CA1237999A - Antibiotics m43a, m43b, m43c and m43d - Google Patents

Abstract

Abstract New antibiotics M43A, M43B, M43C and M43D, new glycopeptide antibiotics of the vancomycin class, are produced by fermentation of Nocardia orientalis NRRL 2450, and NRRL 2452. The products have excellent antibacterial activity comparable to that of vancomycin.

Description

1~3~

IMPROVEMENTS IN AND RELATING TO
ANTIBIOTICS MOE, M43B, MCKEE AND M43D

The present invention concerns novel anti-bionic derivatives of vancomycin, designated MOE, M43B, MCKEE and M43D. The derivatives are products of the fermentation of N. orientals M5-18260 (NRRL 2452) _ and N. orientals M43-05865 (NRRL 2450).
New, improved antibiotics are continually in demand, particularly for the treatment of human disk eases. Increased potency, expanded spectrum of beater-tat inhibition, increased in viva efficacy, and improved pharmaceutical properties (such as greater oral absorb-lion, higher blood or tissue concentrations, longer in viva half life, and more advantageous rate or route of excretion and rate or pattern of metabolism) are some of the goals for improved antibiotics.
In the search for new antibiotics, structural modification of known antibiotics is attempted whenever possible. This approach is limited, however, to mod-fixations which retain the desired activity. Many antibiotics, including the glycopeptides, have such complex structures that even small changes can be difficult to make by chemical means. The discovery of new antibiotics produced by fermentation processes continues, therefore, to be of great importance even in cases where the antibiotic, once recognized, is quite similar to a previously known antibiotic.

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Antibiotics MOE, M43B, MCKEE and M43D are newly discovered members of the glycopeptide group of antibiotics. A closely related member of this group in eludes vancomycin (see, for example, U.S. Patent 3,067,099). In U.S. Patent 3,067,099, McCormick et at.
described the preparation of vancomycin. Three strains of Stre~tomyces orientalist two of which were numbered M43-05865 and M5-18260, were disclosed as being capable of making vancomycin. The cultures were deposited at what was then the Northern Regional Research Labor-tories (now known as the Midwest Area Northern Regional Research Center) at Peoria, Illinois, given the ; accession numbers NRRL 2450 (M43-05865) and NRRL 2452 (M5-18260). Later, the organism designation for the strains was changed from Streptomyces orientals to Nocardia orlentalis.
The vancomycin described in U.S. 3,067,099 became an important, commercially available antibiotic.
The N. orientals culture used to prepare the commercial product was N. orientals strain M5-18260 (NRRL 2452) or its progeny. N. orientals M43~05865 (NRRL 2450) strain produces both antibiotic MOE and vancomycin as major factors.
The structure of a closely related derivative of vancomycin was determined by Sheldrick et at. [GYM.
Sheldrick, PUG. Jones, O. Conrad, DO Williams and GUY. Smith, Nature 271 (5642), 223-225 (1978)]; later, the structure of vancomycin itself was found by Harris et at. [CAM. Harris and TAM. Harris, J. em. Chum. Sock 104, 4293-4295 (1982)~ to be that shown in formula 1:

.

. .

Lo //~
I\
H3C\ \ HO\ I\ OH
H/ COWAN it f Ho Al NO I Rl~4 NIKKO f o It OH

O O
I/ \ O officio ITCH I OH
OH H I- Ho This invention concerns antibiotic M5-18260 or M43-05865 complex, or MOE, M43B, MCKEE or M43D, or a pharmaceutically-acceptable salt thereof, associated with less than 10% by weight of vancomycin.
This invention also concerns new antibiotic complex called MOE, M43B, MCKEE and M43D which have the formula 2:

I, , '' ` , .
, Jo . . , .... . - .. . .
,.

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ION
Jo H3C~H/CH3 R3 ' Jo OH

OH Y OH H

O O

wherein:
R is H or methyl;
R1 and R2 are methyl;
R3 is COWAN or COO;
R4 is glucosyl or vancosaminyl-O-glucosyl;
provided that when R4 is glucosyl, then R must be methyl and R3 must be COWAN; or a pharmaceutically-acceptable salt thereof;
and 3Q associated with less than 10% by weight of vancomycin.

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MOE has structural formula 3:

It I\
H3C~ SHEA f , HO o\ OH
I ONE \ R
OH ox LO

OH
I O I O

WHOOSH
OH

or a pharmaceutically-acceptable salt thereof, also-elated with less than 10% by weight of vancomycin. MOE
is descried in formula 2 when I R1 and R2 are methyl, R3 is COWAN, and R4 is vancosaminyl-0-glucosyl.

.

Jo .. I.

' M43B has structural formula _:

OH
I

HO Coo t \ "I OH
OH

OH funs NO

/
O O

` I' of 0~1 ~13C Liz or a pharmaceutically acceptable salt thereof, also-elated with less than 10% by weight of vancomycin. M43B
is described in formula 2 when R, Al and R2 are methyl, R3 is coo, and R4 is vancosaminyl-O-glucosyl.

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X-6543~ -7-MCKEE has structural formula 5:

.. ÇlH

H3C\ÇH/CH3 ONE f Jo ~jfOH

/
o 0 I t WHOOSH OH

OH

or a pharmaceutically-acceptable salt thereof, also-elated with less than 10% by weight of vancomycin. MCKEE
is described in formula 2 when R, Al and R2 are methyl, R3 is COWAN, and R4 is glucosyl.

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, Jo .

... , . .: .

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M43D has structural formula 6:
Jo I I\
H3C\ SHEA \ HO\ Jo\ OH
OH ONE \ t Ho H So Jo If Jo OH

1 O l O

\ o fife WHOOSH H . OH
6 OH HO Ho or a pharmaceutically-acceptable salt thereof, also-elated with less -than 10% by weight of vancomycin. M43D
is described in formula 2 when R is H, Al and R2 are methyl, R3 is COWAN, and R4 is vancosaminyl-O-glucosyl.

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For convenience, the terms 'vancosaminyl-O-glucosyl' and 'glucosyl' will be used herein to denote a-O-vancosaminyl-~-O-glucosyl and ~-O-glucosyl, respect lively. Also, the terms 'M43 complex' or 'antibiotic(s)' refer to MOE, M43B, MCKEE and/or M43D.
Antibiotics MOE, M43B, MCKEE, and M43D have excellent antibacterial activity. The pharmaceutically-acceptable salts of the derivatives of formula 2 are especially useful.
The N. orientals M5-18260 (NRRL 2452) strain produces very small amounts of MOE, M43B and MCKEE. In addition to vancomycin, the antibiotic complex produced by N. orientals M43-05865 (NRRL 2450), also called the M43 antibiotic complex, contains antibiotic M43~ as a major factor. The ratio of MOE to vancomycin produced : by the M43-05865 (NRRL 2450) strain is approximately 2.5 to 1. In addition, the M43 complex contains a number of minor factors. Among the minor MOE factors are 1) Aye factor A and Aye factor B; 2) antibiotic M43D; 3) the compounds designated agluco~A51568A, aglucovancomycin and agluco-M43A, and desvancosamine-vancomycin; and 4) antibiotic MCKEE and antibiotic M43B. The structural relationships of this group of antibiotics are provided in formula 2 when designated as follows:

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o o o V.. - V V-Jo ox ox l l l o o o I a en E
ox o o o O
V: _ V U U V:
Pi X 04 04 Ox , O / I I O t' N N N N
I / 0=-- . O O O O O O O O O O O O
Z<~= to p! U U U I U U

0---0 ho ho ho ho U
0=0 U U q X

C Z< \ I
O
I I / v o=. Owe G Jo I o v I
I m Jo 5; ¢ ¢ ¢ ¢
I/ \
.
o o I us ox o Jo z; Jo : O

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MOE, M43B, MCKEE and M43D, the new glyco-peptize antibiotics of formula 2, are close to Vance-mizzen in structure and also in activity. They are, therefore, valuable additions to this group of anti-bionics.
MOE, M43B, MCKEE and M43D are shown in formula

2 as zwitterions. Those skilled in the art will recognize, however, that each has one or two carboxyl groups, one or two amino groups and three finlike groups which can react to form various salts. All such forms of MOE, M43B, MCKEE and M43D are part of this invention. The salts are useful, for example, for separating and purifying the antibiotics. In addition, the salts have an improved volubility in water.
MOE, M43B, MCKEE and M43D salts are prepared using standard procedures for salt preparation. For example, the formula 2 zwitterion can be neutralized with an appropriate acid to form an acid addition salt.
The acid addition salts of formula 2 are particularly useful. Representative suitable salts include those salts formed by standard reactions with both organic and inorganic acids such as, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, malefic, fumaric, colic, pamoic, music, D-glu-tamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, Laurie, Starkey, salicylic, methane-sulfonic, benzenesulfonic, sorbic, picnic, benzoic, cinnamic and like acids.
Pharmaceutically acceptable acid addition salts are an especially preferred group of salts of formula 2.

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Antibiotics MOE, M43B, MCKEE and M43D are prepared by culturing Nocardia orientals NRRL 2450 or antibiotics MOE, M43B and MCKEE are prepared by gut-luring N. orientals NRRL 2452, or an MOE, M43B, MCKEE
or M43D-producing variant, mutant or recombinant thereof, under submerged aerobic conditions in a suitable culture medium until a substantial amount of MOE, M43B, MCKEE or M43D is produced. Also a method of producing the M5-18260 or M43-05865 antibiotic complex or MOE, M43B, MCKEE or M43D.as herein before defined comprises cultivating N. orientals NRRL 2450 or NRRL
2452, or an M5-18260 or M43-05865 producing variant, mutant or recombinant thereof, in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic fermentation conditions MOE, M43B, MCKEE or M43D is then separated from the complex. The culture medium used to grow Nocardia orientals NRRL 2450 or NRRL 2452 can be one of a number of media. For economy in production, optimal yield, and ease of product isolation, however, certain culture media are preferred. Thus, for example, pro-furred carbon sources include carbohydrates such as dextrin, dextrose, glucose and glycerol. Preferred nitrogen sources include enzyme digests of cozen, cottonseed meal, soybean grits, protein potency and the like. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, potassium, sodium magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, and like ions.

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Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism. It may be necessary to add small amounts (i.e. 0.2 ml/L) of an anti foam agent such as polypropylene glycol (MOW. about 2000~ to large-scale fermentation media if foaming becomes a problem.
For production of substantial quantities of antibiotics MOE, M43B, MCKEE and M43D, submerged aerobic fermentation in tanks is preferred. Small quantities may be obtained by shake-flask culture. Because of the time lag in antibiotic production commonly associated with inoculation of large Tunis with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism.
The vegetative inoculum is then transferred to a larger tank. The medium used for the vegetative inoculum can be the same as that used for larger fermentations, but other media can also be used.
N. orientals NRRL 2450 or NRRL 2452 can be grown at temperatures between about 25 and about 37C.
Optimum antibiotic production appears to occur at temperatures of about 30C.
As is customary in aerobic submerged culture processes, sterile air is bubbled through the culture medium. For efficient antibiotic production the percent , `

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~37~99 of air saturation for tank production should be about 50% or above [at 30C and about 5 psi (0.34 x 106 dynes/cm2) of back pressure].
Antibiotic production can be followed during the fermentation by testing samples of the broth against organisms known to be sensitive to the antibiotics.
One useful assay organism is Staphylococcus Ayers NRRL
B313. In addition, antibiotic production can be monk-toned by HPLC with W detection.
Following its production under submerged aerobic fermentation conditions, the antibiotics can be recovered from the fermentation medium by filtering the broth to remove Mazola and purifying the filtered broth by a series of adsorptions on suitable adsorbent, such as an ion-exchange resins, chemically modified hydra-phobic inorganic supports used in high performance reversed-phase liquid chromatography, and high porosity polymers, eluding the antibiotics in each case with a suitable solvent such as aqueous acetonitrile.
MOE, My MCKEE and M43D inhibit the growth of a road spectrum of pathogenic bacteria, especially gram-positive bacteria. Table I summarizes the minimal inhibitory concentrations (Micas) at which the anti-bionics inhibit certain organisms, as determined by standard agar-dilution assays. In Table I the activity of MOE and M43D ( each as the phosphate salt) and M43B
and MCKEE (free base) are compared with that of vancomycin (free base).
.

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Table I : In Vitro Activity of MOE, M43B, MCKEE, and M43D

MIX (mcg/ml) Organism Vancomycin MOE
Staphylococcus Ayers NRRL B313 Staphylococcus Ayers V41 Staphylococcus Ayers X400 1 2 Staphylococcus Ayers SUE
Staphylococcus epidermidis EPIC 2 4 lo Staphylococcus epidermidis 222 1 2 Streptococcus Ennui_ C203 Streptococcus E__umoniae Park 1 0.5 0.5 Streptococcus faecium ATTICS 9790 Streptococcus spy group D 9960 4 4 lo Homophiles influenza CAL. 128 64 Haemoph _ s inPluenzae 76 32 64 Escherichia c _ N10 >128 ~64 Escherichia golf ESSAY >64 Escherichia oil THEM >128 >64 Klebsiella pneumonia X26 >128 >64 " " X68 >128 >64 " " KALE >128 >64 Table I keynoted: In Vitro Activity of M43B and MCKEE

MIX ~mcg/ml) Organism M43B MCKEE Vancomycin Staphylococcus Ayers NRRL B313 16 2 Staphylococcus Ayers V4132 4 Staphylococcus Ayers X40032 4 2 Staphylococcus Ayers SUE 4 Staphylococcus epidermidis EPIC 32 8 4 lo Staphylococcus epidermidis 222 32 8 2 Streptococcus pudginess C203 16 2 S eptococcus pneumonia Park 1 2 2 0.25 Streptococcus faecium ATTICS 9790 32 4 2 Streptococcus spy group D 2041 32 8 4 15 Ho flus influenza CAL. >64 ~64 64 Homophiles influenza 76 >64 32 16 Escherichia golf N10 >64 >64 >128 Escherichia Eli ESSAY >64 >64 >128 Escherichia golf THEM >64 >64 >128 20 Klebsiella pneumonia X26 >64 >64 >128 " " X68 >64 >64 >128 " " KALE >64 >64 >128 .

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X 6543M ~17- 1237~9~

Table I keynoted: In Vitro Activity of M43D

MIX (mcg/ml~
Organism Vancomycin M43D
Staphylococcus Ayers NRRL B313 0.5 Staphylococcus Ayers V41 0.5 Staphylococcus Ayers X400 1 2 Staphylococcus Ayers SUE
Staphylococcus epldermidis Evil 2 4 Staphylococcus epidermidis 222 1 2 pudginess C203 0.5 0.5 Streptococcus pneumonia Park 1 0.125 0.25 Streptococcus faecium ATTICS 9790 1 2 Streptococcus so. group D 2041 4 4 influenza CAL. 64 >64 Homophiles influenza 76 128 >64 Escherichia golf N10 >128 >64 __ Escherichia golf ESSAY >128 >64 Escherichia c _ THEM 64 >64 Klebsiella pneumonia X26 >128 >64 " " X68 >128 >64 " " KALE >128 >64 MOE also inhibits the growth of anaerobic bacteria. Table II summarizes the susceptibility of various anaerobic isolates to MOE.

I

Table II: Susceptibility of Anaerobic Bacterial Isolates to MOE
ANAEROBIC BACTERIA MIX gel Vancomycin MOE
Clostridium diffusely 2994 2 Clostridlum perfringens 81 . 1 Clostridium sitcom 1128 4 32 Eubacterium aerofaciens 1235 16 4 Peptococcus asaccharolyticus 1302 2 2 Peptococcus ~_evoti 1281 2 Peptostreptococcus anaerobius 1428 2 0.5 Peptostreptococcus intermediacy 1264 4 8 Propionibacterium ones 79 2 Bacteroides fragilis 111 .64 8 : 20 Bacteroides fragilis 1877 32 <0.125 Bacteroides ragilis 1936B 32 2 Bacteroides thetaiotaomicron 1438 16 8 .
Bacteroides melaninogenicus 1856/28>128 <0.125 Bacteroides melaninogenicus 2736 8 <0.125 Beat rides vulgatis 1211 16 <0.125 Bacteroides corrodes 187432 <0.125 Fusobacterium subsume 1470 2 >0.125.
Fusobacterium necrophorum AYE 2 2 amiss were determined by the agar-dilution method;
endpoints were read after 24-hrs. incubation.

X~6543M -19- 1237~9 MOE and MCKEE have also shown in viva anti-microbial activity against experimental bacterial infections. When two doses of test compound were administered to mice in experimental infections, the activity observed was measured as an EDDY value [effect-live dose in mg/kg to protect 50% of the test animals:
see Warren Wick, et at., J. Bacterial. 81, 233-235 (1961)]. EDDY values observed for MOE and MCKEE are given in Table III.
Table III: EDDY Values for MOE and MCKEE

rganisD Administration EDDY (mg/kg/2) in MOE MCKEE

aureussubcutaneous 1.62 0.5 --pow ones " 1.2 1.36 --Streptococcus Newman " 1.470.88 aye LO Challenge Dose = 125 Pharmaceutical formulations which comprise as active ingredient MOE, M43B, MCKEE or M43D or a forum-ceutically-acceptable salt thereof (an M43 complex), associated with less than 10% by weight of vancomycin and associated with one or more pharmaceutically-acceptable carriers or delineates therefore are assay part "

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of this invention. The antibiotic, preferably as apharmaceutically-acceptable salt, can be formulated for oral or parenteral administration for the therapeutic or prophylactic treatment of bacterial infections. For example, an M43 complex can be admixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The compositions comprising an M43 complex will contain from about 0.1 to about 90% by weight of the active compound, and more generally from about 10 to about 30%. The compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, minutely, dicalcium phosphate, sodium chloride and alginic acid. Disintegrators commonly used in the formulations of this invention include croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid. Tablet binders that can be included are Acadia, methyl cellulose, sodium carboxymethylcellu.lose, polyvinylpyrrolidone (Povidone), hydroxypropyl methyl cellulose, sucrose, starch and ethyl cellulose. Lubricants that can be used include magnesium Stewart or other metallic struts, Starkey acid, silicone fluid, talc, waxes, oils and colloidal silica. Flavoring agents such as peppermint, oil of winter green, cherry flavoring or the like can also be used. It ma be desirable to add a coloring agent to make the dosage form more esthetic in appearance or to help identify the product.

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For intravenous (IV) use, a water soluble form of the antibiotic can be dissolved in one of the commonly used intravenous fluids and administered by infusion. Such fluids as, for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.
For intramuscular preparations, a sterile formulation of a suitable soluble salt form of the antibiotic, for example the hydrochloride salt, can be lo dissolved and administered in a pharmaceutical delineate such as Water-for-Injection, physiological saline or 5%
glucose solution. A suitable insoluble form of the antibiotic may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl owlet.
For oral use, a sterile formulation of a suitable salt form of the antibiotic, for example, the hydrochloride salt, formulated in a delineate such as distilled or deionized water, is particularly useful.
Alternatively, the unit dosage form of the antibiotic can be a solution of the antibiotic or pro-fireball a salt thereof in a suitable delineate in sterile, hermetically sealed ampules. The concentration of the antibiotic in the unit dosage may vary, e.g. from about 1 percent to about 50 percent depending on the paretic-ular form of the antibiotic and its volubility and the dose desired by the physician.
In a further aspect, this invention provides a method for treating or controlling infectious diseases, especially those caused by gram-positive :

I 39~t microorganisms, in animals. This method comprises administering to the animal an effective dose of an M43 complex. An effective dose is generally between about 0.5 and about 100 mg/kg of MOE or M43D or a forum-ceutically acceptable salt thereof; between about 1 and about 200 mg/kg of M43B or MCKEE or a pharmaceutically-acceptable salt thereof. A preferred dose is from about 10 to about 60 mg/kg of MOE or M43D compound; from about 20 to about 120 mg/kg of M43B or MCKEE. A typical lo daily dose for an adult human is from about 250 my to about lo g of MOE or M43D; from about 500 my to about 2.0 g of M43B or MCKEE.
In practicing this method, the antibiotic can be administered in a single daily dose or in multiple doses per day. The treatment regime may require admix-istration over extended periods of time, e.g., for several days or for from two to three weeks. The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the infection, the age and general health of the patient, the tolerance of the patient to the antibiotic and the microorganism or microorganisms involved in the infection.
A convenient method of practicing the treat-mint method is to administer the antibiotic via IVinfusion. In this procedure a sterile formulation of a suitable soluble salt of the antibiotic is incorporated in a solution of a physiological fluid, such as 5%
dextrose solution, and the resulting solution is infused slowly IV. Alternatively, -the piggy-back method of IV
infusion can be used.

~2379~9 In another embodiment, this invention relates to methods of increasing feed-utilization efficiency in poultry, swine, sheep and cattle, of promoting growth rates in cattle raised for meat production and of en-hanging milk production in lactating ruminants. For increasing feed utilization efficiency and promoting growth, an M43 complex is administered orally in a suitable feed in an amount of from about 2 to about 200 grams per ton of total feed. For beef cattle, for example, a range of about 12 to 3000 mg/head/day is suitable. For enhancing milk production in lactating ruminants, oral administration of a daily amount of from about 0.04 to about 16 mg/kg of body weight (or about 25 to about 5000 mg/ruminant/day) is suggested.
The following examples are provided to thus-irate this invention:

Example 1 Isolation of Antibiotic MOE, M43B and MCKEE
, from N. orlentalis NRRL 2452 A. Separation from VancomYCin Using the procedure in U.S. 3,067,099 (Example 2), fourteen 1200 gallon fermenters were processed to give a crud preparation in the hydra-chloride salt form. This preparation was further "k - separated on a Dower 50-2X resin column, selectively ; 30 eluding fractions containing MOE with a 2-percent aqueous ammonium format solution (pi 9.6) as the ` *Trademark I' '` ~`''~ ' .. . .. ..

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X-6543M -24- ~3~99~

fluent, to give a semi-purified preparation containing MOE, M43B and MCKEE.

B. PurificatioIl of MOE

A portion of the M43A-enriched material lo 1.3 g) was suspended in deionized water. The pi of the resulting suspension was adjusted to pi 3 with 10% aqua-out phosphoric acid. The resulting solution, diluted to a volume of lo ml, was passed through a .45 em mom-brine filter (Milks 0.45-~m filter unit, Millipore Corporation, Bedford, MA, 01730). The filtrate was separated in two-ml portions through a chromatography column (37 x 350 mm) containing'~iChroprep~ RP-18 (15-25 Jim (Art. #13901, E. Merck, Darmstadt, Germany) as the stationary phase. The column was eluded with an aqueous acetonitrile gradient containing 12-20% of acetonitrile. The aqueous phase of the gradient con-twined trlethylamine (0.2%) and was adjusted (prior to the acetonitrile addition) to pi 3 with 10% aqueous phosphoric acid. The column effluent was monitored by W activity and fractions were combined accordingly.
The M43A-containing fractions from eighteen such sop-aerations were combined. The acetonitrile was removed by evaporation under high vacuum, and the aqueous solution was concentrated to one-tenth of the original volume.
The concentrated aqueous solution was loaded on a column (15 x 275 mm) containing freshly conditioned Dunn Hod resin (Mitsubishi Chemical Industries Limited, Tokyo 100, Japan). The column was extensively washed with water and then was eluded with water containing , *Trademark **Trademark . .

.

X-6543M ~25- I ~999 25-50% methanol. The methanol was removed under high vacuum, and the resulting aqueous solution was Eva-orated to dryness. MOE was obtained as a dry armor-; chorus solid (0.6 g).
C. Separation of MOE! M43B and MCKEE

A portion (15 g) of material prepared as in Section A was dissolved in water (1.5 L), filtered, and applied to a'CM-Sephadex C-25"(NH4~) column (glass, 6- x 77-cm, packed with 2 L of resin in water). The column was washed with water (2.5 L) at a rate of 5-10 ml/min to remove impurities. The column was then developed using a linear gradient of (a) 8 liters of water in the mixing chambers and (b) 8 liters of EM
NH4HCO3 in the reservoir. Elusion was monitored by assay against Bacillus subtllis in minimal media. Key fractions were also analyzed by analytical HPLC. The M43B- and M43C-containing fractions were combined, desalted on HP-20, concentrated and lyophilized to give 50 my of enriched material. This material was applied ; to a reversed phase silica gel column (LP1-C-18) which was eluded with a CHICANO (agues 0.05M KH2P04, pi

3.2 (87%) solvent system, eluding at a flow rate of 2.~-ml/min. Fractions were monitored by analytical HPLC. The M43B-containing fractions were combined, desalted and lyophilized to give 14.6 my of antibiotic M43B. The M43C-containing fractions were combined, desalted and lyophilized to give 6.2 my of MCKEE.

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2376~3 Eagle 2 Shake-flask Fermentation of N. orientals NRRL 2450 to Produce MOE, M43B, MCKEE and M43D

A lyophilized pellet of Nocardia orientals M43-05865 (NRRL 2450) is dispersed in 1-2 ml of stern-lived water. This solution (<0.1 ml) is used to ionic-late an ajar slant having the following composition:

Ingredient Amount to Dextrin 10 Enzymatic hydrolysate of cozen 2 Beef extract Yeast extract Ajar 20 Distilled waters to 1 liter awns Amine A, Hummock Sheffield Chemical, Lyndhurst NJ
The inoculated slant is incubated at 30C. for

4-6 days. The mature slant culture is covered with sterile distilled water and scraped with a loop to loosen the spores. The resulting spore suspension (1 ml) is used to inoculate 100 ml of a vegetative medium having the following composition:

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' Ingredient Amount (g/L.) Glucose 1 5 Soybean meal 15 Corn steep solids 5 Cook 2 Nail 5 Tap H20 U.S. to 1 liter Thea inoculated vegetative medium is incubated in a 500-ml Erlenmeyer flask for 24-48 hours at 30C.
on a reciprocal shaker with a 2-inch stroke at 108 RPM
or on a rotary shaker operating at 250 RPM.
This incubated vegetative medium ( 5 ml ) is used to inoculate 100 ml of a sterilized (120C. for 30 minutes) production medium having the following come posy lion:

Ingredient Amount (g/L. ) Glucose 10 Edible molasses 20 Petunia 5 Cook 2 Tap H20 U.S. to l liter aBacto (Disco Laboratories, Detroit, MI) . , . . .
. .

- -X-6543M -28- ~37999 The inoculated fermentation medium is incus bated in a 500~ml Erlenmeyer flask at 25-30C. for ; 72-96 hours on either a rotary shaker operating at 250 RPM or a reciprocal shaker operating at 108 strokes ; 5 per minute. The pi of the uninoculated medium varies with the medium used for production, but the production media of Examples 2-5 have an initial pi range of 6.0 to 7.5 and a harvest pi range of 6.5 to 8Ø

B. Isolation of M43 Complex Whole broth (2 L.), prepared as described in Section A, was filtered. The filtrate was treated with a cation exchange resin (Dower WOKS, Ho, NH4+, pi

5.0), using 100-ml of resin and stirring bushes for 30 minutes. The effluent was decanted and discarded.
The resin was washed thoroughly with water, and the water wash was discarded. The resin was then eluded bushes with lo NHgOEI (250 ml and 175 ml per batch).
The equates were combined and concentrated under vacuum to a volume of about 50 ml. An Alcott (2 ml) was removed for assay, and the remaining concentrate was lyophiliYed to give 300 my of M43 complex.

C. Separation of MOE by Analytical HPLC

A portion of M43 complex, prepared as described in Section B, was dissolved in water at a concentration I` of 10 mg/ml. The solution was adjusted to pi 2.3 to increase the volubility of the complex, and the sup-*Trademark I' ;;; , .
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I' 1237~99 pension was then centrifuged. The supernatant was examined by analytical HPLC, using the following con-dictions:
Column: 4.6- x 250-mm stainless steel, repacked by Alter .. " ............ *
Packing: Ultra sphere OHS - 5 micron Solvent: A - CHICANO M KEEP (1:9) pi 3.2 B - CHICANO M KH2P04 (2:8) pi 3-2 Gradient: 0 percent B for 15 mix;, O 40 percent B
for 15-35 mix;, 40 percent B for 35-38 mix;, re-e~uilibrate in 0 percent B until 45 min.
Flow Rate: 1.0 ml/min. Chart Speed: 120 succumb Detection:"LDC Spectromonitor Iota 280 no Sensitivity: 0.1 AUFS
Pumps: "LDC Constametric III"'~
System controlled by"LDC Chromatography Control Module CAM
Sample volume: 10 Al Peak areas were measured by the CAM; then the normalization method of integration was used to give area-percent values for each peak. M43 complex con-twined MOE (area percent = 39.1) and vancomycin (area percent 15.1) and many additional minor factors. Thus, the ratio of antibiotic MOE to vancomycin produced by N. orientals NRRL 2450, as measured by this experiment was 2.6:1.

*Trademark (each instance) .

. .:

;

~37~9~

D. Alternate Separation of MOE by Analytical HPLC
M43 complex is examined by analytical HPLC, using the following system:
Column: Beckman Ultra sphere I particle size), OHS, 25 cm Mobile Phase: Solvent A: CH3CN/TEAP (5:95) " B: " (2:3) [TRAP = 0.5% aqueous triethylamlne adjusted to pi 3 with gone. phosphoric acid Gradient: 9% B to 70% B over a Mooney period;
then hold for 5 min. at 70% B
Flow Rate: 1.0 ml/min.
Detection: W at 254 no MOE Factor Retention Time (min.) Aye factor Byway Aye factor Aye vancomycin 12.23 desvancosamine-A51568A 17.59 M43D 19.96 desvancosamirle-vancomycina 20.38 MOE 24.26 25~ M43Ba 25.46 McKee 29.58 agluco-A51568A 36.97 aglucovancomycin 37.72 agluco-M43A 39.79 trace amount *Trademark Jo i .

' ~L~37~ I

E. Recommended Isolation Procedure for MOE

The following procedure is recommended for isolating antibiotic MOE from MOE complex:
I. Chromatography MOE complex over highly porous polymer (Dunn HP-20, Mitsubishi Chemical Industries, Ltd., Tokyo, Japan):
1) Wash with water and discard the water wash;
2) Wash with 50% aqueous CHICANO and discard this wash;
3) Wash with 50% aqueous CHICANO con-twining 0.1% SCHICK; this equate contains -the MOE.
II. Chromatography the M43A-containing equate (3) over reversed-phase silica gel C18 resin to separate MOE from the remaining factors.
III. Chromatography the separated MOE over HP-20 polymer to desalt the antibiotic, giving pure antibiotic MOE.

F. Separation of M43B, MCKEE and M43D by Analytical HPLC

A portion of the M43 complex prepared as described in Section B is examined by analytical HPLC, using the following system:

Z3~

Column: Beckman Ultra sphere (5 particle size), OHS, 25 cm Mobile Phase: Solvent A: CH3CN/TEAP (5:95) " B: " " (2:3) [TRAP = 0.5% aqueous triethylamine adjusted to pi 3 with gone. phosphoric acid]
Gradient: 9% B to 70% B over a Mooney period, then hold for 5 min. at 70% B
Flow Rate: 1.0 ml/min.
Detection: US at 254 no M43 Factors Retention Time (min.) Aye factor Byway lo Aye factor Aye vancomycin 12.23 desvancosamine~A51568A 17.59 M43D 14.96 desvancosamine-vancomycina 20.38 :

X-6543~ -33- ~37~3~

Keynoted.

M43 Factors Retention Time (min.) MOE 24.26 M43Ba 25.46 McKee 29~58 agluco AYE 36.97 agluco-vancomycin 37.72 agluco-M43A 39.79 trace amounts G. Preparative Separation of_M43D from Vancomycin ; Vancomycin hydrochloride (1.5 g) was dissolved in deionized water. The resulting solution was diluted with deionized water to a volume of 10 ml. This soul-lion was passed through a 0.45-~m membrane filter).
The filtrate was chromatographed in 2-ml portions through a column (37 x 350 mm) containing "LiChroprep RP-18 ~lS-~5 iamb) as the stationary phase. The column *Trademark .

-.
, .

was eluded with an aqueous acetonitrile gradient con-twining 7.5-20% (v/v) of acetonitrile. The aqueous phase of the gradient contained triethylamine (0.2%) and was adjusted (prior to the acetonitrile addition) to pi 3 with 10% aqueous phosphoric acid. The column effluent was monitored by W activity and separated into fractions based on this activity. The M43D-containing fractions from forty such separations were combined.
The acetonitrile was removed by evaporation under high lo vacuum. Inorganic salts were removed by adsorption of M43D on Dunn HP-20 resin). The HP-20 resin was washed with water and subsequently eluded with water containing methanol (25-50% methanol v/v). The methanol was removed under high vacuum, and the resulting aqueous solution was freeze-dried to give M43D as a amorphous white solid (0.107 g).

1) Milks 0.45 Jim filter unit, Millipore Corporation, Bedford, MA 01730 2) Art. ~13901., E. Merck Darmstadt-Germany 3) Mitsubishi Chemical Industries Limited, Tokyo lo, Japan ,., :

Example 3 Characteristics of MOE, M43B, MCKEE and M43D

S MOE has an integer molecular weight of 1475 as determined by fast-atom-bombardment mass spectrum-try. The molecular weight of MOE is 28 units higher than that of vancomycin (mow. 1447).
The molecular structure of MOE, as shown in formula 3, was deduced from X-ray diffraction studies on a closely-related derivative.
M43B has an integer molecular weight of 1476 as determined by fast-atom-bombardment mass spectrometer.
The molecular structure of M43B, as shown in formula 4, is based on proton nuclear magnetic resonance (NOR) studies.
MCKEE has an integer molecular weight of 1332 as determined by fast-atom-bombardment mass spectrum-try. The molecular weight of MCKEE is 143 units lower than that of AYE (mow. 1475). This is consistent with the structural difference between MOE and MCKEE being the presence of an additional vancosamine in MOE.
The molecular structure of MCKEE, as shown in formula 5, was deduced in part from lo NOR studies.
M43D has an integer molecular weight of 1461 as determined by fast-atom-bombardment mass spectrum-try. The molecular weight of M43D is 14 units higher than that of vancomycin (mow. 1447).
The molecular structure of M43D, as shown in formula 6, was deduced in part from lo NOR studies.

..
.:

`

X-6543M 36- ~23799~

The proton NOR assignments for MOE, MCKEE and M43D are summarized in Table Ivy The chemical shifts listed in Table IV were obtained in DMSO do solution at 60C and at 360 MHz proton frequency. The numbering scheme used is shown in formula 7:

~-glucose-vancosamine off Hoyle No JO I H\ N
_ I oh C\ SUE/ +
OKAY H Ho Ho it to I OH\
202I~ E I Ho Ohs/ SHEA
H I/ OH Owl ~37~39~

table IV: Proton NOR Assignments for MOE, MCKEE and M43Da Assignment Chemical Shift -A-NH 6.466.45 6.51 A-2' 4.174.15 4.19 A-l' 5.145.21 5.12 Allah) 5.86 A-2 7.887.86 7.85 A-5 7.287.24 7.30 A-6 7.457.46 7.45 B-NH 8.598.81 7.96 B-l' 5.775.76 5.68 I 5.695.67 5.57 B-6 5.195.21 5.24 C-NH 9.4910.32 7.45 C-2' 4.844.92 4.85 C-1' 5.195.14 5.19 Clue) C-2 7.557.52 7.52 C-3 7.167.18 7.22 C-6 7.617.43 7.30 D-NH 8.398.29 8.34 D-l' 4.444.41 4.45 D-2 6.356.34 6.32 D-4 6.386.45 6.38 .

, . , .

Table IV, keynoted.

: Assignment Chemical Shift E-NH 8.58 8.40 E-l' 4.47 4.45 E-2 7.15 7.16 : E-5 6.70 6.68 E-6 6.77 6.75 Asinine ~6.36.45 6.63 Assign 4.15 4.37 4.45 Awns 2.73 and 2.~8 and 2.16 and " 2.10 2.12 2.34 Asn-C-NH2 7.45 and 7.29 " 6.99 6.72 Lucia 3.20 3.20 2.31 Lowe 4.43 4.72 3.05 Lucy 1.92 and 1.91 and 1.57 and : 1.65 1.59 1.39 ; 25 Lou y 1.53 1.46 1.65 Lucy 0.97 and 0.93 0.87 and 0.90 0.87 0.87 ;

' : ' , ::

~2:3~

Table IV, keynoted.

Assignment Chemical Shift Glucose #1 5.33 5.32 5.35 #2 3.59 3.40 3.60 #3 3.46 3.48 #4 3.30 I ~3.24 Nab #6 3.57 Andy and 3.69 and 3.70 3.70 3.58 Vancosamine #1 5.27 5.24 #2 1.84 and 1.80 and 1.65 1.62 SHEA 1.24 NO
#5 4.64 4.65 SHEA 1.07 Phenols not yet assigned bra = not assigned . .
I`

I,, X-6543M -40- 1237~9~3 Example 4 Antibiotics MOE, M43B, MCKEE and M43D were prepared according to the method of Example 2, but using the following production medium:

Ingredient mount clue.) Glucose 10 Yeast 5 Distillers solubles 5 Clue 4 Cook Tap HO U.S. to 1 liter en MOE, M43B, MCKEE and M43D were prepared by the method of Example 2, but using the following production medium:

Ingredient Amount Casamino acids 5 g/L
Dextrin 5 g/L
Glycerol 5 g/L
Black strap molasses 10 g/L
Yeast 5 g/L
Ox 1 g/L
Mineral Stock 5 ml Tap ~2 U.S. to 1 liter . .. . . .

1~3799~

MOE, M43B, MCKEE and M43D were prepared by the method of Example 2, but using the following production medium:

Ingredient Amount (g/L.) Soybean meal 15 Cozen Nina 3 Glucose syrup 20 Tap HO U.S. to 1 liter en MOE, MCKEE and M43D Tablet Formulation ___ Preparation of tablets containing 250 my of MOE:

MOE, MCKEE or M43D 282.9 of MOE or diphosphate MCKEE or 283.2 my of M43D
: Microcrystalline cellulose 101.1 my Croscarmellose sodium 12.0 my Povidone 12.0 my Magnesium Stewart 3.0 my Starkey acid 4.0 my Purified water 0.16 ml 'I,`' 'I` .

I I

Add MOE, MCKEE or M43D diphosphate, a portion of the microcrystalline cellulose and a portion of the croscarmellose sodium to a suitable container and blend until homogeneous. Prepare a solution of Povidone in waxer, and add the Povidone solution to the blended powders. Granulate the resulting mixture, size if necessary and dry. Add the remaining microcrystalline cellulose and croscarmellose sodium to the dried mixture and blend. Add magnesium Stewart and Starkey acid, and blend the mixture. Compress the resulting powder blend into tablets with a theoretical weight of 415 my. Each tablet contains MOE, MCKEE or M43D diphosphate equip-alert to 250 my of MOE, MCKEE or M43D.

Example 8 MOE, MCKEE or M43D Capsule Formulation Ingredient Weight MOE, MCKEE or M43D
dihydrochloride 262.2 my Corn starch plowable powder 137.65 my Silicone fluid 350 centistokes 2.75 my Corn starch 147.1 my I
Blend MOE, MCKEE or M43D dihydrochloride, starch plowable powder, silicone fluid 350 centistokes and starch powder in a suitable mixer until homogeneous.
Fill into appropriate size hard gelatin capsules to a net fill weight of 550 my. Each capsule contains MOE, MCKEE or M43D dihydrochloride equivalent to 250 my of MOE, MCKEE or M43D.

~2~7~

Example 9 MOE, MCKEE or M43D Suspension Formulation Prepare a sterile insoluble form of MOE, MCKEE or M43D by crystallization or precipitation. Mill or screen to a particle size suitable for suspension.
Suspend the antibiotic in the following vehicle.

Ingredient Amount Lecithin 1%
Sodium citrate 2%
Propylparaben 0.015%
Water for Injection U.S. to desired volume Sterilize the suspension. The suspension may be menu-astride in bulk and filled into vials or may be prepared extemporaneously by adding the vehicle -to the MOE, MCKEE or M43D in the vial.

Claims (21)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing the M5-18260 or M43-05865 antibiotic complex or M43A, M43B, M43C
or M43D of the general formula:

wherein:
R is H or methyl;
R1 and R2 are methyl;
R3 is CONH2 or COOH;
R4 is glucosyl or vancosaminyl-O-glucosyl;

provided that when R4 is glucosyl, then R must be methyl and R3 must be CONH2; or a pharmaceutically acceptable salt thereof; and associated with less than 10% by weight of vancomycin; which comprises cultivating Nocardia orientalis NRRL 2450 or NRRL 2452, or an M5-18260 or M43-05865 producing variant, mutant or recombinant thereof, in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic fermentation conditions.

2. Antibiotic M5-18260 or M43-05865 complex, or M43A, M43B, M43C or M43D, or a pharmaceutically acceptable salt thereof, of general formula 2 as defined in claim 1, wherein:
R is H or methyl;
R1 and R2 are methyl;
R3 is CONH2 or COOH;
R4 is glucosyl or vancosaminyl-O-glucosyl;
provided that when R4 is glucosyl, then R must be methyl and R3 must be CONH2; or a pharmaceutically acceptable salt thereof; and associated with less than 10% by weight of vancomycin, whenever prepared by the process of claim 1 or an obvious chemical equivalent thereof.

3. A process according to claim 1 followed by separation of the complex from the culture medium and associated with less than 10% by weight of vanco-mycin.

4. A process according to claim 3 in which M43A, M43B, M43C or M43D is isolated from the complex and associated with less than 10% by weight of vanco-mycin.

5. A process according to claim 4 for pre-paring M43A of the structure or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin.

6. M43A of formula 3 as defined in claim 5, or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin, whenever prepared by the process of claim 5 or an obvious chemical equivalent thereof.

7. A process according to claim 4 for pre-paring M43B of the structure:
or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin.

8. M43B of formula 4 as defined in claim 7, or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin, whenever prepared by the process of claim 7 or an obvious chemical equivalent thereof.

9. A process according to claim 4 for pre-paring M43C of the structure:
or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin.

10. M43C of formula 5 as defined in claim 9, or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin, whenever prepared by the process of claim 9 or an obvious chemical equivalent thereof.

11. A process according to claim 4 for pre-paring M43D of the structure:
or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin.

12. M43D of formula 6 as defined in claim 11, or a pharmaceutically acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin, whenever prepared by the process of claim 11 or an obvious chemical equivalent thereof.

13. Antibiotic M5-18260 or M43-05865 complex, or M43A, M43B, M43C or M43D, or a pharmaceutically acceptable salt thereof, of the general formula:
wherein:
R is H or methyl;
R1 and R2 are methyl;
R3 is CONH2 or COOH;
R4 is glucosyl or vancosaminyl-O-glucosyl;
provided that when R4 is glucosyl, then R must be methyl and R3 must be CONH2; or a pharmaceutically acceptable salt thereof; and associated with less than 10% by weight of vancomycin.

14. M43A of the structure:
or a pharmaceutically-acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin.

15. M43B of the structure:

or a pharmaceutically acceptable salt thereof, asso-ciated with less than 10% by weight of vancomycin.

16. M43C of the structure:

or a pharmaceutically acceptable salt thereof, asso-ciated with less thans 10% by weight of vancomycin.

17. M43D of the structure:
or a pharmaceutically acceptable salt thereof, associated with less than 10% by weight of vancomycin.

18. A pharmaceutical formulation which comprises, as an active ingredient, M43A as defined in claim 14, or a pharmaceutically acceptable salt thereof, associated with less than 10% by weight of vancomycin and associated with one or more pharmaceutically acceptable carriers or diluents therefor.

19. A pharmaceutical formulation which comprises, as an active ingredient, M43B as defined in claim 15, or a pharmaceutically acceptable salt thereof, associated with less than 10% by weight of vancomycin and associated with one or more pharmaceutically acceptable carriers or diluents therefor.

20. A pharmaceutical formulation which comprises, as an active ingredient, M43C as defined in claim 16, or a pharmaceutically acceptable salt thereof, associated with less than 10% by weight of vancomycin and associated with one or more pharmaceutically acceptable carriers or diluents therefor.

21. A pharmaceutical formulation which comprises, as an active ingredient, M43D as defined in claim 17, or a pharmaceutically acceptable salt thereof, associated with less than 10% by weight of vancomycin and associated with one or more pharmaceutically acceptable carriers or diluents therefor.