WO1999057119A1

WO1999057119A1 - Novel dopamine-like 9-substituted hypoxanthine and methods of use

Info

Publication number: WO1999057119A1
Application number: PCT/US1999/009743
Authority: WO
Inventors: Alvin J. Glasky
Original assignee: Neotherapeutics Inc
Current assignee: Spectrum Pharmaceuticals Inc
Priority date: 1998-05-04
Filing date: 1999-05-04
Publication date: 1999-11-11
Anticipated expiration: 2000-11-04
Also published as: AU3879899A

Abstract

Novel 9-substituted hypoxanthine derivatives with unexpected physiological effects are disclosed. Among these compounds are N-(2-(3,4- dihydroxyphenyl) ethyl)-3- (6-oxohydropurin-9-yl) propanamide, N-(2-hydroxy-2- (3,4-dihydroxyphenyl) )ethyl-3-(6- oxohydropurin-9-yl) propanamide, and N-(1-carboxyl -2-(3,4-dihydroxyphenyl) )ethyl-3-(6- oxohydropurin-9-yl) propanamide. Also disclosed are pharmaceutical compositions and methods for use of the derivatives to treat disorders or conditions treatable by monoamine oxidase inhibitor activity and to regulate calcium channel function.

Description

1 NOVEL DOPAMINE-LIKE 9-SUBSTITUTED HYPOXANTIΠ E AND METHODS OF USE

FIELD OF THE INVENTION

The present invention is broadly directed to purine based compounds. More particularly, the present invention is directed to a novel purine analog having activity mimicking dopamine in addition to other physiological activities and to associated methods of use.

BACKGROUND OF THE INVENTION

The efficacy of many contemporary pharmaceutical compounds intended to treat neurological and physiological conditions is limited by their inability to cross the blood- brain barrier. As a result, molecules that may have neurological activity cannot be administered orally or through injection into the bloodstream because the blood-brain barrier serves as a filter to keep these molecules from leaving the bloodstream and entering the brain and spinal cord. Currently there are three alternative approaches to achieving blood-brain barrier access. The first is to introduce pharmaceutical compounds by direct injection into the brain through the skull. While such treatments have demonstrated some success, the possibility of infection coupled with the complexity and expense of such procedures have limited their practical usefulness. Additionally, there is resistance among many patients to the administration of such injections directly into the skull. The second approach involves the utilization of chemical agents which temporarily break down the blood-brain barrier in order to allow molecules to enter the central nervous system. At present, this approach is in the very early stages of development and carries with it the potential for allowing molecules of all sizes (including undesirable compounds) to cross the blood-brain barrier. This approach, unless and until it can be refined to allow for greater selectivity in crossing the blood-brain barrier, carries with it serious risks. The third approach, pioneered by the present inventor, involves developing small molecules which can mimic the activities of bioactive molecules yet can pass 2 through the blood-brain barrier following oral administration or administration through injection into the bloodstream.

Therefore, there is a need for the development of small molecules that can mimic physiological activities of bioactive molecules and can cross the blood-brain barrier efficiently without requiring complete degradation of the blood-brain barrier.

It is accordingly an object of the present invention to provide such compounds which can either mimic the actions of molecules normally unable to cross the blood-brain barrier or which can stimulate other, unexpected physiological activities.

It is an additional object of the present invention to produce pharmaceutical medicaments configured from such compounds and to provide methods for using these pharmaceutical compositions to treat a variety of physiological, neurological, and psychological disorders and disease conditions.

SUMMARY

I have found that novel 9-substituted hypoxanthine derivatives have unexpected properties and have monoamine oxidase inhibitor activity.

One embodiment of the present invention is a 9-substituted hypoxanthine derivative of formula (I)

O

(CH -

O

(I)

wherein n is an integer from 1 to 6, Ri is selected from the group consisting of H, COOH, and COOWi, where Wi selected from the group consisting of lower alkyl, amino, and lower alkylamino, R₂ is selected from the group consisting of H and OH, and R₃ is selected from the group consisting of H and OH.

Preferably, n is 2. When n is 2, one particularly preferred 9-substituted hypoxanthine derivative is N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9- yl)propanamide, where n=2, Ri is H, R₂ is H, and R₃ is OH. This compound is designated as AIT-203. Another particularly preferred 9-substituted hypoxanthine derivative is N-(2- hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, where n=2, Ri is H, R₂ is OH, and R₃ is OH. This compound is designated as AIT-297. Still another particularly preferred 9-substituted hypoxanthine derivative is N-(l-carboxyl-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, where n=2, Ri is COOH, R₂ is H, and R₃ is OH. This compound is designated as AIT-201.

Another aspect of the present invention is a pharmaceutical composition comprising:

(1) an effective amount of a 9-substituted hypoxanthine derivative according to the present invention as described above; and

(2) a pharmaceutically acceptable carrier. Yet another aspect of the present invention is a method of treating a disease or condition in a mammal treatable by inhibiting the activity of a monoamine oxidase comprising the step of administering an effective amount of a 9-substituted hypoxanthine derivative according to the present invention to the mammal.

Still another aspect of the present invention is a method of regulating calcium channel function in a mammal comprising administering an effective amount of a 9-substituted hypoxanthine derivative according to the present invention to the mammal.

Preferably, the 9-substituted hypoxanthine derivative is N-(2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide.

BRIEF DESCRIPTION OF THE DRAWINGS

The following invention will become better understood with reference to the specification, appended claims, and accompanying drawings, where:

Figure 1 is a diagram of a thin-layer chromatogram of the crude product from the synthesis of N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide of Example 2; chromatography was performed in 20% methanol in ethyl acetate, with visualization by ultraviolet light;

Figure 2 is a diagram of a thin-layer chromatogram of the purified product from the synthesis of N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide of Example 2; chromatography was performed in 30% methanol in ethyl acetate with visualization by ultraviolet light;

Figure 3 is a graph showing the effect of 150 μM of N-(2-(3,4- dihydroxyphenyl)ethyl)-3 -(6-oxohydropurin-9-yl)propanamide, N-(2-hydroxy-2-(3 ,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, and N-( 1 -carboxyl-2-(3 ,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, as well as, for comparison, a 9-substituted hypoxanthine derivative that is a serotonin analogue, N-(2-(5-hydroxyindol-3- 5 yl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, on the catalytic activity of monoamine oxidase A/B; the reaction mixture contained 150 μM substrate (tyramine) and 0.22 mg/mL mitochondrial protein;

Figure 4 is a graph showing the effects of increasing concentrations of the 9- substituted hypoxanthine derivatives tested in the experiment of Figure 3 on the activity of monoamine oxidase A/B; concentrations tested were 0 μM, 2 μM, 10 μM, 20 μM, and 100 μM; the reaction mixture contained 166 μM substrate (tyramine) and 0.24 mg/mL mitochondrial protein;

Figure 5 is a graph showing the effect of increasing concentrations of N-(2- (3 ,4-dihydroxyphenyl)ethyl)-3 -(6-oxohydropurin-9-yl)propanamide, N-(2-hydroxy-2-(3 ,4- dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9-yl)propanamide, and N-( 1 -carboxyl-2-(3 ,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide on the binding of labeled spiroperidol to D₂ dopamine receptors;

Figure 6 is a graph showing the effect of increasing concentrations of N-(2- (3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, and N-(l-carboxyl-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide on the binding of labeled SCH- 23390 to Di dopamine receptors; and

Figure 7 is a graph showing the effect of increasing concentrations of N-(2- (3 ,4-dihydroxyphenyl)ethyl)-3 -(6-oxohydropurin-9-yl)propanamide, N-(2-hydroxy-2-(3 ,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, and N-(l-carboxyl-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide on the binding of labeled MK- 801 to NMDA receptors.

DETAILED DESCRIPTION

These and other objects are achieved by the novel pharmaceutical composition and associated methods of the present invention which provide a novel 9-substituted hypoxanthine based composition which is able to cross the blood-brain barrier and which can impact a variety of physiological and psychological systems. 6

In general, 9-substituted hypoxanthine-based compounds according to the present invention have the structure shown in Formula (I), where n is an integer from 1 to 6, Ri is selected from the group consisting of H, COOH, and COOWi, where Wi selected from the group consisting of lower alkyl, amino, and lower alkylamino, R₂ is selected from the group consisting of H and OH, and R₃ is selected from the group consisting of H and OH. "Lower alkyl," in the context of this disclosure, means a saturated alkyl group of from 1 to 6 carbon atoms. It is generally preferred that n is 2.

O

(CH2)_n-

O

(I)

Particularly preferred compounds according to the present invention are: (1) N- (2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, where n=2, Ri is H, R₂ is H, and R₃ is OH, designated as AIT-203; (2) N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl- 3-(6-oxohydropurin-9-yl)propanamide, where n=2, Ri is H, R₂ is OH, and R₃ is OH, designated as AIT-297; and (3) N-(l-carboxyl-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide, where n=2, Ri is COOH, R₂ is H, and R₃ is OH, designated as AIT-201.

More specifically, the present invention provides a novel purine analog, N-(2- (3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, which, in a broad aspect, can be viewed as analogous to hypoxanthine chemically linked to a dopamine analog. Surprisingly, this compound exhibits functional features of both hypoxanthine and dopamine. As a result, it is able to pass through the blood-brain barrier following oral administration or administration through injection into the bloodstream and, because of the structural similarity of a portion of this compound to dopamine, it exhibits physiological activity mimicking dopamine. Thus, it can function as an orally administered or injectable treatment for Parkinson's disease and for other conditions in which dopamine activity is abnormally lowered or in which the interaction between dopamine and dopamine receptors is blocked. Exemplary dosages in accordance with the teachings of the present invention range from 0.01 mg/kg to 60 mg/kg, though alternative dosages are contemplated as being within the scope of the present invention.

Suitable dosages can be chosen by the treating physician by taking into account such factors as the size, weight, age, and sex of the patient, the physiological state of the patient, the severity of the condition for which the compound is being administered, the response to treatment, the type and quantity of other medications being given to the patient that might interact with the compound, either potentiating it or inhibiting it, and other pharmacokinetic considerations such as liver and kidney function.

Another aspect of the present invention is pharmaceutical compositions. A pharmaceutical composition according to the present invention comprises:

(1) an effective amount of a 9-substituted hypoxanthine-based compound according to the present invention, as described above; and

(2) a pharmaceutically acceptable carrier.

Preferably, the 9-substituted hypoxanthine-based compound is N-(2-(3,4- dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide.

The pharmaceutically acceptable carrier can be chosen from those generally known in the art, including, but not limited to, human serum albumin, ion exchangers, alumina, lecithin, buffered substances such as phosphate, glycine, sorbic acid, potassium sorbate, and salts or electrolytes such as protamine sulfate. Other carriers can be used.

Yet another aspect of the present invention is a method of treating a disease or condition in a mammal treatable by inhibiting the activity of a monoamine oxidase. The method comprises the step of administering an effective amount of the 9-substituted hypoxanthine derivative of the present invention to the mammal. The amount that is an effective amount can be determined from enzyme assays as an amount that produces a detectable inhibition of either monoamine oxidase A or monoamine oxidase B or both enzymes in the enzyme assay used in Example 4.

The most effective mode of administration and dosage regimen for the 9- substituted hypoxanthine derivatives as used in the methods in the present invention depend on the severity and course of the disease, the patient's health, the response to treatment, other drugs being administered and the response to them, pharmacokinetic considerations such as the condition of the patient's liver and/or kidneys that can affect the metabolism and/or excretion of the administered 9-substituted hypoxanthine derivatives, and the judgment of the treating physician. According, the dosages should be titrated to the individual patient.

Among the diseases and conditions for which monoamine oxidase inhibitors are clinically indicated are psychological and psychiatric conditions such as depression, panic disorders, and obsessive-compulsive disorder; chronic pain disorders such as diabetic and other peripheral neuropathic syndromes and fibromyalgia; peptic ulcer and irritable bowel syndrome; chronic fatigue; cataplexy; sleep apnea; migraine, and Parkinson's Disease. Such monoamine oxidase inhibitors may also be useful for other conditions.

The mammal can be a human or another socially or economically important mammal such as a dog, a cat, a horse, a cow, a pig, or a sheep. The method of the present invention is not limited to treatment of humans.

Yet another aspect of the invention is the use of compounds according to the present invention, particularly N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide, in regulating the activity of calcium channels in mammalian tissues. Dysfunction of calcium channels has been implicated in a wide spectrum of human cardiovascular (e.g., hypertension, arrythmia), respiratory (e.g., asthma), and neurological diseases (e.g., stroke, amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease),

Alzheimer's Disease and migraine). Therefore, regulation of calcium channel function is important. The compound N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin- 9-yl)propanamide exhibits both Ca²⁺ agonistic and antagonistic effects. The compound exhibits agonistic effects with vascular smooth muscle, and antagonistic effects with gastrointestinal smooth muscle. Among vascular smooth muscles, the Ca²⁺ agonistic effect is strongest in a small muscular artery and weakest in large elastic arteries. Therefore, this aspect of the invention comprises administering an effective amount of a compound according to the present invention, preferably N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide, to a mammal to regulate calcium channel function.

The invention is illustrated by the following Examples. These examples are provided for exemplification only and are not intended to limit the invention.

Example 1

Condensation of 3-(6-Oxohydropurin-9-yl) Propanoic Acid With N-Hydroxysuccinimide

As a first step in the synthesis of N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6- oxohydropurin-9-yl)propanamide, a derivative of hypoxanthine, 3-(6-oxohydropurin-9-yl) propanoic acid, was activated by condensation with N-hydroxysuccinimide (NHS). The hypoxanthine derivative, 3-(6-oxohydropurin-9-yl) propanoic acid (12.48 g) was reacted with 8.97 g of NHS and 13.62 g of the coupling agent dicyclohexylcarbodiimide (DCC) in 550 ml of dry dimethylformamide (DMF). The resulting mixture was heated with magnetic stirring in an atmosphere of argon in an oil bath (bath temperature 85-90°C) for 4 hours. The reaction mixture was used as such for the reaction of Example 2. 10 Example 2

Synthesis of N-(2-(3 ,4-Dihydroxyphenyl)ethyl -3 -(6-Oxohydropurin-9-vPpropanamide

The dopamine analogue N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6- oxohydropurin-9-yl)propanamide was synthesized by reacting the reaction mixture resulting from Example 1 directly with tyramine hydrochloride to form the amide link. In a flask a suspension of 14.9 g tyramine hydrochloride in 40 ml DMF was heated with 16.7 ml triethylamine with swirling and this mixture was added to the reaction mixture from Example 1. The flask was washed with another 20 ml DMF and added to the reaction mixture. Stirring and heating at 90°C (oil bath) continued. Within 15-20 minutes the reaction mixture became homogeneous. After heating for a total of 2 hours, thin-layer chromatography (TLC) was performed in 20% methanol in ethyl acetate, with visualization by ultraviolet light. The results are shown in Figure 1.

The product was cooled to room temperature and then in an ice/water bath for 30 minutes. The precipitated dicyclohexylurea was filtered off. The filtrate was evaporated to dryness. The residue was heated with methanol with magnetic stirring for 2 hours, filtered, and washed with methanol. The residue was heated with heated with 300 ml saturated aqueous NaHCO₃ with magnetic stirring for 30 minutes to remove any unreacted acid. The product was filtered, washed with water, and dried under vacuum at 60°C overnight. A total of 20 g of the product was recrystallized from 300 ml DMF and 200 ml acetonitrile. The recrystallization mixture was kept in the freezer overnight, filtered, washed with acetonitrile and dried at 60°C under high vacuum for 18 hours.

The yield of product was 13.4 g. Thin-layer chromatography was performed in 30% methanol in ethyl acetate with visualization by ultraviolet light. The results are shown in Figure 2, with a single spot being visualized with an Rf of 0.6. The product had a melting point of 260-263°C. 11 Example 3

Alternative Synthesis of N-(2-Hydroxy-2-(3 ,4-Dihydroxyphenyl))ethyl-3 -(6-Oxohydropurin-9- vDpropanamide

An alternative synthesis of N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide was carried out as follows: A quantity (0.300 g; 0.9111 mmol) of 3-(l,6-dihydro-6-oxo-9H-purin-9-yl)propanoic acid, 4-nitrophenyl ester and (R)-(-)- norepinephrine hydrochloride (0.190 g; 0.924 mmol) were placed into a 10-ml round bottom flask with 2 ml dimethylsulfoxide and a magnetic stirring bar. Triethylamine (128 mg; 1.26 mmol) was added and the solution was stirred at room temperature for one hour. Chloroform (10 ml) was added and a copious yellow precipitate formed immediately. The solution was stirred for several minutes and was filtered by vacuum. The resulting solid was washed with chloroform and allowed to dry. After drying, 359 mg of a yellow solid, N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, was obtained. The yield was about 110%.

Example 4

Inhibition of Monoamine Oxidase A and B by N-(2-(3,4-Dihvdroxyphenyl)ethyl)-3-(6-

Oxohydropurin-9-yDpropanamide. N-(2-Hydroxy-2-(3,4-Dihydroxyphenyl))ethyl-3-(6-

Oxohydropurin-9-yr)propanamide. and N-(l-Carboxyl-2-(3,4-Dihydroxyphenyl) ethyl-3-(6-

Oxohydropurin-9-yl)propanamide

Experiments were carried out to determine whether the dopamine analogues N- (2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, and N-(l-carboxyl-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide inhibited monoamine oxidases A and B. These experiments were performed as follows: 12

Mice (3-5 mice) were sacrificed by cervical dislocation. The brains were isolated and placed immediately in 10 ml cold isolation medium (IM) (500 μM EDTA, 5 mM HEPES, 0.25 M sucrose, and 10 mg L bovine serum albumin (BSA)). The brains were minced and rinsed twice with 10 mL IM. The brains were then homogenized (5-6 strokes) in 10 mL IM. A volume (20 mL) of IM was then added and the homogenized preparation was centrifuged 8-10 minutes at 600 x g at 4°C. The supernatant was transferred to a new tube. The pellet was resuspended in 30 mL IM and centrifuged again for 8 minutes at 600 x g at 4°C. The supernatant was transferred to a new tube and the pellet discarded. Both supernatants were centrifuged for 10 min at 12,000 x g at 4°C. The supernatant was discarded, the pellet was dislodged with a glass rod, and both pellets were resuspended in 5 mL IM and combined. A volume of IM (20 mL) was added and the suspension centrifuged for 10 min at 12,000 x g at 4°C. The supernatants were discarded. The pellets were dislodged with a glass rod and resuspended in 2-4 mL IM and stored on wet ice during the course of the experiments.

The compounds tested were predissolved in dimethyl sulfoxide (DMSO) and further diluted with water as required.

For the enzyme assays, 1.0 mL to 1.5 mL 50 mM sodium phosphate, pH 7.4 containing 5 μM scopoletin and 2 U/mL horseradish peroxidase) were aliquoted in the wells of a Falcon 24-well plate (reaction mixture or RM). Mitochondrial protein (0.22-0.24 mg/mL final concentration) was added, and the reaction mixtures were preincubated approximately 6 minutes with 16 μM pargyline (monoamine oxidase B inhibitor) and 166 μM clorgyline (monoamine oxidase A inhibitor) or 2-150 μM of the tested serotonin analogues or the corresponding volume of water containing 1-5% DMSO respectively at 37°C. The preincubation was carried out in the Cytofluor® 4000 (PerSeptive Biosystems) and the baseline fluorescence at λ_ex=360 nm and λem=460 nm was recorded. After 6 min, the reading was interrupted and 150-166 μM tyramine (monoamine oxidase A and monoamine oxidase B substrate) or the corresponding amount of water were added according to the plate layout. The reading was continued for approximately 30 minutes at 3-minute intervals. The rate of the fluorescence decrease during this time was used to determine the effect of the tested 13 compounds on the catalytic activity of both enzyme isoforms. Each plate contained a positive control containing the inhibitors for monoamine oxidase A or B respectively as well as a negative control without enzyme substrate. All determinations were in duplicates on the plate.

Determination of the protein concentration was carried out according to the manufacturer's instructions (Sigma, St. Louis, MO) for the bicinchoninic acid (BCA) protein determination.

The results are shown in Figures 3 and 4. In Figures 3 and 4, the compounds are designated as follows: N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9- yl)propanamide is designated as AIT-203; N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide is designated as AIT-297; and N-(l-carboxyl-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide is designated as AIT-201. For comparison, a 9-substituted hypoxanthine derivative that is a serotonin analogue, N-(2-(5- hydroxyindol-3-yl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, designated AIT-203, is also included in the experiments of Figures 3 and 4. For the results presented in Figure 3, the reaction mixture contained 150 μM substrate (tyramine) and 0.22 mg/mL mitochondrial protein. For the results presented in Figure 4, the reaction mixture contained 166 μM substrate (tyramine) and 0.24 mg/mL mitochondrial protein.

The ICso is estimated at 6.0 μM for N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6- oxohydropurin-9-yl)propanamide, 6.0 μM for N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3- (6-oxohydropurin-9-yl)propanamide, and 8.0 μM for N-(l-carboxyl-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide. Lyles et al. (J. Pharm. Pharmac. 26: 921-930 (1974)) reported an IC₅₀ of approximately 1.5 μM using rat heart mitochondria and tyramine as substrate. This indicates a significant effect of these dopamine analogues on the catalytic activity of monoamine oxidase A and B. This is particularly important because a drug currently available for treatment of Parkinson's disease, a disease of dopaminergic neurons, selegiline, which has the chemical name (R)-(-)-N-2-dimethyl-N-2- propynyl-phenylethylamine hydrochloride, is a monoamine oxidase type B inhibitor. Moreover, this result is unexpected. 14

Example 5

Lack of Dopamine Agonism or Antagonism by N-(2-(3.4-Dihydroxyphenyl)ethvD-3-(6-

Oxohydropurin-9-yl)propanamide. N-(2-Hydroxy-2-(3.4-Dihydroxyphenyl))ethyl-3 -(6- Oxohydropurin-9-vDpropanamide, and N-(l-Carboxyl-2-(3,4-Dihydroxyphenyl))ethyl-3-(6-

Oxohydropurin-9-yl)propanamide

The three compounds tested, the dopamine analogues N-(2-(3,4- dihydroxyphenyl)ethyl)-3 -(6-oxohydropurin-9-yl)propanamide ( AIT-203 ), N-(2-hydroxy-2- (3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide (AIT-297), and N-(l- carboxyl-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide (AIT-201 ), exhibited neither dopamine agonism or dopamine antagonism when tested in systems employing dopamine Di, D₂, or NMDA receptors.

Methods

Bovine Striatal Membrane Preparation. Bovine striata were obtained from a local abbatoir, dissected, and stored at -80°C until use. The striata were homogenized at 0°C in 10 volumes homogenization buffer containing 0.25 M sucrose, 50 mM Tris, pH 7.4, 1 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride (PMSF), using a motor-driven Dounce homogenizer. The tissue was then centrifuged at 1 lOOxg at 4°C for 10 min. The resultant supernatant was collected and stored on ice. The pellet was resuspended in 5 volumes of homogenization buffer and centrifuged at 1 lOOxg at 4°C for 10 min. The resulting supernatant was collected and pooled with the previous supernatant. The pooled supernatants were mixed and centrifuged at 35,OOOxg for 20 min at 4°C. The resultant pellet was collected nd resuspended in 5 volumes of storage buffer containing 50 mM Tris, pH 7.4, 1 mM EDTA, 5 mM MgCl₂, and 0.1% ascorbic acid. The membrane suspension was centrifuged at

35,000xg at 4°C for 30 min and the resultant pellet was resuspended in 2 volumes of storage 15 buffer. The membrane preparation was stored at -80°C and the concentration of protein was then determined by the Coomassie Blue dye-binding method (Bio-Rad, Richmond, CA).

Dopamine D^ Receptor Binding. The method employed was substantially as previously described (S.K. Gupta & R.K. Mishra, "Desensitization of Di Dopamine Receptors Down-Regulates the G_sα Subunit of G Protein in SK-N-MC Neuroblastoma Cells," J. Mol. Neurosci. 4: 117-123 (1993); S.K. Gupta & R.K. Mishra, "Up-Regulation ofDi Dopamine Receptors in SK-N-MC Cells After Chronic Treatment with SCH 23390," Neurosci. Res. Commun. 15: 157-166 (1994)). Bovine striatal membranes (175 μg) were incubated in an 0.5 ml volume of assay buffer containing 50 mM Tris, 1 mM EDTA, 5 mM MgCl₂, 0.1% ascorbic acid, and 0.8 nM [³H]-SCH 23390 (84.0 Ci/mmol) for 90 minutes at 25°C. Non-specific binding was determined in the presence of 1 μM cis-flupenthixol. Dose-response curves were generated for the compounds being tested over a concentration range of 100 pM to 100 μM. The assay was terminated by the addition of 4 ml ice-cold wash buffer (50 mM Tris, pH 7.4, and 5 mM MgCl₂), followed by rapid vacuum filtration through Whatman GF-B filter paper using a Brandell M24 cell harvester. Non-specific binding was removed by washing the filter paper four times with 4 ml of ice-cold wash buffer. Radioactivity was determined by counting the ³H dpm in 5 ml scintillation fluid (BCL, Amersham) using a Beckman LS5000TA liquid scintillation counter.

Dopamine D₂ Receptor Binding The method employed was essentially as previously described (P.W. Baures et al., "Design, Synthesis, X-Ray Analysis, and Dopamine Receptor-Modulating Activity of Mimics of the 'C5' Hydrogen-Bonded Conformation in the Peptidomimetic 2-Oxo-3-(R)-[(2(S)-Pyrrolidinylcarbonyl)amino]- 1 -Pyrrolidineacetamide," 1 Med. Chem. 37: 3677-3683 (1994)). Bovine striatal membranes (175 μg) were incubated in an 0.5 ml volume of assay buffer containing 50 mM Tris, 1 mM EDTA, 5 mM MgCl₂, 0.1% ascorbic acid, 50 nM ketanserin and 0.5 nM [³H]-spiroperidol (93.0 Ci mmol) for 60 minutes at 25°C. Non-specific binding was determined in the presence of 1 μM butaclamol. Dose- response curves were generated for the compounds being tested over a concentration range of 100 pM to 100 μM. The assay was terminated by the addition of 4 ml ice-cold wash buffer (50 mM Tris, pH 7.4, and 5 mM MgCl₂), followed by rapid vacuum filtration through 16

Whatman GF-B filter paper using a Brandell M24 cell harvester. Non-specific binding was removed by washing the filter paper four times with 4 ml of ice-cold wash buffer. Radioactivity was determined by counting the ³H dpm in 5 ml scintillation fluid (BCL, Amersham) using a Beckman LS5000TA liquid scintillation counter.

Glutamate NMDA Receptor Binding. The method employed was essentially as previously described (J.E. Savelli et al., "Modulation of N-Methyl-D-Aspartate (NMDA) Antagonist-Induced Darting Behaviour by the Peptidomimetic PAMTA," Brain Res. 682: 41- 49 (1995)). Bovine striatal membranes (200 μg) were incubated in an 0.5 ml volume of assay buffer containing 10 mM HEPES, 1 mM DTA, 100 μM glutamate, 30 μM glycine and 2.5 nM [³H]-MK-801 (22.0 Ci/mmol) for 120 minutes at 25°C. Non-spcific binding was determined in the presence of 30 μM MK-801. Dose-response curves were generated for the compounds being tested over a concentration range of 100 pM to 100 μM. The assay was terminated by the addition of 4 ml ice-cold wash buffer (50 mM Tris, pH 7.4, and 5 mM MgCl₂), followed by rapid vacuum filtration through Whatman GF-B filter paper using a Brandell M24 cell harvester. Non-specific binding was removed by washing the filter paper four times with 4 ml of ice-cold wash buffer. Radioactivity was determined by counting the ³H dpm in 5 ml scintillation fluid (BCL, Amersham) using a Beckman LS5000TA liquid scintillation counter.

Testing in the 6-Hydroxy-Dopamine Lesioned Rat Model of Parkinson's

Disease. Lesions induced by 6-hydroxy-dopamine (6-OHDA) were made in the substantia nigra of rats and development of dopamine receptor supersensitivity was assessed after i.p. injections of 0.25 mg/kg apomorphine. The compounds were tested at a dose of 0.25 mg/kg; when these compounds were given in combination with apomorphine, they were given 15 minutes prior to the injection of apomorphine.

Results. The results of the dopamine Di, dopamine D₂, and NMDA receptor tests are shown in Figures 6 (dopamine DI), 5 (dopamine D2), and 7 (NMDA), and summarized in Table 1, which gives the IC50 values for these compounds toward these receptors in bovine striatum. These results indicate that these compound do not displace [³H] 17 radioligands specifically bound to these receptors, which indicates that they do not interact with these receptors.

TABLE 1

IC50 values for various 9-substituted hypoxanthine derivatives toward dopamine Di, dopamine D₂, and NMDA receptors in bovine striatum.

Receptor [³H]-Ligand Competing Drug Affinity, log Non-Specific (M) Binding

Dopamine Di [³H]-SCH23390 cis-flupenthixol -8.4 10%

AIT-201 7-4

AIT-203 7-4

AIT-297 7-4

Dopamine D₂ [³H]-Spiroperdol Butaclamol -8.3 25%

AIT-201 7-4

AIT-203 7-4

AIT-297 7-4

Glutamate [³H|-MK-801 MK-801 -8.6 21%

NMDA AIT-201 7-4

AIT-203 7-4

AIT-297 7-4

The results in the rat model of Parkinson's disease are summarized in Table 2. These results that the compounds were ineffective either as antagonists or as agonists in this model.

18 TABLE 2

Testing of 9-substituted hypoxanthine derivatives in the 6-hydroxy-dopamine lesioned rat model of Parkinson's disease.

Rat # Compound Compound Alone Apomorphine-Induced Rotations Tested Baseline Compound

Pretreatment

40 AIT-201 No turns or 55 61 locomotion

37 AIT-201 No turns or 91 77 locomotion

35 AIT-203 No turns or 66 53 locomotion

36 AIT-203 No turns or 47 55 locomotion

39 AIT-297 No turns or 41 37 locomotion

34 AIT-297 No turns or 40 38 locomotion

Thus, unexpectedly, these compounds did not act as either agonists or antagonists of dopamine in these systems.

Example 6

Ca²⁺ Agonism and Antagonism of N-(2-Hvdroxy-2-(3,4-Dihvdroxyphenyl))ethyl-3-(6-

Oxohydropurin-9-yl)propanamide

The compound N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide (AIT-297) was tested for Ca²⁺ agonism and Ca²⁺ antagonism in smooth muscle preparations. 19

The following results were obtained:

(1) In vascular tissues devoid of functional epithelium, the compound N-(2- hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide elicited a contraction, in a concentration dependent manner (10-400 μM) with a maximal magnitude of 200-250%, 25-50%, and 10-20% of the maximal concentration induced by 80 mM KCl in physiological saline solution (PSS or Krebs solution) in dog mesenteric artery, dog carotid artery, and rat aorta, respectively. Such a contraction in unstimulated vascular smooth muscle tissues was not obsrved in Ca²⁺-free PSS, suggesting the requirement of extracellular Ca²⁺ in sustaining the vasoconstriction.

(2) Washout of N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide restored the original level of resting tension without compromising the contraction to 80 mM KCl, suggesting that the effect of N-(2-hydroxy-2- (3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide was entirely reversible.

(3) In the presence of 100 μM of N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9-yl)propanamide, the concentration-response profile of KCl contraction shifted to the left, which is consistent with N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide being a L-type Ca²⁺-channel agonist.

(4) The contraction due to N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide was totally blocked by 10 μM verapamil, but only little affected by 1 μM nifedipine. This finding is extremely interesting and may suggest agonistic effect at a selective site of the L-type Ca²⁺ channels.

(5) The effect of N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide on the lower esophageal sphincter (LES) smooth muscle of the dog was totally different from that on the vascular smooth muscle. The compound N-(2- 20 hydroxy-2-(3 ,4-dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9-yl)propanamide inhibited the tone of LES by 50-80% in a concentration-dependent manner when tested at a range of 10- 400 μM. The spontaneous muscle tone of LES following stretch is known to be sensitive to the removal of extracellular Ca²⁺ and L-type Ca²⁺ channel antagonists. Thus N-(2-hydroxy-2- (3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide appears to be a Ca²⁺ antagonist in this case.

(6) The field-stimulated response of isolated LES strips was also modified by 200 μM of N-(2-hydroxy-2-(3 ,4-dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9- yl)propanamide in such a manner that the rapid relaxation response (release of nitric acid due to stimulation of NCNA nerve) was unaltered, but the subsequent contractile response, most likely due to the release of acetylcholine from the cholinergic nerves, was enhanced. These responses may be related to the N-type Ca²⁺ channels.

These results indicate that N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide has tissue selectivity in two respects. First, the Ca 2+ agonistic effect of this compound was associated with vascular smooth muscle, while the Ca 2+ antagonistic effect was associated with gastrointestinal smooth muscle. Second, among vascular smooth muscles, the Ca²⁺ agonistic effect of this compound was strongest in dog mesenteric artery, a small muscular artery, and relatively low in dog carotid artery and rat aorta, the large elastic arteries. Thus, N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6- oxohydropurin-9-yl)propanamide exhibits selectivity by tissue type and size of artery.

ADVANTAGES OF THE INVENTION

The 9-substituted hypoxanthine derivatives of the present invention cross the blood-brain barrier efficiently and selectively and have unexpected activity as monoamine oxidase inhibitors. These derivatives therefore have use in treating conditions and diseases treatable with monoamine oxidase inhibitors. 21

Although the present invention has been described with considerable detail, with reference to certain preferred versions thereof, other versions and embodiments are possible. Therefore, the scope of the invention is determined by the following claims.

Claims

22 What is claimed is:

1. A 9-substituted hypoxanthine derivative of formula (I)

O

(CH2)_n-

O

(I)

2. The 9-substituted hypoxanthine derivative of claim 1 where n is 2.

3. The 9-substituted hypoxanthine derivative of claim 2 wherein the compound is N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9-yl)propanamide, where n=2, Ri is H, R₂ is H, and R₃ is OH.

4. The 9-substituted hypoxanthine derivative of claim 2 wherein the compound is N-(2-hydroxy-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, where n=2, Ri is H, R₂ is OH, and R₃ is OH.

5. The 9-substituted hypoxanthine derivative of claim 2 wherein the compound is N-(l-carboxyl-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide, where n=2, R_Ϊ is COOH, R₂ is H, and R₃ is OH. 23

6. A pharmaceutical composition comprising:

(a) an effective amount of the 9-substituted hypoxanthine derivative of claim 1; and (b) a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6 wherein the 9-substituted hypoxanthine derivative is the compound of formula (I) wherein n is 2.

8. The pharmaceutical composition of claim 7 wherein the 9-substituted hypoxanthine derivative is N-(2-(3,4-dihydroxyphenyl)ethyl)-3-(6-oxohydropurin-9- yl)propanamide.

9. The pharmaceutical composition of claim 7 wherein the 9-substituted hypoxanthine derivative is N-(2-hydroxy-2-(3 ,4-dihydroxyphenyl))ethyl-3 -(6-oxohydropurin- 9-yl)propanamide.

10. The pharmaceutical composition of claim 7 wherein the 9-substituted hypoxanthine derivative is N-(l-carboxyl-2-(3,4-dihydroxyphenyl))ethyl-3-(6-oxohydropurin- 9-yl)propanamide.

11. A method of treating a disease or condition in a mammal treatable by inhibiting the activity of a monoamine oxidase comprising the step of administering an effective amount of the 9-substituted hypoxanthine derivative of claim 1 to the mammal.

12. The method of claim 11 wherein the 9-substituted hypoxanthine derivative is the compound of formula (I) wherein n is 2.

13. The method of claim 12 wherein the 9-substituted hypoxanthine derivative is N-(2-(3 ,4-dihydroxyphenyl)ethyl)-3 -(6-oxohydropurin-9-yl)propanamide. 24

14. The method of claim 12 wherein the 9-substituted hypoxanthine derivative is N-(2-hydroxy-2-(3 ,4-dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9-yl)propanamide.

15. The method of claim 12 wherein the 9-substituted hypoxanthine derivative is N-( 1 -carboxyl-2-(3 ,4-dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9-yl)propanamide.

16. A method of regulating calcium channel function in a mammal comprising administering an effective amount of the 9-substituted hypoxanthine derivative of claim 1 to the mammal.

17. The method of claim 16 wherein the compound is N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3 -(6-oxohydropurin-9-yl)propanamide .

18. The method of claim 17 wherein the N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide acts as a Ca²⁺ agonist.

19. The method of claim 17 wherein the N-(2-hydroxy-2-(3,4- dihydroxyphenyl))ethyl-3-(6-oxohydropurin-9-yl)propanamide acts as a Ca²⁺ antagonist.