DE1965304A1 - Benzdiazepinone compounds and processes for their preparation - Google Patents

Description

DR. ELISABETH JUNG. DR. VOLKER VO8GIUS, DiPL.-ING. GERHARD.CÖLDEWEY

• MONK M. CLEMENtSTRASSE 30. TELEPHONE 345087. TELEGRAM ADDRESS: INVENT / MONCHEN. TELEX 5-29686

29th Dec 1969

u.z .: E 899 (Vo / Mü / kä) Case 557

PUJISAWA PHARMACEUTICAL CO., Osaka, Japan

"Benzdiazepinone Compounds and Processes for Their Manufacture" -

Priority: December 30, 1968, Japan, No. 83/69

June 26, 1969, Japan, No. 50780/69

The invention relates to new benzdiazepinone compounds and processes for their preparation. The basic compound is produced in a microbiological way, its derivatives in a chemical way. The compounds have antibiotic properties, phage and virus effects, and they can be used to treat tumors.

The invention includes the antibiotic active substance in diluted forms, such as raw concentrates, and in pure crystalline forms. The antibiotic and its derivatives have high activity against various microorganisms such as gram-positive and gram-negative bacteria, mycobacteria, fungi and bacteriophages. A strong virucidal effect on some viruses has been observed in vitro. Another important antibiotic property of the compounds is their ability to promote growth and growth

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To call the development of certain transplantable and induced tumors. Because of these antibiotic properties, sine the compounds of great value as drugs in treatment various diseases.

The microorganism used to make the antibiotic is a newly discovered species of Streptomyces isolated from a soil sample collected in Musashi-Koganei, Japan. His culture of the following with Streptomyces achromogenes var. tomaymyceticus named organism at the A.T.C.C, Rockville, Maryland, V.St, v.A., Under the iir. 21353 deposited.

The manufacture of the antibiotic of the invention is not limited to the use of the specific organism mentioned and to the use of the organisms which the Waehstura peculiarities described by way of illustration. and have microscopic features, descr -.... -: r, Expressly intended to include the use of mutants caused by various methods, such as X-ray irradiation, UV irradiation, treatment Bit StiokstoJXLost and exposure? 21 phages can be extracted from the desired organism. "*""- ■

Likewise, all microorganisms should pay attention to their appearance or physiological behavior caused by transformation, transduction, or genetic recombination other genetic engineering experiences using jucleic acids from the. named-species can be obtained, with the resulting Microorganism has the ability to produce the product described here or the biological conversion described here continue to perform.

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For the isolation and characterization of the Kikrο organism One part of the soil sample shaken in sterile, distilled water and painted on Krainsky-Agariaediuni. Kach seven days Incubation at 30 C, colonies of Streptomyces achroEoger.es var. Tomaymyceticus A.T.C.C. 21353 from the medium isolated and then grown on Bennet agar medium.

Lie morphological characteristics of Streptomyces achromogenes var. Tomayiayceticus ATCC. 21353 after 10-14 days of breeding

c
Good at 30 C on Czapek agar are given below. The conidium is spherical to oval with a smooth surface. The result is a branched and straight or slightly curved, long aerial mycelium with sparse growth,

There were a number of breeding characteristics and physiological characteristics of the new strain Streptomyces & shrcocg «n2s var. Tcmayinyceticus. Nutrient medium «. are naciister.end stated Lie observations were made after IG - 14 days of breeding at 3O. ^U C open. “Unless otherwise stated, the breeding time and breeding experiment described here were used.

Ccapek agar: gray to pale yellowish, colony-like Growth ;; scanty vigilance; of a powdery, white aerial mycelium; no soluble pigment.

Starch ammonium agar: Schv / aeh grayish growth with powdery, dark gray: air cells no soluble pigment .. weak diastatic effect.

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ülucose-asparagine-agar: white to light ivory colored, colony-like growth; no growth or sparse growth of a powdery, white aerial mycelium; no soluble pigment.

Calcium malate agar; Cream-colored growth with powdery white to dark gray aerial mycelium; no soluble pigment, calcium nitrate is made soluble.

Tyrosine Agar: Sparse, colorless or light brownish, vegetative Growth without formation of aerial mycelium and soluble pigment.

Broth agar: Cream-colored, colony-like growth without Aerial mycelium formation; Manufacture of brownish, soluble pigment. No production of hydrogen sulfide after 7 days of incubation.

Bennett Agar: light cream-colored, colony-like growth; no formation of aerial mycelium and soluble pigment. After incubation at 37 C a brownish, colony-like "growth occurs, with powdery, dark gray aerial mycelium and a brown, soluble pigment.

Glucose Broth: Cream-colored, colony-like growth with brown, soluble pigment; Not a single word of aerial mycelium.

Glucose czapek solution; Surface growth sparse, colorless, Colony-like with sparse growth of a powdery, white aerial mycelium and no formation of soluble pigment. Kitrat will not or only weakly reduced to l.'itrite.

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Schrägkul ^r tur:

Gelatine / cream color Growth without the formation of aerial mycelium and soluble pigment after incubation for 21 days at 15 -

20 C. A slight liquefaction of gelatin is observed.

Litmus milk: The culture grows as a cream-colored ring on the surface. It becomes a dull grayish-brown, soluble pigment educated. Slight peptonization but no coagulation.

Potato slices: greyish-cream-colored, vegetative growth M with a wrinkled surface? sparse growth of a powdery, white aerial mycelium; Formation of dark brown, soluble pigment.

Cellulose agar: There will be no growth with ammonium = · or titrations observed as nitrogen sources.

The recovery from carbon sources was increased after 7 days of incubation investigated at 30 C according to the method of Pridham and Gottlieb.

(a) Well-utilized substrates include: glucose, xylose, Mannose, fructose and mannitol. ~ "

(b) The substrates that are used in moderation include: arabinose, Rhamnose, sucrose, lactose, trehalose, raffinose and Inositol.

(c) The substrates that are rarely recycled include: Salicin »

The antibiotic of the invention is produced when Streptomyces achromogenes var. Tomaymyceticus is in a culture medium is grown under controlled aerobic submerged conditions. A variety of culture media can be used in cultivation will. It was found according to the invention that the best

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Results are obtained using an aqueous nutrient medium containing an assimilable carbon source and an assimilable nitrogen source or a proteinaceous material. Can be used as assumable sources of carbon! polyvalent alcohols and mono-, di- and polysaccharides, such as glucose, fructose, sucrose, sugar, brown sugar, starch, Kais starch, galactose, dextrin, glycerine or molasses. Unchanged proteins and protein degradation products can be used as protein materials, in particular products which are used in the
Protein hydrolysis arise. The following sources of assimilable nitrogen and protein can be used: corn steep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, peanut meal, cottonseed meal, corn meal, milk solids,
Casein split up by the pancreas, the soluble residues resulting from the distillation of spirits, animal peptone liquid solids. . "· '■?, -" iöcliaxtrakt, peptone, fish meal, yeast extract and Kno- Qh ^ nL.-sbl and. pieces of iron and inorganic materials such as nitrate and ammonium salts. These carbon and nitrogen sources, which are preferably used in combination, do not have to be used in pure form, as less pure materials,
the traces of growth factors and considerable amounts of
Containing mineral nutrients, very suitable. Possibly
they can be used together with mineral salts such as sodium chloride and - 'potassium chloride, and buffer substances such as calcium carbonate and
CaIeiu phosphate can be used. If necessary, an agent to prevent foam formation, such as liquid paraffin, a higher alcohol or silicone, is added to the nutrient medium.

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Achromogenes to achieve a maximum growth and development of Streptomyces var. Tomaymyceticus to 5.5 δ of the nutrient medium prior to inoculation with your organism to a _pH-value, G ,. preferably from 6.0 to 7.0. According to the invention, it was found that the culture medium is advantageously kept in the p "range between 6.0 and 6.5 during the growth period of the organism and the production of the antibiotic. The best antibiotic yields are obtained at a cultivation temperature of 25 - 37 C and a cultivation time of 40 - 80 hours. After this farmer ran out, a considerable amount of "antibiotic" was created.

As with the technical production of other antibiotics, the aerobic submerged process is also preferred here. For the production of smaller quantities of the antibiotic, the submerged process can be carried out in small bottles, which are shaken or stirred in the most suitable way. Large tanks or barrels, such as are commonly used in the fermentation industry, can be used to grow large volumes of the nipped culture medium. For the production of large quantities, the use of the vegetative form of the organism for inoculating the tanks or barrels is preferred in order to avoid growth retardation in the production of the antibiotic. Accordingly, it is desirable to first prepare a vegetative inoculum of the organism by inoculating a relatively small amount of the nutrient medium with the spore shape of the organism and then aseptically transfer the vegetative inoculum to large tanks or barrels. The vegetative inoculum can be used in the same 'oaer in a different nutrient medium as it is used for production

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of the antibiotic used.

Moving and aerating a breeding mixture can be done in different ways can be achieved. The movement can be achieved by a propeller stirrer or similar mechanical movement devices, by a rotary or shaking mixer, by various pumping devices. directions or by passing a stream of sterile air through the medium. Ventilation can be done by blowing a stream of sterile air into the cultivation mixture or by spraying, squirting or pouring the fermentation mash into or through it, an appropriate atmosphere can be achieved.

After filtering or centrifuging the fermentation mash, the antibiotic of the invention can be obtained from the culture medium by conventional extraction or adsorption processes used in the production of other antibiotics. The extraction can preferably be carried out with polar organic solvents, for example with alcohols such as methanol, ethanol, propanol, isopropanol and buxanol, alkyl esters of fatty acids such as ethyl acetate; Ketones such as acetone ; chlorinated hydrocarbons such as chloroform; una Pyriain. Other solvents of a similar character can also be used. A mixture of these solvents is advantageously used. Another way to get the antibiotic from the fermentation mash is to use an adsorbent such as diatomaceous earth, activated alumina, silica gel, activated carbon and silica. The antibiotic can be easily eluted by using a polar organic solvent in which it dissolves. A suitable method for obtaining the antibiotic from the extract or eluate comprises evaporating the solvent

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up to a relatively small volume and the precipitation of the antibiotic by adding a solvent in which the Antibiotic is insoluble. The antibiotic is then purified by recrystallization or chromatography. Suitable Solvents for recrystallization are hydrous acetone, hydrous methanol and other solvents in which the antibiotic has a certain solubility. Suitable adsorbents can be used to obtain the antibiotic can also be used with success for chromatographic purification. Suitable for the production of the antibiotic can also be used Eluents can be used.

The isolated according to the procedures essentially described above Antibiotic · is in the form of a powder.

The derivatives of the antibiotic are prepared by treating the antibiotic isolated from the extract or eluate with an alcohol, Mercaptan or dialkylamine treated.

The derivatives of the invention have the general formula I,

HO- * 9 H
υΊο R.
ι
-CH 1 CHCH ₅ CH-O- Il - Ν
\ O ) ~ " ν 3 ί

in which R is a lower alkoxy-, an aryl-lower-alkyloxy-, a lower alkylthio, an aryl-lower-alkylthio or a di-lower-alkylamino radical is.

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To produce a derivative of the general? Or ::. El I solves the powdery antibiotic in an alcohol, mercaptan or dialkylamine of the general formula R-H, in which R is a lower Alkoxy-, aryl-lower-alkoxy-, lower alkylthio-, aryl-lower-alkylthio- or Di-nieaer-Alkyliminorest, and leaves cool the solution to form crystalline material. Lower aliphatic alcohols are suitable as alcohols and mercaptans and mercaptans with 1 to β carbon atoms as well as alcohols and mercaptans with 7 or 8 substituted by an aromatic radical Carbon atoms. Specific examples of alcohols and mercaptans that can be used are Metnanol, Ethanol, Prcpanol, Isopropanol, Benzyl- ' alcohol, methyl mercaptan, ethyl mercaptan, propyl mercaptan, isopropyl mercaptan and benzyl mercaptan.

The dialkylamines contain 1 to 6 carbon atoms per alkyl radical. Specific Examples are diethylamine, dietnylamine, dipropylamine, Methylethylamine and methylpropylamine. So can others here alcohols, mercaptans and dialkylamines not specifically mentioned can be used.

To achieve a maximum yield of the derivatives, the reaction is advantageously carried out in an inert solvent. Examples of such solvents are methylene chloride, Chloroform, carbon tetrachloride and ethyl acetate. The reaction temperature is not particularly critical. Preferably the reaction is carried out at 25 to 35.degree.

The remainder of the alcohols, mercaptans and dialkylamines mentioned are introduced into the 11-position of the ring system.

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Lie compounds of the general formula I can easily under IiIiisinierung the best HH converted into a compound of the formula II will.

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The Siiiainierung of the substituent H in the II-position of the ring system can be achieved by converting the compound of the general formula I in a solvent such as η-hexane, acetonitrile, Acetone, chloroform or ethyl acetate dissolves, in addition an excess of these solvents can be used. This elimination reaction occurs, presumably in liaumteiLpsaa ^ ur carried out; higher temperatures cause an acceleration the reaction and a reduction in the reaction time. The product can be separated out from the solution by customary methods such as filtration, decantation or centrifugation. ™

By applying the process that is understood to produce aer Compound of the general formula 1 is given, the compound of the formula II can easily be converted into the compound of the general. Formula I can be converted.

The compounds of the formulas I and II can be converted into a compound of the following general formula III by acylation will,

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= CHCH,

(III)

in which R ¹ -, a lower alkylcarbonyl, an aryl-lower-alkylcarbonyloaer an arylcarbonyl radical and Y is the group -NH-CHR- or -K = GH-, where R has the meaning given above. The acylation reaction can be carried out by reacting the compound of general formula I or II with an acylating agent in a solvent such as pyridine. All acylating agents which can provide an acyl radical which reacts with a hydroxyl group in the 8-position can be used. These include the free carboxylic acids, the acid halides, acid anhydrides and esters. Specific examples of acylating agents which can be used are acetic acid, propionic acid, benzoic acid, p-bromobenzoic acid, their chlorides and bromides, their anhydrides and their methyl and ethyl esters. The reaction with these 'acylating agents is preferably carried out at room temperature or with the solution being cooled. Processes in which the reaction mixture is cooled or the acylating agent is poured into an ice-water-cooled mixture lead to the formation of the acylated compound. These acylated compounds can be made to crystallize by pouring the reaction mixture into water, for example, filtering off the precipitated crude product, purifying it by chromatography, if necessary, and recrystallizing it from a solvent such as acetonitrile or methanol.

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The compounds of the formulas I and II can also be obtained by alkylation be converted into a compound of the general formula IV,

(IV)

in which R "is a lower alkyl radical with 1 to 4 carbon atoms, preferably 1 or 2 carbon atoms, and Y has the meaning given above. For the alkylation, the compound is mixed with an alkylating agent in a solvent such as methanol Alkylating agents which react with the hydroxyl group in the 8-position of the compound are, for example, diazoalkanes and dialkyl sulfates. Specific examples of alkylating agents that can be used are diazomethane, diazoethane and dimethyl sulfate. During the alkylation reaction, the reaction mixture is preferably cooled. The reaction mixture is, for example, placed in a refrigerator When the reaction is complete, the mixture is evaporated to dryness and the residue is dissolved in methanol. Ether is added to the methanol solution, and the ether solution is cooled to ⁰ ° C. The alkylated compound crystallizes out in the process.

The compounds of the general formulas, in which Y is the group -N = CH- III and IV, can access _the} 'be converted into compounds I described manner above in connection with the preparation of the compounds of the general formula in which Y represents the group -M-CHR, where R has the meaning given above.

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All of the above formulas are represented by the following general formula reproduced,

CH, 0

= CHCH

in which R- ^ a hydrogen atom, a lower alkyl, a lower Alkylcarbonyl, an aryl-lower-alkylcarbonyl or an arylcarbonyl radical and Y has the meaning given above, and where all of the alkyl radicals mentioned have 1-6 carbon atoms and the arylalkyl radicals Contains 7 or 8 carbon atoms.

The antibiotic and its derivatives prepared therefrom by the methods indicated have high activity against a number of microorganisms. The investigations were carried out with the H-methoxy compound of the formula I ₁ ie the 1,2,3,10,11, lla-hexahydro-2-ethylidene-7,11-dimethoxy-8-hydroxy-5H-pyrrolo / ~ 2, lc // l, 4_7-benzodiazepin-5-one, performed. The activity of the compound is expressed by the minimum inhibitory concentration (MIK), which was determined by the usual serial dilution method. The tests were carried out for bacteria with a glucose broth medium and for fungi and yeast with a Sabouraud medium. The Untersuchungsmediuni was incubated for 24 to 72 hours at 30 ⁰ C and the MIC values are / expressed by the concentration of the compound injig ml, which prevented the growth of the organism.

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The following results were obtained.

To a 11- Γρ, Άϊΐΐ Sine

MIK, µg / ml

Jtaphylocoecus aureus 2G9-P Bacillus subtilis A.T.C.C. 6633 Corynebacterium xerosis iSarcina lutea Escherichia coli Pseudomonas aeruginosa Proteus vulgaris Aspergillus niger Periiei 11 ium chrysogenum Q-176 Saccharomyces cerevisiae i'orula utilis Candida aibicans

6.2

12.5

25.0

25.0

100.0 100.0 100.0

50.0

25.0

• 50.0

50.0

50.0

The effects of the H-methoxy compound on bacteriophages in in vitro investigations are given below. In the investigations, 1 ml of a suspension containing 2 χ IC particles of the test phage per ml in 0.1 Cl m? Ris-HCl buffer (p ^ -V / ert 7.2) added to each dilution (1 ml) of the samples of the compound to be examined in the named buffer. C. 1 ml of the mixture was incubated for 1 hour at 37 C. and poured into a Petri dish with 1.5 / J 1.5 ½ ’agar. The phage count was carried out using the drop method with the corresponding VJirtstanur. carried out, the amount that just 50 ^c ß> of the phage inactivated is expressed in ^ ug / ml.

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Sestphagen concentration for inactivation

from $ 50 of phage activity, µg / ml

Escnerichia coli Τ -, - phage 0.1

Escnerichia coli ^r i? 2 ~ -P ^na S ^e 3i 2

Escnerichia coli T ^ phage ■ 0.2

Escherichia coli T. Pha ^ e 3.2

Escnerichia coli Λ phage 1.0

Eschericnia coli β phage 12.5

Escherichia coli HS-2 phage 12.5

Bacillus subtilis M-2 phage 0.2

Bacillus subtilis SP-10 phage 0.2

Lactobacillus acidophilus J -, - phage 50.0

Pseudomonas aeruginosa P ^ -Phage 12.5

Some of the compounds of the invention also possess antiviral and antitumor activity.

In vitro, the H-kethoxy compound was active against the DNA virus Herpes simplex hominis. In these experiments doses of 0.1. and 0.05 ng / ml in distilled water containing 10% dimethyl sulfoxide in test tubes with equal parts of a virus

-3

Suspension in Hank's solution at a dilution of 10 'mixed! "' Oh different contact times at 22 ⁰ C were ml doses of 0.2, an amount previously in Hank's Lo. Solution of an LDg, - was administered intraperitoneally to a randomly selected group of 10 male mice of the strain NMRJ weighing 15-19 g. A Kcntrollgruppe of 10 mice were 0.2 ml of the mixture of the virus suspension, and a solution of 10 i> DMSO in distilled water without the H-Kethoxy compound administered.

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The 100 # mortality of the control group decreased with 1 - 4 hour contact time to $ 20 and to 0 after 6 hours Contact time. This means that the virulence of this DNA virus is partly or entirely due to the 11-methoxy compound gets destroyed.

The 11-methoxy compound also shows complete inhibition of various transplantable ascites tumors such as Ehrlich's carcinoma and Cr. Sarcoma 180 in mice, strain NHRJ, and Yoshida sarcoma, strains AH 66 R and AH 130, in 'Wistar rats. When administered intratumorally, it is also effective against Walker's solid Carcino sarcoma. The leukemia strains L1210-S and L-1210-R (6-mercaptopurine-resistant) are partially inhibited. The prolongation of survival time with the well-tolerated dose of 0.125 mg / kg with 4 intraperitoneal administrations on 4 consecutive days is $ 26-50 for the L 1210-R and $ 51-75 for the L 1210-R .

In all these studies with transplantable ascites tumor strains to mice or rats a certain amount \ transplant of cells or cell material in Hank's solution. These doses caused tumors in the amount of $ 100. Arbitrarily chosen groups of 8 animals per dose in rats or animals per dose in mice were treated with a dose of the 11-methoxy compound for the first time 4 hours after the transplantation and with the same dose in each case on the following 3 days. Solutions in triethylene glycol with 90 $ distilled water were used. A single dose for mice consisted of 0.5 ml per 20 g and for rats 1 ml per 100 g, these doses ultimately being converted to kg.

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The activity of the H-methoxy compound was dose-dependent, adder 99% inhibition was still observed at a dose of 0.0625 mg / kg i.p. in ascites tumors Ehrlich's carcinoma and Cr. Sarcoma 180 (Haus) and 0.1 mg / kg i.p. and from 0.05 mg / kg i.p. with Yoshida sarcoma AH 66 R or Yoshida sarcoma AH 130 (rat).

All the doses administered were well tolerated by the test animals.

The compounds of the invention can be used as medicinal substances in the form of medicinal preparations which use the antibiotic or its derivatives in admixture with a pharmacologically acceptable, organic or inorganic, solid or liquid carrier suitable for oral or parenteral administration. The medicinal preparations can be in solid form, such as capsules, tablets or coated tablets, or liquid form, such as solutions, suspensions or emulsions. If necessary, auxiliaries can be added to the preparations, for example preservatives, stabilizers, wetting or emulsifying agents, salts to change the osmotic pressure and buffers. The dosage of the compounds varies from compound to compound and also depends on the age and condition of a patient, but is a daily dose of about 20 mg / kg of the compound is generally effective.

The examples illustrate the invention.

example 1

Vegetative growth and growth grown on inclined agar Streptomyces achromogenes var. Tomaymyceticus A.T.C.G. 21353 were in a 500 ml bottle, the 100 ml of the following Containing nutrient medium, spent.

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Weight -f 3 1 1 1 0 , 25

hey ingredients lactose

Pieiscnextract Yeast Extract Polypeptone I Sodium Chloride

This medium was sterilized and inoculated with the above agar. It was shaken at 30 ° C. for 3 days.

In a 2-ton stainless steel tank, 1,000 liters of a Kährmediunks of the following composition was charged.

Assistance parts wt .- $

Lactose 3

Meat extract 1

Yeast extract 1

Polypeptone 1-

i>. sodium chloride 0.25

Potassium chloride 0.25

Potassium dihydrogen phosphate 1.5

! »Atrium hydrogen phosphate (12 HgO) C, 43

The p - value of the medium was set to o, l. Las Kährmediun was sterilized by heating at about 120 ° G for 3C for 3 minutes under pressure. The medium was cooled and approximately 1 ml of the inoculation medium described above was added aseptically. The organism was cultured in the medium at a temperature of 30 ^{° C. for 50-60 hours.}

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During the growing season, the fermentation mash was made with a propeller mixer at 350 r.p.m. stirred and aerated with sterile air in an amount of 1000 liters / minute.

After the cultivation was complete, the mycelium was centrifuged off. The supernatant was stirred with 5 kg of activated charcoal for 30 minutes. The mixture was then filtered and the activated charcoal twice with 100 ^; Liters of a mixture of pyridine, ammonia, ethanol and water in a ratio of 10: 3: 80: 10 eluted with heating at 45 ° C for 30 minutes. The extract was evaporated under reduced pressure at 50 ° C. and freeze-dried. 1.6 kg of powdery product were obtained. The powder was washed in about 10 liters of η-hexane and dissolved in v / ater. The p “value of the solution was adjusted to 2-3. The acidified solution was extracted 4 times with 5 liters of chloroform each time. The chloroform extract was washed with 5 $ aqueous Hatriumbicarbonatlösung, evaporated, dried over sodium sulfate and concentrated under reduced pressure at 50 ⁰ C. An oily residue was formed which was taken up in petroleum ether, as a result of which a powder separated out. By filtration

The petroleum ether solution gave about 20 g of a powder which was dissolved in 100 ml of ethyl acetate, adsorbed on silica in a column and eluted with about 8 liters of ethyl acetate. The eluate was evaporated almost to dryness and then about 30 ml of methanol were added. Each 2 days of standing at -20 ⁰ C divorce "crystals, which were filtered off. This gave 1.8 g of crude product which was then dissolved in about 30 ml of warm methanol. The mixture was left in the methanol solution for 2 days at -20 ° C. This gave 1.2 g of pure, crystalline 1,2,3,10,11,11a

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O 9 N 21 , 05 9 , 21 21 , 25 , 05.

2-ethylidene-7, ll-dimethoxy-8-hydroxy-5H-pyrrolo / 2, l-c // l, 4 / -benzdiazepin-5-one from P. 145 - 146.5 ° C (decomposition).

C H

C ₁₆ H ₂₀ N ₂ O ₄ 'calculated: 63.16 6.58

Found: 62.95 6.66

The UV absorption spectrum of this compound in methanol shows maxima at 224 mu (£ = 36000) and at 320 mu (£ = 3600) and Shoulders at 237 mu (f = 30000) and 260 mju (£ = 9000), as out Pig. I emerges.

The IR absorption spectrum in Nujöl shows bands at 3340, I64O, 1570, 1510, 1425, 1290, 1265, 1210, 1190, 1180, 1070, 830, 800 and 765 cm "as shown in Pig.

According to another embodiment of the work-up, 100 liters of the supernatant were adjusted to the p ^ value 2 with hydrochloric acid and extracted 3 times with 30 liters of chloroform each time. The chloroform extracts were combined and evaporated to approximately 10 liters. Then 10 liters of methanol were added. The solution was further evaporated ml to about 300, wherein like slowly methanol was added. The methanol solution was placed in a refrigerator. The precipitate formed was filtered and washed with ethyl acetate. The resulting powder was dissolved in warm methanol and allowed to stand in the cold. The resulting crystalline precipitate was recrystallized from methanol. About 2 g of 1,2,3,10,11,1la-hexahydro-2-ethylidene-7,11-dimethoxy-8-hydroxy-5H-pyrrolo / 2, lc // 1,4 / benzdiazepine-5 were obtained -on.

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Example 2

100 liters of the fermentation mash prepared in Example 1 were with Hydrochloric acid adjusted to the p ^ value 2 and 3 times with 30 Litei each Ethyl acetate extracted. The extract was evaporated to dryness and dissolved in a small amount of chloroform. The solution was passed through a column filled with silica gel. The silica gel column was treated with a 3: 1 ethyl acetate / chloroform mixture eluted. The eluate was evaporated to dryness and η-hexane was added to the residue. The precipitate formed was filtered off, dissolved in chloroform and, as indicated above, chromatographed. The eluate was evaporated and that obtained in the process Powder dissolved in ethanol and left to stand in the cold. The crystalline material obtained was recrystallized from ethanol. Pale yellow needles of 1, 2, 3, 10, 11, 11a-hexahydro-2-ethylidene-7-methoxy-8-hydroxy-11-ethoxy-5H-pyrrolo were obtained /2,l-c/A4./benzdiazepin-5-on from F. 134 - 136 ° C (decomposition).

ber .: 64 C. 6th H 20th 0 N 80 C ₁₇ H ₂₂ N ₂ O ₄ found: 63 , 13 7th , 97 20th , 10 8th, 44. , 85 , 02 , 77 8th,

The compound has UV absorption maxima in ethanol at 225 mu (£ * 38000) and 325 mji (f = 6700) and shoulders 235 mu (£ β 35000) and 262 mu (£ = 11000), as shown in Fig. 3 going on.

The IR absorption bands in Nujol are at 3350, I64O, I600, 1570, I513, 1425, 1290, 1265, 1210, 1190, 1160, 1130, 1070, 890, 835, 800, 765 and 710 cm " ¹ , as shown in Fig 4 shows.

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Example 3

A solution of Ig! ^^, lOjll.Tla-Hexahydro ^ fll-dimethoxy-S-hydroxy-5H-pyrrolo / 2, lc // I, 4_ / benzdiazepin-5-one in an excess of chloroform or ethyl acetate was added to a Smaller volume evaporated. ^ The residue was treated with n-hexane. The precipitate formed was filtered off and washed with ice-cold ether. About 700 mg of 1,2,3,11a-tetrahydro-2-ethylidene-7-methoxy-8-hydroxy-5H-pyrrolo / 2, l-c_7 / l, 4 / benzdiazepin-5-one as a pale yellow powder from P. 108-112 ⁰ G

(Decomposition). ber .: 66 C. 5 H- 17th 0 10 N found: 66 , 16 6th , 92 17th , 63 10 , 29 C ₁₅ H ₁₆ N ₂ O ₅ , 04 , 02 , 55 , 41. Example 4

A solution of 100 mg l, 2,3,10, ll, lla-Kexahydro-2-Ethylidcn-8-hydroxy-7, ll-üimethoxy-bH-pyrrolo / 2, l-c7 / 1,4 / -benzdiazepine- 5-one in 5 ml of pyridine was treated dropwise with 0.2 ml of acetic anhydride while cooling. The reaction mixture was allowed to stand at room temperature for 20 hours and poured into ice water. The avowed corresponds% precipitate was filtered off, washed with water, dissolved in 1 ml methanol and allowed to stand in the cold. The resulting precipitate was recrystallized from methanol. Pale yellow needles were obtained from! ^^, lO.lljlla-Hexahydro ^ -äthyliden-7, ll-dimethoxy-e-acetyloxy-SH-pyrrolo / ^, lc // l, 47benzdiazepin-5-one from P. 132-133 ° C. (Yield $ 85 of theory).

CK ON C ₁₈ H ₂₂ N ₂ O ₅ calc .: 62.41 6.40 23.10 8.09 found: 62.30 6.53 23.24 7.95.

0 0 98 ^{30/1 95 3 BAD} Q * IQINal

Example 5

A solution of 300 mg!, ^^, lOjl ^ lla-Hexahydro ^ -athyliden-7, ll-dimethoxy-e-hydroxy-SH-pyrrolo / i ', 1-cJ / l , 4 / benzdiazepin-5-one in 400 mg of p-brorabenzoic anhydride were added to 5 ml of pyridine. The reaction mixture was left to stand for 20 hours and the precipitate formed was filtered off, washed with chloroform, a 5% aqueous sodium bicarbonate solution and with 2 η hydrochloric acid and evaporated to a smaller volume. The residue was adsorbed on a silica gel column and eluted with an 8: 1 chloroform / ethyl acetate mixture. The eluate was evaporated. Crude 1,2,3,10, ll, lla-hexahydro-2-ethylidene-T ^ l-dimethoxy-ep-bromobenzoyloxy-SH-pyrrolo / ^ lc / / li4_ / benzdiazepin-5-one, which with acetonitrile was treated, white needles of! ^^, Ha-Tetrahydro ^ -äthyliden -? - methoxy-8-p-brombenzoyloxy-5H-pyrrolo / 2, lc // l, 4_ / benzdiazepin-5-one from P. 204-205 ° C (yield 10 # of theory).

CH 0 N Br C ₂₂ H ₁₉ N ₂ O ₄ Br calc .: 58.02 4.17 14.07 6.15 17.58

found j 58.12 4.25 14.00 6.50 17.58

Example 6

A solution of 100 mg l, 2,3,10, ll, lla-hexahydro-2-ethylidene-

/ 1.47 7, ll-dimethoxy-e-hydroxy-SH-pyrrolo / iZ, l-cJpDenzdiazepin-5-one was added dropwise with an ethereal diazomethane solution. The reaction mixture was left in the cold for 20 hours and then evaporated to dryness. The residue was dissolved in 10 ml of methanol and then 10 ml of ether were added ^{at 0 ° C.} Pale yellow needles of 1,2,3,10,11,1a-hexahydro-7,8,11-trimethoxy-5H-pyrrolo / 2, lc // i, 4 / benzdiazepin-5-one were obtained

009830/1953

BATH ORIGINAL

.25-

from P. 88 ⁰ C. (yield 90 $ of theory).

G H 0. - N

C ₁₇ H ₂₂ N ₂ O ₄ calc .: 64.13 6.97 20.10 8.80 found: 64.33 7.05. 20.24 8.60.

Example 7

A solution of 0.54 g of l, 2,3, lla-tetrahydro-2-ethylidene-7-methoxy-8-hydroxy-5H-pyrrolo / 2, l-c // l, 4 / benzdiazepin-5-one 0.26 g of benzyl mercaptan was added to 5 ml of methylene chloride. After stirring for 5 hours, the reaction mixture was left at room temperature for 4 days ditched. The methylene chloride was evaporated off under reduced pressure at a temperature below 50.degree. That the resulting yellow powder was purified by silica gel thin layer chromatography. 0.14 g of 1,2,3,10,11,1a-hexahydro-2-ethylidene-7-methoxy-8-hydroxy-11-benzylthio-5H <-pyrrolo were obtained / äjl-cy / l ^ / benzdiazepin-S-on. It was recrystallized from benzene. M.p. 143-145 ° C (decomposition).

H N

6.11 7.07 6.00 6.52.

A solution of 0.27 g of 1,2,3,1la-tetrahydro-2-ethylidene-7-methoxy-8-hydroxy-5H-pyrrolo / 2, l-c // i, 47benzdiazepin-5-one 1.5 ml of ethyl mereaptane were added in 5 ml of methylene chloride. the The solution was left to stand for 6 days and then evaporated under reduced pressure. The residue was dissolved in water and the aqueous solution extracted with methylene chloride. The methylene chloride solution was washed with water and over magnesium sulfate dried. After distilling off the solvent

009830/1953

O ₃ S ber .: '66 G C ₂₂ H ₂₄ N ₂ found j 66 , 72 8th , 70 example

one held 0.2 g! ^^, lO.ll.lla-Hexahydro ^ -äthyliden-T-raethoxy-8-hydroxy-ll-ethylthio-5H-pyrrolo / 2 _r lc // l »4 / benzdiazepine-5- on in the form of yellow crystals from P. 70-74 C (decomposition).

0, p ber .: 8th N ^C 17 ^H 22 ^N 2 • found: 8th , 65 9 ', 15. example

A solution of 0.54 g! ^^
1.2 g of dimethylamine were added to oxy-8-hydroxy-5H ~ pyrrolo / 2, lc // l, 4_ / benzdiazepin-5-one in 5 ml of methylene chloride. After stirring for 5 hours, the reaction mixture was allowed to stand for 4 days. Thereafter, the methylene chloride layer was separated, washed with water and dried over magnesium sulfate. After removal of the solvent under reduced pressure, 0.3 g of 1,2,3,10,11,1la-hexahydro-2-ethylidene-7-methoxy-8-hydroxy-lldimethylamino-5H-pyrrolo / 2,1-cJ remained '/1,4./ benzdiazepin-5-one in the form of a pale yellowish brown powder. This was purified by silica gel thin layer chromatography. A pure yellowish-brown, crystalline material with a melting point of 65-68 ° C. (decomposition) was obtained.

Example 10

A solution of 0.27 g! ^^, lla-Tetrahydro ^ -äthyliden ^ -methoxy-8-hydroxy-5H-pyrrolo / ~ 2, l-c // l, 4 ./- benzodiazepin-5-one in 5 ml of methylene chloride, 1.5 ml of methanol were added and the mixture was stirred at room temperature for 5 days. The reaction mixture was then Vigorously evaporated under reduced pressure. The residue was placed in a refrigerator. The formed crystals of 1,2,3,10,11, lla-hexahydro-2-ethylidene-7,11-dimethoxy-8-hydroxy-

009830/1953

5H-pyrrolo / 2, l-c / A * 4 / -benzodiazepin-5 - one are filtered off and dried. P. 145 - 146.5 ° C (decomposition).

Example 11

An injection preparation was made from the following components:

Compound of Example 10 0.02 g

Ethanol "10 ml

distilled water ■ 100 ml

Adjust to Pu 7.5 with 1 η sodium hydroxide solution.

009830/1953

Claims (23)

in which R _{1 is} a hydrogen atom, a lower alkyl, a lower alkylcarbonyl, an aryl-lower-alkylcarbonyl or an arylcarbonyl radical and Y is the group -N = CH- or -NH-CHR-, where R is a lower alkoxy , is an aryl-lower-alkoxy-, a lower alkylthio-, an aryl-lower-alkylthio- or a di-lower alkylamino radical, as indicated by - '" (a) Streptomyces achromogenes var. 'tomaymyceticus A; T.C; C. 21353 or closely related mutants of this microorganism in one Culture medium under aerobic submerged conditions until in the nutrient medium substantial amounts of an antibiotic Enrich the substance / and optionally isolate this substance or ■. " (b) the anti- 'isolated or not isolated from the fermentation mash biotic with an alcohol, to the compound of the general formula '(I). HQ CH, 0 (D or
in which R is a lower alkoxy / an aryl-lower-alkoxy radical is, · possibly ■ - ·. -, (c) the compound of the general formula (I) -in one not alcoholic solvent to a compound of general Formula (II) implemented. . " 009830/1953 original ■ ζ -, CH (II) CHCH- or where appropriate (d) a compound of the general formula I or II with an acylating agent to give a compound of the general formula (Hl) implements R ', C- (III) in which R ¹ - ^ is a lower alkylcarbonyl, an aryl-niecarer-aikyl · carbonyl or an arylcarbonyl radical and Y has the meaning given above, and optionally (e) a compound of the general formula I or II rr.with a Reacts alkylating agent to a compound of general formula (IV) (IV) una * 3 in the K "^ a lower alkyl radical / Y the group -N = CH- or -NH-CHR-, where R has the meaning given above, and optionally 009830/1953 Bass - 28 - Claims Iy Benzdiazepinone compounds of the general formula • (ν) , = CHCH in the R-, a hydrogen atom ,, a .lower alkyl, a lower Alkylcarbonyi -, - an aryl-lower-alkylcarbonyl- or a Arylcarbonylrest and Y is the group -N = CH or -IiH-CHR, Wherein R is a lower alkyloxy, .ein aryl-lower-alkyloxy-, a lower Alkylthio-, an aryl-lower-alkylthio- or a di-lower-alkylamino-ester is.. · 2. A compound according to claim 1, characterized in that R-, a water st off atom and Y is the group -N = CH-. '. ■ "■ ' 3 .. Compound according to claim 1, characterized in that it is calibrated. -et ^ that R- ^ is the p-Brombenzyl oxy group and Y is the group -N = CH-. 4-. A compound according to claim 1, characterized in that R _{1 is} a hydrogen atom and Y is the group '-NH-CHR-', where R is the methoxy group. 009830/1953 INSPECTED 5. A compound according to claim 1, characterized in that R _{1 is} a hydrogen atom and Y is the group -NH-CHR- is -t, where R is the ethoxy group. 6. A compound according to claim 1, characterized e i chn.e t that R-, the acetyl group and Y the group -NH-CHR-, where. R is the metoxy group. 7. Compound 'according to claim 1, since dur-ch characterized in that R ₁ ' is the p.-Bro'mbenzoylgruppe 'and Y is the group -NH-CHR-, where -R is the methoxy group. 8. Compound according to claim I ^ d a d u .r c h characterized, · -.that R-,. the methyl group and Y the group -NH-CHR-, where R is the methoxy group. ·. 9. A compound according to claim 1, characterized in that R _{1 is a} hydrogen atom and Y is the -NH-CHR- group, where R is the benzylthio group. 10. Compound according to claim 1, characterized ge kenn- ^ ζ eic h η et that R _{1 is} a hydrogen atom and. Y is the group -NH-CHR-, w.obei- R is the dirnethylamino group. 11.. Process for the preparation of the compounds of the general formula 'V · "'" ■ '. '' · · ■ · '· · ·'' (V) “CHCHj 009830/1953 (f) a compound of the general formula (V) in the ¥ die where R has the above meaning, Group -NH-CH-R-, / converts in a non-alcoholic solvent to a compound of the general formula VI, • H .' (VI) = CHCH leads. in which R * has the meaning given above, and if applicable '-. . (g) the compound of the general formula (II) with an alcohol, thioalcohol or dialkylamine to form a compound of the general formula Pormel (L) converts, in which R is a lower alkoxy, an aryl-lower-alkoxy-, a lower alkylthio-, an aryl-lower alkylthio- or a di-lower-alkylamino radical. 12. The method according to claim 11 ', characterized geken η ze i without t that the cultivation of the microorganism 50 60 hours at a temperature of about 25 - 55 ⁰ C by- 13. The method according to claim 11, characterized in that g e k e η η ζ e i c h η et that to obtain the antibiotic the Fermentation mash with a p ^ -value of 2-3 with a polar, organic Solvent extracted. 14. The method according to claim ll (b) or (g), characterized ge Indicates that the alcohol used is methanol. ■ · ■ - .. ■ -. ' ·. . · ■ ".- ORIQtNAL INSPECTED 009830/1953 15. The method according to claim 14, characterized in that that one uses ethanol as alcohol * 16. The method according to claim ll (c) or (f), characterized by dadur.ch, that n-hexane is used as a non-alcoholic, organic solvent. 17. The method according to claim II (d), 'thereby · ge - ke η η ζ ei "c" h η et, '- that diazomethane is used as the alkylating agent. "' · '. ·. ■ "'"' .- A 18. The method according to claim ll (e), characterized in that, that acetic anhydride is used as acylating agent ... . . · ... 19. 'Method according to claim'll (e), d a d u r c h g e ■ * ■' · ke η η z ^: eic hn et that p-bromobenzoic anhydride is used as acylating agent ... · -. "· 20. 'The method according to claim II (g), dadurchge-. k e η ri z- ei., c h. η e. t that one is called OJhioalkahol methyl mercaptan used. . · ... 21. · The method according to claim II (g), .durchgek e η η ζ e i c h.n et, - that'mah as thio alcohol Beiizylmercap- tan used. '·' ■ -. '■ ■ ·. '·' ·. '. ' 22. Method according to claim. 11 (g), thereby identified z. e.i c h η e t that dialkylamine is dimethylamine .used. . ·. · '.. ......... ...... ORiGINALINSPECTEO 009830/1953 23. The method according to claim 11 (c) or (f), characterized marked that one as non-alcoholic, organic solvent acetonitrile used. ORIGINAL INSPECTED 009830/1953 ^0B Blank page