed with water. The washed solution was dried and the chloroform was evaporated to give 8H mg of 3 "(d-methyl-pyrrol-2-ylmethylamino) -tolypomycinone.

Example 27 JRimisbus o 1- (N-methyl-pyrrolidin-2-yl-motylamino) -tolypolysinone (comp. 29) 2-Aminomethyl-1H-methyl-pyrrole was added to a suspension of platinum dioxide in 150 ml of acetic acid. The mixture was then stirred under an atmosphere of hydrogen gas for 5 hours to reduce the pyrrole ring. The acetic acid was then evaporated in vacuo, the residue dissolved in methanol and the remaining acidity neutralized with sodium methoxide. The solid precipitate was then filtered off and the solvent was evaporated from the methanol solution. The residual oil was distilled to give 5 g of pure 2-aminomethyl-U-methyl-pyrrolidine (b.p. 90-10 ° C / 18 mm Hg).

1.5 g of 2-aminoacetyl-N-methyl-pyrrolidine in 1.5 ml of acetonitrile were added with mechanical stirring to 900 mg of tolpomycinone in 5 ml of large acetone-22 591 01. The mixture was stirred for 2 hours, after which 500 mg of manganese dioxide was added. After 10 minutes, the manganese dioxide was filtered off and 100 ml of chloroform was added to the filtrate. This mixture was then washed with pure water and water containing 5% citric acid, after which the solution was dried over anhydrous sodium sulfate. The solvent was then evaporated in vacuo. The residual solid was chromatographed through a column of silica gel 60 Merck (inner diameter 3 cm, height 10 cm) using two different solvents. The first comprised chloroform / ethyl acetate (containing 1% oxalic acid) / methanol in a volume ratio of 1 + 0: S ?: 5 * this eluted the remaining to-lypomycinone and some impurities. The second solvent, comprising only methanol, eluted compound 29 · Fractions containing compound 29 / Rf (one) = 0.19, by TLC on silica gel 60 F25U Merck, eluent chloroform / ethyl acetate / methanol, volume ratio ä0: 5:! J7 The combined fractions were dried, the solvent was evaporated in vacuo, the solid residue was dissolved in 1.5 ml of chloroform and reprecipitated with n-hexane to give 130 mg of crude 3 * (H-methylpyrrole). din-2-ylmethyl amino) tolpomycinone a.

Example 28 Preparation of 3- (pyrazinylmethylamino) -tlypomycinone (Compound 30) 22 mg of pyrazinamide and 22 g of phosphorus pentoxide were mixed and heated in vacuo to give 7 g of eyanopyrazine (b.p. 92-9 ° C / 18 mm Hg). , IR tape in 22h0 cm -1). This cyanopyrazine was dissolved in 30 ml of anhydrous ether, and the solution was added to a suspension of 3.0 g of lithium aluminum hydride in 100 ml of anhydrous ether with stirring at room temperature. Mechanical stirring was continued for 13 hours, after which the mixture was heated to reflux for 3 hours. The reaction was quenched by cooling the mixture in an ice bath and then adding 3.2 mL of water, 2.0 mL of 20% aqueous sodium hydroxide, and 11 mL of water. The solid which precipitated was filtered off and the ether was evaporated from the filtrate in vacuo. The residual oil was distilled to give 1.5 β aminomethylpyrazine (b.p. 97 ° C-99 ° C / 10 mm mmlg).

500 mg of aminomethylpyrazine in 3 ml of chloroform was added mechanically with stirring to 500 mg of tolypomycinone in 3.5 ml of chloroform at room temperature. The reaction was monitored by TLC (silica gel 60 F25 / Merck, eluent ethyl acetate / methanol, 23.5: 1.5 v / v). Compound 30 has an Rf (one) of 0.78. After 2 hours, 300 mg of manganese dioxide was added to the reaction mixture and then filtered after 10 minutes. The chloroform phase was then washed with water and citric acid, then dried over anhydrous sodium sulfate and the chloroform was evaporated. The residual solid was chromatographed on a column of silica gel 60 Merck using ethyl acetate (containing 0.5 P oxalic acid) / methanol 23.5: 1.5 by volume as eluent (column inner diameter 3 cm, height 12 cm). The fractions containing compound 30 were combined and some of the solvent was evaporated therefrom. Then, 100 ml of chloroform was added, and the chloroform solution was washed with water. The washed chloroform solution was then dried and the chloroform was evaporated. The remaining solid was dissolved in 1.5 ml of chloroform, which was mixed with 110 mg of crude 3 "(pyrazinylmethylamino) tolpomycinone with n-necane.

Example 29 Preparation of 3 - [[5- (1,2,4-triazol-1-yl) -ethylamino] -N-tolpomycinone (Compound 31) 100 mg of 1- (N, N-aminoethyl) -1,2,2-triateol / obtained according to Ainsworth and JoneB, J.Am.Chem.Soc., 77, 62I-62U (1955) 7 in 10 ml of a 2: 1 by volume mixture of acetonitrile / methanol was added to a solution in which was 300 mg toly-pomycinone in 3 ml acetonitrile. After 12 hours, 300 mg of manganese dioxide was added to the reaction mixture, which was allowed to stand for 30 minutes and then filtered. To the filtrate was added 100 ml of chloroform, and then washed with water. The organic phase was dried and the solvent was evaporated. The solid was collected in 2 ml of chloroform and chromatographed on a silica gel layer 60 F25 ^ Merck, 200 mm thick, using chloroform / methanol (23: 2 by volume) as solvent. U5 mg of crude N- (N- (1,2,5-triazol-1-yl) ethylamino] -7-lipomycinone Rf (one) = 0.587 were obtained.

Example 30 Preparation of 1- (N- (1,2, U-triazol-1-yl) -propylamino] -tholypomylinone (Compound 32) 530 mg of 1- (N-aminopropyl) -1,2,1-triazole was obtained from Ainsworth. and Jones, J.Am.Chem.Soc., 77, 62I-62U (1955) 1 in 2 ml of methanol and 0.1 ml of triethylamine were slowly added to 100 mg of tolpomycinone in 6 ml of acetonitrile. The reaction was monitored by TLC on silica gel layer 60 F251 * Merck, eluting with chloroform / methanol 23: 2 by volume.

The Rf (one) for compound 32 is 0.18.

After 12 hours, 100 mg of manganese dioxide was added and kept in contact with the reaction mixture for 30 minutes, after which time it was filtered off. The filtrate was washed with water and citric acid and dried over anhydrous sodium sulfate; the solvents were then evaporated. The remaining solid was passed through a chromatography column of silica gel 60 Merck (inner diameter 2, U cm, height 15 cm) using ethyl acetate (containing 1 oxalic acid) / methanol (23: 2 by volume) as eluent. Fractions containing 32 were combined and washed with water after the addition of 100 μl of chloroform. The organic phase was dried and the solvent was evaporated in vacuo. The solid was collected in chloroform, from which 30 mg of essentially pure 3 "- (1,2-.alpha.-triazol-1-yl) -propylamino?

Example 31 3- # U.2.3. Preparation of U-tetrazol-1-yl) -propylamino) -tolypomycinone (Compound 33) 200 mg 1- ((E-aninopropyl) -1,2,3,1 * -tetratolol / prepared according to Ainsworth and Jones, J. Am Chem.Soc., 77, 62I-62U (1955)? in a mixture of acetonitrile and methanol (2: 1 by volume) and two drops of triethylamine were added to 300 mg of tolpomycinone dissolved in 1 * ml of acetonitrile. The reaction was stopped after 17 hours. The Rf (one) for compound 33 was 0.71 (TLC on silica gel 60 F25 ^ Merck, eluent ethyl acetate / methanol, 23: 2 by volume).

100 ml of chloroform was added to the mixture, which was then washed with water. The chloroform phase was dried over anhydrous sodium sulfate and then the solvents were evaporated. The residual solid was chromatographed on a column of silica gel (inner diameter 2.1 x cm, height 20 cm) using ethyl acetate (containing 1% oxalic acid) / methanol in a volume ratio of 23.5: 1.5 as eluent. Fractions containing 33 were combined and washed with water after adding 100 ml of chloroform. The organic phase was dried and then the solvent was evaporated. The remaining solid was dissolved in 1.5 ml of chloroform and reprecipitated with n-hexane. 60 mg of crude 3 - [- ((1,2,3,4'-tetrazol-1-yl) -propylamino] -polypomycinone were obtained.

Example 32 Preparation of 3- (U-methylpiperid-3-ylmethylamino) -tolypomycinone (Compound 3 * 0) 210 mg of H-methyl-3-aminomethylpiperidine was prepared by Villyams,

According to Dmitriev, Dokl.Rossiisk and Sel'Skokhoz, Akad. 99, 553_7 (1961 »)? dissolved in 2.5 ml of chloroform was added to a solution of 300 mg of tolypomycinone in 2 ml of chloroform. The reaction was monitored by TLC on a silica gel plate 25 59101 60 F25 * t Merck using a mixture of chloroform and methanol, 21: U by volume as eluent. Rf (one) for compound 31 * was 0.1 * h.

After 2 hours, 150 mg of manganese dioxide was added and the mixture was allowed to react for 30 minutes. The solid was filtered off and the filtrate was washed with water and citric acid. The organic phase was dried and the solvents were evaporated.

The residual solid was chronographed on a column of silica gel 60 Merck (internal diameter 2 cm, height 12.5 cm) using chloroform / methanol (containing 0.2% oxalic acid) in a volume ratio of 87.5: 12.5 as solvent and eluent. The fractions containing 3 * 4 were combined, washed with water and dried, after which the solvent was evaporated. The remaining solid was collected in 1 ml of chloroform, from which it was reprecipitated with n-hexane. 100 mg of 3 '(J-methylpiperid-3-ylmethylamino) tolypomycinor were obtained.

Example 33 Preparation of 3- (pyrid-2-ylmethylamino) -tolypomycinone (Compound 35) 270 mg of 2-aminomethylpyridine in 1 ml of chloroform was slowly added to 100 mg of tolpomycinone in 1+ ml of chloroform. The reaction was monitored by K-chromatography on a layer of silica gel 60 F25 ** Merck using chloroform / methanol 32: 3 by volume as eluent. The Rf (one) for compound 35 was 0.7.

After 1 hour, ** 00 mg of manganese dioxide was added and the mixture was stirred for 30 minutes; the manganese dioxide was then filtered off. The chloroform phase was washed with water and citric acid and then dried, after which the chloroform was evaporated in vacuo. The resulting solid was collected in 2 ml of chloroform and purified on a silica gel plate 60 F25 ** Merck, 2 mm thick, using ethyl acetate as eluent. Compound 35 was eluted from silica gel with methanol, the solid was filtered off and the solvent was evaporated from the organic solution. The remaining solid was collected in 1 ml of chloroform, from which 6 mg of pure 3- (pyrid-2-ylmethylamino) -tolypomycinone with n-hexane was reprecipitated.

26 59101 Preparation of 3-3-cyclohexylaninotolypolyquinone (reference compound) 300 [mu] g of tolypomycinone dissolved in 6 ml of dioxane were added at low temperature And with mechanical stirring 0.300 ml of cyclohexylamine dissolved in 6 ml of dioxane. The solution was then stirred for 30 minutes at room temperature. While stirring the solution, 60 ml of chloroform was added, followed by 60 ml of a 2-point aqueous solution of potassium ferricyanide. The pH of the solution was adjusted to Ϊ by the addition of 0.5H hydrochloric acid.

The organic phase was separated and the aqueous phase was extracted with 20 ml of chloroform. The combined organic extract and organic phase were dried and the solvent was evaporated in vacuo. TLC (silica gel 60 Merck F25 10) gave the product as a purple head spot in Hf (one) * 1.20. The product was chromatographed on 30 g of silica gel 60 Merck and eluted with ethyl acetate to give 130 mg.

The minimum inhibitory concentration values for the compounds prepared above against different strains of microorganisms were then determined. The experiments were performed on Brain 7. ...

Heart Infusien broths inoculated with 1 x 10 of the administered microorganisms and incubated for 1 hour. Results were determined after 2b hours of incubation at 37 ° C. For comparison, similar experiments were performed against Tolypomycin Y and tolypomy-synon. The minimum inhibitory concentrations, mcg / ml, for compounds 1, 2, 1 *, 6-9 »11-1 ^, 16-23 and 26-33 against a limited number of microorganisms are given in Table 1 and the results for compounds 3, 5» 10, 12, 15 »2U and 25 and Tolypomycin Y against a larger number of microorganisms are given in Table 2.

Table 1 5 9101 27

Staph, aureus Staph, aureus Strept. pyogenes

Compound 209 P FDA 14 / A ATCC 1233Ö "* I

__ ____________: _. ___________________________ I

1 0, 005 0, 005 - 0,005 - 0,025 0,025 2 0, 1 - 0,25 4 0,005 6 <0,005

7 0.005 - I

0,025 .- 8 0,005 - 0,025 - - 5 0,1 - 0,25 · 11 0,025 '- - 13 0,25 - 0,5 - .-.

14 <0, 005 - * 1G <0,005 - .- 17 <0,005 · - · - 18 0,05 - 0,025 10 0, 005 - 0, 025 20 <0,005 - 21 0, 1 - 0 .05 and 22 <0,000 - - *

I I

___________________________ J __________; _______________ \ 28 59101

Table 1 (cont.)

Ilacillus ccrcus Sr.reinci Jutea Klebsiella pneumonia e j

Compound "ÄTCCTgK ATCC 9341 'OUavIrtni I

1 0, 1 - 0, 25 <0,005 25 - 50 2 4 _ - - 6 - -> 100 7 - - 8 - - 25-50 9 - - i ----------—... ........ ... -. | - ... - i 11 · - - 25 13 -. - - 1 ““ ——————— ΠΓ '- ”· - - 1' -“ V-. I - «· - · 14 - - 50 - 100 · 1G - - f> 100 17 - - J 12,5 | .18 - - 12, 5

19 - - 25 I

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59101 29

Table 1 (recast)

Bsclie r ichja_ coli Salmonella

Compound kiL / 35 p. Tiphy B

0243 K Sclavo 1 50 - 100 50 - 100 2 - - 4 - - β 100 · 25 - 50 7 - - 8 6, 25 - 12,5 C, 25 - 12,5 0 11 12,5-25. 12, 5-25 13 - 14 25 12.5 16> 100 100 - 50 17 25 '12.5 18 50 - 25 12, 5 19 12.5 3 20 12.5 - G, 25 12, 5 - G, 25 21 100 100 - 50 22> 100 50

Table 1 (rev.) 30 59101

Stnnh. aureus Staph, aureus Strent. pyoffencs

Compound aim * FDA 14 / A ATCC 12300 '23 <0.005 2G 0; 005 - 0, 025 27 0, 005 - 0, 025 - 28 '<0,005 29 0,005 - 0,025 - 30 <0,005 - - 31 0,05-0,1 - 32 0, 005 - 0,025 - 33 0,005 - · - 0,025 34 0, 005 -0.025 35 <0.005 - j

Vert.x.X 1 0.1 - 0.25 j -

Tolyponorcin * 0.025 0.05 L 0.5-1

Tolyponysinoui 0.25-0.5 · 0.25 and 0.5-1 3C · 3 ~ cyclohexylamino-tolypomycinone

Table 1 (cont'd) 59101 31 2- I. - - -......... . - ........... ..............., j * ... I ... ..........

Bacillus cereus Sarcirui lutoa Klebsiella

Compound ATCC ~ 9631 'ATCC 93-11 pneumoniae

Oltav Iani 23 - j. . . 100.

26 - - 100 - 50 ....

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Vert.yhd. *! _ | _ |

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ToluponysinoNo 59101-

Table 1 (cont.) 32. Escherichia coli ft? 1 monel ia

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Mice were experimentally infected with Staphylococcus aureus Colliva and the PD (protective dose) 50 for certain oral compounds described above was evaluated experimentally. The PD 50 for 5, [3- (2-morpholinoethyl amino) -tolypomycinone] was found to be 1.05 mg / kg LD50 calculated for intraperitoneal administration to mice and was found to be 31.9 mg / kg (safe limits: 203.2 - U8.2) for compound 15, (3- (2-tetrahydrofuryl) -methylamino-lypomycinone) '. It was not possible to calculate the LD50 for oral use because mortality did not occur even at very high doses in the order of 1000 mg / kg.

Claims (2)

A process for the preparation of antibacterial 3 '(substituted aminoHolypomycinones, 3- (substituted amino) -1H-dihydrotolypomycinones and 3,16-di (substituted amino) -10,17-dihydrothypyycinones of the formula:? 3 "3 / CH OH OH,. (II) CH30 3 J- o \ J \ OH OH QO 3 NH- (ClI2) nR 0 0-1 0 ®3 wherein R is H, OH, mono- or dialkylamino, a phenyl group which may be substituted by hydroxy, lower alkoxy or a lower alkylamino group or a 5 "or 6-membered heterocyclic group which may be substituted by a lower alkyl, nitro or hydroxy group; n is an integer 1-H; and Q is one of CH, CH, CH 2, 5 1 3, 3 - CH = C "- CHn - CH - -CH" -C- NH (CH 2) n R (17) (1-5) (17) ( 16) (17) (16) (R means the same as above) 39 591 01, characterized in that (a) for the preparation of compounds of the formula II in which Q 1 is CH CJ 1? 3 'J - CH = C - or - C1I2 - C - HI: (CH) R 2 n (17) (io) (li) (1C) is subjected to a tolpomycinone of the formula CU3 CK3 Λ CH3C ° Ö J. OH OH CHO OH 0 il CH II N Yti i I oo o - [-, · c «30 to react with an amine of formula (I) CHg) R» or (d) to prepare compounds of formula II wherein ^ is CH3 CH - CH - CH - or - CH - C - Cl M1 (CH 2) n R (17) (Ιό) (17) (iC) is reacted with 16,17-dihydrotolypomycinone of the formula äO 59101 cn3 c, 0.1 OH S OH OHO ί) o-- ch3 0 with the formula 110H (CHO) H, wherein the reaction according to a) or b) is carried out in the presence of an oxidant such as potassium ferricyanide or manganese dioxide hl 591 01 Förfarar for the preparation of antibacterial 3 "(substituted amino) -tolypornycinoner, 3- (substituted amino) -1,6,17-dihydrotolypomycinone and 3,10-amino (substituted amino) -1,17-dihydrotolypomycinone with the form Oi, CH 2 ch 3CO ^ 0H (11) Ι'Η, Ο] 0 \ / \ OH OH Q ™ 1J, - "3 ° / ^ HH" (CH2 'n "0 3 --- 1 o CH, j color R betecknar n, OH , mono- or dialkylamino, phenyl group, hydrogen group substituted with hydroxy, alkyl alkoxy or alkylamino group, or 6-membered heterocyclic group, alkyl group substituted with alkyl, nitro or hydroxy group ; n är ett heltal 1-U; and Q is selected from the group consisting of CH, CH- CH. i J i -5 t J - CH “C - - CH2 - CH - - CH2 - C - NH (CH_) K 2 n (17) (lo) dl) (l») (17) (16) (d har samma betydelse som ovan) 59101 k2 kanne tecknat aärav, att (a) Tor iramst illning av suciana föreningar med formuleln II, värv U betecknar en. en, I O 1 J - en = c - - ci! ~ c - 2 i HI! (CiI) (17) (16) (17) (16) * '· omsättes tolypouycinon med formulaeln CH3 CHi / | «3« OH 0 !! Cii3 ° J γ °, s -A. y OH 0 H 0 O o - [- (CK3 0 med en Amin med formuleln H l'J (CH) H, eller 2. n (b) for framställning av sodana föreningar med formuleln II, värv Q betecknar Ci! , CU, o .3 - c :: - ca - - c:; p -c- k (cn) n. Li (17) (lu) (17) (16) omens