Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
  • C07D471/14—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
• • A—HUMAN NECESSITIES
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  • A61K9/00—Medicinal preparations characterised by special physical form
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  • A61K9/2013—Organic compounds, e.g. phospholipids, fats
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  • A61K9/2022—Organic macromolecular compounds
  • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
  • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/4841—Filling excipients; Inactive ingredients
  • A61K9/4858—Organic compounds
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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  • A61K9/4866—Organic macromolecular compounds
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P25/08—Antiepileptics; Anticonvulsants
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/22—Anxiolytics
• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention relates to organic compounds useful for therapy or prophylaxis in a mammal, and in particular to new pyrido diazepine derivatives that exhibit activity as GABAA ⁇ l receptor positive allosteric modulators (PAMs) and are thus useful for the treatment or prophylaxis of GABAA l receptor related diseases or conditions.
• PAMs GABAA ⁇ l receptor positive allosteric modulators
• GABA gamma-aminobutyric acid
• GABA A receptors which are members of the ligand- gated ion channel superfamily
• GABA B receptors which are members of the G- protein linked receptor family.
• the GABA A receptor complex which is a membrane-bound heteropentameric protein polymer is composed principally of ⁇ , ⁇ and ⁇ subunits.
• GABA A receptors are ligand-gated chloride channels and the principal mediators of inhibitory neurotransmission in the human brain.
• GABA A receptor subunits There are 19 genes encoding for GABA A receptor subunits that assemble as pentamers with the most common stoichiometry being two ⁇ , two ⁇ and one ⁇ subunit. GABA A subunit combinations give rise to functional, circuit, and behavioral specificity. GABA A receptors containing the ⁇ l subunit (GABA A ⁇ l) are of particular interest due to their enriched expression in the limbic system and unique physiological and pharmacological properties.
• the GABA A ⁇ l subunit-containing receptors while less abundant (around 5-10 % of total expression of GABA A receptors in the brain) than ⁇ 2 subunit-containing receptors exhibit an enriched brain mRNA and protein distribution in key brain areas such as extended amygdala (central, medial, and bed nucleus of the stria terminalis), lateral septum, hypothalamus, and pallidum/nigra. These structures form the interconnected core of a subcortical limbic circuit regulating motivated social and affective behaviors. In abnormal or disease conditions, hyper-recruitment of this circuit promotes anxiety, arousal, aggression, fear and defense while inhibiting foraging and social interactions.
• a GABA A ⁇ l positive allosteric modulator may be an effective treatment as a symptomatic or disease- modifying agent.
• a CNS disorders of particular interest in the context of the present invention is autism spectrum disorder (ASD), including its core symptoms and associated comorbidities, such as anxiety and irritability, social anxiety disorder (social phobia) and generalized anxiety disorder.
• ASD is a complex, heterogeneous neurodevelopmental disorder characterized by impairments in two core domains: impairments in social interaction and communication, and presence of repetitive or restricted behaviors, interests, or activities (American Psychiatric Association 2013).
• GABRG1 positron emission tomography study
• MRS magnetic resonance spectroscopy
• Mutations in 4p12 gene cluster have been linked to increased risk of anxiety, substance abuse and eating disorders - providing a link between GABRGl/4p12 and affective dysfunction.
• MRS studies found altered GABA levels in ASD and in particular some recent studies showed reduced GABA and altered somatosensory function in children with ASD.
• a reduced number of inhibitory interneurons were found from postmortem tissues of ASD and TS patients.
• reduced GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67 were found in parietal and cerebellar cortices of patients with autism.
• Compounds of the present invention are selective GABA A ⁇ l receptor positive allosteric modulators (PAMs) that selectively enhance the function of ⁇ l -containing GABA A receptors by increasing GABAergic currents (influx of chloride) at a given concentration (e.g. EC20) of gamma amino butyric acid (GABA).
• PAMs GABA A ⁇ l receptor positive allosteric modulators
• the compounds of the present invention have high PAM efficacy and binding selectivity for the ⁇ l -containing subtypes ( ⁇ 5 ⁇ l, ⁇ 2 ⁇ l, ⁇ l ⁇ l) relative to the ⁇ 2-containing subtypes (e.g. ⁇ l ⁇ 2, ⁇ 2 ⁇ 2, ⁇ 3 ⁇ 2 and ⁇ 5 ⁇ 2).
• compounds of the present invention are strongly differentiated from classical benzodiazepine drugs such as Alprazolam, Triazolam, Estazolam, and Midazolam, which are selective for the ⁇ 2-containing GABA A subtypes and possess low affinity for the ⁇ l- containing subtypes.
• selective GABA A ⁇ l PAMs will restore GABAergic signaling in key brain regions (e.g. extended amygdala: central, medial, and bed nucleus of the stria terminalis, lateral septum, hypothalamus, and pallidum/nigra) without the side-effects of non-selective GABA A modulators (e.g. benzodiazepines).
• the selective GABA A ⁇ l PAMs described herein and their pharmaceutically acceptable salts and esters are useful, alone or in combination with other drugs, as disease-modifying or as symptomatic agents for the treatment or prevention of acute neurological disorders, chronic neurological disorders and/or cognitive disorders, including autism spectrum disorders (ASD), Angelman syndrome, age-related cognitive decline, Rett syndrome, Prader-Willi syndrome, amyotrophic lateral sclerosis (ALS), fragile-X disorder, negative and/or cognitive symptoms associated with schizophrenia, tardive dyskinesia, anxiety, social anxiety disorder (social phobia), panic disorder, agoraphobia, generalized anxiety disorder, disruptive, impulse-control and conduct disorders, Tourette’s syndrome (TS), obsessive-compulsive disorder (OCD), acute stress disorder, post-traumatic stress disorder (PTSD), attention deficit hyperactivity disorder (ADHD), sleep disorders, Parkinson’s disease (PD), Huntington’s chorea, Alzheimer’s disease (AD), mild cognitive impairment (MCI), dementia, behavioral and psychological symptoms (BPS) in
• the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the variables are as defined herein.
• the present invention provides a process of manufacturing the compounds of formula (I) described herein, wherein said process is as described in any one of Schemes 1 to 11 herein.
• the present invention provides a compound of formula (I) as described herein, when manufactured according to the processes described herein. In a further aspect, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, for use as therapeutically active substance.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, and a therapeutically inert carrier.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, for use in a method for treating or preventing acute neurological disorders, chronic neurological disorders and/or cognitive disorders in a subject.
• alkyl refers to a mono- or multivalent, e.g., a mono- or bivalent, linear or branched saturated hydrocarbon group of 1 to 6 carbon atoms (“C 1 -C 6 -alkyl”), e.g., 1, 2, 3, 4, 5, or 6 carbon atoms. In some embodiments, the alkyl group contains 1 to 3 carbon atoms, e.g., 1, 2 or 3 carbon atoms.
• alkyl include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, iso-butyl, sec-butyl, tert-butyl, and 2,2- dimethylpropyl. Particularly preferred, yet non-limiting examples of alkyl include methyl and ethyl.
• alkoxy refers to an alkyl group, as previously defined, attached to the parent molecular moiety via an oxygen atom. Unless otherwise specified, the alkoxy group contains 1 to 6 carbon atoms (“C 1 -C 6 -alkoxy”). In some preferred embodiments, the alkoxy group contains contains 1 to 4 carbon atoms. In still other embodiments, the alkoxy group contains 1 to 3 carbon atoms. Some non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and tert-butoxy. A particularly preferred, yet non-limiting example of alkoxy is methoxy.
• halogen refers to fluoro (F), chloro (Cl), bromo (Br), or iodo (I).
• halogen refers to fluoro (F), chloro (Cl) or bromo (Br).
• Particularly preferred, yet non-limiting examples of “halogen” or “halo” are fluoro (F) and chloro (Cl).
• cycloalkyl as used herein refers to a saturated or partly unsaturated monocyclic or bicyclic hydrocarbon group of 3 to 10 ring carbon atoms (“C 3 -C 10 -cycloalkyl”).
• the cycloalkyl group is a saturated monocyclic hydrocarbon group of 3 to 8 ring carbon atoms.
• “Bicyclic cycloalkyl” refers to cycloalkyl moieties consisting of two saturated carbocycles having two carbon atoms in common, i.e., the bridge separating the two rings is either a single bond or a chain of one or two ring atoms, and to spirocyclic moieties, i.e., the two rings are connected via one common ring atom.
• the cycloalkyl group is a saturated monocyclic hydrocarbon group of 3 to 6 ring carbon atoms, e.g., of 3, 4, 5 or 6 carbon atoms.
• cycloalkyl examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and spiro[2.3]hexan-5-yl.
• cycloalkyl examples include cyclopropyl, cyclobutyl and cyclopentenyl.
• heterocyclyl or “heterocycloalkyl” refers to a saturated or partly unsaturated mono- or bicyclic, preferably monocyclic ring system of 3 to 14 ring atoms, preferably 3 to 10 ring atoms, more preferably 3 to 8 ring atoms wherein 1, 2, or 3 of said ring atoms are heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
• 1, 2, or 3 of said ring atoms are heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
• 1 to 2 of said ring atoms are selected from N and O, the remaining ring atoms being carbon.
• Bicyclic heterocyclyl refers to heterocyclic moieties consisting of two cycles having two ring atoms in common, i.e., the bridge separating the two rings is either a single bond or a chain of one or two ring atoms, and to spirocyclic moieties, i.e., the two rings are connected via one common ring atom.
• heterocyclyl groups include azetidin-3-yl; azetidin-2-yl; oxetan-3-yl; oxetan-2-yl; piperidyl; piperazinyl; pyrrolidinyl; 2-oxopyrrolidin-l-yl; 2-oxopyrrolidin-3-yl; 5- oxopyrrolidin-2-yl; 5-oxopyrrolidin-3-yl; 2-oxo-l -piperidyl; 2-oxo-3 -piperidyl; 2-oxo-4- piperidyl; 6-oxo-2-piperidyl; 6-oxo-3 -piperidyl; 1-piperidinyl; 2-piperidinyl; 3-piperidinyl; 4-piperidinyl; morpholino (e.g., morpholin-2-yl or morpholin-3-yl); thiomorpholino, pyrrolidiny
• hydroxy refers to an -OH group.
• haloalkyl refers to an alkyl group, wherein at least one of the hydrogen atoms of the alkyl group has been replaced by a halogen atom, preferably fluoro.
• haloalkyl refers to an alkyl group wherein 1, 2 or 3 hydrogen atoms of the alkyl group have been replaced by a halogen atom, most preferably fluoro.
• Non-limiting examples of haloalkyl are fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, 2-fluoroethyl, and 2,2-difluoroethyl.
• a particularly preferred, yet non-limiting example of haloalkyl is trifluoromethyl.
• hydroxyalkyl refers to an alkyl group, wherein at least one of the hydrogen atoms of the alkyl group has been replaced by a hydroxy group.
• hydroxyalkyl refers to an alkyl group wherein 1, 2 or 3 hydrogen atoms, most preferably 1 hydrogen atom of the alkyl group have been replaced by a hydroxy group.
• Preferred, yet non-limiting examples of hydroxyalkyl are hydroxymethyl, hydroxyethyl (e.g. 2- hydroxyethyl), hydroxypropyl (e.g., 2-hydroxypropyl), and 3 -hydroxy-3 -methyl-butyl.
• salts refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.
• the salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, in particular hydrochloric acid, and organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, lactic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcy stein and the like.
• salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts and the like.
• Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tnpropylamine, ethanolamine, lysine, arginine, N-ethylpipendine, piperidine, polyimine resins and the like.
• Particular pharmaceutically acceptable salts of compounds of formula (I) are hydrochlorides, fumarates, formates, lactates (in particular derived from L-(+)-lactic acid), tartrates (in particular derived from L-(+)-tartaric acid) and trifluoroacetates.
• the compounds of formula (I) can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereioisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
• the asymmetric carbon atom can be of the "R” or "S” configuration.
• treatment includes: (1) inhibiting the state, disorder or condition (e.g. arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); and/or (2) relieving the condition (i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms).
• the benefit to a patient to be treated is either statistically significant or at least perceptible to the patient or to the physician.
• a medicament is administered to a patient to treat a disease, the outcome may not always be effective treatment.
• prophylaxis or “prevention” as used herein includes: preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject and especially a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition.
• subject includes both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. In a particularly preferred embodiment, the term “subject” refers to humans.
• uM means microMolar and is equivalent to the symbol ⁇ M.
• the abbreviation uL means microliter and is equivalent to the symbol ⁇ L.
• the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: is selected from:
• R 1 is selected from hydrogen, C 1 -C 6 -alkyl, hydroxy-C 1 -C 6 -alkyl-NH-C(O)-, and a group ; and R 1a is hydrogen; or
• R 1 and R 1a taken together with the carbon atoms to which they are attached, form a C 3 -C 10 -cycloalkyl;
• R 1b is selected from hydrogen, halogen, hydroxy, oxo, C 1 -C 6 -alkyl, and C 1 -C 6 - alkoxy;
• R 1c is selected from hydrogen, hydroxy, and oxo;
• R 2 is C 1 -C 6 -alkyl
• R 3 is chloro or bromo
• R 4 is selected from halogen, C 1 -C 6 -alkyl, halo-C 1 -C 6 -alkyl, and C 3 -C 10 -cycloalkyl;
• R 5 is halogen
• L is selected from a covalent bond, carbonyl, -C(O)NH-,-NHC(O)-, - CH 2 NHC(O)-; and A is selected from 3-14-membered heterocycloalkyl and C 3 -C 10 -cycloalkyl.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein In one embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• R 1 is selected from C 1 -C 6 -alkyl, hydroxy-C 1 -C 6 -alkyl-NH-C(O)-, and a group ; and R 1a is hydrogen; or
• R 1 and R 1a taken together with the carbon atoms to which they are attached, form a C 3 -C 10 -cycloalkyl; and R 1b , R 1c , A, and L are as defined herein.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• R 1 is selected from C 1 -C 6 -alkyl, hydroxy-C 1 -C 6 -alkyl-NH-C(O)-, and a group ;
• R 1a is hydrogen; and
• R 1b , R 1c , A, and L are as defined in claim 1.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• R 1 and R 1a taken together with the carbon atoms to which they are attached, form a C 3 -C 10 -cycloalkyl; and R 1b , R 1c , A, and L are as defined in claim 1.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• R 1 is selected from C 1 -C 6 -alkyl, hydroxy-C 1 -C 6 -alkyl-NH-C(O)-, and a group R 1b is C 1 -C 6 -alkyl; R 1c is hydroxy;
• L is carbonyl
• A is a 3-14-membered heterocycle.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein
• R 1 is selected from 2-hydroxyethyl-NH-C(O)-, 2-hydroxypropyl-NH-C(O)-, methyl, and a group ;
• R 1b is methyl;
• R 1c is hydroxy;
• L is carbonyl
• A is a azetidinyl.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 1 is hydroxy-Ci- C 6 -alkyl-NH-C(O)-.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 1 is 2- hydroxyethyl-NH-C(O)-.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 2 is methyl.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 3 is chloro.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 4 is haloalkyl.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 4 is CF 3 .
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 5 is halogen.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 5 is fluoro or chloro. In a preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 5 is fluoro.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein R 5 is chloro.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein:
• R 1 is selected from C 1 -C 6 -alkyl, hydroxy-C 1 -C 6 -alkyl-NH-C(O)-, and a group R 1b is C 1 -C 6 -alkyl; R 1c is hydroxy;
• R 2 is C 1 -C 6 -alkyl
• R 3 is chloro
• R 4 is halo-C 1 -C 6 -alkyl
• R 5 is halogen
• L is carbonyl
• A is a 3-14-membered heterocycle.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein:
• R 1 is selected from methyl, 2-hydroxyethyl-NH-C(O)-, 2-hydroxypropyl-NH-
• R 1b is methyl
• R 1c is hydroxy
• R 2 is methyl
• R 3 is chloro
• R 4 is CF 3 ;
• R 5 is fluoro; is carbonyl; and A is azetidinyl.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is selected from:
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is selected from:
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is (7S)-11-chloro-9-(2,6-difluorophenyl)-N-(2-hydroxyethyl)-7- methyl- 12-(trifl uoromethy 1 )-2, 3 , 5,8,13 -pentazatricyclo [8.4.0.02,6]tetradeca- 1(10), 3, 5, 8, 11, 13 -hexaene-4-carboxamide.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is (7S)-11-chloro-9-(2,6-difluorophenyl)-N-(2-hydroxyethyl)-7- methyl- 12-(trmuoromethyl)-2, 5 ,8,13 -tetrazatncyclo [8.4.0.02, 6]tetradeca- 1(10), 3, 5, 8, 11, 13 -hexaene-4-carboxamide.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is (7S)-11-chloro-9-(2,6-difluorophenyl)-3,7-dimethyl-12- (trifluoromethyl)-2, 4, 5 ,8,13 -pentazatricyclo [8.4.0.02,6]tetradeca- 1(10), 3, 5, 8, 11, 13- hexaene.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is [(7S)-11-chloro-9-(2,6-difluorophenyl)-7-methyl-12- (trifluoromethyl)-2, 3,5,8,13 -pentazatricyclo [8.4.0.02,6]tetradeca- 1(10), 3, 5, 8, 11, 13- hexaen-4-yl] -(3 -hydroxy-3 -methyl-azetidin- 1 -yl)methanone.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is (7S)-11-chloro-9-(2,6-difluorophenyl)-N-[(2S)-2- hydroxypropyl] -7-methyl- 12-(trifluoromethyl)-2, 3,5,8,13- pentazatricyclo [8.4.0.02,6]tetradeca- 1(10), 3, 5, 8, 11, 13 -hexaene-4-carboxamide .
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (I) is (7S)-11-chloro-9-(2,6-difluorophenyl)-N-[(2R)-2- hydroxypropyl] -7-methyl- 12-(trifluoromethyl)-2, 3,5,8,13- pentazatricyclo [8.4.0.02,6]tetradeca- 1(10), 3, 5, 8, 11, 13 -hexaene-4-carboxamide .
• the present invention provides pharmaceutically acceptable salts of the compounds of formula (I) as described herein, especially pharmaceutically acceptable salts selected from hydrochlorides, fumarates, lactates (in particular derived from L-(+)- lactic acid), tartrates (in particular derived from L-(+)-tartaric acid) and trifluoroacetates.
• the present invention provides compounds according to formula (I) as described herein (i.e., as “free bases” or “free acids”, respectively).
• the compounds of formula (I) are isotopically-labeled by having one or more atoms therein replaced by an atom having a different atomic mass or mass number.
• isotopically-labeled (i.e., radiolabeled) compounds of formula (I) are considered to be within the scope of this disclosure.
• isotopes that can be incorporated into the compounds of formula (I) include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, chlorine, and iodine, such as, but not limited to, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl , 123 I, and 125 I, respectively.
• Certain isotopically-labeled compounds of formula (I), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies.
• the radioactive isotopes tritium, i.e. 3 H, and carbon-14, i.e., 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
• a compound of formula (I) can be enriched with 1, 2, 5, 10, 25, 50, 7S, 90, 95, or 99 percent of a given isotope.
• substitution with heavier isotopes such as deuterium, i.e. 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.
• Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
• the preparation of compounds of formula (I) of the present invention may be carried out in sequential or convergent synthetic routes. Syntheses of the compounds of the invention are shown in the following schemes. The skills required for carrying out the reactions and purifications of the resulting products are known to those skilled in the art. The substituents and indices used in the following description of the processes have the significance given herein before and in the claims, unless indicated to the contrary.
• the compounds of formula (I) can be manufactured by the methods given below, by the methods given in the examples or by analogous methods. Appropriate reaction conditions for the individual reaction steps are known to a person skilled in the art.
• the time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents. However, a period of from 0.5 h to several days will usually suffice to yield the described intermediates and compounds.
• the reaction sequence is not limited to the one displayed in the schemes, however, depending on the starting materials and their respective reactivity the sequence of reaction steps can be freely altered. Starting materials are either commercially available or can be prepared by methods analogous to the methods given below, by methods described in references cited in the description or in the examples, or by methods known in the art.
• the compounds of formula (I) can be manufactured by the methods given below, by the methods given in the examples or by analogous methods.
• Appropriate reaction conditions for the individual reaction steps are known to a person skilled in the art.
• the reaction sequence is not limited to the one displayed in schemes 1 - 11, however, depending on the starting materials and their respective reactivity the sequence of reaction steps can be freely altered.
• Starting materials are either commercially available or can be prepared by methods analogous to the methods given below, by methods described in references cited in the description or in the examples, or by methods known in the art.
• a compound of formula (la) can be prepared in one or two steps starting from lactams of formula (II). Following thionation reaction using Lawesson’s reagent or P 2 S 5 , lactams (II) are converted to corresponding thiolactams (III). Their reaction with hydrazides via a Pellizzari type process yields 1,2,4-triazoles of general formula (la). Alternatively, compounds (la) can be directly accessed by reaction of lactams (II) with an hydrazide using bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-CI) in presence of a base (NaH) in tetrahydrofuran.
• Scheme 2 synthesis of pyrido-diazepines (la), wherein R 4 is Me or c-Pr.
• pyrido-diazepines (la), wherein R 4 is methyl or cyclopropyl can be obtained by a palladium-catalyzed Suzuki-Miyaura cross-coupling reaction between 2- chloropyridines (IV) and boron reagents such as trimethylboroxine or cyclopropylboronic acid, using an inorganic base (e.g. K 2 CO 3 or K 3 PO 4 ) in 1,4-dioxane or toluene at elevated temperatures.
• an inorganic base e.g. K 2 CO 3 or K 3 PO 4
• Triazoles of formula (lb) can be prepared according to a process described in Scheme 3.
• Scheme 3 synthesis of pyrido-diazepines of formula (lb) wherein R 1 is Me, as described above and in the claims.
• 1,2,4-triazoles (lb) can be prepared, starting from thiolactames (III), by treatment with ammonia in methanol to form amidines (V). Following their sequential reaction with triethyl or trymethyl orthoacetate, treatment with ammonia in methanol, and final ring closure by reaction with sodium hypochlorite in water and methanol, final derivatives (lb) were obtained.
• Electrophilic amination of lactams (II) using and O-(diphenylphosphinyl)hydroxylamine yields intermediates of formula (VI). Their thermal cyclocondensation reaction with imidates provides 1,2,4-triazoles (VII).
• Final derivatives of formula (lb) can be obtained by either saponification of the ethyl ester (VII) to the carboxylic acid (VIII) under basic conditions (e.g. NaOH or LiBr, Et 3 N), followed by standard amide coupling with amines HNR 5 R 6 (e.g. HATU, DIPEA or PyBOP, DIPEA), or by direct reaction of the ester (VII) with amines HNR 5 R 6 in ethanol.
• basic conditions e.g. NaOH or LiBr, Et 3 N
• standard amide coupling with amines HNR 5 R 6 e.g. HATU, DIPEA or PyBOP, DIPEA
• N-protected protected triazoles (IX) can be accessed by a Curtius rearrangement when carboxylic acids (VIII) are heated with diphenylphosphoryl azide in presence of a base (e.g. Et 3 N). Removal of N-Boc protecting group can be accomplished with mineral acids (e.g. HCl) or organic acids (e.g. trifluoroacetic acid) to yield amines of formula (X), which, in turn, can be coupled with a carboxylic acid R 8 CO 2 H (e.g. POCI 3 in pyridine) to provide final derivatives (lb).
• 4-chlorobutanamides (XI) can be cyclized in presence of a base (e.g. Et 3 N) to form 5-membered lactams of formula (lb).
• imidazoles of formula (Ic) can be prepared according to a process described in Scheme 7.
• thiolactams (III) can be reacted with an aminoalcohol of general formula HOCH 2 CH(NH 2 )R 1 to form substituted amidines (XII).
• Final compounds (Ic) are obtained in two steps synthesis by Dess-Martin oxidation of alcohols (XII) to corresponding aldehydes, followed by thermal cyclization.
• the alcohol (XII) can be oxidized with TEMPO and phenyl- ⁇ 3 -iodanediyl diacetate (BAIB), followed by cyclization with POCI 3 and pyridine to provide imidazoles (Ic).
• imidazoles of general formula (Ic) can be prepared via ester intermediates
• 5,6-dichloropyridin-3-amine can be protected with a suitable protecting group such as tert-butyloxycarbonyl by treatment with di- tert-butyl dicarbonate in presence of a base (e.g. diisopropylethylamine), followed by treatment with trifluoroacetic acid in dichloromethane to provide tert-butyl N-(5,6-dichloro-3- pyridyl)carbamate.
• a suitable protecting group such as tert-butyloxycarbonyl by treatment with di- tert-butyl dicarbonate in presence of a base (e.g. diisopropylethylamine), followed by treatment with trifluoroacetic acid in dichloromethane to provide tert-butyl N-(5,6-dichloro-3- pyridyl)carbamate.
• Amides (XXIII) can be obtained by coupling with N-Boc protected L-amino acids upon exposure to phosphoryl chloride (POCI 3 ) in pyridine. Removal of N-Boc protecting group can be effected with mineral acids (e.g. HC1) or organic acids (e.g. trifluoroacetic acid) to yield amines of formula (XXIII).
• Final intramolecular condensation reaction promoted by acidic media (e.g. silica in toluene or pivalic acid in ethanol) and heat (80-110 °C) provides desired lactam building block of formula (II).
• pyridines can undergo a Buchwald-Hartwig amination reaction with primary amides of formula (XXV), using a palladium catalyst (e.g. Pd 2 (dba) 3 ), a suitable ligand (e.g. Xantphos), and a base such as cesium carbonate.
• Amides (XXVI) can be deprotonated at low temperature ( n-BuLi in tetrahydrofuran at -78°C) to undergo a 1,2-carbonyl addition reaction with commercially available aldehydes (XVIII) to yield alcohols of formula (XXVII).
• Final oxidation to corresponding ketones (XXII) can be accomplished using TEMPO and sodium hypochlorite.
• racemization at the chiral center occurs to various extents (20-100%), depending on specific reaction conditions adopted.
• chiral purification e.g. by HPLC or SFC
• the present invention provides a process of manufacturing the compounds of formula (I) described herein, wherein said process is as described in any one of Schemes 1 to 11 above.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, when manufactured according to the processes disclosed herein.
• the compounds of formula (I) and their pharmaceutically acceptable salts possess valuable pharmacological properties that make them useful for the treatment or prevention of diseases or condi ctions that are associated with the GABAA ⁇ l receptor.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, for use as therapeutically active substance.
• the present invention provides a method for treating or preventing acute neurological disorders, chronic neurological disorders and/or cognitive disorders in a subject, said method comprising administering an effective amount of a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, to the subject.
• the present invention provides the use of a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, in a method for treating or preventing acute neurological disorders, chronic neurological disorders and/or cognitive disorders in a subject.
• the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, for use in a method for treating or preventing acute neurological disorders, chronic neurological disorders and/or cognitive disorders in a subject.
• the present invention provides the use of a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of prevention of acute neurological disorders, chronic neurological disorders and/or cognitive disorders.
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are selected from autism spectrum disorders (ASD), Angelman syndrome, age-related cognitive decline, Rett syndrome, Prader-Willi syndrome, amyotrophic lateral sclerosis (ALS), fragile-X disorder, negative and/or cognitive symptoms associated with schizophrenia, tardive dyskinesia, anxiety, social anxiety disorder (social phobia), panic disorder, agoraphobia, generalized anxiety disorder, disruptive, impulse-control and conduct disorders, Tourette’s syndrome (TS), obsessive- compulsive disorder (OCD), acute stress disorder, post-traumatic stress disorder (PTSD), attention deficit hyperactivity disorder (ADHD), sleep disorders, Parkinson’s disease (PD), Huntington’s chorea, Alzheimer’s disease (AD), mild cognitive impairment (MCI), dementia, behavioral and psychological symptoms (BPS) in neurodegenerative conditions, multi-infarct dementia, agitation, psychosis, substance-induced psychotic disorder, aggression, eating disorders, depression, chronic apathy, anhedonia, chronic fatigue, seasonal affective
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are selected from Alzheimer’s disease, mild cognitive impairment (MCI), age-related cognitive decline, negative and/or cognitive symptoms associated with schizophrenia, bipolar disorders, autism spectrum disorder (ASD), Angelman syndrome, Rett syndrome, Prader-Willi syndrome, epilepsy, post-traumatic stress disorder (PTSD), amyotrophic lateral sclerosis (ALS), and fragile-X disorder.
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are selected from autism spectrum disorder (ASD), Angelman syndrome, Alzheimer’s disease, negative and/or cognitive symptoms associated with schizophrenia and post-traumatic stress disorder (PTSD).
• ASD autism spectrum disorder
• Angelman syndrome Angelman syndrome
• Alzheimer’s disease negative and/or cognitive symptoms associated with schizophrenia and post-traumatic stress disorder (PTSD).
• PTSD post-traumatic stress disorder
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are selected from autism spectrum disorder (ASD), Rett syndrome, Angelman syndrome, post-traumatic stress disorder and fragile-X disorder.
• ASD autism spectrum disorder
• Rett syndrome Rett syndrome
• Angelman syndrome post-traumatic stress disorder
• fragile-X disorder fragile-X disorder
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are selected from autism spectrum disorder (ASD), and Angelman syndrome.
• ASD autism spectrum disorder
• Angelman syndrome selected from Angelman syndrome
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are autism spectrum disorder (ASD).
• ASD autism spectrum disorder
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are Angelman syndrome.
• said acute neurological disorders, chronic neurological disorders and/or cognitive disorders are autism spectrum disorder (ASD), targeting core symptoms and associated comorbidities, such as anxiety and irritability, social anxiety disorder (social phobia) and generalized anxiety disorder.
• ASD autism spectrum disorder
• core symptoms and associated comorbidities such as anxiety and irritability
• social anxiety disorder social phobia
• generalized anxiety disorder such as anxiety and irritability
• the present invention provides pharmaceutical compositions comprising compounds of formula (I) or their pharmaceutically acceptable salts as defined herein and one or more pharmaceutically acceptable excipients.
• Exemplary pharmaceutical compositions are described in the Example section below.
• the present invention relates to pharmaceutical compositions comprising compounds of formula (I) or their pharmaceutically acceptable salts as defined above and one or more pharmaceutically acceptable excipients for the treatment or prevention of acute neurological disorders, chronic neurological disorders and/or cognitive disorders.
• the compounds of formula (I) and their pharmaceutically acceptable salts can be used as medicaments (e.g. in the form of pharmaceutical preparations).
• the pharmaceutical preparations can be administered internally, such as orally (e.g. in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions), nasally (e.g. in the form of nasal sprays) or rectally (e.g. in the form of suppositories).
• the administration can also be effected parentally, such as intramuscularly or intravenously (e.g. in the form of injection solutions or infusion solutions).
• the compounds of formula (I) and their pharmaceutically acceptable salts can be processed with pharmaceutically inert, inorganic or organic excipients for the production of tablets, coated tablets, dragees and hard gelatin capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used, for example, as such excipients for tablets, dragees and hard gelatin capsules.
• Suitable excipients for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid substances and liquid polyols, etc.
• Suitable excipients for the production of solutions and syrups are, for example, water, polyols, saccharose, invert sugar, glucose, etc.
• Suitable excipients for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils, etc.
• Suitable excipients for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols, etc.
• the pharmaceutical preparations can contain preservatives, solubilizers, viscosity-increasing substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
• the dosage can vary in wide limits and will, of course, be fitted to the individual requirements in each particular case.
• the pure enantiomers can be separated by methods described herein or by methods known to the man skilled in the art, such as e.g., chiral chromatography (e.g., chiral SFC) or crystallization.
• the reaction mixture was allowed to warm up to - 20 °C, before being quenched by addition of saturated aqueous ammonium chloride (250 mL). The mixture was stirred at 0 °C for 15 min, then further saturated aqueous ammonium chloride (60 mL) was added. The mixture was extracted with methyl tert-butyl ether twice, dried (MgSO 4 ) and concentrated in vacuo. The crude was purified by flash chromatography (silica, 0-40 % ethyl acetate in heptane) to afford the title compound (9.21 g, 40 %) as a light yellow solid.
• aqueous sodium hypochlorite (10-15 wt.%, 16 ml, 26 mmol) was added dropwise and the reaction mixture was stirred at 0 °C for 2 h.
• the aqueous layer was extracted with dichloro methane.
• the combined organic layers were washed with saturated aqueous sodium carbonate and brine, dried (Na2SO4) and concentrated in vacuo.
• the residue was purified by flash chromatography (silica, 0-30 % ethyl acetate in heptane) to afford the title compound (4.99 g, 63 %) as a white solid.
• reaction mixture was concentrated in vacuo and the residue was dissolved in methanol (1 mL).
• Sodium hypochlorite solution (448 mg, 0.372 mL, 0.904 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 30 min, then diluted with water and extracted with di chloromethane. The organic layers were combined, washed with brine, dried (Na 2 SO 4 ) and concentrated in vacuo.
• Example 6 azetidin-l-yl-[(7S)-11-chloro-9-(2,6-difluorophenyl)-7-methyl-12-(trifluoromethyl)- 2,3,5,8,13-pentazatricyclo[8.4.0.02,6]tetradeca-1(10),3,5,8,11,13-hexaen-4- yl] methanone a) (3S)- 1-amino-6-chloro-5-(2,6-difluorophenyl)-3-methyl-7-(trifluoromethyl)-3H- pyrido [3 ,4-e] [1,4] diazepin-2-one
• Example 10 [(7S)-11-chloro-9-(2,6-difluorophenyl)-7-methyl-12-(trifluoromethyl)-2,3,5,8,13- pentazatricyclo[8.4.0.02,6]tetradeca-1(10),3,5,8,11,13-hexaen-4-yl]-(3-hydroxy-3- methyl-azetidin-l-yl)methanone
• Example 13 l-[(7S)-11-chloro-9-(2,6-difluorophenyl)-7-methyl-12-(trifluoromethyl)-2,3,5,8,13- pentazatricyclo[8.4.0.02,6]tetradeca-1(10),3,5,8,11,13-hexaen-4-yl]pyrrolidin-2-one a) 4-chloro-N-[(7S)-11-chloro-9-(2,6-difluorophenyl)-7-methyl-12-(trifluoromethyl)-
• Example 15 (7S)-11-chloro-9-(2,6-difluorophenyl)-N-(2-hydroxyethyl)-7-methyl-12- (trifluoromethyl)-2,3,5,8,13-pentazatricyclo[8.4.0.02,6]tetradeca-1(10),3,5,8,11,13- hexaene-4-carboxamide a) (7S)- 11 -chloro-9-(2,6-difluorophenyl)-7-methyl- 12-(trifluoromethyl)-2, 3,5,8,13- pentazatricyclo[8.4.0.02,6]tetradeca-l(10),3,5,8,11,13-hexaene-4-carboxylic acid
• the affinity of compounds at GABA A ⁇ l subunit-containing receptors was measured by competition for [ 3 H]RO7239181 (67.3 Ci/mmol; Roche) binding to membranes from HEK293F cells (ThermoFisher R79007) expressing human (transiently transfected) receptors of composition ⁇ 5 ⁇ 2 ⁇ l, ⁇ 2 ⁇ 2 ⁇ l, ⁇ 1 ⁇ 2 ⁇ l.
• the 28 amino acid long signal peptide (Metl to Ala28)of the human GABA A ⁇ 2 subunit was substituted by the 31 amino acid long signal peptide (Metl to Ser31) of human GABA A ⁇ 5 subunit.
• Cell disruption was performed by stirring the suspension in a Parr vessel #4637 at 435 psi for 15 minutes, and then the suspensions were centrifuged at lOOOxg for 15 minutes at 4°C (Beckman Avanti J-HC; rotor JS-4.2).
• the supernatant (SI) was transferred in a 21 Schott flask and the pellet (Pl) was resuspended with Mannitol Buffer up to 17Sml.
• the resuspended pellet was transferred into a 250ml Corning centrifugal beaker and centrifuged at 1500xg for 10 minutes at 4°C (Beckman Avanti J-HC; rotor JS-4.2).
• the supernatant (SI) was then transferred in the 21 Schott flask and the pellet was discarded.
• the supernatants (SI) were centrifuged in 500ml Beckman polypropylene centrifugal beaker at 15’000xg for 30 minutes at 4°C (Beckman Avanti J-20 XP; rotor JLA-10.500).
• the pellet (P2) was resuspended with Mannitol Buffer 1 : 1 and frozen at -80°C.
• the supernatant (S2) was centrifuged in 100 ml Beckman polypropylene centrifugal tubes at 48000xg for 50 minutes at 4°C (Beckman Avanti J-20 XP; rotor JA-18).
• the supernatant (S3) was discarded and the pellet (P3) was resuspended with 1 : 1 Mannitol Buffer.
• the P2 and P3 protein concentration was determined with the BIORAD Standard assay method with bovine serum albumin as standard and measured on the NANO-Drop 1000.
• the membrane suspension was aliquots (500pl per tube) and stored at -80°C until required.
• Membrane homogenates were resuspended and polytronised (Polytron PT1200E Kinematica AG) in Potassium Phosphate 10 mM, KCl 100 mM binding buffer at pH 7.4 to a final assay concentration determined with a previous experiment.
• Radioligand binding assays were carried out in a volume of 200 pL (96-well plates) which contained 100 pL of cell membranes, [ 3 H]RO7239181 at a concentration of 1.5 nM ( ⁇ 5 ⁇ 2 ⁇ l) or 20-30 nM ( ⁇ 1 ⁇ 2 ⁇ l, ⁇ 2 ⁇ 2 ⁇ l) and the test compound in the range of [0.3- 10000] x 10' 9 M.
• Nonspecific binding was defined by 10 x 10' 6 ( ⁇ 5 ⁇ 2 ⁇ l) and 30 x 10 -6 M RO7239181 and typically represented less than 5% ( ⁇ 5 ⁇ 2 ⁇ l) and less than 20% ( ⁇ 1 ⁇ 2 ⁇ l, ⁇ 2 ⁇ 2 ⁇ l) of the total binding.
• a microwave tube was charged with 7-bromo-6-chloro-5-(2,6-difluorophenyl)-1,3 - dihydro-l,4-benzodiazepin-2-one (450 mg, 1.17 mmol), trimethylboroxine (205 mg, 228 pL, 1.63 mmol), potassium carbonate (242 mg, 1.7S mmol) and tetrakis(triphenylphosphine)palladium (0) (67.4 mg, 58.4 pmol).
• Degassed 1,4-dioxane 8.1 mL
• H2O 2.7 ml
• the suspension was reacted in microwave at 130 °C for 30 min to give complete conversion.
• the affinity of compounds at GABA A ⁇ 2 subunit-containing receptors was measured by competition for [ 3 H]Flumazenil (81.1 Ci/mmol; Roche) binding to HEK293F cells expressing human (transiently transfected) receptors of composition ⁇ 1 ⁇ 3 ⁇ 2.
• Radioligand binding assays were carried out in a volume of 200 pL (96-well plates) which contained 100 pL of cell membranes, [ 3 H]Flumazenil at a concentration of 1 nM and the test compound in the range of [0.1 - 10 -3 - 10] x 10 -6 M. Nonspecific binding was defined by 10' 5 M Diazepam and typically represented less than 5% of the total binding.
• Assays were incubated to equilibrium for 1 hour at 4 °C and harvested onto GF/C uni-filters (Packard) by filtration using a Packard harvester and washing with ice-cold wash buffer (50 mM Tris; pH 7.5). After anhydrousing, filter-retained radioactivity was detected by liquid scintillation counting. Ki values were calculated using Excel-Fit (Microsoft) and are the means of two determinations.
• the compounds of the accompanying examples were tested in the above described assay, and the preferred compounds were found to possess large Ki value for displacement of [ 3 H]Flumazenil from the ⁇ 1 ⁇ 3 ⁇ 2 subtype of the human GABA A receptor of 100 nM or above. Most preferred are compounds with a Ki ⁇ 1 ⁇ 3 ⁇ 2 (nM) > 300.
• the compounds of the invention are binding selectively for the ⁇ l subunit- containing GABA A receptors relative to ⁇ 2 subunit-containing GABA A receptors.
• compounds of the present invention have ⁇ 2/yl selectivity ratio defined as “Ki ⁇ 1 ⁇ 3 ⁇ 2 (nM) / Ki ⁇ 2 ⁇ 2 ⁇ l (nM)” above 10-fold, or LogSel defined as “Log [Ki ⁇ 1 ⁇ 3 ⁇ 2 (nM) / Ki ⁇ 2 ⁇ 2 ⁇ l (nM)]” above 1.
• Representative test results, obtained by the above described assay measuring binding affinity to HEK293 cells expressing human (h) receptors, are shown in the Table 1 below.
• Xenopus laevis oocytes at maturation stages V-VI were used for the expression of cloned mRNA encoding GABA A receptor subunits.
• Xenopus oocytes microinjection Oocytes were plated in 96-well plates for microinjection using the Roboinject automated instrument (MultiChannelSystems, Reutlingen, Germany). Approximately 50 nL of an aqueous solution containing the RNA transcripts for the subunits of the desired GABA A receptor subtype was injected into each oocyte. RNA concentrations ranged between 20 and 200 pg/pL/subunit and were adjusted in pilot experiments to obtain GABA responses of a suitable size and a maximal effect of Flunitrazepam, Triazolam and Midazolam, reference benzodiazepine positive allosteric modulators (PAM) at the GABA A receptor benzodiazepine (BZD) binding site.
• PAM benzodiazepine positive allosteric modulators
• Electrophysiological experiments were performed using the Roboocyte instrument (MultiChannelSystems, Reutlingen, Germany) on days 3 to 5 after the micro-injection of mRNA.
• the oocytes were constantly superfused by a solution containing (in mM) NaCl 90, KCl 1, HEPES 5, MgCh 1, CaCl 2 1 (pH 7.4).
• Oocytes were impaled by two glass microelectrodes (resistance: 0.5-0.8 M ⁇ ) which were filled with a solution containing KCl 1M + K-acetate 1.5 M and voltage-clamped to -80 mV.
• the recordings were performed at room temperature using the Roboocyte two-electrode voltage clamp system (Multichannelsystem).
• the digitized current traces of the first and second GABA response were superimposed and, if necessary, rescaled to equal maximal amplitudes.
• the ratio between the two responses during the time interval of test compound application was calculated point by point.
• the extremum of the resulting “ratio trace” was taken as the efficacy (“Fold increase”) of the compound expressed as "% modulation of GABA EC20" (100* (Fold increase- 1)).
• Benzodiazepines reference compounds (classical marketed benzodiazepines) and reference thieno-diazepines listed below were tested for their affinity towards the GABA A receptor ⁇ 1 ⁇ 2 ⁇ l and ⁇ 2 ⁇ 2yl subtypes as well as in the GABA A receptor ⁇ 1 ⁇ 3 ⁇ 2 subtype. The results are shown in Table 3.
• RE-A is disclosed in Drug Design and Discovery (1993), 10(1), 45-55 (Synthesis and anticonvulsant activity of 1,3 -dihydro-5-phenyl-2H-pyrido[3,4-e]- 1,4-diazepin-2-ones).
• Capsules comprising compounds of formula (I) are manufactured as follows:
• a compound of formula I lactose and corn starch are firstly mixed in a mixer and then in a comminuting machine. The mixture is returned to the mixer; the talc is added thereto and mixed thoapproximatively. The mixture is filled by machine into suitable capsules, e.g. hard gelatin capsules.
• Injection solutions comprising compounds of formula (I) are manufactured as follows:

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• 2022
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• 2024
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