HK1166472B - Treatment of dyskinesia related disorders - Google Patents

Description

Treatment of dyskinesia-related disorders

Technical Field

Aspects of the invention relate to methods of treating parkinson's disease while maintaining a low dyskinesia induction profile and methods of reversing dyskinesias comprising administering a therapeutically effective amount of a compound disclosed herein. The invention also relates to the use of said compounds for the preparation of medicaments for the treatment of said diseases or other movement disorders, such as Huntington's disease, and to pharmaceutical compositions of said compounds.

Background

The use of dopamine replacement agents in the treatment of Parkinson's Disease (PD) symptoms has undoubtedly succeeded in improving the quality of life of patients. L-DOPA, which has been used for many years and remains the gold standard for PD therapy, alleviates motor symptoms of PD characterized by bradykinesia (bradykinesia), rigidity, and/or tremor. It is understood that L-DOPA acts as a prodrug, which is metabolized biologically to Dopamine (DA). DA in turn activates dopamine receptors in the brain, which fall into two categories: d1 and D2 receptors. The D1 receptor can be divided into₁And D₅The D2 receptor can be divided into₂、D₃And D₄A receptor. However, dopamine replacement therapy does have limitations, especially after long-term treatment. Over the years, the duration of response to a dose of L-DOPA has become progressively shorter and the patient's period of response to the drug has been complicated by the appearance of a series of side effects.

Such side effects may be manifested as dyskinesias, which can be observed when patients are undergoing dopamine replacement therapy or even when patients are weaning from therapy. Dyskinesia is an abnormal involuntary movement disorder. Abnormal movements may be manifested as chorea (involuntary, rapid, irregular, rhythmic movements that may affect the face, arms, legs, or torso), tossing (involuntary movements similar to chorea but with more violent and powerful features), dystonia (sustained muscle contractions, usually producing twisting and repetitive movements or abnormal postures or positions), and/or athetosis (repetitive involuntary, slow, bending, twisting movements, which are particularly severe in the hands).

Patients afflicted with PD may cycle between "on" periods (which are complicated by dyskinesias) and "off" periods (during which they are severe parkinson's patients). As a result, they may experience heavy weakness despite the fact that L-DOPA is still a potent antiparkinson drug throughout the disease process (Obeso et al Neurology 2000, 55, S13-23). Dopamine agonists such as bromocriptine, lisuride, pramipexole, ropinirole and pergolide are less effective than L-DOPA, especially in moderate to severe PD. However, their side effect profile is different from that of L-DOPA. It is noteworthy that DA agonists do cause less dyskinesia than L-DOPA, but this has limited value for dyskinetic PD patients, since many of them have moderate to severe PD and therefore they require the efficacy of L-DOPA.

Dyskinesias and other movement disorders resulting from dysfunction of the basal ganglia are of great socio-economic importance. There have been numerous attempts to develop drugs for the prevention and/or treatment of movement disorders, despite the limited success of these efforts. Therefore, there is a need to provide new drugs for the treatment of movement disorders.

The 6-hydroxydopamine (6-OHDA) injury model of Parkinson's disease in rats provides a very valuable tool for studying preclinical PD and evaluating new treatment options (Schwarting and Huston, prog. neurobiol.1996, 50, 275-. One of the most widely used 6-OHDA paradigms is the evaluation of rotational behaviour in rats with discontinuous degeneration of the dopaminergic nigrostriatal pathway (Ungerstedt and aburthinsont, Brain res.1970, 24, 485). In this model, 6-OHDA was injected unilaterally into the nigrostriatal pathway, new striatum, or the forebrain medial bundle (MFB), creating a functional imbalance between dopaminergic nigrostriatal systems. Administration of drugs that directly stimulate dopamine receptors (such as the dopamine metabolic precursor L-DOPA and the dopamine agonist apomorphine) produces a rotational behavior that is directed away from the body side in which the 6-OHDA is injected.

In addition to motion-related defects, the 6-OHDA model may be used to replicate other features of PD. The development of sensitized rotational behaviour and Abnormal Involuntary Movement (AIM) has been observed in rats injected with 6-OHDA in new striatum or MFB and treated chronically with L-DOPA, thus providing another animal model to study L-DOPA-induced dyskinesia (Lundblad et al eur. j neurosci.2002, 15, 120-. In this model, L-DOPA, but not bromocriptine, induced the progressive development of AIM during long-term treatment. Based on these observations, rats injured by 6-OHDA have been considered to exhibit motor deficits that have substantial functional similarities to parkinson's dyskinesia and can be used to evaluate therapeutic potential to provide a dyskinesia treatment.

In an attempt to identify new therapies for the treatment of dyskinesias and other associated movement disorders, applicants have unexpectedly discovered that (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol [ herein referred to as compound 10] is an effective D1/D2 agonist; (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene [ herein compound 11 ]; and (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one [ herein compound 12] have beneficial characteristics in rats with unilateral 6-OHDA injury. They induce less dyskinesia than L-DOPA and apomorphine and are more effective in reducing L-DOPA induced dyskinesia than D2 agonists (as exemplified by pramipexole). Thus, compounds 10, 11 and 12 have the potential to become the first PD drugs with L-DOPA-like efficacy and beneficial characteristics not only in inducing dyskinesias but also as drugs for reversing dyskinesias.

Thus, the compounds identified above are expected to be useful in the treatment of dyskinesias and other associated movement disorders such as Huntington's disease. Furthermore, the present invention contemplates the use of the corresponding racemic trans mixture. The invention also provides a method of treating parkinson's disease with low dyskinesia-inducing characteristics, which method comprises administering a therapeutically effective amount of said compound. In one aspect, treatment of Parkinson's disease is as effective as L-DOPA treatment. Also provided are methods of reversing dyskinesias or treating Parkinson's disease comprising administering the compounds and pharmaceutical compositions thereof.

Summary of The Invention

One aspect of the present invention relates to the use of (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of parkinson's disease while maintaining a low dyskinesia induction profile.

Another aspect relates to the use of racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol in the manufacture of a medicament for the treatment of parkinson's disease while maintaining a low dyskinesia induction profile.

A separate aspect of the invention relates to the use of (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of parkinson's disease.

Another aspect relates to the use of racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol in the manufacture of a medicament for the treatment of parkinson's disease.

A separate aspect of the invention relates to the use of (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for reversing dyskinesia.

Another aspect relates to the use of racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol in the manufacture of a medicament for reversing dyskinesia.

Another aspect relates to a pharmaceutical composition comprising (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol or a pharmaceutically acceptable salt thereof for use in the treatment of parkinson's disease while maintaining low dyskinesia induction characteristics.

An independent aspect of the invention relates to a pharmaceutical composition comprising racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol in the manufacture of a medicament for the treatment of parkinson's disease while maintaining a low dyskinesia induction profile.

Another aspect relates to a method of treating parkinson's disease while maintaining low dyskinesia induction characteristics, comprising administering a therapeutically effective amount of (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol or a pharmaceutically acceptable salt thereof.

A separate aspect relates to a method of treating parkinson's disease while maintaining a low dyskinesia induction profile, comprising administering a therapeutically effective amount of racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol.

Another aspect relates to a method of reversing dyskinesia comprising administering a therapeutically effective amount of (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol or a pharmaceutically acceptable salt thereof.

Another aspect relates to a method of reversing dyskinesia comprising administering a therapeutically effective amount of racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol, or a pharmaceutically acceptable salt thereof.

One aspect of the present invention relates to the use of (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of parkinson's disease while maintaining a low dyskinesia induction profile.

A separate aspect of the invention relates to the use of (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reversing dyskinesia.

Another aspect relates to a pharmaceutical composition comprising (6aR, 10 aR-7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof for use in the treatment of parkinson's disease while maintaining low dyskinesia induction characteristics.

An independent aspect of the invention relates to a pharmaceutical composition comprising (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of parkinson's disease while maintaining low dyskinesia induction characteristics.

Another aspect relates to a method of treating parkinson's disease while maintaining low dyskinesia induction characteristics, comprising administering a therapeutically effective amount of (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof.

Another aspect relates to a method of reversing dyskinesia comprising administering a therapeutically effective amount of (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof.

One aspect of the present invention relates to the use of (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of parkinson's disease while maintaining a low dyskinesia induction profile.

A separate aspect of the invention relates to the use of (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of parkinson's disease.

A further aspect relates to the use of (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for reversing dyskinesia.

Another aspect relates to a pharmaceutical composition comprising (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one, or a pharmaceutically acceptable salt thereof, for use in treating parkinson's disease while maintaining low dyskinesia induction characteristics.

An independent aspect of the invention relates to a pharmaceutical composition comprising (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of parkinson's disease while maintaining low dyskinesia induction characteristics.

Another aspect relates to a method of treating parkinson's disease while maintaining low dyskinesia induction characteristics, comprising administering a therapeutically effective amount of (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof.

Another aspect relates to a method of reversing dyskinesia comprising administering a therapeutically effective amount of (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof.

Detailed description of the invention

The compounds of the present invention contain two chiral centers (used in the formula below)^*Is shown)

The compounds of the invention may exist in two different diastereomeric forms (cis and trans isomers), both of which may exist in two enantiomeric forms. The invention relates only to the trans racemate and the (4aR, 10aR) -enantiomer.

As indicated previously, the present invention is based on the following findings: (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol (referred to herein as "Compound 10") reversed L-DOPA/benserazide and apomorphine-induced dyskinesia in 6-OHDA-injured rats. The corresponding trans racemates are also within the scope of the present invention.

In addition, the compounds of the present invention contain two chiral centers (used in the following formulas)^*Is shown)

The compounds of the invention may exist in two different diastereomeric forms (cis and trans isomers), both of which may exist in two enantiomeric forms. The invention relates only to the trans racemate and the (6aR, 10aR) -enantiomer.

As indicated previously, the present invention is based on the following findings: (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene (herein referred to as "Compound 11") reversed L-DOPA/benserazide and apomorphine-induced dyskinesia in 6-OHDA-injured rats.

Furthermore, the present invention is based on the following findings: (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one (herein compound 12) has beneficial characteristics in rats with unilateral 6-OHDA injury. It induces less dyskinesia than L-DOPA and apomorphine and more effectively reduces L-DOPA induced dyskinesia than D2 agonists (as exemplified by pramipexole).

The present invention is explained in more detail below, but the description is not intended to be an exhaustive list of all the different ways in which the invention can be carried out or all the features of the invention can be added.

Definition of

As used herein, "movement disorder" refers to a condition characterized by abnormal involuntary movement associated with disorders in the region of the brain known as the basal ganglia. Dyskinesia may be the appearance of "L-DOPA-induced dyskinesia" and is a complication in the treatment of parkinson's disease (the most common basal ganglia disease). Dyskinesias can manifest physically in two forms: chorea and dystonia. Chorea consists of involuntary, continuous, purposeless, stiff, rapid, brief, non-continuous, and irregular movements of one part of the body that flow to another. Dystonia refers to a sustained muscle contraction that causes twisting and repetitive motion or abnormal posture.

"treating" or "treatment" refers to inhibiting a disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter), or both, and inhibiting at least one physical parameter that may not be discernible to the patient. Furthermore, "treating" refers to delaying the onset of a disease or disorder, or at least symptoms thereof, in a patient who may be exposed to or susceptible to the disease or disorder, even if the patient has not experienced or exhibited symptoms of the disease or disorder.

"therapeutically effective amount" refers to an amount of a compound that, when administered to a patient to treat a disease or condition, is sufficient to effect such treatment of the disease or condition. The "therapeutically effective amount" will vary depending on the compound, the disease or condition and its severity and the age and weight of the patient to be treated.

As used herein, the phrase "while maintaining low dyskinesia characteristics" refers to dyskinesia characteristics as seen in a patient who has been treated by continuous dopaminergic stimulation. Treatments involving continuous dopaminergic stimulation are described in Stocchi and Olanow, Neurology 2004, 2004, 62, S56-S63; and Hilary et al, Journal of neurology 2004, 251, 11, 1370-.

The (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol used herein as an effective D1/D2 agonist is referred to as compound 10.

The (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene used herein is referred to as compound 11.

(4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one, as used herein, is referred to herein as compound 12.

Compound 10, 11 or 12 may be used as monotherapy for the treatment of dyskinesias (i.e. use of the compound alone); as an adjunct to the compositions for the prevention of the side effects of dyskinesias caused by said compositions (e.g. as an adjunct for the treatment of L-DOPA or apomorphine in parkinson's patients) or alternatively said compounds may be administered in combination with other therapies which also reduce dyskinesias (e.g. opioid receptor antagonists, (α 2-adrenoceptor-antagonists, cannabinoid CBI-antagonists, NMDA receptor-antagonists, cholinergic receptor antagonists, histamine H3-receptor agonists and globus pallidus/subthalamic nucleus damage/deep brain stimulation).

The invention also relates to the simultaneous, separate or sequential use in the treatment of parkinson's disease while reducing dyskinesia induced by L-DOPA or dopamine agonists, which comprises administering a therapeutically effective amount of compound 10, 11 or 12 or a pharmaceutically salt thereof.

In one embodiment, the movement disorder is associated with a basal ganglia-related movement disorder.

In another embodiment, the movement disorder is associated with parkinson's disease.

One embodiment relates to dyskinesias associated with idiopathic parkinsonism or post-encephalitic parkinsonism.

In one embodiment, the dyskinesia is associated with off-dystonia as in parkinson's disease.

In a separate embodiment, the dyskinesia arises as a side effect of a therapeutic agent for the treatment of parkinson's disease.

In another embodiment, the movement disorder is associated with dopamine replacement therapy. In one embodiment, the dopamine replacement therapy agent is selected from the group consisting of rotigotine, ropinirole, pramipexole, cabergoline, bromocriptine, lisuride, pergolide, L-DOPA and apomorphine.

In one embodiment, the dyskinesia is determined as a result of repeated administration of L-DOPA.

As previously described, the present invention provides a pharmaceutical composition comprising compound 10, 11 or 12, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of parkinson's disease while maintaining low dyskinesia induction characteristics, and a pharmaceutical composition comprising racemic trans-1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol in the manufacture of a medicament for the treatment of parkinson's disease while maintaining low dyskinesia induction characteristics.

In one embodiment, the pharmaceutical composition further comprises a MAO-B inhibitor.

In one embodiment, the MAO-B inhibitor is selegiline (selegine). In a separate embodiment, the MAO-B inhibitor is rasagiline.

In another embodiment, the present invention is directed to pharmaceutical compositions comprising a therapeutically effective amount of compound 10, 11 or 12, or a pharmaceutically acceptable acid addition salt thereof, and one or more pharmaceutically acceptable carriers, diluents, and excipients.

In a particular embodiment of the invention, the mammal is a human subject.

A therapeutically effective amount of compound 10, 11 or 12 (calculated as the daily dose of compound 10, 11 or 12 above as the free base) is suitably between 0.01 and 125 mg/day, more suitably between 0.05 and 100 mg/day, for example preferably between 0.1 and 50 mg/day.

In a specific embodiment, the daily dose of compound 10, 11 or 12 is between 1.0 and 10 mg/day.

In another embodiment, the daily dose of compound 10, 11, or 12 is less than about 1.0 mg/day.

In a separate embodiment, the daily dose of compound 10, 11, or 12 is about 0.10 mg/day.

In another embodiment, the invention provides an oral formulation comprising 0.001mg to 125mg of compound 10, 11 or 12.

In another embodiment, the invention provides an oral formulation comprising 0.001mg to 0.100mg of compound 10, 11 or 12.

In another embodiment, the invention provides an oral formulation comprising 0.01mg to 1.0mg of compound 10, 11 or 12.

In another embodiment, the invention provides an oral formulation comprising 0.10mg to 10mg of compound 10, 11 or 12.

Pharmaceutically acceptable salts

Compounds 10, 11 or 12 form pharmaceutically acceptable acid addition salts with a wide variety of organic and inorganic acids. These salts are also part of the present invention. As is well known in the art, a pharmaceutically acceptable acid addition salt of compound 10, 11 or 12 is formed from a pharmaceutically acceptable acid. These salts include the pharmaceutically acceptable salts listed in Journal of pharmaceutical science, 1977, 66, 2-19 and are known to those skilled in the art. Typical inorganic acids used to form these salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphorous, metaphosphoric, pyrophosphoric, and the like. Salts derived from organic acids such as aliphatic mono-and dicarboxylic acids, phenyl substituted alkanoic, hydroxyalkanoic and hydroxyalkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids may also be used. Thus, such pharmaceutically acceptable salts include chloride, bromide, iodide, nitrate, acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-ethoxybenzoate, isobutyrate, phenylbutyrate, alpha-hydroxybutyrate, butyne-1, 4-dicarboxylate, hexyne-1, 4-dicarboxylate, decanoate, octanoate, carnosite, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, methanesulfonate, nicotinate, isonicotinate, oxalate, phthalate, acetate, benzoate, and the like, Terephthalate (terapthalate), propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, benzenesulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1, 5-sulfonate, p-toluenesulfonate, xylenesulfonate, tartrate and the like.

Pharmaceutical composition

Methods of preparing solid pharmaceutical compositions are also well known in the art. Thus, tablets may be prepared by mixing the active ingredient with conventional adjuvants, fillers and diluents and subsequently compressing the mixture in a convenient tabletting machine. Examples of adjuvants, fillers and diluents include microcrystalline cellulose, corn starch, potato starch, lactose, mannitol, sorbitol, talc, magnesium stearate, gelatin, lactose, gums and the like. Any other adjuvants or additives such as colorants, fragrances, preservatives and the like may also be used provided they are compatible with the active ingredient.

In particular, the tablet formulation of the present invention can be prepared by direct compression of compound 10, 11 or 12 mixed with conventional adjuvants or diluents. Alternatively, compression of the tablet may be carried out using wet or melt pellets of compound 10, 11 or 12, optionally mixed with conventional adjuvants or diluents.

Solutions of compounds 10, 11 or 12 for injection can be prepared by dissolving the active ingredient and possible additives in a portion of the solvent for injection, preferably sterile water, adjusting the resulting solution to the desired volume, sterilizing the resulting solution and filling in suitable ampoules or vials. Any suitable additive commonly used in the art may be added, such as tonicity agents, preservatives, antioxidants, solubilizers, and the like.

Brief Description of Drawings

FIG. 1: crystal structure of compound ent-10. The absolute configuration was determined by anomalous scattering of the "heavy" bromine atoms.

FIG. 2: by hD₅Dopamine-acting intracellular Ca in transfected CHO-Ga16 cells²⁺The dose-response curve of the released concentration-dependent stimulus.

Experimental part

Analytical LC/MS data were obtained on a PE Sciex API 150EX instrument equipped with an atmospheric pressure photoionization device and a Shimadzu LC-8A/SLC-10A LC system. Purity was determined by integration of UV (254nm) and ELSD traces. The MS instrument is from peskier (api) equipped with an APPI source and operated in cationic mode. The Residence Time (RT) of the UV-tracer is indicated in min. Solvent a was prepared from 0.05% TFA in water, while solvent B was prepared from 0.035% TFA and 5% water in acetonitrile. Several different methods were used:

the method 25 comprises the following steps: API 150EX and Shimadzu LC10AD/SLC-10A LC system. Column: dC-184.6X30mm, 3 μm (Atlantis, Waters). Column temperature: at 40 ℃. Gradient: with a reverse phase of the ion pair. Flow rate: 3.3 mL/min. Injection volume: 15 μ L. Gradient: from 2% B in A to 100% B in 2.4min, then 2% B in A, 0.4 min. Total run time: 2.8 min.

The method 14 comprises the following steps: API 150EX and Shimadzu LC8/SLC-10A LC system. Column: C-184.6X30mm, 3.5 μm (Symmetry, Waters). Column temperature: and (4) room temperature. Gradient: with a reverse phase of the ion pair. Flow rate: 2 mL/min. Injection volume: 10 μ L. Gradient: from 10% B to 100% B in A within 4 min; then 10% B in A, 1 min. Total run time: and 5 min.

Determination of the X-ray crystal structure was performed as follows. The crystals of the compound were cooled to 120K using a Cryostream nitrogen cooling system. Data were collected on a Siemens SMART Platform diffractometer with a CCD area sensitive detector. Solving the structure by direct method and passing all data pairs F²Is improved by full matrix least squares. The hydrogen atoms in the structure can be found in the electron density difference spectrum. The non-hydrogen atoms are precisely defined anisotropically. All hydrogen atoms are used in O-H-0.84, C-H-0.99-1.00, N-H-0.92-0.93The calculated position of the running model (rating model) of (1). For all hydrogen atoms, the thermal parameter [ U (h) of the connecting atom ═ 1.2U]。FThe lack x-parameter is in the range of 0.0(1) to 0.05(1), indicating that the absolute structure is correct. The programs used for Data collection, Data simplification, and consolidation are SMART, SAINT, and SADABS [ see "SMART and SAINT, Area Detector Control and Integration Software", version 5.054, Bruker analytical X-Ray Instruments Inc., Madison, USA (1998), Sheldrick "SADABS, Program for empirical Correction of Area Detector Data" version 2.03, University ofGermany (2001)]. The procedure SHELXTL was used [ see Sheldrick "SHELXTL, Structure determination programs", 6.12 th edition, Bruker Analytical X-Ray Instruments Inc., Madison, USA (2001)]Solution structures and molecular patterns.

Synthesis of Compounds of the invention (Compounds 10 and 11)

Starting from compound 1 (the synthesis of which is described in that document) prepared as described in Taber et al, j.am.chem.soc., 124(42), 12416(2002), compound 8 was prepared in 8 steps as described herein. This material can be resolved by chiral SFC as described herein to give compound 9 and ent-9. After cleavage of the Boc protecting group, a n-propyl group can be introduced at the nitrogen atom by reductive amination. Can be obtained by treatment with 48% HBr or by reaction with BBr₃The reaction deprotects the resulting masked catecholamine under standard conditions to give compound 10 and ent-10. May use 10 and CH₂Further reaction of ClBr or related reagents in the presence of a base gives the compounds of the present invention (compound 11).

Synthesis of Compound 10 and ent-10

7-iodo-1, 2, 6-trimethoxy-naphthalene (compound 2).

To a stirred solution of compound 1(26.2 g; prepared as described in Taber et al, J.am.chem.Soc., 124(42), 12416 (2002)) in anhydrous THF (200mL) was slowly added sec-butyllithium (1.2M in cyclohexane, 110mL) under an argon atmosphere at-78 ℃. The solution was stirred at-78 ℃ for 3 h. A solution of iodine (30.5g) in dry THF (50mL) was added over 10 min. The resulting mixture was then stirred at-78 ℃ for another 10 min. By adding saturated NH₄Cl (100mL), water (240mL) and Et₂The reaction mixture was quenched with O (240 mL). The organic layer was washed with 10% aqueous sodium sulfite (100mL) and dried (Na)₂SO₄) And concentrated in vacuo. The crude material was purified by distilling off unreacted starting materials. The residue was further purified by silica gel chromatography (EtOAc/heptane) to give an impure solid material which was purified by precipitation from EtOAc/heptane to give 11.46g of compound 2.

(E/Z) -3- (3, 7, 8-trimethoxy-naphthalen-2-yl) -acrylonitrile (Compound 3).

To a suspension of Compound 2(3.41g) in anhydrous acetonitrile (10.7mL) in a microwave reaction vial was added acrylonitrile (1.19mL), Pd (OAc)₂(73mg) and triethylamine (1.48 mL). The vial was sealed and the mixture was heated under microwave irradiation at 145 ℃ for 40 min. This step was carried out two more times (using a total of 10.23g of compound 5). The crude reaction mixtures were combined, the catalyst was filtered off and the filtrate was concentrated in vacuo. The residue is taken up in Et₂The layers were separated between O (300mL) and 2M HCl (150 mL). The organic layer was washed with brine (100mL) and dried (Na)₂SO₄) And concentrated in vacuo. The crude material (7.34g) was purified by silica gel chromatography (EtOAc/heptane) to give 5.23g of compound 3 as a mixture of olefin isomers.

3- (3, 7, 8-trimethoxy-naphthalen-2-yl) -propionitrile (Compound 4)

Compound 3(5.23g) was dissolved in CHCl₃(15mL) and 99% EtOH (100 mL). 10% Pd/C (0.8g) was added and the solution was hydrogenated using a Parr shaker at 3bar hydrogen pressure for 45 min. The catalyst was filtered off and the filtrate was passed through a small piece of silica gel (eluent: 99% EtOH). Yield: 4.91g of Compound 4as a white solid.

[3- (3, 7, 8-trimethoxy-1, 4-dihydro-naphthalen-2-yl) -propyl ] -carbamic acid tert-butyl ester (compound 5).

Compound 4(5.0g) was dissolved in 99% EtOH (150mL) and the mixture was heated to reflux under a nitrogen atmosphere. Sodium metal (5g) was added in small portions over 3 h. The mixture was refluxed for an additional 2h, then stirred at room temperature for 2 days. It was then heated to reflux again, more sodium metal (3.68g) was added and the mixture was refluxed overnight. After cooling on an ice/water bath, the reaction was quenched by the addition of solid ammonium chloride (20g) and water (25 mL). The resulting mixture was filtered and the filtrate was concentrated in vacuo. The residue was partitioned between diethyl ether (50mL) and water (50 mL). The aqueous layer was neutralized with 37% HCl and extracted with diethyl ether (2 × 50 mL). The combined organic extracts were washed with brine (50mL) and dried (MgSO)₄) And concentrated in vacuo to give an oil. This material was dissolved in THF (50mL) at room temperature and Boc was used₂O (2.34g) and Et₃N (1.78 mL). After 6 days, the volatiles were removed in vacuo and the residue was purified by silica gel chromatography (EtOAc/heptane). This gave impure compound 5(1.52 g).

Racemic 6, 7-dimethoxy-2, 3, 4, 4a, 5, 10-hexahydro-benzo [ g ] quinoline hydrochloride (Compound 6)

Compound 5(1.52g, from above step) was dissolved in MeOH (20 mL). 37% HCl (3.5mL) was added and the mixture was refluxed for 4 h. Volatiles were removed in vacuo and toluene was used to azeotropically remove water. This gave impure compound 6(0.89g) as a yellow oil.

Racemic trans-6, 7-dimethoxy-3, 4, 4a, 5, 10, 10 a-hydro-2H-benzo [ g ] quinoline-1-carboxylic acid tert-butyl ester (Compound 8).

Dissolve Compound 6(0.89g) in MeOH (10mL) and add NaCNBH₃(0.19 g). The reaction was stirred at rt overnight. The crude mixture was cooled on an ice/water bath and then used in Et₂It was quenched with 2MHCl in O (1 mL). The mixture is taken up in Et₂The layers were separated between O (50mL), water (50mL) and 2M NaOH (10 mL). The aqueous layer was extracted with diethyl ether (3 × 50 mL). The combined organic layers were dried (MgSO)₄) And concentrated in vacuo to afford the impure free amine (compound 7). This material was dissolved in THF (25mL) and Boc at room temperature₂O (0.68g) and Et₃N (0.86mL) for 1 h. The crude mixture was concentrated in vacuo and the residue was purified by silica gel chromatography (EtOAc/heptane) to give 1.18g of slightly impure racemic compound 8.

SFC separation of the enantiomers of racemic trans-6, 7-dimethoxy-3, 4, 4a, 5, 10, 10 a-hydro-2H-benzo [ g ] quinoline-1-carboxylic acid tert-butyl ester (Compound 9 and ent-9)

Compound 8(19.7g) was resolved into its enantiomers using chiral SFC on a Berger SFC multigramII instrument equipped with a Chiralcel OD 21.2x 250mm column. A solvent system: CO 2₂EtOH (85: 15), method: constant gradient, flow rate of 50 mL/min. Fraction collection was performed by UV 230nm detection. The fast eluting enantiomer (4aR, 10aR enantiomer; compound 9): 9.0g of a white solid. Slow eluting enantiomer (4aS, 10aS enantiomer, compound ent-9): 8.1g of a white solid.

(4aS, 10aS) -6, 7-dimethoxy-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline hydrochloride (Compound ent-9')

Compound ent-9(0.52g) was dissolved in MeOH (15mL) at room temperature and used in Et₂Treatment with 5M HCl in O (7.5mL) for 2 h. The mixture was concentrated in vacuo and the solid was dried in vacuo to give compound ent-9' as a white solid. LC/MS (method 14): RT 1.31 min.

(4aR, 10aR) -1-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol hydrobromide (Compound 10)

Compound 9(0.5g) was dissolved in 99% EtOH (5mL) at room temperature and used in Et₂O (4mL) was treated overnight with 2M HCl. The crude mixture was concentrated in vacuo and the residue was partitioned between EtOAc and 10% aqueous NaOH (5 mL). The aqueous layer was extracted with EtOAc and the combined organic layers were washed with brine and dried (MgSO)₄) And concentrated in vacuo. The residue was dissolved at room temperatureDissolved in 99% EtOH (5mL) and treated with propionaldehyde (0.52mL), NaCNBH₃(0.45g) and AcOH (3 drops) overnight. The crude mixture was taken up in saturated NaHCO₃The layers were separated between aqueous (12.5mL), water (12.5mL) and EtOAc (2X25 mL). The combined organic layers were washed with brine and dried (MgSO)₄) And concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH/EtOAc). The resulting intermediate was treated with 48% HBr (3mL) at 150 ℃ under microwave conditions for 1h, and the crude mixture was then stored overnight at 4 ℃. The precipitated material was isolated by filtration and dried in vacuo. Yield of compound 10: 103mg of solid. LC/MS (method 25): RT 0.77 min.

(4aS, 10aS) -1-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol hydrobromide (Compound ent-10).

Starting from compound ent-9' (0.5 g; HCl salt was liberated by delamination between EtOAc and 10% aqueous NaOH solution prior to the reductive amination step) according to the procedure described for compound 10. Yield of Compound ent-10: 70mg of solid. LC/MS (method 25): RT 0.70 min. A small sample of compound ent-10 was dissolved in MeOH and allowed to slowly crystallize at room temperature over a 2 month period. The white crystals formed were collected and analyzed by X-ray analysis (see fig. 1). The absolute configuration of compound ent-10 was determined by X-ray crystallography and the stereochemistry of compounds 9 and 10 and therefore their derivatives was determined unambiguously.

(6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene hydrochloride (Compound 11)

Compound 1 is reacted under argon atmosphere0(7.80g)、Cs₂CO₃(18.6g)、CH₂BrCl (2.2mL) and DMF (180mL) were heated to 100 ℃ for 1 h. The crude reaction mixture was added to a separatory funnel and diluted with ice/water (300 mL). Et used for the resulting mixture₂O (3 × 300 mL). The combined organic layers were washed with brine (200mL) and dried (MgSO)₄) And concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc/MeOH) to give a light red solid, which was dissolved in MeOH (25mL) and added to Et₂2M HCl and Et in O (20mL)₂O (100mL) precipitated it as the hydrochloride salt. The precipitated product was isolated by filtration and dried in vacuo. Yield of compound 11: LC/MS (method 111): RT 0.70 min. ELSD 100%. UV 97.0%. MH⁺：274.0。

(4aR, 10aR) -n-1-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one (Compound 12)

The synthesis of compound 12 can be prepared as described in EP patent No.1274411, the contents of which are incorporated herein by reference. In the above patent, compound 12 is referred to as (-) -GMC 6650.

Experimental part

Example 1: catechol-containing active metabolites of Compounds 11 and 12 converted to Compound 10 upon in vivo administration

The active metabolite (i.e., compound 10) was found to act as a potent agonist at the D1 and D2 receptors in vitro. As discussed in more detail below, the data generated by in vivo experiments indicate that the active metabolite has superior characteristics to other dopamine agonists and is the same as the efficacy seen with L-DOPA/apomorphine treatment.

Example 2: pharmacological testing of Compound 10

D₁cAMP assay

Stable expression of human recombinant D as determined₁The ability of the compounds to stimulate or inhibit D1 receptor-mediated cAMP formation in CHO cells of the receptor. Cells were seeded at a concentration of 11000 cells/well in 96-well plates and experiments were performed 3 days later. On the day of the experiment, 1mM MgCl in pre-warmed G-buffer (in PBS (phosphate buffered saline)) was added₂、0.9mM CaCl₂And 1mM IBMX (3-isobutyl-1-methylxanthine)) the cells were washed once and the assay was initiated by adding 100. mu.l of a mixture of test compound diluted in 30nM A68930 and G buffer (antagonism) or 100. mu.l of test compound diluted in G buffer (agonism).

The cells were incubated at 37 ℃ for 20 minutes and were incubated by adding 100. mu.l S buffer (0.1M HCl and 0.1mM CaCl)₂) The reaction was terminated and the plates were left at 4 ℃ for 1 h. 68 microliters of N buffer (0.15M NaOH and 60mM NaOAc) was added and the plates were shaken for 10 minutes. Transfer 60 microliters of reaction to cAMP FlashPlates (DuPont NEN) containing 40 microliters of 60mM sodium acetate (pH 6.2) and add 100 microliters IC mix (50mM sodium acetate, pH 6.2, 0.1% sodium azide, 12mM CaCl₂1% BSA (bovine serum Albumin) and 0.15. mu. Ci/mL¹²⁵I-cAMP). After incubation for 18h at 4 ℃, the plates were washed once and counted in a Wallac TriLux counter. In this assay, Compound 10 proved to function as D₁The effect of an agonist.

D₂cAMP assay

In human D as determined₂Stimulation or inhibition of D by the Compounds in CHO cells transfected with the receptor₂The receptor mediates the ability to inhibit cAMP formation. Cells were seeded at 8000 cells/well in 96-well plates and experiments were performed 3 days later. On the day of the experiment, the cells were incubated in pre-warmed G buffer (1 mM MgCl in PBS)₂、0.9mM CaCl₂And 1mM IBMX) are addedCells were washed once and the assay was initiated by adding 1 μ M quinpirole, 10 μ M forskolin and 100 μ l of a mixture of test compound in G buffer (antagonism) or 10 μ M forskolin and 100 μ l of a mixture of test compound in G buffer (agonism).

Cells were incubated at 37 ℃ for 20min and washed by adding 100. mu.l S buffer (0.1M HCl and 0.1mM CaCl)₂) The reaction was terminated and the plates were left at 4 ℃ for 1 h. 68 microliters of N buffer (0.15M NaOH and 60mM sodium acetate) was added and the plates were shaken for 10 minutes. Transfer 60 microliters of reaction to cAMP FlashPlates (DuPont NEN) containing 40 microliters of 60mM NaOAc (pH 6.2) and add 100 microliters IC mix (50mM NaOAc, pH 6.2, 0.1% sodium azide, 12mM CaCl₂1% BSA and 0.15. mu. Ci/ml¹²⁵I-cAMP). After incubation for 18h at 4 ℃, the plates were washed once and counted in a Wallac TriLux counter. In this assay, Compound 10 proved to function as D₂The effect of an agonist.

D₅Measurement of

At hD₅Concentration-dependent stimulation of intracellular Ca by dopamine in transfected CHO-Ga16 cells²⁺And (4) releasing. To the cells, fluoro-4 (a calcium indicator dye) was added and held for 1 h. Calcium response (change in fluorescence) was monitored by FLIPR (fluorescence imaging reader) for 2.5 min. Peak responses (EC) from two wells for each data point₅₀) Mean, and plotted against drug concentration (for dopamine, see figure 2). In this assay, Compound 10 proved to function as D₅The effect of an agonist.

6-OHDA rat model

Dopamine agonists may be active at the D1 receptor, the D2 receptor, or both. The spin response in rats with unilateral 6-OHDA injury can be used to assess the ability of compounds to stimulate both receptor types and induce spin (Ungerstedt and Arbutnott, Brain Res., 1970, 24, 485; Setler et al Eur. J. Pharmacol., 1978, 50(4), 419; and Ungerstedt et al. "Advances in Dopamine Research" (Kohsaka, Ed.), Pergamon Press, 1982, Oxford, p.219). 6-OHDA (6-hydroxydopamine) is a neurotoxin used by neurobiologists to selectively kill dopaminergic neurons at injection sites within the brains of experimental animals. In the 6-OHDA model, one side (unilateral) of the brain is destroyed by injecting 6-OHDA into the anterior brain medial tract in front of the substantia nigra, the nigra striatal dopamine cells. Such unilateral injections combined with stimulation of dopamine agonists such as apomorphine will induce rotational behavior as if only one side of the brain is stimulated. The experiment consisted of determining the lowest effective dose (MED) at which the compound in question induced rotation. Once the MED is determined, a second experiment is performed to determine the MED (MED) at which the compound overcomes the nemorubide blockade_Nemopride). Nemorubide is a D2 antagonist that blocks the D2 receptor, so any observed rotation may depend on activity at the D1 receptor. Finally, once the MED is known_NemoprideUsing the MED_NemoprideA third experiment was performed by dosing and observing the effect of the D1 antagonist (SCH 23390 alone), the D2 antagonist (nemorubide alone) and finally the combined treatment of SCH 23390 and nemorubide. This third experiment confirmed that: the activity of a compound as either antagonist alone at both receptors was only partially inhibitory to the rotational response induced by the test compound and the combined treatment completely blocked all rotations in rats [ Arnt and Hyttel, Psychopharmacology, 1985, 85(3), 346; and Sonsalla et al, j. pharmacol exp. ther., 1988, 247(1), 180]. Proof of principle compounds using apomorphine as a mixed D1/D2 agonist validated this model.

In this model, compound 10 has 'apomorphine' -like characteristics compared to apomorphine having a D1/D2 ratio of about 3, wherein the D1/D2 ratio is about 2-4. Furthermore, the observed time of action of the compound was about 18h, which is significantly higher than that seen with L-DOPA/apomorphine. No D1 component was observed for the D2-agonist (exemplified by pramipexole and rotigotine).

Optimization model

Apomorphine and L-DOPA reverse motor deficits in a mouse model of severe dopamine depletion. Both apomorphine and L-DOPA stimulated the D1 and D2 dopamine receptors. Pramipexole (an agonist of the D2 receptor) was ineffective in this model.

The experiment was performed as follows: mice treated first with MPTP (2x15mg/kg subcutaneously) and with stable lesions and mice treated with vehicle were used as normal controls. MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) is a neurotoxin that causes permanent parkinson's disease symptoms by killing certain neurons in the substantia nigra of the brain. It was used to study disease in monkeys and mice. On the day of the experiment, mice were treated with AMPT (250mg/kg subcutaneously) and then returned to the rearing cage, and after 1.5 hours they were placed in individual cages of a sports apparatus. AMPT (α -methyl-p-tyrosine) is a drug that transiently decreases the activity of catecholamine (in this case, specifically dopamine levels) in the brain. Three hours after AMPT injection, attempts were made to rescue the motor deficit with compound 10 and activity was recorded for an additional 1.5 hours. The first 30min data collected after rescue treatment was "contaminated" as evidenced by increased levels in vehicle controls, due to stressful animals treated and injected, and therefore data analysis was performed using the last 1 hour of recorded data. Various dopaminergic compounds were tested for their ability to reverse the motor deficits produced in this model. Both L-DOPA/benserazide and apomorphine restored locomotion in the mice in a dose-dependent manner. Benserazide is a DOPA decarboxylase inhibitor that cannot cross the blood brain barrier; it is used to prevent the metabolism of L-DOPA to dopamine outside the brain. In contrast, the D2 agonists pramipexole and bromocriptine do not restore locomotion in mice.

This model was used to evaluate whether compound 10 exhibited the same superiority as L-DOPA and apomorphine over the D2 agonist. Dose response experiments with compound 10 were performed and there was a dose-dependent trend to reverse the hypokinesia defect induced by severe endogenous dopamine depletion. Final experiments comparing the effects of apomorphine, pramipexole and compound 10 directly were performed in this model and confirmed that compound 10 was able to regain locomotion and outperform pramipexole in MPTP treated mice.

Induction of dyskinesia model using naive 6-OHDA rats

The induction of dyskinesia by compound 10(s) (s.c.; n.7; group 1) compared to L-DOPA/benserazide (6mg/kg/15mg/kg s.c.; n.7; group 2) and apomorphine (1mg/kg s.c.; n.6; group 3) was tested using 20 male Sprague Dawley rats with unilateral 6-OHDA lesions. Benserazide is a DOPA decarboxylase inhibitor that cannot cross the blood brain barrier; it is used to prevent the metabolism of L-DOPA to dopamine outside the brain. Three weeks after 6-OHDA surgery, an animal's rotational response induced by 2.5mg/kg amphetamine was tested, which induced ipsilateral gyrus (amphetamine increased intracerebral dopamine levels through intact neurons on the uninjured side causing the animal to rotate in the opposite direction) compared to the animal's response to direct agonists such as L-DOPA and apomorphine, which act primarily on the injured side of the brain. All animals included in this study met the criteria of greater than 350 revolutions per 60 min. Rats were then randomly assigned to three treatment groups to balance the rotational response of the animals to phenylpropylamine in each group.

In the actual dyskinesia experiments, rats received a once daily subcutaneous injection of the test compound and were observed for 3h after injection. Each animal was observed for 1 minute every 20min for the entire 3h period using the Abnormal Involuntary Movement Scale (AIMS) as previously described, observing the presence of dyskinesias (Lundblad et al, eur. j neurosci, 15, 120, (2002)). Rats received drug for 14 consecutive days and scored animals at 1, 2, 3, 4, 5, 8, 10 and 12. Two-way repeated measures ANOVA showed significant treatment effects, time effects and treatment-time interactions (p < 0.001, in all cases). Post hoc comparisons using the Holm-Sidak method showed that: animals treated with compound 10 had significantly lower dyskinesias (score of about 30) than animals treated with L-DOPA or apomorphine (score of about 70). There was no difference between the L-DOPA and apomorphine treatment groups. After this experiment, all rats were injected subcutaneously with compound 10 from day 15-19 to determine how example I affected the severity of dyskinesia observed in the apomorphine and L-DOPA groups. Dyskinesia scores were performed on day 19 of the experiment (5 days corresponding to compound 10). The data show that the partial reversal of L-DOPA and apomorphine-induced dyskinesia approaches the level of compound 10-induced dyskinesia (compound 10 did not cause an increase in dyskinesia in group 1 compared to a score of about 30 observed after 12 days of treatment).

Dyskinesia rat model

A separate dyskinesia study was conducted in the reversal of L-DOPA-induced dyskinesia with pramipexole or compound 10. Briefly, 18 animals were treated with L-DOPA/benserazide (6/15mg/kg subcutaneously) for 7 days. Animals were observed on days 1, 3 and 5 and were scored for AIMS. The animals were then divided into 3 groups of 6 animals each using a day 5 score. Group 1 continued daily L-DOPA treatment. Group 2 was treated with compound 10 (subcutaneous administration). Group 3 was treated with pramipexole (0.16mg/kg subcutaneously). Daily treatment was continued for 10 days and the amount of dyskinesias was scored on days 1, 5, 9, 10. Two-way repeated measures anova showed that animals treated with compound 10 had significantly less dyskinesia than the pramipexole group and the L-DOPA/benserazide group. The pramipexole group had significantly less dyskinesia than the L-DOPA/benserazide group. Thus, compound 10 has superior characteristics over pramipexole in reversing L-DOPA-induced dyskinesia.

anti-Parkinsonism Effect in MPTP-treated common marmosets

The experiment was performed using 6 MPTP-treated marmosets (2.0mg/kg daily for 5 consecutive days dissolved in sterile 0.9% saline solution). All animals were previously treated with L-DOPA (12.5mg/kg p.o. plus carbidopa 12.5mg/kg p.o.) daily for 30 days to induce dyskinesia. Prior to the study, all subjects exhibited stable motor deficits, including significant reduction in basal motor activity, poor motor coordination, abnormal and/or rigid posture, reduced alertness, and head examination movements. Domperidone was administered 60 minutes prior to any test compound. Domperidone is an anti-dopaminergic drug that inhibits nausea and vomiting. The motor activity evaluation is carried out by adopting a test cage, the test cage is composed of 8 photoelectric switches, the 8 photoelectric switches are composed of 8 infrared beams, the beams are strategically placed in the cage, and the interruption of the beams is recorded as one-time counting. The total number of beam counts per unit time period is then plotted against time or expressed as the area under the curve for total viability (AUC). The assessment of incapacity of movement was performed by a trained observer blinded to the treatment.

As already described, L-DOPA (12.5mg/kg, p.o.) increases locomotor activity and reverses locomotor disability (Smith, et al. mov. dis.2002, 17(5), 887). The dose selected for this stimulation is at the top of the dose response curve for this drug. Compound 10 (subcutaneously administered) produced a dose-related increase in locomotor activity and a reversal of locomotor disability that tended to occur with a greater response than that produced by L-DOPA (12.5mg/kg, p.o.). Compared to L-DOPA, the two test compounds produced prolonged exercise with no reversal and the same efficacy as L-DOPA. Compound 10 produced prolonged exercise, in comparison to L-DOPA, which was not reversible and was as potent as L-DOPA.

Example 3: pharmacological testing of Compound 11

D₁cAMP assay

Stable expression of human recombinant D as determined₁Stimulation or inhibition of D by the Compounds in CHO cells of the receptor₁The ability of the receptor to mediate cAMP formation. Cells were seeded at a concentration of 11000 cells/well in 96-well plates and experiments were performed 3 days later. On the day of the experiment, 1mM MgCl in pre-warmed G-buffer (in PBS (phosphate buffered saline)) was added₂、0.9mM CaCl₂And 1mM IBMX (3-isobutyl-1-methylxanthine)) the cells were washed once and the assay was initiated by adding either 30nM A68930 and 100. mu.l of a mixture of test compound diluted in G buffer (antagonism) or 100. mu.l of test compound diluted in G buffer (agonism).

Cells were incubated at 37 ℃ for 20min and washed by adding 100. mu.l S buffer (0.1M HCl and 0.1mM CaCl)₂) The reaction was terminated and the plates were left at 4 ℃ for 1 h. Add 68. mu.l of N buffer (0.15M NaOH and 6)0mM NaOAc) and plates were shaken for 10 min. Transfer 60 microliters of reaction to cAMP FlashPlates (DuPont NEN) containing 40 microliters of 60mM sodium acetate (pH 6.2) and add 100 microliters IC mix (50mM sodium acetate, pH 6.2, 0.1% sodium azide, 12mM CaCl₂1% BSA (bovine serum Albumin) and 0.15. mu. Ci/mL¹²⁵I-cAMP). After incubation for 18h at 4 ℃, the plates were washed once and counted in a Wallac TriLux counter. In this assay, the active metabolite or Compound 10 was found to be D₁An agonist.

D₂cAMP assay

In human D as determined₂Stimulation or inhibition of D by the Compounds in CHO cells transfected with the receptor₂Receptor-mediated ability to inhibit cAMP formation. Cells were seeded at 8000 cells/well in 96-well plates and experiments were performed 3 days later. On the day of the experiment, the cells were incubated in pre-warmed G buffer (1 mM MgCl in PBS)₂、0.9mM CaCl₂And 1mM IBMX) and the assay was initiated by adding 1 μ M quinpirole, 10 μ M forskolin and 100 μ M mixture of test compound in G buffer (antagonism) or 10 μ M forskolin and 100 μ M mixture of test compound in G buffer (agonism).

Cells were incubated at 37 ℃ for 20min and washed by adding 100. mu.l S buffer (0.1M HCl and 0.1mM CaCl)₂) The reaction was terminated and the plates were left at 4 ℃ for 1 h. 68 microliters of N buffer (0.15M NaOH and 60mM sodium acetate) was added and the plates were shaken for 10 minutes. Transfer 60 microliters of reaction to cAMP FlashPlates (DuPont NEN) containing 40 microliters of 60mM NaOAc (pH 6.2) and add 100 microliters IC mix (50mM NaOAc, pH 6.2, 0.1% sodium azide, 12mM CaCl₂1% BSA and 0.15. mu. Ci/ml¹²⁵I-cAMP). After incubation for 18h at 4 ℃, the plates were washed once and counted in a Wallac TriLux counter. In this assay, the active metabolite or Compound 10 was found to be D₂An agonist.

D5 determination

At hD₅Transfection of CHO-Ga16 cells by pluripolyConcentration-dependent stimulation of intracellular Ca by dopamine²⁺And (4) releasing. To the cells, fluoro-4 (a calcium indicator dye) was added and held for 1 h. Calcium response (change in fluorescence) was monitored by FLIPR (fluorescence imaging reader) for 2.5 min. Peak responses (EC) from two wells for each data point₅₀) Mean and plotted against drug concentration. In this assay, the active metabolite or Compound 10 was found to be D₅An agonist.

6-OHDA rat model

Dopamine agonists may be active at the D1 receptor, the D2 receptor, or both. The ability of compounds to stimulate both receptor types and induce spin can be assessed using spin responses in rats with unilateral 6-OHDA lesions (Ungerstedt and Arbutnott, Brain Res.24, 485 (1970); Setler et al, Eur.J.Pharmacol., 50(4), 419 (1978); and Ungerstedt, et al, "Advances in dopamine Research" (Kohsaka, Ed.), Pergamon Press, 1982, Oxford, p.219). 6-OHDA (6-hydroxydopamine) is a neurotoxin used by neurobiologists to selectively kill dopaminergic neurons at injection sites within the brains of experimental animals. In the 6-OHDA model, one side (unilateral) of the brain is destroyed by injecting 6-OHDA into the anterior brain medial tract in front of the substantia nigra, the nigra striatal dopamine cells. Such unilateral injections combined with stimulation of dopamine agonists such as apomorphine will induce rotational behavior as if only one side of the brain is stimulated. The experiment consisted of determining the lowest effective dose (MED) at which the compound in question induced rotation. Once the MED is determined, a second experiment is performed to determine the MED (MED) at which the compound overcomes the nemorubide blockade_Nemopride). Nemorubide is a D2 antagonist that blocks the D2 receptor, so any observed rotation may depend on activity at the D1 receptor. Finally, once the MED is known_NemoprideUsing the MED_NemoprideA third experiment was performed by dosing and observing the effect of the D1 antagonist (SCH 23390 alone), the D2 antagonist (nemorubide alone) and finally the combined treatment of SCH 23390 and nemorubide. This third experiment confirmed that: compounds which are antagonists of either alone are only partially tested for inhibition of activity at both receptorsCombination treatment completely blocked all rotations in rats in response to compound-induced rotations (Arnt and Hyttel; psychopharmacogology, 85(3), 346 (1985); and Sonsalla et al, j. pharmacol exp. ther., 247(1), 180 (1988)). Proof of principle compounds using apomorphine as a mixed D1/D2 agonist validated this model.

In this model, active metabolite or compound 10 and compound 11 have 'apomorphine' -like characteristics compared to apomorphine having a D1/D2 ratio of about 3, wherein the D1/D2 ratio is about 2. Furthermore, the observed time of action of the compound was about 18h, which is significantly higher than that seen with L-DOPA/apomorphine. No D1 component was observed for the D2-agonist (exemplified by pramipexole and rotigotine).

Optimization model

Apomorphine and L-DOPA reverse motor deficits in a mouse model of severe dopamine depletion. Both apomorphine and L-DOPA stimulated the D1 and D2 dopamine receptors. Pramipexole (an agonist of the D2-like receptor) was ineffective in this model.

The experiment was performed as follows: mice previously treated with MPTP (2x15mg/kg subcutaneously) and with stable lesions and mice treated with vehicle were used as normal controls. MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) is a neurotoxin that causes permanent parkinson's disease symptoms by killing certain neurons in the substantia nigra of the brain. It was used to study disease in monkeys and mice. On the day of the experiment, mice were treated with AMPT (250mg/kg subcutaneously) and then returned to the cage, where they were placed in individual cages of a sports apparatus after 1.5 hours. AMPT (α -methyl-p-tyrosine) is a drug that transiently decreases the activity of catecholamine (in this case, dopamine levels, among others) in the brain. Three hours after AMPT injection, an attempt was made to rescue the motor deficit with the active metabolite or compound 10 and viability was recorded for an additional 1.5 hours. As evidenced by the increased levels in the vehicle controls, the first 30min data collected after the rescue treatment was "contaminated" due to the stressful animals treated and injected, and therefore, the data was analyzed using the last 1 hour of recorded data. Various dopaminergic compounds were tested for their ability to reverse the motor deficits produced in this model. Both L-DOPA/benserazide and apomorphine restored locomotion in the mice in a dose-dependent manner. Benserazide is a DOPA decarboxylase inhibitor that cannot cross the blood brain barrier; it is used to prevent the metabolism of L-DOPA to dopamine outside the brain. In contrast, D2 agonists (pramipexole and bromocriptine) did not restore locomotion in mice.

This model was used to evaluate whether active metabolite or compound 10 exhibited the same superiority as L-DOPA and apomorphine over the D2 agonist. Dose response experiments were performed and there was a dose-dependent trend to reverse the hypokinesia defect induced by severe endogenous dopamine depletion. A final experiment was performed comparing the effects of apomorphine, pramipexole and compound 10 directly. Compound 10 has been shown to restore locomotion and outperform pramipexole in MPTP treated mice.

Dyskinesia rat model

The effect of active metabolites with L-DOPA/benserazide on dyskinesia assessed as Abnormal Involuntary Movement (AIM) in 'parkinson' rats was determined using the rat dyskinesia model reported in the literature (Lundblad et al, eur. j neurosci., 2002, 15, 120).

Design of research

Throughout the study, animals received L-DOPA/benserazide (6mg/kg and 15mg/kg subcutaneously) or the active metabolite (compound 10) once daily at t ═ 20min.0-180min (group B). Animals were scored for dyskinesias. Days 1-14: all animals were dosed with L-DOPA/benserazide (group a) or active metabolite (compound 10) (group B).

On days 1, 3, 5, 8 and 12, animals were scored for dyskinesia by recording dyskinesia using an abnormal involuntary movement metric (AIMs) according to AIM-score as already described (Lundblad et al, eur. j neurosci, 2002, 15, 120). Day 15-26: group A animals were treated with the test drug (same as group B) instead of L-DOPA/benserazide. Days 15, 16, 17, 19, 22, 24 and 26: animals were scored according to AIM-score.

Reversion of L-DOPA-induced dyskinesia in 6-OHDA rats

After 8 days of treatment, the dyskinesia score of group a animals was 10-12, which remained constant until day 12. In contrast, group B animals had significantly less dyskinesia (score 2-4). For group B, the degree of dyskinesia did not change during the study. After the group a animals were converted from L-dopa/benserazide to the test drug, their dyskinesia levels gradually declined to those observed in the other group. Thus, compound 11 induced significantly less dyskinesia than L-DOPA and was able to reduce dyskinesia induced by L-DOPA.

anti-Parkinsonism Effect in MPTP-treated common marmosets

The experiment was performed using 6 MPTP-treated marmosets (2.0mg/kg daily for 5 consecutive days dissolved in sterile 0.9% saline solution). All animals were previously treated with L-DOPA (12.5mg/kg p.o. plus carbidopa 12.5mg/kg p.o.) daily for 30 days to induce dyskinesia. Prior to the study, all subjects exhibited stable motor deficits, including significant reduction in basal motor activity, poor motor coordination, abnormal and/or rigid posture, reduced alertness, and head examination movements. Domperidone was administered 60 minutes prior to any test compound. Domperidone is an anti-dopaminergic drug that inhibits nausea and vomiting. The motor activity evaluation is carried out by adopting a test cage, the test cage is composed of 8 photoelectric switches, the 8 photoelectric switches are composed of 8 infrared beams, the beams are strategically placed in the cage, and the interruption of the beams is recorded as one-time counting. The total number of beam counts per unit time period is then plotted against time or expressed as the area under the curve for total viability (AUC). The assessment of incapacity of movement was performed by a trained observer blinded to the treatment.

As already described, L-DOPA (12.5mg/kg, p.o.) increases locomotor activity and reverses akinesia (Smith et al, mov. disc.2002, 17(5), 887). The dose selected for this stimulation is at the top of the dose response curve for this drug. Compound 11 (orally administered) and compound 10 (subcutaneously administered) produced a dose-related increase in locomotor activity and a locomotor disability reversal that tended to occur with a greater response than that produced by L-DOPA (12.5mg/kg, p.o.). Compared to L-DOPA, the two test compounds produced prolonged exercise with no reversal and the same efficacy as L-DOPA.

In vitro hepatocyte assay

Cryopreserved pooled male rat hepatocytes (Sprague Dawley) and pooled human hepatocytes from 10 donors (male and female) were purchased from In Vitro Technologies inc. Cells were thawed in a water bath at 37 ℃, viable cells were counted and seeded in 96 plates in a total of 100 microliters in Dulbecco's modified Eagle medium (high glucose) containing 5mM Hepes buffer, containing 250.000 cells/mL rat and 500.000 cells/mL human hepatocytes, respectively, in each well. Incubation began 15min after preincubation, and was terminated at time points 0, 5, 15, 30, and 60min for rat hepatocytes and 0, 30, 60, 90, and 120min for human hepatocytes. The incubation was stopped by adding an equal amount of ice-cooled acetonitrile containing 10% 1M HCl. After centrifugation, 20 microliters of supernatant was injected into HPLC Column Atlantis dC 183 μm, 150x 2.1mm i.d. (Waters, MA, USA). The mobile phase had the following composition: a: 5% acetonitrile, 95% H₂O，3.7ml/l 25％NH₃Aqueous solution, 1.8mL/L formic acid. Mobile phase B: 100% acetonitrile and 0.1% formic acid. The flow rate was 0.3 ml/min. The gradient was run at 0% -75% B in 5min-20min and the eluate was analyzed using a Q-tof micro mass spectrometer (Waters, MA, USA). The formation of the product/metabolite was confirmed by accurate mass determination and comparison with synthetic standards that yielded the same retention time. In this assay, the metabolism of compound 11 to compound 10 was confirmed.

Example 4: pharmacological testing of Compound 12

D₁cAMP assay

Stable expression as determined byHuman recombinant D₁Stimulation or inhibition of D by the Compounds in CHO cells of the receptor₁The ability of the receptor to mediate cAMP formation. Cells were seeded at a concentration of 11000 cells/well in 96-well plates and experiments were performed 3 days later. On the day of the experiment, 1mM MgCl in pre-warmed G-buffer (in PBS (phosphate buffered saline)) was added₂、0.9mM CaCl₂And 1mM IBMX (3-isobutyl-1-methylxanthine)) the cells were washed once and the assay was initiated by adding 100 μ l of a mixture of 30nM A68930 and test compound diluted in G buffer (antagonism) or 100 μ l of test compound diluted in G buffer (agonism).

Cells were incubated at 37 ℃ for 20min and washed by adding 100. mu.l S buffer (0.1M HCl and 0.1mM CaCl)₂) The reaction was terminated and the plates were left at 4 ℃ for 1 h. 68 microliters of N buffer (0.15M NaOH and 60mM NaOAc) was added and the plates were shaken for 10 minutes. Transfer 60 microliters of reaction to cAMP FlashPlates (DuPont NEN) containing 40 microliters of 60mM sodium acetate (pH 6.2) and add 100 microliters IC mix (50mM sodium acetate, pH 6.2, 0.1% sodium azide, 12mM CaCl₂1% BSA (bovine serum Albumin) and 0.15. mu. Ci/mL¹²⁵I-cAMP). After incubation for 18h at 4 ℃, the plates were washed once and counted in a Wallac TriLux counter. In this assay, the active metabolite (i.e., Compound 10) was found to be D₁An agonist.

D₂cAMP assay

In human D as determined₂Stimulation or inhibition of D by the Compounds in CHO cells transfected with the receptor₂The receptor mediates the ability to inhibit cAMP formation. Cells were seeded at 8000 cells/well in 96-well plates and experiments were performed 3 days later. On the day of the experiment, the cells were incubated in pre-warmed G buffer (1 mM MgCl in PBS)₂、0.9mM CaCl₂And 1mM IBMX) and the assay was initiated by adding 1 μ M quinpirole, 10 μ M forskolin and 100 μ M mixture of test compound in G buffer (antagonism) or 10 μ M forskolin and 100 μ M mixture of test compound in G buffer (agonism).

Cells were incubated at 37 ℃ for 20min and washed by adding 100. mu.l S buffer (0.1M HCl and 0.1mM CaCl)₂) The reaction was terminated and the plates were left at 4 ℃ for 1 h. 68 microliters of N buffer (0.15M NaOH and 60mM sodium acetate) was added and the plates were shaken for 10 minutes. Transfer 60 microliters of reaction to cAMP FlashPlates (DuPont NEN) containing 40 microliters of 60mM NaOAc (pH 6.2) and add 100 microliters IC mix (50mM NaOAc, pH 6.2, 0.1% sodium azide, 12mM CaCl₂1% BSA and 0.15. mu. Ci/ml¹²⁵I-cAMP). After incubation for 18h at 4 ℃, the plates were washed once and counted in a Wallac TriLux counter. In this assay, the active metabolite (i.e., Compound 10) was found to be D₂An agonist.

D₅Measurement of

At hD₅Intracellular Ca stimulation by dopamine concentration dependence in transfected CHO-Ga16 cells²⁺And (4) releasing. To the cells, fluoro-4 (a calcium indicator dye) was added and held for 1 h. Calcium response (change in fluorescence) was monitored by FLIPR (fluorescence imaging reader) for 2.5 min. Peak responses (EC) from two wells for each data point₅₀) Mean and plotted against drug concentration (for dopamine, see figure 1). In this assay, the active metabolite (i.e., Compound 10) was found to be D₅An agonist.

6-OHDA rat model

Dopamine agonists may be active at the D1 receptor, the D2 receptor, or both. The spin response in rats with unilateral 6-OHDA injury can be used to assess the ability of compounds to stimulate both receptor types and induce spin (Ungerstedt and Arbutnott; Brain Res., 24, 485 (1970); Setler et al Eur.J. Pharmacol., 50(4), 419 (1978); and Ungerstedt et al, "Advances in Dopamine Research" (Kohsaka, Ed.), Pergamon Press, 1982, Oxford, p.219). 6-OHDA (6-hydroxydopamine) is a neurotoxin used by neurobiologists to selectively kill dopaminergic neurons at injection sites within the brains of experimental animals. In the 6-OHDA model, one side of the brain (unilateral) was destroyed by injecting 6-OHDA into the anterior brain medial bundle in front of the substantia nigraNigrostriatal dopamine cells. Such unilateral injections combined with stimulation of dopamine agonists such as apomorphine will induce rotational behavior as if only one side of the brain is stimulated. The experiment consisted of determining the lowest effective dose (MED) at which the compound in question induced rotation. Once the MED is determined, a second experiment is performed to determine the MED (MED) at which the compound overcomes the nemorubide blockade_Nemopride). Nemorubide is a D2 antagonist that blocks the D2 receptor, so any observed rotation may depend on activity at the D1 receptor. Finally, once the MED is known_NemoprideUsing the MED_NemoprideA third experiment was performed by dosing and observing the effect of the D1 antagonist (SCH 23390 alone), the D2 antagonist (nemorubide alone) and finally the combined treatment of SCH 23390 and nemorubide. This third experiment confirmed that: the activity of a compound as either antagonist alone at both receptors was only partially inhibitory to the rotational response induced by the test compound and the combined treatment completely blocked all rotations in rats [ Arnt and Hyttel, Psychopharmacology, 1985, 85(3), 346; and Sonsalla et al j. pharmacol exp. ther., 1988, 247(1), 180]. Proof of principle compounds using apomorphine as a mixed D1/D2 agonist validated this model.

In this model, compounds 10 and 12 have 'apomorphine' -like characteristics with a D1/D2 ratio of about 2-4, compared to apomorphine having a D1/D2 ratio of about 3. Furthermore, the observed time of action of the compound was about 18h, which is significantly higher than that seen with L-DOPA/apomorphine. No D1 component was observed for the D2-agonist (exemplified by pramipexole and rotigotine).

Optimization model

Apomorphine and L-DOPA reverse motor deficits in a mouse model of severe dopamine depletion. Both apomorphine and L-DOPA stimulated the D1 and D2 dopamine receptors. Pramipexole (an agonist of the D2 receptor) was ineffective in this model.

The experiment was performed as follows: mice previously treated with MPTP (2x15mg/kg subcutaneously) and with stable lesions and mice treated with vehicle were used as normal controls. MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) is a neurotoxin that causes permanent parkinson's disease symptoms by killing certain neurons in the substantia nigra of the brain. It was used to study disease in monkeys and mice. On the day of the experiment, mice were treated with AMPT (250mg/kg subcutaneously) and then returned to the rearing cage for 1.5 hours before they were placed in individual cages of a sports apparatus. AMPT (α -methyl-p-tyrosine) is a drug that transiently decreases the activity of catecholamine (in this case, dopamine levels, among others) in the brain. Three hours after AMPT injection, an attempt was made to rescue the motor deficit with compound 10 and viability was recorded for an additional 1.5 hours. As evidenced by the increased levels in the vehicle controls, the first 30min data collected after the rescue treatment was "contaminated" due to the stressful animals treated and injected, and therefore, the data was analyzed using the last 1 hour of recorded data. Various dopaminergic compounds were tested for their ability to reverse the motor deficits produced in this model. Both L-DOPA/benserazide and apomorphine restored locomotion in the mice in a dose-dependent manner. Benserazide is a DOPA decarboxylase inhibitor that cannot cross the blood brain barrier; it is used to prevent the metabolism of L-DOPA to dopamine outside the brain. In contrast, the D2 agonists pramipexole and bromocriptine did not restore locomotion in mice.

This model was used to evaluate whether compound 10 exhibited the same superiority as L-DOPA and apomorphine over the D2 agonist. Dose response experiments were performed and there was a dose-dependent trend to reverse the hypokinesia defect induced by severe endogenous dopamine depletion. A final experiment was performed comparing the effects of apomorphine, pramipexole and compound 10 directly. Compound 10 was shown to restore locomotion and outperform pramipexole in MPTP treated mice.

Dyskinesia rat model

The effect of compound 12 on dyskinesias detected as Abnormal Involuntary Movement (AIM) in 'parkinson' rats relative to L-DOPA/benserazide was determined using the rat dyskinesia model reported in the literature (Lundblad et al, eur. j neurosci., 2002, 15, 120).

Design of research

Throughout the study, animals received L-DOPA/benserazide (6mg/kg and 15mg/kg subcutaneously) or compound 12 (group B) once daily at t ═ 20min.0-180 min. Animals were scored for dyskinesias. Days 1-14: all animals were dosed with L-DOPA/benserazide (group a) or compound 12 (group B).

On days 1, 3, 5, 8 and 12, animals were scored according to AIM-score by recording dyskinesia using an abnormal involuntary movement measure (AIMs), as already described. Day 15-26: animals of group A were treated with compound 12 (same as group B) instead of L-DOPA/benserazide. Days 15, 16, 17, 19, 22, 24 and 26: animals were scored according to AIM-score.

Results

After 8 days of treatment, the dyskinesia score of group a animals ranged from 70-80, which remained constant until day 15. In contrast, group B animals had significantly less dyskinesia (score 10-25). For group B, the degree of dyskinesia did not change during the study. After 1210 days of conversion of group A animals from L-dopa/benserazide to compound, their dyskinesia levels gradually declined to a score of 30-35. Thus, compound 12 induced significantly less dyskinesia than L-DOPA and was able to reduce dyskinesia induced by L-DOPA.

anti-Parkinsonism Effect in MPTP-treated common marmosets

Experiments were performed using 6 MPTP-treated marmosets (2.0mg/kg for 5 consecutive days, dissolved in sterile 0.9% saline solution). All animals were previously treated with L-DOPA (12.5mg/kg p.o. plus carbidopa 12.5mg/kg p.o.) daily for 30 days to induce dyskinesia. Prior to the study, all subjects exhibited stable motor deficits, including significant reduction in basal motor activity, poor motor coordination, abnormal and/or rigid posture, reduced alertness, and head examination movements. Domperidone was administered 60 minutes prior to any test compound. The locomotor activity evaluation was performed using a test cage consisting of 8 photoelectric switches consisting of 8 infrared beams strategically placed within the cage and recording the interruption of the beam as a count. The total number of beam counts per unit time period is then plotted against time or expressed as the area under the curve for total viability (AUC). The assessment of incapacity of movement was performed by a trained observer blinded to the treatment.

As already described, L-DOPA (12.5mg/kg, p.o.) increases locomotor activity and reverses akinesia (Smith et al, mov. disc.2002, 17(5), 887). The dose selected for this stimulation is at the top of the dose response curve for this drug. Compound 12 (orally administered) and compound 10 (orally administered) produced a dose-related increase in locomotor activity and an inability to reverse locomotor, which tended to occur with a greater response than that produced by L-DOPA (12.5mg/kg, p.o.). Compared to L-DOPA, the two test compounds produced prolonged exercise with no reversal and the same efficacy as L-DOPA.

Claims (12)

1. Use of (4aR, 10aR) -1-n-propyl-1, 2, 3, 4, 4a, 5, 10, 10 a-octahydro-benzo [ g ] quinoline-6, 7-diol, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for reversing dyskinesia.
2. Use of (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of Parkinson's disease while maintaining low dyskinesia induction characteristics.
3. Use of (6aR, 10aR) -7-n-propyl-6, 6a, 7, 8, 9, 10, 10a, 11-octahydro-1, 3-dioxa-7-aza-cyclopenta [ a ] anthracene or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reversing dyskinesia.
4. Use of (4aR, 10aR) -1-n-propyl-2, 3, 4, 4a, 5, 7, 8, 9, 10, 10 a-decahydro-1H-benzo [ g ] quinolin-6-one or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for reversing dyskinesia.
5. 5. The use of any one of claims 1-4, wherein the movement disorder is associated with a basal ganglia-related movement disorder.
6. 6. The use of claim 5, wherein the movement disorder is associated with Parkinson's disease.
7. 7. The use of claim 6, wherein the dyskinesia is associated with idiopathic Parkinson's disease or post-encephalitic Parkinson's disease.
8. 8. The use of claim 7, wherein the dyskinesia is associated with "off-stage" dystonia in Parkinson's disease.
9. 9. The use of claim 8, wherein the dyskinesia arises as a side effect of a therapeutic agent to treat Parkinson's disease.
10. 10. The use of claim 9, wherein the movement disorder is associated with dopamine replacement therapy.
11. 11. The use of claim 10, wherein said dopamine replacement therapy agent is selected from the group consisting of rotigotine, ropinirole, pramipexole, cabergoline, bromocriptine, lisuride, pergolide, L-DOPA, and apomorphine.
12. 12. The use as claimed in claim 11, wherein the dyskinesia is established as a result of repeated administration of L-DOPA.