6-Substituted-2(1h)-Quinolinones and Related Compounds Having Use as Cardiotonic, Antihypertensive, and Antithrombotic Agents

To a slurry of 1.6 g of NaH (60% dispersion in Nujol; 0.036 mol) in 10 ml of of THF is added dropwise with stirring a solution of 2.3 g (0.012 mol) of 6-acetyl-3,4-dihydro-1-methyl-2(1H)-quinolinone (IV) in a mixture of 50 ml of tetrahydrofuran and 10 ml of N,N-dimethylformamide at room temperature.

After the addition is over, the reaction mixture is stirred at room temperature for an additional hour when a clear solution is obtained. Dimethyl carbonate (8 ml) is added to the reaction mixture which is then heated gradually to 100.degree. C. and maintained there for 17 hours. The reaction mixture is cooled, filtered, and the residue is washed with ether. The solid residue is dissolved in water, acidified, and the oil is extracted with methylene chloride. Methylene chloride is stripped leaving behind an oil which is crystallized from ethylacetate to give 1.3 g of 1,2,3,4-tetrahydro-1-methyl-.beta.,2-dioxo-6-quinolinepropanoic acid, methyl ester III, mp 175-176.degree. C.

To a slurry of 0.7 g of sodium hydride (60% dispersion in Nujol; 0.017 mmol) in 10 ml of N,N-dimethylformamide is added dropwise a solution of 1.3 g (0.005 mol) of 1,2,3,4-tetrahydro-1-methyl-.beta.,2-dioxo 6-quinolinepropanoic acid, methyl ester III as prepared above in Preparation 1, in 25 ml of N,N-dimethylformamide at room temperature.

After the addition is completed, a solution of 2.8 g (0.02 mol) of iodomethane in 5 ml of N,N-dimethylformamide is added and stirring is continued for four hours. The reaction mixture is evaporated under reduced pressure and the residue is treated with water. The aqueous solution is extracted with methylene chloride. The extract is washed successively with cold 2 N sodium hydroxide solution and evaporated leaving behind 1.1 g of a viscous gum, 1,2,3,4-tetrahydro-.alpha.,.alpha., 1-trimethyl-.beta.,2-dioxo-6-quinolinepropanoic acid, methyl ester II which was used as such for the synthesis of 6-(4,5-dihydro-4,4-dimethyl)-5-oxo-1H-pyrazol-3-yl)-3,4-dihydro-1-methyl-2 (1H)-quinolinone I in the following Example 1.

A mixture of 1.1 g of 1,2,3,4-tetrahydro-.alpha.,.alpha.-dimethyl-.beta., 2-dioxo-6-quinoline propanoic acid, methyl ester (II) as prepared above in Preparation 2, and 0.5 g of 80% hydrazide hydrate in 15 ml of ethanol is heated under reflux for four hours. The reaction mixture is cooled, filtered, washed with small volume of cold ethanol, and air dried to give 0.7 g of the product 6-(4,5-dihydro-4,4-dimethyl)-5-oxo-1H-pyrazol-3-yl)-3,4-dihydro-1-methyl-2 (1H)-quinolinone (I), mp 235-236.degree. C.

Anal. calcd. for C.sub.15 H.sub.17 N.sub.3 O.sub.2 ;

Found: C, 66.40; H, 6.32; N, 15.49;

C, 66.39; H, 6.38; N, 15.31.

The usefulness of the compounds of the present invention as cardiotonic agents is demonstrated by their effectiveness in standard pharmacological test procedures, for example, in causing a significant increase in the myocardial contractility in the pentobarbital-anesthetized dog with low or minimal changes in heart rate and moderate changes in blood pressure. This test procedure is described in the following paragraphs.

This screen consists of determining the effects of increasing intravenous doses of compound on myocardial contractility (dP/dt max of left ventricular blood pressure), heart rate, aortic blood pressure, and vascular resistance of the pentobarbital-anesthetized dog.

Adult mongrel dogs of either sex are anesthetized with pentobarbital, 35 mg/kg, IV, and are subsequently maintained under anesthesia with a continuous infusion of pentobarbtal, 3.5 mg/kg/hour. The trachea is intubated but the animals are permitted to breathe spontaneously. A cannula is inserted into the femoral vein for administrating test agents. A Millar catheter tip pressure transducer or a fluid filled catheter is inserted into the ascending aorta via the femoral artery for measuring aortic blood pressure. A Millar catheter tip pressure transducer is passed into the left ventricle via the left carotid artery for measuring left ventricle blood pressure. Needle electrodes are placed subcutaneously for recording a lead II electrocardiogram (ECG).

Left ventricular and aortic blood pressures are recorded on a strip chart recorder. Heart rate, using a biotachometer triggered from the R wave of the ECG, and the first derivative of left ventricular blood pressure (dP/dt), obtained with a differentiator amplifier coupled to the corresponding pressure amplifier, are also recorded. Changes in vascular resistance is also recorded showing increased resistance as a peripheral effect. A period of at least 30 minutes is utilized to obtain control data prior to administration of test compound.

Depending on solubility, the compounds are dissolved in 0.9% saline solution or in dilute HCl or NaOH (0.1 or 1.0 N) and are diluted to volume with normal saline. Ethanol of dimethylacetamide can be used as solvents if adequate dilutions can be made. Appropriate vehicle controls are administered when needed.

Each dose of the test compound is administered in a volume of 0.1 ml/kg over a period of one minute.

When tested by the above-described Anesthetized Dog Procedure, the compounds of the present invention when administered intravenously at a rate of about 0.01 to 0.31 mg/kg/min cause dose related significant increases in cardiac contractility with only low or minimal changes in heart, moderate changes in blood pressure and increases in vascular resistance.

Accordingly, the present invention also includes a pharmaceutical composition for increasing cardiac contractility and/or treating hypertension comprising an effective amount of a compound of the formula I as defined above together with a pharmaceutically acceptable carrier.

The present invention further includes a method for incresing cardiac contractility and/or treating hypertension in mammals suffering therefrom comprising administering to such mammals either orally or parenterally a corresponding pharmaceutical composition containing a compound of the formula I as defined above in appropriate unit dosage form.

The usefulness of the compounds of the present invention as antithrombotic agents is demonstrated by their effectiveness in inhibiting human platelet aggregration in standard in vitro platelet aggregation test using human platelet-rich plasma.

Blood is collected from volunteers who have not injested aspirin or other nonsteroidal antiinflammatory drugs within the preceding two weeks and have not eaten within nine hours before blood draw. Blood is collected in 4.5 ml portions in Vacutainer Number 6462S silicone-coated tubes containing 0.5 ml of 3.8% trisodium citrate. Usually six portions of 4.5 ml are drawn from each volunteer. The blood collected from three or four volunteers is pooled prior to centrifugation. The pooled blood is put in 50 ml polyethylene tubes and centrifuged at 80 xg (ca 600 rpm) in an International Model K centrifuge with number 240 rotor for 20 minutes at room temperature. A portion (approximately two-thirds) of the supernatant platelet-rich plasma (PRP) is removed and set aside, and the remaining blood sample is recentrifuged at 1400 xg (ca 2800 rpm) for 15 minutes to prepare platelet-poor plasma (PPP). The platelet content of the PRP is determined with a Coulter Thrombocounter. The PRP is adjusted to a count of 250,000 platelets per microliter using the PPP.

Test drugs are dissolved in small amounts of dimethyl sulfoxide (DMSO) followed by dilution with saline (final concentration of DMSO is 1%). Other lower concentrations are prepared by serial dilution in saline.

Platelet-rich plasma adjusted to 250,000 platelets per microliter is distributed in 0.36 ml aliquots into silicone-coated cuvettes of 0.312 inch diameter. Addition of drug solution or saline (0.02 ml) is followed by addition of aggregating agents (ADP or collagen suspension, 0.02 ml). Extent of aggregation (ADP stimulus) or rate of aggregation (collagen stimulus) is determined using the Payton Scientific Dual Channel Aggregation Module, Model 300B. Appropriate concentrations of aggregating agents are determined by an initial brief titration.

The height in millimeters of aggregation curves for control (no drug addition, saline only) aggregations are compared with the heights of curves obtained after drug addition at various appropriate concentrations. Heights after drug addition are finally expressed as "percent of control" values. These values are plotted versus drug concentration on semilog paper. Estimates of IC.sub.50 values can then be made from the resulting curves.

The major slopes (i.e., the slope of the longest straight line portion) of collagen-induced aggregation curves are determined and compared to the slopes obtained for control aggregation curves (saline and aggregating agent only added). Values obtained are expressed as "percent of control" values. These values are plotted versus drug concentration on semilog paper. Estimates of IC.sub.50 values are made from the resulting curves.

When tested by the above procedure, 6-(4,5-dihydro-4,4-dimethyl)-5-oxo-1H-pyrazol-3-yl)-3,4-dihydro-1-methyl-2 (1H)-quinolinone had a very potent IC.sub.50 of 3.5.times.10.sup.-7 M against collagen-induced aggregation and had an IC.sub.50 of 4.5.times.10.sup.-6 M against ADP-stimulated aggregation.

Accordingly, the present invention also includes a method for treating thrombosis in mammals suffering therefrom comprising administering to such mammals either orally or parenterally a corresponding pharmaceutical composition containing a compound of the formula I as defined above in "Appropriate Unit Dosage Form."

For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solublizers, lubricants, suspending agents, binders or tablet disintegrating agents; it can also be encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active compound. In the tablet the active compound is mixed with carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5 or 10 to about 70 percent of the active ingredient. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" in intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component (with or without other carriers) is surrounded by carrier, which is thus in association with it. Similarly, cachets are included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions. As an example may be mentioned water or water propylene glycol solutions for parenteral injection. Liquid preparations can also be formulated in solution in aqueous polyethylene glycol solution. Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, i.e., natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions. These particular solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit. Alternately, sufficient solid may be provided so that after convension to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volume of the liquid form preparation as with a syringe, teaspoon, or other volumetric container. When multiple liquid doses are so prepared, it is preferred to maintain the unused portion of said liquid doses at low temperature (i.e., under refrigeration) in order to retard possible decomposition. The solid form preparations intended to be converted to liquid form may contain, in addition to the active material, flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The liquid utilized for preparing the liquid form preparation may be water, isotonic water, ethanol, glycerine, propylene glycol, and the like as well as mixtures thereof. Naturally, the liquid utilized will be chosen with regard to the route of administration, for example, liquid preparations containing large amounts of ethanol are not suitable for parenteral use.

Preferably, the pharmaceutical preparation is in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form.

The quantity of active compound in a unit dose of preparation may be varied or adjusted from 10 mg to 500 mg preferably to 5 to 100 mg according to the particular application and the potency of the active ingredient. The compositions can, if desired, also contain other compatible therapeutic agents.

In therapeutic use as described above, the mammalian dosage range for a 70 kg subject is ordinarily from 0.01 to 10 mg/kg of body weight per day or preferably 0.1 to 5 mg/kg of body weight per day. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. ##STR1##