MC360A1 - Process for the preparation of new compounds of the tetracycline family - Google Patents

Description

PATENT D 1 lITErïIOI

The preparation process of new compounds from the tetracycline family.

t,

Company known as:

AMERICAir CTAHAM1D COMPACT

The present invention relates to a process for preparing new compounds of the tetracycline family and more particularly it relates to a process for preparing new substituted 6-deoxytetracy-5 clines and 6-demethyl-6-deoxytetracyclines which can be represented by the following general formula;

in which R^ and Rg represent a hydrogen atom, or an azide, lower alkanoyloxyl, haloalkanoyloxyl, or lower alkanoyloxyl group, but cannot both represent hydrogen atoms, and R^ is a hydrogen atom or a methyl group. The invention also relates to a process for preparing non-toxic salts formed by adding acids to novel tetracycline derivatives. It is understood that throughout this description, the term "lower alkanoyloxyl" includes the formate radical.

The new tetracyclines of the invention are generally yellow solids relatively soluble in water, methanol, ethanol, acetic acid, etc., and relatively insoluble in benzene, toluene, ether, petroleum ether, etc. The compounds can generally be crystallized by a mixture of alcohol and ether, by a mixture of alcohol and benzene, etc. As is characteristic of tetracyclines in general, the compounds of the invention are amphoteric solids that exhibit wide melting ranges above 150°C.

The new tetracyclines of the invention are biologically active and possess the broad-spectrum antibacterial activity of previously known tetracyclines. The antibacterial activity of some of these compounds, defined by the amount required to inhibit

The growth of various "typical bacteria" was determined in the usual way by the streak plate technique on agar with dilution, commonly used for testing new antibiotics. The table below shows the

9

Minimum inhibitory concentrations, in micrograms per cubic centimeter, for various organisms tested. For comparison, the antibacterial activity of tetracycline against the same organisms is also indicated.

TABLE I.

Organization

Te-

tra- 7-acetoxy-6- 7-ethoxythio- 7-azido-6- 9-azido-6-cy- demethyl-6- carbonyltliio" demethyl-6- demethyl-6-

cli- desoxytetra- 6-demethyl-6~ desoxytetra- desoxytetra-

ne cycline desoxytetracycline cycline c3rcline

Mycobacterium ranae

Micobacterium smegmatis ATCC 607

.6taphyl o co c eus aureus 209P

Streptococcus

804-5

Bacillus

ATCC 6633

Streptococcus pyogenes. 0205

Streptococcus alpha no. 11

Staphylococcus albus no. 69 Streptococcus beta no. 80

Staphylococcus aureus NY 104

—l 1 " 1

Bacillus

Cereus No. 5 Proteus

Vul&aris 9484

Escherichia coli ATCC 9657

Salmonella gçallinarum

BschericMa coli n° 22

0.5 0.25 250 250 250

0.5 15 62 15 2

■4

0.5 1 15 51 8 4

0.5 4 51 15 8

8

15

8

8

15 8 Ï5 8 8 31 250 250 250

0.25

0.25

4

8

4

0.5 2 15 31 8

2

4 4

1

2 15 15

8 4 1 8

31 62 8

From the above, it can be seen that in many respects the antibacterial spectrum of the new derivatives is closely parallel to that of tetracycline, but that in addition, some of the new compounds have a biological activity 1.5-2.5 times greater than that of tetracycline against Taphylococcus aureus, the measurement being made by the usual turbidimetric test (Annals of the New York Academy of Sciences, jjl, 218 (1948)).

Furthermore, the new derivatives are extremely effective in vitro and in vivo against certain tetracycline-resistant bacterial strains. For example, the compounds of the invention are 30-80 times more effective than tetracycline against Streptococcus alpha No. 11, Staphylococcus albus No. 69, and Streptococcus beta-hemolytica strain No. 80, which are resistant to tetracycline.

Furthermore, some of these compounds exhibit high in vitro activity against the resistant microorganisms Staphylococcus Smith and Staphylococcus Rose. The Staphylococcus Smith is derived from a strain studied at the Rockefeller Institute and was isolated by Sharp and Dohme. This Staphylococcus Smith is coagulase-positive, tellurite-negative, and susceptible to tetracycline, penicillin, streptomycin, erythromycin, carbomycin, neomycin, chloramphenicol, and novobiocin in vitro. Attempts have been made to identify this strain using bacteriophage, but such identification has proven impossible.

6.-

Staphylococcus aureus, Rose variety (ATCC No. 14154), was clinically isolated from an abscess in a patient who was unresponsive to tetracycline treatment. This organism was found to be resistant in vitro to clinically used tetracyclines. Staphylococcus Rose is positive for coagulase and tellurite and resistant to tetracycline, penicillin, streptomycin, and erythromycin. It is susceptible to carbomycin, neomycin, chloramphenicol, and novo-10 biocin in vitro. Staphylococcus aureus gave the following results in bacteriophage diagnosis:

Staphylococcus aureus 9 Bacteriophage motif variety Rose 80/81

Some of the new tetracyclines of the invention have been tested in vitro against Staphylococcus Smith and Staphylococcus Rose, alongside clinically used tetracycline types. The table below shows the antibacterial activities thus obtained, expressed as minimum inhibitory concentrations in micrograms per cubic centimeter.

15

20

TABLE II.

10

Staphylococcus Staphylococcus Smith Rose

Tetracycline 0.25 250

Demethyltetracycline. 0.25 125

Demethylchlorotetracycline 0.12 125 7~azido-6-demethyl~6~

deoxytetracycline 0.25 2 9-azido-6-demethyl-6~

deoxytetracycline 0.5 2 7-acetoxy-6-demethyl-6-

deoxytetracycline 0.06 8

As previously stated, the antibacterial activity of the new derivatives is equal to, and in many respects superior to, that of tetracycline. Therefore, physicians can administer the new compounds in the same manner and at approximately the same dosages as currently used tetracyclines. Furthermore, since the new compounds exhibit the broad-spectrum antibacterial activity characteristic of previously known tetracyclines, they can be used in the treatment of various infections caused by Gram-positive and Gram-negative bacteria, where treatment of these infections with tetracycline, chlorotetracycline, or desmethyl-chlorotetracycline is indicated.

The new compounds of the invention can be prepared from the corresponding tetracycline diazonium salts. For example, the hydrochloride of 6'-demethyl-6-deoxytetracycline-7-diazonium sulfate can be prepared by nitrifying 7-amino-6-demethyl-6-deoxytetracycline sulfate in suspension in a suitable mineral acid, for example methanolic hydrochloric acid.

The new derivatives of the invention are then prepared by reacting the appropriate tetracycline diazonium salt with hydrazoic acid or a lower alkylxanthic acid such as ethylxanthic acid or a lower saturated aliphatic carboxylic acid such as acetic acid, or a lower haloalkylcarboxylic acid such as dichloroacetic acid, under the conditions required to replace the diazonium radical with a lower azidef aoetoxyl, formyloxyl or alkylxanthate radical.

Generally, the reaction is carried out in water, in alcoholic hydrochloric acid or in an excess of reagent such as formic or acetic acid, at temperatures ranging from 0 to 50°C, advantageously at room temperature, for a time ranging from a few minutes to several hours or more.

However, when reacting a tetracycline diazonium salt with an alkanoic acid to prepare an inferior alkanoloxytetracycline, the reaction is preferably induced using ultraviolet radiation. Ultraviolet radiation of 200 to 450 mJ has been found to be effective for this purpose.

To isolate and purify the compounds of the invention, common methods are used such as precipitation, solvent evaporation, lyophilization, crystallization, column chromatography, etc.

The new tetracyclines of the invention are amphoteric compounds, and therefore acid addition salts can easily be prepared. Generally, preferred acids are non-toxic acids acceptable from a pharmaceutical point of view, for example...

Mineral acids such as hydrochloric acid, sulfuric acid, etc., are used, although organic acids such as trichloroacetic acid may also be used. To prepare the acid addition salt derived from the new tetracyclines, the amphoteric compound can be treated with approximately two or more equivalents of the chosen acid. Preferably, the tetracycline is suspended in a suitable solvent during acidification.

The invention will be described in more detail with regard to the specific examples that follow.

EXAMPLE 1

Preparation of 7-azido-6-demethyl-6-deoxy sulfate

tracycline.

To a cold solution of 100 mg (0.19 millimole) of 7-amino-6-demethyl-6-deoxytetracycline sulfate (J.A.0.8. 82, 1253? (1960)), in 1 cm3 of hydrochloric acid at ice bath temperature, 0.1 cm3 of butyl nitrite is added. The reaction mixture is stirred at this temperature for 30 minutes and slowly poured into 100 cm3 of ether; 95 mg of 6-demethyl-6-deoxytetracycline-7-diazonium sulfate hydrochloride is obtained.

To a solution of 1.0 g (1.7-1 millimole) of 6-demethyl-6-deoxytetracycline sulfate hydrochloride in 30 cm³ of a 0.1 N hydrochloric acid solution in methanol, 0.113 mg (1.75 millimole) of sodium azide is added. The solution is stirred at room temperature for 2 hours and then slowly poured

The solid is dissolved in 1 liter of ether. The solid is filtered and dried (yield 0.9 c). 100 mg of this substance is dissolved in 2.0 cm³ of methanol, and ether is added until the solution becomes cloudy. The solution is filtered, and ether is added again until the solution becomes cloudy. The solution is filtered again, and when the filtrate is left to stand at 0°C for 2 days, it precipitates with 20 mg of crystalline 7-azido-6-demethyl-6-deoxytetracycline sulfate.

y HCl 0.1 n

Ultraviolet s X 255, 34-Oj log£.4j33? 3y92«

^max „

— +

infrared s 4?73 yU (spreading ~N~N=N). Rf ; 0?77 (butanol/phosphate system pH 2).

Yield t 80 fo*

PC

Rotation /~^C_7 ]q -185° ,

EXAMPLE 2.

Preparation of 7-etho:sythiocarbonylthio-6-desmethyl-6~desozytetracycline

A mixture of 100 mg (0.171 millimoles) of 6'-desmethyl-6-desoisotetracycline-7-dic,-sonium sulfate hydrochloride and 50 mg (0.314 millimoles) of potassium ethylsranthate is stirred at room temperature for 5 minutes in 30 cm³ of water. The solution is dried without freezing, yielding 0.12 g of product. This material is purified by column chromatography.

N max.

4.25; 4.10.

Rf. 0.70 (butanol/phosphate system pH 2).

Yield s 75

Rotation /"cX 7 ^ -96.5°

EXAMPLE 5 o

Preparation of 7-acetoxy-6-demethyl-6-deoxytetracycline by 5 1/2 hours a solution of 600 mg (1.026 millimole) of β-demethyl-β-deoxytetracycline-7-diazonium sulfate hydrochloride in a mixture of 200 cm3 of glacial acetic acid and 2 cm3 of water. The solution is dried by freezing and the residue is purified by column chromatography.

Preparation of 9-azido hydrochloride ~ 6 ~ demet Iiy 1-6-deoxytetracycline.

A 1.0 g (0.002 mol) solution of 9-amino-6-demethyl-6-deoxytetracycline hydrochloride (J.A.O.S.--■■82. 125^ (i960} ) flnnn 2^ otïi'S rie FOI înethanol iaue

We irradiate with an ultraviolet lamp pen-

Ultraviolet

Infra-red; 5.7 yu.

Rf 0.68 (butanol/phosphate system pH 2). Yield s 50

EXAMPLE 4.

Cooled to 0-5°C, 1.0 cm⁵ (0.009 ounce) of butyl nitrite is added. The solution is stirred at this temperature for 15 minutes, then poured into 1500 cm³ of cold ether. The separating solid is filtered, washed with ether, and dried. 0.98 g of 6-denethyl-6-deoxytetracycline-9-diazonium oxide hydrochloride is obtained.

To a solution of 5.0 g (0.105 mol) of 6-→desmethyl-6-deoxytetracycline-9-diazonium oxide hydrochloride in 125 cm³ of 0.1l methanolic hydrochloride, 750 mg (0.11 mol) of sodium azide is added. The solution is stirred at room temperature for 45 minutes. The separating solid is filtered and dried; yield: 2.5 g. The filtrate is poured into 1500 cm³ of ether, yielding an additional 1.5 g of product. The product is crystallized with a mixture of methanol and ether.

V HCl 0.1 n Ç

Ultraviolet; ' . . 260? 345, log C-4>3? 4#1*

' >• mak.

Infra-red; 4?75 yu.

Rf: 0.74 (butanol/phosphate system pH 2).

Yield; 85

Rotation ï /"^^ 7 ^ -230°.

EXAMPLE 5.

Preparation of 9-formyloxy-6-demethyl-6-deoxytetracycline hydrochloride

A solution of 100 mg of 6-demeth-1-6-deoxytetracycline-9-diazonium oxide hydrochloride in 10 cm³ is irradiated with an ultraviolet lamp for 2 hours.

forraic acid. The reaction mixture is poured into 200 cm 3 of dry ether and the solid is filtered. yield ; 60 mg.

v HCl 0,ln c

Ultraviolet; yv 270; 345? log C-4.85I 4.13.

5' ^-max.

Infrared; 5.75 ya.

Rf s 0.42 (butanol/phosphate system pH 2).

Yield i 60 fo.

10 EXAMPLE 6.

Preparation of 9-ethoxythiocarbonylthio-6-demethyl-6-deoxytetracycline.

A solution of 1.0 g (0.002 mole) of 6-desmethyl oxide hydrochloride is shaken at room temperature.

-6-deoxytetracycline-9-diazonium and 0.75 g (0.004 mol) of potassium ethylxanthate in 20 cm³ of 0.1 N hydrochloric acid and 30 cm³ of water. Once nitrogen evolution has ceased, the pH of the solution is adjusted to 4.5. The separating solid is filtered and dried; yield;

0.51 G' The material is purified by column chromatography.

\HC1 0,ln <r

265? 345? log C- 4.34? 4.10.

max.

25 Rf: 0.86 (butanol/phosphate system pH 2).

Yield s 50 fo.

Rotation ; / CK J ^ -51°.

EXAMPLE 7.

Preparation of 9-ethoxythiocarbonylthio-6-deoxytetra-

cyclino «

357 mL of butyl nitrite is added to a 500 g (0.0078 ounce) solution of 9-amino-6-deoxytetracycline (J.A.C.S. 82, 1253 (1960)) in 0.1 N methanehydroxy HCl at 5-10°C. The solution is stirred for 40 minutes at this temperature, then poured into one liter of cold anhydrous ether. The resulting 6-cleoxytetracycline-9-diazonium disulfate is filtered, washed with ether, and dried.

To a solution of 1.76 g (0.011 mol) of potassium ethyl xanthate in 40 cm³ of water, 1.00 g (0.0015 mol) of 6-deoxytetracycline-9-dia-zoniutn disulfate is added. The mixture is stirred for 1 hour at room temperature. The liquid that rises to the surface is decanted, the solid is washed with water, and it is dried by freezing.

After purification by column chromatography and neutralization, the product gives a yield of 25

v HCl 0,lft c

Ultraviolet; \. 255? 275? 300? log O 3?21?

' \max.

3.23? 3.10.

Rf; 0.81 (butanol/phosphate system pH 2).

Yield; $25.

EXAMPLE 8.

Preparation of 7-formyloxy-6-demethyl-6-cle s oxy tetracycline sulfate.

We irradiate for 3 hours at the temperature

ambient with an ultraviolet lamp a 1% solution of 6-desmethyl-6-desoxytetracycline sulfate

cline-7-diazonium in fornic acid (98-100 $>). After freezing and ether washing, the reaction mixture gives a yield of 60 fo,

v HCl 0.ln Ultraviolet; \263»34-0»

' \ max.

Rf î'0.45 (butanol/phosphate system pH 2).

Yield s 60 fo.

Infrared s 5"75 yu.

7-Dichloracetic sulfate tosy-6-denethyl-6-deoxytetracycline,

A solution of 0.100 g (0.000175 mol) of 6-desmethyl-6-deoxytetracycline-7-diazoniuri sulfate hydrochloride in 10.0 cm³ of dichloroacetic acid was lyophilized under an ultraviolet lamp. The reaction mixture was then lyophilized, the residue dissolved in anhydrous ethyl ether, filtered, and dried.

EXAMPLE 9.

We irradiate for 6 hours at the temperature

Rf s 0.82 (butanol/phosphate system pH 2).

Yield i 80 fo.

Infra-red; 5.7 yu.

Claims (1)

SUMMARY general Method for preparing compounds of formula N(ch3)2 t 2 OH Ô OH OH 'Ô in which R^ and R^ represent a hydrogen atom or an azide, lower alkanoyloxyl, haloalkanoyloxyl or lower aleoxythiocarbonylthio group, but cannot all represent hydrogen atoms, and R^ is a hydrogen atom or a methyl group, characterized by s 1° - 011 reacts a corresponding 6-deoxytetracy-cline-diazonium compound with a compound that provides hydrazoic acid, or with a lower alkylxanthic acid, a lower saturated aliphatic carboxylic acid, or a lower haloalkylcarboxylic acid. 2° - An alkali metal azide is reacted with the diazonium derivative. 3° - An alkali alkylxanthiate is reacted with the diazonium derivative. 4° - A saturated aliphatic or haloaliphatic acid is reacted with the diazonium derivative, activating the reaction by means of ultraviolet rays. 17.- 5° - The reaction is carried out at a temperature of 0-50°C. 6° - Process for preparing addition salts of 5-acid derivatives of compounds according to point 1, characterized by the fact that the free base is reacted with an acid. (Seventeen pages) by proxy of; MERICMT CYAHAMID COMPANY ORIGINAL l in _M— pages containing References word added word crossed out nuis By proxy of R A U Summer Industrial 28, Bouf. Princess Chorloifa, JS8. MONTE - CARLO