CA1116522A - Antitumor agents - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
This disclosure describes compositions of matter useful as inhibitors of transplanted mouse tumor growth and the method of inducing the regression and/or palliation of leukemia and related cancers in mammals therewith, the active ingredients of said compositions of matter being certain 1,4-bis(substituted-amino)anthraquinones and the non-toxic acid-addition salts thereof.

Description

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l3l~lrJl ~lJMM~i~Y 01;`'1'11~ INVEN~ N
_ _____ __ __ ._ __ 'l'his inventiorl relates to novel compositions of matter useful for amel.iorating cancer diseases in mammals.
More particularly, i.t relates to therapeutic compositions containing certain 1,4-bis(substituted-amino)anthrayuinones or the non-to~ic acid-addi~ion salts thereof which inhibit the growth oF transplanted mouse tumors. The invention includes the new compositions of matter and the method of inducing the regression and/or palliation of leukemia and related cancers in mammals therewith. The 1,4-bis(sub-stituted-amino)-anthraquinones of the present invention may be represented by the following structural formula:

O Nil-Q-N~ 1 ~ R 2 Nll-Q~
~R2 wherein Q is - CH2CH2-r and- ~ 1 is aminor methylamino!

ethylamino, dimethylamino, diethylamino ! piperidino or morpholino.

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6~ Z~3 ~so include~d within the ~urvi.ew of the present .i.nvention are the leuco bases and tautomers tihereof which may be represented by the followillg structural formulae ~/here.ill the coml?lete side chains at the l-l?osition and the 4-pOSitiOIl are not dep:icted for sake of hrevity:

O Ni3-Q-N' ~ 1~ 1 (III, leuco hases) O N~l~Q-N' lS ~ I (IV, ~automeric form) O N-Q-N

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~j.,~ 1 In one aspect, the present invention relates to a pharmaceutical composition in dosage unit form comprising about one to about 400 mg. of a compound of the fo~nula:

N~l-Q-N
~ R2 ~ Q-N ~

the tautomeric forms and the pharmacologically ac oeptable acid-addition salts thereof~ ~herein Q is -CH2-CH2-; and -N ~ 1 is amino, methylamino, ethylamino, dimethylamino, diethylamino, piperidino or morpholino; in a~ix-ture with a non-aqueous phanmaoe utically acceptable carrier.

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DETAILED DESCRIPTION OF THE INVENTION
-The active compounds of the present invention are obtainable as reddish brown to blue black crystalline mater-ials having characteristic melting points and absorption spectra and which may be purified by leaching with lower alkanols since the free bases are insoluble in water and some of them are insoluble in most organic solvents. The organic bases of this invention (I, II, III and IV) form non-toxic acid-addition salts with a variety of pharmaco-logically acceptable organic and inorganic salt-forming reagents. Thus, acid-addition salts, formed by admixture of the organic free base with two or more equival~nts of an acid~ suitably in a neutral solvent, are formed wi~h such acids as sulfuric, phosphoric, hydrochloric, hydro-bromic, sulfamic, citric, lactic, malic, succinic, tartaric, acetic, benzoic, gluconic, ascorbic, and the like. For pur-poses of this invention the free bases are equivalent to their non-toxic acid-addition salts. The acid-addition salts of the organic bases of the present invention are, in general, crystalline solids, rela~ively soluble in water, methanol and ethanol but relatively insoluble in non-polar organic solvents such as diethyl ether, benzene, toluens, and the like~
The active compounds of the present invention may be readily prepared in accordance with the following reaction scheme:

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~ ¦ J -~ 1l2~ e~N~

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(I) wherein Rl, R2 and Q are as hereinabove defined. In accor-dance with this reaction scheme, quinizarin (Yl (o~r leuco-quinizarin) is condensed with an appropriate alkylene diamine (VI) in water or N,N,N',N'-te~ramethylethylenediamine as sol-vent at the reflux temperature for 1-15 hours to produce the corresponding bases. The leuco bases may be readily oxidized to the fully aromatic derivatives (I) by a variety of methods such as air oxidation or treatment with hot nitrobenzene, or treatment with chloranil, hydrogen peroxide, or sodium per-borate.
- The active compounds of the present invention possess the property of inhibiting the growth of trans-planted mouse twnors as established by the following tests.
I.~mphocytic leukemia P388 test (intra~ itoneal) __ The animals used are BDFl or CDFl~mice all of one sex, weighing a minimwn of 17 grams and all within a 3-gram weight range. There are 5 or 6 animals per test group. The t~lor transplant is by intraperitoneal injection of 0~1 ml.
of dilute ascitic fluid containiny 106 cells of lymphocytic leukemia P388. The tes-t compounds are administered intra-peritoneally on days one, 5 and 9; or 1-9 (relative to tumor inoculation) at various dosesO The animals are weighed and survivors are recorded on a regular basis for 30 days. The median survival time and the ratio of survival time for trea-ted (T~/control (C) animals are calculated. The positive control compound is 5~-fluorouracil, dosed at 60 mg./kg. of body weight. The results of this test with typical compounds of the present invention appear in Table I below. The criterion for efficacy is T/C x 100~ 125%.

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Lymphocytic leukemia P388 test (oral) The procedure used i5 the same as for the previ-ously descri~ed test for lymphocylic leukemia P388 except that the test compounds are administered orally at various doses rather than intraperitoneally. The results of this test with a typical compound of the present invention appear in ~able II below. The criterion for efficacy is T/C x 100 ~ 125%.

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_ Lymphocytic leukemia L1210 test The procedure is the same as fo.r the Lymphocytic leukemia P388 intraperitoneal test except that the tumor transplant consists of lymphocytic leukemia L1210 inoculated at a concentration of 105 cells/mouse with a mean survival time being calcula~ed. The results with a representative compound of this invention appear in Table III below.

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I _ -1 11~ C,) U7,_ Melanotic Melanoma sl6 The animals used are C57BC/6 mice, all of the same sex, weighing a minimum of 17 grams and all within a 3-gram weight range. There are normally 10 animals per test group.
A one-gram portion of melanotic melanoma B16 tumor is homo-genized in 10 ml. of cold balanced salt solution and implanted intraperitoneally into each of the test mice as a 0.5-ml~ ali-quot of the homogenateO The test compounds are administered intraperitoneally on days one through nine (relative to tumor inoculation) at various doses. The animals are weighed and survivors are recorded on a regular basis for 60 days. The median survival time and the ratio of survival time for treat-ed (T)/control (C) animals are calculated. The positive con-trol compound is 5-fluorouracil. The results of this test with typical compounds of the present invention appear in Table IV below. The criterion for efficacy is T/C x 100 125%.

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The active ingredients of the present invention inhibit transplanted mouse tumor growth and induce regression and/or palliation oE leukemia and related cancers in mammals when administered orally in amounts ranging from about 5 mg.
to about 200 mg. per kilogram of body weight per day. A
preferred dosage regimen for optimum results would be from about 5 mg. to about 50 mg. per kilogram of body weight per day, and such dosage units are employed that a total of from about 350 mg. to about 3.5 grams of the active compound for a suhject of about 70 kg. of body weight are administered in a 24-hour period. This dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exi~encies of the therapeutic situation. A decided practical advantage of this invention is that the active compound may be admini-stered in any convenient manner such as the oral or buccal routes or it may be incorporated directly in the diet.
The active ingredients of the present invention may be orally administered, ~or example, with an inert diluent or with an assimilable edible carrier, or they may be enclos2d in hard or soft shell gelatin capsules, or they may be com-pressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administra-tion, the active compounds may be incorporated with excipi-ents and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspension, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1~ of the active compound. The percentage of the compositions and preparations may, of course, be varied and ;52~

may conveniently be between about 2 to about 60% of the weight of the unit. The amount of ac~ive ingredient in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or prepara-tions according to the present invention are prepared so that an oral dosage unit form contains between about 5 and 200 milligrams of active compoundO
The tablets, troches, pills, capsules and the like may also contain the following: ~ binder such as gum traga-canth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, po-tato starch, alginic acid and the like; a lubri-cant such as magnesium stearate; and a sweetening agent sueh as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry fla voring. When the dosage unit foxm is a capsule, it may con~
tain, in addition to materials of the above type, a li~uid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
For instance, tablets, pills, or capsules may be coated with shellac t sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring sueh as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutieally pure and substantially non-toxic in the amounts employed.
In addition, the active ingredients may be incorporated into sustained-release preparations and formulations.
The active ingredients of the present invention may also be administered parenterally or intraperitoneally.

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Solutions of the acti~e ingredient as a free base or salt can be prepared in water suitably mixed wlth a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporanous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. ~he carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol~ propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vege~able oils The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of micro-organisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable ko include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the in-jectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
~terile injectable solutions are prepared by in~
corporating the active ingredient or ingredients in the re-quired amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporatiny the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the re~uired other ingredients from those enumerated ahove. In the case of sterile powder~ for the preparation of sterile injectable solutions, the preferred methods of prepar-ation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-~iltered solution thereof.
As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media~ coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use o~ such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatable with the active ingredient, its use in the present compositions is contemplated. Supplementary active ingredients can also be incorporated into the inventive com-positions.
It is especially advantageous to formulate paren-teral compositions in dosage unit form for ease of admini-stration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discreet units suited as unitary dosages for the mammalian subjects to be tre~ted; each unit containing a predetermined quan-tity of active material calculated to produce the desired therapeutic effect in associa-tion with the required pharma~
ceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on ~a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as disclosed in detail in this specificatlon.
The dosage of the principal active ingredient for the treatment of the indicated conditions depends upon the age, weight and condition of the subject being treated; the particular condition and its severity; the particular form of the active ingredient and the route of administration.
daily dose of from about one to about 100 mg./kg. of body weight given singly or in divided doses of up to S times day embraces the effective range for the treatment of most conditions for which the compound is effective and substantial-ly non-toxic. For a 75 kilogram subject, this translates into between about 75 and about 7500 mg./day. If the dosage is divided, for example, into 3 individual dosages, these will range from about 25 to aboùt 2500 mg. of the active ingredient.
The preferred range is from 2 to about 50 mg./kg. o~ body weight/day with about 2 to about 30 mg./kg./day being more preferred.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically-acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can~
for example, contain the principal active ingredient in amounts ranging from about 0.1 to about 400 mg., with from about one to about 30 mg. being preferred~ Expressed in proportions, the active ingredient is generally present in from about 0.1 to about 400 mg./ml. of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of ad-ministration of the said ingredientsO
Regression and palliation of cancers are attained, for example, using intraperitoneal administration. A single intravenous dosage or repeated daily dosages can be admini-stered. Daily dosages up to about 5 or 10 days are often sufficient. It is also possible to dispense one daily dosage or one dose on alternate or less frequent days. As can be seen from the dosage regimens, the amount of principal active ingredient administered is a sufficient amount to aid regres-sion and palliation of the leukemia or the like, in the ab-sence of excessive deleterious side effects of a cytotoxic nature to the hosts harboring the cancer~ As used herein, cancer means blood malignancies such as leukemia, as well as other solid and non-solid malignancies such as the melano-carcinomas, lung carcinomas, and mammary tumors. By regres-sion and palliation is meant arresting or retarding the growth of the tumor or other manifestation of the disease compared to the course of the disease in the absence of treatment.
The invention will be described in greater detail in conjunction with the following specific examples.

Exam~le 1 Preparation of 1,4-bis(2- imeth~laminoethylamino)-anthra~uinone A mixture of 3 g. of quini7arin, 10 g. of N,N-di-methylethylenediamine and 17.5 ml. of ~ater is stirred and heated under reflux for 3.5 hours. The mixture is cooled and the solid is collected and washed with water giving 2.96 g. of the desired product as a dark blue solid, mp. 170~
-172C .
Alternatively, the above product may be prepared by stirring and heating under reflux for 5 hours a mixture of 2.4 g. of quinizarin, 2.82 g. of N,N-dimethylethylene-diamine and 9 ml. of N,N,N',N'-tetramethylethylenediamine.
The product is recovered as described above.
Example 2 Preparation of 1,4-bis(2-morpholinoethylamino)-anthraquinone A 9.60 g. portion of quinizarin, 46.90 g. of N-- -(2-aminoethyl)morpholine and 56 ml. of water are reacted as described in Example 1 giving 9.92 g. of the desired product as a blue-black solid, mp. 15~-159C.
Example 3 P e aration of 1 4-bis(2-dieth~laminoethylamino)-r p anthraq~linone A mixture of 42 ml. of N,N,N',N'-tetramethylene-diamine, 17.43 g. of N,N-diethylethylenediamine and 12.01 g.
of quinizarin is stirred and heated under reflux for 15 hours.
The resulting solution is evaporated to dryness and a chloro-form solution of the residue is filtered through 300 g. of alumina. The blue filtrate is chromatographed on silica

- 2~ -gel in a Nylon~ll) film column, developing with a chloroform:-methanol (6:]) mixture. The major blue band is cut out and then eluted with a chloroform:methanol:triethylamine (6:2:1) mixture. The elua-te is evaporated. The residue is crystal-lized from hexane and washed with petroleum ether, giving 1.43 g. of the desired product as blue-black plates, mp.

Example 4 -Preparation of leuco-1,4-bis(2-aminoethylamino)-.
~' A 125-ml. portion of ethylenediamine is de-aerated by bub~ling nitrogen through it for 15 minutes. A 12~0-gO
portion of leucoquinizarin is added and the mixture is heated with stirring under nitrogen at 50C. for one hour. The mix-ture is allowed to cool. The solid is collected and washed successively with ethyl acetate, acetonitrile and petroleum ether, giving 8.07 g. of the desired product as green-gold crystals, mp. 162-165C. (dec.) at a heating rate of 9 per minute.
Example 5 Preparation of 1,4-bis(2-aminoethylamino)-anthraquinone _ Air is bubbled into a mixture of 7.0 g. of leuco--1,4-bis(2-aminoethylamino)anthraquinone and 87.5 ml. of ethylenediamine while heating at 50C. for one hour. The mixture is diluted with 87.5 ml. of acetonitrile, allowed to cool and the solid is collected and washed with acetonitrile giving 5.43 g. of the desired product as a dark blue solid, mp. 170-171C.

Example 6 Preparation of 1,4-bis[2-(methylamino)ethylamino]-anthraquinone A mixture of 2.4 g. of leucoquiniæarin and 25 g. of de-aerated N-me~hylethylenediamine is heated at 50C. with stirring under nitrogen for one hour. Heating at 50C. is continued as air is bubbled into the mixture for 40 minutes.
The mixture is evaporated to dryness, then re-evaporated twice from 25 ml. portions of ethanol. Crystallization of the residue from ethanol-ether at -70C. gives 2.32 g. of crude solid which is recrystallized twice from carbon tetra-chloride giving 1.92 g. of the desired product as dark blue crystals, mp. 131-132C.
EXample Preparation of 1,4-bis(2-piperidinoethylamino)-anthraquinone .. _ _ ~
A mixture o 4~07 g. of quinizarin, 21.74 g~ of N-(2-aminoethyl)piperidine and 26 ml. of water is stirred under reflux for 2 hours and then allowed to stand overnight.
The gummy solid is collected and washed with water by centri-fugation, giving 1.99 g. of blue-black solid. This solid is dissolved in 15 ml. of chloroform and chromatographed by an abbreviated wet-column procedure on 100 g. of alumina, eluting with chloroform. A total of 180 ml~ of eluate is collected in eight separate cuts from the time the eluate turns blue until a black band nears the bottom of the column. Cuts 1-6 are combined and evaporated giving 1.42 g. of blue-black crystals which are recrystallized from ethanol giving 1.35 g.
of the desired product as blue-black needles, mp. 140U-141C.
Product dried in vacuo at 78C. melted at 156~157C.
-65'~

xample 8 Preparation of leuco-1,4-bis(2-dimethylamino-____ __ _ _ ethylamino)anthraquinone A solution of 26.44 g. of N,N-dimethylethylene-diamine in 75 ml. of N,N,N',N'-tetramethylethylenediamine is de-aerated by bubbling through N2 for 15 minutes. Then, 12.11 g. of leucoquinizarin is added and the resulting mixture is stirred under N2 while heating at 48-50C.
for 21 hours. After cooling overnight under nitrogen~
the solid is collected by filtration and washed three times by slurrying with acetonitrile and then twice with petroleum ether. There is thus obtained 12.52 g. of dark green crystals, mp. 150-157C.; or on the hot stage miscroscope, mp. 153--154C.
Example_~
Preparation of 50 mg. Tablets Per Tablet Per 10,000 Tablets -0.050 gm. 1,4-bis~Z (methylamino)- ~ O 500 gm~
ethylamino]anthraquinone 0.080 gm. Lactose ~ OO~O~O~ O 800 gm.
O.010 gm. Corn Starch (for mix) .O~.O~O~100 gm.
0.008 gm. Corn Starch (for paste1................ 75 gm.
0.148 gm. ~7~ gm.
0.002 gm. Magnesium Stearate (1%) ~O~ 15 gm.
0.150 gm. 1490 gm.
The 1,4-bis~2-(methylamino)ethylamino]anthraquinone, lactose and corn starch (for mix) are blended together. The corn starch (for paste) is suspended in 600 ml~ of water and 5 heated with stirring to form a paste. This paste is then used to granula~e the mixed powders. Additional water is used if necessary. The wet granules are passed through a No~ 8 hand screen and dried at 120F. The dry granules are then passed through a No. 16 screen. The mixture is lubricated with 1%

magnesium stearate and compr~ssed into tablets in a suitable tableting machine~
Example 10 Preparation of Oral Suspension Ingredient Amount Leuco-1,4 bis(2-aminoethylamino~anthraquinone.~..... 500 mg.
Sorbitol solution (70% N.F.) .......................... ~.~ 40 ml~
Sodium banzoate .~.............. ~..... ~...... ~.~....... ...150 mg.
Saccharin ...... ~.............. ~...................... ....10 mg.
Red dye ........ ~.............. 0.~..................... ....10 mg.
Cherry flavor ............ ........................ ..... ....50 mg.
Distilled water.~.gs... ad....... ~.... ~... ~............. ....100 ml.
The sorbitol solution is added to 40 ml. of dis-tilled water and the leuco-1,4-bis(2-aminoethylamino)-anthraquinone is suspended therein. The saccharin, sodium benzoate, flavor and dye are added and dissolved. The volume is adjusted to 100 ml. with distilled water. Each ml. of syrup contains S mg. of leuco-1,4-bis(2-aminoethyl-amino)anthraquinone.
Example 11 Preparation of Parenteral Solution .
In a solution of 700 ml. of propylene glycol and 200 ml. of water for injection is suspended 20~0 grams of 1,4-bis~4-aminobutylamino)anthraquinone with stirring.
After suspension is complete, the pH is adjusted to 5~5 with hydrochloric acid and the volume is made up to 1000 ml. with water for injection. The formulation is sterilized, filled into 5.0 ml. ampoules each containing 2.0 ml. (representing 40 mg. of drug) and sealed under nitrogen.

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Exampl.e 12 Preparation of leuco-1,4-bis(3-aminopropylamino)-anthraquinone When 1,3-propanediamine is used instead of ethyl-enediamine in the procedure of Example 4 the product is the title compound.
Example 13 Preparation of leuco-1,4-bis[2-dimethylaminopropyl~
amino]anthraquinone Using 15.32 g. of 2-dimethylaminopropyl amine instead of N,N-dimethylethylenediamine in the procedure of Example 8 gives the title compound after a reaction time of one hour at 50.
Example 14 Leuco-1,4-bis[2-(2-methylaminoethylamino)ethyl-amino]anthraquinone A solution of 14.10 g. (0 12 mole) of l-methyl--diethylenetriamine in 100 ml. of ethanol is de-aerated by bubbling nitrogen through it for 15 minutes; then 9.69 g.
(0.04 mole) of leucoqu.inizarin is added gradually with stir~
ring. The mixture is stirred under nitrogen and heated with an oil bath at 50C. for 21 hours. The mixture is allowed to cool, the product is collected by filtration and washed with acetonitrile and then with petroleum ether to give the title compound as a dark green solid.
Example 15 Preparation of leuco~l,4,bis(2-methylaminoethylamino)-anthraquinone To a de-aerated (See Example 8) solution of 8.89 g.
of N-methylethylenediamine in 80 ml. of N,N,N',N'-tetra-methylethylenediamine is added 9.68 g. of leucoquinizarin.
The mixture is stirred and heated at 50C. under nitrogen for one hour, then allowed ~o cool. The solid is collected, washed with toluene and then with ether, giving 9.0 g. of a green-black solid, m.p. 105-109~C.

Claims (3)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition in dosage unit form comprising from about one to about 400 mg. of a compound of the formula:

the tautomeric forms and the pharmacologically acceptable acid-addition salts thereof, wherein .OMEGA. is -CH2CH2-; and is amino, methylamino, ethylamino, dimethylamino, diethylamino, piperidino or morpholino; in admixture with a non-aqueous pharmaceutically acceptable carrier.

2. The composition according to Claim 1, wherein -NR1R2 is piperidino, and in the leuco base form.

3. The composition according to Claim 1, wherein -Q- is ethylene, -NR1R2 is ethylamino, and in the aromatic dihydrochloride form.