PL170235B1 - Method of obtaining 9-(3-cyanophenyl)-3,4,6,7,9,10-heksahydro-1,8-(2h,5h)acridinodione - Google Patents

Abstract

9- (3-cyanophenyl) production method 3,4,6,7,9,10-helkiaiydrro 1,8- / 2H, 5H / acridine a dione or a pharmaceutically acceptable thereof an acid addition salt characterized in that 3-Ammo-2-cyclohexen-1-one is reacted with 3-cyanobenzaldehyde or its acetal or a hemiacetal or a reactive derivative.

Description

The present invention relates to a method for the preparation of a novel compound useful as a cell opener of potassium channels in mammals such as man. More specifically, the present invention relates to a process for the preparation of 9- (3-cyanophenyl) -3,4,6,7,9,10-hexahydro-1,8- (2H, 5H) acridinedione represented by the formula 1 and hereinafter referred to as the compound, applicable for the treatment of urinary incontinence in mammals (including man).

DE 2003148 discloses a group of 1,4-dihydropyridine derivatives which are said to have a broad and multifaceted pharmacological spectrum of action. The main effects of the compounds have been found to include a strong action to reduce muscle contraction as evidenced by smooth muscles of the gastrointestinal tract, urogenital tract and respiratory system. Other main effects reported are on the heart (heart relief) and blood pressure reduction in animals with normal or high blood pressure so that they can be used as antihypertensive agents. This compound has unexpected properties in that it has the ability to selectively act on the bladder without significantly affecting the cardiovascular system, as shown by measurements of heart rate and blood pressure. Thus, advantageously, the compound can be used to treat urinary incontinence in elderly patients for whom cardiovascular effects, such as blood pressure lowering effects, are particularly undesirable.

As stated above, the invention includes a process for the preparation of 9- (3-cyanophenyl) 3,4,6,7,9,10-hexahydro-1,8- (2H, 5H) acridinedione.

Also included within the scope of the invention are the acid addition salts of the above compound prepared with a sufficiently strong acid to provide a physiologically acceptable anion.

In the process of the present invention, a compound is prepared by reacting a 3-amino-2-cyclohexen-1-one of the formula III with a benzaldehyde of the formula II or its acetal or hemiacetal or a reactive derivative thereof. The reaction can be carried out as reported by Chaaban et al. In J. Chem. Soc. Perkin I, 1593 (1978) or Eynde et al in Tetrahedron, Vol. 48, No. 7, pp. 1263-1268, 1992.

When benzaldehyde or its acetal or hemiacetal is used in the reaction, the reaction is conveniently carried out in the presence of an acid catalyst, for example hydrochloric acid, sulfuric acid, acetic acid or p-toluenesulfonic acid. The reaction is conveniently carried out at a temperature ranging from 0-100 ° C, preferably from 25-40 ° C. Suitable solvents for the reaction include alcohols, for example ethanol.

When a reactive benzaldehyde derivative is used in the reaction, it may be, for example, N- (alpha-chlorophenylmethyl) pyridimium chloride. Thus, benzaldehyde can be treated with thionyl chloride in the presence of a halogenated hydrocarbon solvent such as dichloromethane, and the resulting N- (alpha-chlorophenylmethyl) pyridimic chloride can then be reacted with a compound of formula 3.

If not commercially available, necessary starting materials for processes such as those described above can be prepared by methods selected from standard chemistry techniques.

170 235, a technique that is analogous to the known syntheses of structurally similar compounds or techniques that are analogous to the procedures described above or the procedures described in the examples.

When used to treat urinary incontinence, the compound is generally administered in the form of a suitable pharmaceutical composition which contains the compound together with a pharmaceutically acceptable diluent or carrier. The composition is adapted to the particular route of administration selected, with oral administration being preferred. They can be obtained by traditional methods, using known excipients and binders, and they can be in various dosage forms. They can, for example, be in the form of tablets, capsules, solutions or suspensions for oral administration.

The following are exemplary pharmaceutical dosage forms containing the compound for therapeutic or prophylactic use in humans:

(a) Tablets mg / tablet Relationship 50.0 Mannit, Pharmacopeia St. US (USP) 223.75 Croscarmellose sodium 6.0 Corn starch 15.0 Hydroxypropyl methylcellulose (HPMC), USP 2.25 Magnesium stearate 3.0 (b) Capsules Relationship 10.0 Mannite, U488.5 Croscarmellose sodium 15.0 mg / capsule Magnesium stearate 1.5 The above formulations can be obtained by procedures well known in the art of pharmacy.

The tablets may be fully coated with conventional agents, for example they may have a coating of cellulose acetate phthalate.

Treatment with a compound of the invention may be curative or therapeutic by administering the compound at the onset of the disease or developing urinary incontinence in the patient. Treatment can also be prophylactic or future proof by administering the compound pre-emptive to incontinence that may develop, for example in a patient who has suffered from urinary incontinence in the past.

It is known that bladder tissue is excitable and that urinary incontinence can be caused by uncontrolled or unsteady contractions. It is further known that by acting to open the potassium channels, compounds which open the potassium channel can act to relax smooth muscle. Without wishing to be bound by theory, it is believed that the compound of the invention acts by opening potassium channels in bladder cells and thereby relaxing bladder smooth muscle tissue thereby preventing or correcting uncontrolled bladder contractions which may result in urinary incontinence.

The administered dose of the compound will vary according to the rules with respect to the route of administration, the severity of the incontinence state, and the size and age of the patient. In general, a compound of formula 1 will be administered to a warm blooded animal (including a human) such that an effective dose is obtained, generally a daily dose of greater than 0.005, for example in the range of about 0.01 to about 10 mg / kg body weight. Preferably the compound is administered orally within this dose range. The compound was found to be active and selective in rats when dosed orally at 0.1, 0.3, 1.0 and 3.0 mg / kg. The compound has also been found to be active and selective in dogs when administered orally at a dose of 3 mg / kg. It will be appreciated that the precise dose range into which the compound may be dosed to obtain selective action will depend on the particular species treated. This dose can be determined by traditional methods. In general, selective action is expected to be achieved with oral doses of 3 mg / kg or less, for example 1 mg / kg or less. In some cases, a selective effect may be obtained at a dose of 0.3 mg / kg or less, for example 0.1 mg / kg or less.

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It should be noted that the compound of formula I may be co-administered with other therapeutic or prophylactic agents and / or medicaments compatible with medicine. The compound according to the invention does not show any adverse side effects in animals for laboratory tests, repeatedly exceeding the minimum effective dose.

The activity of compounds of formula I as smooth muscle relaxants, useful as therapeutic agents for the treatment of urinary incontinence, can be demonstrated using appropriately designed in vitro tests such as the test described below. The compound of the invention exhibits an IC50 of 4.2 0.4 micromolar in the test. IC50 is a well known term and represents the concentration of a test compound which produces a 50% in vitro reduction in contraction of bladder tissue described in the following test.

Male Hartley's albino guinea pigs (450-500 g) are killed by twisting their necks. The lower abdominal cavity opens and locates the bladder. Once located, it is cleansed of surrounding connections and adipose tissue. Two pelvic nerves on the ventral surface of the bladder are severed, then the bladder substance is removed above the entrance of the ureters. The bladder is washed in Krebs-Henseleit buffer solution (composition in mmoles: NaCl 118.0, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCh 2.5, NaHCOs 25 and D-glucose 11.1) a it then places it on a buffer-soaked gauze in a petri dish. The bladder dome is cut off and discarded.

A longitudinal intra-abdominal incision is made with scissors and the bladder is laid flat on the gauze. The stripes are stripped from the edge of the dome and the edge of the base and discarded. The remaining detrusor in the mid-section is cut into two wide (horizontal) stripes approximately 2.0 mm wide. These two stripes are cut in half at the center of the dorsal portion to form four stripes of similar dimensions. Thus, each strip comprises a dorsal portion and a ventral portion of the bladder.

Each individual strip is tied at one end directly to the supporting glass rod and a length of 4-0 black braided surgical suture is tied to the other end. Glass rods are secured in a 20 ml tissue bath and the length of the surgical suture is attached to a forced displacement transducer (GrassFT03 model).

Tissues are bathed in a Krebs-Henseleit buffer solution. The bath solution is heated to 37 ° C and saturated with 5% CO2 and 95% O2 gas by vigorously bubbling the bubbles. The solution should have a pH of approximately 7.4.

The transmitters are connected to a polygraph (Grass 7E model) and integrated into the system interfacial by the process signal from the Modular Instrument Micro 5000 and Biowindow Data Acquisition Software (running on Microsoft OS / 2 with a compatible IBM PC).

The polygraph is calibrated at 5 mV / cm and the calibration is checked for linearity with 5 and 0.5 g weights.

Tissue was incubated in the buffer solution for 15 minutes without preload and then 30 minutes with the voltage applied. The pre-load applied was 2 g, which was relaxed to about 1 g. The tissue was washed at 15 minute intervals with the tension adjusted to 2 g just before washing. After a 45 minute equilibration period, a control dose of 15 mmol KCl (total bath concentration) was applied. The tissue was washed after 10 minutes and two more times at 15 minute intervals with up to 2 g tension applied before each wash.

When the tissue relaxes to a steady state after final washing, 15 mmol KCl are added again. As soon as the tissue reaches a steady state, baseline data is obtained on the Biowindow Data Acquisition System. This is done by averaging 5 minutes of measurement data at 32 Hz. Once a baseline is obtained, test compounds are dosed cumulatively with half-log overgrowths. The contact time for each dose is 10 minutes with a final 5 minutes which is the time period for obtaining dose response data. If 30 μmoles of the test compound do not remove the mechanical activity of the detrusor, then 30 μmoles of cromakalim (cromakalim) are dosed in order to establish the maximum dose. The activity of the compounds is expressed in% of maximum relaxation of the tension inducing agonist.

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It should further be noted that the efficacy of the compound according to the invention can be demonstrated in standard in vivo tests. The following is a description of such a standard assay used to evaluate the ability of a test compound to relax smooth muscle.

Male Wistar rats weighing 450-550 g are anesthetized with 20 mg / kg i.p. of Nembutal and 80 mg / kg of Ketamine. A tube is inserted into the trachea to prevent obstruction of the airways. Body temperature is maintained with a heating pad. Arterial blood pressure and heart rate are measured by a pressure transducer connected to a polyethylene (PE50) tube that is inserted into the right carotid artery. A drug delivery tube is inserted into the right jugular vein. The urinary bladder is exposed through an incision in the center of the abdomen and is emptied of urine by light pressure with the hand. The catheter (PE 50) is immersed through the apex of the bladder dome about 3-4 mm of its lumen and tied with surgical suture (4-0 silk) to prevent leakage. The bladder catheter connects to a pressure transducer to measure bladder pressure. The bladder is returned to the abdomen and the incision is sutured except where the catheter is. The bladder is allowed to equilibrate for approximately 15 minutes. After the equilibration period, the rats are poured with saline directly into the bladder at a rate of 0.05 ml / minute throughout the experiment. Thereafter, bladder pressure is monitored for bladder contractions to begin. After the contractions have started, the animal is then allowed to establish the contraction pattern for approximately 30-45 minutes prior to drug administration.

The test compounds are administered intravenously in a strictly defined dose of 3 mg / kg. The comparator drug cromakalim (Smithkline-Beecham) was evaluated in this model and was administered intravenously at a dose of 0.05-0.5 mg / kg.

The above in vivo test enables the assessment of both blood pressure and cystometric activity of the tested compounds. Blood pressure is measured just after drug injection and 5, 15 and 30 minutes later. Soaking spasms are induced by the slow and continuous infusion of saline directly into the bladder. The average change (in seconds versus control) in the duration of the intramystolic interval (time between contractions) over a period of more than about 20 minutes is given for each compound.

The following is a description of the in vivo assay which complements the above-described assay and which can be used to ascertain that the test compound is active and furthermore that the test compound exhibits bladder selectivity without significant effects on the cardiovascular system when administered orally. . The compound is active and selective in this test at an oral dose of 0.1, 0.3, 1.0 and 3.0 mg / kg body weight.

Male Wistar rats / 400-500 g / are anesthetized with 50 mg / kg Nembutal i.p. Each rat has its abdomen and front and back of the neck shaved and the skin disinfected with povidone iodine. For carotid catheterization, the left carotid artery is exposed through a small incision. The exposed area is washed with a 2% lidocaine HCl solution to relax the vessel. The catheter, filled with 0.9% saline, is inserted approximately 2.4 cm into the artery with its tip remaining in the aortic arch. The distal end of the catheter extending from the nape of the neck is filled with heparin (1000 units / ml) and heat sealed. For bladder catheterization, the bladder is exposed through a slit in the center of the abdomen. A trocar is pulled through the abdominal muscle about 1 cm from the top end of the cut and then a subcutaneous channel is made to emerge through the skin on the back of the neck. A saline-filled catheter is passed through the trocar. Small holes are formed in the bladder dome by burning with Accu-Temp. The catheter is placed into the bladder and secured with a 4-0 silk ligature. The catheter is rinsed with saline and checked for patency. The outer end of the catheter is sealed to prevent urine leakage. Abdominal muscles and skin are sewn up. Both catheters are threaded through a stainless steel (Instech) anchored button which is then sutured to the subcutaneous muscle at the point of exit outward. The leather is sewn over the button. The animals are allowed to recover from sleep.

24-48 hours after surgery, each rat is placed in a metabolism cage and connected via an anchored button to the tether spring and Instech articulation system to protect the catheters from damage and allow the animal to rest.

170 235 cage movement. The cervical catheter attaches to a Gould P23XL blood pressure transducer. The bladder catheter connects to the brine infusion pump and pressure transducer with a PE50 tubing and a 4-way closure valve. Peak load compensation with a urine reservoir is placed under the cage to measure urine output.

The rats are weighed, the oral dosing is successful (dosing needle inserted but no fluid is pumped in) and by blistering saline infusion (0.18 ml / minute) is started and continued throughout the experiment. Changes in blood pressure, heart rate, intravesical pressure, and urine output are recorded using either a Grass Polygraph or Gould TA4000 recording system. Animals are allowed to equilibrate until a soaking pattern is established (approximately 45-90 minutes). At this point, the baseline level of each parameter in the experiment is recorded and the rats are dosed by oral gavage with the appropriate dose of the compound (in 75% PEG 400-saline vehicle) at concentrations such that the volume is 1 ml / kg body weight. The effect of the compound on the parameters of the experiment is monitored for 5 hours after the administration of the compound.

Experimental results for both contraction intervals and heart rate are expressed as mean ± S.E.M. / Standard Error of Measures = standard error of measurement /% change from baseline, except that each animal served itself as a control. Mean arterial pressure is expressed as mean ± S.E.M. mmHg changes from baseline.

It should further be noted that the compound also exhibits activity and selectivity when tested in vivo in dogs.

A sterile foley catheter was introduced into well-trained, awake hounds and the residual urine was collected. Using a 4-way valve, the catheter was connected to a Grass Model pressure transducer and bladder pressure was recorded on a dynograph. Chronic catheters were surgically implanted in the carotid artery. Connecting the carotid catheter to a pressure transducer and a recorder allowed for the simultaneous monitoring of blood pressure and heart rate along with the pressure in the bladder obtained from a bladder foley catheter.

After a 15 minute equilibration period, sterile saline was infused into the bladder via a 4-way closure valve by 30 ml single injections until a sustained increase in bladder pressure (10-12 mmHg) was observed. A series of smaller (10-15 ml) infusions was then administered until the bladder began to contract spontaneously. The total volume of the infusion varied from approximately 100 ml to 200 ml depending on the individual animal. Simultaneously, baseline systolic and diastolic blood pressure lines as well as heart rates were obtained. A control period of continuous spontaneous bladder contractions was recorded for 30 to 60 minutes in order to establish a baseline measurement from the intervals between contractions (interstitial interval, seconds) to determine the number of contractions per hour.

Test compounds were then administered by oral gavage suspended in an aqueous suspension vehicle. Blood pressure was measured continuously. Heart rate measurements were taken every 15 minutes for the first two hours after dosing and every 30 minutes at the third and fourth hours. Bladder pressure and interstitial intervals were maintained for four hours after oral administration.

Each dog was left undisturbed for at least one week between experiments to recover. Only calm and cooperative dogs were selected for training and adaptation to conditions of light bladder restraint and catheterization. None of the animals showed any distress or discomfort as a result of the treatment used in this model.

In this trial, the compound was active and selective when administered at a dose of 3 mg / kg.

The invention is now illustrated by the following example in which, unless otherwise stated:

(i) temperatures are given in ° C; operations were carried out at ambient or room temperature, that is, at a temperature in the range of 18-25 ° C;

(Ii) the temperatures reported are uncorrected and (decomposition) indicates decomposition; the melting points given are those for materials prepared as described; in some preparations, polymorphism may occur when isolating materials with different melting points:

(iii) all products were substantially pure by TLC and had satisfactory nuclear magnetic resonance (NMR) and microanalytical data;

(iv) yields are given for illustration only;

(v) the chemical symbols have their usual meanings; the following abbreviations are also used · m.p. (melting point), ml (milliliter), g (grams), and (vi) NMR data are reported as delta values in parts per million (ppm) relative to trimethylsilane (TMS) as internal standard, determined at 300 MHz under using d6-dimethyl sulfoxide as a solvent; traditional abbreviations are used to denote the signal shape;

(vii) mass spectra (MS) were determined with an electron energy of 70 electron volts by chemical ionization using a directly exposed probe; only the parent weight is given.

Example 1 9- (3-Cyanophenyl) -3,4,6,7,9,10-hexahydro-1,8- (2H, 5H) acridinedione.

To a stirred mixture of 3-amino-2-cyclohexen-1-one (4.45 g) and 25 ml of ethanol was added 32 ml of 1N HCl followed by 3-cyanobenzaldehyde (2.62 g), and the mixture was stirred at room temperature overnight. The resulting solid was filtered, washed well with water and then with ethanol. After drying at 50 ° C / 27 Pa the title compound was obtained as a pale yellow solid (3.89 g), mp. 303-7 ° C (dec) MS (Cl, CH4) 3.19 (M + 1).

Analysis for C20H18N2O2: Calcd. C 75.44; H5.71; N 8.80; found C 75.42; H 5.70; N 8.66.

NMR: 1.80-1.93 (m, 4), 2.19-2.22 (m, 4), 2.50-2.54 (m, 4), 4.91 (s, 1), 7.37-7.42 (m, 1), 7.48-7.54 (m, 3), 9.55 (s, 1); MS: m / z = 3.19 (M + 1).

Example II. 9- (3-Cyanotropyl) -3,4,6,7,9,10-hexahydro-1,8- (2H, 5H) -acridinedione.

A stirred solution of thionyl chloride (2.86 g) in 24 ml of methylene chloride under nitrogen atmosphere was cooled to -10 ° C in a bath and treated with a solution of pyridine (1.90 g) in 12 ml of methylene chloride as a slow stream is poured on. A solution of 3-cyanobenzaldehyde (2.62 g) in 10 ml of methylene chloride was then added in a slow stream and the mixture was stirred in a bath at -10 to 0 ° C for 1 hour. Then 3-amino-2-cyclohexen-one (6.67 g) was added in one portion and washed with 10 ml of methylene chloride. After stirring overnight at ambient temperature, the methylene chloride solvent was removed in vacuo and the resulting orange solid was triturated with water. The solid was collected by vacuum filtration, washed well with water then ethanol then dried in vacuo at 60 ° C / 27 Pa to give the title compound (3.86 g) as a pale yellow solid; mp 302-5 ° C (decomp). MS (Cl, CH4): 319 (M + 1). The NMR spectrum was identical to the product of Example I.

170 235

MODEL 1

CHO

CN

PATTERN 2

- NH2

MODEL 3

Publishing Department of the Polish Patent Office. Circulation of 90 copies. Price PLN 2.00

Claims (1)

Patent claim A method for the preparation of 9- (3-cyanophenyl) -3,4,6,7,9,10-hexahydro-1,8- (2H, 5H) acridinedione or a pharmaceutically acceptable acid addition salt thereof, characterized in that 3-amino -2-cyclohexen-1-one is reacted with 3-cyanobenzaldehyde or its acetal or hemiacetal or a reactive derivative.