DIHYDROPHENAZINE DERIVATIVE, PROCESS FOR PRODUCING THE SAME AND DRUG FOR TUBERCLE BACILLI AND ATYPICAL ACID-FAST BACILLI TECHNICAL FTFT.n

The present invention relates to a dihydrophenazine derivative, a process for producing the same and a drug for tubercle bacilli and atypical acid-fast bacilli, which contains this derivative. BACKGROUND ART

A dihydrophenazine derivative having an antitubercular activity is disclosed, for example, in (i) V.C. Barry et al., J. Chem. Soc, 4495, (1958), (ii) British Patent 1,233,375) and (iii) Japanese Patent Publication No. 46-9248. The above dihydrophenazine derivative, however, does not have sufficient antibacterial activity to tubercle bacilli and atypical acid-fast bacilli.

It is, accordingly, an object of the present invention to provide a novel compound having an improved antibacterial activity to tubercle bacilli and atypical acid- fast bacilli.

Γ_TSΓ.T.OSURE OF THE TNVFNTTON

The present invention provides a novel dihydrophenazine derivative represented by the following general formula (1):

wherein R is a lower alkyl group or a hydroxy-lower alkyl group; and a pharmaceutically acceptable salt thereof.

The dihydrophenazine derivative (1) and a salt thereof show excellent antibacterial and bactericidal activities to tubercle bacilli and a wide range of atypical acid-fast bacilli (which mean mycobacteria other than tubercle bacilli), particularly to M. chelonea which causes tubercle bacilli and M. avium complex (mycobacteria which develop as immune system is depressed by being infected with AIDS), infectious diseases, tuberculoid diseases, lymphadenitis and subcutaneous septic wound, whose patients have been on the rise.

Accordingly, the dihydrophenazine derivative (1) of the present invention and a salt thereof are effective for tuberculose due to tubercle bacilli, and infectious diseases

of lung, lymphnodus, soft tissue, arthrosis, peritenon, bone, etc. due to atypical acid-fast bacilli.

Particularly, the dihydrophenazine derivative (1) of the present invention and a salt thereof provide a novel drug which is used for a drug therapy to tuberculose and atypical mycobacteriosis, and they are particularly preferred as one of the drug in polypharmacy therapy in which the development of resistant bacteria is prevented.

The dihydrophenazine derivative (1) and drug containing the dihydrophenazine derivative (1) as the active ingredient are well absorbed in vivo, and have a long duration of drug efficacy and less side effect (chromatosis to skin, photo anaphylaxis, hepatotoxicity, etc.), and show excellent effect to resistant bacteria of other antimicrobial drugs. Furthermore, the drug of the present invention has high stability and excellent absorption-excretion, and also have high therapeutic effect to infection and good transition to pulmonary tissue.

BEST MODE FOR HARRYING OUT THK TNVKNTTON Examples of the lower alkyl groups in the general formula (1) include alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s- butyl, t-butyl, pentyl and hexyl.

Examples of the hydroxy-lower alkyl groups include hydroxy-lower alkyl groups having 1 to 6 carbon atoms, such as

hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxy-l- i methylethyl, 4-hydroxybutyl, hydroxy-t-butyl, 5-hydroxypentyl and 6-hydroxyhexyl.

The dihydrophenazine derivative (1) of the present invention is produced, for example, by the following reaction scheme. Reaction scheme (I):

wherein R ¹ is as defined above.

Specifically, the dihydrophenazine derivative (1) can be synthesized by reacting a 2,10-dihydrophenazine derivative represented by the general formula (2) with cyclooctylamine (3) in a solvent or no solvent, as shown in the reaction scheme (I).

The solvent may be any one which exerts no influence on the reaction. There are, for example, lower alcohols such as methanol, ethanol and isopropanol; ethers such as dioxane, tetrahydrofuran and diethyl ether; and aromatic hydrocarbons such as benzene, toluene and xylene.

The reaction is normally conducted at 50 to 200 "C, preferably 80 to 150 °C, for about 3 to 30 hours, preferably about 10 to 25 hours.

The 2,10-dihydrophenazine derivative (2) as the starting material in the reaction scheme (1) is synthesized, for example, by the following process.

Reaction scheme (II)

(6)

(7)

wherein R is as defined above, and X is a halogen atom.

That is, a nitrobenzene derivative (4) is reacted with N-phenylformamide (5), and then the resulting compound

(6) is reduced by a catalytic reduction process, or reduced with a catalyst such as zinc, iron, tin, etc. in the presence of an acid to obtain an o-anilinoaniline derivative (7). Reaction scheme (III):

(9)

wherein R ¹ and X are as defined above.

The above o-anilinoaniline derivative (7)is reacted with dihalogenonitrobenzene (8) to obtain a phenylenediamine derivative (9).

Reaction scheme (IV) :

/

wherein R ¹ and X are as defined above.

The above phenylenediamine derivative (9 ) is reacted with p-chloroaniline (10), and then the resulting compound (11) is reduced by a catalytic reduction process, or reduced with a catalyst such as zinc, iron, tin, etc. in the presence of an acid. The resultant is cyclized to form a phenazine ring, followed by oxidation in the presence of air to obtain a 2, 10-dihydrophenazine derivative represented by the general formula (2) .

The dihydrophenazine derivative (1) includes a salt to which a pharmaceutically acceptable acid is added.

Examples of the salt include inorganic acid salts such as hydrochloride, hydrobromide, nitrate, sulfate and phosphate; and organic acid salts such as methanesulfonate, p- toluenesulfonate, acetate, citrate, tartrate, maleate, fumarate, maleate and lactate.

The dihydrophenazine derivative ( ) includes geometric isomers (e.g. cis-isomer, trans-isomer, syn-anti isomer) and optical isomers.

The dihydrophenazine derivative (1) is normally used in the form of a normal medical drug.

Next, the drug containing the dihydrophenazine derivative (1) of the present invention or a salt thereof as the active ingredient will be described.

The drug is that obtained by formulating the dihydrophenazine derivative (1) into the form of a normal

medical formulation, and is prepared by using diluents or excipients which are normally used, such as fillers, extenders, binders, moistening agents, disintegrators, surfactants and lubricants. The form of the drug can be selected from various forms depending on the therapeutic purpose. Typical examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories and injections (e.g. solutions, suspensions). When shaping into the form of tablets, a variety of conventional carriers can be used. There are, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, potassium carbonate, kaolin, crystalline cellulose and silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and polyvinyl pyrrolidone; disintegrators such as dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch and lactose; disintegration inhibitors such as sucrose, stearin, cacao butter and hydrogenated oil; absorption accelerators such as quaternary ammonium base and sodium lauryl sulfate; humectants such as glycerin and starch; absorbents such as starch,

lactose, kaolin, bentonite and colloidal silicic acid; and lubricants such as purified talc, stearate, borax and polyethylene glycol.

The tablets can be optionally formulated into tablets subjected to normal tablet coating (e.g., sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets), double tablets or multilayer tablets.

When shaping into the form of pills, a variety of conventional carriers can be used. There are, for example, excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin and talc; binders such as gum arable, tragacanth powder, gelatin and ethanol; and disintegrators such as laminarane and agar.

When shaping into the form of suppositories, a variety of conventional carriers can be used. There are, for example, polyethylene glycol, cacao butter, higher alcohol, esters of higher alcohol, gelatin and semisynthetic glyceride.

When preparing as injections, it is preferred that solutions, emulsions and suspensions are sterilized and are isotonic with blood. When shaping into the form of these solutions, emulsions and suspensions, a variety of conventional diluents can be used. There are, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, sodium chloride,

glucose or glycerin whose amount is sufficient to prepare an isotonic solution, may be contained in the medical formulation. Normal solubilizing agents, buffering agents and soothing agents and, if necessary, colorants, preservatives, perfumes, flavors, sweeteners and other medicines may also be contained.

When shaping into pastes, creams and gels, for example, there can be used vaseline, paraffin, glycerin, cellulose derivative, polyethylene glycol, silicon and bentonite as the diluent.

The amount of the dihydrophenazine derivative (1), which is contained in the drug, or a salt thereof is not specifically limited, and can be selected within a wide range. The amount is preferably from 1 to 70 % by weight based on the total composition.

The administration method of the drug is not specifically limited, and it may be appropriately selected from methods which have hitherto been known according to various dosage forms, age and sex of the patient, symptom of diseases and other conditions. In the case of tablets, pills, solutions, suspensions, emulsions, granules and capsules, an oral administration can be selected.

In the case of injections, the drug can be intravenously administered as it is or after mixing it with a normal fluid replacement such as glucose, amino acid, etc. If

necessary, the drug can also be intramuscularly, intracutaneously, subcutaneously or intraperitoneally administered as it is. In the case of suppositories, the drug is intrarectally. The dose of the drug is not specifically limited, and it may be appropriately selected according to direction for use, age and sex of a patient, symptom of diseases and other conditions. The drug is normally administered 1 to 4 times per day with a dairy dose of 1 to 100 mg/kg, preferably 2 to 20 mg/kg.

FIET.P OF INDUSTRIAT. APPLTflABTT.TTY

The dihydrophenazine derivative and the pharmaceutically acceptable salt thereof have excellent antibacterial and bactericidal activities to tubercle bacilli and a wide range of atypical acid-fast bacilli, including M. avium complex (mycobacteria which develop as immunological function reduces by infection with AIDS), M. chelonea, etc.

The dihydrophenazine derivative and the pharmaceutically acceptable salt thereof are also superior in characteristics such as duration time of drug efficacy, stability and absorption-excretion.

Furthermore, it is found that the dihydrophenazine derivative or the pharmaceutically acceptable salt thereof also has advantage that, when being used in combination with r-interferon, the tuberculostatic action is enhanced.

^• RXAMPT.ES

The invention will be further illustrated by he following Reference Examples and Examples. Reference Example 1 Synthesis of 4- (4-chloroanilino .-3-nitrotr.lιip.ne

A mixture of 4-fluoro-3-nitrotoluene (4.0 g, 25.8 mmol), 4-chloro-N-formylaniline (4.2 g, 27.1 mmol), potassium carbonate (17.8 g, 0.13 mol) and N,N-dimethylformamide (DMF) (10 ml) was heated with stirring on an oil bath at 130 °C for 5 hours. After disappearance of the raw material was confirmed by thin-layer chromatography (TLC) (stationary phase: silica gel ("silica gel 60" manufactured by Merk Co., hexane:ethyl acetate = 5:1)), the reaction solution was added to water (200 to 300 ml). After stirring vigorously, a precipitated orange crystal was filtered and then washed with a mixed solution of methanol and water (1:1). Then, the crystal was dried under reduced pressure at 50 to 60 °C to obtain the titled compound (5. 2 g, 19.7 mmol), yield 77 %. Melting point: 111.9-113.1 °C. Reference Example 2

Synthesis of 3-amino-4-(4-chloroanilino oluene

4-(4-Chloroanilino)-3-nitrotoluene (5.2 g, 19.7 mmol) obtained in Reference Example 1, ammonium chloride (5.3 g, 98.6 mmol), ethanol (50 ml) and water (10 ml) were mixed, and then zinc (6.45 g, 98.6 mmol) was slowly added while

stirring vigorously at the reaction temperature of not more than 40 °C. After the color of the reaction solution turned into green and the disappearance of the raw material was confirmed by TLC (the same as that of Reference Example 1), the reaction solution was filtered to remove zinc. The residue was washed with dichloromethane, and then the wash and filtrate were mixed. The filtrate was concentrated under reduced pressure and, after adding water, the precipitated crystal was filtered, washed with water and dried under reduced pressure at 40 to 50 °C to obtain the titled compound (4.5 g, 19.1 mmol), yield 97 %.

Melting point: 132.2-133.1 °C. Reference Example 3 Synthesis of N-4-C4-chlorophenyl)-N' - (5-c_hloro-2 _J .4- dinitrophenyl)-4-methyl-l,2-phenylenediamine

A mixture of 3-amino-4-(4-chloroanilino)toluene (17.5 g, 75.2 mmol) obtained in Reference Example 2, 1,3- dichloro-4,6-dinitrobenzene (18.7 g, 79.0 mmol), ethanol (100 ml) and triethylamine (14 ml) was refluxed with stirring for 3 hours. After the disappearance of the raw material was confirmed by TLC (the same as used in Reference Example 1), the reaction solution was stirred with ice cooling. The precipitated crystal was filtered, washed with water, washed with methanol (about 500 ml) and then dried under reduced pressure at 50 to 60 °C to obtain the titled compound (28.5 g,

65. 8 mmol ) , yield 87 % .

Melting point: 206.0-207.3 °C Reference Example 4

Synthesis of N-4-_ 4-chlorophenyl )-N' -[5-_ 4-(__hloroanilino)-2 _f 4- dinitrophenyl]-4-methyl-l _r 2-phenylenediπmine

A mixture of N-(4-chlorophenyl)-N'-(5-chloro-2,4- dinitrophenyl)-4-methyl-l,2-phenylenediamine (23.5 g, 54.2 mmol) obtained in Reference Example 3, p-chloroaniline (20.8 g, 0.16 mmol) and n-butanol (200 ml) was refluxed with stirring overnight. After the disappearance of the raw material was confirmed by TLC (stationary phase: the same as that of Reference Example 1, dichloromethane:hexane = 2:1), the reaction solvent was distilled off under normal pressure so that the amount was reduced by about half. Then, the reaction solution was cooled and, after adding methanol

(200 ml), the mixture was stirred. The precipitated crystal was filtered and then washed with methanol (500 ml). The resulting crystal was dried under reduced pressure at 50 to 60 "C to obtain the titled compound (27.8 g, 53.0 mmol), yield 98 %.

Melting point: 190.1-191.0 °C. Reference Example 5 Synthesis of 3-_ 4-chloroanilino)-10-(4-chlorophenyl)-7-methyl-

To N-(4-chlorophenyl)-N'-[5-(4-chloroanilino)-2,4-

dinitrophenyl]-4-methyl-l,2-phenylenediamine (42.6 g, 81.2 mmol) obtained in Reference Example 4, acetic acid (400 ml) was added, and the mixture was stirred to cause suspension. To this suspension, zinc (53.1 g, 0.81 mmol) was added at the reaction temperature of not more than 40 °C over about 1.5 hours, followed by stirring for 30 minutes. After the disappearance of the raw material was confirmed by TLC (stationary phase: the same as that of Reference Example 1, dichloromethane:methanol = 10:1), the reaction solution was filtered to remove zinc. The residue was washed with acetic acid (200 ml), and then the wash and filtrate were mixed. After the filtrate was stirred overnight in the presence of air, the acetic acid was distilled off under reduced pressure. To the resulting residue (crystal), methanol (300 ml) and water (30 ml) were added and, after stirring, the crystal was filtered by subjecting to an ultrasonic wave treatment. Then, the crystal was washed with methanol, washed with water and then dried under reduced pressure at 70 to 80 °C to obtain the titled compound (34.8 g, 68.6 mmol), yield 85 %. Melting point: 192.5-193.7 °C (with decomposition)

Example 1

Synthesis of 3-.4-chloroanilino)-10-(4-chlorophenyl)-2- cyclooc:tylimino-7-methyl-2 _r 10-dihydrophenazine

A mixture of 3-(4-chloroanilino)-10-(4- chlorophenyl)-7-methyl-2,10-dihydrophenazine acetate (3.0 g,

5.9 mmol) obtained in Reference Example 5, 1,4-dioxane (15 ml) and cyclooctylamine (2.3 g, 17.8 mmol) was refluxed with stirring for 24 hours. After disappearance of the raw material was confirmed by TLC (the same as used in Reference Example 5), 1,4-dioxane was distilled off. To the resulting residue, methanol (30 ml) was added and, after stirring, a crystal was filtered and then washed with methanol (20 ml) . Then, the crystal was dissolved in dichloromethane (40 ml) and a 4N hydrogen chloride-ethyl acetate (5 ml) was added, followed by stirring for 30 minutes. Furthermore, ethyl acetate (20 ml) was added and the dichloromethane was distilled off under reduced pressure at room temperature. The precipitated crystal was filtered, recrystallized from ethanol (100 ml) and then dried under reduced pressure at 80 to 90 °C to obtain hydrochloride of the titled compound (3.0 g, 5.1 mmol), yield 85 %, purity 99.6% due to high performance liquid chromatography (HPLC) (stationary phase: YMC ODS-A A-302, eluent: aqueous 70 % acetonitrile solution-0.05 % trifluoroacetic acid) . Melting point: 205.2-206.5 °C

Then, hydrochloride of the titled compound (800 mg, 1.4 mmol) was dissolved in methanol (50 ml) and an aqueous IN sodium hydroxide solution (3 ml) was added, followed by stirring for 15 minutes. Water (5 ml) was added to the solution, which was stirred additionally. A precipitated

crystal was filtered, washed with methanol, water and hexane in this order, and dried under reduced pressure at 80 to 90 °C to obtain the titled compound (668 mg, 1.2 mmol), yield 89 %, purity due to HPLC 99.8 %. Melting point: 193.3-195.0 °C

Example 2

Synthesis of 3-(4-chloroanilinn .-10-.4-πhlnrnphenyl ^* _-2- r.yπlooctylimino-7-hydroxymethyl- _r 10-dihydrnphpnr. τnp.

The titled compound was obtained in the same manner as in Example 1, except that 3-(4-chloroanilino)-10-(4- chlorophenyl)-7-hydroxymethyl-2, 10-dihydrophenazine acetate was used as the starting material. Formulation Example

Hereinafter, Formulation Examples will be described. Formulation Example 1

3-(4-Chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-methyl-2,10-dihydrophenazine 200 mg

Glucose 250 mg Distilled water for injection q.s.

Total amount 5 ml

3-(4-Chloroanilino)-10-(4-chlorophenyl)-2-

cyclooctylimino-7-methyl-2,10-dihydrophenazine obtained in Example 1 and glucose were dissolved in distilled water for injection, and the solution was poured into a 5 ml ampul. After substituting with nitrogen, the ampul was sterilized under pressure at 121 °C for 15 minutes to produce an injection of the above composition. Formulation Example 2

3-(4-Chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-methyl-2,10-dihydrophenazine 100 g

Abisel (trade name, manufactured by Asahi 40 g

Kasei Co., Ltd. )

Corn starch 30 g

Magnesium stearate 2 g TC-5 (trade name, manufactured by Sinetsu Kagaku

Kogyo Co., Ltd., hydroxypropylmethyl cellulose) 10 g

Polyethylene glycol 6000 3 g

Castor oil 40 g

Ethanol q.s.

Total amount 255 g

After 3-(4-chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-methyl-2,10-dihydrophenazine obtained in Example 1, abisel, corn starch and magnesium stearate were

mixed and pulverized, the mixture was compressed with a punch having a sugar coating R of 10 mm. The resulting tablets were coated with a film coating agent comprising TC-5, polyethylene glycol 6000, castor oil and ethanol to produce film-coated tablets of the above composition. Formulation Example 3

3-(4-Chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-methyl-2,10-dihydrophenazine 2 g Purified lanolin 5 g

White beeswax 5 g

Vaseline 88 g

Total amount 100 g

White beewax was heated to a liquid state, to which were added 3-(4-chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-methyl-2,10-dihydrophenazine obtained in Example 1, purified lanolin and vaseline. The mixture was heated to a liquid state, and was stirred until it began to solidify to obtain an ointment of the above composition. Antibacterial test

The minimum inhibitory concentration (MIC) with respect to the 3-(4-chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-methyl-2,10-dihydrophenazine obtained in

Example 1 and the 3-(4-chloroanilino)-10-(4-chlorophenyl)-2- cyclooctylimino-7-hydroxymethyl-2,10-dihydrophenazine obtained in Example 2 were determined by an agar dilution plate method.

The antibacterial test was conducted according to the method described in Chemotherapy, 29, 76-79, (1981), and the vial microbe cell number of the respective bacilli was adjusted to about 1 x 10 ⁶ /ml (O.D. 600 βm, 0.07 to 0.16).

The results of the antibacterial test are shown in Table 1.

Table 1

(Unit: g/ml)

Strain Example 1 Example 2

M. tuberculosis H37Rv 0.39 0. 1

M. tuberculosis Kurono 0.39 0. 2

M. avium IF03082 1 . 56 -

M. intracellulare ATCC15984 0.78 0. 2

M. Kansasii NIHJ1619 0.2 0. 2

M. marinum W2 0. 2 0.05

M. scrofulaceum ATCC19981 1 . 56 1. 56

M. gordonae ATCC14470 0.78 0. 1

M. nonchromogenicum ATCC19530 1. 56 3. 13

M. fortuitum NIHJ1615 1 . 56 3. 13

M. chelonea NIHJ1609 6.25 12. 5