NO134219B - - Google Patents

Description

The present invention relates to a method by producing

position of 7(3-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxy-methyl)-7_methoxy-3-cephem-4-carboxylic acid (I), and salts, esters and amides thereof. The compound of formula I is structurally related to the cephalosporin series of compounds, but while cephalosporin C

only contains a D-5-amino-5-carboxyvaleramido group in the 7-still-

none , the process compound also has a 7-methoxy substituent,

and while cephalosporin C is substituted with acetoxymethyl in 3_still-

none on the ring, the process compound herein has a carbamoyloxy-methyl group.

Compound I is designated herein as "antibiotic 842A". IN

Norwegian patent 132,242 describes the preparation and properties of a compound, "antibiotic Al6886l", which is later stated to have

flour I above. According to patent 132,242, when cultivating a new microorganism, Streptomyces clavuligerus NRRL 3585, a mixture of at least two closely related antibiotics (Al6886l and AI6880II) is obtained

and, if desired, antibiotic Al6886l can be isolated, optionally in the form of a salt. Cultivation of Streptomyces lactamdurans NRRL 3802 according to the present invention leads to the formation of a single, specific

obtained distinct antibiotic 842A (or Al6886l), thereby avoiding the obvious disadvantages of obtaining a mixture of closely related compounds which must be separated by time-consuming and expensive methods in order to obtain a reasonably pure antibiotic Al6886l. The present method is thus simpler and cheaper than the method according to patent 132,242.

The present invention also includes the production of

the pharmacologically acceptable salts, esters and amides of compounds

compounds of formula I. These include organic and inorganic

salts such as acid addition salts, metal salts, quaternary salts

and amine salts derived from tertiary organic nitrogenous bases. Suitable esters and amide derivatives include the mono- and diesters

such as those derived from alkanols', cycloalkanols, aromatic alcohols and aralkanols, and mono- and di-amides such as those derived from ammonia, lower alkylamines', di-lower alkylamines, aralkylamines and heterocyclic amines. Examples of these derivatives and methods for producing them are discussed below.

Antibiotic 84 2 A : This antibiotic is 7|3 - (D-5-aiiiino-5 - carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid (I), and its salts, esters and amides. This connection has the following plan formula:

This product (I) is produced by a new strain of Actinomycet.es and a sample of this microorganism, designated MA-2908, is deposited in the culture collection of Merck & Co., Inc., Rahway, New Jersey. A sample of this culture is also deposited in the culture collection of the Northern Utilization Research and Development Branch of the U.S. Department of Agriculture, Peoria Illinois, and has been given the cult number NRRL 3802. Besides being antibiotically active, this product (I) is also an intermediate in the production of the corresponding 3-hydroxy-, 3-acyloxy- and carbamoyloxy derivatives. Below, the product i, i.e. 73-(D-5-amino-5-carboxyvaleramido|-3-(carba-moyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid) will be referred to as Antibiotic 842A, or simply 842A.

Activity

A significant difficulty in antimicrobial therapy is the propensity of most antibiotics to enzymatic degradation. Penicillin G e.g. is effective against a wide range of gram-positive and gram-negative microorganisms, but in the presence of penicillinase it breaks down into a form that is ineffective against most pathogens.

One way to solve this problem has been the development of new antibiotics that contain the "cephem" ring properties of cephalosporin C. Cephalosporin C has a. intrinsic resistance to penicillinase and is active against both gram-negative and gram-positive bacteria, but it is only moderately active, and there are enzymes other than penicillinase that are effective in destroying its activity. These enzymes are called cephalosporinases. The process products (I) show resistance not only to penicillinase, but also to cephalosporinases. They show activity against both gram-negative and gram-positive bacteria, but the degree of activity and the range of organisms against which they are effective are not identical.

Antibiotic 842A is characterized by an increased activity against gram-negative microorganisms. Unlike cephalosporin C, which has a relatively low antibacterial activity, this product exhibits a considerable in vivo gram-negative effect with a potency that is generally greater than that of cephalothin. This activity includes effectiveness in vivo against Proteus morganii and an effectiveness against the following Gram-negative bacteria: Escherichia coli, Proteus vulgaris, Proteus mirabilis, Proteus morganii, Salmonella schottmuelleri, Klebsiella pneumoniae AD, Klebsiella pneumoniae B and Paracolobactrum arizoniae.

In addition to a generally increased gram-negative effect and an increased potency compared to cephalothin and a greater resistance to cephalosporinases, 842A is characterized by a low degree of toxicity and provides a rapid blood count. Within 6 hours after administration, approx. 100% eliminated in the urine. Moreover, it is more resistant to enzymatic degradation than cephalosporin C, and resistance to it develops slowly, and it is bactericidal. Given orally it protects against infections caused by Paracolobactrum arizoniae 3270, Proteus vulgaris 1810 and Salmonella schottmuelleri 3010, and administered subcutaneously it is from two to ten times more effective than cephalothin against the same infections.

The microorganism

The microorganism that forms Antibiotic 842A is a previously unknown strain of Actinomycetes. The original isolate was obtained as a single colony from soil on an agar slant and grown in a medium with the following composition:

After several days of growth, it turned out that no sporulation could be detected.

Taxonomy: The microorganism (strain MA-2908) producing 842A has been identified as a new phylum of Actinomycetes. The taxonomy used in this determination is described in "Bergey<*>'s Manual of Determinative Bacteriology", J. edition and in "The Actinomycetes", Vol. 2, "Classification, Identification and Description of Genera and Species", S. A. Waksman (1961 ). Using this method, the culture was found to belong to the genus Streptomyces, and it has many of the attributes of the known species Streptomyces

.fradiae. Biochemically, it is essentially a complete side piece to the latter. Morphologically, however, there are important differences. For example, the color of the aerial mycelium of S. fradiae is "seashell pink", while the culture MA-2908 is usually "cream" colored. Moreover, the vegetative growth in the MA-2908 culture shows pigment differences on the different media used, and, as stated below, no sporulation was detected on standard taxonomic media. On the basis of these differences, the culture has been assigned a new species name: Streptomyces lactamdurans. Table I below describes the biochemical attributes of the Streptomyces lactamdurans species and of the known Streptomyces fradiae. All the readings in Table I were made after 3 weeks of incubation at 28°C where otherwise not stated; The pH of the medium used in these investigations was approximately neutral, i.e. 6.8 to 7.2. The physiological tests were performed at the end of the seventh and twenty-first day.

Morphology: Sporophores were not detected when the culture was grown on the media indicated in the description of the culture characteristics, although repeated observations were made up to 8 weeks. However, stained microscopic preparations showed long filaments, many of which were segmented into subunits of different sizes,

generally broadly shaped and approx. 0.9 x 1.7 micron in size.

The aerial mycelium was short, straight, little branched. It appears to be of the same size as the vegetative mycelium, 0.9 M> wide. It is light, powdery and scrapes off easily.

The vegetative mycelium is gram positive, not acid fast. It clings to, and in some media, is embedded in the agar. There is some breaking up of rods in shaking flask cultivation, but this is not extensive. Vegetative mycelium from shake flasks and stationary flasks (inoculum medium, ^—6 days, 28°C) showed some "buds" and short, thickened, almost club-shaped segments on the mycelium, but these were not numerous q<y>g their possible significance is unknown .

Tomato paste - oatmeal - agar

Vegetative growth - reverse side, "orange" matte, tort utseer.de, wrinkled.

Aerial mycelium - spread, "cream".

No soluble pigment.

Czapek-Dox-agar

Vegetative growth - matte, "deep cream"

Aerial mycelium - powdered, '"creamish white"

No soluble pigment

Glycerol- asparagine- agar

Vegetative growth - matte, reverse side - "golden yellow" to "orange" Aerial mycelium - powdered, "cream" with "pale peach tones" Soluble pigment - "pale amber"

Egg albumin agar

Vegetative growth - dull, "cream" to "yellow"

Aerial mycelium - powdered, "cream"

No soluble pigment

Calcium Maleate Agar

Vegetative growth - matte back - "yellow" edged mec. "orange"

Aerial mycelium - powdered, "white" to "cream" edged with "peach" No soluble pigment

Nutrient tyrosine agar

Vegetative growth - dull, "tan" to "orange"

Aerial mycelium - scattered, "cream" with "white"

No soluble pigment

Tyrosine crystals cleaved

Molasses sticky yeast hydrolyzate agar

Vegetative growth - matte back - "orange"

Aerial mycelium - powdered, "creamish white"

No soluble pigment.

Weathering agar

Vegetative growth - dull to "golden yellow"

Aerial mycelium - powdered, "cream"

No soluble pigment

Litmus milk

Scattered growth ring - 10 vegetative growths - no aerial mycelium

Peptonization: alkaline reaction;

pH 7.3 - 7, h (control pH - 6.7)

Skimmed milk

Scattered growth ring - "tan" to "orange" Vegetative growth - no aerial mycelium "Light tan" soluble pigment Peptonization - alkaline reaction

pH 7.2 (control pH - 6.6)

Skimmed milk agar

Vegetative growth - dull, "orange" Aerial mycelium - moderate, ".cream" to "pale coral" "Light tan" soluble pigment Hydrolysis of casein

Gelatin stick

Spread "cream" to "orange" cob vegetative. growth suspended throughout the rudder

No soluble pigment

Complete liquefaction

Nutritional gelatin agar

Vegetative growth - dull, "orange" Aerial mycelium - scattered, powdered "cream" No soluble pigment

Liquification of gelatin

Nutrient starch agar

Vegetative growth - dull, "orange" Aerial mycelium - scattered, powdery, "pinkish cream" No soluble pigment

Moderate hydrolysis of starch

Synthetic starch agar

Vegetative growth, - matte, "backside - "cream" edged with "orange"

Aerial mycelium - powdered, "white" edged with "peach" No soluble pigment

Moderate hydrolysis of starch

Loeffler's blood serum agar

Vegetative growth - "cream" colored to "orange"

Aerial mycelium - nothing

No soluble pigment

No dilution

Peptone-. iron - yeast extract - agar

Vegetative growth - "cream"

Aerial mycelium - scattered - "whitish"

No soluble pigment

Microaerophilic growth

(Yeast extract dextrose puncture - <1>+0 mm deep puncture)

Good surface growth and along the upper l/h of the puncture line.

Temperature - Yeast extract - dextrose - slant cultures

Good growth at 28°C

Scattered growth at 37°C

No growth at 50°C

Yeast extract - dextrose agar

Vegetative growth - matte "golden yellow"

Aerial mycelium - powdered, "cream" to "pale flesh pink"

No soluble pigment

Potato plug

Vegetative growth - dry, dull, "cream" to "orange" Aerial mycelium - spreading, "creamish"

No soluble pigment

Reduction of nitrates to nitrites - negative

All readings were taken after 3 weeks of incubation at 28°C where otherwise not stated. Physiological tests were carried out on 7

and 21 days.

The morphological differences between Streptomyces lactamdurans and Streptomyces fradiae are indicated in Table II. The observations were carried out on the media indicated in Table II at growth intervals of one week, three weeks and eight weeks. The aerial mycelium of S. lactamdurans is short and straight with little branching. The. seems to be. about the same size as the vegetative mjicelium, i.e. 0.9 j.. Um wide. It's light, powdery and scrapes off - easily. The vegetative mycelium is gram-positive, it is not acid-fast. It clings to and in some media is embedded in the agar. There is some fragmentation in staves during shaking flask cultivation, but it is not extensive. Vegetative mycelium from shake flasks and stationary flasks (inoculum medium h to 6 days, 28°C) showed some "buds" and short, thickened, almost club-shaped segments on the mycelium, but these were not numerous. All the readings in Table II were taken after 3 weeks of incubation at 28°C where otherwise not stated. The pH of the media used in these investigations was approximately neutral, i.e. 6.8 to 7.2. The physiological tests were carried out at the end of the 7th and 21st day. The colors used in the description are in accordance with the definitions in the "Color Harmony Manual", first edition, 1958; Container Corporation of America.

Carbon utilization: Streptomyces lactamdurans (MA-2908) was also examined for its ability to utilize or assimilate different carbon sources by growing the microorganism in a primary synthetic medium (T. G. Pridham and D. Gottlieb, 1948) containing 1% of the carbohydrate at 28° C for 3 weeks. Table III shows the utilization or assimilation of these carbon sources by Streptomyces lactamdurans (MA-2908) • The explanation of the symbols in Table III is as follows: + indicates good growth, ± indicates poor growth and - indicates no growth on the particular carbon source.

The characteristics listed in Tables I, II and III were used to assign Streptomyces lactamdurans (MA-2908) to a species classification using the keys given in "Bergey<*>'s Manual of Determinative Bacteriology", 7th edition, page 694 -829 (1957) and in "The Actinomycetes", Vol. 2: pages 61-292 (196l). A comparison of the detailed characteristics of Streptomyces lactamdurans with known species shows that the organism corresponds biochemically to Streptomyces fradiae. As shown above, there are, however, important morphological differences such as e.g. by the color of the aerial mycelium of S. fradiae which is "seashell pink" compared to the "cream" color of M^-2908- Furthermore, while the vegetative growth of S. fradiae shows pigment differences on the different media, no sporulation was observed in MA-2908- On the basis of these differences and the characteristics described in the preceding tables, the microorganism that produces antibiotic 842A (MA-2908) was given the new species name Streptomyces lactamdurans.

According to Norwegian patent 132,242, antibiotic A16886 can be produced by cultivating an organism isolated from soil samples from South America.

The organism was isolated from said soil samples by suspending parts of the soil samples in sterile distilled water and by streaking the suspensions onto nutrient agar. The inoculated nutrient agar plates were incubated at 25-35°C for several days. At the end of the incubation period, colonies of the antibiotic Al6886-producing organism were transferred to agar slant cultures by means of a sterile platinum loop. The agar slant cultures were then incubated to provide suitable amounts of inoculum for the production of antibiotic A16886.

The actinomycete used according to patent 132,242 for the preparation of antibiotic Al688° is stated to have been difficult to classify in the genus Streptomyces because of its atypical spore morphology. However, cell wall assay data indicate that the culture should be considered a species in the Streptomyces genus. Consequently, the organism was considered a new species and was given the name Streptomyces clavuligerus.

This organism characteristically produces an extensive network of short,'; multiaxially branched aerial hyphae that eventually divide into spores. Short clover-shaped lateral branches are formed, which usually produce from 1 to 4 spores each. No substrate conidia are formed. Electron micrographs show smooth-walled spores. Cell wall preparations contain the L,L isomers of diamine-pimelic acid and glycine in addition to the main constituents, aspartic acid, glutamic acid and alanine. Spores are gray in quantity and the primary mycelium is pale yellow to yellowish brown. No soluble pigment is produced. The culture has an optimal temperature range between 26° and 30°C. No growth occurs at 37°C. Morphologically, this culture resembles certain strains of Thermomonospora and Micromonospora.

The new organism capable of producing antibiotic A16886 has been permanently deposited without restriction as to availability in the culture collection of The Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture (formerly: Northern Regional Research Laboratories), Peoria, Illinois 6l6o4, and is available to the public under culture No. NRRL 3585.

The characteristics of Streptomyces clavuligerus NRRL 3585 are indicated in the following tables. The methods recommended by the International Streptomyces Project (Shirling et al., "Methods for Characterization of Streptomyces Species", "Intern. Bull Systemic Bacteriol." 16, 313-340 (1966)) for the characterization of Streptomyces species have been used with certain supplementary tests. The color names were determined in accordance with the ISCC-NBS method described by Kelly et al. in "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names" (U.S. Department of Commerce Circ. 553, Washington D.C. 1955). The numbers in brackets refer to Resner and Backus' color series (Tresner et al.: "System of Color Wheels for Streptomyces Taxonomy", "Appl. Microbiol." 11, 335-338 (1937)) and color table designations are underlined. Maerz and Paul *'s color descriptions (Maerz et al.: "Dictionary of Color" (McGraw-Hill Book Co., Inc. New York 1950)) are indicated in brackets. Cultures were grown at 30°C for 14 days unless otherwise specifically stated.

Table V shows the results of carbon utilization experiments carried out on the organism NRRL 3585. The following symbols are used in the table:

+ = growth and utilization

= no growth, no utilization

= likely exploitation

(-) = questionable exploitation

When comparing the properties of Streptomyce lactamdurans NRRL 3802 and the properties stated in patent 132,242 for Streptomyces clavuligerus NRRL 3585, it appears that these are two different strains of the Streptomyces genus.

In vitro and in vivo studies

Antibiotic 842 A, in vitro: The determination of the in vitro biological properties was carried out by the disc-plate agar diffusion method. These tests were performed by placing 7 mm discs, wetted with the antibiotic solution, on the surface of Petri dishes containing 5 ml of "Difco" nutrient agar and 0.2% yeast extract inoculated with 5 or 10 ml of standard cell suspension (OD = 0.22 at 66o nm) per 150 ml

medium and incubated at 25°C or 37°C for 16 hours as indicated. The method and basis for these tests are described in the publication "Cross Resistance Studies and Antibiotic Identification", Applied Micro-biology, Vol. 6: pages 392-398 .(1958). The following tables show the results of these tests on antibacterial spectrum and cross-resistance, and indicate the test organisms used and the conditions used.

Antibiotic 81+ 2A. in vivo: When 8^2 A is given subcutaneously to mice

it is generally more active than cephalothin and about the same as cephaloridine, ampicillin or chloromycetin in protecting against infection by gram-negative organisms. It is remarkably non-toxic and is quickly excreted in the urine with approx. 79% of the subcutaneously injected 8^2A recovered within h hours.

In in vivo studies, antibiotic 842A protects against infection by 3 species of Proteus, 2 of Salmonella, 1 strain of Escherichia coli, 2 of Klebsiella and also against Paracolobactrum arizoniae, Aerobacter aerogenes, Pasteurella multocida and Diplococcus pneumoniae E<>>+ 00.

The results of these tests are described in Table Vllb.

8Lf2A fermentation: Antibiotic 8k2A is formed during the aerobic fermentation of suitable aqueous nutrient media under controlled conditions by inoculation with the organism Streptomyces lactamdurans. In general, many media which are a source of carbon and nitrogen can be used in the production of 8U2A.

The exact amount of the carbohydrate and nitrogen sources will depend on the other components included in the fermentation medium, but in general the amount of carbohydrate is approx. 1-6% by weight of the medium, and the amount of available nitrogen, either alone or in combination, is usually from approx. 0.2% to approx. 6% by weight of the medium. The media described below are examples of those that are suitable for. production of Antibiotic 8<*>+2A. These media are only typical examples of media that can be used.

The fermentation is carried out at temperatures from approx. 20°C to 37°C, but for optimal results it is preferred to carry out the fermentation at a temperature from approx. 2h at 32°C. pH values of the nutrient media suitable for the cultivation of Streptomyces lactamdurans (MB-2908) during the formation of Antibiotic 8k2A should be in the range V from approx. 6.0 to approx. 8.0.

Fermentation of Antibiotic 8k2A on a small scale is conveniently carried out by inoculating a suitable nutrient medium with the antibiotic-producing strain and allowing the fermentation to proceed at a constant temperature of approx. 28°C on a shaker for several days. At the end of the incubation period, the mycelium is removed and the supernatant liquid is analysed.

In practice, this fermentation is carried out in a sterilized flask over a one-, two-, three- or four-stage germ development. The nutrient medium for the germ stages can be any suitable combination. tion of carbon and nitrogen sources such as any of the media IX-XI above. The germ flask is shaken in a chamber with a constant temperature of approx. 28°C in from 1 to approx. 3 days and the growth obtained is used to inoculate either a second-stage germ or the production medium. Intermediate stages of seed flasks, when used, are developed in essentially the same way, i.e. the contents of the flasks are used to inoculate the production medium, the inoculated flasks are shaken at a constant temperature for several days and at the end of the incubation period the contents of the flasks are centrifuged to remove the mycelium. The remaining liquid or broth is then concentrated and purified to obtain Antibiotic 81+2A.

When working on a larger scale, it is preferred to carry out the fermentation in suitable tanks equipped with a stirrer and device for aerating the fermentation medium. According to this method, the nutrient medium is prepared in the tank and sterilized by heating at temperatures up to approx. 120°C. After cooling, the sterilized medium is inoculated with the producing culture and the fermentation is allowed to proceed for several days, which e.g. from 2 to k days, while the nutrient medium is stirred and/or aerated and the temperature is kept at approx. 28°C. With changes in the inoculum development and changes in the production medium, it is also possible to achieve a multifold improvement in production and increase in the power of this antibiotic.

8i+ 2A- determinant. method using Vibrio percolans: Determinations were carried out by the disc-plate method using 12.7 mm filter paper discs. The determination plates were prepared using "Difco" nutrient agar plus 2.0 g/l "Difco" yeast extract at 10 ml per disc. An overnight growth of the target organism, Vibrio percolans (MB-1272) in nutrient broth and 0.2% yeast extract was diluted in sterile saline to a suspension

with h0% transmittance at a wavelength of 660 nm. This suspension was added in an amount of 20 ml/l to the medium for tilting the plates.

The determination plates were kept at <!>+°C until use (maximum 5 days). After application of the antibiotic-saturated determination plates, the plates were incubated at 28°C for from 8 to 2 hours. Inhibition zones were read as mm diameter. They were used to determine relative strengths, or by comparison with a purified ■ reference standard, the strength in <u.g/ml. When such a determination is carried out quantitatively, from 1 to 2/£g/ml of antibiotic can be detected.

Bacterial inactivation in 842A: An in vitro study was designed to determine the resistance of Antibiotic 8k2A to bacterial inactivation compared to cephalosporin C, cephaloridine and cephalothin. These investigations showed that Antibiotic 842A was more stable than the latter against certain microorganisms.

The transforming bacteria used in this investigation were

two organisms known to completely inactivate cephalosporin C, namely Alcaligenes faecalis (MB-9) and Alcaligenes viscosus (MB-12).

(a) Preparation of bacterial cells: Alcaligenes viscosus (MB-12) and A. faecalis (MB-9) cells were prepared as follows: The contents of an L tube were mixed with a few ml of nutrient broth containing 0.2% yeast extract. A ladleful of this suspension was spread over the surface of a nutrient agar slant culture and incubated for 18 hours at 37°C. All slant cultures were stored at 15°C and used within one week of incubation. A bowlful of the surface growth from each slant culture was aseptically transferred to 50 ml of nutrient broth containing 0.2% yeast extract and incubated on a shaker for 18 hours at 28°C. The culture was then centrifuged at 4000 rpm. in

10 minutes. The supernatant was decanted and the remaining pellet of cells was washed twice with sterile 0.1 N phosphate buffer, pH 7.5 (6.8 g of potassium phosphate, i.e. KH 2 PO 1 + and 7.1 g of sodium hydrogen phosphate per liter of distilled water ). The washed cells were then resuspended; in 1/10 of the original volume of a h mg/ml solution of antibiotic in 0.1 M phosphate buffer. The trial mixture was then incubated without shaking in a water bath set at 37°C for h hours. The test mixtures were then centrifuged at 2000 rpm. for 10 minutes and this gave a clear supernatant which was decanted into sterile tubes and immediately frozen in dry ice until ready for biodetermination, usually within 3 hours. Control samples were incubated on noy-

in much the same way except for the absence of cells.

(b) Degree of Antibiotic 842A inactivation: The supernatants were tested for antibacterial activity as follows: 7 mm paper discs were moistened with the supernatants and placed on the surface of nutrient agar-yeast extract (0.2%) plates as previously was inoculated with the relevant test organism. B. subtilis (MB-964) determination plates were inoculated as follows: 5 ml of a suspension of washed spores in 0.9% saline was added to each 150 ml of a 2% nutrient agar-yeast extract (45°C) of which 5 ml then transferred to 15 x 100 mm Petri dishes. All assay dishes were stored at 5°C and used within 3 days. The assay dishes were incubated overnight at 25°C to measure the zones of inhibition around the test disks.

Cell-free control samples of each antibiotic were determined at 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 dilutions to obtain a standard reference curve. Readings of trial antibiotics were determined at full strength after incubation in the presence of the washed bacterial cells. All samples were performed in triplicate.

(c) Results: Percent inactivation was calculated by averaging the three zones of inhibition obtained for each trial and determining the amount of antibiotic remaining in the trial solution as shown by the standard curve. This value is then subtracted from the initial concentration (4 mg/ml) and the remainder is divided by the initial concentration and multiplied by 100 to obtain percent inactivation. Table VIII shows the inactivation obtained for cephalosporin C and Antibiotic 842A under the above conditions."

The ability of Antibiotic 842A and Cephalosporin C to resist the transforming action of four other cultures was also determined. These cultures are Escherichia coli 236, Proteus morganii 251, Proteus morganii 356 and Proteus mirabilis 2kl. Each of these is gram-negative and resistant to Cephalosporin C. When carrying out the determination, individual mixtures of the organisms and one of the antibiotic substances were sampled after 4 hours of incubation and analyzed for residual antibiotic activity. The method is the same determination method as described above against Alcaligenes faecalis MB-9 and A. viscosus MB-12. The following tables show the percentage inactivation of cephalosporin C and 842A on B. subtilis (MB-964) by this method:

The preceding data show that Antibiotic 842A is apparently more resistant than Cephalosporin C to the inactivation of A. faecalis, A. viscosus, Escherichia coli 236, Proteus morganii 251, Proteus morganii 236 and Proteus mirabilis 241.

Antibiotic: 842A is obtained by the present fermentation method as an amphoteric compound with an apparent isoelectric point of *approx. pH 3.5, it is unstable above pH 9.0, but is relatively stable at pH 1.5*

As Antibiotic 842A and its salts effectively inhibit the growth of various species of Salmonella, it can be used as a disinfectant in various household and industrial applications. For example, 842A exhibits

activity against Salmonella schottmuelleri 3010, S. gallinarum and S. typhosa and this property indicates its utility as a sanitary

agent in the household and in industrial applications.

Purification; Antibiotic 842A can be purified by adsorption on an ion-exchange resin such as e.g. on resins consisting of quaternary ammonium or sulfonic acid exchange media. The adsorbed antibiotic is eluted from the resin adsorbate with aqueous solutions or with an aqueous alcoholic solution of a suitable salt such as ammonium chloride, sodium chloride and the like. Suitable ion exchange resins which can be used include e.g. the polystyrene core sulfonic acid resins (45% or 53% water) or the polystyrene trimethyl-benzylammonium resins (43% water) which are known as "Dowex 50" or "Dowex 1" respectively. If desired, the eluate obtained by the preceding method can be further purified by a second or third adsorption and elution step. All the eluates thus obtained are then concentrated to obtain the purified 842A product.

Preparations

Antibiotic 842A can be used alone or in combination as the active ingredient of any of a number of pharmaceutical preparations. These antibiotics and their corresponding salts can be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. They can be administered orally, intravenously or intramuscularly. Suitable carriers that can be used in the preparations include e.g. mannitol, sucrose, glucose or sterile liquids such as water, saline,

glycols and oils of petroleum, animal, vegetable or synthetic origin such as e.g. peanut oil, mineral oil or sesame oil. In addition to the carrier, the preparations can also include other ingredients such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity-increasing agents, flavorings and the like. In addition, the preparation can also include other active ingredients that provide a wider spectrum of antibiotic activity.

The dose to be administered depends to a large extent on the disorder being treated and the patient's weight. The parenteral route is preferred for general infections and the oral route for infections of the digestive tract. In general, a daily dose consists of from approx. 15 to approx. 175 mg of active ingredient per kg body weight in the patient with one or more administrations per day. The preferred daily dose for Antibiotic 842A is in the range from approx. 40 to 80 mg of active ingredient per kg body weight.

The preparations can be administered in several unit dose forms, such as e.g. in solid or liquid orally ingestible dosage form. The preparations, will per unit dose, either in liquid or solid form, generally contain from approx. 15 mg to approx. 700 mg of the active ingredient, but in general it is preferred to use a dose in the range from approx. 80 mg to 320 mg. For parenteral administration, the unit dose is usually the pure compound in a sterile water solution or in the form of a soluble powder intended for dissolution.

Synthetic methods

The process compound can also be used as starting materials in the production of derivatives in which the 3-carbamoyloxy group of 842A is replaced with a number of different substituents. Generally, this exchange or substitution is accomplished by simply treating 842A with a reagent capable of transferring the 3-carbamoyloxymethyl group to the desired 3-substituent. In practice, however, before the replacement of the 3-carbamoyloxy radical, it is often necessary to protect the free amino and carboxy groups by treating 842A with a nitrogen-blocking agent such as an N-lower alkoxycarbonylphthalimide, and an esterification agent to obtain the corresponding 76(D -5-phthaloylamino-5-carboxyvaleramido)-3-(carbamoyloxy-methyl)-7-methoxy-3-cephem-4-carboxylic acid diester.

The process compound of formula (I) forms a wide range of pharmacologically acceptable salts with inorganic and organic bases. These include e.g. metal salts such as those derived from alkali metal and alkaline earth metal hydroxides, carbonates and bicarbonates, and salts derived from primary, secondary and tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylenediamines, N,N-diaryl -lower alkylenediamines, aralkylamines, amino-substituted lower alkanols, N,N-di-lower alkylamino-substituted lower alkanols, amino-, polyamino- and guanidino-substituted lower alkanoic acids and nitrogen-containing heterocyclic amines. Representative examples of these salts include salts derived from sodium hydroxide, sodium carboriate, sodium bicarbonate, potassium carbonate, potassium hydroxide and calcium carbonate and salts derived from such amines as tri-methylamine, triethylamine, piperidine, morpholine, quinine, lysine, prot-amine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol, theophylline and N-methylglucamine.

The aforementioned salts can be monosalts such as the monosodium salt obtained e.g. by treating one equivalent of sodium hydroxide with one equivalent of product (I), or mixed disalts obtained by treating one equivalent of the monosalt with one equivalent of a different base. Alternatively, these disalts can be obtained by treating one equivalent of a base with a divalent cation such as calcium hydroxide, with one equivalent of product (I). Furthermore, the invention encompasses the preparation of mixed salts and esters such as those obtained by treating the product (I) with an equivalent of sodium hydroxide and then

with an equivalent of lactic acid.

The salts produced according to the invention are pharmacologically acceptable non-toxic derivatives which can be used as the active ingredient in suitable unit dose pharmaceutical forms. Moreover, they can be combined with other drugs to obtain preparations with a broad activity spectrum. Moreover, these salts and also the corresponding ester and amide derivatives are used as starting materials in the preparation of the carboxylic acid product of formula I. These salts can also be used in the preparation of other pharmaceutically acceptable salts.

In addition to salts, the process product (I) can also be converted to the corresponding mono- and diesters and mono- and diamides,

like for example. the pivaloyloxymethyl or dibenzhydryl esters, or alkyl, cycloalkyl, aryl or aralkyl esters such as e.g. methyl, ethyl, cyclohexyl, phenyl and benzyl esters, or amides, diamides, N-lower alkyl amides, N,N-di-lower alkyl amides, W-aralkyl amides, N,N-diaralkyl amides or heterocyclic amides such as N-methyl and N- ethyl amide, N,N-dimethylamide, N,N-diethylamide, N-benzylamide, N,N-dibenzylamide, piperidide, pyrrolidide or morpholide.

The methods for producing the above-mentioned esters and amide derivatives include reaction of the carboxylic acid product (I) or the corresponding acid halide with methanol, ethanol, cyclohexanol, phenol, benzyl alcohol or dibenzhydrol. In a similar manner, the amide derivatives can be obtained by treating the corresponding acid halide with ammonia or with the appropriate alkylamine, dialkylamine, aralkylamine or heterocyclic amine. ■ These and other conventional methods for producing the esters and amides are generally known.

The following examples further illustrate the invention.

Example

Antibiotic 8U - 2A

Step A: Shaker flask production

A lyophilized tube of Streptomyces lactamdurans (MA-2908) was opened aseptically. The stirrup was then used to inoculate a 250 ml scooped Erlenmeyer flask containing 50 ml nutrient medium I by breaking the stirrup in sterile gas and transferring the pellet aseptically into the flask. Medium I has the following composition:

Medium I:

This seed flask was shaken at 28°C on a 220 rpm rotary shaker with a 50 mm stroke for 3 days. 5 ml portions (10% inoculum) of this growth were then transferred using sterile pipettes to four second-stage seed flasks of the same size containing the same medium as above, and these flasks were shaken as above. The contents of the second-stage seed flasks were then pooled aseptically in a flask and used to inoculate 11 two-liter scooped Erlenmeyer flasks, each containing 350 ml of Medium II with 2-3% inoculum using sterile pipettes. Medium II has the following composition:

The production flasks were then shaken at 28°C on a 14-5 rpm shaker with a 50 mm stroke for four days. At the end of the incubation period, the contents of ten such flasks were combined and a sample was centrifuged to remove the mycelium.

The presence of Antibiotic 842A, i.e. the product 76-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy~3-cephem-4-carboxylic acid (I), in the fermentation broth was determined by agar diffusion assays carried out with 12.7 mm filter paper discs permeated with the fermentation broth and placed on the surface of determination plates containing 10 ml nutrient agar ("Difco") plus 0.2% yeast extract ("Difco") medium inoculated with the bacterial inoculum. The inhibition zones were measured in mm after overnight incubation at 28°C. The analysis of fermentation fluid coughed up after fermentation for 4 days showed an inhibition zone of 31.5 mm diameter on plates inoculated with Vibrio percolans (MB-1272).

Step B: Adsorption on an ion exchange resin

The filtered fermentation broth was adjusted to pH 7.0 with dilute hydrochloric acid and 2900 ml was adsorbed onto 100 ml of a strongly basic anion exchange resin with a styrene-divinylbenzene matrix ("Dowex 1 x 2" in the chloride form) at 10 ml/min. The consumed liquid was collected in 500 ml fractions. The resin column was washed with water and eluted with 3% ammonium chloride in 90% methanol. The eluate was collected in 100 ml fractions.

Disk-plate determinations against Vibrio percolans (MB-1272) were carried out on all fractions and the zone diameters are indicated below.

These provisions show that approx. 60% of the activity is in what is consumed and approx. 18% is in the eluates. Furthermore, they show that the resin capacity is only 2 fractions or ten column volumes of the fermentation liquid. Eluate fractions 1-4 were combined and concentrated to remove methanol. Spent fractions 1-6 were combined, whereby 1960 ml of solution was obtained. 1860 ml of the solution was adjusted from pH 7.2 to 8.0 with dilute sodium hydroxide and adsorbed onto 100 ml of a strongly basic anion exchange resin with a styrene-divinylbenzene matrix ("Dowex 1x2" in the chloride form) at 14 ml/min. The consumed liquid was collected in 4 equal fractions and analyzes show that 5% of the activity was present. The column was washed with water and eluted with 5% aqueous sodium chloride. The eluate was collected in 50 ml fractions and analyzed. The analyzes showed that 90% of the activity was present in fractions 3 - 16 and these were therefore combined.

Step C: Adsorption on a cation exchange resin

A 50 ml portion of the concentrate prepared in step B was diluted to 500 ml, the pH was adjusted from 8.8 - 2.0 with dilute hydrochloric acid and adsorbed on 25 ml of a strongly acidic sulfonate type cation exchange resin with a styrene-divinylbenzene matrix ("Dowex 50 x 2" in the hydrogen form) at 2.5 ml/min. The column was washed with 25 ml of water, then eluted with 2% pyridine until the pH of the column effluent rose to pH 7 (54 ml). Analyzes of the consumed fraction and the eluate showed 9% of the activity in the consumed and 90% in the eluate. The eluate was identified as the pyridinium salt of Antibiotic 842A.

The '<8>42A product is amphoteric with an apparent isoelectric point at ca. pH 3.5- The product is unstable above pH 7, but stable at pH 1.5. The eluate thus obtained was adjusted to pH 8.0 with dilute sodium hydroxide and evaporated under vacuum to remove pyridine. The product thus obtained was identified as the mono-sodium salt of Antibiotic 842A. The molecular weight is <1>+68 based on the empirical formula.

Analysis for C-^H^N^SO^Na:

Calculate. C 4l.0%; H 4.5%; N 12.0%; S 6.8;

0 30.8%; Na 4.9%.

Found: C 39.31%; H ^.76%; N 11.16%; S 6.46%;

0 3^.12%; Na 4.19%.

In in vitro studies, this product, i.e. Antibiotic 842A, inhibits the growth of the following gram-negative bacteria: Escherichia coli, Proteus vulgaris., Alcaligenes faecalis, Brucella bronchiseptica, Salmonella gallinarum, Vibrio percolans and Xanthomonas vesicatoria. In addition, the product inhibits the growth of the following gram-positive bacteria:

Staphylococcus aureus, Sarcina lutea and Bacillus subtilis.

In in vivo studies in mice, Antibiotic 842A inhibits the following activities. Administration was by subcutaneous injection. After the end of the experimental period, usually 7 days after administration, the amount of product required to protect 50% of the mice (ED^q) from this otherwise lethal injection was calculated:

In addition to the above-mentioned in vivo trials with the product, a clinical isolate of Proteus morganii 356, which is resistant to cephalosporins and capable of degrading cephalosporin C, was used in a mouse protection test carried out in the same manner as stated above. ED^q for these trials is as follows:

Example 2

Antibiotic 8V2A

Shaker flask production

The inoculum was prepared as described in Example 8. The contents of two second-stage seed flasks were combined and the fermentation liquid used to inoculate (at 1 ml/flask) 62" 250 ml Erlenmeyer flasks each containing 50 ml of Medium "IH. Medium IH has the following composition:

Medium III;

The flasks were shaken at 28°C on a 220 rpm shaker with 50 mm

stroke for 5 days. At the end of the incubation period, the contents of 60 such flasks were combined and a sample was centrifuged to remove the mycelium.

The presence of Antibiotic 8h2A was determined by the method described in Example 1 via agar diffusion determinations carried out on a 12.7 mm filter paper determination disc. After inhibition for h days, an analysis of the fermentation liquid gave an inhibition zone of 33 mm against Vibrio percolans (MB-1272).

Example 3

Antibiotic 842A

Fermentation:

Step 1: A lyophilized tube of Streptomyces lactamdurans • (MA-2908) was used to inoculate 50 ml of sterile Medium I in a shoveled 200 ml Erlenmeyer flask.

Medium I:

The inoculated flask was placed on a 220 rpm rotary shaker with a 50 mm stroke and incubated for 72 hours at 28°C.

Step 2: An inoculum of 10.0 ml of the vegetative growth formed was then used to inoculate a 2 liter scooped Erlenmeyer flask containing 500 ml thereof. sterilized Medium I described 'above. The inoculated flask was placed on a 220 rpm rotary shaker and incubated for 48 hours at 28°C.

Step 3: The contents of the inoculum flask were then used to inoculate a 190 l stainless fermenter containing 160 l of the same medium I described above. The inoculated medium was incubated at 28°C for 48 hours with agitation while there

an air flow of 85 l/min was maintained. through the fermentation liquid. During the fermentation period Me small amounts of "Polyglycol 2000" added to combat foaming.

Step 4: An inoculum of 43 1 of the growth obtained was then used to inoculate a 760 1 stainless steel fermenter containing 467 1 of a sterile medium IV with the following composition: ;

The fermentation was carried out at a temperature of 28°C with stirring while an air flow of 280 l/min was maintained for 72 hours. During the fermentation, small amounts of an antifoam agent, "Polyglycol 2000", were added to prevent increased foaming. The batch was coughed up and the activity was determined by the disc-plate method. The fermentation liquid was then filtered through diatomaceous earth at a pH of 7.8 and the product thus obtained was identified as 842A. disc-plate-

assays of a 1:10 dilution gave a zone of inhibition of 21.5 mm against Vibrio percolans (MB-1272).

Example

Purification; Monosodium salt of 7p-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cephem- 4- carboxylic acid

Adsorption on carbon: Four fermentation batches prepared according to Example 1 were each adsorbed on 100 ml of a strongly basic anion exchange resin with a styrene-divinylbenzene matrix ("Dowex 1x2" in the chloride form) and diluted with 1% aqueous sodium chloride. The eluate was collected in 50 ml fractions and analyzed. The eluate fractions from all four batches were adjusted to pH 5 with dilute hydrochloric acid and combined, whereby 4300 ml of solution was obtained. 4200 ml of this solution was stirred with 42 g of carbon ("Darco G-60") for half an hour. The carbon was collected by filtration and washed with water. The filtrate and washing liquid were without activity. The carbon cake was eluted twice with one liter portions of 60% aqueous acetone by stirring the mixture for 1/2 hour and filtering each time. The eluates were evaporated under vacuum to 108 ml and 100 ml, respectively. Analyzes showed that the first eluate contained 76% of the activity,

eighteen times as strong as the starting material, and that the other contained ' 17% of the activity, fourteen times the strength of the starting material. The two concentrates were combined and further concentrated to 61 ml and adjusted from pH 4 to pH 5 with dilute sodium hydroxide. This concentrate contained 40 mg/ml dry matter and gave a 25 mm zone against MB-1272 at a dilution of 1:100 (400 mcg/ml). The product was identified as the monosodium salt of 7B-(D-5-amino-

5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cef em-k-carboxylic acid (I).

Example 5

Purification; Monosodium salt of 7p-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cephem- 4- carboxylic acid

Adsorption on gel: A 22 ml portion of the eluate obtained according to Example 1, step B, was adjusted to pH 7.0 with dilute sodium hydroxide and chromatographed on a column containing 388 ml of "BioGel P-2". The column was developed with water, the effluent was checked with a differential refractometer and 5 ml fractions

•collected automatically and bioanalyzed. The bioactivity was shown

in fractions K7 - 63, while sodium chloride appeared in fractions 62 - 72. Fractions 50 - 60 were combined, analyzed and evaporated to dryness yielding 10.8 mg of residue which was identified as the monosodium salt of 7β-(D-5-amino -5-carboxyvaleramido)-3-(carb-amoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid (I). When determined with Vibrio percolans, this product gave a 25 mm zone at 8 mcg/ml.

Example ' 6

7p-(D-5-amino-5-carboxyvaleramido)-3~(carbamoyloxymethyl)-7- methoxy- 3- cephem- 4- carboxylic acid

Modified fermentation procedure:

Step A: Slant cultures

A lyophilized tube with Streptomyces lactamdurans (MA-2908) was opened aseptically and the organism transferred to a medium of the following composition:

Medium V:

1% "Blackstrap Molasses"

1% "National Brewer's" yeast

Water to volume

The slant cultures were incubated for 7 days at 28°C. When stored cold, the slant cultures were stable for more than 13 weeks.

Stage B: Germ stage: Two-stage sister

First germ: The first germ was inoculated directly, from the slant culture from, Stage A to h0 ml 1% "Primary Dried Yeast N.F." pH 7.0 in a 250 ml scooped Erlenmeyer flask. The flasks were then shaken on a 220 rpm rotary shaker with a 50 mm stroke at 28°C for a period of

two to three days.

Second germ: A 2.5% inoculum from the first germ stage was added to a flask containing a 2% Fleischmann S-150" yeast autolysate, pH 7.0. The growth in this stage is characteristically light and the incubation, continued as in the first stage, was not extended beyond 48 hours.

Step C: Production medium

The production medium contained per liter of distilled water: 30 g "Distiller's Solubles", 7.5 g "Primary Dried Yeast N.F." and 0.25% by volume "Mobilpar-S" antifoam agent. The medium was set to

pH 7.0 with a small amount of concentrated sodium hydroxide solution, placed in Erlenmeyer flasks and treated in an autoclave for 15-20 minutes at 121°C. After cooling, a 2.5% inoculum of the germ obtained in step B was added to the medium. The incubation time can vary from approx. 50 hours to 100 hours, but an incubation time of approx. 72 hours is preferred. The volume of medium in each flask can vary from 30 to 50 ml, but 40 ml is routinely used. The amount of inoculum can vary from 1% to 5%, but in practice 2.5% is generally used.

Step D: Analysis

When the fermentation is finished, the cells are removed by centrifugation and the fermentation liquid is diluted with phosphate buffer, pH 7.0. The concentration of 7β-(D-5-amino-5-carboxyvaleramido)*-3-(carbamoyl-oxymethyl)-7-methoxy-3-cephem-4-carboxylic acid in the fermentation broth was determined by the standard biological disc determination method. The test organism used was Vibrio percolans (ATCC 8461). Filter paper discs were immersed in the diluted fermentation fluids and placed on the surface of agar-containing Petri dishes inoculated with the target organism Vibrio percolans (ATCC 8461). Discs that had previously been dipped in standard solutions containing known concentrations of 842A were also placed on these Petri dishes. The slices were incubated overnight at 28°C and the diameter of the inhibition zones was noted. The concentration of 842A and the fermented liquid was calculated by interpolation from the standard curve indicating the relationship between zone diameters and known concentrations of standard 842A solutions. By this method it was calculated that Streptomyces lactamdurans MB-2908 produced 78.6 Mg/ml 842A

by the modified fermentation method.

Example 7

Purification; monosodium salt of 7B-(D-5-amino-5-carboxyvaleramido)-3-( carbamoyloxymethyl)- 7- methoxy- 3- cephem- 4- carboxylic acid

1.0 g of monosodium salt of 7B-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid prepared according to example 5 was dissolved in 20 ml of 1%-lg aqueous n-butanol and chromatographed on a column containing 2530 ml of "Sephadex G-10", a modified dextran in bead form. The column was developed with 1% lg aqueous n-butanol at 10 ml/min and 10.5 ml fractions were collected automatically. The effluent was monitored with a recording refractometer and the fractions were bioanalysed. The bioactivity was evident in fractions 99 - 122, and these were combined and evaporated to dryness, whereby 670 mg of product containing mainly the monosodium salt of 7B-(D-5-amino-5-carboxyvaleramido)~3-(carb-amoyloxymethyl)- 7-Methoxy-3-cephem-4-carboxylic acid.

The column was later calibrated by chromatography on a mixture of "Blue Dextran 2000" and sodium chloride under identical conditions. "Blue Dextran 2000" was detected in fractions 85 - 93 and sodium chloride was detected in fractions 10 - 155, which shows that the bioactive product can be separated from contaminants of this nature.

Claims (1)

Procedure for the preparation of 78-(D-5-amino-5-carboxy-valeramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylic acid with the formula: and salts, esters and amides thereof, when cultivating a microorganism of the genus Streptomyces in an aqueous nutrient medium under aerobic conditions, characterized in that Streptomyces lactamdurans NRRL 3802 is used as the microorganism, and that the product obtained is optionally transferred to a salt, an ester or an amide thereof in a manner known per se.