NOVEL 1,2-DIHYDROPYRIDO-(3,4-B)PYRAZINES

Abstract: 1,2-Dihydropyrido[3,4-b]pyrazines are provided which possess anticancer activity. The compounds have the structure: wherein x has a value of 1, 2 or 3; Y is CH₂ or N(CH₃); R₁ is a lower alkyl group; e.g., an alkyl group containing up to six car­ bon atoms such as methyl, ethyl, propyl, butyl, etc.; R₂ is a member selected from the group consisting of hydrogen, alkyl radicals having from about one to about 12 carbon atoms, pref­ erably from about one to about 6 carbon atoms; alkenyl radi­ cals having from about two to about 15 carbon atoms, prefer­ ably from about two to about 10 carbon atoms; cycloalkyl radi­ cals having from about three to about 20 carbon atoms, prefer­ ably from about three to about 15 carbon atoms; aralkyl and alkaryl radicals having from about six to about 20 carbon atoms, preferably from about six to about 15 carbon atoms; a halogen radical, e.g., chlorine, fluorine, bromine and iodine; a hydroxyl group; an amino group; an alkoxy or aryloxy group; an alkylthio group or an arylthio group having from about one to about 20 carbon atoms, preferably from about one to about 15 carbon atoms; a sulfonic acid group or alkyl- or arylsulfonyl group having from about one to about 20 carbon atoms, prefer­ ably from about one to about 15 carbon atoms; an alkyl- or arylsulfinyl group having from about one to about 20 carbon atoms, preferably from about one to about 15 carbon atoms; an alkyl- or aryl mono- or diamino group having from about one to about 20 carbon atoms, preferably from about one to about 15 carbon atoms; a hydrocarbyl group, such as defined above, carrying halogen, hydroxyl, amino, alkoxy or aryloxy; and, when taken together with the aromatic ring to which it is at­ tached, a fused ring structure such as naphthyl; and R₃ and R₄ are either both hydrogen or one is hydrogen and the other is a lower alkyl group; provided that when each of R₂, R₃ and R₄ are hydrogen, Y is CH₂.

Description: NOVEL 1,2-DIHYDROPYRIDO [3,4-b]PYRAZINES Background Of The Invention This invention relates to novel 1,2-dihydropyrido[3,4-b]pyrazines, also known as 1-deaza-7,8-dihydropteridines. This invention also relates to a process for making such compounds and to novel intermediates obtained in said process. The antimitotic chemical agents commonly known as spindle poisons are plant products of which the best known are colchicine, podophyllotoxin, and the vinca alkaloids. [L. Wilson, J.R. Bamburg, S.B. Mizel, L.M. Grisham and K.M. Creswell, Federation Proceedings, 33, 158 (1974)]. Two members of the latter, vincristine and vinblastine, are currently used clinically in the treatment of neoplasms. Although these agents produce a number of biochemical actions such as the inhibition of macromolecular synthesis, their primary effect is to prevent mitosis by interfering with the function of microtubules, which results in the accumulation of cells in metaphase. In addition, several benzimidazol-2-yl carbamates have been introduced as fungicides, antheelmintics and antitumoral agents. [L.C. Davides and W. Flach, J. Cell Biol. 72, 174 (1977) . These compounds also present mitosis and their biological activity can probably be attributed to interference with the formation or functioning of microtubules. The development of procedures for the preparation of 1-deazapteridines is reported by J.A. Montgomery and N.F. Wood, J. Org. Chem., 29, 734 (1964); R.D. Elliott, C. Temple, Jr. and J.A. Montgomery, J. Org. Chem., 33, 533 (1968); R.D. Elliott, C.Temple, Jr., J.L. Fryc and J.A. Montgomery, J. Org. Chem., 36, 2818 (1971); and R.D. Elliott, C. Temple, Jr. and J.A. Montgomery, J. Med. Chem., 17, 553 (1974). These references disclose the preparation and use of various 1,2-dihdro[3,4-b]pyrazine derivatives. Thus, the 1964 J. Org. Chem. reference discloses the compounds: EMI2.1 The 1968 J. Org. Chem. reference discloses the compound: EMI2.2 The 1971 J. Org. Chem reference discloses the compounds: EMI3.1 The J. Med. Chem. reference discloses that a dihydro-ldeazapteridine precursor of l-deazamethotre ate showed activity against leukemia L1210 in mice. An ahstract presented at the 28th Southeast Regional Meeting of the American Chemical Society in Gatlinburg, Tennessee, October 27-29, 1976 discloses that the compound EMI3.2 showed cytotoxicity in the KB cell culture screen and activity against leukemia L1210 in mice. An abstract of a paper by B.J. Bowdon, G.P. Wheeler, C.G. Temple and J.A. Montgomery in AACR Abstracts, Vol. 22, March 1981 (page 25) discloses that a compound designated as "NSC-181928" was active against several neoplasms. NSC-181928 is the designation for the compound EMI4.1 Summary Of The Invention It has now been found that certain 1,2-dihy dropyridof3,4-b]pyrazines which are not disclosed in any of the references discussed in the preceding section possess anticancer activity. The compounds of this invention have the structure: EMI4.2 wherein x has a value of 1, 2 or 3; Y is CH2 or N(CH3); R1 is a lower alkyl group, e.g., an alkyl group containing up to six carbon atoms such as methyl, ethyl, propyl, butyl, etc.; R2 is a member selected from the group consisting of hydrogen, alkyl radicals having from about one to about 12 carbon atoms, preferably from about one to about 6 carbon atoms; alkenyl radicals having from about two to about 15 carbon atoms, preferably from about two to about 10 carbon atoms; cycloalkyl radicals having from about three to about 20 carbon atoms, preferably from about three to about 15 carbon atoms; aralkyl and alkaryl radicals having from about six to about 20 carbon atoms, preferably from about six to about 15 carbon atorns; a halogen radical, e.g., chlorine, fluorine, bromine and iodine; a hydroxyl group; an amino group; an alkoxy or aryloxy group; an alkylthio group or ari arylthio group having from about one to about 20 carbon atoms, preferably fror about one to about 15 carbon atoms; a sulfonic acid group or alkyl- or arylsulfonyl group having from about one to about 20 carbon atoms, preferably from about one to about 15 carbon atoms; an alkyl- or arylsulfinyl group having front about one to about 20 carbon atoms, preferably from about one to about 15 carbon atoms; an alkyl- or aryl mono- or diamino group having from about one to about 20 carbon atoms, preferably from about one to about 15 carbon atoms; a hydrocarbyl group, such as defined above, carrying halogen, hydroxyl, amino, alkoxy or aryloxy; and, when taken together with the aromatic ring to which it is attached, a fused ring structure such as naphthyl; and R3 and R4 are either both hydrogen or one is hydrogen and the other is a lower alkyl group; provided that when each of R2 t R3 and R4 are hydrogen, Y is CU2. Compounds of Formula I wherein R3 is hydrogen may be prepared by aminating a lower alkyl ester of 6-amino-4-chloro-S-nitropyridin-2-ylcarbamate having the structure: EMI5.1 with the oxime of an alpha-ajnino ketone having the structure: EMI6.1 to give a compound having the structure: EMI6.2 wherein R1, R2, R4, x and Y are the same as previously defined, further provided that R2 may be a nitro group. The compound of Formula IV is hydrolyzed, e.g., by acid hydrolsis to give the corresponding ketone having the formula: EMI6.3 wherein R1, R2, R4, x and Y are the same as previously defined, further provided that R2 may be a nitro group. The compound of Formula V is converted to the compound of Formula I by catalytic hydrogenation. An intermedi- ate product formed during hydrogenation has the formula: EMI7.1 wherein R1, R2, R4, x and Y are the same as previously defined. Compounds of Formula I wherein R4 is hydrogen may be prepared by aminating the compound of Formula II with an alphaamino alcohol having the structure: EMI7.2 to give a compound having the structure: EMI7.3 which is oxidized to give a ketone having the structure: EMI8.1 wherein R1, R2, R3, x and Y are the same as previously defined, further provided that R2 may be a nitro group. The compound of Formula VA is converted to a compound of Formula I by catalytic hydrogenation. Detailed Description Of The Invention The compounds of the invention form pharmaceutically acceptable salts with both organic and inorganic acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicyclic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic, and the like. The salts are prepared by contacting the free base form with an equivalent amount of the desired acid in the conventional manner. The free base forms may be regenerated by treating the salt form with a base. For example, dilute aqueous base solutions may be utilized. Dilute aqueous sodium hydroxide, potassium carbonate, ammonia, and sodium bicarbonate solutions are suitable for this purpose. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but the salts are otherwise equivalent to their respective free base forms for purposes of the invention. Also embraced within the purview of the present invention are therapeutic compositions of matter useful for ameliorating cancer diseases in mammals and containing the l-deaza-7,8-dihydropteridines of this invention or pharmaceutically acceptable salts thereof. The active ingredients of the therapeutic compositions and the novel compounds of the present invention inhibit transplanted mouse tumor growth whe administered 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 subject 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 exigencies of the therapeutic situation. A decided practical advantage is that the active compound may be administered in any convenient manner such as by the oral, intravenous, intramuscular or subcutaneous routes. The active compounds may be orally administered, for example, with an inert diluent o with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, trochees, capsules, elixirs, suspensions, syrups, wafers and the li];e. Such compositions and preprations should contain at least 0.1Vi of active compound. The percentacge of the compositions and preparations may, of course, be varied and may conveniently be between about 2 and about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 5 and about 200 milligrams of active compound. The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid 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, 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 such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparations and formulations. The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmaceutically acceptable salt can be prepared in water suitahly mixed with 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 usc, these preparations contain a preservative to prevent the growth of microoryanisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous 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. The 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 vegetable 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 microorganisms 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 to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation 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-filtered solution thereof. As used herein, 11pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical 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 herein disclosed in detail. 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 compound 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 compound 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 administra tiop of the said ingredients. A preferred lower alkyl ester of 6-amino-4 chloro-5-nitropyridin-2-ylcarhamtate is the ethyl ester, i.e., ethyl 6 -amino-4 -chloro-5 -nitropyridin-2-ylcarba- mate. This compound is prepared according to the procedure described by R.D. Elliott, C. Temple, Jr. and JA. Montgomery, J. Org. Chem., 31, 1890 (1966). Oximes of alpha-amino ]cetones, i.e., compounds of Formula III, may be prepared by known prior art procedures. Thus, they can be prepared by condensing the corresponding alpha-amino ketones with hydroxylamine hydrochloride in a refluxing mixture of pyri- dine and ethanol to give the oxime derivatives [R.D. Elliott, C. Temple, Jr. and J.A. Montgomery, J. Org. Chem., 35, 1676 (1970)]. The compounds of Formula III can also be prepared by alkylation of phthalimide with the corresponding aipha-bromo-ketone, treatment of the alpha (phthalimido)--ketone product with hydroxylamine, and removal of the phthaloyl protecting group from the resulting oxime with hydrazine [k.D. Elliott, C. Temple, Jr. and J.A. Montgomery, J. Org. Chem., 35, 1676 (1970)]. Examples of these two procedures for the preparation of compounds of Formula III follow: Method I. l-Amino-3-[EN-(4-methoxyphenyl)-N- methyl]amino]propane Oxime A mixture of l-bromo-3-(phthalimido)propanone (1.00 g., 3.54 mmol), N-methyl-p-anisidine (487 mg, 3.54 mmol), and NaHCO3 (298 mg, 3.54 mmol) in DMAC (N,N-dimethyl acetamide) (10 ml) was stirred at room temperature for 20 hours, slowly diluted with H2O (3 ml) and stirred for 30 minutes. The yellow precipitate was collected by filtration, washed with DMAC-H2O (10:3) then H20 and dried in vacuo over P205: yield 927 mg. Dilution of the filtrate with additional H20 gave a second crop of product: yield 131 mg. Recyrs- tallization of the combined crops from ethanol gave the diaminopropanone: yield 834 mg. Mass spectrum: m/e 338 (M) + A solution of this product (600 mg, 1.77 mmol), hydroxylamine hydrochloride (184 mg, 2.65 mmol) and pyridine (2 ml) in ethanol (8 ml) was refluxed for 2 hours and evaporated to dryness in vacuo. The residue was washed with II2O (2 x 5 ml), then triturated with ethanol (2.5 ml) until a homogeneous yellow powder formed. The mixture was cooled in an ice bath and the oxime was collected by filtration, washed with cold ethanol and dried in vacuo over P2O5: yield 516 mg. Mass spectrum: m/e 353 (M)+. A solution of the oxime (400 mg, 1.13 mmol) in ethanol (15 ml) at 700C was treated dropwise with a solution of 95% hydrazine (39.2 l. 1.16 mmol) in ethanol (2 ml). The resulting solution was heated at 400C for 17 hours, cooled to 250C, treated with 1 N IICl (1.13 ml), stirred for 1 hour, cooled to 0 C and filtered to remove phthalhydrazide. The filtrate was evaporated to dryness in vacuo ( < 40"C) and the residue was stirred with 1120 (2 ml) and again evaporated to dryness in vacuo. An extract of the residue in 1120 (5 ml) at 60 C was cooled to 250C, filtered, cooled in an ice bath, treated with concentrated Nil4O11 (76 ul) and stirred at 0 C until a homogeneous tan solid formed. The alpha-aminoketone oxime was collected by filtration, washed with cold H20 and dried in vacuo over P205: yield 124 mg. Mass spectrum: m/e 223 (M) Method II. l-Amino-4-phenyl-2-butanone Oxime A solution of crude 1-amino-4-phenyl-2-butan- one hydrochloride (9.84 g, 49.2 nunol) (Degraw, J., Isakotellis, P., Kisliuk, R., and Gaumont, Y., J. Heterocyclic Chem.r 1971, 8, 105), hydroxylamine hydrochloride (6.84 g, 98.4 mmol) and sodium acetate 3H20 (13.4 g, 98.4 mmol) in 50% ethanol (250 ml) was heated at 75-800C for 30 minutes, filtered, treated with a hot solution of picric acid (11.7 g, 51.1 mmol), cooled to 250C, filtered and allowed to stand for 2 days. The crystalline picrate was collected, washed with 2:1 water-ethanol and dried in vacuo: yield 9.62 g, mp, 1510C (Kofler Heizbank). The mother liquor was evaporated to dryness in vacuo, and the residue was crystallized from hot water (500 ml) to give an additional amount of the picrate: yield, 3.62 g, mp 151 C. A solution of the picrate in 3:1 ethanol-water (400 ml) was treated with washed BioRad AG1-X8 (Cl ) ion exchange resin (100 g) and stirred for 18 hours. The solution was filtered and the resin was washed with 3:1 ethanol-water. The filtrate and wash were treated with additional resin (40 g), stirred for 2 hours and filtered. The almost colorless solution was evaporated with ethanol (3 x 200 ml). The residue was stirred with ethanol (100 ml), filtered and the precipitate was rinsed with additional ethanol (40 ml). The filtrate and wash were diluted with diethyl ether (600 ml) to give a crystalline hydrochloride which was collected, washed with diethyl ether and dried in vacuo (P205): yield, 5.36 g. Alpha-amino alcohols of Formula IIIA are prepared by the following procedure: Method III. l-Amino-3-[[N-(4-chlorophenyl)- N-methyl] amino] 2-propanol A solution of epichlorohydrin (11 ml) and 4-chloro-N-methylaniline (11 g, 78 mmol) in a mixture of ethanol (10 ml) and water (7 ml) was refluxed for 2 hours, diluted with water (20 ml), and extracted with diethyl ether (3 x 50 ml). The combined extracts were evaporated to dryness, the residue was treated with a solution of NAOMI (5 g) in water (10 ml) for 1 hour, and the resulting mixture was extracted with diethyl ether (4 x 25 ml). The combined extracts were dried (MgSO4) and evaporated to dryness in vacuo to give 1-[[N-(4- chlorophenyl) -N-methyl] amino] -2 ,3-epoxypropane: yield, 11 g (72%). A solution of this sample in a mixture of ethanol (50 ml) and liquid NH3 (20 ml) was heated in a glass-lined stainless steel bomb at 1000C for 3 hours. The resulting reaction solution was evaporated to dryness, and the dried residue was recrystallized from C6H5: yield, 5.3 g (448). The oximes and alpha-amino alcohols set forth in Table I were prepared by Method I, Method II or Method III, as indicated in Table I. The first column of Table I sets forth the structure of the group EMI17.1 in Formula III for the oximes prepared by Methods I and II and in Formula IIIA for the alpha-amino alcohol prepared by Method III. TABLE I Alpha-Aminoketone Oximes (III) and Alpha-Aminoalcohols (IIIA) Analysis Yield, Calcd, % Found, % Compounda Method %b M.p. C Formula C H N C H N 4-CH3OC6H4N(CH3)CH2 I 28 c C11H17N3O. 0.37H2O 57.46 7.78 18.27 57.39 7.70 18.38 C6H5CH2CH2 II 50 193 C10H14N2O.HCl. 0.3H2O 54.82 6.72 12.79 54.63 7.00 12.58 4-ClC6H4N(CH3)CH2 III 32 117-9d C11H17N3O. 0.37H2O 55.95 7.04 13.05 56.29 7.18 13.17 aR3 and R4 = H bOverall yield cIndefinite melting point dResolidification and melting at 123 C A compound of Formula II is laminated with a compound of Formula III under nitrogen in refluxing ethanol containing triethylamine as an acid acceptor to give a compound of Formula IV. An example of this procedure follows: Example 1 Ethyl 6-Amino-4-[3-[(3-[[N-4-methoxyphenyl) N-methyl]amino]-2-oxopropylamino]-5 nitro-2--pyridinecarbamate Oxime (IV: R1 = C2H5; R2 = 4-CH3O; R4 = H; Y = N(CH3)) A solution of ethyl 6-amino-4-chloro-5-nitro- 2-pyridinecarbamate (5.00 g, 19.2 mmol), l-amino-3-[[N (4-methoxyphenyl)-N-methyl]amino]propanone oxime (4.29 g, 19.2 mmol) and triethylamino(l.94 g, 19.2 mmol) in CH3OH (75 ml) was refluxed under N2 for 15 hours and cooled to 250C. The yellow crystalline precipitate was collected by filtration, washed with ethanol and dried in vacuo. A solution of the crude product (6.46 g) in DMAC (260 ml) was diluted with 1120 (430 ml) to give a precipitate which was collected by filtration, washed with H2O and dried in vacuo over P205: yield 6.03 g. + Mass spectrum: m/e 447 (M) Additional compounds were prepared similarly wherein the oxime of Example 1 was replaced with other oximes. The properties of these compounds are set forth in Table II. The first column of Table II sets forth the structure of the group: EMI19.1 in the starting alpha-aminoacetophenone oxime, see Formula III wherein R1 is C2H5 and q is H, and in the final product, see Formula IV, wherein R1 is C2 115 and R4 is H. TABLE II Ethyl 4-(Substituented)amino-6-amino-5-nitro pyridine-2-carbamate Oximes Analyses Reaction Calcd, % Found, % Time, Yield, Compound Hours % M.p. C Formula C H N C H N 4-CH3OC6H4N(CH3)CH2 15a 75 190 C19H25N7O6 51.00 5.63 21.91 50.97 5.89 21.87 C6h5CH2CH2 24b 84 187 C18H22N6O5 53.72 5.51 20.89 53.61 5.56 20.77 Treatment of a compound of Formula IV with a 1:1 mixture of 1 N hydrochloric acid and dioxane at 60 C hydrolyzes the oxime function to give a compound of Formula V. An example of this procedure follows: Example 2 Ethyl 6-Amino-4-[2-oxo-4-phenylbutyl) aminc]-5-nitro-2-pyridinecarbamate (V: R1 1 C2H5; R2 = U; R4 = II; Y = CH2) A solution of the oxime of the title compound (6.17 g, 15.4 mmol) in warm dioxane (60 ml) was treated with 1 N HCl (120 ml), stirred at 550C for 1 hour and cooled in an ice bath. The precipitated hydrochloride was collected, washed with cold water, then suspended in water (300 ml) and neutralized with 1 N NaOH. The yellow product was collected, washed with water and dried in vacuo (P205): yield, 5.08 g (83%); m.p. 1550C. Anal. Calcd. for C181121N505: C, 55.80; H, 5.46; N, 18.08. Found: C, 55.42; , 5.49; N, 18.12. Amination of the compound of Formula II with the compound of Formula IIIA in refluxing ethanol containing triethylamine as an acid acceptor gives a compound of Formula VII. Oxidation of a compound of Formula VII gives a ketone of Formula VA. Two examples of this procedure follow: Example 3 A. Ethyl 6-Amino-4-[3-[[N-[4-chlorophenyl) N-methyl]amino]-2-hydroxypropyl amino]-5-nitro]-2-pyridinecarbamate (VII: R1 = C2H5; R2 = 4-Cl; R3 = H; Y = N(CH3)) A solution of ethyl 6-amino-4-chloro-5-nitro- 2-pyridinecarbamate (10.0 g), l-amino-3-[[N-(4-chloro phenyl) -N-methyl] amino] -2-propanol (8.25 g), and triethylamine (10.7 ml) in methanol (120 ml) was heated at 600C for 18 hours and evaporated to dryness in vacuo. The dark residue was washed with diethyl ether (1.5 1) to give a yellow solid. This solid was washed with water (50 ml) and recrystallized twice from a mixture of ethanol and hexane: yield, 4.92 g. Similar treatment of the residue obtained from the ether wash from above gave a slightly impure sample of product: yield, 5.89 g. Total yield, 10.81 g (64%); m.p., 1810C. Anal. Calcd. for C18H23ClN605: C, 49.26; H, 5.28; N, 19.15. Found: C, 49.16; H, 5.46; N, 19.22. B. Ethyl 6-Amino-4-[3-[[N-(4-chlorophenyl)- N-methyl] amino] -2-oxopropyl- amino]-5-nitro-2-pyridinecarbamate (VA: R1 = C2H5; R2 = 4-Cl; R3 = H; Y = N(CH3)) A solution of ethyl-6-amino-4-[3-[[N-(4chlorphenyl)-N-methyl]amino]-2-hydroxypropylamino]-5nitro-6-pyridinecarbamate (1.76 g) and acetic anhydride (8 ml) in dimethyl sulfoxide (40 ml) was stirred at room temperature for 20 hours, diluted with water (200 ml), and neutralized to p11 5.2 with 1 N NaOH. The solid that deposited was collected by filtration and dissolved in CHCl3. Evaporation of this solution to dryness and trituration of the resulting solid successively with water and ethanol gave the product: yield, 0.36 g (20%); m.p. 123-50C. Anal. Calcd. for C18H21ClN6O5.O.5H2O: C, 48.49; II, 4.97; N, 18.85. Found: C, 48.74; H, 4.65; N, 18.89. Example 4 A. Ethyl 6-Amino-4-[3-[[N-methyl-N phenyl]amino]-2-hydroxyoxopropyl amino]-5-nitro-2-pyridinecarbamate (VII: R1 = C2H5; R2 = II; R3 = H; Y = N(CH3)) This compound was prepared by the procedure described in Example 3A, substituting 1-amino-3-[(N- methyl-N-phenyl)amino]-2-propanol [0. Eisleb, German Patent 473,219 (1926); Chem. Zentra., 100 (II), 350 (1929)] for 1-amino-3-[[N-(4-chlorophenyl)-N-methyl] amino]-2-propanol: yield, 738; m.p., 88-900C. Anal. Calcd. for C18H24N605: C, 53.46; H, 5.98; N, 20.78. Found: C, 53.63; H, 5.93; N, 20.81. B. Ethyl 6-Amino-4-[3-[(N-methyl-N phenyl)amino]-2-oxopropyl amino] -5-nitro-2-pyridinecarbamate (VA: R1 = C2H5; R2 = H; R3 = H; Y = N(CH3)) Crystalline o-phosphoric acid (3.08 g, 31.5 mmol) was added to a stirred solution of ethyl 6-amino4-[3-[(N-methyl-N-phenyl)amino]-2-hydroxypropylamino] 5-nitro-2-pyridinccarbamate (3.18 g, 7.87 mmol) and N,N'-dicylohexylcarbodiimide (4.86 g, 23.6 mmol) in dry dimethyl sulfoxide (40 ml). The mildly exothermic reaction was kept below 250C by water bath cooling. After 2.5 hours, the deposit of dicyclohexylurea was filtered off and washed with dimethyl sulfoxide (25 ml). The filtrate was cooled in an ice bath and diluted slowly wi.th water (100 ml) to precipitate the product as a bright yellow solid that was washed thoroughly with water and dried in vacuo over P205: yield, 2.89 g (80%); m.p. #80 C with presoftening. Anal. Calcd. for C18H22N605 H2 .5(CU3)2S0: C, 49.67; H, 5.92; N, 18.29. Found: C, 49.82; H, 5.69; H, 18.06. The catalytic hydrogenation of a compound of Formula V or VA with a three-fold amount of Raney nickel in a large volume of ethanol (i.e., more than one liter per gram) at atmospheric pressure at room temperature or with intermittent warming (e.g., to no higher than 600C.) with a water bath gives the intermediate compound of Formula VI which is cyclized in situ with the elimination of water to give a compound of Formula I. Such reaction is shown in Example 5. The compounds of Formula I can also be prepared directly by hydrogenation of a compound of Formula IV in the presence of Raney nickel as shown in Example 6. Example 5 Ethyl 5-Amibno-1,2-dihydro-3-(2-phenylethyl) pzrido(3,4-b)pyrazine-7-carbamate (I: R1 = C2H5; R2 = 4-CH3O; R3 = H; R4 = H; Y = CH2) A solution of ethyl 6-amino-4-[2-oxo-4-(phen- ylbutyl)amino]-5-nitro-2-pyridinecarbamate (300 mg, 0.775 rrmol) in N,N-dimethyl acetamide (7 ml) was hydrogenated in the presence of RaIley nickel (890 mg, weighed wet, washed with ethanol) for 20 hours to give an H2 uptake of 58 ml (3.07 mnol). The reaction mixture was filtered under N2 and evaporated in vacuo at 250C. The residual syrup was stirred with water (10 ml) to give a white powder which was collected, washed with water and dried in vacuo (P205): yield, 230 mg. The properties are set forth in Table III. Example 6 Ethyl 5-Amino-1,2-dihydro-3-[[N-(4-methoxy phenyl)-N-methyl]aminomethyl]pyrido [3,4-b] pyrazine-7-carbamate (I: R1 = C2H5; R2 = 4-CH3O; R3 = H; R4 = H; Y = N(CH3)) A suspension of the oxime of ethyl 6-amino-4 [3-[[4-(N-methoxyphenyl)-N-methyl]amino]-2-oxopropylamino]-5-nitro-2-pyridinecarbonate (672 mug, 1.50 mmol) and Raney nickel (2 g, weighed wet, washed with ethanol) in ethanol (90 ml) was hydrogenated at room temperature and atmospheric pressure with vigorous stirring. The hydrogen uptake (124 ml, 5.66 mmol) was complete in 2.75 hours. The mixture was filtered under N2 and the catalyst extracted with boiling ethanol (3 x 30 ml). The combined filtrate and extract were concentrated in vacuo to about 7 ml and cooled in an ice bath to deposit the product as a pale yellow crystalline solid: yield 421 mg. Mass spectrum: m/e 384 (M) The properties are set forth in Table III. The compounds set forth in Table III were prepared by the procedure of Example 5 of Example 6, as indicated in Table III. The properties of these compounds are set forth in Table III. The first column of Table III sets forth the structure of the group: EMI25.1 in the startling material, see Formulas IV, V and VA wherein R2 is C2U5 and R3 and R4 are hydrogen, and in the final product, see Formula I wherein R1 is C 2115 and R3 and R4 are hydrogen. TABLE III Ethyl 3-Subsituted 5--Amino-1,2-dihydropyrido [3,4-b]pyrazine-7-carbonates Procedure Reaction Analyses of Time, Yield, Calcd, % Compound Example Hours % M.p. C Formula C H N C H N 4-ClC6H5N(CH3)CH2 5 20 16 a C18H21ClN6O2 0.5 H2O 0.5C2H6O 54.22 5.99 19.97 54.06 5.96 19.83 4-CH3OC6H4N(CH3)CH2 6 3 73 187 C19H24N6O3 59.36 6.29 21.86 59.16 6.29 21.88 C6H5CH2CH2 5 20 88 163 C18H21N5O2 63.70 6.24 20.64 63.35 6.25 20.47 aindefinite The 1,2-dihydropyrido[3,4-b]pyrazines of this invention are powerful inhibitors of the proliferation of cultured lymphoid luekemia L1210 cells as shown in Table IV. The concentration causing a 50% inhibition of proliferation of the cells during 24 hours is similar to that observed for vincristine, vinblastine, and colchicine. Also, the addition to the test medium of inosine, thymidine, glycine, citrovorum factor, individually and in combinations, and elevated concentrations of amino acids and vitamins did not overcome the inhibitions. In addition to cell cytotoxicity, the 1,2-di hydropyrido[3 ,4-b]pyrazines showed activity against lymphocytic leukemia P388 cells (106) implanted intraperitoneally in mice. Ethyl 5-amino-1,2-dihydro-3-[(N methyl-N-phenyl)aminomethyl]pyrido[3,4-b]pyrazine-7- carbamate and ethyl 5-amino-1,2-dihydro-3-[L(N-(L- methoXyphenyl)-N-methyl]aminomethyl]pyrido[3,4-b]pyra- zine-7-carbamate are also active in mice against P388 cells that were resistant to vincristine. The 1,2-dihydropyrido[3,4-b]pyrazines of this invention at concentrations that prevented any increase in the cell number during a 24 hour period had little effect upon the synthesis of DNA, RNA, and protein by cultured L1210 cells during exposure for four hours. This result and those described above led to the determination of the effect of the 1,2-dihydropyri.do[3,4-b]- pyrazines upon cell division. Exposure of cultured L1210 cells to the 1,2-dihydropyrido[3,4-b]pyrazines inhibited cell division as measured by the mitotic index (MI) (Table IV), which is the fraction of the cell population that is made up of metaphase cells. Subsequent experiments showed that these agents caused the accumulation in metaphase of human epidermoid carcinoma &num;2 cells, P388 cells, and P388 cells resistant to vincristine grown in suspension culture and of colon tumor &num;26 cells and colon tumor &num;38 cells grown on plastic surfaces. Table IV sets forth biological data for l-deaza-7,8-dihydropteridines of this invention and for three prior art compounds. The first column of Table IV sets forth the structure of the group R in the formula in the heading of the table for the l-deaza7, 8-dihydropteridines tested. The data in Table IV shows that the 1 deaza- 7,8-dihydropteridines of this invention are active against leukemia in laboratory animals. TABLE IV Bilogical Data-1-Deaza-7,8-dihydropteridines EMI29.1 P388(c) 106 Tumor cell implant, i.p. L1210(a) Mitotic Index(b) Schedule, Compound(R) ID M 12h( M) 24h( M) Days %IIS(mg/kg) Nocodayole 27x10-3 0.19(0.3) Vincristide 1x10-3 0.62(0.3) 1 100(2.7) 4-CH3OC6H4N(CH3)CH2 7.9x10-3(d) 0.64(0.03) 1 80(50)(e) 0.49(0.3)) 1(f) 133(25) 0.49(0.03) 1(g) 150(25) 4-ClC6H4N(CH3)CH2 14.5x10-3 0.71(0.03) 1 65(100) C6H5CH2CH2 13x10-3 0.33(0.3) 1 29(100) 4-CH3O2CC6H4N(CH3)CH2(h) 58x10-3 0.61(0.3) 1-9 30(25) 0.44(0.1) (Continued) TABLE IV Biological Data - 1-Deaza-7,8-dihydropteridines (Continued) (a) Concentration of agent that inhibits proliferation of cultured lymphoid luekemia L1210 cells to 50% control growth during 48 hours. G.P. Wheeler, B.J. Bowdon, J.A. Werline, A.M. Adamson and C.Temple, Jr., Cancer Res., 42, 791 (1982). (b) Fraction of the cell population of cultured lymphoid luekemia L1210 cells in mitosis [ref in a]. (c) Lymphocytic leukemia P388. R.I. Geran, N.H. Greenbert, M.M MacDonald, A.M. Schumacker, and B.J. Abbott, Cancer Chemother. Rep., 3 (2) (1972). (d) Average of 2-determinations. (e) 2-cures. (f) Methotrexate-resistant line of P388 (designated tumor P7 by The Drug Evaluation Branch, National Cancer Institute). In mice with 106 cell implant (IP), methotrexate at a dose of 2mg/kg on the qd 1-9 against the methotrexate-resistant line of P388. (g) Vincristine-resistant line of P388. L.J. Wilkoff and E.A. Dulmadge, J. Natl. Cancer Inst., 61, 1521 (1978). (h) R.D. Elliott, C. Temple, Jr., J.L. Frye, and J.A. Montgomery, J. Org. Chem., 36, 2818 (1971).

Claims: WE CLAIM: 1. A 1,2-dihydropyrido[3,4-b]pyrazine having the formula: EMI31.1 wherein x has a value of 1,2 or 3; Y is CH2 or N(CH3); R1 is a lower alkyl group; R2 is a member selected from the group consisting of hydrogen, alkyl radicals having from about one to about 12 carbon atoms, alkenyl radicals having from about two to about 15 carbon atoms, cycloalkyl radicals having from about three to about 20 carbon atoms, aralkyl and allcaryl radicals having from about six to about 20 carbon atoms, a halogen radical, hydroxyl, amino, alkoxy, aryloxy, alkylthio and arylthio radicals having from about one to about 20 carbon atoms, sulfonyl, alkylsulfonyl and arylsulfonyl radicals having from about one to about 20 carbon atoms, alkylsulfinyl and arylsulfinyl radicals having from about one to about 20 carbon atoms, alkylamino and arylamino radicals having from about one to about 20 carbon atoms, or a hydrocarbyl group carrying halogen, hydroxy, amino, alkoxy or aryloxy, and, when taken together with the aromatic ring to which it is attached, a fused ring structure; and R3 and P4 are either both hydrogen or one is hydrogen and the other is a lower alkyl group; provided that when each of R2, R3 and R4 are hydrogen, Y is CU2; and pharmaceutically acceptable salts thereof. 2. A compound as defined in claim 1 wherein R1 is ethyl. 3. A compound as defined in claim 2 wherein R3 and R 4 are each hydrogen and the group EMI32.1 is selected from those hiving the following structures: C6H5N(CH3)CH2; 4-CH3OC6H4N(CH3)CH2; 4-ClC6H4N(CH3)CH2 and C6H5CH2CH2. 4. Ethyl 5-amino-1,2-dihydro-3-[[N-(4methoxyphenyl)-N-methyl]aminomethyl]pyrido[3,4-b]pyra zine-7-carbamate. 5. Ethyl 5-amino-1,2-dihydro-3-(2-phenylethyl) pyrido[3,4-b-]pyrazine-7-carbomate. zine-7-carbamate. 6. Ethyl 5-amino-1,2-dihydro-3-(2-phenylethyl) pyrido[3,4-b-]pyrazine-7-carbomate. 7. A compound having the formula: EMI32.2 wherein x has a value of 1, 2 or 3; Y is CU2 or N(C133); R1 is a lower alkyl group; R3 2 is a member selected from the group consisting of hydrogen, , alkyl radicals having from about one to about 12 carbon atoms, alkenyl radicals having from about two Lo about 15 carbon atoms, cycloakyl radicals having from about three to about 20 carbon atoms, aralkyl and alkaryl radicals having from about six to about 20 carbon atoms, a halogen radical, hydroxyl, amino, thiol, nitro, alkoxy, aryloxy, alkylthio and arylthio radicals having from about one to about 20 carbon atoms, sulfonyl, alkylsulfonyl and arylsulfonyl radicals having from about one to about 20 carbon atoms, alkylsulfinyl and arylsulfinyl radicals having from about one to about 20 carbon atoms, alkylamino and arylamino radicals having from about one to about 20 carbon atoms, or a hydrocarbyl group carrying halogen, hydroxyl, amino, alkoxy or aryloxy, and, when taken together with the aromatic ring to which it is attached, a fused ring structure; and R4 is hydrogen or a lower alkyl group. 8. A compound as defined in claim 7 wherein R1 is ethyl. 9. A compound as defined in claim 8 wherein R4 is hydrogen and the group EMI33.1 is selected from those having the following structures: C6H5N(CU3)CH2; 4 CH3OC6H4N(CH3)CH2 and C6H5CU2CU2. 10. A compound having the formula: EMI33.2 wherein x has a value of 1,2 or 3; Y is CH2 or N(CH3); R1 is a lower alkyl group; R2 is a member selected from the group consisting of hydrogen, alkyl radicals having from about orie to about 12 carbon atoms, alkenyl radicals having from about two to about 15 carbon atoms, cycloalkyl radicals having from about three to about 20 carbon atoms, aralkyl and alkaryl radicals hiving from about six to about 20 carbon atoms, a halogen radical, hydroxyl, amino, thiol, nitro, alkoxy, aryloxy, alkylthio and arylthio radicals having from about one to about 20 carbon atoms, sulfonyl, alkylsulfonyl and arylsulfonyl radicals having from about one to about 20 carbon atoms, alkylsulfinyl and arylsulfinyl radicals having from about one to about 20 carbon atoms, alkylamino and arylamino radicals having from about one to about 20 carbon atoms, o a hydrocarbyl group carrying halogen, hydroxyl, amino, alkoxy or aryloxy, and, when taken together with the aromatic ring to which it is attached, a fused ring structure; and R3 is hydrogen or a lower alkyl group. 11. A compound as defined in claim 10 wherein R1 is ethyl. 12. A compound as defined in claim 11 wherein R3 is hydrogen and the group EMI34.1 is selected from those having the following structures: C6H5N(CH3)CH2 and 4-ClC6H4N(CH3)CH2. 13. A compound having the formula: EMI35.1 wherein x has a value of 1,2 or 3; Y is CH2 or N(CU3); R1 is a lower alkyl group; R2 is a member selected from the group consisting of hydrogen, alkyl radicals having from about one to about 12 carbon atoms, alkenyl radicals having from about two to about 15 carbon atoms, cycloalkyl radicals having from about three to about 20 carbon atoms, aralkyl and alkaryl radicals having from about six to about 20 carbon atoms, a halogen radical, hydroxyl, amino, thiol, nitro, alkoxy, aryloxy, alkylthio and arylthio radicals having from about one to about 20 carbon atoms, sulfonyl, alkylsulfonyl and arylsulfonyl radicals having from about one to about 20 carbon atoms, alkylsulfinyl and arylsulfinyl radicals having from about one to about 20 carbon atoms, alkylamino and arylamino radicals having from about one to about 20 carbon atoms, or a hydrocarbyl group carrying halogen, hydroxyl, amino, alkoxy or aryloxy, and, when taken together with the aromatic ring to which it is attached, a fused ring structure; and R3 and R4 are either both hydrogen or one is hydrogen and the other is a lower alkyl group. 14. A compound as defined in claim 13 wherein R1 is ethyl. 15. A compound as defined in claim 14 wherein R3 and R4 are each hydrogen and the group EMI36.1 is selected from those hiving the following structures: C6H5N(CH3)CH2, 4-ClC6H4N(CH3)CH2 and C6H5CH2CH2. 16. A process for preparing a 1,2-dihydropyrido[3,4-b]pyrazine as defined in claim 1 which comprises aminating a lower alkyl ester of 6-amino-4chloro-5-nitropyrimidin-2-ylcarbamate with the oxime of an alpha-amino ketone having the structure: EMI36.2 to obtain a lower alkyl ester of a 6-amino-5-nitro-4 [(2-oxoethyl) amino] pyridin-2-ylcarbamate oxime having the structure: EMI36.3 hydrolyzing said compound to obtain the corresponding ketone having the structure: EMI37.1 and subjecting said ketone to catalytic hydrogenation, wherein R R2, R4, x and Y are as defined in claim 1, provided that R2 may also be nitro in the intermediate products. 17. A process for preparing a 1,2-dihydropyrido[3,4-b]pyrazine as defined in claim 1 which comprises aminating a lower alkyl ester of 6-amino-4- chloro-5-nitropyrimidin-2-ylcarbamate with the oxime of an alpha-amino ketone having the structure: EMI37.2 to obtain a lower alkyl ester of a 6-amino-5-nitro-4 [(2-oxoethyl)amino]pyridin-2-ylcarbamate oxime having the structure: EMI38.1 and subjecting said compound to catalytic hydrogenation, wherein R1, R2 Rq, x and Y are as defined in claim 1, provided that R2 may also be nitro in the intermediate products. 18. A process for preparing a 1,2-dihydropyrido[3,4-b]pyrazine as defined in claim 1 which comprises aminating a lower alkyl ester of 6-amino-4- chloro-5 nitropyridin-2-ylcarbamate with an alpha-amino alcohol having the structure: EMI38.2 to give a compound having the structure: EMI38.3 oxidizing said compound to give a ketone having the structure: EMI39.1 and subjecting said ketone to catalytic hydrogenation, wherein R1, R2, R3, x and Y are as defined in claim 1r provided that R2 may also be nitro in the intermediate products. 19. A pharmaceutical composition in dosage unit form comprising an effective amount of a compound as defined by claim 1 in association with a pharmaceutical carrier.

Inventors: ELLIOTT, ROBERT D., MONTGOMERY, JOHN A., TEMPLE, CARROLL G., JR., WHEELER, GLYNN P.
Applicants: SOUTHERN RESEARCH INSTITUTE
Application year: 1983
Main classification: C07D471
IPC codes: A61P35, C07C239, C07C67, C07D213, C07D471, A61K31