DE2118636A1 - 3-nucleotides of lincomycin and their analogues, processes for their preparation and their use in therapeutic preparations - Google Patents

Description

HC HTSAN WXLTE

DR. JUP. Dr! - CHEW. WALTER AT

ALFR "l .. ^ pl-.ILR

DR. J. S. ül L-CriΕΛΑ. H.-J. WOLFF DR. JL-R. hAN3 CHk. AX

623 FRAMKFURT AM MAJN-HÖCHST

ADELÜNSIRASSE 58

2118636 April 16, 1971

Our no. 17 007

The Upjohn Company Kalamazoo, Mich., V.St.A.

3-nucleotides of lincomycin and their analogs, methods for their manufacture and their use in therapeutic preparations.

The invention relates to novel antibacterial compounds of the general formula

1 CHt

H \

HO i

V ι /

OH

109345/1941

in the

Y is an -SR radical, where R is an alkyl radical with 1 to 6 carbon atoms, a -S-CH ₂ -CH ₂ -O-

CjL Jj or a -S-CH ₂ -CH ₃ -OH-ReSt,

HO
R. H or a cis or trans lower alkyl radical

with 1 to 8 carbon atoms,
R ₀ H, a CH, - or C ₀ H ^ radical, X an OH radical, a chlorine, bromine or iodine atom

or an -OR, radical, where R, is an alkyl radical

represents with 1 to 6 carbon atoms, in each case

in the (R) - or (S) configuration and Z is a nucleoside 5'-phosphate group, the mucleo-

sid be adenosine, guanosine, cytidine or uridine

can mean,

their salts and processes for their preparation.

In particular, the invention relates to novel 3-hucleotides of lincomycin as well as their analogues and celesticetins.

Examples of alkyl radicals with 1 to 8 carbon atoms are the "-! Ethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl radicals as well as their isomers.

The novel compounds are prepared by adding a compound of the formula I in which Z in the 3-position of the molecule is hydrogen ( hereinafter referred to as the "parent compound") to the fermentation medium of a Strentomyces fermentation and this compound to a above described novel 3-nucleotide transferred. The process according to the invention also supplies, in varying amounts, phosphate esters of the parent compounds. These 3-phosphorus esters are easy to distinguish from the 3-nucleotides according to the invention,

1 ÜÖ845 / 1941

INSPECTE0

ι ο 6 ο 6

L

because the 3-phosphates have no UV absorption maximum and are not hydrolyzed to the parent compound by snake venom diesterase (animal venom phosphodiesterase) or spleen diesterase. As will be described in detail below, the 3-nucleotides in the recovery process according to the invention are determined by ultraviolet analysis and by testing with snake venom diesterase. The poison diesterase splits, for example, clindamycin adenylate into clindamycin and adenosine 5 ¹ -dhosphate.

The initial presence of 3-nucleotides in fermentation broths is determined by the alkaline test described below Phosphatase detected. However, this test does not differentiate between 3-phosphates and 3-nucleotides, so that the 3-nucleotides remained undetected, other investigations would not be carried out as described above.

Although the compounds according to the invention have no antibacterial activity against S. aureus and Sarcina lutea in vitro , they are activated against S. aureus , for example, when used in vivo. This activation in vivo can probably be compared with the fact that the lincomycin parent compound is formed on contact of the 3-nucleotide lincomycin compound with alkaline phosphatase in vitro.

The lincomycin compounds referred to below as the starting or parent substance can be found in various patents, Processes disclosed in publications and patent applications, namely:

Lincomycin U.S. Patent 3,086,912

with reference to formula I,

in the

Y = -SCH ₃ - to -SC ₆ H ₁₃ -ReSt U.S. Patent 3.380.9 ^ 2

1 0 9 8 k 5/1 9 4 1

2118836

R ₁ = cis or trans-alkyl USA patent 3.38O.992 with up to 8 carbon atoms

R ₂ = hydrogen or alkyl USA patent 3,380,992 with up to 8 carbon atoms

X = (S) OH- or OR, -rest USA Patent 3,380,992

X = (R) or (S) Cl- or BeIg. Patent 676.202 Br-Atom U.S. Patent 3,496,163

X = - (R) or (S) J-Atom U.S. Patent 3,496,163 Celesticetin U.S. Patent 2,928,844

Desalicetin U.S. Patent 2,851,463

4'-Depropyl-4 ¹ -äthyllincomycin, where in the formula IY denotes the -SCH-z radical, R. trans-Ethyl, R ₂ denotes the CH_ radical and X denotes the (R) OH radical, is according to the process according to Examples 1 and 2 of US Pat. No. 3,359,164, in which this compound is designated Lincomycin B, obtainable.

l'-Demethyl-l'-ethyllincomycin, where in the formula IY the -SCH-z radical, R. the trans-n-propyl radical, R ₂ the ethyl radical and X the (R) OH radical means, is by the process according to Example 1 and 2 of US Pat. No. 3,359,163, in which this compound is referred to as Lincomycin C, obtainable.

I ¹ -Demethyllincomycin, where in the formula IY the -SCH, -Rest, R _{1 is} the trans-n-propyl radical, R ₂ H and X is an (R) OH radical, according to the method according to Example 1 is the U.S. Patent 3,329,568, in which this compound is referred to as Lincomycin D, is available.

From the series of the aforementioned compounds is 7 (S) -chloro-7-deoxylincomycin currently also known under the general name "clindamycin".

As already stated, the lincomycin parent compounds or their analogues as well as the Celesticetin there-

109845/1941

2118838

by converting into the 3-nucleotides of the formula I that the parent compound is incorporated into a Streptomyces fermentation. For example, clindamycin nucleotides are obtained after adding clindamycin hydrochloride to a fermentation of Streptomyces coelicolor Müller, NRRL 3532.

The fermentation for the preparation of the compounds according to the invention can be submerged in an aqueous nutrient medium aerobic conditions. For the recovery of limited amounts of 3 ~ nucleotides, of course surface cultures and flasks can also be used. The organism used for fermentation is in a culturing medium grown that is a source of carbon, e.g. an assimilable Carbohydrate, as well as a source of nitrogen, e.g. contains an assimilable nitrogen compound or proteinaceous substance. Preferred carbon sources are: Glucose, brown sugar, sucrose, glycerin, starch, corn starch, lactose, dextrin, molasses and the like. Preferably sources of nitrogen to be used are corn water, yeast, autolyzed Brewer's yeast with milk solids, soy flour, cotton seed flour, corn flour, milk solids, pancreatic casein digestive juice (pancreatic digest of casein), grain fermentation residues in distilleries (distillers' solubles), animal peptone liquids, meat and bone pulp, and the like. It is advantageous to use these carbons and nitrogen sources to combine with each other. Trace metals such as zinc, magnesium, cobalt, manganese, iron and the like need the GSr substrate not to be added, since tap water and unpurified ingredients are used as components of the medium will.

The compound η according to the invention can be produced at any temperature which requires satisfactory growth dor 51t v <ip tornyceb-Ku 1 fcur -.iienlLch IfJt ₃ heirjpiol «much

/. ; ί ο 5 3 5

18 and 40 ⁰ C ,, preferably between about 20 and 37 ° C.

If an above-described Streptomyces fermentation is used to produce nucleotides of lincomycin or one of its analogous compounds defined here or of celesticetin, the lincomycin or celesticetin parent compound (non-nucleotide) can be added before the inoculation of the fermentation medium. However, the parent compound can also be added in small increments during the fermentation cycle, as long as the addition in such a fermentation cycle does not take place too late in order to achieve the desired transfer of all of the added parent substance. The time and the amounts of the addition of the parent compound can easily be determined for each fermentation by adding the parent compound until a certain toxicity on the fermentation is observed, such as, for example, an inhibition of the 3-nucleotide formation. If the parent compound remains at the end of the fermentation cycle, smaller amounts of the parent compound should also be used in subsequent fermentations and / or the time of addition should be changed.

Since the antibacterial effectiveness of the parent compound against §. Lutea in vitro is lost after it has been converted into a 3-nucleotide, the presence of such residual antibacterial effectiveness in vitro in a culture or in a culture extract 24 hours after addition of the parent compound is proof of this that the ability of the culture or extract of the culture to convert the parent compound, was exceeded, or that the content of added compound was too high and überfiihrungsprozess retardant acted on the ^Γ τ ϊ kroorfanismus wherein. Pie above mentioned antibacterial :? '.'irknamkeit _Ln jn_ivro lets α I lurch normal mi.krobiolnrisnhf! Plate sample rare -len .fflikroor ^ inlninun Sarcina Tuten erinl ttoln.

BATH ORIGINAL

* · ** ■ <r μ

₇ _ "..8636

The isolation and purification of the compounds according to the invention can be carried out by various methods, for example by solvent extraction, by liquid-liquid distribution in a Craig device, by ion exchange liquid extraction or adsorption on a suitable adsorbent such as carbon and column chromatography. According to a preferred recovery process, the new 3-nucleotide compounds are isolated from a fermentation broth by filtration as described. The filtrate then passes through a suitable absorbent such as activated carbon or Amberlite XAD-2 (a nonionic, macroporous mixed polymer of styrene crosslinked with divinylbenzene resin, Rohm and Haas Company). This resin is obtained by suspension polymerization of styrene / divinylbenzene copolymers in the presence of a substance that is a good solvent for the copolymer (cf. JACS-. 8jj_, 306, 1962) to remove water-soluble impurities that interfere with the subsequent chromatography could remove. The resin is eluted with a mixture of water and water-miscible organic solvents such as water and lower alcohols, water and lower ketones and the like. The eluate from the charcoal or amberlite XAD-2 resin then passes through a chromatography column containing an anion exchange resin such as Dowex-1- (X-Jj) in the acetate form (Dow Chemical Company, Midland, Michigan). Fractions are collected from the chromatography column and checked for their effectiveness against the microorganism S_ before and after their treatment with alkaline phosphatase to be described below. lutea checked. Fractions which, after testing with alkaline phosphatase, show the highest activity against S. lutea , are combined, concentrated and then subjected to countercurrent distribution in a Craig device, using a solvent system of n-butanol and water (1: 1 vol./ Vol.) Is used. Those fractions that have ο maximum Tlltraviolettabsorption and

109845/1941

hydrolyzed by snake venom phosphodiesterase, are collected and give a preparation that contains a mixture of 3 nucleotides. This mixture can by appropriate Separation process can be separated into the individual 3-nucleotides.

In a preferred separation method for obtaining the individual 3-nucleotides from a mixture thereof, DEAE-Sephadex column chromatography (Pharmacia Pine Chemicals, Inc., Pscataway, NJ, USA or Pharmacia, Uppsala, Sweden) is carried out. The column is eluted with tris (hydroxymethyl) aminomethane (THAM) acetate. The fractions are examined by testing before and after their treatment for their effectiveness against S. lutea with alkaline phosphatase and by ultraviolet spectral analysis at the original pH of the fraction and at an acidic pH (approx. 2.0). Fractions with biological activity against i3. lutea after treatment with alkaline phosphatase and with ultraviolet absorption are combined. Each such cluster contains a single 3-nucleotide along with other undesirable substances. The THAM acetate buffer is removed from these accumulations by passing these collection fractions over a column containing Amberlite XAD-2 packed in water. The column is washed with water and then eluted with aqueous methanol (approx. 80% aqueous methanol). Fractions of approximately 20 ml each are collected and analyzed by ultraviolet spectrum. Fractions with UV absorption are combined and concentrated to dryness. The remaining residue is dissolved in a lower alcohol, for example in methanol. After mixing the solution with ether "a precipitate of a 3-nucleotide compound according to formula I is obtained. According to this formula I, the 3-nucleotide component of the new compounds consists of the 3- (5'-cytidylate),? - ( 5'-Adenyla1 :), 3- (5'-uridylate) and 3- (5 ^f -guanylate).

5 /;

-9- 2118836

The nucleotides can also be determined by partition chromatography Can be separated from each other via diatomaceous earth (Dicalite), whereby solution systems consisting of water and one with water are not miscible solvents can be used.

Lincomycin-3-nucleotides and the 3-nucleotides of the lincomycin analogues are practically ineffective against bacteria in vitro. These new 3-nucleotide compounds are therefore detected by testing their bioactivity against £ 5. lutea after the samples have been treated with alkaline phosphatase. The reaction mixture consists, for example, of 0.5 ml (0.5 M) Tris buffer with a pH value of 8.0, 0.5 ml alkaline phosphatase (1 mg / ml) in Tris buffer (0.5 M) with a pH of 8.0 and 0.05 ml (about 50 pg) of lincomycin-3 nucleotides. This reaction mixture is incubated at 28 ° C. overnight.

Streptomyces which can be used for the production of the compounds according to the invention are, for example: S. coelicolor 19 * 15 , NRRL 3531; S, coelicolor Müller, MRRL 3532 and S. venezuelae , NRRL 3527. These cultures are available without restriction from the Northern Utilization and Research Division, Agricultural Research Service, US Department of Agriculture, Peoria, Illinois, USA.

The compounds according to the invention are amphobic compounds, which can exist in different ionic forms depending on the pH of the environment a At low pH the compounds exist in the form of the acid addition salt, at a higher pH in the zwitterionic form and at an even higher pH in the form of a Metal salt. The acid addition salts include those strong organic or inorganic acids whose pK is that of des Equals or is less than phosphate, such as hydrochloric acid, sulfuric acid, phosphoric acid and the like.

109845/1941

2 ^: I 8636 - ίο -

Acid and metal salts are e.g. the alkali metal salts (with Na and K as well as ammonia) and the alkaline earth metal salts (with including calcium, magnesium, zinc and aluminum) as obtained by neutralizing an acid form with the appropriate Base such as ammonium hydroxide, sodium and potassium hydroxide or alkoxides, calcium or magnesium hydroxide, etc. obtained will. The acidic and neutral salts also include amine salts, such as those obtained by neutralizing an acid form e.g. a basic amine. Examples of basic amines are mono-, di- and trimethylamines, mono-, di- and Triethylamine, mono-, di- and tripropylamine (in iso- and normal form), ethyldimethylamine, benzyldiethylamine, cyclohexylamine, Benzylamine, dibenylamine, N, N'-dibenzylethylenediamine, Bis (o-methoxyphenylisopropyl) amine and similar lower aliphatic, lower cycloaliphatic and lower araliphatic Amines, the lower aliphatic and lower cycloaliphatic radicals with up to and including 8 carbon atoms; heterocyclic amines such as piperidine, morpholine, pyrrolidine, piperazine and the lower alkyl derivatives, their alkyl radicals has up to 8 carbon atoms such as 1-methylpiperidine, ^ -ethylmorpholine, 1-isopropylpyrrolidine, 1, ^ - dimethylpiperazine, 1-n-butylpiperidine, 2-methylpiperidine and 1-sthyl-2-methylpiperidine; Amines with water-solubilizing or hydrophilic groups such as mono-, di- and triethanolamines, ethyl diethanolamine, n-butyl monoethanolamine, 2-amino-1-butanol, 2-amino-2-ethyl-1, 3-propanediol, 2-amino-2-methyl-1-propanol, tris (hydroxymethyl) aminomethane, Phenylmonoethanolamine, p-tert-amylphenyl-diethanolamine and Galactamine, N-methylglucamine, N-methylglucosamine, ephedrine, Phenylephrine, epinephrine and procaine, tetraethylammonium hydroxide and guanidine. The different shapes can be interchangeable can be used, but for most purposes the zwitterionic form,

1 0 9 8 4 ^{5/1 9 A 1 0RIGINAL 1NSP6CTED}

CH.

NH

HO / I

(oz ©

in which R ₁ , R ₂ , X, Y and Z have the meaning given above, and the ammonium salt form is preferred.

The subject of the invention is a preparation for the therapeutic treatment of humans and animals, which are affected by responsive, pathogenic microbe organisms (bacterial and other microparasites) are infested, and for preventive purposes Treating disease-prone subjects by administering the 3-nucleotide ester or a pharmacologically acceptable one Salt.

The mixtures according to the invention, like lincomycin and celesticetin, are suitable for the treatment of humans, birds and animals with various clinical pictures. They provide a means for the oral and parenteral administration of the therapeutically active ingredient for systemic treatment and can be used for the therapy of tonsillitis, pneumonia, otitis, conjunctivitis, boils, carbuncles and other bacterial infectious diseases in humans. At Tieron the compounds can be used prophylactically. Rats at; ^j pi el nwoi ne can use it during the shipment

INSPECTED

be protected from Streptococcus viridans . Animals for meat needs can be treated prophylactically in favor of increased weight gain.

Mammals infected by a parasitic protozoa species of the class Sporazoa from the genus Coccidia (a parasitic microorganism which causes the disease coccidiosis) can be treated by administration with the compounds according to the invention, for example: cattle infected with E. zurnii, E. bovis , E. illipsordalis , sheep and goats infected with E. parva , E. faurei , pigs infected with E. debliecki , E. scabra. and Isospora suic , dogs and cats infected with Isospora biqemina , Isospora felis, E. canis , E. felini , poultry infected with E. tenella , rabbits infected with E. steedae »E. perforans , and mink infected with E. mustelae.

The mixtures are also useful for treating diseases caused by members of the genus Mycoplasma . The most widely known forms are the PPLO (pleuropneumonia-like organisms) such as M. hominis, M. salivarium , M. mycoides , M. hyopneumonia , M. hyorhinis , M. gallisepticum , M. arthriditis and other species found in humans and Animals including domestic animals such as sheep, hundred cattle, pigs, poultry (e.g., chickens, turkeys, ducks, and geese), and experimental animals (e.g. rats and mice).

The compounds are used to treat infections of the kidneys and other infections when L-forms of gram-negative and gram-positive bacteria, such as the L-forms of P. mirabilis , are present.

The present 3-nucleotides and salts become more solid in form and liquid dosage units for oral and parenteral

109845/1941

Administration provided, e.g. as tablets, capsules, Powders, granules, pills, sterile parenteral solutions and suspensions and oral solutions and suspensions and oil / water emulsions.

Powders are made into a suitable one by crushing the 3 ~ nucleotides Fineness and mixing made with equally finely divided extenders. Edible carbohydrates can be used as an extender such as starch or lactose can be used. Preferably a sweetener or sugar as well as a flavoring agent is present. Dry granulates for treatment with water are produced using water-soluble extenders. A Powder mixture of a finely divided 3-nucleotide and a water-soluble extender such as sucrose, glucose or the like. is made with a binding agent such as acacia gum mucilage (aciiacia mucilage) or gelatin solution and then pushed through a sieve to form granules that are allowed to dry. A thickening agent or suspending agent, such as methyl cellulose, as well as a wetting agent and a flavoring oil are also preferably added.

For the production of capsules, a powder mixture is produced as described above and filled into shaped gelatin shells, It is best to use the powder mixture as an adjuvant before filling add a lubricant such as talc, magnesium stearate or calcium stearate.

For the production of tablets, a powder mixture is produced, which is granulated wet or dry or granulated with a Lubricant is added and pressed into tablets. The powder mixture is crushed by mixing the appropriately 3-nucleotide with an excipient or carrier such as starch, lactose, kaolin, dicalcium phosphate and the like. Prepared. To the The powder mixture is granulated with a binding agent such as corn syrup, gelatin solution, methyl cellulose solution or acacia

109845/1941 ^ ^INSPE0TED

Gummy mucilage moistened and pressed through a sieve. Instead of After granulation, the powder mixture can also be compressed, i.e. sent through the tablet machine and the resulting Break large tablets (pellets) into granules. To prevent them from sticking to the tablet molds, the Make granules lubricious by adding stearic acid, iron stearate salts, talc or mineral oil. The lubricious mixture is then pressed into tablets. The tablets can preferably be provided with a protective coating consisting of a sealant Cover a layer of shellac, a coating of sugar and methyl cellulose and a smooth coating of carnauba wax.

Liquids for oral use are provided in unit dosage form such as syrups and elixirs, each Teaspoon of the preparation contains before a certain amount of 3-nucleotide to be administered.

A syrup is obtained by dispersing the 3-nucleotide in an appropriate, tasty aqueous sucrose solution. Elixirs are similarly made using a hydro-alcoholic vehicle. Elixirs are suitable preferably for use as carriers when a solution of a compound is desired which is low in water Shows solubility and good solubility in an aqueous-alcoholic medium.

Forms of sterile liquid dosage units can be prepared for parenteral administration. When preparing the parenteral form, a measured amount of the 3-nucleotide is placed in an ampoule, which is sterilized and sealed with its contents. An ampoule with sterile water can be added as a carrier component zxxv preparation of a suspension or solution (depending on the water solubility of the compound) before administration. Can be advantageous in the sterile

109845/1941

- 15 -? 1 1863B

Water a suspending agent, local anesthetic and buffering agent can be dissolved.

A parenteral suspension with prolonged action is obtained by having the 3-nucleotide in a for Parenteral administration acceptable vegetable oil suspended with or without additional adjuvants.

The term "unit dosage form" used in the description and claims refers to based on physically separate units suitable as dosage units for humans, each with a specific one contain the desired dose, calculated amount of active ingredient in association with the necessary pharmaceutical excipient or vehicle. The specifications for the new unit dosage forms of the invention are dictated by and are straightforward depending on

a) the unique properties of the active ingredient and the special therapeutic effect to be achieved,

b) the limitations imposed by the technique of mixing such an active ingredient for therapeutic use as detailed in the description - these are features of the invention. Examples of suitable dosage unit forms according to the invention are tablets, capsules, powder packs, granules, wafers, teaspoonfuls and tablespoons, dropperfuls, ampoules, vials, separate multiples of all the forms mentioned above, and other types described here. The formulated with suitable pharmaceutical carriers dosage forms unit ⁷ included in preferred embodiments, each dosage unit 25 to 500 mg of 3-nucleotide or its pharmacologically acceptable salts, and 5 to 65 f w / v for parenteral preparations to be used.

The amount of the 3-nucleotide or its salts to be administered

1098 / «5/1941 ORi (SlWAL INSPECTED

range depends on the age and weight of the patient, the particular condition being treated and the one being administered chosen path. For. systemic treatment will be doses of about 1 mg / kg / day to about 50 mg / kg / day preferred.

The following examples are intended to explain the invention without restricting it. Unless otherwise noted, all are Percentages based on weight and all specified solvent mixture ratios based on volume.

example 1

Clindamycin-3-nucleotide

A. Fermentation

Several 500 ml Erlenmeyer flasks were inoculated with a soil culture of Streptomyces Coelicolor Müller, NRRL 3532, each containing 100 ml of sterile seed medium with the following composition:

Glucose monohydrate 25 g / l Pharmamedia 25 g / l

Tap water (q.s.) rest

Pharmamedia is a cottonseed flour for industrial purposes (from Trader's Oil Mill Company, Fort Worth / Texas)

The shake flasks were left on for 3 days at 28 ° C for culture Rotary shaker.

With 5 ml of the inoculum thus prepared several 500 ml Erlenraeyer flasks were inoculated, each 100 ml sterile

2118B3S

Fermentation media contained the following composition:

Yeast extract 2.5 g / l NZ amine B ⁺ 5.0 g / l Glucose monohydrate 20th g / l Sodium nitrate 3 g / i Dipotassium phosphate 1 g / l Magnesium sulfate 0.5 g / l Potassium chloride 0.5 g / l Perrosulfate 0.01 g / l tap water rest

⁺ (Difco Laboratories, Detroit / Michigan; a peptone in powder form that is obtained through the pancreatic digestion of casein.)

2k hours after the inoculation, 100 mg / 1 clindamyein hydrochloride was added to the nutrient solution in the fermentation flask.

The Gärkolben remained the development 2k hours at 28 ⁰ C on a rotary shaker. After the transformation reaction in the fermentation flask, S_. lutea measured the decrease in clindamycin effectiveness. In about 2k hours, almost 100 % of the added clindamycin was converted into an in vitro antibacterially inactive form. The sample with ^. lutea was carried out as follows: The sample material was adjusted to pH 6-8 on agar with phosphate buffer (pH 7.0; 0.1 M). A volume unit (0.08 ml) of the solution containing the substance to be tested was placed on a test disk measuring 12.7 mm, which was then placed on an agar plate germinated with the test microorganism. The dish was incubated at 37 ^° C. for 18-2k hours. The in vitro antibacterial effectiveness is evident in one of the discs

1 0 9 8 4 5/1 9 A 1

21 13H3S

surrounding zone of growth inhibition (grown inhibition). The antibacterial effectiveness can be expressed quantitatively in Mg (mcg) parent compound (or lincomycin or clindamycin) per ml by the linear ratio of log. Dosage to zone diameter (log dose to zone diameter) can be expressed in a known manner in comparison to the standard. The presence of clindamycin-3-nucleotides is determined by first incubating the inactive fermentation solution with alkaline phosphatase at pH 8.0 in Tris buffer and then testing the reaction mixture against S. lutea as described above.

B. Extraction

1) Filtration and absorption on nonionic resin

The fermentation described above was correspondingly larger Scale for the production of ^ 90 1 fermentation broth, the Containing clindamycin-3-nucleotide, in a fermentation tank transfer. In order to obtain the clindamycin-3-nucleotides from the entire fermentation broth, broth was first filtered with 10 kg diatom filter aid. The filter cake was washed with water. The aqueous washing solutions were with the combined clear broth and the combined solutions to remove water-soluble impurities that reduce the effectiveness the subsequent chromatography with an adsorbent such as charcoal or amberlite XAD-2 (Rohm and Haas Company) treated. To produce the absorption column, about 22 kg of adsorbent (Amberlit XAD-2) were suspended in water. The slurry was poured into a glass column (inner diameter 50.8 mm), allowed to settle under atmospheric pressure, and then was let it run off. The clear broth combined with the wash, described above, was dispensed at one flow rate of about 1 liter per minute passed through the column. The column was washed with water, 100 liters of the stam

ORIGINAL IMSPECTED ■> - ■ ■ "'■ ^' 10 3 8 4 5/1941

- 19 - 21 1 β R 3 R

Wash used water was poured away. The column was then filled with 120 liters of 60% aqueous methanol (Eluate I) and 100 liters of 95% aqueous methanol (eluate II) eluted. Eluate I was further treated to obtain the clindamycin-3-nucleotides, Eluate II was poured away.

2) absorption on ion exchange resin

The eluate I described above was chromatographed on an anion exchange chromatography column. The column was washed with 22 kg Dowex-1 (X ¹ O in the acetate form (Dow Chemical Company, Midland / Michigan) packed. Eluate I was adjusted with concentrated ammonium hydroxide to pH 10.0 and the alkaline solution passed through the chromatographic column The used liquid from the column was evaporated to dryness. The yield was 877 g of the material containing clindamycin-3-nucleotides, named "Material A." The column was then washed with 100 liters of water and 70 liters % aqueous acetic acid eluted, the acetic acid eluates were concentrated and the concentrate was freeze-dried to give 89.4 g of a material containing clindamycin-3-nucleotide called "material B".

C. Purification

1) Absorption on nonionic resin

Most of the above "Material A" (776 g) was dissolved in 1.5 liters of water. The solution was concentrated with Ammonium hydroxide adjusted to pH 7.5 and passed through a column which contained 2 1 Amberlite XAD-2 resin. The pillar was washed with 6 l of water. The aqueous washing solutions were collected in three 2 liter fractions (W-I, W-2, W-3). the Column was then eluted with 90% aqueous methanol. It

1 0 9 B U b / 1 U 4 1 ° «W / MAL iNSP _{£ oreo}

2 1 1 8B3R

20 ml fractions were collected and tested for effectiveness against Σ3 before and after treatment with alkaline phosphatase. lutea checked. The fractions with the numbers 61,250 were combined and concentrated to dryness. 52 g of a material were obtained which contained clindamycin-3-nucleotides and which was designated “ADA-IO.1”.

The above-described fractions W-2 and W-3 were combined with the fractions 1 - 60 from the column of Amberlit XAD-2 described above and passed together again through the same Amberlit XAD-2 column, which was first mixed with 15 l of water ( WI obtained as described above) and then eluted with 4 l of absolute methanol. 3 cuts were made: methanol fraction 1 = 1 liter, methanol fraction 2 = 1 liter, methanol fraction 3 = 3 liters. These fractions were examined for their activity against S. lutea before and after treatment with alkaline phosphatase. It was concentrated to dryness from methanol fraction 2. Yield 12.63 g of a material which contained clindamycin-3-nucleotide and was named "ADA-II.1".

The methanol fraction 3, concentrated to dryness, gave 0.7 g of a material which contained clindamycin 3-nucleotides and was designated "ADA-II. Z" .

The above prepared preparations "ADA-IO.1", "ADA-11.1" and "ADA-Il.2" were combined to form the preparation ADA-37.1 (61.7 g). This preparation, which contained clindamycin 3-nucleotides, was produced by the double countercurrent distribution described below further cleaned.

2) double countercurrent distribution

Part of the preparation ADA-37.1 described above

109845/1941 ORIGINAL INSPECTED

- 2a -

(21 g) was dissolved in 100 ml of the upper and 100 ml of the lower phase of a solution system composed of n-butanol / water (1: 1, volume / volume). The solution was introduced into the central tubes of an all-glass double countercurrent manifold (100 tubes). After 48 steps (transfers), the upper and lower phases were collected in 50 ml fractions. A total of 100 steps were carried out. The fractions collected and the material in the tubes of the double countercurrent distributor (DGSV) were tested for effectiveness against £> before and after treatment with alkaline phosphatase. lutea checked.

Under the same conditions as described above, 2 further double countercurrent distributions of 21 g each were made of the preparation ADA-37.1.

The following collection fractions were used in all three distributions educated:

Collective fraction I collector of the lower phase

Political groups No. 20-50

Collection fraction II Remaining in the DGSV device

lower and upper phase

Collection fraction III collector of the upper phase

Fractions No. 5 - 35

The collection fraction I from all three distributions was concentrated to dryness. The resulting residue was in absolute Dissolved methanol and mixed the solution with ether. The resulting precipitate was filtered off and dried. The yield was 7 »12 g. This preparation was not pursued any further.

The collection fractions II and III from all three distributions

109845/1941 * ^

were treated like collection fraction I. The yield of collection fraction II was 13.6 g and was designated "ADA-39.2", collection fraction III gave 0.9 g of a material which was designated "ADA-39.3". Ineffectiveness against S. lutea before and effectiveness against S. lutea after treatment with alkaline phosphatase proved that the preparations ADA-39.2 and ADA-39-3 consisted of largely pure clindamycin-3 "nucleotides. The presence of clindamycin after treatment with Phosphatase was detected by thin layer chromatography.

D) Separation of clindamycin-3-nucleotides by Chromatography

The clindamycin-3-nucleotides thus obtained were subjected to chromatography at DEAE - Sephadex broken down into the individual clindamycin-3-nucleotides. The resin was through about an hour Slurry 500 g DEAE - Sephadex (A-25) with water. Then the resin was filtered off and 2 hours with Stirred 0.5N aqueous sodium hydroxide solution. Then the resin was filtered again, washed with water until the pH value the wash liquor was almost neutral, then stirred for 2 hours with 0.5N aqueous acetic acid and finally washed, until the pH was neutral.

The resin produced in this way was placed in a glass column and allowed to settle under atmospheric pressure. The column was-aminomethane hydroxymethyD solution washed with ¹ J 1 water, then with 4 1 0.1? Aqueous tris ((THAM).

The starting material (ADA-39.2, 13 »0 g) was in 100 ml of water dissolved and the pH value adjusted to pH 9.0 with concentrated ammonium hydroxide. This solution was then added to the column (on the head part), which were eluted successively as follows:

109845/1941

23-2: 18636

1) 15 1 0.05 molar THAM acetate (preparation: 6.05 e THAI τ were dissolved in 800 ml of water, the pH was adjusted to 8.0 with glacial acetic acid and then the volume was brought to 1 liter)

2) 40 liters of 0.1 molar THAM acetate buffer, pH 8.0

3) 20 liters of 0.2 molar THAM acetate buffer, pH 8.0 l ») 20 liters of 0.3 molar THAM acetate buffer, pH 8.0.

20 ml fractions were collected. The following factions were obtained with the individual buffers:

0.05 molar buffer - fractions 1 0.1 molar buffer - fractions 723-2920 0.2 molar buffer - fractions 2921 - 3985 0.3 molar buffer - fractions 2985 - 5000

Selected fractions were determined by testing the effectiveness against S. lutea before and after the treatment with alkaline phosphatase and by UV spectra of the liquid flowing out of the column - once as it was obtained and once at an acidic pH (approx ) - examined.

The following collection fractions were formed: SaBooelfraktion 1

Fractions 850-965
Volume approx. 2300 ml

109845/19 4

neutral, pH 7.0

acidic, pH 2.0 basic, pH 11.0

18636

X »max. (A)

270 (3.72) 279 (5.40)

271 C3.72)

Collective fraction II

Factions 1240-1535 7.0 λ Max. (a) volume approx. 5200 ml , 0 261 (11 , 4) ITV neutral, pH 11.0 255 (11 .25) UV » acidic, pH 2 258 (11 .25) basic, pH

Collection fraction III

Factions 1550-1680 7.0 X Max . (a) volume 2600 ml , 0 262 (3 .60) TTV neutral, pH 11.0 262 (3 .64) uv acidic, pH 2 261 (2 .82) basic, pH

Collection fraction IV

Factions 1771-2125 7.0 X Max . (a) 278 volume 7000 ml 254 (3 , 74): shoulder 278 UV: neutral, pH , 0 254 (3 , 66): shoulder 11.0 264 (3 , 20) acidic, pH 2 basic, pH

a) ISOLATION OF CLINDAMYCIN-3- (5'-CYTIDYLATE) PRESENT IN COLLECTING FRACTION I BY CHROMATOGRAPHY

The column was made from 150 ml of Amberlite XAD-2. the

109845/1941

aforementioned saramel fraction I became at a rate of 6 ml / minute passed through the column. The discharge was collected in 116 fractions of 20 ml each. None of the fractions showed a UV maximum and were discarded. The column was then washed with 900 ml of water (Fractions 117-16l). The wash liquor was also discarded. The column was then eluted with 80 Ji aqueous methanol. The UV analysis of the fractions yielded the following results:

Fraction No. A. max. (a)

162 No UV maximum

163 No UV maximum

164 No UV maximum

165 No UV maximum

166 No UV maximum

167 271 (9.9)

168 271 (161.8)

169 271 (168.0)

170 271 (61.6)

171 271 (19.4)

172 271 (3.6)

173 271 (1.0)

174 271 (0.3)

175 271 (0.15)

Fractions I67-172 were pooled. The solution was evaporated to an aqueous concentrate and freeze-dried. 750 mg of clindamycin 3- (5'-cytidylate) were obtained.

500 mg of this preparation were dissolved in 5 ml of methanol and the solution was mixed with Xther. ¹ JOO mg of clindamycin 3- (5 ^f -cytidylate) of the following formula were obtained:

109845/1941

CH.

CH

H-C-Cl

CONH ΌΗ

NS.

OH

CH ₂ CH ₂ CH

NH,

.0

Z =

ti

P-O-CH OH

II

OH OH

109845/1941

118B3G

Analytical data for C ₂₇ H ₁₁₅ N ₅ O ₁ . calculated:

found:

C 44.48; H 6.17; N 9.60;
0 26.39; S 4.39; Cl 4.87; P 4.25.

C 45.62, 45.86; H 6.99, 7.63; N 9.80; S 3.61; Cl 3.90, 4.04; P 3.44.

Molecular weight

Calculated: 729.5

found: 742 (vapor pressure osmometry in methanol).

Potentiometric titration

In water: pKa ^f 7 * 7

Equivalent weight (eg. Wt.) 587

Specific rotation

, +61 (c, 1, water)

Infrared spectrum : The infrared spectra in both

Mineral oil gauze as well as KBr tablets are the following:

fNSPECTED

109845/1941

In mineral oil gauze

Gang fre-

Inten- band fre-

Inte «- gang fre-

Internal

- 1 - 1 - 1

quenz (em) sity quenz (cm) sity quenz (cm) sity

3340 (oil) S. In 1490 S. 992 S. 3240 (oil) S. 1455 (oil) S. 967 M. 2930 (NS) S. 1375 (oil) S. 933 M. 2860 S. 1365 (Sch) S. 886 S. 2730 M. 1282 S. 853 M. 1717 (NS) M. 1215 S. 805 (Sch) M. 1650 S. 1095 S. 787 S. 1610 S. IO7O S. 720 (oil) S. 1520 S. 1050 S. KBr tablets

Gang free Inten Gang fre- ' Inten Gang fre- Inten frequency (cm ~) sity frequency (cm) sity frequency (cm) sity 3400 S. 1530 (Sch) M. 1045 S. 3210 S. 1520 S. 990 M. 3100 (Sch) S. 1490 S. 965 M. 2955 S. 1455 M. 930 M. 2925 S. 1395 M. 882 M. 2870 M. 1380 M. 850 M. 2790 M. 1286 M. 800 (Sch) M. 1640 S. 1215 S. 785 M. 1615 S. 1083 S. 700 M. 1575 (Sch) M. 1065 S.

The band intensities in the infrared spectra given here are denoted by "S", "M" and "W" respectively and are approximately related to the background absorption in the environment

109845/1941

of the gangs indicated. An "S" band has the same intensity like the strongest band in the spectrum. "M" bands have one Intensity between one third and two thirds of the intensity of the strongest band; have the "W" bands an intensity less than a third of the intensity of the strongest band.

These assessments are made based on a percentage permeability scale. The note "Sch" refers on a "shoulder".

UV spectrum in water at the following pH values:

, 0 /\Max. Enzymes α , 16 ε 600 pH 2 , 0 279 13th , 37 9 835 pH 7 , 0 269 9 , 10 6th 638 pH 11 with 271 9 6th Reactions

Crude alkaline phosphatase

Treatment with alkaline phosphatase gave clindamycin, which was determined by thin layer chromatography (silica gel, ethyl acetate-acetone-water [8: 5: 1, Vol / VolD) was identified.

Animal venom diesterase

Treatment with animal venom diesterase gave clindamycin identified by thin layer chromatography (as above). In addition to clindamycin , cytidine-5'-phosphate was formed.

109845/1941

b) ISOLATION OF PRESENT IN COLLECTIVE FRACTION II
CLINDAMYCIN-3- (5'-ADENYLATE) BY CHROMATOGRAPHY

The column was XAD-2 prepared from ¹ ml of Amberlite IOO. the
Collection fraction II was passed through the column at a rate of 15 ml / minute. The column was washed with 4 liters of water. Neither the wash liquor nor the discharge showed UV maxima; both were therefore discarded. The column was eluted with 80 # aqueous methanol. The UV analysis of the fractions had the following results:

Parliamentary group no.

10 12 14

15 16 17 18

19 20 21 22

23

24

25 26 27 28 29 30

31 32 33

Amax. (a)

260 No maximum 26O (0.18) 26O (0.46) 260 (0.47) 260 (230.0) 260 (632) 260 (628) 260 (540) 260 (405) 260 (280) 260 (230) 260 (135) 26O (80) 260 (55) 260 (31.5) 260 (26.4) 260 (12.8) 260 (9.0) 5.5 (260) 3.65 (260) 2.60 (260) 2.0 (260) 1.55 (260)

109845/1941

Fractions 15-21 were combined and the solution was with 1500 ml of acetone mixed. The precipitate was collected and dried to give 2.1 g of clindamycin 3- (5'-adenylate) as follows Structural formula

NS.

III

CH ₂ CH ₂ CH

NH,

N '

Z *

O tr,

• P-O-CH-i. 2

OH

OH OH

109845/1941

Clindamycin ^ - (5'-adenylate) has the following chemical and physical properties:

Analytical data
For C ₂₈ H ₁₁₅ N ₇ O ₁

calculated: C M, 63; H 6.05; N 13.07; S 4.28; Cl 4.72; P 4.11

found: C, 44.77; H 6.66; N 12.57; S 4.65; Cl 4.38; P 3.52. ί

Molecular weight

For C ₂₈ H ₄₅ N ₇ O ₁₁ PSCl

calculated: 753.5

found: 726 (vapor pressure osmometry in methanol)

Potentiometric titration

In water: pKa '7.6

Equivalent weight 620

Specific rotation: Ca] ^, + 62.9 ° (c, 1.04, water) Infrared spectrum in mineral oil gauze and KBr tablets:

10.9 8 45/1 94

In mineral oil gauze

Bandenfre- Inten- Bandenfre- Inten- Bandenfre- Inten-

- 1 - 1 - 1

rate (cm) sity rate (cm) sity rate (cm) sity

3300 S. 1470 (NS) S. 1065 S. 3240 S. 1450 (oil) S. 1050 S. 2940 (oil) S. 1445 (NS) S. 987 S. 2920 (oil) S. 1435 (NS) S. 965 S. 2845 (oil) S. 1417 S. 927 M. 1680 (Sch) S. 1373 (oil) S. 885 S. 1653 S. 1363 (NS) S. 853 M. 1635 S. 1327 S. 817 M. 1595 S. 1298 S. 795 S. 1570 S. 1245 (NS) S. 717 (oil) S. 1510 S. 1215 S.

In KBr tablets

Bandenfre- Inten- Bandenfre- Inten- Bandenfre- Inten-

■ * 1 - 1 - 1

rate (cm) sity rate (cm) sity rate (cm) sity

3340 S. 1637 S. 1313 S. 3270 S. 1593 S. 1085 S. 3220. S. 1570 M. 1065 S. 2950 M. 1510 M. 1045 S. 2920 S. 1470 M. 986 M. 2865 M. 1453 M. 927 M. 1682 S. 1415 M. 885 M. I675 S. 1375 M. 852 M. I66O S. 1325 M. 815 M. 1650 S. 1295 M. 795 M. 1645 S. 1245 (Sch) _λ M. 717 M. 10 9 8 4 5/1941

2113636

UV spectrum in water at the following pH values:

2 , 0 Xmax. 16 C. £ 628 pH 7th , 0 257 16 , 76 12th 56Ö pH 11 , 0 261 16 , 67 12th 711 pH 261 , 87 12th

Reactions with enzymes
Crude alkaline phosphatase

Treatment with alkaline phosphatase gave clindamycin, which was determined by thin layer chromatography (silica gel, Ethyl acetate-acetone-water C 8: 5: 1, vol / vol 3 was identified

Motorbike poison diesterase

Treatment with animal venom diesterase gave clindamycin and Adenosine 5'-phosphate.

Effectiveness in vivo

Clindamycin-3- (5'-adenylate) is antibacterially ineffective against S. lutea in vitro. In vivo it shows a weight of 30 mg / kg (mice, SQ, S. aureus ).

bacterially ineffective. In vivo , however, it shows a CD, - ₀ -Effective-

c) ISOLATION OF CLINDAMYCIN-3 "(5'-URIDYLATE) PRESENT IN COLLECTING FRACTION III BY CHROMATOGRAPHY

The column was made from 150 ml of Amberlite XAD-2. The collection fraction III was passed through the column at a rate of 10 ml / minute. The column was filled with 1 liter of water

109845/1941

- ^ ■) ίΐ 4 <Γ. Λ ft: 1

c \ ι ö 6 3 b

to wash. Neither the aqueous wash liquor nor the discharge showed UV maxima. The column was then washed with 80% aqueous methanol eluted. The UV analysis of the fractions had the following results:

Fraction No * Xmax. (a)

2 No maximum

4,261 (0.25)

5,261 (0.97)

6,261 (107)

7,261 (248)

8 261 (75)

9 261 (18)

10 261 (2.5)

11 261 (0.87)

Fractions 6-9 were combined and the solution was concentrated to dryness. The residue was dissolved in methanol and the solution was mixed with ether, a precipitate being formed; to give 7 mg clindamycin ¹ IO-3 "(5 '-uridylat) with the following structural formula

R ₁ = CH ₂ CH ₂ CH

IV

109845/1941

ο 5 ^f ζ = -po-Ch

OH

OH OH

Clindamycin-3- (5'-ui »idylate) has the following chemical and chemical properties physical properties:

Analytical data

calculated:

found:

Molecular weight

C M, 27; H 6.33; N 7.68; 0 28.49; S 4.39; Gl 4.86; P 4.24

C 44.62; H 6.19; N 7.79; S 4.04; Cl 4.32; P 4.22.

calculated: found:

732.5

764 (vapor pressure osmometry in methanol)

109845/1941

Potentiometric titration

In water pKa ¹ 7.6

Equivalent weight

Specific rotation : Ca] ² ^, + 79.5 ° (c-, 0.99, water) Infrared spectrum in mineral oil gauze and KBr tablets:

In mineral oil gauze

Gang fre- Inten Gang free Inten Gang free Inten frequency (cm) sity frequency (cm) sity frequency (cm) sity 3330 S. 1660 (Sch) S. 1060 S. 3080 S. 1545 S. 990 S. 2950 (oil) S. 1515 S. 885 S. 2920 (oil) S. 1455 (oil) S. 850 S. 2840 (oil) S. 1375 (oil) S. 810 S. 1750 (Sch) M. 1260 S. 763 S. 1680 S. 1215 S. 720 (oil) S.

In KBr tablets

Band frequency ^, Inten band frequency ₁ Intenquence (cm) 4% $ & p _r frequency (cm ") sity

Band frequency (cm) sity

3410 (NS) S. 1685 3260 S. 1512 3100 M. 1458 2960 S. 1380 2920 S. 1260 2865 M. 1215 2800 (NS) M. 1085 1700 S. 1060

S MMM SSS S

990
965
883
850
808
760
705

M M M M M M M

1098A5 / 1941

- 38 - 2 ': 1 8636

UV spectrum in water at the following pH values:

λmax. a_ £

pH 2.0 261 11.18 8 189

pH 7.0 262 11.44 8 379

pH 11.0 262 8.90 6519

Reaction with enzymes

Crude alkaline phosphatase

Treatment with alkaline phosphatase gave clindamycin, which was determined by thin layer chromatography (silica gel, ethyl acetate-acetone-water [8: 5: 1, VoI / VoI3) was identified.

Animal venom diesterase

Treatment with animal venom diesterase resulted in clindamycin and uridine 5 * phosphate.

Effectiveness in vivo

Clindamycin-3- (5'-uridylate) is antibacterially ineffective against Sarcina lutea in vitro. In vivo , however, it shows an activity with a CD, _ ₀ value of 37 mg / kg (SQ, mice, S_. Aureus).

d) ISOLATION OF CLINDAMYCIN- 3- (5 ^f -GUANYLATE) PRESENT IN COLLECTIVE PRACTICE IV BY CHROMATOGRAPHY

The column was made from 200 ml of Amberlite XAD-2. The collecting group IV was applied at a rate of 20 ml / minute

109845/1941

**> 1 Ί Λ

8636

passed through the column. The column was washed with 3 liters of water. Neither the aqueous wash liquor nor the discharge showed UV maxima. The column was filled with 80 ϊ aqueous methanol eluted; the UV analysis of the fractions gave the following results:

Parliamentary group no.

2 H 6 7 8

10 11 12 13 14

7V. Max. (a) maximum ( ⁺ Shoulder) No maximum 278 No (0.56); NS 278 254 (260); NS 278 254 (740); NS 278 254 (400); NS 278 254 (168); NS 278 254 (50); NS 278 254 (11.5); NS 278 254 (3.2); NS 278 254 (1.16); NS 254

Fractions 7-10 were combined and the solution mixed with 1 liter of acetone. The resulting precipitate was 1.2 g Clindaraycin-3- (5'-guanylate) with the following structural formula:

H-C-Cl

CONH

-CH

NT i / l

109845/1941

CH ₂ CH ₂ CH

OH

S 5
-PO-CH
t

OH

OH OH

Clindamycin - ^ - (5 * -guanylate) has the following chemical and physical properties:.

Analytical data

C ₂₈ H ₁₁₅ N ₇ O ₁₂ PSCl

calculated: . C 43.71; H 5.85; N 12.74;

0 25.00; S 4.16; Cl 4.61; P 4.03

found: C, 43.69; H 6.34; N 11.62;

S 3.63; Cl 4.15; P 3.81.

Molecular weight

calculated:
found:

769.5

750 (vapor pressure osmometry in methanol)

1098A5 / 19A1

Potentiometric titration

In water: Ir pKa x 7.6 Inten Inten 0, water) Inten Inten sity sity i KBr tablets: sity sity S. S. S. Specific turning university ?: S. Equivalent weight 721 S. M. Gang fre- M. Infrared spectrum S. ία} ² g, + 69 ° M. M. -1
frequency (cm) M. S. '(c, 1, S. M. 1050 (Sch) M. Gang free S. in mineral oil gauze and S. M. 987 S. frequency (cm ~) S. ι Mineral oil gauze M. M. 963 M. 3330 S. Gang free S. M. 925 M. 3220 S. frequency (cm) S. S. 885 M. 2920 (oil) S. 1530 S. S. 855 M. 2845 (oil) S. 1457 S. 795 M. 1684 S. 1409 In KBr tablets 780 1675 1375 (oil) Gang fre- 717 (oil) Inten 1635 Inten 1365 -1
frequency (cm) 680 sity 1630 sity 1315 1570 S. 1595 S. 1250 (Sch) 1530 Gang free S. 1565 S. 1215 1450 frequency (cm) M. S. 1080 (Sch) 1405 IO65 M. Gang free S. IO65 13 80 1045 M. frequency (cm) S. 1355 985 M. 3420 S. 1255 (Sch) 925 M. 3240 (Sch) S. 1210 885 M. 2950 S. 1080 855 2920 S. 109845/1941 800 2865 78Ο 1685 1635 I63O 1595

W spectrum in water at the following pH values:

_ / JLmax. & £

pH 1.0 pH 7.0 pH 11.0

256
277 NS 14.49
9.69 11
7th 150
456 254
273 NS ■ 16.18
11.21 12th
8th 45O
626 259
266 13.95
13.78 10
10 734
603

Reaction with enzymes

Crude alkaline phosphatase

Treatment with alkaline phosphatase gave clindamycin, which was determined by thin layer chromatography (silica gel, ethyl acetate-acetone-water [8: 5: 1, vol / vol]) was identified.

Animal venom diesterase

Treatment with venom diesterase yielded clindamycin and guanosine 5'-phosphate.

Effectiveness in vivo

Clindamycin-3- (5'-guanylate) is antibacterially ineffective against Sarcina lutea in vitro. In vivo , however, it shows an activity with a CD _{c /} ~ value of 26 mg / kg (SQ, Mäuae, S. aureus ).

tju - -

10984S / 1941

Example 2;

If the microorganism Streptomyces venezuelae NREL 3527t was used instead of the microorganism S. coelicolor Miller MRL 3532 used in Example 1, the clindamyein 3-nucleotides according to Example 1 were obtained.

Example 3:

Lincomycin was used instead of clindamycin in the fermentation medium in Example 1, lincomycin 3-nucleotide with the same nucleotide proportions as in example 1 received.

Example 4:

With l'-demethyl-clindamycin instead of clindamycin in the fermentation medium of Example 1 were l'-demethylclindamycin-3-nucleotides obtained with the same nucleotide proportions as in Example 1.

Example 5:

.With l'-demethyl-4'-depropyl-4'-pentyl-clindamycin instead of clindamycin in the fermentation medium of Example 1 were 1 · -demethyl-4'-depropyl-4'-pentyl-clindamycin-3-nucleotides with the same nucleotide proportions as in example 1 received.

109845/194

Example "-6 j.

With 4 ^f -depropyl-4'-ätb.yl-lineomycin instead of clindamycin in the lermentation medium of Example 1, 4'-deprapyl-4'-ethyl-lincomycin-3-nucleotides with the same kueleotide proportions as in Example 1 were obtained.

Example 7 t

With l'-demethyl-l'-ethyl-lincomycin instead of clindamycin 1'-Demethyll'-ethyl-.lincomycin-3-nucleotides were in the fermentation medium of Example 1 with the same nucleotide proportions as obtained in example 1.

Example 8t

With l'-demethyl-lincomycin instead of clindamycin in the fermentation medium of Example 1, l ^f -desmethyl-lincomycin-3-nucleotides with the same nucleotide proportions as in Example 1 were obtained.

Example 9t

With celesticetin instead of clindamycin in the fermentation medium of Example 1 were Celesticetin-3-nucleotLde obtained with the same nucleotide proportions as in Example 1.

109845/1

In the following examples, as in the previous examples, the nucleotide fractions of the compounds of the examples are the same as described in Example 1, namely gytidylate, adenylate, uridylate and guanylate.

Example 10;

Lincomycin-3-nucleotide ammonium salt.

Am lincomycin-3-nucleotide in zwitterionic form was dissolved in the smallest possible amount of water and diluted with the same amount of methanol. The solution was cooled in an ice-water bath, then saturated with ammonia gas and concentrated to dryness at 30 ° C. under a strong vacuum. The residue was dissolved in the smallest possible amount of methanol and diluted with five times the volume of ether, with the lincomycin-3-nucleotide precipitated as the ammonium salt.

Example 11;

Aqueous preparation to be administered orally in the form of drops.

One lincomycin-3 nucleotide 100 g

Propyl paraben 0.25 g

Methyl paraben 0.75 g

Sorbic acid 1.0 g

4 η aqueous sodium hydroxide in the for setting up pH 7.5 required amount

Deionized water to

fill up to 1000 ml

109845 / 19A 1

Example 12?

Syrup.

An aqueous preparation was made from the following ingredients for oral administration, which is 400 mg in each 5 ml Lincomycin-3-nucleotides contained, from the following components manufacturedj

Lincomycin-3-nucleotide 800 g

Methyl paraben, (U.S.P.) 7.5 g

Propyl paraben (U.S.P.) 2.5 g

Sorbic acid 10 g

Saccharin Sodium 6.5 g

Glycerin 3000 ml

Tragacant powder 100 g

Orange oil flavor 10 g

P.D. and C. orange dye 7.5 g

4N aqueous sodium hydroxide in the required amount to adjust to pH 7.5

Deionized water to make up to 10,000 ml

Instead of a lincomycin-3-nucleotide according to the example 11 and 12 can also be a 7 (S) -chloro-7-deoxylincomycin-3-nucleotide or water-soluble salts of a 7 (S) -chloro-7-deoxylincomycin-3-nucleotide, e.g. the alkali metal salts including the ammonium salt, used will.

109845/1941

The aqueous formulations of Examples 11 and 12 are particularly suitable as pediatric preparations and can be administered orally at the same dosage as lincomycin.

Example 13:

Capsules.

1000 double hard gelatin capsules for oral use, each 350 mg of a l'-demethyl-clindamycin-S-nucleotide were made from the following ingredients in the specified quantities:

a l'-demethyl ~ clindamycin-3-nucleotide 550 g

Corn starch 50 g

Talc 25 g

Magnesium stearate 2§5 g

The substrates were mixed thoroughly and then made into capsules in the usual manner.

These capsules are well suited for the systemic treatment of infections in adults by oral administration of 1 capsule every 4 hours.

According to the foregoing method, capsules with a corresponding l ^l -Demethyl-clindamycin-3-nucleotide in amounts of 50, 125, 250 and 500 mg prepared by reacting, instead of the 350 g of a l'above-demethyl-clindamycin

109845/1941

3-nucleotides of this nucleotide in amounts of 125 »250 and 500 g used.

Example 14t
Tablets.

1000 tablets for oral use with 500 mg each of s 1'-demethyl-4 · -depropyl-4'-pentyl-clindamycin-3-nucleotides were made from the following ingredients in the specified quantities:

a 1 · -demethyl-4 t-pyy

elindamyein-3-nucleotide 500 g

Lactose 50 g

Corn starch, 65 g

Magnesium stearate 3 g

Liquid (light) paraffin 3 g (light liquid petrolatum)

The ingredients were mixed well and granulated. The pellets were pressed through a sieve with a mesh size of 1.0 mm (number sixteen screen) to break them open. The granular material was then compressed into tablets with an active ingredient content of 500 mg each.

These tablets work well for the systemic treatment of infections in adults by all Hours a tablet is administered orally.

This procedure can be used when using

109845/1941

only 200 g active ingredient tablets with one active ingredient content of 200 mg.

Example 15:

Preparation for parenteral administration.

A sterile aqueous preparation for intramuscular administration with 300 mg of a Celesticetin-3-nucleotide per milliliter was made from the following ingredients in the specified quantities:

a Celesticitin-3-nucleotide 300 g Benzyl alcohol 9 g

Water for injection to make up to 1000 ml

The sterile material was dispersed in the sterile carrier liquid of benzyl alcohol and water and in Bottled vials that have been sealed.

Example 16:
Animal feed.

1000 g of a feed mixture were made from the following ingredients and in the specified quantities:

a 4'-depropyl-4 ^l -ethyl- 20 g

lincomycin-3 nucleotide

109845/1941

2 Ü8638

Soybean meal 390 g

Fish meal 4 bottles) g

Wheat germ oil 50 g

Sorghum molasses 140 g

These ingredients were mixed and pressed into tablets. You can test animals like had, Mice, guinea pigs or rabbits should be given as a preventive measure upon dispatch.

In larger animals, the preparation in for the Correct dosage of the active ingredient corresponding to the appropriate amount to be added to the regular feed.

Example 17;

Preparation for parenteral administration.

A sterile aqueous solution that can be used intramuscularly Preparation with 300 mg of a Linco: aycin-3-nucleotide in 1 milliliter was made up of the following ingredients and in produced in the specified quantities:

one lincotaycin-3-nucleotide 300 g

Benzyl alcohol 9 g

"Water for injection to make up

to 1000 ml

The sterile material was in the sterile carrier liquid from benzyl alcohol and water and distributed in

109845/1941

2118638

Filled vials that were sealed.

Example 18:

7-Deoxy-7 (S) -methoxy-lincomycin hydrochloride.

Part 18 A: Methyl-N-acetyl-7-deoxy-7 (S) -methoxy-a-thiolincosaminide.

CH

HN '.

HO / j

OH

OH

CH

■ • z

AeNH

OHe

HO /.
'\ 0H

SMe

OH

VI

VII

2.35 g of methyl 6,7-aziridino-6-desamino-7-deoxyethiolincosaminide (VI) was kept in suspension in 25 ml of methanol with stirring. The suspension was then with

109845 / 1.941

211863S

2.04 g of acetic anhydride are added. After stirring for one hour at room temperature, the solvent was ^{removed on a rotary evaporator at 40 °} C. and 7 mm pressure. The remaining solids were chromatographed on a 4.8 × 94 cm silica gel column with 1 MeOH: 10 CHCl * as the solvent system. The silica weighed 750 g. After 1000 ml forerun, 50 ml fractions were collected. Fractions 31-85 were combined and concentrated to dryness; They gave 3.2 g of methyl-N-acetyl-7 (S) -methoxy-7-deoxy-a-thioline cosaminide (VII) as a colorless amorphous pesticide with a molecular weight of 309 determined by mass spectrometry (calculated molecular weight 309.38).

The aziridino starting compounds of the formula VI were obtained by elimination of hydrogen halide from methyl ~ 7 (S) -chloro-7-deoxy-a-thiolinoosaminide (Belgian patent specification 705,427, U.S.A patent application 692,272 obtained. The elimination of hydrogen halide was carried out with anhydrous sodium carbonate by heating under reflux in dimethylformamide (Belgian patent 732 352, U.S. patent application 725 531.

Part 18 B: Methyl-7-deoxy-7 (S) -methoxy-a-thioline cosaminide (VIII)

(Methyl-O-S-dideoxy ^ -O-methyl-o-amino-1-thio-II-threo-a-D-galacto-octopyranoside).

109845/1941

2118638

OH

NH,

OMe

VIII

A solution of 3 »g of 2-methyl-7-deoxy-7 (S) -methoxya-thiolincosaminid (VII) in 25 g of hydrazine hydrate was heated overnight with stirring and under mild reflux in an oil bath at 145 ⁰ C. By removing the solvent as completely as possible from the colorless solution by distillation from an oil bath at 100 ° C./15 mm pressure and finally under strong vacuum, methyl 7-deoxy-7 (S) -methoxy-a-thiolincosaminide was obtained as a colorless syrup . This syrup was chromatographed on 750 g of silica gel in a column 4.8 × 97 cm with 1 MeOH: 10 CHCl as the solvent system. After 1.4 1 forerun, 50 ml dractions were collected. Fractions 281-600 were combined and concentrated to dryness. The yield was 2.06 g of methyl 7-deoxy-7 (S) -methoxy-a-thiolincosaminide (VIII), which was obtained after recrystallization from acetonitrile in the form of colorless needles with the following properties:

Melting point: 154-155 ° C;

1098A6 / 1941

£ ^a -7 _D + 260 ° (ο, 0.5634, H ₂ O).
Analysis:

For C ₁₀ H ₂₁ O ₅ HS

calculated: C, 44.92; H 7.92; N 5 * 24; S 12.00; OMe 11.61; found: 0 45.20; H 7.96; N 5.08; S 12.19; OMe 11.86.

Molecular weight: calculated 267.35;

found (mass spectrometry): 267.

Part 18 Ci 7-deoxy-7 (S) -methoxy-lincomycin-hydroehlorid.

Me

Pr

Il

Pr α n-propyl

CH,
j

HO

OMe

, 0H)! J V ÖMe

OH

.HCl

IX

A suspension of 2.7 g of trans-1 -methyl-4-propyl-L-2-pyrrolidine-carboxylic acid hydrochloride in 90 ml of acetonitrile, 2.89 g of triethylamine were added with stirring.

109845/1941

Stirring was continued until all of the solid had dissolved, then the reaction mixture was cooled to -5 ° C. in an ice / methanol bath and formed a precipitate of triethylamine hydrochloride. During the dropwise addition of 1.78 g of isobutyl chloroformate, the reaction temperature was kept at -5 ° to -3 G. More triethylamine hydrochloride settled out and stirring was continued at -5 ° C. for 20 minutes. The reaction mixture obtained was treated with 1.74 g of methyl 7-deoxy-7 (S) -methoxy-a-thiolincosaminide (VIII) dissolved in 10 ml of water. When the solids were dissolved, the temperature rose to about ⁰ ° C. stirring was continued for 2 hours without further icing of the cooling bath. After removal of the solvent on a rotary evaporator at 40 ° C./15 mm pressure, a brown, viscous residue remained, which was dissolved in dilute hydrochloric acid. This solution (pH 2) was extracted twice with chloroform; the combined extracts were washed once with water. The aqueous phase, which contained the washing water, was adjusted to pH 11 with sodium hydroxide (50% aqueous solution), saturated with sodium chloride and extracted three times with chloroform. The combined chloroform extracts were dried over anhydrous sodium sulfate and concentrated to dryness, 1.76 g of a dark yellow amorphous solid being obtained. The solid was chromatographed on 750 g of silica gel in a column 4.8 × 94 cm with 1 MeOH: 15 CHCl ₅ as the solvent system. After 1.3 1 forerun, 50 ml fractions were collected. Fractions 60-80 were combined and, after concentration to dryness, gave 7-deoxy-7 (S) -methoxy-lincomycin in the form of the free base as an almost colorless syrup. This free base was used in

109845/1941

added thin aqueous hydrochloric acid. Filtering and freeze-drying this solution gave 801.4 mg of 7-deoxy ^ iSj-methoxy-lincomycin hydrochloride obtained as a colorless amorphous solid with the following properties:

C α JQ + 117 ° (c, 0.9626, H ₃ O);

Analysis:

For C ₁₇ H ₂₆ O ₆ N ₂ S.HCl

Calculated: C, 49.93; H 8.16; N 6.13; S 7.02; found (5.14 # H ₃ O considered):

C 49.44; H 7.99; N 6.20; S 6.48.

Molecular weight

calculated for anhydrous free base: 420.57; found (mass spectrometry): 420.

The 7-deoxy-7 (S) -methoxy-lincomycin thus obtained can be converted into the corresponding new 3-nucleotides according to the invention leads over will.

Example 19:

Alternative process acid production of methyl-7-deoxy-7 (S) -methoxy-a-thiolineosaminide (VIII).

Part 19 A: Methyl-N-acetyl-6, 7-aziridino-6-desamino-7-deoxy-2,3 »4-tri-0-acetyl-a-thiolincosaminide (X).

109845/1941

CH,

Ac-

AeO / 0

fa *)

¹ Λ / SMe

I.
OAc

A solution of 2.0 g of methyl 6,7-aziridino-6-des amino-7-deoxy-a-thiolincosaminide (VI) in 20 ml of pyridine were added ver with stirring with 10 ml of acetic anhydride and the reaction mixture turned off over Naeht at room temperature. The volatile material - was from the Reaction mixture on a rotary evaporator at 40 C / 7 mm and finally as complete as possible under a strong vacuum removed, leaving a colorless solid. The residue was dissolved in chloroform for disposal of the pyridine stirred with aqueous cadmium chloride and filtered. The chloroform layer was washed with water twice and dried over anhydrous sodium sulfate. After removing the solvent on a rotary evaporator 3.1 g of methyl N-acetyl-6,7-aziridino-6-desamino-7-deoxy-2,3,4-tri-0-acetyl-X-thiolincosaminide were obtained at 40 ° C./7 mm (X) as a colorless crystalline solid. After recrystallization from ethyl acetate / techn. Hexane (Skelly solve B) the product became more colorless prismatic Needles received. Characteristics:

109845/1941

2113636

Melting point: 173t5 - 175 ° C

[ 'a_7 _D + 222 ° (c, 0.912, CHCl ₃ );

Analysis;

For C ₁₇ H ₂₅ OgNS

calculated: C, 50.61; H 6.25; N 3.47; S 7.95; Found: C, 50.43; H 6.33; N 3.41; S 8.31.

Molecular Weight:

calculated: 403 »45;

found (mass spectrometry): 403.

Part 19 B: Methyl-N-acetyl-2,3,4-tri-0-acetyl-7-deoxy-7 (S) -methoxy-α-thiolincosaminide (XI).

A mixture of 5 g of methyl-N-acetyl-2,3,4-0-triacetyl-6,7-aziri dino-o-desamino ^ -deoxy-a-thiolincosaminide (X), 50 ml of methanol and 5 ml of glacial acetic acid was 6 hours heated under moderate reflux in an oil bath at 130 ° C. Compartment removal of the solvent from the colorless Solution on a rotary evaporator at 40 C / 7 mm left a crystallizing, light yellow syrup. the Crystals were taken up in methylene chloride solution, washed first with saturated aqueous sodium bicarbonate and then with water and then over anhydrous Dried sodium sulfate. Removal of the solvent as indicated above gave 5.31 g Methyl-N-acetyl-2,3,4-tri-0-acetyl-7 (S) -methoxy-7-deoxy-a-thioline cosaminide (XI) as colorless crystals.

109845/194 1

Crystallization from ethyl acetate / hexane (Skellysolve B) gave fine, colorless needles with the following properties:

Melting point: 235-236 ° C;
faJ _J} + 20 ° (c, 0.9952, GHGl ₃ );

analysis

For C ₁₉ H ₂₉ O ₉ NS

calculated: C, 49.64; H 6.71; N 3.22; S 7.36; OMe 7.13; Found: C, 49.77; H 6.92; N 3.65; S 7.90; OMe 7.38.

Molecular weight

calculated: 435.49;

found: (mass spectrometry): 435

Hydrazinolysis according to the procedure of Part 18B gave methyl-7-deoxy-7 (S) -methoxy-α-thiolincosaminide (VIII).

Example 20:

Modification of example 18

Part 20 A: Methyl-N-acetyl-2,3,4-tri-0-acetyl-7-deoxy-7 (S) -methoxy-a-thiolincosaminide (XI) and methyl-K-acetyl-2,3-di-0-acetyl-7-deoxy-7 (Sj-methoxy-a-thilincosaminide (XII).

1098AB / 19 A 1

CH,

OH-

AcKH ¹

AcO

OHe

AcNH

OMe

SNe

SMe

OAc

OAc

XII

26.61 g of methyl-N-acetyl-7-deoxy-7 (S) -methoxy-ethioline cosarainide (VII) in 100 ml of pyridine were admixed with 50 ml of acetic anhydride with stirring and the reaction mixture was stored overnight at room temperature, then the volatile one Material removed by distillation on a rotary evaporator at 40 ^° C./7 mm and finally under strong vacuum * The residue was dissolved in chloroform and washed with saturated aqueous sodium bicarbonate. The aqueous layer was washed with chloroform. The pyridine was removed from the combined chloroform extracts by stirring with aqueous cadmium chloride.

The precipitate was filtered off and washed well with chloroform. The separated chloroform layer was washed twice with water and dried over anhydrous sodium sulfate and the solvent was ^{removed on a rotary evaporator at 40 0} g / 7 mm. A pale yellow syrup was obtained which crystallized on standing. After recrystallization

109845/1941

2118836

sation from ethyl acetate / hexane, the product was in the form of kielner, colorless, flattened needles obtained with the following properties;

Melting point 245-247 ° C;
£ "" J _D + 202 ° (c, 0.7142, CHCl 3);

analysis

For C ₁₈ H ₂₉ O ₉ JiS

calculated: C, 49.64; H 6.71; N 3.22; S 7.36; OMe 7.13; Found: C, 49.24; H 6.75; N 3.34; S 7.52; OMe 7.17.

Molecular weight

calculated: 435 * 49;

found (mass spectrometry): 435.

This material was purified by Craig countercurrent distribution with EtOH 1: 1 H ₃ O: 1 ÄtOAc: 1 cyclohexane as a solvent syst em, a content of 70 $ »Methyl-N-acetyl-2,3,4r-tri-0-acetyl- 7-deso3cy-7 (S) -methoxy-a-thiolincosaminide (XI) and 3O56 methyl-N-aeetyl ^^ - di-O-aeetyl ^ -deoxy-7 (S) -methoxy-a-thiolincosaminide (XII) . After 500 steps, the fractions from tubes 225-310 were combined (K value 1.14) and concentrated to dryness. After recrystallization from ethyl acetate / hexane, methyl-N-acetyl-2,3,4-tri-0-acetyl-7-deoxy-7 (S) -methoxy-athioline cosaminide (XI) was obtained in SOrm of fine colorless needles, which with the Product from Part 19B was identical.

109845/1941

The fractions of the tubes 115-220 (K value 0.59) are summarized and after evaporation resulted in Dryness and recrystallization from ethyl acetate / hexane hydrocarbon f en methyl-1-acetyl-2,3-di-Q-aeetyl-7-aesoxy-7 (S) - »itt9thoxy-a-thiolineosaminide (XII) in the form of colorless lumpy (chunky) ladein with the following properties:

Melting point; 189-19 ° C;

fcc J _D + 275 ° (c, 1.0188, GHCl ₃ );

analysis

For C ₁₆ H ₂₇ O ₈ NS

"Calculated: C 48.84; H 6.92; N 3.56; S 8.15; OMe 7.89; found s C 48.71; H 7.11; N 3.93; 3 7.96; OMe 7.98.

Molecular weight

calculated 393 * 46;

found (mass spectrometry) s 393-

Part 20 B: Acetylation of methyl-B-acetyl-2,3 ~ di-0-

acethyl-7-deoxy-7 (S) -methoxy-a-thiolinco sami nid (XII).

A solution of 200 mg of methyl-H-aeetyl-2,3-di-O-acetyl-7-deoxy-7 (S) -metlioxy'-a-thiolincosaminide (XII) in 20 ml of pyridine was added 10 ml of acetic anhydride with stirring added and the reaction product overnight

10S845 / i 941

Turned off at room temperature. The solvent was removed from the colorless solution on a rotary evaporator at 40 ° C / 7 mm and finally at 40 ° C / high vacuum. The syrupy residue was dissolved in chloroform, washed with dilute aqueous HCl (0.5 normal), twice with water, with saturated sodium carbonate solution and again twice with water and dried over anhydrous sodium sulfate. After removing the solvent on a rotary evaporator at 40 ° C / 7 am, methyl-N-acetyl-2,3 _f 4-tri-0-acetyl-7-desoiy-7 (S) -methoxy-a-thiolincosaminide (XI) obtained as a colorless syrup which crystallized on standing.

After hydrazinolysis of the products from Part 20 A and 20 B, methyl 7-deoxy-7 (S) -methoxy-a-thioline cosaminide was obtained (VIII).

Example 21:

Alternative to example 18.

Part al A: Ethyl-H-acetyl-6,7-aziridino-6-desamino-7-deoxy-tt-thiolineosaminide (XIII).

XIII

109845/194

A suspension of 2.3 g of methyl 6,7-aziridino-6-desamino-7-deoxy-a-thiolincosaminide (Vl) in 25 ml of isopropyl alcohol was added while stirring with 2.04 g of acetic anhydride offset. Most of the solid appeared to go into solution for replacement with new solid. The reaction mixture was left overnight at room temperature geriikrt and then filtered; the backlog was with Isopropyl alcohol and washed in a vacuum oven at 60 C / 15 m dried. 2.28 g of methyl N-acetyl-6,7-aziridino-ö-desamino-o-desamino ^ -deoxy-a-thiolincosaminide were obtained as colorless platelets with the following properties:

Scisaelz point: 145 ⁰ C;

C α J _D + 255 ° (c, 0.7916, H ₂ O);

analysis

for C ₁₁ H ₁₉ O ₅ NS

calculated: C, 47.63; H 6.9; N 5.05; S 11.56; found s 0 47.57; H 6.71; N 5.23; S 11.29.

Molecular weight

calculated: 277.34;

found (mass spectrometry): 277.

Part 21 B: Methyl-N-acetyl-.7-deoxy-7 (S) -methoxy-a-thioline cosaminide (VII).

Mach treatment of methyl-N-aeetyl-6, 7-aziridino-6-

109845/1941

desamino-7-deoxy-a-thiolincosaminide (XIII) with methanol and acetic acid under reflux, methyl-B-acetyl-7-deoxy-7 (S) -methoxy-urethiolincosaminide was obtained (VII), which was identical to the product in Part 18 A.

Example 22:

7-deoxy-7 (S) -ethoxy-lincomycin-hydrochlori d.

Part 22 A: Methyl-N-acetyl-7-deoxy-7 (S) -ethoiy-a-thiolincosaminide (XIV).

AcNH

OÄt

ho / r- °

KpH / \ L__j /

SMe ™

OH

Treatment of the methyl-N-acetyl-6,7-aziridino-6-desamino-7-deoxy-a-thiolincosaminide (XIII) under moderate reflux with ethanol and acetic acid gave methyl-N-acetyl-7-deoxy-7 (S) -ethoxy-1-thio-α-lincosaminide. (XIV) as a syrup with the molecular weight determined by mass spectrometry 323 and the calculated mol, weight 323 _t 41- ·

109846/1941

Part 22 B:

MetJh.yl-ii-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7 (s) -ethoxy-a-thiolincosaniinide (XV) and

Methyl-N-acetyl - ^ - desöxy- ?! S) -ethoxy-2, 3-dio-aeetyl-a-thiolineosaminide (XVI).

CH,

AcNH

OÄt

AcM

OAt

HO

- Q

/ SMe

OAc

OAc

Was methyl-I \ r-acetyl-7-deoxy-7 (S) -ethoxy-a-thioline-kosaaiinide treated according to the procedure in Part 20 A
(XIV) with acetic anhydride and pyridine were obtained
Methyl -N-acetyl-2,3,4-tri-0-acetyl-7-deoxy-7 (S) -Ö-ethyla-thiolincosaminide (XV) and a smaller amount of H-acetyl-2,3-di-0 -acetyl-7-aesoxy-7 (S J-ethoxy-a-thiolincosiaeiiinid
(XVI), The products isolated with a Craig device in 500 steps with a solvent system of ethanol / Waaaer / ethyl acetate / cyclohexane (ratio 1: 1: 1: 1) have the following properties:

Mixture: melting point 197-199 ° C.

109845/1941

faJj ₃ + 247 ° (c, 0.665, CHCl ₅ );
analysis

For C ₁₉ H ₃₁ O ₉ HS

Calculated: C, 50.76; H 6.95; IT 3.12; S 7.13; (Et 10.02; Found: C, 50.42; H 7.07; N 3.18; S 7.37; OÄt 11.85.

XV neat (E = 1.59); Melting point 254-255 ° C; £ aJ _B + 199 ° (c, 8638, CHCl ₃ ).

analysis

For C ₁₉ H ₃₁ O ₉ SS

Calculated: C, 50.76; H 6.95; H 3.12; S 7.13; Oat 10.02; found: C, 50.75; H 7.06; N 3.37; S 7.31; OÄt 10.25.

Molecular weight

calculated: 449.52;
found (mass sp.) ϊ 449;

pure (K = 0.87) | Melting point 215.5-216.5 ° C;

²⁶¹ * ( ^c »1.0448, CHCl ₃ )

analysis
Pure

109845/1941

Calculated: C, 50.11; H 7.17; N 5.44; S 7.87; Found: C, 50.17; H 7.30; Ή 3.50; S 7.62.

Molecular weight

calculated: 407.48;
found (mass col.): 407.

Part 22 C: Methyl 7-deoxy-7 (S) -ethoxy-a-thiolincosaminide (XVII).

OÄt

SMe

XVII

- Subjected the products of Part 22 B, i.e. the mixture, the pure compound XV and the pure compound XVI hydrazinolysis gave methyl-7-deoxy-7 (S) -ethoxya-thiolincosaminide (XVII) having the following characteristics:

Melting point: 194-196 ° C;

+ 252 ° (c, 0.7438,

1 09845/1941

L ! I ο ο 3 ο

analysis

calculated: C 46.95; H 8.24; If 4.98; S 11.40; found: C, 46.66; H 8.09; N 5.26; S 11.33.

Molecular weight calculated: 281.37; found (mass col.): 281.

Part 22 D:

7-deoxy-7 (S) -ethoxy-lineomycin hydrochloride (XVIII).

Me

Pr

Me = methyl Ät = ethyl Pr = propyl

OÄt

Y \

I OH Λ

SMe

OH

XVIII

.HCl

Following the procedure in Part 18 G, methyl-7-deoxy-

109845/1941

211863S

7 (S) -ethoxy-a-thiolincosaminide (XVII) in 7-deoxy-7 (S) -ethoxy-lincomycin hydrochloride, "who has the following properties:

Melting point: colorless, amorphous pesticide;

/ ~ <x_7 _D + 109 ° (e, 0.9824, H ₂ O) -T

analysis

Neat C ₂₀ H ₅₈ O ₆ F ₂ S ^ HCl

calculated: C 50.99; H 8.35; F 5.95;

Cl 7.53; S 6.81; Oat 9.57;

found (5 * 07 $ water taken into account):

C 50.54; H 8.19; »5.63; Cl 7.61; ö 6.95; Oat 10.16.

Example 23:

Part 23 A: Methyl-N-acetyl-7-deoxy-7 (S) -propoxy-ethiolincosaminide (XIX), methyl-M-aeefyl-2,3,4-tri-0-ac ethyl-7-deoxy-7 (S) -propoxy-ccthiolincosaminide (XK) and. Meth.yl-U-aeetyl-7-deoxy-7 (S) -propoxy-2,3-di-0-acetyl-ethiolincosaminide (XXI).

109845/1941

OPr

HD A \

\ OH 1 ^X j]

OH

AcNH

AcO /

OPr

OAC)

SMe OAc

XIX

XX

AcBH

OPr

HO Ί — O

NOAe β

OAc

XXI

Bach treatment of methyl-N-acetyl-ö.T-aziridino-odesamino-7-deoxy-α-thi olincosajninid (XIII) with propanol and acetic acid under moderate reflux was methyl-N-acetyl-7-

109845/1941

desoxy-7 (S) -propoxy-oc-thiolincosaminide and from it after Acetylate with acetic anhydride in pyridine according to the procedure in Part 22 B Methyl -N-acetyl-2,3,4-tri-O-acetyl-7 (S) -propoxy-7-deoxy-QC-thiolincosaminide (XX) which contains a small amount of methyl N-acetyl-2,3-di-0-acetyl-7 (S) -propoxy-7-deoxy-a-thiolincosaminide (XXI) was obtained.

Characteristics:

Mixture: melting point 240-242 ⁰ C;
ZTaT ₁ ) + 207 ° (c, 0.9054, CHCl3);

analysis
For C

Calculated: C, 51.81; H 7.17; N 3.03; S 6.92; found: C, 51.41; H 7.33; N 3.16; S 6.92.

Molecular weight

calculated: 463 »60;

found (mass spectrometry): 463;

XX pure:
Melting point 241.5-242.5 ° C;

[_ cc J _D -1-193 ° (c, 09254, CHCl ₃ )

analysis

For C ₂₀ H ₃₃ O ₉ NS

109845/1941

calculated: 0.51.81; H 7.17; N 3.03; S 6.92; found: C, 51.77; H 7.02; N 3.37; S 6.84.

Molecular weight

calculated: 463.60;

found (mass spectrometry): 463.

Part 23 B: Methyl 7-deoxy-7 (S) -propoxy-a-thiolincosaminide (XXII).

CH

OPr

SMe

XXII

Upon hydrazinolysis of the aforementioned products (Part 23 A), methyl 7-deoxy-7 (S) -propoxy-a-t-molincosaminide was obtained (XXII) obtained.

Part 23 C: 7-Deoxy-7 (S) -propoxy-lincomycin hydrochloride (XXIII).

109846 /

Il

OPr

-NH

i
OH

XXIII

After acylation with trans-1-itüethyl-4-propyl-L-2-pyrrolidinecarboxylic acid according to the procedure in Part 18 C, T-Deoxy-TiSj-propoxy-linetHaycin-hydrocüLorid (XXIII) obtain.

Example 24;

Part 24 Δ:

7 (S) -isopropoxy-a-t-molincosaminide (XUV)

109845/1941

OH,

AcNH

0 (iPr)

AcO

XXIV

Isopropyl alcohol was used in the procedure of Part 19B instead of methanol, methyl-N-aeetyl-2,3 was obtained , A-tri-O-aeetyl-T-deoxy-T (S) -i s op rop oxy-ct-thi olincosaainid (2X1V) has the following characteristics:

Melting point: 253-254 C; C ^ JQ +! 32 ° < ^c »0.535,

analysis

For C

20 ^H 33 ⁰ 9 ^HS

Calculated: C, 51.81; H 7.17; N 3.03; S 6.92; found: C, 51.96; H 7.07; N 3.19; S 6.61.

109845/1941

Molecular weight

calculated: 463.6;

found (mass spectrometry): 463.

Part 24 B: Methyl-7-deoxy-7- (S) -isopropoxy-a-thiolineosaminici (XXV).

O (iPr)

jjp _r _

SMe

XKV

After hydrazinolysis of compound XXIV (Part 24 A), methyl-7-deoxy-7 (S) -isopropoxy-a-thicün cosaminide was obtained with the following properties:

Melting point: 215 ° G;

+ 225 ° (c, 0.376,

analysis

109845/1941

-Tl-

calculated: G 48.79; H 8.53; N 4.74; S 10.86; found: G 48.52; H 8.55; H 5.26; S 10.84.

Mol ekul argewi rushes

calculated: 295.40;

found (mass spectrcanetry): 295

Part 24 C: 7-Deoxy-7 (S) -isopropoxy-lincomycin hydrochloride (XXVI).

HO

OH

"0

O (iPr)

.HGl

SMe

OH

XXVI

Following the procedure in Part 18 C, the connection was made XXVI (Part 24 G) converted into 7-deoxy-7 (S) -isopropoxy-lincomycin hydrochloride with the following properties:

1 098487T941

Melting point - amorphous;

/ ~ a7 _D + 81 ° (0.898, H ₂ O);

analysis

Me C ₂₁ H ₄₀ O ₆ N ₂ S-HCl

calculated: C, 51.99; H 8.52; N 5.78; S 6.61; Gl 7.31; Found: (4.36 % water included): C 51.72; H 8.33; IT 5.59; S 6.35; Cl 7.29.

Molecular weight

calculated (free base): 448.62;

found: 448.

Efficacy: about the same as lincomycin.

Example 25 A: Methyl-N-acetyl ^^^ - ti-i-O-aeetyl - ^ - deoxy-

7 (S) -cyclohexyloxy-a-tluLolineosaininid (XXVII)

- O-cyclohexyl

AcNH j

AcO } - °.

KOAc ^v

'M ι. me XXVII

OAc

1098A5 / 1941

The use of cyclohexanol in place of methanol in the procedure in Part 19B gave methyl N-acetyl-2,3,4-tri-O-aeetyl-7-deoxy-7 (S) -cyclohexyloxy-ethioline cosaminide (XXVII) with the following properties:

Melting point: 266-268 ° C;
°

^α - ^ + 163 (c, 1.055, GHCl ₃ ).

analysis

PUr G ₂₅ H ₃₇ O ₉ NS

calculated: C, 54.85; H 7.41; N 2.78; S 6.37; Found: C, 54.93; H 7.53; N 2.87; S 6.65.

Molecular weight

calculated; 503 »6;

found: (feeding spectrometry): 503.

Part 25 B: * iethyl-7 (S) -cyelohexyloxy-7-deoxy-α-1hi olincosaminide (XXVIII).

Bra ₂
HO

O cyclohexyl

(O"

XXVIII

} SMe i

OH

109845 / 19A1

After hydrazinolysis of the compound XXVII (Part 25 A) was methyl-TiSi-cyclohexyloxy-T-deoxy-a-thiolincosaminide (XXVIII) obtained.

Part 25 G: 7 (S) -cyclohexyl-7-deoxy-lincomycin hydrochloride.

CH

NH

- O-cyclohexyl

.HGl

- SMe

• Following the procedure in Part 19 G, methyl-7 (ö) -cyclohexyloxy-7-deoxy-a-thiolincosaininide is obtained (XXVIII) in 7 (S) -Gy el ohexyl oxy-7-deoxy-l incomycin hydro ohl orid transferred.

Example 26;

Part 26 A: Methyl-N-acetyl-7-deoxy-7 (S) -2'-hydroxyethoxy-a-thiolincosaminide (XXIX) and methyl-N-acetyl-2,3,4-tri-0-acetyl-7 (S) -2'-acetoxyethoxy-7-deoxy-a ~ thiolincosaminide (XXX).

109845/1941

211863S

-SI

CH,

HO

OCH ₂ CH ₂ OH

AcNH

K OB. λ

and

OCH ₂ CH ₂ OAc

SMe

OH

XXIX

XXX

According to your procedure in Part 18 A, when 2-hydroxyethanol was used instead of methanol, methyl N-acetyl-7-deoxy -? (S) -2'-hydroxyethoxy-a-thiolincosaminide (XXIX) was obtained. This was acylated according to the procedure in Part 20 A, except that it was heated on the steam bath
used to obtain a fully acylated product. Methyl-N-acetyl-2,5,4-tri-0-acetyl-7 was obtained
(S) -2'-aceoxyethoxy-7-deoxy-a ~ thiolincoseminide.

Properties: Melting point: 223-225 ° C;

f (xJ _D + 172 ° (c, 1.010, C analysis
For Ο «,

109845/1941

calculated: G 49.69; H 6.55; N 2.76; S 6.32; found ί G 49.56; H 6.63; M 2.90; S 6.63-

Molecular weight
calculated: 507.55 j
found: 507.

Part 26 B: Methyl-7-deoxy-7 (S) -2'-hyaroxyethoxy-a-thioline cosaminide (XXXI).

GH,

OCH ₂ CH ₂ OH

NH,

HO /

XXXI

SMe

OH

After hydrazinolysis of Wethyl-li-acetyl-2,3,4-tri-0-acetyl-7-deoxy-7 (S. ) -2 '-acetox.yäthoxy-α-thiolincosaminide (XXXI) one obtained methyl-7-deoxy-7 (s) -2'-hydroxyethoxyoc-thiolincosaminide with the following properties:

Melting point: 178.5-179.5 ° C;

109845/1941

2118638

£ <xJ _D + 245 ° (c, 0.662, H ₂ O).

analysis

Mir C ₁₁ H ₂₅ O ₆ NS

calculated: C, 44.45; η 7.8μ; S 10.78; N 4.71; found: C, 44.40; H 7.99; S 10, bl; N 4.60.

Molecular Weight: 297.37;

found (mass spectrometry): 297.

Part 26 C: Mexhyl-7-aesoxy-7 (S) -2'-hydroxyethoxylincomycin hydrochloride (XXXIl).

Me

, N CH,

\ Pr. , / 1 OCH ₂ CH ₂ OH

C NH

Q HO \ 0 .HCl

\ ] avie

OH
XXXII

Following the procedure in Part 18 C, methyl 7-deoxy-7 (fcj) -2'-hydroxyethyxy-a-thiolincosaminide is obtained (XXXI) in 7-Des-

1 09845/1941

oxy-7 (S) -2 ⁱ -hydroxyäthox.y-lincomycin-hydrochloride with the following properties:

Melting point: amorphous;

jTaJ + 105 ° (c, 1.102, H ₀ O).

D ^ά

analysis

For C ₂₀ H ₅₈ O ₇ N ₂ S * HGl

Calculated: C, 49.32; H 8.07; N 5.75; S 6.58; Cl 7.28; found ($ 2.11 water included):

C 49.61; H 7.85; N 5.54; S 6.46; Cl 7.26.

Molecular weight

calculated (free base): 450.59; found (mass spectrometry): 450.

Efficacy: about 1/3 of the lincomycin.

Example 27:

Part 27 A: Methyl-N-aeetyl-7-deoxy-7 (S) -2'-methoxyethoxyoc-thiolincosaminide (XXXIII) and methyl-N-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7 (S) -2'-methoxy ethoxy-oc-thiolincosaminide (XXXIV)

109845/194

CH ₃ CH ₃

' _{0CH CH me} -J OCH ₂ CH ₂ OMe

1 -TLTTT j

AcNH -

^WÄ ~ \

U .. I / SMe OMe

XXXIII XXXIV

If, in the process of Part 18A, 2-methoxyethanol was used instead of methanol, methyl-N-acetyl-7-deoxy-7 (S) -2'-methoxyethoxy-a-thioline cosimide was obtained (XXXIII). This product was acetylated using the procedure in Part 20-A except that heat was applied the steam bath to recover a fully acetylated product. Methyl N-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7 (S) -2'-methoxyethoxy-a-thiolincosaminide was obtained (XXXIV).

Characteristics:

Melting point: 222-223 ° 0;
£ a J _B + 177 ° (c, 1.079, CHCl ₃ ).
analysis

"D (I · ψ% / * t XT f \ 10Ό

109845/1941

2118638

Calculated: C, 50.09; H 6.94; N 2.92; S 6.69; OMe 6.47; Found: C, 50.13; H 7.00; N 2.77; S 6.33; OMe 7.28.

Molecular weight

calculated: 479 »54;

found (mass spectrometry): 479.

Part 27 B: Methyl 7-deoxy-7 (S) -2'-methoxyethyl-a-thiolincosaminide (XXXV).

CH

OCH ₂ CH ₂ OMe

HO, / \,

Γ OH 1

«/ SMe

OH

After hydrazinolysis of methyl-M-acetyl-2,3,4-tri-O-acetyl-7-deoxy-7 (S) -2 '~ methoxyethoxy-a-thioline-GOsaminide (XXXIV), methyl-7-deoxy-7 was obtained (S) -2 ^t -methoxyäthoxya-thiolincosaminid (XXXV) with the following properties:

Melting point: 178-179 ° C;

L <* 7 _D + 231 ° (c, 0.827, H ₂ O)

109845/1941

analysis

Pure G ₁₂ H ₂₅ O ₆ NS

calculated: C, 46.28; H 8.09; N 4.50; S 10.30; found: G 46.57; H 8.32; N 5.01; S 10.70.

Molecular weight

calculated: 3H »40;

found (mass spectrometry): 311.

Part 27 C: kethyl-7-deoxy-7 (S) -2'-methoxyethoxy-lin comycin hydrochloride (XXXVI).

Me

Pr!

"-I OGH ₂ CH ₂ OMe

.HCl

C MH

ο ^Η °,

OH

-0

i ^me χχχνί

OH

Following the procedure in Part 18 C, methyl 7-deoxy-7 (b) -2'-methoxyethoxy-a-thiolincosaminide was obtained (XXXV) in

7-deoxy-7 (ö) -2 ^f- methoxyethoxy-lincomycin hydrochloride transferred.

109845/1941

2118838

Characteristics:

Melting point - amorphous; / ~ a7 _D + 87 ° (c, 0.575

analysis

For C ₂₁ H ₄₀ U ₇ N ₂ S

calculated: C, 50.53; H 8.25; N 5.59; S 6.40; Cl 7.08; found (4.17 # H ₃ O considered):

C 50.47; H 8.60; N 5.26; S 5.86; Cl 7.50.

Molecular weight

calculated (free base): 464.62; found (mass spectrometry): 464.

Efficacy: about 1/3 that of lincomycin.

Example 28:

Methyl-7-deoxy-7 (Sj-hydroxy-a-thiolincosaminide (XXXVII) (Methyl-ö-amino-ojS-dideoxy-L-threo-a-D-galactoocto pyranoside).

CH ₃

OH

HO, / 'Λ »XXXVII

OH / I

I SlVIe

OH

109845 / 19A1

Part 28 A:

Methyl N-acetyl-7-deoxy-7 (3) -hydroxy-athioline cosaminide (XXXVIII)

(Methyl-o-acetamido-δ ^ -dideoxy-L-threo-a-D-galacto-octopyranoside).

CH ₃

OH

AoNH

^H 9 / ~~ \ OH

XXXVIII

t SMe OH

A solution of 2.35 g of methyl 6,7-aziridino-6-desamino-7-deoxy-oc-thiolincosaminide (VI) in 25 ml of water, 2.04 g of acetic anhydride were added and the solution Stored overnight at room temperature. After concentrating the solution to dryness on a rotary evaporator 40 ° G / 7 mm, a colorless syrup was obtained, which was placed on 750 g of silica gel in a 4.8 χ 98 cm column with 1 MeOH: 7 CHCl-z as the solvent system chromatographed. After 550 ml forerun, 50 ml fractions were collected. Fractions No. 90-160 were combined and taken to dryness constricted. The yield was 2.3 g of methyl N-acetyl-7-deoxy-7 (s) -hydroxy-a-thiolincosaminide as a colorless pest, which is made from methanol in the form of colorless rods crystallized.

10984 5 / ΊΠ1

2118R36

Characteristics:

Melting point: 21b - 219 ⁰ C;
Γ * ■ 7-η + 260 ° (e, 1.0296,

analysis

For C ₁₁ Hp ₁ OgNo

calculated: C, 44.7'5; H 7.17; N 4.74; S 10.86; Found: C, 44.89; H 7.02; M 5.16; S 10.64.

Molecular weight

calculated: 295 »36;

found (ivias spectrometry): 295.

Part 28 B: Deacetylation.

The crystallized material from Part 28 A was combined with the mother liquors and added to the rotary evaporator 40 ° C / 7 mm narrowed to the Trocicne. 2.01 g of solid were obtained, which was stirred and refluxed overnight 40 ml of hydrazine hydrate was heated. After removing the solvent from the colorless solution on a rotary evaporator at 7 mm pressure and at 120 ° C. in an oil bath, a colorless, crystalline residue remained, which was recrystallized from methanol became. Methyl 7-deoxy-7 (S) -hydroxy-oc-thiolineosaminide is obtained (XXXVII) as colorless platelets with the following properties:

Melting point: 211-212 ° C;

109845/1941

Z "a_7 _D + 280 ° (c, 0.7728, HgO).

analysis

For CqH4q0p-NS

calculated: G 42.67; H 7.56; H 5.53; S 12.66; found: C, 42.81; H 7.69; N 5.85; S 12.73.

Molecular weight

calculated: 253.32;

found (hate spectrometry): 253

Example 29:

Methyl -H-acetyl-2,3 ^ -tri-ü-acetyl- ^ Sj-ethoxy - ^ - deoxycc-thiolincosaminide (XV) and

Methyl-N-acetyl-2 »3,4-tri-0-acetyl-7 (S) -acetoxy-7-deoxy-thiolincosaiainide (XXXIX).

CH, CH,

OÄt

OAc

AcNH —I and ^AcNH !

AcO ₁ '■ ° AcO J °

¹ 1 öf-ie JJ SMe

OAc OAc

XV. XXXIX

109845 / 19A1

If, in the process of Part 19 B, ethanol was used instead of methanol, methyl-N-acetyl-2,3,4-tri-0-acetyl-7 (S) -ethoxy-7-deoxy-a-thiolincosaminide (XV ), [ identical to the product in Part 22 Bj, and in a smaller amount N-acetyl-2,3 »4-tri-0-aoetyl-7 (S) -acetoxy-7-deoxy-oc-thiolincosaminide (XXXIX), that through counter-current distribution. according to Craig in 500 steps with the solvent system 1 ÄtOH: 1 EUO: 1 ÄtOAc: 1.5 cyclohexane was separated. The smaller component (XXXIX) was obtained from tubes 14U - 200 (K = 0.52) and the larger component (XV) from tubes No. 260-330 (K = 1.43). Crystallization of the smaller component (XXXIX) from ethyl acetate gave colorless needles with the following properties:

Melting point; 312-313 ° C.
Z> 7 _D + 182 ° (c, 0.5898, CHGl ₃ ).

analysis

For C ₁₉ H ₂₉ O ₁₀ NS

calculated: C, 49.22; H 6.31; N 3.02; S 6.92; Found: C, 49.17; H 6.51; N 3.08; S 6.81.

Molecular weight

calculated: 463.50;

found (mass spectrometry): 463.

After hydrazinolysis of the smaller constituent, methyl-7-deoxy-7 (S) -hydroxy-a-thiolincosaminide identical to the product of Part 28 B was obtained (XXXVII).

109845/1941

Example 30:

Part 30 A: 2'-hydroxyethyl-N-acetyl-2 ', 2,3,4-tetra-O-

acetyl-7-0-methyl-l-thio ~ <x - lineosaminide (XL)

CH ^ O
AcMH

AcO

OAo

OAc

I / S-CH ₂ -CH ₂ -O-Ac

1 »0 g of 2'-hydroxyethyl-1-thio-a-celestosaminide (example 3 eier U.S. Patent 3,255,174) was in 25 ml

Pyridine and 12 ml of acetic anhydride dissolved and stored overnight. Removal of the solvent in vacuo gave a colorless oil which was dissolved in chloroform and washed with water, dilute aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate and water. The mixture was dried over anhydrous sodium sulfate and the solvent was removed in vacuo. 2 _f O3 g of syrup were obtained, which after crystallization from ethyl acetate / techn. Hexane (Skellysolve B) 2 ¹ -

Hydroxyethyl-N-acetyl-2, 2,3,4-tetra-O-acetyl-7-O-methyl-. 1-thio-a-lincosaminide (Formula XL) in the form of flattened, colorless prisms.

ORJGiNAL INSPECTED

109845/1941

Melting point: 143 -

analysis

- 94 -

144 ° C;

For ^G 2i ^H 33 ° ll ^NS

calculated 0 49.68; H 6.54; N 2.76; 8 6.32 ^ found: 0 49.66; H 6.50; N 2.91; S $ 6.34

+ 216 ° (c, 0.7746, CHCl3).

Part 30 B: Methyl-N-acetyl-2, 3, A-

1-thio-a-una-β-lincosaminide, (XLI and XLII).

CH ₃ O
AcHH

AcO

and

OAc

' NS

OAc

CH ₃ O-

AcNH-

AcO h -O

NS,

OAc

XLI

XLII

A solution of 10 g of 2 ^l -hydroxyethyl-N-acetyl-2 ^l , 2,3,4-tetra-O-acetyl-l-thio-a-celestosaminide (prepared according to the method of Part 30 A) in 200 ml of chloroform

109845/1941

ORIGINAL INSPECTED

a solution of 5.05 g (1.62 ml) of bromine in 100 ml of chloroform was added within about 30 minutes from a pressure equalization dropping funnel with stirring and under anhydrous conditions. At first the bromine color disappeared immediately, later a deep orange-red hue developed. After further half an hour's stirring at room temperature the solvent on a rotary evaporator at 40 ⁰ C / 7 mm was removed, a yellow-orange, syrupy residue was obtained. This was redissolved in chloroform, the solvent was removed in vacuo and the process was repeated as long as the distillate became colorless and a yellowish amorphous residue of 1-bromo-TO-methyl-β-lincosamine tetra-acetate of the formula

CH.

CH, 0 -
AcNH -

AcO

0.

XT ¹ *

OAc

OAc

was obtained.

The residue was dissolved in 200 ml of dry dimethyl formamide, mixed with 4.5 g of thiourea and the reaction mixture (colorless solution) was stirred overnight at room temperature. The isothio uronium salts were formed of the formulas:

c- ^ CmU 0 9 8 4 5/1 9 A 1 ORIGINAL INSPECTED

CH,

CH, 0

and

AcNH

AcOT

-0

NOAc

OAc

OAc

XLIV

XLV

Without isolation of the salts and after cooling in an ice bath, 100 ml of water and then 8.3 g were slowly added anhydrous potassium carbonate, 10.6 g sodium bisulfate and 28 g (12.3 ml) of methyl iodide were added. The mixture became three Vigorously stirred with a magnetic stirrer for hours, the cooling bath was removed after 20 minutes.

Volatile substances were removed in vacuo at 40 ^° C. and finally at 80 ^° C. / <1 mm. The yellow residue was dissolved in a chloroform / water mixture, the aqueous layer was extracted with chloroform and the combined chloroform extracts were washed twice with water and dried over anhydrous sodium sulfate. After removal of the solvent in vacuo, a colorless amorphous residue remained (6.48 G). Thin-layer chromatography (acetone / hexane hydrocarbons 1: 1) showed a larger zone of the product with a small zone with a slightly higher R ^

109845/1941

This material was in an acetone / hexane hydrocarbon system (1: 1.5) on 1.2 kg silica gel (column size 5.8 × 90 cm) chromatograph. After 500 ecm forerun 50 cc fractions were automatically collected and after the substances had eluted, IXinns cht chromatography was performed carried out. Fractions No. 145 - 173 including, corresponded to the material with a higher R ^ value, Fractions No. 185-310 inclusive corresponded to the main product, Fractions No. 174-184 were a mixture from both.

After removing the solvent from the combined fractions 145-173 in vacuo, 570 mg remained of a colorless syrup which, after crystallization from ethyl acetate / hexane hydrocarbons, methyl-N-acetyl-2,3,4-tri-0-acetyl-7-0-methyl-1-thio-a-lincosaminide in the form of small, colorless prisms. Melting point 212-213 ° C, which is when mixed with a Sample according to Example 31 (Part 31 G) with a melting point of 211.5-213 ° C not lowered, not even different from it by infrared, NMR and mass spectrum, nor by optical rotation.

After removing the solvent in vacuo from the combined fractions _{185-310, 4 f} 23 g of a pale yellow, amorphous solid were obtained which, after crystallization, methyl-N-acetyl ^^^ - tri-O-acetyl ^ -O-methyl-1- thio-ß-lincosaminid formed in the form of colorless prisms of melting point 187-188 ⁰ C.

analysis

109845/1941

calculated: G 49.64; H 6.71; Ii 3.22; S 7.36; MeO 7.15; found ι G 49.73; H 6.95; N 3.18; S 7.64; MeO 7.41.

f OiJ-Q + 24 ° (c, 0.7484, CHGl ₃ );

Molecular weight

calculated: 435.49;

found! (Mass spectrometry, M):

435.

The total yield of introducing the SMe group (i.e. α- and ß-anomers) was $ 49.2 (6.7 $ α, 42.5 $ B), the ratio of α to β being 1: 6.35. The β-anomer can be fed back into the process of part 30B and thus the overall yield of the preferred a-anomer can be increased.

Part 30 Cs

In repetition of the procedure of part 30 B, hexamethylphosphoric triamide £ (Me ₂ K ") J? = 0_7 was used instead of methyl formamide. The total yield was 65» 5 #, of which 22.7 # α and 42.8 <$> Q , which corresponded to a ratio a / ß of 1: 1.9.

Part 30 D-I: Methyl 7-0-methyl-1-thio-a-lincosaminide (XLVI).

R-O

NH

2
HO

CH,

3H

S-alkyl

OH

109845/1941

XLVI

ORIGINAL INSPECTED

1.46 g of ethyl-7-U-methyl-1-thio-a-lincosaminide tetraacetate were dissolved in 50 ml of hydrazine hydrate and heated under moderate reflux at 15 g in an oil bath for 24 hours. Bas volatile solvents T "; then urde as far as possible by distillation at HO ⁰ C / 15 mm removed to give a colorless crystalline residue, which was triturated with anhydrous acetonitrile. The solid is filtered off and dried. After crystallization from a concentrate of 95 # ethanol, 430 mg of methyl-TO-methyl-1-thio-a-lincosaminide hemihydrate (Polymorph I) were obtained in the form of flattened, colorless needles, melting point 126-126.5 ° C.

analysis

For C ₁₀ H ₂₁ O ₆ HS.1 / 2 H ₃ O

calculated: 0 43.46; H 8.03; N 5.07; S 11.60; OMe 11.23;

Mole weight (anhydrous) 267.35;

found: C, 43.63; H 8.30; N 5.18; S 11.67; OMe 11.74. pKa x 7.1;

+ 263 ° (c, 0.8284, Hgo);

Molecular weight (mass spectrometry, M ⁺ ): 267.

Part 30 D-2:

In repeating the procedure of Part 30 DI crystallization was slow i "ethanol induced in a more dilute solution in 95th Colorless ones emerged

109845/194

2118638

Methyl 7-0-methyl-1-thio-a-lincosaminide hemihydrate platelets; Melting point 162-163 ° C (Polymorph II).

Both polymorphic forms showed identical chromatographic behavior (R ~ 0.2 for thin-layer ear oma- ■ tography on silica gel in methanol / chloroform in a vol.

1:15).

I and II

II and II
I and II

Melting point
Melting point
Enamel

126 -

- 163 ° C

rain of the form IL / so the form I at temperatures below yfe C in the form II over -

Part 30 E: T-O-methyl-lincomycin hydrochloride (XLVII)

approx.

HO . ·

OH

NS,

OH

XLVII

.HCl

109845/1941

A mixture of 3.08 g of 4- trans- propyl-hygric acid hydrochloride and 75 ml of acetonitrile was magnetically stirred in a 500 ml three-necked flask equipped with a drying tube and a thermometer which reached the surface of the liquid. After adding 3 »31 g of triethylamine, the solid was rapidly dissolved to form a pale brown (pale tan) solution.

After cooling to -5 ° in an ice / methanol bath, a colorless precipitate of triethylammonium chloride was formed severed. Without removing this precipitate, 2jO2 g (1.94 ml) of isobutyl chloroformate became such Added speed so that the temperature was kept between -5 ° C and 8 ° C. After that it was 15 minutes -5 ° C. Then the above mixed anhydride solution rapidly 2.0 g of methyl 7-0-methyl-1-thio-ctlincosaminide in 25 ml of water was added, a pale brown solution being obtained, which was stirred at 0 ° C. for 45 minutes became. Thin layer chromatography on silica gel (Ethyl acetate / eetone / water in a volume ratio of 8: 5sl) showed only a trace of residual amino sugar as well a large new zone with R ^ = 0.4. The volatile one Solvent was removed in vacuo at 40 C, the pale aqueous residual solution by addition of aqueous Sodium hydroxide (N) adjusted to pH 10 and the mixture extracted three times with 100 ml of chloroform and the combined extracts were washed with water and dried over anhydrous sodium sulfate. After removal of the solvent in vacuo at 40 ° C., a pale brown amorphous plague remained (2.32 g).

Chromatography was carried out on 450 g of silica gel (column size 3.8 χ 95 cm) with methanol / chloroform im

109845/1941

113636

Volume ratio 1: 15, at which 25 ml fractions were automatically collected after 250 ml forerun. After removal of the solvent in vacuo, 2.20 g of 7-0-methyllincomycin were obtained from fractions 44-70 as a colorless syrup. This syrup was stirred in 5 nil water and concentrated by adding Hydrochloric acid to achieve a pH of 3, dissolved. The solution was filtered under suction, the sinter washed with 3 ml of water and the filtrate and wash liquors cooled in the egg s / m ethanol bath. 200 ml of acetone and then 100 ml of ether were added with stirring. It formed itself a colorless crystalline precipitate, which is drawn off and dried in a vacuum desiccator at room temperature became. 1.71 g of solid were obtained in small, elongated, colorless platelets. Melting point 155-157 ° C.

analysis
For C ₁

HCl

Calculated: C, 49.93; H 8.16; N 6.13; S 7.02; Cl 7.76;

OMe 6.79;

Molecular weight (free base) 420.57;

found? (4.83 # water taken into account)

C 50.09; H 8.22; N 6.02; S 7.20; Cl 7.46;

OMe 7.03.

[ α 7 _D + 145 ° (c, 1.063, H ₂ O); pKa «7.6.

109845/1941

Molecular weight (mass spectrometry, M of the free Base): 420.

The 7 (R) -0-feethyl-lincomycin thus obtained can after the present novel process can be further processed to the corresponding 3-nucleotide.

Example 31 i

Part 31 A-I: Methyl-N-acetyl-2-O-acetyl ~ 3f4-0-isopropylidene-1-thio-a-lincosaminide (XLVIII).

0 ^H0

Il

CH ₅ C-NH

XLVIII

5 g of methyl 6-N, 7-0-ethylidine-3,4 ~ 0-isopropylidene-1-thio-a-lineosaminide (Example 1 C of US Pat. No. 3,337,527) were used for acetylation via Efach at room temperature in a Mixture of 25 ml of pyridine and 12 ml of acetic anhydria turned off. The solvent was removed in vacuo on a rotary evaporator at 40 ° C., a pale yellow syrup being obtained. The syrup was dissolved in chloroform, washed with water, saturated aqueous Na-

109845/1941

trium carbonate and washed again with water and dried over anhydrous sodium sulfate. Thin-layer chromatography (Silica gel, methyl ethyl ketone / acetone / water in a volume ratio of 75: 25: 10) showed that no starting material was present and a new zone with a slightly higher R value had been formed. After distance the solvent in vacuo at 40 ° C. became methyl 2-0-acetyl-6N, 7-0-ethylidine-3,4-0-isopropylidene-1-thio-alincosaminide obtained as an almost colorless syrup that could not be made to crystallize.

75 ml of water with the pH value of 7 were under magnetic Added stirring. The mixture was heated on a steam bath. After 6 hours the solvent became removed at 40 C in vacuo, 'whereby 5.95 g of a colorless crystalline solid were obtained, which was 600 g Silica gel (column 4.8 79 cm) in methanol / chloroform im Volume ratio 1: 7 was chromatographed. To At 650 ecm forerun, 25 ml fractions were automatically collected. The elution was followed by thin-layer chromatography. The desired product was in fractions 35-41. After removing the solvent were 1.5.7 g of a colorless amorphous solid were obtained. By Recrystallization from acetone / techn. Hexane hydrocarbons gave colorless needles of methyl-N-acetyl-2-0-acetyl-3,4-0-isopropylidene-1-thio-a-lincosaminide; Melting point 178-179 ° C

analysis

For C ₁₆ H ₂₇ O ₇ NS

103845/1941

118636

Calculated: C, 50.92; H 7.21; N 3.71; S 8.49;

Molecular weight 377.46;

found: G 50.50; H 7.20; N 3.77; S 8.50.

Molecular weight (mass spectrometry, M ⁺ ): 377. Part 31 A-2:

In repetition of the procedure according to Part 31 A-I, the solvent was now after a heating -

time of 2 hours (instead of 6 hours) away. The yield of methyl-N-acetyl - ^ - O-acetyl - ^^ - isopropiliden-1-thio-a-lincosaminide increased to $ 60.5

Part 31 B: Methyl-N-acetyl ^ -O-acetyl - ^ - O-methyl - ^, 4-0-isopropylidene-1-thio-a-lincosaminide (XLVIX).

CH,

Il

HO

.0

XLVIX

S-alkyl

OAo

1.0 g (1 mol) of methyl-N-acetyl-2-0-aoetyl-3,4-0-isopropyl-

109848/1941

2113638

iden-l-thio-a-lincosaminide, 37.6 g (16.5 ml, 100 nol) Methyl iodide and 3.1 g (5 moles) of silver oxide were heated and stirred under moderate reflux for 16 hours. The methyl iodide was removed in vacuo at 40 C, the yellow-gray powder that remained was washed thoroughly with methylene chloride extracted. Removal of the solvent in vacuo gave 1.09 g of a yellow syrup. This crude product was a countercurrent distribution (500 steps) t in ethyl acetate / ethanol / water / cyclohexane in the volume ratio 1: 1: 1: 2 using equal volumes of the upper and lower phases. It was a main peak of K = 0.34 was found, which corresponded to the theoretical curve.

After removing the solvent from the united fractions of the material of K = 0.34 were 250 mg of a syrup obtained which crystallized on standing. After recrystallization from ethyl acetate / techn. Hexane hydrocarbons were 160 mg of methyl-N-acetyl-2-0-acetyl-7-0-methyl-3,4-0-isopropylidene-1-thio-a-lincosaminide in the form of blunt, colorless needles with a melting point of 152-154 °. By a second recrystallization the pure product was obtained from the same solvent mixture; Melting point 152.5 - 154 C.

analysis

For C ₁₇ H ₂₉ O ₇ NS

Calculated: C, 52.15; H 7.47; N 3.58; S 8.19;

Molecular weight 391.48; Found: C, 52.24; H 7.48; N 3.92; S 7.98.

ORIGiWAL INSPECTED

109846/1941

2118638

Molecular weight (mass spectrometry, M ⁺ ): 391. £ OiJ-Q + 188 ° (c, 1.185,

Part 31 G: Methyl-N-acetyl-7-O-methyl-1-thio-a-lincosaminide and its tri-acetate.

CH ₃

RO

Il

CH, C-NH

HO

1 S-alkyl

OH

100 mg methyl-N-acetyl ^ -O-acetyl ^ j / V-O-isopropylidene-1-thio-a-lincosaminide were stirred overnight at room temperature with 20 ml of water and 5 ml of aqueous hydrochloric acid (N). The solution was neutralized with 3 g of silicon excess carbonate touched. The solids were filtered off and washed with water. The piltrat and the suds were concentrated to dryness on a rotary evaporator at 60 G / 7 mm, whereby methyl-N-acetyl-7-O-methyl-l-thio-oc-lincosaminide was obtained as a colorless, non-crystallizing syrup. This was used for further characterization converted into the tri-acetate.

109845/1941

5 ml of pyridine and 3 ml of acetic anhydride were added. The mixture was stirred until the syrup had dissolved. The mixture was then stored at room temperature overnight. The solvent was then ^{removed as completely as possible at 40 °} C. / <1 mm, a dark yellow crystalline mixture being obtained which was dissolved in chloroform, washed with aqueous 0.1η hydrochloric acid, water, saturated aqueous sodium bicarbonate and water and washed over anhydrous Sodium sulfate was dried. After removal of the solvent in vacuo, methyl-N-acetyl-2, 3 "4" -tri-0-acetyl-7-C-methyl * .l-thio-cc-lincosaminide was obtained as an almost colorless, crystalline solid which from ethyl acetate / techn. Hexane hydrocarbons crystallized out in small, colorless prisms.

Melting point: 211.5 - 213 ° C *
analysis

calculated: C, 49.64; H 6.71; N 3.22; S 7.36; MeO 7.13;

Molecular Weight: 435.49;

found: G 49.72; H 6.77; N 3.36; S 7.27; MeO 7.08.

[ α J _B + 229 ° <e, C, 7174, CHCl ₃ ); Molecular weight (mass spectrometry, M ⁺ ): 435.

Instead of methyl iodide, ethyl, propyl, butyl, isobutyl, sec. Butyl and tert-butyl iodide were used Preparation of the corresponding 7-0 (lower) alkyl analogs used.

98A5 / 1941

Instead of the 4-propylhygric acid hydrochloride (l-methyl-4- trans -propyl-L-2-pyrrolidine-carboxylic acid hydrochloride), the hydrochlorides of other L-2-pyrrolidine carboxylic acids of the formula

R ₁

OH

where R «is a hydrogen atom or a lower alkyl radical, such as the methyl or ethyl radical, and R _{1 is} a hydrogen atom or a lower alkyl radical, for example the methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, Octyl and their isomeric forms, or a lower cycloalkyl radical, such as a cyclopentyl, cyclohexyl or cyclohexylmethyl radical, for the preparation of compounds of the following formula

ti

CH, O-

109845/1941

2! 18636

in which R ₁ and R 1 are as defined above, are used. The resulting lincomycin compounds can be converted into the corresponding new 3-nucleotides by the novel method according to the invention.

ORIGINAL INSP5CTPD

109845/1941

Claims (31)

- Ill - Patent application marriage: Compound of formula R, CH ₅ <I öb3b ti NH OH in which Y is an -SR radical, where R is an alkyl radical having 1 to 6 carbon atoms, a S- It _Ί _ο -c —.- · HO Location**- , fine -S-CH ₂ -CH ₂ -OH ιQn Πι as well as their isomeric forms, 109845/1941 R _{1 is} a hydrogen atom or a cis or trans- _{lower alkyl radical with 1 to 8 carbon atoms, R 2 is} a hydrogen atom, a CH, - or CgHpp radical, X is an OH radical, a chlorine, bromine or iodine atom, an -OR -z radical, where R, represents an alkyl radical having 1 to 6 carbon atoms, in each case in the (R) or (S) configuration, and Z is a nucleoside 5'-phosphate group, the nucleoside of which is adenosine, guanosine, cytidine or Uridine is, mean, and its salts. 2. The zwitterionic form of the compound of claim 1. 3. A compound according to claim 1 of the formula HO GH, HO / ι / l \ oz OH in which Y, R ₁ and R _{2 have} the meaning given in claim 1, and Z is a nucleoside-5'-phosphate group, the nucleoside of which is adenosine, guanosine, cytidine or uridine , and salts thereof. 109846/1941 4. A compound according to claim 3 of the formula GH, n-C 3 ^H 7 HO ft HO / f> OZ K SCH OH in the Z an IMucleoside-S'-phosphate group, the nucleoside of which Adenosine, guanosine, cytidine or uridine means and their salts. 5. A compound according to claim 3 of the formula CH, HO NH HO , OZ 109845/1941 I M α - 114 - in the Z is a nucleoside 5'-phosphate group, the nucleoside of which Is adenosine, guanosine, gytidine or uridine, and R * mean a CEU residue. 6. A compound according to claim 1 of the formula GH, Halo HO / " ⁰ OZ OH in which the "halo radical" means a chlorine or a bromine atom and Y, R ₁ , R ₂ and Z have the meaning given in claim 1, as well as their salts. 7. A compound according to claim 6 of the formula: 109845/1941 INSPECTED * 3 C. Il O I. N nC _x I
ι ^y N - 115 - Cl HO OZ SR, OH in the Z a nucleoside-5'-phosphate group, the nucleoside of which Adenosine, g-uanosine, cytidine or uridine, and R * is a CH ^ radical, as well as their salts. 8. A compound according to claim 6 of the formula Cl in the Z is a nucleoside 5 ¹ -phosphate group, the nucleoside of which 109845 / 19Λ1 Adenosine, - guanosine, cytidine or uridine, and mean a CH ~ residue. 9. A compound according to claim 1 of the formula CH Halo OH in which the "Halo" radical is a chlorine or a bromine atom, R is a CH ^ radical, R ^ a pentyl radical and Z is a NucleOsid-5 ^l -phosphate group, the nucleoside of which is adenosine, g-uanosine, cytidine or uridine is, mean, and their salts. 10. A compound according to claim 9 of the formula: 109845/1941 .ο CH 01 HO / ["O OH in the IU a CH ^ radical, R ₁ a pentyl radical, and Z a nucleoside 5'-phosphate group, the nucleoside of which is adenosine, guanosine, gytidine or uridine, and their salts. 11. A compound according to claim 9 of the formula CH, Cl 109845 / 19Λ1 211 8638 - 118 - in the Z a h ucleοsid-5'-phosphate group, the nucleoside of which is adenosine, g-uanosine, gytidine or uridine ,. R., a GH ^ radical, and R, a pentyl radical. 12. A compound according to claim 1 of the formula Halo in which the ^{! f} halo "radical is a chlorine or a bromine atom, R is a CEU radical, R-, a pentyl radical, R is a hydrogen atom and Z is a nucleoside-5 ^? -phosphate group, the nucleoside of which is adenosine, guanosine, gytidine or uridine, as well as their salts. 13. A compound according to claim 12 of the formula: 109845/1941 ORlQlMAL INSPECTED 211 3636 - 119 - Il GH. NH HO Cl } OZ J SR OH in each of R ~ a GH ^ radical, R ₁ a pentyl radical and Z a nucleoside 5 ¹ -phosphate group, the hucleoside of which is adenosine, g-uanosine, cytidine or uridine, as well as their oil salts. 14. A compound according to claim 12 of the formula ^H © Il CH Eq NH HO / \ 0 ί SR ₃ OH 109845/1941 in which R ^ is a CH ^ radical, R _{1 is} a pentyl radical and Z is a nucleoside 5'-phosphate group, the nucleoside of which is adenosine ₅ g-uanosine, cytidine or uridine. 15 · Compound according to claim 1, wherein Y of the formula a -SCEz radical, R ₁ a propyl radical, R ₂ a CH-z radical, X a chlorine atom and Z a nucleoside 5 ¹ -phosphate group, the nucleoside of which is cytidine , mean. 16. A compound according to claim 1, wherein Y of the formula is a -SCH ^ radical, R _{1 is} a propyl radical, R _? a CH, residue, X a chlorine atom and Z a nucleoside-5'-phosphate group, the nucleoside of which is adenosine, mean » 17. A compound according to claim 1, wherein Y of the formula is a -SCH ^ radical, R _{1 is} a propyl radical, Rp is a CEU radical, X is a chlorine atom and Z is a nucleoside-5'-phosphate group, the nucleoside of which is uridine . 18 ο A compound according to claim 1, wherein Y of the formula "is a -SCfi ^ radical, R _{1 is} a propyl radical, R" is a CH, - Radical, X is a chlorine atom and Z is a nucleoside-5 ¹ -phosphate group, the nucleoside of which is g-uanosine. 19. Method of making a connection according to claim 1, characterized in that one Compound of formula s 109845/1941 GH H( Γ / OH OH in which R ,, Rp, X and Y have the meaning given in claim 1, incorporated into the fermentation medium of a Streptomyces fermentation. 20. The method according to claim 19, characterized in that one for the preparation of compounds of formula CH, It HO / Cl NS. OH in the Z a mucleoside-S'-phosphate group, the nucleoside of which 109845 / 19Λ1 2113636 Is adenosine, g-uanosine, gytidine or üriciin, means a compound of the formula GH, n-C G 0 GH, Cl - OH OH incorporated into a fermentation of Streptomyces coelicolor Müller, NRBL 3532. 21. The method according to claim 19 »characterized in that that for the preparation of compounds of the formula s 109845/1941 2113636 CH, Eq It HO f OZ '1 - ι oh OH in which Z is a nucleoside 5 ¹ -phosphate group, the nucleoside of which is adenosine, guanosine, cytidine or uridine, a compound of the formula CH Cl -N ■ ". η ( JMiI OH
- j , 11-C ₅ H _{7 /} Kj i Il
O HO .Γ * " ⁰ ·, ¹ OH incorporated into a fermentation of Streptomycea venezuelae, NRRL 3527. 109845/1 22. A method for isolating a compound of the formula NH in which R-,, Rp, Z, X and Y have the meaning given in claim 1 * from a Streptomyces fermentation medium, characterized in that one 1. Filter the fermentation medium; 2. the filtrate is absorbed on a suitable agent to remove water-soluble contaminants; 3. the eluate obtained from the absorbent to a » Anion exchange resin chromatographed; 4. the fractions obtained from the anion exchange resin are subjected to countercurrent distribution, and 5. the individual 3-nucleotides chromatographically separates. 109845/1941 23 · Therapeutic preparation containing a compound according to claim 1 or its pharmacologically acceptable Salts as an active ingredient together with a pharmaceutical carrier. 24. Therapeutic preparation in the form of a dosage unit containing about 25 to about 500 mg of a compound according to claim 1 or its pharmacologically acceptable Salts as an essential active ingredient together with a pharmaceutical carrier. 25. Therapeutic preparation containing 5 to about 82 % of a compound according to claim 1 or its pharmacologically permissible salts as the essential active ingredient together with a pharmaceutical vehicle. 26. Sterile preparation for parenteral administration, containing about 5 to about 82 ^ (w / v) of a compound according to claim 1 or pharmacologically thereof permitted salts as the essential active ingredient together with a sterile vehicle. 27 »Pharmaceutical preparation for the treatment of responsive microbial infectious diseases Humans and animals, containing an effective amount of a compound according to claim 1 or its pharmacologically acceptable Salts in association with a pharmaceutical carrier. 28. Pharmaceutical preparation according to claim 27 in the form of a dosage unit, characterized in that 109845/1 2 1 ί ββ3β that there is about 25 to about 500 mg of the active compound or their pharmacologically acceptable salts. 29. A pharmaceutical preparation according to claim 27, characterized in that the dose of the effective Ver binding per day is about 1 mg / kg to about 50 mg / kg. 30, Pharmaceutical preparation for the preventive treatment of responding infectious microbial diseases, containing an effective amount of a compound according to claim 1 or its pharmacologically acceptable salts together with a pharmaceutical carrier. 31 ο Pharmaceutical preparation according to claim 30 in the form of a dosage unit containing about 25 to about 500 mg of the active compound of the formula H. Q CH ₅ HO / Ί 0 OH OHKSINAL INSPECTED 109845/1 in which R ,, L ₁ Z, X and Y have the meaning given in claim 1, or their pharmacologically acceptable salts. For: The Upjohn Company Lawyer ^{i! m} ' ^iUf 109845/1941 “.- ■