DK154084B - METHOD OF ANALOGUE FOR THE PREPARATION OF 1-SUBSTITUTED 6,7-METHYLENDIOXY-4 (1H) -OXOCINNOLINE-3-CARBOXYL ACID OR SALTS AND INTERMEDIATE USE OF THE PROCEDURE - Google Patents

Description

The present invention relates to an analogous process for the preparation of antimicrobial, novel 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acids of the general formula I set forth in claim 1, or salts thereof with alkali metal. or alkaline earth metal compounds, ammonia or amines with pKa values above 7.5. The invention further relates to an intermediate product for use in the process.

The process according to the invention is characterized by the characterizing part of claim 1. Thus, the subject compounds are prepared by hydrolyzing a carbonitrile intermediate of the general formula

(II) wherein R is as defined in claim 1 to the corresponding carboxylic acid of formula I. The hydrolysis can be carried out either with an acid or a base. It is preferred to perform the hydrolysis with concentrated hydrochloric acid in a reaction medium consisting of acetic acid, wherein the molar ratio of hydrochloric acid to acetic acid is between 1: 4 and 4: 1, and the molar concentration ratio of acetic acid to nitrile is between 5: 1 and 20: 1.

The intermediate of formula II can be prepared by a multi-step synthesis from the known compound 2'-nitro-4 ', 5'-methylenedioxyacetophenone. This compound can be reduced using platinum as a catalyst in a Parr hydrogenation apparatus at approx. 3.5 atm at a temperature of 30 - 40 ° C to form 2'-amino-45'-methylenedioxyacetophenone. It will be obvious to those skilled in the art that other catalysts, e.g. palladium on carbon or Raney nickel can also be used and that pressures and temperatures may be used in addition to those indicated or that the reduction can be carried out by a suitable chemical reduction process.

The second reaction step consists of a diazotation and cyclization of the amino compound to a cinnoline derivative, diazotizing the aminoacetophenone with an equivalent amount of a nitric acid-forming nitrite in the presence of an excess of 5-10 molar equivalents of a hydrochloric acid solution at a temperature of 0 ° C. thereafter, cycling is completed over a period of 3 - 24 hours at a temperature of 0 - 80 ° C, the preferred being 3-4 hours at 80 ° C. In the reaction, the known compound 6,7-methylene-dioxycinnoline is formed. 4-ol [Schofield and Simpson, J. Chem.

Soc., 519 (1945)] of the formula:

This cinnoline derivative can be brominated with bromine in the presence of approximately equimolar amounts of potassium acetate in an excess of 5-10 mole equivalents of acetic acid at the reflux temperature of the reaction mixture. The resulting compound, 3-bromo-6,7-methylene-dioxycinnolin-4-o], is converted to 6,7-methylenedioxycinnolin-4-ol-3-carbonitrile by, for example, reacting the bromo derivative with an equimolar amount or with an excess of up to 2 molar equivalents of copper (I) cyanide. The reaction is usually carried out in an excess of 5 - 20 molar equivalents of dimethylformamide at atmospheric pressure for 4 - 24 hours at a temperature of between approx. 50 ° C and up to the reflux temperature of the reaction mixture, and it is preferred to carry out the reaction for 4 - 6 hours at the reflux temperature.

The compound 6,7-methylenedioxycinnolin-4-ol-3-carbonitrile can then be reacted with methyl, ethyl, n-propyl, isopropyl, n-butyl or allyl bromide, chloride, iodide or sulfate in the usual manner. in a polar organic solvent, e.g. in C ^-Cg alkanol, C₂-C ^ alkanediol, diethylene glycol, triethylene glycol, dimethylformamide, dimethylacetamide or N-methylpyrrolidone in the presence of a basic reagent to give the corresponding 1-methyl, ethyl, n-propyl, isopropyl, n-butyl or allyl-6,7-methylene-dioxy-4 (1H) -oxocinnoline-3-carbonitrile compounds of formula II, which are the intermediates of the invention.

The invention also relates to the preparation of the salts of the subject 1-substituted 6,7-methylenedioxy-4 (1H) -oxo-cinnoline-3-carboxylic acids with alkali metals, such as e.g. lithium, sodium and potassium; alkaline earth metals, such as magnesium, calcium, stronthium and barium, ammonium and amines having a pKa greater than 7.5, such as e.g. triethylamine and pyrrolidine.

The salts can be formed by reacting the free acid with a base, a basic salt or an amine, such as e.g. sodium bicarbonate, calcium carbonate, ammonium hydroxide and triethylamine. Of the preferred salts are the sodium, potassium, calcium and ammonium salts.

The 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acids and their salts are essentially white, high-melting crystalline compounds. The free acids are soluble in most polar organic solvents, whereas the monovalent cationic salts are soluble in aqueous solvents.

The compounds prepared by the process of the invention and their salts are effective against microbial infections in warm blooded animals when the compounds are administered in daily amounts of 25-500 mg / kg body weight.

Use of the alkali metal and ammonium salts of the subject 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline carboxylic acids is preferred because of their greater water solubility, chemical stability and efficiency, and because the salts are easy to prepare and suitable for use. mix in different food items.

It is preferred to administer 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid orally as a salt via suitable foodstuffs in which the compound is present in amounts of 25 - 2500 g / ml. tonnes of total food, with the exact concentration depending on the amount of food required to deliver an effective dose of the active compound when consuming a normal amount of food. The addition of the active compounds to the food is usually done by preparing a suitable food premix which is then mixed with the rest of the food. However, one can also put the desired amount of compound into the food at once.

A suitable food premix can be prepared by grinding the active compounds into a powder and mixing it with a suitable carrier such as e.g. lucerne grains, solvent extracted soybeans, cornmeal and peeled hydrobiotite. The premix is then mixed with the desired food when administration of the active compound is desired. The food premix can only be diluted with a food supplement or concentrate until the desired concentration of the active compound is obtained and the treated food or concentrate can either be given simultaneously with the rest of the ration or mixed in the final food.

An alternative method for preparing the premix may consist of dissolving the active compound in a solvent, e.g. water or prepare a slurry of the compound in a suitable agent such as e.g. an edible vegetable oil or an edible glycol and then put such a solution or slurry to the premixed carrier while stirring.

The subject 1-substituted carboxylic acids and their salts can be administered at once or in multiple doses either parenterally or orally. At parenteral dosing, the active compound can be dissolved in an aqueous agent at a concentration of 25 - 200 mg / ml, the exact concentration depending on the required effective dose of the active compound and the volume of solution which can be suitably injected into the animal.

The compounds may also be administered orally in a normal manner, e.g. in the form of filled capsules, liquids, suspensions and pressed tablets. Such oral dosage forms usually contain the desired amount of the active compound together with suitable fillers, binders, solvents and protective agents.

For example, 1-ethyl-6,7-methyldioxy-4 (1H) -oxocinnoline-3-carboxylic acid are pressed into tablets for oral administration as follows: 5.0 g of 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid is mixed with 22.0 g of lactose, 2.9 g of starch and 0; 1 g of magnesium stearate and the mixture is pressed into tablets of such size that each tablet contains 50 mg of the active compound.

The process of the invention is further illustrated by the following example.

EXAMPLE

Preparation of starting material (a) 2, -amino-4,1,5'-methylenedioxyetheaphenone 90 g (0.43 mol) of 2'-nitro-4 ', 5'-methylenedioxyethiophenone was suspended for approx. 300 ml of absolute ethanol and 200 mg of platinum dioxide were added. The mixture was placed in a Parr hydrogenation apparatus and reduced with hydrogen at 3.5 atm. at a temperature of 30 - 40 ° C for 3-4 hours until no uptake of hydrogen was observed. The solvent was distilled off under vacuum and the residue was dissolved in 500 ml of warm chloroform. The mixture was filtered and the filtrate evaporated to dryness. The evaporation residue was recrystallized in an 80:20 mixture of ethanol and water to give ca. 70 g of amber crystals of 2'-amino-4 ', 5' -methylenedioxyetophenone, m.p. 167 -168 ° C. The yield was 90.7 °.

Analysis: C ^H ^ NONO,, molecular weight: 179;

Calculated: C 60.33, H 5.06, N 7.82.

Found: C, 60.60; H, 5.33; N, 7.75.

(b) 6,7-methylenedioxycinnolin-4-ol

70 g (0.41 mole) of 2'-amino-4 ', 5'-methylenedioxyethophenone was added to 800 ml of concentrated hydrochloric acid at room temperature. The resulting mixture was cooled to 0 ° C and a solution of 35 g (0.5 mole) of sodium nitrite in 100 ml of water was added dropwise keeping the temperature at 0 ° C.

under stirring. Stirring was continued for 30 minutes after all sodium nitrite was added. The resulting reaction mixture was filtered and the filtrate was heated to 80 ° C for 4 hours and then allowed to stand overnight at room temperature, whereby the product precipitated and this was recrystallized in 600 ml of an 80:20 mixture of dimethylsulfoxide and water. 65 g of 6,7-methylene-dioxycinnolin-4-ol were obtained in the form of light brown crystals. The product melted at approx. 338 ° C during decomposition. The yield was 83.6 / ¾.

Analysis: ^ 9 ^ 6 ^ 2 ^ 35 mole weight: 190.

Calculated: C 56.84, H 3.18, N 14.73.

Found: C 56.81, H 2.93, N 14.50.

(c) 3-Bromo-6,7-methylenedioxycinnolin-4-ol 60 g (0.3 mol) of 6,7-methylenedioxycinnolin-4-ol was added to a solution consisting of 30 g (0.3 mol) of anhydrous potassium acetate in 400 ml of acetic acid. The resulting mixture was heated to reflux, whereupon a solution of 48 g (0.3 mole) of bromine in 100 ml of acetic acid was added dropwise with stirring over 2 hours. Then, the resulting reaction mixture was stirred for a further 30 minutes under reflux. The reaction mixture was now allowed to cool to room temperature and then poured into 1 L of ice water. The resulting precipitate was isolated by filtration and washed with 300 ml of a 5¾ sodium bicarbonate solution. Recrystallization in an 80:20 mixture of dimethyl sulfoxide and water yielded 76 g of brown crystals of 3-bromo-6,7-methylenedioxycinnolin-4-ol which melted at 330 ° C during decomposition. The yield was 94.3¾.

Analysis: Cgh ^Br BrO₂; molecular weight: 269.

Calculated: C 40.17, H 1.87, N 10.41, Br. 29.70.

Found: C, 40.31; H, 1.85; N, 10.60; Br. 29.90.

(d) 6,7-methylenedioxycinnolin-4-ol-3-carbonitrile 100 g (0.37 mol) of 3-bromo-6,7-methylenedioxycinnolin-4-ol was added to a slurry of 63 g (0.70 mol) copper (I) cyanide in 1100 ml of dry dimethylformamide. The resulting mixture was refluxed for 4 to 5 hours with simultaneous stirring. The solution became homogeneous upon boiling. Upon completion of the reaction, a heavy green precipitate formed. The resulting reaction mixture was cooled to room temperature and poured into a solution consisting of 160 g of iron (III) chloride, 120 ml of concentrated hydrochloric acid and 250 ml of water. The resulting mixture was heated to 60 ° C for 15-20 minutes, poured over 2 L of crushed ice and filtered. The yellow product was recrystallized from an 80:20 mixture of dimethyl sulfoxide and water to give 75 g (93.8%) of 6,7-methylene-dioxycinnolin-4-ol-3-carbonitrile in the form of brown crystals melting at 330 - 340 ° C under decomposition.

Analysis: ^ 10 ^ 5 ^ 3 ^ 35 mole weight: 215.

Calculated: C, 55.82; H, 2.34; N, 19.53.

Found: C, 55.74; H, 3.04; N, 18.63.

(e) 1-Ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carbonitrile 70 g (0.36 mol) of 6,7-methylenedioxycinnolin-4-ol-3-carbo-nitrile was suspended in 500 ml. dry dimethylformamide. 18 g (0.36 mol) of dry sodium hydride (prepared by washing 50% sodium hydride in mineral oil with n-hexane) was added with stirring to the above slurry. The mixture became homogeneous as hydrogen developed.

After cessation of hydrogen evolution, draft newspaper was added with stirring 63 g (fl, 40 moles) of ethyl iodide at ambient temperature. After the addition was complete, stirring was continued for 1 hour. The resulting reaction mixture was then heated to 100 ° C for 2 hours, cooled to room temperature, poured into 11 liters of ice water and acidified with hydrochloric acid. The precipitate formed was filtered off and recrystallized in an 80:20 mixture of dimethyl sulfoxide and water. 50 g (62.5% yield) of 1-ethyl-6,7-anethylene-dioxy-4 (1H) -oxocinnoline-3-carbonitrile in the form of lysis bromine crystals melting at 268 - 269 were obtained. ° C ..

Analysis: ^ 12 ^ 9 ^ 3 ^ 3 'im ° i' / æ9t: 243.22.

Calculated: C 59.26, H 3.73, N 17.28.

Found: C 59.05, H 3.74, N 17.10.

Preparation of 1-ethyl-6,7-methylenedioxy-4 (1H) -caxo-cinnoline-3-carboxylic acid (final product) 23 g (0.095 mol) of 1-ethyl-6,7-methylenedioxy-4 (11H) -roxo cinnolin-3-carbonitrile was added to a mixture of 200 ml of concentrated hydrochloric acid and 200 ml of acetic acid. The resulting reaction mixture was refluxed for 18 hours. Excess acid was removed under vacuum and the residue was dissolved in 150 ml of a 5% sodium hydrogen carbonate solution. The resulting solution was treated with 5 g of charcoal and filtered. The filtrate was acidified by the addition of hydrochloric acid and the resulting precipitate was filtered off. 23 g (corresponding to 91.6¾) of 1-ethyl-6,7-methylenedioxy-4 (1H) -oxo-cinnoline-3-carboxylic acid were obtained in the form of light brown crystals, melting at 261 - 262 ° C during decomposition. .

Analysis: C12-10N2 ° 5 'm ° m: 262.

Calculated: C 54.96, Η 3.84, N 10.68.

Found: C 54.77, H 3.84, N 10.61.

Using the above-described procedure for hydrolysis of corresponding 1-substituted nitriles, the following compounds were obtained: 1-methyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, m.p. 308 ° C (decomposition).

Analysis calculated for n 8 8 2 5: ^ 53.23, H 3.29, N 11.29.

Found: C, 53.01; H, 3.27; N, 11.59.

1-n-propyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, m.p. 229 - 230 ° C.

Analysis calculated for ^ 13 ^ 12 ^ 2 ^ 5: ^ 4.38, N 10.14.

Found: C 56.31, H 4.45, N 10.36.

1-isopropyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, m.p. 304 -305 ° C (decomposition).

Analysis calculated for C 56.52, H 4.38, N 10.14.

Found: C 56.31, H 4.29, N 10.24.

1-allyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, m.p. 214 - 215 ° C.

Analysis calculated for 8 ^ 3 ^, 10 10 55, 55 8 8, 93, H 5.68, N 10.22.

Found: * 8 56.68, H 3.86, N 10.25 1-n-Butyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, m.p. 210 - 211 ° C.

Analysis calculated for: C 57.93, Η 4.86-, N 9.65

Found: C 57.71, H 4.98. N 9.71

The disclosed 1-substituted 6,7-methylenedioxy- * 4 ((1H) -oxoinnolin-3-carboxylic acids produced by the process of the invention and their salts exhibit a pronounced antimicrobial activity. Of the organisms that are pathogenic before warm-blooded animals, and where significant activity has been shown in vitro, may be mentioned iMy coplasma gallisepticum as well as the gram-negative bacteria Escherichia coli, Salmonella dublin and Vibriocoli.

Representative compounds (such as sodium salts) prepared by the process of the invention have the following minimum inhibitory concentrations (expressed as µg per ml) against the organisms listed above in the usual tube dilution experiments.

TABLE I

In vitro antimicrobial activity

Minimum inhibitory concentration yug / ml opposite: M.galli-

Compound E. coli S. dublin \ 1. coli septicum 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, sodium salt 12.5 6.25 25., 3.12 l-isopropyl-6,7-methylene dioxy -4 (1H) -oxocinnoline-3-carboxylic acid, sodium salt 25.0 12.5 50 - 0.25.0 1-allyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, sodium salt 50 , 0 12.5 50.0 50.0

The present 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acids and their salts are effective therapeutic agents in microbial infections in warm-blooded animals. They are both effective at parenteral and oral administration, as evidenced by experiments with chickens. Eg. the sodium salt of 1-ethyl-6,7-methylene-dioxy-4 (1H) -oxocinnoline-3-carboxylic acid is injected into the throat of day-old chickens in amounts of approx. 50 mg per chicken. Two groups of 5 chickens were treated in this way. In addition, two groups of 5 chickens were used as control animals. The four groups of 5 chickens were administered intramuscularly 0.1 ml of a Salmonella typhimurium culture. On the 6th day, the number of surviving animals in each group was counted. The number of surviving animals indicates the activity of the compound towards the test organism. The results are shown in Table II. The sodium salt was also administered by oral route by mixing 200 g / ton of the salt in chicken feed, which was constantly used for feeding after hatching. Four groups of 9 chickens were given the indicated food. Four groups of 9 chickens received the same ration, but without the active compound. The 8 groups of 9 chickens got the microorganism by giving each chicken group 50 ml of a S. typhimurium meat soup culture instead of drinking water, the intake being over 24 hours. The number of surviving animals is counted on the 10th day. The oral activity of the compound against the test organism was indicated by the difference in the number of surviving animals in the treated and untreated groups. Table III shows the results of these experiments.

TABLE II

Antimicrobial activity of 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, sodium salt, injected subcutaneously into day-old chicks

Number of surviving dives <(6 days) /

No. Treatment total tantalum animals 1 1-ethyl-6,7-methylenedioxy-4 (1H) - oxocinnoline-3-cariboxylic acid, sodium salt, 50 mg 4/5

0 W

2 3./5

Total 7 '/ 10 l Infected Control Animals' 0/5 2a "" 0/5

Total 0/10

TABLE III

Antimicrobial Activity of 1-Ethyl-6,7-Methylem: d: 1Dxy-4 (1H) -oxocinnoline-3-cax: Boxyl Acid, Sodium Salt, Mixed in 1 Feed for Unburdened Chickens

Number of survivors: r (10 days) /

No. Treatment total number of animals 1 1-ethyl-6,7-methylenedioxy-4 (1H) - oxocinnoline-3-carboxylic acid, sodium salt, 200 g / ton 6/9 2 "0/9 3" 8/9 4 "Total 5/9 19/36 la Infected control animals 0/9 2a "" 0/9 3a "" 3/9 4a "" 1/9

Total 4/36

Furthermore, em injection of 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid protected sodium salt in the throat of 6-week-old roast chickens weighing approx. 600 g, the treated animals against administered doses of E. coli. Ten groups of 10 animals were used for the experiment. Six of the 10 groups were injected subcutaneously with the active compound. The last four groups served as infected control animals. Doses of 50, 100 and 200 mg / kg are administered. Immediately after treatment, each chicken's trachea was injected with 0.5 ml of a 10-fold diluted 24-hour tryptose nutrient medium of an E. coli strain, with half of the groups being administered the Gross strain and the other half the Glantz strain. Seven days after treatment, the surviving chickens were slaughtered and autopsied. Each chicken was examined for trachea, pericarditis and periheptatitis. The average injury rate for each group was determined by assigning each chicken number on the following scale: 4 = death before the day the surviving animals were slaughtered; 3 = trachea plus pericarditis plus periheptatitis; 2 = tracheal injury plus either pericarditis or periheptatitis; 1 = tracheal injury; 0 = no damage. All the numbers for a group were added together and the sum was divided by the number of chickens in the group. A mean injury score of 4.0 means that all the chickens died before the end of the trial, while 0.0 means that none of the chickens died before slaughter or showed any damage. The number difference between the treated and untreated chicks indicates the activity of the compound towards the test organism. The results of these experiments are listed in Table IV.

TABLE IV

Anti-infective activity of 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acid, sodium salt, injected subcutaneously in 6-week-old chickens

Number of Cooling Aids in E. Cnli Damage

Treatment experimentally, the number of 1-ethyl-6,7-methylene in (oxy-4 (1H) -oxocinnoline-3-carboxylic acid, sodium salt, 50 mg / kg 10 Grosss 0.9 "10 Glantz 1.9 l 6,7-methylenedioxy-4 (1H) -oxoinnolin-3-carboxyl; acid, sodium salt, 100 mg / kg Gross 1.0 "Glantz 0.6 l-Ethyl-6,7-methylenedioxy-4 (1H) -oxocinnolin-3-carboxylic acid, sodium salt, 200 mg / kg 10 Grosa 0.0 "10 Glantz 0.5

Infected Control Animals 10 Gross 1.7 "" 10 Gross 2.6 "" 10 Glanitz 1.7 "" 10 Glanttz 2.3

The disclosed 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acids and their salts have low mammalian toxicity. For example, if the sodium salt of 1-ethyl-6,7-methylenedioxy-4 (1H) -oxocinnolin-3-earboxylic acid is administered to mice at doses as high as 200 mg / kg intraperitoneally or 500 mg / kg orally, no one dies. of the mice.

Claims (3)

An analogous process for the preparation of 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline-3-carboxylic acids of the general formula (I) wherein R represents methyl, ethyl, n-propyl, isopropyl, n-butyl or allyl, or salts thereof with alkali metal or alkaline earth metal compounds, ammonia or amines having pKa values above 7.5, characterized by hydrolyzing an nitrile of the formula (II) wherein R is as defined above and, if desired, transferring a formed compound of formula I into a salt by reaction with an alkali metal or alkaline earth metal compound with ammonia or with an amine having a pKa value greater than 7.5 . Analogous process according to claim 1, characterized in that R is ethyl. A 1-substituted 6,7-methylenedioxy-4 (1H) -oxocinnoline-3-nitrile for use as a starting material in the process of claim 1 or 2, characterized in that it has the general formula (II) wherein R is methyl, ethyl, n-propyl, isopropyl, n-b: utyl or allyl.