IL32324A - Phenazine derivatives and their preparation - Google Patents

Description

32324/2 Tnaam τ***" ηπ^ιη Phenazine derivatives and their preparation SPARAMEDICA A.G.

- RAN 4410/57 This invention relates to phenazine derivatives of the general formula wherein R is lower alkyl, preferably methyl, ethyl or propyl, and to a process for the manufacture thereof.

The preferred compound is a cupric complex of 6-methoxy- 1-phenazinol 5*10-dioxide, i.e. a compound of formula I, wherein R is methyl.

The process in accordance with the invention for preparing the novel compounds of formula I comprises treating a solution of a β-lower alkoxy-l-phenazinol 5*10-dioxide with a solution of a cupric salt.

The cupric complex can be isolated as crystals by precipitation from the mother liquors. The selection of a relative solubilities which can be readily done by those of ordinary skill in the art. As the solvent, it is preferred to utilize a solvent in which both the 6-lower alkoxy-l-phenazinol 5,10-dioxide and the cupric salt reagents are more soluble than the complex reaction product formed therefrom. Exemplary solvents which can be conveniently employed are the inert organic solvents such as acetonitrile, dimethylformamide , dioxane, etc. Any cupric salt can be employed as starting material. However, since, in the formation of the cupric complex of formula I there is also formed the acid corresponding to the anionic moiety of the cupric salts and the cupric comple is less stable under strongly acidic conditions, it is preferred to utilize a cupric salt of a weak acid. As a suitable cupric salt there can be utilized a cupric salt formed from any of the weak acids, e.g., acids having a pKa of about 4.2 or greater. Exemplary acids answering this description are the organic acids such as the lower alkanoic acids, e.g., acetic acid, propionic acid, etc., benzoic acid and the like. There can also be employed cupric salts of mineral acids such as, for example, cupric sulfate. When employing a mineral acid salt,it is preferred to utilize a buffered solvent system in order to avoid highly acidic conditions. The cupric complex of formula I cen be readily prepared by simply combining a saturated solution of the 6-lower alkoxy-l-phenazinol 5*10-dioxide starting material in a suitable solvent with a saturated solution of a cupric salt in the same solvent at room temperature and separating the cupric complex which precipitates. Temperatures above room temperature can be employed in order to facilitate the precipitation of the complex can also be facilitated by cooling. The separation of the precipitated crystalline complex is accomplished by the usual techniques for solvent removal such as, for example, by filtration, solvent displacement followed by filtration, etc.

The 6-lower alkoxy-l-phenazinol 5,10-dioxide starting materials employed in the preparation of the copper complexes of this invention are known compounds or analogs which can be readily prepared by selectively alkylating the known compound 1,6-phenazinol 5>10-dioxide (iodinin). The selective alkylation can be readily accomplished by treating 1 ,6-phenazinediol 5,10-dioxide with an alkylating agent such as di-lower alkylsulfate in the presence of an aqueous alkali such as sodium hydroxide. This process in its preferred mode is accomplished by first preparing the sodium salt of the 1,6-phenazinediol 5,10-dioxide and thereafter treating with an alkylating agent in a nonaqueous solvent.

The novel cupric complexes of formula I have been found to possess broad spectrum anti-microbial activity. In particular, the cupric complex of 6-methoxy-l-phenazinol 5*10-dioxide has demonstrated a high level of activity against a wide variety of both Gram-positive and Gram-negative bacteria such as Streptococcus pyogenes, Streptococcus agalactiae, Diplococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhosa, Salmonella schottmuelleri, Pasteurella multocida, against fungi and yeasts such as Candida albicans, Trichophyton mentagrophytes, Microsporum canis, Microsporum audouini, Ustilago zeae, Pusarium oxysporum, Botrytis paeoniae, Aspergillus flavus and Aspergillus niger; against protozoa such as Trichomonas vaginalis and Trichomonas foetus; and against helminths such as Syphacia obvelata and Ascaris suum. The novel cupric complexes of this invention are also active against plant pathogens and can be employed as foliar fungicides or soil fungicides. These compounds have, for example, demonstrated a high degree of activity against Phytophthora infestans, the causative organism in late blight of tomatoes and against Xanthomonas vesicatoria, the causative agent of bacterial leaf spot of tomatoes and against Piricularia oryzae, the pathogen associated with rice blast.

The novel compounds of this invention are particularly useful in the treatment of plant and animal diseases of microbial origin by virtue of their low mammalian toxicity and phyto toxicity. When the novel compounds of this invention are employed in the treatment of microbial, e.g. bacterial, helminth and/o protozoal infections as well as ectoparasitic infections or infestations, they are conveniently utilized in composition with suitable carrier materials for use as chemo-therapeutic agents in combatting mammalian . diseases. They are formulated by uniformly distributing in a vehicle that is chemically compatible with the particular compound selected and are non-inhibiting with respect to the active ingredient and essentially non-injurious to body tissue under the conditions employed in amounts ranging from about 0.1 to about 0.5^ by weight of the chemotherapeutic composition, e.g., gel, cream, ointment, suspensions, suppository or the like. It will be understood that the compounds of this invention when employed in forms suitable for topical administration may be utilized in diverse formulations: for example, solid formulations including finely divided powders and granular materials; liquid formulations including suspensions, concentrations, slurries, tinctures, aerosols and the like, depending on the application intended and the formulation media desired. They may be employed as creams, gels, jellies, ointments, pastes, etc. When utilizing formulations in which the compounds of this invention have partial solubility, e.g., those containing propylene glycol, the molar ratio of the myxin and copper changes in the dissolved complex since some of the materials present, e.g., solvent, can act as a ligand. This invention is intended to include such compositions .

A still further aspect of this invention involves the use of the novel cupric complexes for a variety of industrial antimicrobial applications. Thus, the chemical, physical and biological properties of these complexes make them suitable for the following uses: mildew proofing fabrics such as textiles, canvas and other woven and non-woven materials; as paint fungicides; as a fuel additive for the control of microbial growth; as preservative agents for clothes and shoes; as topical antiseptics; as disinfectant agents; plant and tree spray, anti-mycotic agents for soaps; mildew proofing agents in plastics, cardboard, fiberboard, paper, etc.; slimicides in pulp liquors, etc.; additives in cosmetics, shampoos, deodorants, mouthwashes, etc.

For the purpose of the present invention, the term "anti-microbial activity" is meant to include all the various activities mentioned above.

In one of its more particular aspects, the cupric complexes of this invention, particularly the compound 6-methoxy-l-phenazinol 5>10-dioxide, cupric complex, have demonstrated a remarkably high degree of activity both in vitro and in vivo against pathogens, particularly the bacteria and yeasts which are the etiologic agents of bovine mastitis. Known anti-mastitis agents are not completely satisfactory since their effectiveness is limited by their having relatively narrow spectra of antimicrobial activity and also by the fact that they may cause irritation. Thus, the compounds of this invention, owing to their potent broad spectrum anti-microbial activity and their non-irritating properties, are particularly effective as anti-mastitis agents. The compounds, especially 6-methoxy-l-phenazinol 5> 10-dioxide, cupric complex, have been found in actual application to the udder of ruminants, especially cows, to be non-irritating, potent anti-mastitis agents. The compounds of formula I when used as anti -mastitis agents can be applied directly to the udder of the animal by topical administration, e.g., inserts, or by intramammary infusion.

Thus, in a more specific embodiment this invention comprises the use of a compound of formula I as an anti-mastitis agent and compositions comprising a compound of formula I together with carrier materials suitable for topical or intramammary administration. For use as anti-mastitis agents the compounds of formula I are effectively employed in amounts between about mg and about 50 mg per dose administered twice daily for about, one or two days or until a therapeutic response is obtained which can occur even after a single administration. A preferred dosage range is between about 5 and ½0 mg per dose administered in a suitable vehicle in 20 ml dose amounts. The optimal dose is a single dose containing 20 mg of active sub-stance.

Intramammary infusion preparations typically contain oil dispersible or water dispersible vehicles. The vehicles are composed of inert ingredients, e.g., surface active agents, antioxidants and the like with vegetable oil or water carriers. Typical ingredients which are pharmaceutically acceptable for use in compositions used for intramammary infusion administration are sodium citrate, citric acid, magnesium aluminum silicate ,carboxymethyl cellulose, polyoxyethylene (20)sorbitan monooleate, sorbitol, glycerin, microcrystalline cellulose, propylene glycol, benzyl alcohol, polyoxyethylene (20) sodium monostearate, butylated hydroxyanisole , butylated hydroxy-toluene, lecithin, finely divided silica, hydrogenated castor oil, methyl paraben, propyl paraben, chlorbutanol, aluminum monostearate, sorbitan monostearate, modified 1-hydroxy stearin, glycerol monostearate, polyoxyethylene sorbitol tristearate, dichlorodifluoromethane, carbon tetrafluoride and dimethyl polysiloxane.

In vivo tests on mice infected with mastitis isolates have been shown to demonstrate the anti-mastitis effectiveness of known anti-rnastitis agents and such in vivo test results have been well correlated with anti-mastitis activity in animals susceptible to mastitis infections, e.g., dairy cows. The test procedure involves determination of activity in the presence of milk to insure ef ectiveness in the presence of organic fluids. The in vivo test method employed in the determination of the anti-mastitis activity of the compounds of formula I is as follows: Material and Methods Medium Sterile milk containing less than 1 bacteria/5 ml is prepared.

Cultures Bacterial strains used to contaminate the milk are field isolates of bovine mastitis and are maintained on tryptose agar. Twenty-four hours growth on tryptose agar is used for contaminating the milk. The final concentrations of the organisms in the milk are as follows: 7 a) Streptococcus agalactiae - 5 x 10 organisms per ml o b) Staphylococcus aureus - 2 x 10 organisms per ml o c) Escherichia coli - 2 x 10 organisms per ml d) Pseudomonas aeruginosa - 5 x 10 organisms per ml The subcutaneous injection into 18 to 20 gram. mice of 0.5 ml of milk contaminated with the strains indicated above agalactiae are preconditioned with triamcinolone acetonide in order to show well defined lesions.

Testing Procedure A 50 mg amount of 6-methoxy-l-phenazinol 5, 10-dioxide, cupric complex is ground into 10 ml of sterile distilled water, pH 7 to 7· 2, using a Teflon grinder and further dilutions containing 1, 0.2, 0.04 and 0.006 mg of compound per ml are prepared in sterile distilled water. A 0.5 ml amount of each dilution of the compound is added to a tube containing 4.5 ml of contaminated milk (l tube/dilution) so that the final concentrations of the compound in the miik are 500, 100, 20, 4 and 0.8 micrograms per ml. After vigorous shaking to assure a uniform distribution of the compound throughout the milk, 0.5 ml of each combination (contaminated milk plus compound) is injected subcutaneously into 4 white mice of IS to 20 gram weight. Normal mice are used for the Staphylococcus, Coli and Pseudomonas infections while preconditioned mice (injected subcutaneously 24 hours in advance with 1 mg of triamcinolone acetonide) are used for the Streptococcus infection. Each group of mice is kept in a separate cage and receives normal pelleted food and water ad libitum. The animals are observed twice a day for two days after injection and sacrificed on the third day. A record is kept for signs and deaths. An autopsy is performed on all mice which die during test and on all survivors sacrificed on the third day of the test. Activity of the compound against the 4 bacterial strains is evaluated as follows: Streptococcus - absence of pus and necrosis at the site of the injection; Staphylo^ coccus - absence of necrosis at the site of the' injection and of gelatinous edema and necrosis at the site of injection, lack of signs and death.

Controls Controls are run with every test to establish: 1. sterility of milk, saline and distilled water used in the test; 2. bacterial count of the contaminated milk; J. purity of the strains used to contaminate the milk; 4. titration of the infecting inoculum in mice; · lack of pathogenic effect of non-contaminated milk.

Evaluation of the Results Using the method described, the following criteria and parameters are obtained: 1. protection of animals from local lesions induced by inoculation of milk contaminated with Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa; 2. protection from death and clinical symptoms induced by Staphylococcus and Pseudomonas.

Results Using this procedure, the activity of the anti-mastitis formulations is established against four strains of bacteria. The following are the results of the testing of an exemplary compound useful in the compositions of this invention. Tables I and II show the anti-bacterial efficacy both in vitro and in vivo.

Table I In vitro activity of 6-alkoxy-l-phenazinol ,10-dioxide, cupric complexes MINIMUM INHIBITORY CONCENTRATION 6-methoxy-l- 6-ethoxy-l- phenazinol phenazinol ,10-dioxide, 5,10-dioxide, cupric complex cupric complex Organism Streptococcus agalactiae less than 0.16 less than 4 Staphylococcus aureus 0.16 less than 4 Escherichia coli 2.4 20 Pseudomonas aeruginosa 12 100 Table II In vivo anti-bacterial activity as EDC~ mcg/ml S. S. E. P.

Compound agalactiae aureus coli aeruginosa 6-methoxy-l-phenazinol 0.8 6o 12 60 5, 10-dioxide , cupric c In addition to their use as anti-mastitis agents, the compounds of formula I are useful in the treatment of a wide variety of animal and plant infections. The compounds of formula I have been found to be particularly useful in the treatment of localized animal infections caused by a variety of organisms.

The novel cupric complexes of this invention can be effectively employed in medical therapy such as the treatment of bacterial, yeast and/or mycotic infections of man and animals where the skin, hair, nails and/or other areas of the body are infected.

Thus, in still another of its specific embodiments this invention involves the use of compounds of formula I and novel compositions containing these compounds for the treatment of infections of the skin, ear, eye and genitals of animals. Thus, the anti-bacterial activity of these compounds against skin infections caused by Staphylococcus aureus and Pseudomonas aeruginosa has been shown by the following in vivo tests.

Anti-bacterial activity Scarification of rabbit skin followed by infection with Staphylococcus aureus and Pseudomonas aeruginosa. Two rabbits/ infection. Treatment - 4 days, total treatments - 7. Therapeutic efficacy is obtained by compounding clinical observations after 1, 2 and 3 days of treatment and 3 days after the end of treatment and comparing the treated with the non-treated and the placebo-treated areas.

THERAPEUTIC EFFICACY PER CENT S. P.

Compound aureus aeruginosa 6-methoxy-l-phenazinol ,10-dioxide, cupric complex - 0.5 per cent 70 48 0.1 per cent 57 78 The activity against yeast has been demonstrated by New Zealand white rabbits conditioned with triamcinolone acetonide were infected by scarification with a strain of Candida albicans and treated for two cycles of 5 days with experimental formulations. Therapeutic efficacy is evaluated by scoring the lesions for improvement at weekly intervals and comparing the treated with the non-treated and placebo-treated areas of the skin.

THERAPEUTIC EFFICACY PER CENT Per 1 week 2 week 1 week after Compound Cent treatment treatment treatment 6-methoxy-l-phenazlnol 5*10-dioxide, cupric complex - gel 0.5 73 58 100 0.1 47 4 0.02 60 92 89 Gel placebo 40 0 11 6-methoxy-l-phenazinol 5*10-dioxide, cupric complex - cream 0.1 50 44 33 Cream placebo 33 22 12 6-methoxy-l-phenazinol 5» 10-dioxide, cupric complex in petro-latum base 0.5 74 89 100 Petrolatum placebo 0 11 33 Anti -fungal activity has been demonstrated in animals by the following test.

Albino guinea pigs conditioned with triamcinolone acetonide were infected by multiple stabbing with a spore suspension of Trichophyton mentagrophytes and treated for two cycles of 5 days with experimental formulations. Therapeutic efficacy is evaluated by scoring the lesions for clinical improvement at weekly intervals and comparing the treated with the non-treated and placebo-treated areas of the skin.

ANTI-TRICHOPHYTON MENTAGROPHYTES ACTIVITY THERAPEUTIC EFFICACY PER CENT 1 week 2 weeks Per 2 week after after Compound cent treatment treatment treatment 6-methoxy-l-phenazinol ,10-dioxide, cupric complex - gel 0.5 96 99.5 98.5 0.1 75 77 71 0.05 65 65 61 6-methoxy-l-phenazinol ,10-dioxide, cupric complex - cream 0.5 87 86 9 0.1 42 82 81 Cream placebo 23 32 6-methoxy-l-phenazinol ,10-dioxide, cupric complex in petrolatum base 0.5 78 89 79 Petrolatum placebo -11 -20 ■30 Foliar fungicide activity of the novel complexes of this invention has been demonstrated in standard tests for foliar fungicide activity. Exemplary results of such tests with 6-methoxy-l-phenazinol 5, 10-dioxide , cupric complex are shown in the following table.

Concentration Plant Pathogen Compound (PPM) per cent control : lant injury* 6-methoxy-l-phenazinol 5» 10-dioxide, cupric complex 100 Phytophthora infe 7^:0 Xanthomonas vesicatoria 82:0 Piricularia ory 66:0 * 0 indicates no visible effect The activity against protozoa has been demonstrated by the following tests in animals infected with Trichomonas vaginalis : Groups of 7 to S albino mice weighing IS to 20 grams were infected subcutaneously on the abdominal surface with approximately 500, 000 cells. The animals were treated by infiltration into the site of infection with dose levels of the compounds of formula I of from 2 meg. to 1000 meg. per ml. of solution on the day of the infection and the day after infection. The mice were examined the third day after infection for lesions at the site of the infection. The number of animals with lesions and the number of lesion-free animals were determine for each dose level tested.

The CD50 was calculated using the method of Reed and uench [American Journal Hygiene, Vol. 27 , page 93 (1933 ) ] .

Cumulative Dose No No Compound meg. /ml. s.c. lesion Lesion lesion Lesion 6-methoxy-l- 1000 35 5 121 5 phenazinol 5 , 10- 400 22 10 36 15 dioxide, cupric 200 19 12 64 27 complex 100 11 5 32 40 11 5 34 37 11 5 23 42 7 1 12 43 4 3 5 5 43 2 2 6 2 54 The CD50 is 43 meg. /ml. s.c. based upon the data from th' tests .

Example 1 Preparation of 6-methoxy-l-phenazinol 5, 10-dioxide, cupric complex To a solution of 6 g of 6-methoxy-l-phenazinol 5 » 10-dioxide in 3 1 of acetonitrile was added with stirring a 75°C solution of 3 6 cupric acetate monohydrate in 900 ml acetonitrile. The mixture was allowed to cool and was kept at room temperature for hours. The precipitated cupric complex was collected by filtration and washed thoroughly on the filter, first with acetonitrile and then with ether. Thus, the complex was obtained in the form of dark green, fine crystals. Pertinent absorption bands in the IR spectrum: 13 5 cm 1 (in Fluorolube); 1059, 776, 578 cm"1 (in Nujol). U.V. absorption maxima in DMSO: 287 πΐμ - ( £ = 68,500) 300 πΐμ - ( ί. = 63,200) 356 πΐμ - ( £ = 10,000) 408 πιμ - ( ε. = 10,400) 610 Πΐμ - ( S. = 9,500) Analysis Calculated for C, 5 .03; H, 3.14; N, 9. 69; Cu, 10. 99 Found: C, 53.71 ; H, 3· 31 ; N, 9. 66; Cu, 10. 92 Example 2 In analogy to the procedure described in Example 1 above, there was also prepared 6-ethoxy-l-phenazinol 5, 10-dioxide, cupric complex starting with 6-ethoxy-l-phenazinol 5, 10-dioxide.

Analysis Calculated for - N, 9. 25 Found: N, 9. 12 Example 3 A 6 ml suspension is prepared by mixing together the following: 6-methoxy-l-phenazinol > 10-dioxide , cupric complex 10 mg Ethylenediamine 57 nig Distilled monoglycerides 60 mg Polyethylene glycol 400 q.s.

Infuse 6 ml into teat canal of each infected quarter. Repeat in 12 and/or 24 hours if necessary.

Example 4 ml suspensions are prepared using the following formulations: a) 6-methoxy-l-phenazinol 5»10-dioxide , cupric complex 20 mg Carboxymethyl cellulose 2 per cent Methylparaben 0.1 per cent Propylparaben 0.025 per cent Distilled water q.s. b) 6-methoxy-l-phenazihol 5» 10-dioxide , cupric complex 30 mg Carboxymethyl cellulose 2 per cent Methylparaben 0.1 per cent Propylparaben 0.025 per cent Polyoxyethylene (20) sorbitan mono- oleate 2 per cent Distilled water q.s. c) 6-methoxy-l-phenazinol 5> 10-dioxide , cupric complex 40 g Carboxymethyl cellulose 2 per cent Chlorbutanol 0.5 per cent Distilled Water q.s. d) 6-methoxy-l-phenazinol 5, 10-dioxide cupric complex 10 mg Carboxymethyl cellulose 2 per cent Chlorbutanol 0.5 per cent Polyoxyethylene (20) sorbitan monooleate 2 per cent Distilled water q.s.

Lactating cows are treated with 20 ml of suspension in each infected quarter immediately after milking. The suspension is allowed to remain in the quarter until the next milking. The treatment is repeated at 24 hour intervals if necessary.

Example 5 6-methoxy-l-phenazinol 5, 10-dioxide , cupric complex -anti mastitis preparation - 0.1 per cent Grams/Liter 6-methoxy-l-phenazinol 5» 10- dioxide, cupric complex 1.00 Polyoxyethylene (20) sorbitan monooleate 60.00 Sorbitan monooleate 40.00 Benzyl Alcohol 10.00 Grams/Liter Sodium acetate 2.00 non-ionic hydroxypropyl cellulose 19.0 Distilled water 1 liter Procedure : The o-methoxy-l-phenazinol 5,10-dioxide, cupric complex and benzyl alcohol were triturated in a suitable size stainless steel container to form a paste. Polyoxyethylene (20) sorbitan monooleate and sodium acetate were slowly added in that order with constant stirring. The hydroxypropyl cellulose was dispersed in 850 ml of distilled water at 50°C and then cooled to 30°C. This was then added to the previous ingredients and stirred until homogeneous. The preparation was then brought to final volume with distilled water.

Example 6 6-methoxy-l-phenazinol 5> 10-dioxide, cupric complex-topical cream - 0.1 per cent Grams per Kilo Mg. per Gram Part I: Stearyl Alcohol 125.00 Petrolatum 100.00 Nipasol 0.50 polyoxyethylene glycol 40.00 The polyoxyethylene glycol should be of a quality that is soluble in woler and ethanol, insoluble in cottonseed oil and mineral oil and milky in propylene glycol.

Grams per Kilo Mff. per Gram Part II: 6-methoxy-l-phenazinol 5>10-dioxide, cupric complex 1.00 Distilled water 612.00 Propylene Glycol 120.00 Nipagin 2.00 Procedure ; 1. The stearyl alcohol, petrolatum, nipasol and pol oxy- ethylene glycol were heated in a suitable size stainless steel container to 75°C. 2. The distilled water, propylene glycol and nipagin were heated in a separate suitable size stainless steel container to 75 °C. and stirred well. The 6-methoxy-l-phenazinol 5,10-dioxide, cupric complex was then added and stirred well. 3. Part II was slowly added to Part i while slowly mixing with continuous stirring. The stirring was continued at low speed, using a Lightning Mixer, until the cream cooled to 35 °C The cream was packaged in suitable size opal glass ointment jars.

Creams suitable for topical application according to the process of this invention will ordinarily contain from about 0.1 to about 1 per cent by weight of active ingredient, preferably between about 0.1 and about 0.5 per cent by weight.

Claims (21)

Having now particularly described and ascertained the nature of our said invention and in what manner the same is to be performed, we declare that what we claim is:

1. Process for the manufacture of phenazine derivatives the general formula wherein R is lower alkyl, which comprises treating a solution of a 6-lower alkoxy-1-phenazinol 5»10-dioxide with a solution of a cupric salt.

2. Process according to Claim 1, wherein a saturated solution of the 6-lower alkoxy-l-phenazinol in an inert organic solvent is treated with a saturated solution of the cupric salt in the same inert organic solvent.

3. · Process according to any one of Claims 1 and 2, wherein there is employed a cupric salt of an acid having a pKa of about 4.2 or greater.

4. Process according to any one of Claims 1-J5, wherein

5. Process according to any one of Claims 1-4, wherein a 6-lower alkoxy-l-phenazinol 5»10-dioxide is used as starting compound, in which lower alkoxy represents methoxy, ethoxy or propoxy .

6. Process according to Claim 5, wherein 6-methoxy-l-phenazinol 5,10-dioxide is used as starting compound. ( M formula I in Claim 1

7. Process for the manufacture of phenazine as hereinbefore particularly described, especially with reference to Examples 1 and 2.

8. Process for the manufacture of preparations having anti-microbial properties, characterized in that a phenazine derivative of the general formula wherein R is lower alkyl, is mixed, as active substance, with nontoxic, inert, solid or liquid carriers, commonly used in such preparations.

9. Process according to Claim 8 for the manufacture of an effective amount of the pheiiazine derivative is mixed with a pharmaceutically acceptable carrier commonly used in anti-mastitis preparations.

10. Process according to any one of Claims 8 and 9 wherein a phenazine derivative of formula I is employed, in which R is methyl.

11. Compositions having anti-microbial properties, containing a phenazine derivative of the general formula wherein R is lower alkyl, and a carrier.

12. Compositions according to Claim 11 for controlling mastitis in cattle containing as the active ingredient an effective amount of the phenazine derivative together with a pharmaceutically acceptable carrier.

13. Compositions according to any one of Claims 11 and 12, wherein a phenazine derivative of formula I is present, in which R is methyl.

14. Phenazine derivatives of the general formul wherein R is lower alkyl, whenever prepared according to the process claimed in any one of Claims 1-7 or by an obvious chemical equivalent thereof.

15. Compounds according to claim 14, wherein R is methyl, ethyl or propyl, whenever prepared according to the-process claimed in claim 5 or by an obvious chemical equivalent thereof.

16. A compound according to claim 14 wherein R is methyl, whenever prepared according to the process claimed in claim 6 or by an obvious chemical equivalent thereof.

17. A compound according to claim l6 in crystalline form, whenever prepared according to the process claimed in claim 6 or by an obvious chemical equivalent thereof.

18. Phenazine derivatives of the general formul wherein R is lower alkyl.

19. Compounds according to claim l8, wherein R is methyl, ethyl or propyl.

20. A compound according to claim 18, wherein R is methyl.

21. A compound according to claim 20 , in crystalline form. For the Applicants DR. EiNHOLD COHlTMID PARTNERS