HK1162172A - Novel 3-phenyl-azetidine derivatives useful as modulators of cortical catecholaminergic neurotransmission - Google Patents

Description

Novel 3-phenyl-azetidine derivatives as modulators of cortical catecholaminergic neurotransmission

Technical Field

The present invention relates to novel 3-phenyl-azetidine derivatives useful for modulating extracellular levels of catecholamines, dopamine and norepinephrine, in cerebral cortical areas of the mammalian brain, and more particularly useful for treating central nervous system disorders.

In other aspects, the invention relates to pharmaceutical compositions comprising the 3-phenyl-azetidine derivatives of the invention and the use of these compounds in therapeutic applications.

Background

The cerebral cortex contains several major areas involved in higher-order functions, such as thought, sensation, memory and planning. Biogenic amines, namely dopamine, norepinephrine and 5-hydroxytryptamine, are important for cortical function in mammals. The ascending dopamine and norepinephrine pathways innervate the cortex. The 5-hydroxytryptamine neurons of the CNS project to virtually all areas of the brain, including the cerebral cortex. Primary and secondary dysfunctions in the activity of these pathways lead to a dysregulation of dopamine, norepinephrine and 5-hydroxytryptamine receptor activity in these brain regions, which in turn leads to the manifestation of psychiatric and neurological symptoms.

Biogenic amines of the cortex regulate several aspects of cortical function that control emotion, anxiety, motivation, cognition, attention, arousal, and wakefulness. Therefore, the catecholamines dopamine and norepinephrine exert a strong role in the prefrontal cortex area, the integrity of which is essential for the so-called execution of cognitive functions involving, for example, attention, planning of activities and control of impulses. Norepinephrine is a major component of the pathway that regulates anxiety and fear, and is therefore believed to be disregulated in anxiety disorders such as panic disorder, Generalized Anxiety Disorder (GAD), and specific phobias. With respect to mood and emotional function, the use of compounds to specifically promote norepinephrine and 5-hydroxytryptamine neurotransmission in the treatment of depression and anxiety strongly contributes to the widely accepted concept: these neurotransmitters are all involved in the regulation of emotional function.

In general, compounds that specifically affect biogenic amine transmission, more specifically monoamines, norepinephrine, dopamine and 5-hydroxytryptamine, are successfully used to alleviate affective, cognitive or attention symptoms in patients suffering from, for example, depression, anxiety and Attention Deficit Hyperactivity Disorder (ADHD).

Furthermore, the monoamine system in the cortex is known to be directly or indirectly involved in the core symptoms of schizophrenia. Based on biochemical synthetic and genetic findings, as well as neuropsychological findings in schizophrenia indicating dysfunction of specific cortical regions, it has been proposed that when different pathological causes accumulate on cortical function, resulting in cortical microcirculation disorders, the disorder appears, with clinical manifestations of symptoms of schizophrenia. This cortical microcirculation is regulated by several neurotransmitters, including glutamate, GABA and dopamine.

GB 1266587 describes certain azetidinol (azetidinol) derivatives for use as analgesics, GB 1236078 describes certain substituted azetidinols for use as antidepressants, and US 3481920, US 3494964 and US 3668196 describe certain substituted azetidinol derivatives for use as CNS agonists. However, the 3-phenyl-azetidine derivatives of the present invention have not been reported.

Summary of The Invention

It is an object of the present invention to provide new pharmaceutically active compounds which are particularly suitable for the treatment of disorders in the central nervous system. It is a further object to provide compounds that modulate dopamine and norepinephrine neurotransmission in the brain of mammals, including the human brain. Still another object is to provide novel compounds with cortical enhancing properties. It is a further object to provide compounds which have a therapeutic effect after oral administration. Still a further object is to provide compounds with better pharmacodynamic properties, such as kinetic behavior, bioavailability, solubility and efficacy. It is a further object to provide compounds which are superior in terms of efficacy or side effects to the compounds currently known for the treatment of several disorders related to CNS dysfunction.

The present invention relates to the unexpected discovery of the pharmacological effects of the compounds of the present invention on monoamines in the cerebral cortex, and the use of these compounds in the treatment of certain CNS disorders. Compounds of the invention regioselectively increase catecholamine levels in the frontal cortex as demonstrated by pharmacological testing in rats in vivo. As catecholamines specifically modulate the effects of cortical function involving cognition, attention and emotion, the compounds of the invention may be useful in the treatment of disorders characterized by dysfunction in these regions. Thus, the compounds may be useful in the treatment of cognitive disorders, ADHD, depression and anxiety. The compounds may also be used in the treatment of schizophrenia, which is characterized by dysfunction of the cerebral cortex manifested by cognitive and psychiatric disorders.

In a first aspect thereof, the present invention provides a compound represented by formula 1

Any stereoisomer thereof or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof; wherein R is¹、R²、R³And X is as defined below.

In its second aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

In another aspect, the present invention provides the use of a compound of the invention, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a pharmaceutical composition for the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is responsive to modulation of catecholamines in the cerebral cortex.

In still another aspect, the present invention relates to a method for the treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, including a human, which disorder, disease or condition is responsive to modulation of catecholamines in the cerebral cortex, which method comprises the steps of: administering to such a living animal in need thereof a therapeutically effective amount of a compound of the present invention, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

Other aspects of the invention will be apparent to those skilled in the art from the following detailed description and examples.

Detailed Description

3-phenyl-azetidine derivatives

In a first aspect thereof, the present invention provides 3-phenyl-azetidine derivatives of formula 1:

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein

X is OH or OCH₃；

R¹Is F or Cl;

R²h, F or Cl;

R³is H, CH₃Or CH₂CH₃；

And with the proviso that if X is OH, then R³Is not H.

In a preferred embodiment, the 3-phenyl-azetidine derivative of the invention is a compound of formula 2:

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein

R^o、R^m、R^pAnd R^qOne of them represents R¹；

R^o、R^m、R^pAnd R^qOne of the remaining 3 of (1)Represents R²；

R^o、R^m、R^pAnd R^qThe remaining 2 of (a) represent H; and is

X and R³As defined above.

In a more preferred embodiment, R^mRepresents R¹，R^oRepresents R²And R is^pAnd R^qRepresents H.

In another embodiment, R^mRepresents R¹，R^pRepresents R²And R is^oAnd R^qRepresents H.

In yet another embodiment, R^mRepresents R¹，R^qRepresents R²And R is^oAnd R^pRepresents H.

In another preferred embodiment, the 3-phenyl-azetidine derivative of the present invention is a compound of formula 1 or formula 2, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein X is OH or OCH₃。

In a more preferred embodiment, X is OH.

In another more preferred embodiment, X is OCH₃。

In a 3 rd preferred embodiment, the 3-phenyl-azetidine derivative of the present invention is a compound of formula 1 or formula 2, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein R is a pharmaceutically acceptable salt thereof¹Is F or Cl.

In a more preferred embodiment, R¹Is F.

In another more preferred embodiment, R¹Is Cl.

In a4 th preferred embodiment, the 3-phenyl-azetidine derivative of the present invention is a compound of formula 1 or formula 2, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein R is a pharmaceutically acceptable salt thereof²Is H, F or Cl.

In a more preferred embodiment, R²Is H.

In another more preferred embodiment, R²Is F.

In a 3 rd more preferred embodiment, R²Is Cl.

In a 5 th more preferred embodiment, the 3-phenyl-azetidine derivative of the present invention is a compound of formula 1 or formula 2, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein R is a pharmaceutically acceptable salt thereof³Is H, CH₃Or CH₂CH₃Or a deuterated analog thereof.

In a more preferred embodiment, R³Is H or D.

In another more preferred embodiment, R³Is CH₃Or CD₃。

In a 3 rd more preferred embodiment, R³Is CH₃。

In a4 th more preferred embodiment, R³Is CH₂CH₃Or CD₂CD₃。

In a 5 th more preferred embodiment, R³Is CH₂CH₃。

In yet another more preferred embodiment, the 3-phenyl-azetidine derivative of the invention is

3- (2, 3-difluorophenyl) -1-ethyl azetidin-3-ol;

3- (3, 4-difluorophenyl) -1-ethyl azetidin-3-ol;

3- (3, 5-difluorophenyl) -1-ethyl azetidin-3-ol;

3- (3-chloro-5-fluorophenyl) -1-ethyl azetidin-3-ol;

3- (3, 4-difluorophenyl) -1- (ethyl-D5) azetidin-3-ol;

3- (3-chloro-2-fluorophenyl) -1-ethyl azetidin-3-ol;

3- (2, 3-difluorophenyl) -1-ethyl azetidin-3-ol-1-oxide;

3- (2, 3-difluorophenyl) -3-methoxyazetidine;

3- (2, 3-difluorophenyl) -1-ethyl-3-methoxyazetidine; or

3- (2, 3-difluorophenyl) -1- (ethyl-D5) azetidin-3-ol;

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof.

In yet another more preferred embodiment, the 3-phenyl-azetidine derivative of the invention is

3- (2, 3-difluorophenyl) -1-ethyl azetidin-3-ol;

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof.

In another more preferred embodiment, the 3-phenyl-azetidine derivative of the invention is

3- (2, 3-difluorophenyl) -1-ethyl azetidin-3-ol fumarate;

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof.

Any combination of two or more embodiments as defined above is considered to be within the scope of the present invention.

Pharmaceutically acceptable salts

The compounds of the invention may be provided in any form suitable for the desired administration. Suitable forms include pharmaceutically (i.e. physiologically) acceptable salts as well as prodrug or prodrug forms of the compounds of the invention.

Examples of pharmaceutically acceptable salts include, but are not limited to, non-toxic inorganic and organic acid addition salts such as hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, pamoate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, p-toluenesulfonate and the like. Such salts may be formed by methods well known and described in the art.

Other acids, not considered pharmaceutically acceptable, such as oxalic acid, may be used to prepare salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

Examples of pharmaceutically acceptable cationic salts of the compounds of the invention include, but are not limited to, sodium, potassium, calcium, magnesium, zinc, aluminum, lithium, choline, lysine salts of the compounds of the invention containing an anionic group(lysine) and ammonium salts, and the like. Such cationic salts can be formed by methods well known and described in the art.

In the context of the present invention, of compounds containing N "Salts "are also considered to be pharmaceutically acceptable salts. Preferably "Salts "include alkyl-Salt, cycloalkyl-Salts and cycloalkylalkyl-And (3) salt.

Examples of prodrugs or prodrug forms of a compound of the invention include examples of suitable prodrugs of a substance of the invention, including compounds modified at one or more reactive or derivatizable groups of the parent compound. Of particular interest are compounds modified at the carboxyl, hydroxyl or amino group. Examples of suitable derivatives are esters or amides.

The compounds of the present invention may be prepared in soluble or insoluble form with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.). Soluble forms may also include hydrated forms such as the monohydrate, dihydrate, hemihydrate, trihydrate, tetrahydrate and the like. In general for the purposes of the present invention, a soluble form is considered to be equivalent to an insoluble form.

Stereoisomers

One skilled in the art will appreciate that the compounds of the present invention may exist in different stereoisomeric forms, including enantiomers, diastereomers, and cis-trans isomers.

The present invention includes all such isomers and any mixtures thereof, including racemic mixtures.

Racemic forms can be resolved into the optical antipodes by well-known methods and techniques. One way of separating enantiomeric compounds (including enantiomeric intermediates) is-in the case of compounds which are chiral acids-by using optically active amines and liberating diastereomeric resolved salts by treatment with an acid. Another method for resolving racemates into the optical antipodes is based on chromatography on optically active substrates. The racemic compounds of the invention can thus be resolved into their optical antipodes, for example by fractional crystallization of, for example, D-or L- (tartrate, mandelate or camphorsulfonate) salts.

The compounds of the present invention may also be resolved by reacting a compound of the present invention with an optically active carboxylic acid, such as a carboxylic acid derived from (+) or (-) phenylalanine, (+) or (-) phenylglycine, (+) or (-) camphoric acid, to form diastereomeric amides, or by reacting a compound of the present invention with an optically active chloroformate or the like to form diastereomeric carbamates.

Other methods for resolving optical antipodes are known in the art. Such methods include those described by Jaques J, Collet A, & Wilen S in Enantiomers, Racemates, and solutions, John Wiley and Sons, New York (1981).

Optically active compounds can also be prepared from optically active starting materials.

N-oxide

In the context of the present invention, N-oxide means an oxidized derivative of a tertiary amine, including the nitrogen atom of aromatic N-heterocyclic compounds, non-aromatic N-heterocyclic compounds, trialkylamines and triallylamines. For example, the N-oxide of a pyridyl-containing compound may be a 1-oxy-pyridin-2, -3, or-4-yl derivative.

The N-oxides of the compounds of the present invention may be prepared by oxidation of the corresponding nitrogen-containing basic substance using a conventional oxidizing agent (e.g. hydrogen peroxide) in the presence of an acid such as acetic acid at elevated temperature, or by reaction with a peracid in a suitable solvent, for example peracetic acid in dichloromethane, ethyl acetate or methyl acetate, or 3-chloroperoxybenzoic acid in chloroform or dichloromethane.

Deuterated analogs

The compounds of the present invention may be provided in the form of their deuterated analogs. Deuterium forms a bond with carbon that vibrates at a lower frequency and is therefore stronger than a C-H bond. The "deuterium" (deuterium) form of the drug can therefore be more stable and less prone to degradation and be maintained in the organism for a longer period of time.

The deuterated analogs of the present invention can be fully or partially deuterated derivatives. Preferred deuterated derivatives of the invention have fully or partially deuterated alkyl groups, in particular-CD₃(methyl-D3) or-CD₂CD₃(Ethyl-D5).

In the context of the present invention, when a particular position is designated as having deuterium (described as "D" or "deuterium"), it will be understood that the abundance of deuterium at that position is substantially greater than the natural abundance of deuterium (i.e., at least 50.1% incorporation of deuterium), which is 0.015%.

In a preferred embodiment, the abundance of deuterium at this position is at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporation), which is 0.015%. In other preferred embodiments of the invention, the deuterium abundance at this position is at least 3500 times (52.5% deuterium incorporation), at least 4000 times (60% deuterium incorporation), at least 4500 times (67.5% deuterium incorporation), at least 5000 times (75% deuterium incorporation), at least 5500 times (82.5% deuterium incorporation), at least 6000 times (90% deuterium incorporation), at least 6333.3 times (95% deuterium incorporation), at least 6466.7 times (97% deuterium incorporation), at least 6600 times (99% deuterium incorporation), or at least 6633.3 times (99.5% deuterium incorporation).

Labelled compounds

The compounds of the invention may be used in their labeled or unlabeled form. In the context of the present invention, a labeled compound has one or more atoms replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. The label can allow convenient quantitative detection of the compound.

The labeled compounds of the invention can be used as diagnostic tools, radiotracers or monitoring agents in various diagnostic methods, and for in vivo receptor imaging.

The labelled isomer of the invention preferably comprises at least one radionuclide as label. Positron emitting radionuclides are all candidates for use. In the context of the present invention, the radionuclide is preferably selected from²H (deuterium),³H (tritium),¹¹C、¹³C、¹⁴C、¹³¹I、¹²⁵I、¹²³I and¹⁸F。

the physical method for detecting the labelled isomer of the present invention may be selected from Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Magnetic Resonance Spectroscopy (MRS), Magnetic Resonance Imaging (MRI) and computer assisted axial X-ray tomography (CAT) or combinations thereof.

Preparation method

The compounds of the present invention may be prepared by conventional methods for chemical synthesis, such as those described in the preparation examples. The starting materials for the processes described herein are well known or readily prepared by conventional methods from commercially available chemicals.

Conventional methods may also be used to convert one compound of the invention to another compound of the invention.

The reaction end product described herein may be isolated by conventional techniques, e.g., by extraction, crystallization, distillation, chromatography, and the like.

Those skilled in the art will appreciate that in order to prepare the compounds of the present invention in other ways-and in some cases in a more convenient manner-the individual process steps described previously may be performed in a different order, and/or the individual reactions may be performed at different stages throughout the pathway (i.e., the chemical transformations may be performed as different intermediates with respect to those described previously in connection with the particular reaction).

Biological activity

The compounds according to the invention have modulatory properties for norepinephrine and dopamine and to some extent 5-hydroxytryptamine, and they and their pharmaceutical compositions are useful for the treatment of a number of central nervous system disorders, including psychiatric disorders. In particular, the compounds and their pharmaceutical compositions are useful for the treatment of CNS disorders in which the cortical monoamine energy system is dysfunctional by direct or indirect cause. In another embodiment, the compounds according to the invention may be used for the treatment of affective and cognitive disorders, including neurodegenerative and developmental disorders. Furthermore, compounds having a modulating effect on the dopaminergic system may also be used for improving motor and cognitive functions.

In particular embodiments, the compounds of the invention are contemplated for use in the treatment, prevention or alleviation of dementia, age-related cognitive impairment, childhood developmental disorders, ADHD, cerebral palsy, huntington's disease, Gilles de la Tourette's syndrome, depression, bipolar disorder, schizophrenia, schizophreniform disorder, Generalized Anxiety Disorder (GAD), specific phobias, panic disorder, sleep disorders, bipolar disorders, drug-induced psychotic disorders, iatrogenic psychosis, iatrogenic hallucinations, non-iatrogenic hallucinations, mood disorders, anxiety disorders, depression, obsessive-compulsive disorders (obsessive-compulsive disorders), mood disorders related to aging, alzheimer's disease, dementia related to alzheimer's disease, age-related cognitive impairment, brain injury, substance abuse, cognitive impairment, mental alertness, mental impairment, mental depression, mental alertness, mental retardation, mental depression, Disorders characterized by misuse of food (misuse of food), sleep disorders, sexual dysfunction, eating disorders, obesity, headache, pain in conditions characterized by increased muscle tone, dyskinesia, parkinson's disease, parkinsonism syndrome, dyskinesia, levodopa-induced dyskinesia, dystonia, neurodevelopmental disorders, neurodegenerative disorders, tics, tremor, restless legs, narcolepsy, and behavioral disorders (behavioural disorders).

Pharmaceutical composition

In another aspect, the present invention provides novel pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention.

The present invention relates to pharmaceutical compositions comprising the compounds of the invention, and their use in the treatment of CNS disorders. Both organic and inorganic acids may be used to form non-toxic pharmaceutically acceptable acid addition salts of the compounds according to the invention. Suitable acid addition salts of the compounds of the present invention include those formed with pharmaceutically acceptable salts such as those mentioned above. Pharmaceutical compositions comprising a compound according to the invention may also contain substances useful for facilitating the preparation of pharmaceutical preparations or the administration of preparations. Such agents are well known to those skilled in the art and may be, for example, pharmaceutically acceptable adjuvants, carriers and preservatives.

In clinical practice, the compounds according to the invention are usually administered orally, rectally, nasally or by injection in the form of pharmaceutical preparations containing the active ingredient, either as the free base or as a pharmaceutically acceptable non-toxic acid addition salt, for example hydrochloride, lactate, acetate or sulfamate, in association with a pharmaceutically acceptable carrier. The carrier may be a solid, semi-solid or liquid formulation. Typically the active substance will constitute from 0.1 to 99% by weight of the preparation, more particularly from 0.5 to 20% by weight of the preparation for preparations intended for injection, and from 0.2 to 50% by weight of the preparation for preparations suitable for oral administration.

To prepare pharmaceutical preparations containing a compound according to the invention in dosage unit form for oral administration, the selected compound may be mixed with a solid excipient, such as lactose, sucrose, sorbitol, mannitol, starches such as potato starch, corn starch or amylopectin, cellulose derivatives, binders such as gelatin or polyethylene-pyrrolidine, lubricants such as magnesium stearate, calcium stearate, polyethylene glycol, waxes, paraffins and the like, and compressed to tablets. If coated tablets are desired, the core material (cores) (prepared as described above) may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatin, talc, titanium dioxide, and the like. Alternatively, the tablets may be coated with a polymer known to those skilled in the art, which polymer is dissolved in a readily volatile organic solvent or mixture of organic solvents. Dyes may be added to these coatings to easily distinguish tablets containing different active substances or different amounts of active compound.

For the preparation of soft gelatin capsules, the active substance can be mixed with, for example, vegetable oils or polyethylene glycols. Hard gelatin capsules may contain granules of the active substance using the excipients mentioned for tablets, such as lactose, sucrose, sorbitol, mannitol, starches (e.g. potato, corn or amylopectin), cellulose derivatives or gelatin. Liquid or semi-solid drugs can also be filled into hard gelatin capsules.

Examples of tablet and capsule formulations suitable for oral administration are as follows:

dosage units for rectal use may be solutions or suspensions, or may be prepared in the form of suppositories containing the active substance mixed with a neutral fat base, or gelatin rectal capsules containing the active substance mixed with vegetable or paraffin oils. Liquid preparations for oral administration may be in the form of syrups or suspensions, for example, solutions containing from about 0.2% to about 20% by weight of the active substance described herein, the remainder being the mixture of sugar and ethanol, water, glycerol and propylene glycol. Optionally, such liquid preparations may contain coloring agents, flavoring agents, saccharin and carboxymethylcellulose as thickening agents, or other excipients known to those skilled in the art.

Aqueous solutions of water-soluble pharmaceutically acceptable salts of the active substances can be prepared by injection of solutions for parenteral use, preferably at a concentration of 0.5% to about 10% by weight. These solutions may also contain stabilizers and/or buffers and may conveniently be presented in ampoules of different dosage units. The use and administration to the patient to be treated will be apparent to those of ordinary skill in the art.

For intranasal or inhalational administration, the compounds of the invention may be delivered in the form of a solution, dry powder or suspension. Administration may be by pump spray container, by squeezing or aspirating the container by the patient, or by aerosol spray from a pressurized container or nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The compounds of the invention may also be administered by dry powder inhalers, as a finely divided powder in combination with a carrier material, such as a saccharide, or as microspheres. The inhaler, pump spray or aerosol spray may be single dose or multi-dose. The dosage can be controlled by a valve that delivers a measured amount of the active compound.

The compounds of the present invention may also be administered in a controlled release formulation. The compound is released at a rate required to maintain a constant pharmacological activity over a desired period of time. Such dosage forms provide a supply of drug to the body at a predetermined time, thus maintaining the drug level in the therapeutic range for a longer period of time than conventional non-controlled formulations. The compounds may also be formulated in controlled release formulations, wherein the release of the active compound is targeted. For example, by the pH sensitivity of the formulation, the release of the compound may be localized to a particular region of the digestive system. Such formulations are well known to those skilled in the art.

Additional details of formulation and administration techniques can be found in the latest edition of Remington's pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

The compositions may be administered in varying doses depending on the disorder, the patient being treated, and the route of administration. The dosage will also depend on the relationship between absorption potential and frequency and route of administration. The dose may be administered 1, 2 or 3 or more times per day. The compounds of the invention may be administered to a subject in a dosage range of 0.01mg to 500mg per kg body weight per day, although variations will necessarily occur depending on the body weight, sex and condition of the subject being treated, the disease state being treated and the particular route of administration chosen. However, dosage levels ranging from 0.1mg to 10mg per kg body weight per day (single or divided doses) are most suitable for use in humans to treat the disease. Alternatively, the dose level is such that the serum concentration of the compound is between 0.1nM and 10. mu.M.

Examples

The invention is further illustrated in the following examples, which are not intended to limit the scope of the invention in any way.

Example 1

3- (2, 3-difluorophenyl) -1-ethylazetidin-3-ol

1, 3-dichloro-2- (2, 3-difluorophenyl) propan-2-ol (6.0g, 24.89mmol) was dissolved in acetonitrile (90ml) and transferredTo 6 different microwave vials, each containing an equivalent amount (1.0g, 2.49 mmol). Sodium bicarbonate (1.04g, 12.44mmol (total 6.27g, 74.67mmol)) was added to each tube and the tube was sealed. Ethylamine (2.0M, 2.07ml, 4.15mmol (12.44 ml total, 24.89mmol) in tetrahydrofuran was added through a septum, the mixture was heated at 120 ℃ under microwave radiation for 30min, then ethylamine (2.0M, 1.03ml, 2.07mmol (6.22 ml total, 12.44mmol) in tetrahydrofuran was added, the mixture was heated at 120 ℃ under microwave radiation for 30min, a solution in which water (100ml) and t-butyl methyl ether (100ml) were mixed was added, the organic phase was collected, the aqueous phase was extracted with t-butyl methyl ether (100ml), the mixed organic phase was dried (Na)₂SO₄) And evaporation. The crude product was purified by flash column chromatography (ethyl acetate/methanol, 1: 0 to 9: 1) on silica gel to give the title compound (2.53g, 48%). Conversion of amine to fumarate, recrystallization from ethanol/diethyl ether/diisopropyl ether: m.p.148-150 ℃. MS M/z (relative intensity, 70eV)213(M +, 1), 156(39), 141(75), 127(56), 58 (bp).

Example 2

3- (3, 4-difluorophenyl) -1-ethylazetidin-3-ol

A mixture of 3- (3, 4-difluorophenyl) azetidin-3-ol (0.3g, 1.62mm0l), triethylamine (0.68ml, 4.87mmol) and iodoethane (0.19ml, 2.44mmol) in tetrahydrofuran (15ml) was stirred at ambient temperature for 15 hours. The solvent was evaporated, aqueous hydrochloric acid (50mL. 10%) was added and the mixture was washed with tert-butyl methyl ether (2X 50 mL). The aqueous phase was made basic by the addition of aqueous sodium hydroxide (5M) and extracted with tert-butyl methyl ether (2X 50 ml). The combined organic phases were dried (Na)₂SO₄) Evaporation under reduced pressure gave the crude product (0.26 g). Purification by flash column chromatography (ethyl acetate/methanol 1: 1) on silica gel gave 0.2g (58%) of the title compound. Conversion of amine to fumarate, recrystallization from methanol/diethyl ether: m.p.177-179 ℃. MS M/z (relative intensity, 70eV)213(M +, 1), 141(86), 127(45), 114(58), 58 (bp).

Example 3

3- (3, 5-difluorophenyl) -1-ethylazetidin-3-ol

1, 3-dichloro-2- (3, 5-difluorophenyl) propan-2-ol (1.0g, 4.15mmol) was dissolved in acetonitrile (10ml), sodium bicarbonate (1.39g, 16.59 mmol) and ethylamine (70%, 0.33ml, 4.15mmol) in water were added, the mixture was refluxed for 10 hours, water (50ml) and tert-butyl methyl ether (50ml) were added, the organic phase was collected, the aqueous phase was extracted with tert-butyl methyl ether (50ml), the combined organic phases were extracted with aqueous hydrochloric acid (10%, 50ml), the aqueous phase was basified, extracted with tert-butyl methyl ether (2X 50ml), dried (Na)₂SO₄) And evaporated to give 0.35g of crude product. Purification by flash column chromatography (ethyl acetate/MeOH 1: 0 → 1: 1) on silica gel afforded the title compound (0.2g, 23%). Conversion of amine to fumarate, recrystallization from ethanol/diethyl ether/diisopropyl ether: m.p.140-141 ℃. MS M/z (relative intensity, 70eV)213(M +, 1), 156(36), 141(37), 127(64), 114(50), 58 (bp).

Example 4

3- (3-chloro-5-fluorophenyl) -1-ethyl azetidin-3-ol

A mixture of 3- (3-chloro-5-fluorophenyl) azetidin-3-ol (0.41g, 2.03mmol), potassium carbonate (0.56g, 4.06mmol) and iodoethane (0.31g, 2.03mmol) in acetonitrile (5ml) was heated at 120 ℃ for 10min under microwave irradiation. Water (50ml) and ethyl acetate (50ml) were added to the solution, and the organic phase was collected. The aqueous phase was extracted with ethyl acetate (2X 50 ml). The combined organic phases were dried (Na)₂SO₄) And evaporating under reduced pressure to obtain a crude product. Purification by flash column chromatography (ethyl acetate) on silica gel gave 0.28g (60%) of the title compound. Conversion of amine to fumarate, recrystallization from ethanol/diethyl ether/diisopropyl ether: m.p.166-168 ℃. MS M/z (relative intensity, 70eV)229(M +, 1), 172(23), 130(24), 109(33), 58 (bp).

Example 5

3- (3, 4-difluorophenyl) -1- (ethyl-D5) azetidin-3-ol

A mixture of 3- (3, 4-difluorophenyl) azetidin-3-ol (0.02g, 0.11mmol), potassium carbonate (0.037g, 0.27mmol) and iodoethane-d 5(0.017g, 0.11mmol) in acetonitrile (5ml) was heated at 120 ℃ for 30min under microwave radiation. The crude product was analyzed by GCMS. Analysis showed 30% conversion. MS M/z (relative intensity, 70eV)218(M +, 1), 141(58), 127(31), 114(39), 63 (bp).

Example 6

3- (3-chloro-2-fluorophenyl) -1-ethyl azetidin-3-ol

A mixture of 1, 3-dichloro-2- (3-chloro-2-fluorophenyl) propan-2-ol (0.88g, 3.42mmol), sodium bicarbonate (0.92g, 10.95mmol) and ethylamine in tetrahydrofuran (2.0M, 1.82ml, 3.64mmol) in acetonitrile (15ml) was heated at 120 ℃ for 30min under microwave radiation. Ethylamine in tetrahydrofuran (2.0M, 0.91ml, 1.82 mmol) was added and the mixture was heated at 120 ℃ under microwave irradiation for 30min, aqueous sodium carbonate (50ml, 10%) and ethyl acetate (50ml) were added and the organic phase was collected, the aqueous phase was extracted with ethyl acetate (50ml), the combined organic phase was dried (MgSO4) and evaporated, the crude product was purified by flash column chromatography (ethyl acetate/isooctane, 1: 4 to 1: 1) on silica gel to give the title compound (0.28g, 35%). the amine was converted to fumarate and recrystallized from ethanol/diethyl ether/diisopropyl ether: M.p.172-173 ℃ C. MS M/z (relative strength, 70eV)229(M +, 1), 172(29), 157(56), 109(41), 58 (bp).

Example 7

3- (2, 3-difluorophenyl) -1-ethylazetidin-3-ol-1-oxide

3-Chloroperoxybenzoic acid (77%, 0.98g, 4.37mmol) was added portionwise to a stirred solution of 3- (2, 3-difluorophenyl) -1-ethylazetidin-3-ol (0.622g, 2.91mmol) in dichloromethane (30 mL). The resulting mixture was stirred at ambient temperature for 5 hours. The reaction mixture was poured onto a basic alumina column, which was washed with dichloromethane and eluted with methanol. Purification by HPLC on waters OBD C18, 5 μm (MeOH/33mM NH3, 10: 90 to 30: 70) gave the title compound (0.160g, 23.9%). The N-oxide was converted to the hydrochloride salt and recrystallized from methanol/acetonitrile. The product was analyzed by LCMS (Qtrap, Applied Biosystems, Q1 MS): MS (m + 1)/z; 231(M +1, bp).

Example 8

3- (2, 3-difluorophenyl) -3-methoxyazetidine

Trifluoroacetic acid (3mL, 40mmol) was added to a stirred solution of tert-butyl 3- (2, 3-difluorophenyl) -3-methoxyazetidine-1-carboxylate (0.7g, 2.34mmol) in dichloromethane (20 mL). The resulting mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated and the crude product was purified on a Biotage Isolute SCX-3SPE cartridge (washed with methanol, eluted with methanol/triethylamine, 4: 1). The solvent was evaporated to give (0.16g, 64%) of the title compound. Conversion of the amine to the oxalate, recrystallization from ethanol/diethyl ether/diisopropyl ether: m.p.141-142 ℃. MS M/z (relative intensity, 70eV)199(M +, 1), 170(bp), 169(97), 140(82), 127 (47).

Example 9

3- (2, 3-difluorophenyl) -1-ethyl-3-methoxyazetidine

A mixture of 3- (2, 3-difluorophenyl) -3-methoxyazetidine (0.75g, 4.05mmol), potassium carbonate (0.99g, 8.1mmol) and iodoethane (0.388ml, 4.05mmol) in acetonitrile (25ml) was stirred at ambient temperature for 1 hour. Water (50mL) and ethyl acetate (50mL) were added, the organic phase was collected, and the aqueous phase was extracted with ethyl acetate (2X 50 mL). The combined organic phases were dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/methanol 1: 1) on silica gel to give the title compound (0.58g, 63%). Conversion of the amine to the oxalate, recrystallization from ethanol/diethyl ether/diisopropyl ether: m.p.147-148 ℃. MS M/z (relative intensity, 70eV)227(M +, 4), 170(66), 169(bp), 140(63), 127 (39).

Example 10

3- (2, 3-difluorophenyl) -1- (ethyl-D5) azetidin-3-ol

A mixture of 3- (2, 3-difluorophenyl) azetidin-3-ol (0.025g, 0.135mmol), 1-iodoethane-D5 (0.0108ml, 0.135mmol) and potassium carbonate (0.0373, 0.27mmol) in acetonitrile (5ml) was heated at 110 ℃ for 10min with microwave radiation. The crude product was analyzed by GCMS. Analysis showed-90% conversion. MS M/z (relative intensity, 70eV)218(M +, 1), 156(22), 141(43), 127(35), 63 (bp).

Preparation 1

1, 3-dichloro-2- (2, 3-difluorophenyl) propan-2-ol

N-hexyllithium (2.3M in hexane, 41.7mL, 0.096mol) was added dropwise to a solution of 1-bromo-2, 3-difluorobenzene (19.5g, 0.10mol) in dry ether (120mL) at-78 ℃ under nitrogen. The mixture was stirred for 1min, after which a solution of 1, 3-dichloroacetone (12.2g, 0.096mol) in dry ether (30mL) was added dropwise. The resulting mixture was stirred at-78 ℃ for 1 hour, then returned to ambient temperature and stirred for 1 hour. Aqueous hydrochloric acid (50mL. 10%) was added, and the mixture was extracted with ethyl acetate (2X 50 mL). The combined organic phases were washed with brine and dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/isooctane, 1: 9 to 1: 1) on silica gel to give the title compound (11.8g, 48%). MS M/z (relative intensity, 70eV)241(M +, 1), 193(33), 191(bp), 141(11), 127 (68).

Preparation 2

Tert-butyl 3- (3, 4-difluorophenyl) -3-hydroxyazetidine-1-carboxylate

N-hexyllithium (2.3M in hexane, 3.0ml, 7.01mmol) was added dropwise to a solution of 4-bromo-1, 2-difluorobenzene (1.35g, 7.01mmol) in dry ether (50ml) at-78 ℃ under nitrogen. The mixture was stirred for 10min, after which a solution of 1-tert-butyloxycarbonyl (Boc) -azetidinone (azetidone) (1.0g, 5.85mmol) in dry ether (10mL) was added dropwise. The resulting mixture was stirred at-78 ℃ for 10min, then returned to ambient temperature and stirred for 10 min. Adding saturatedAqueous ammonium chloride (50mL) and the mixture extracted with tert-butyl methyl ether (2X 50 mL). The combined organic phases were dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/isooctane 1: 1) on silica gel to give the title compound (1.06 g). MS M/z (relative intensity, 70eV)285(M +, 1), 156(60), 141(43), 127(80), 57 (bp).

Preparation 3

3- (3, 4-difluorophenyl) azetidin-3-ol

A mixture of tert-butyl 3- (3, 4-difluorophenyl) -3-hydroxyazetidine-1-carboxylate (1.06g, 3.72mmol) and trifluoroacetic acid (3ml) in dichloromethane (30ml) was stirred at ambient temperature for 1.5 h. The solvent was evaporated and the crude product was purified on a Biotage Isolute SCX-3SPE cartridge (washed with methanol, eluted with methanol/triethylamine, 4: 1) to give the title compound (0.52 g). MS M/z (relative intensity, 70eV)185(M +, 1), 156(50), 141(bp), 127(58), 114 (90).

Preparation 4

1, 3-dichloro-2- (3, 5-difluorophenyl) propan-2-ol

A mixture of 1-bromo-3, 5-difluorobenzene (5.0g, 25.9mmol), magnesium turnings (0.82g, 31.1mmol) and small pieces of iodine in anhydrous tetrahydrofuran (50mL) was stirred at ambient temperature under nitrogen for 1 hour, after which a solution of 1, 3-dichloroacetone (3.46g, 25.9mmol) in anhydrous tetrahydrofuran (20mL) was added dropwise. The resulting mixture was stirred at ambient temperature for 1 hour. Aqueous hydrochloric acid (50ml, 5%) was added and the phases separated and the aqueous phase extracted with ethyl acetate (2X 50 ml). The combined organic phases were washed with brine and dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/isooctane, 1: 9 to 1: 1) on silica gel to give the title compound (2.78g, 45%). MS M/z (relative intensity, 70eV)241(M +, 1), 193(32), 191(bp), 127(68), 77 (39).

Preparation 5

Tert-butyl 3- (3-chloro-5-fluorophenyl) -3-hydroxyazetidine-1-carboxylate

A mixture of 1-bromo-3-chloro-5-fluorobenzene (1.47g, 7.01mmol), magnesium turnings (0.82g, 31.1mmol) and small pieces of iodine in anhydrous tetrahydrofuran (50mL) was gently heated under nitrogen until the reaction started, stirred at ambient temperature for 1 hour, after which a solution of 1-tert-butoxycarbonyl-3-azetidinone (1.0g, 5.85mmol) in anhydrous ether (10mL) was added dropwise. The resulting mixture was stirred for 15min, water (70mL) and saturated aqueous ammonium chloride (20mL) were added and the mixture was extracted with ethyl acetate (2X 50 mL). The combined organic phases were dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/isooctane 1: 4 to 1: 1) on silica gel to give the title compound (1.25 g). MS M/z (relative intensity, 70eV)285(M +, 1), 156(60), 141(43), 127(80), 57 (bp).

Preparation 6

3- (3-chloro-5-fluorophenyl) azetidin-3-ol

A mixture of tert-butyl 3- (3-chloro-5-fluorophenyl) -3-hydroxyazetidine-1-carboxylate (1.25g, 4.14mmol) and trifluoroacetic acid (3ml) in dichloromethane (50ml) was stirred at ambient temperature for 1 hour. The solvent was evaporated and the crude product was purified on a Biotage Isolute SCX-3SPE cartridge (washed with methanol and eluted with methanol/triethylamine, 4: 1) to give the title compound (0.74 g). MS M/z (relative intensity, 70eV)201(M +, 1), 172(47), 157(53), 130(76), 109 (bp).

Preparation 7

1, 3-dichloro-2- (3-chloro-2-fluorophenyl) propan-2-ol

N-hexyllithium (2.5M in hexane, 3.2mL, 8.0mmol) was added dropwise to a solution of 1-bromo-3-chloro-2-fluorobenzene (1.4g, 6, 7mmol) in dry ether (25mL) at-78 ℃ under nitrogen. The mixture was stirred for 10min, after which a solution of 1, 3-dichloroacetone (1.1g, 7.3mmol) in dry diethyl ether (10ml) was added dropwise. The resulting mixture was stirred at-78 ℃ for 0.5 hour,then returned to ambient temperature and stirred for 2 hours. Saturated aqueous ammonium chloride (25ml) was added and the mixture was extracted with ethyl acetate (3X 25 ml). The combined organic phases were dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/isooctane, 1: 9 to 1: 1) on silica gel to give the title compound (0.88g, 51%). MSm/z (relative intensity, 70eV)257(M +, 1), 209(64), 207(bp), 143(44), 77 (13).

Preparation 8

Tert-butyl 3- (2, 3-difluorophenyl) -3-hydroxyazetidine-1-carboxylate

N-butyllithium (2.5M in hexane, 4.7ml, 11.8mmol) was added dropwise to a solution of 3-bromo-1, 2-difluorobenzene (2.3g, 11.8mmol) in dry ether (20ml) at-78 ℃ under nitrogen. The mixture was stirred for 1 hour, after which a solution of 1-tert-butoxycarbonyl-3-azetidinone (2.0g, 11.7mmol) in dry diethyl ether (20mL) was added dropwise. The resulting mixture was stirred at-78C for 15min, then returned to ambient temperature and stirred for 2 hours. Aqueous ammonium chloride (50mL, 50%) and diethyl ether were added, the organic phase was collected, and the aqueous phase was extracted with ethyl acetate (2X 50 mL). The combined organic phases were dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography (ethyl acetate/isooctane 1: 4 to 1: 1) on silica gel to give the title compound (1.9 g). MS M/z (relative intensity, 70eV)285(M +, 1), 156(55), 141(69), 127(bp), 57 (88).

Preparation 9

3- (2, 3-difluorophenyl) azetidin-3-ol

Trifluoroacetic acid (2ml) was added to a solution of tert-butyl 3- (2, 3-difluorophenyl) -3-hydroxyazetidine-1-carboxylate (0.80g, 2.80mmol) in dichloromethane (30 ml). The reaction mixture was stirred at ambient temperature for 1 hour. The solvent was evaporated and the crude product was purified on a Biotage Isolute SCX-3SPE cartridge (washed with methanol and eluted with methanol/triethylamine 4: 1) to give the title compound (0.52 g). MS M/z (relative intensity, 70eV)185(M +, 1), 141(68), 127(bp), 114(82), 63 (40).

Preparation 10

Tert-butyl 3- (2, 3-difluorophenyl) -3-methoxyazetidine-1-carboxylic acid ester

Sodium hydride (60% dispersion in mineral oil, 0.31g, 7.7mmol) was added to a solution of tert-butyl 3- (2, 3-difluorophenyl) -3-hydroxyazetidine-1-carboxylate (1.1g, 3.86mmol) in N, N-dimethylformamide (15 mL). The mixture was stirred for 20 min. Methyl iodide (0.29mL, 4.63mmol) was added. The mixture was stirred for 30min, aqueous lithium chloride (50ml, 5%) was added and the aqueous phase was extracted with ethyl acetate (2X 50 ml). The combined organic phases were dried (Na)₂SO₄) Filtration and evaporation gave the title compound (1.55 g). MS M/z (relative intensity, 70eV)299(M +, 1), 223(21), 169(62), 140(49), 57 (bp).

Preparation 11

1, 3-dichloro-2- (3, 4-difluorophenyl) propan-2-ol

Preparation according to preparation 1: 1-bromo-3, 4-difluorobenzene (5g, 25.9mmol), dehydrated ether (100mL), n-hexyllithium (2.3M in hexanes, 11.2mL, 25.9mmol) and 1, 3-dichloroacetone (3.29g, 25.9 mol). Yield: 4.54g (73%). MS M/z (relative intensity, 70eV)241(M +, 1), 193(32), 191(bp), 141(14), 127 (64).

Preparation 12

1, 3-dichloro-2- (3-chloro-5-fluorophenyl) propan-2-ol

Preparation according to preparation 1: 1-bromo-3-chloro-5-fluorobenzene (5g, 23.9mmol), dry ether (100mL), n-hexyllithium in hexane (2.3M, 10.4mL, 23.9mmol), and 1, 3-dichloroacetone (3.03g, 23.9 mol). Yield: 4.49g (73%). MS M/z (relative intensity, 70eV)257(M +, 1), 209(65), 207(bp), 211(11), 143 (28).

Preparation 13

Tert-butyl 3- (2, 3-difluorophenyl) -3-methoxyazetidine-1-carboxylic acid ester

Tert-butyl 3- (2, 3-difluorophenyl) -3-hydroxyazetidine-1-carboxylate (0.7g, 2.45mmoL) was dissolved in N, N-dimethylformamide (20mL) and sodium hydride (60% dispersion in mineral oil, 0.2g, 4.90mmoL L) was added at 0 ℃. The mixture was stirred at 0 ℃ for 15min, at ambient temperature for 20min, after which methyl iodide (0.41mL, 2.94mmol) was added. The mixture was stirred at ambient temperature for 1 hour, a mixture of aqueous lithium chloride (5%, aq, 20mL) and ethyl acetate (20mL) was added and the organic phase collected. The aqueous phase was extracted with ethyl acetate (2X 20mL) and the combined organic phases were dried (Na)₂SO₄) Filtration and evaporation. Purification by flash column chromatography (ethyl acetate/isooctane 2: 8 to 1: 1) on silica gel gave the title compound (0.7g, 95%). MS M/z (relative intensity, 70eV)299(M +, 1), 223(60), 198(35), 169(bp), 170 (bp).

Example 11

Biological activity

The compounds according to the invention were evaluated using the following tests.

In vivo testing: behavior

Behavioral activity was measured using 8 Digiscan activity monitors (RXYZM (16) TAO, Omnitech Electronics, Columbus, OH, USA) connected to an Omnitech Digiscan analyzer and an Apple Macintosh computer equipped with a data interface plug-in (NB DIO-24, National Instruments, USA). Each activity monitor contained a square metal frame (W × L ═ 40cm × 40cm) fitted with a light sensor. During the course of the behavioral activity measurements, rats were placed in clear acrylic cages (W.times.LXH, 40.times.40.times.30 cm) which were placed in turn in the activity monitor. Each activity monitor was equipped with 3 rows of infrared light sensors, each row including 16 sensors. Row 2 was placed at a 90 ° angle along the front and sides of the cage floor, and row 3 was placed 10cm above the floor to measure vertical activity. The light sensors were placed at 2.5cm intervals. Each activity monitor was fitted with a box containing weak room light and a fan with the same sound and light attenuation.

Computer software using object oriented programming (National instruments, Austin, TX, USA).

Behavioral data for each activity monitor, representing the position of the animal at each time (horizontal center of gravity and vertical activity), was recorded at a sampling frequency of 25Hz and used with the classic (custom write) LABView^TMAnd collecting the application programs. The data from each recording session is stored and analyzed with respect to distance traveled. Each activity recording period lasted 60min, starting at about 4min after injection of the assay compound. Similar behavioral recording methods were used for rats that were not treated with drug and pre-treated with drug. Rats pre-treated with d-amphetamine were given a dose of 1.5mg/kg i.p. 10min before the recording period in the activity monitor. Rats pre-treated with MK-801 were given a dose of 0.7mg/kg i.p. 90min before the recording period in the activity monitor. Results are expressed in arbitrary length units in the form of counts/60 minutes or counts/30 minutes. Statistical comparisons were performed using student t-tests against the control group. In MK-801 or amphetamine-pretreated animals, statistical comparisons were made against MK801 or d-amphetamine controls, respectively.

Calculating ED for reducing amphetamine-induced hyperlocomotion by curve fitting₅₀The value is obtained. For most compounds, in a single experiment (one single experiment), evaluations were based on 16 amphetamine-pretreated animals at doses ranging from 0, 11, 33 and 100 μmol/kg s.c, while in separate experiments supplementary doses were employed. The calculation was based on the distance during the last 45 minutes of the 1 hour measurement. Distance was normalized by amphetamine-control and fit to the synthetic function "End (End) - (End-control)/(1 + (dose/ED) minimized by least squares₅₀)^{Oblique Rate of change})". 4 parameters (control, end, ED)₅₀And slope) were fitted using the following constraints: ED (electronic device)₅₀Greater than 0, 0.5 < slope < 3, endEnd-0% of control. The restriction of the locking tip serves to focus on potential rather than efficacy. To estimate the confidence level of the parameter, the fitting was repeated 100 times with randomly evenly distributed squared weights (0 to 1) for each measurement. Presented ED₅₀The range covers 95% of these values.

In vivo assay: neurochemistry

After the period of behavioral activity, the rats were decapitated and their brains were quickly removed and placed on ice-pre-chilled petri dishes. The limbic forebrain, striatum, frontal cortex and remaining hemispheric portion of each rat were dissected and frozen. Each brain part was then analyzed for monoamine content and their metabolites.

By HPLC separation and electrochemical detection, monoamine transmitter substances (NA (norepinephrine), DA (dopamine), 5-HT (5-hydroxytryptamine)) and their amine (NM (isoproterenol), 3-MT (3-methoxytyramine)) and acid (DOPAC (3, 4-dihydroxyphenylacetic acid), 5-HIAA (5-hydroxyindoleacetic acid), HVA (homovanillic acid)) metabolites were quantified in brain homogenates.

The analytical method is based on 2 chromatographic separations specific for amines or acids. The 2 chromatographic systems share a common autosampler with 10-way valves and 2 sample loops for simultaneous injection in the 2 systems. The 2 Systems were equipped with reversed-phase columns (Luna C18(2), dp 3 μm, 50X 2mm i.d., Phenomenex) and electrochemical measurements were carried out at 2 potentials on glassy carbon anodes (MF-1000, Bioanalytical Systems, inc.). The column effluent enters the detection cell or waste discharge port through a T-connection. This is done by 2 solenoid valves which block the waste or detector outlet. By preventing the chromatographic front from reaching the detector, a better detection state is achieved. The aqueous mobile phase of the acid system (0.4mi/min) contained 14mM citric acid, 10mM sodium citrate, 15% (v/v) MeOH, and 0.1mM EDTA. The detection potentials relative to the Ag/AgCl reference were 0.45 and 0.60V. The aqueous ion-pair mobile phase of the amine system (0.5ml/min) contained 5mM citric acid, 10mM sodium citrate, 9% (v/v) MeOH, 10.5% v/v MeCN, 0.45mM decanesulfonic acid and 0.1mM EDTA. The detection potentials relative to the Ag/AgCl reference were 0.45 and 0.65V.

Calculation of increased ED of DOPAC in striatum by Curve fitting₅₀The value is obtained. For most compounds, in a single experiment, evaluations were based on 20 animals, covering dose ranges of 0, 3.7, 11, 33 and 100 μmol/kg s.c., with supplementary doses in separate experiments. DOPAC levels were normalized to controls and fitted to the function "terminal- (terminal-control)/(1 + (dose/ED) by least squares minimization₅₀)^{Slope of})". 4 parameters (control, end, ED)₅₀And slope) were fitted using the following constraints: ED (electronic device)₅₀> 0, 0.5 < slope < 3, 350 < end < 400% of control. To estimate the confidence level of the parameter, the fitting was repeated 100 times with randomly evenly distributed squared weights (0 to 1) for each measurement. Presented ED₅₀The range covers 95% of these values.

In vivo assay: oral bioavailability

Experiments were performed 24 hours after arterial and venous catheter implantation. Test compounds were administered orally at 12.5 μmol/kg or intravenously using an intravenous catheter at 5 μmol/kg, with n-3 per group. Arterial blood samples were then taken at 0, 3, 9, 27, 60, 120, 180, 240, 300 and 360 minutes within 6 hours after administration of the test compound. For each rat, oral bioavailability was calculated as the ratio of the AUC (area under the curve) obtained after oral administration to the AUC obtained after intravenous administration. The parameter AUC was calculated as follows:

AUC: the area under the plasma concentration versus time curve from time zero to the final measured concentration (Clast) was calculated by the log/linear gradient method.

The levels of test compounds were measured by liquid chromatography-mass spectrometry (LC-MS) (Hewlett-Packard 1100MSD Series). The LC-MS module includes a quaternary pump system, a vacuum degasser, a thermostatted autosampler, a thermostatted column chamber, a diode array detector, and an API-ES spray chamber. Data processing was performed using the HP ChemStation rev.a.06.03 system. The instrument is set up: MSD mode: selective Ion Monitoring (SIM) MSD polarity: positive gas temperature: 350 ℃ dry gas: 13, 0l/min of spray gas: 50psig capillary voltage: 5000V fragmenter voltage: 70V

And (3) analyzing the column: zorbax eclipse XDB-C8(4.6 ANG. 150mm, 5 μm) at 20 ℃. The mobile phases were acetic acid (0.03%) (solvent a) and acetonitrile (solvent B). The flow rate of the mobile phase was 0.8 ml/min. Isocratic elution was started with 12% solvent B for 4, 5min, then increased linearly to 60% over 4, 5 min.

The extraction method comprises the following steps: plasma samples (0.25-0.5ml) were diluted to 1ml with water and 60pmol (100. mu.l) of internal standard (-) -OSU6241 was added. By adding 25. mu.l of saturated Na₂CO₃The pH was adjusted to 11. After mixing, the sample was extracted with 4mi dichloromethane by shaking for 20 min. The organic layer was centrifuged and transferred to a smaller tube and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 120. mu.l of mobile phase (acetic acid (0.03%): acetonitrile, 95: 5) for LC-MS analysis (injection of 10. mu.l). Selective ion (MH) was monitored for each example⁺) And MH⁺296 corresponds to (-) -OSU6241((3- [3- (ethylsulfonyl) phenyl)]-1-propylpiperidine).

Standard curves in the range of 1-500pmol were prepared by adding the appropriate amount of test compound to a blank plasma sample.

In vitro assay: metabolic stability in rat liver microsomes

According toL1980 slightly modified to isolate rat liver microsomes, e.g., 0.1MNa/K O PO containing 0.15M KCl, pH 7.4, 3mL/g liver was added prior to homogenization₄Buffer (buffer 1), the homogenate was centrifuged for 20 minutes instead of 15 minutes, the supernatant was ultracentrifuged at 100.000g instead of 105.000g, and the pellet produced by ultracentrifugation was suspended in 20% v/v 87% glycerol in buffer 1 at 1mL/g liver.

mu.L of 0.2 or 1mM test substance was diluted in water, 10. mu.L of 20mg/mL rat liver microsomes was mixed with 149. mu.L of buffer 1 at 37 ℃, and the reaction was started by adding 40. mu.L of 4.1mg/mL NADPH. After incubation in a heater (LAB-LINE, MULTI-BLOK heater or LAB4you, TS-100Thermo shaker at 700 rpm) at 37 ℃ for 0 or 15 minutes, the reaction was stopped by adding 100. mu.L of pure acetonitrile. The protein pellet was removed by centrifugation at 10.000g for 10min (Heraeus, Biofuge fresco) at 4 ℃ followed by removal of the pellet. The compounds to be tested were analysed using HPLC-MS (Hewlett-Packard 1100MSD Series) using either a Zorbax SB-C18 column (2.1 mm, 5 μm) with 0.03% formic acid and acetonitrile as mobile phase (gradient) or Zorbax Eclipse XDB-C18(3 mm, 75mm, 3.5 μm) with 0.03% acetic acid and acetonitrile as mobile phase (gradient). The 15min turnover was calculated as the percentage of test compound eliminated after 15 minutes, expressed as a percentage of the 0min level, i.e. 100 [ test compound concentration at 0 min-concentration at 15min ]/concentration at 0 min.

According to1980 the preparation of liver microsomes was performed. Protocols for incubation with liver microsomes are mentioned in Crespi and Stresser, 2000 and Renwick et al, 2001. Microdialysis

Male Sprague-Dawley rats weighing 220-320g were used in the experiments. Prior to the trial, animals were housed in groups of 5 animals per cage, with free access to water and food. After arrival, animals were housed for at least one week prior to surgery and use in trials. Each rat was used only once for microdialysis.

We used a modified version of the type I probe (Santiago and Westerink, 1990) (Waters et al, 1994). The dialysis membrane we used was AN69 polyacrylonitrile/sodium methallylsulfonate copolymer (HOSPAL; o.d./i.d.310/220 μm: Gambro, Lund, Sweden). In the dorsal striatum we used a probe with a dialysis membrane exposed to a length of 3mm, the corresponding length in the prefrontal cortex being 2.5 mm. The rats were operated under isoflurane inhalation anesthesia with the rats being loaded into a Kopf stereotaxic apparatus. Calculating the coordinates relative to the pro-halogen point according to (Paxinos & Watson, 1986); back striatum AP +1, ML + -2.6, DV-6.3; pf cortex, AP +3.2, 8 ℃ ML. + -. 1.2, DV-4.0. The dialysis probe was placed in a bore hole under stereotactic guidance and bonded with phosphatine dental cement.

Rats were housed individually in cages 48 hours prior to the dialysis trial to recover them from surgery and minimize the risk of interaction between the drug and the anesthetic in the next trial. During this period the rats had free access to food and water. On the day of the experiment, rats were connected to the micro-perfusion pump by a swivel (swiwel) and placed in cages where they were free to move within limits. According to (Moghaddam & Bunney, 1989), the perfusion medium is a solution containing mmol/l: NaCl; 140, CaCl 2; 1.2, KCl; 3.0, MgCl 2; 1.0 and ascorbic acid; 0.04 of ringer solution. The pump was set to a perfusion rate of 2. mu.l/min and 40. mu.l samples were collected every 20 min. Each sample was analyzed on 2 HPLC systems. On an autosampler (CMA 200) with a ten-way valve (Valco C10WE), 2 sample loops (4 μ Ι and 20 μ Ι) were connected in series, and each brain dialysis sample was loaded into both loops simultaneously. For the determination of Dopamine (DA), Noradrenaline (NA), isoproterenol (NM), 3-methoxytyramine (3-MT) and 5-hydroxytryptamine (5-hydroxytryptamine, 5-HT), 20 μ l of the sample was introduced into the column switching system (reversed phase combined with reversed phase ion pair) at the time of injection, while for the chromatography of the acidic monoamine metabolites 3, 4-di-hydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), 4 μ l of the sample was introduced into the reversed phase column. The currents generated by the 2 EC detectors were converted to digital data and evaluated on a PC using Chromeleon software (Dionex). Method sample turnaround time was 4.5min, 2 parallel samples were typically analyzed simultaneously on the system.

After the experiment, the rats were disconnected from the perfusion pump and decapitated. Their brains were removed quickly and placed in fresh solution (Kebo-lab, Sweden) for subsequent examination of probe localization. In Goldeburg, Sweden (Sweden) approved the methods used in these experiments.

The results of these tests are listed in table 1 below.

TABLE 1

Maximum effect compared to baseline value at 50. mu. mol/kg s.c. (percentage of control. + -. SEM)

Microdialysis was performed in conscious and freely moving rats.

Dopamine ═ DA; noradrenaline (NA); 5-hydroxytryptamine ═ 5-HT; striae; PFC ═ prefrontal cortex.

Reference to the literature

Crespi CL and DM Stresser: fluorimetric screening for metabolic based drug-drug interactions.J.pharm.Tox.meth.200044325-331;

L：Effects of Clophen A50，3-methylcholantrene，pregnenolone-16aq-carbonitrile and Phenobarbital on the hepafic microsomal cytochrome P-450-dependent monooxygenaser system in rainbow trout，salmo gairdneri，of different age and sex.Tox.Appl.Pharm.198054(3)420-430；

renwick AB et al: metabolim of 2, 5-bis (trifluoromethyloxy) -7-benzoyloxy-4-trifluoromethylcarbonyl by human pharmaceutical CYP: evidence for selection towards CYP3A4.xenobiotica 200131(4) 187-204;

Moghaddam B & Bunney BS：1onic Composition of Microdialysis Perfusing Solution Alters the Pharmacological Responsiveness and Basal Outflow of Striatal Dopamine.J.Neurochem.198953652-654；

Paxinos G & Watson C：The Rat Brain in Stereotaxic Coordinates.New York，Academic Press，1986。

Santiago M & Westerink BHC：Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes.Naunyn-Schmiedeberg′s Arch.Pharmacol.1990342 407-414；

waters N, Lofberg L, Haadsna-Svensson S, Svensson K, Sonesson C and Carlsson A: differential effects of dopamine D2 and D3 receptors in ligand to dopamine release, in vivo receptor displacement and behaviour.J.neural.Transm.Gen.Sect.199498 (1): 39-55.

Claims (13)

1. A 3-phenyl-azetidine derivative of formula 1:

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein

X is OH or OCH₃；

R¹Is F or Cl;

R²h, F or Cl;

R³is H, CH₃Or CH₂CH₃；

With the proviso that if X is OH, then R³Is not H.

2. The 3-phenyl-azetidine derivative according to claim 1, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein X is OH or OCH₃。

3. The 3-phenyl-azetidine derivative according to any one of claims 1-2, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein R is¹Is F or Cl.

4. The 3-phenyl-azetidine derivative according to any one of claims 1-3, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein R is²Is H, F or Cl.

5. The 3-phenyl-azetidine derivative according to any one of claims 1-4, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, wherein R is³Is H, CH₃Or CH₂CH₃Or a deuterated analog thereof.

6. 3-phenyl-azetidine derivatives according to claim 1, which are

3- (2, 3-difluorophenyl) -1-ethyl azetidin-3-ol;

3- (3, 4-difluorophenyl) -1-ethyl azetidin-3-ol;

3- (3, 5-difluorophenyl) -1-ethyl azetidin-3-ol;

3- (3-chloro-5-fluorophenyl) -1-ethyl azetidin-3-ol;

3- (3, 4-difluorophenyl) -1- (ethyl-D5) azetidin-3-ol;

3- (3-chloro-2-fluorophenyl) -1-ethyl azetidin-3-ol;

3- (2, 3-difluorophenyl) -1-ethyl azetidin-3-ol-1-oxide;

3- (2, 3-difluorophenyl) -3-methoxyazetidine;

3- (2, 3-difluorophenyl) -1-ethyl-3-methoxyazetidine; or

3- (2, 3-difluorophenyl) -1- (ethyl-D5) azetidin-3-ol;

any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition comprising a therapeutically effective amount of a 3-phenyl-azetidine derivative of any one of claims 1-6, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient or diluent.

8. Use of a 3-phenyl-azetidine derivative according to any one of claims 1-6, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament.

9. Use according to claim 8 for the preparation of a pharmaceutical composition for the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is responsive to modulation of catecholamines in the cerebral cortex.

10. The use according to claim 9, wherein the disease, disorder or condition is dementia, age-related cognitive impairment, childhood developmental disorder, ADHD, cerebral palsy, huntington's disease, Gilles de la Tourette's syndrome, depression, bipolar disorder, schizophrenia, schizophreniform disorder, Generalized Anxiety Disorder (GAD), specific phobias, panic disorder, sleep disorder, bipolar disorder, drug-induced psychotic disorder, iatrogenic psychosis, iatrogenic hallucinations, non-iatrogenic psychosis, non-iatrogenic hallucinations, mood disorder, anxiety disorder, depression, obsessive-compulsive disorder, mood disorder related to aging, alzheimer's disease, dementia disorder related to alzheimer's disease, age-related cognitive impairment, brain injury, substance abuse, disorder characterized by misuse of food, sleep disorder, Sexual dysfunction, eating disorder, obesity, headache, pain in a condition characterized by increased muscle tone, movement disorder, parkinson's disease, parkinsonism syndrome, dyskinesia, levodopa-induced dyskinesia, dystonia, neurodevelopmental disorder, neurodegenerative disorder, convulsion, tremor, restless legs, narcolepsy, or behavioral disorder.

11. A method for the treatment, prevention or alleviation of a disease or a disorder or a condition of a living animal body, including a human, which disease, disorder or condition is responsive to modulation of catecholamines in the cerebral cortex, which method comprises the steps of: administering to such a living animal in need thereof a therapeutically effective amount of a 3-phenyl-azetidine derivative according to any one of claims 1-6, or any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof.

12. A 3-phenyl-azetidine derivative according to any one of claims 1-6, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament.

13. A 3-phenyl-azetidine derivative according to any one of claims 1-6, any of its stereoisomers or any mixture of its stereoisomers, or an N-oxide thereof, or a deuterated analog thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is responsive to modulation of catecholamines in the cerebral cortex.