HK1230197B - Compounds for treating spinal muscular atrophy - Google Patents

Description

Compounds for treating spinal muscular atrophy

Introduction

The present invention provides compounds that act as SMN2 gene splicing regulators, their preparation, pharmaceutical compositions containing the same, and their use as drugs for treating spinal muscular atrophy (SMA).

In particular, the present invention relates to compounds of formula (I) and pharmaceutically acceptable salts thereof

wherein A, R ¹ , R ² and R ³ are as described herein.

Background Art

Spinal muscular atrophy (SMA) in its broadest sense describes a variety of inherited and acquired central nervous system (CNS) diseases characterized by progressive motor neuron damage in the spinal cord and brainstem, resulting in muscle weakness and wasting. The most common form of SMA is caused by mutations in the survival motor neuron (SMN) gene and manifests with a wide range of severity affecting infants through adults (Crawford and Pardo, Neurobiol. Dis., 1996, 3:97).

Infantile SMA is the most severe form of this neurodegenerative disorder. Symptoms include muscle weakness, low muscle tone, inability to cry, limp or tendency to fall, difficulty sucking or swallowing, accumulation of secretions in the lungs or throat, difficulty eating, and increased susceptibility to respiratory infections. The legs are often weaker than the arms, and developmental milestones such as holding the head or sitting up cannot be reached. Generally, the earlier the symptoms appear, the shorter the life expectancy. Symptoms appear soon after due to deterioration of the motor neuron cells. Severe forms of the disease are fatal, and there is no known cure for all forms. The progression of SMA is directly related to the rate of deterioration of the motor neuron cells and the severity of the resulting weakness. Infants with severe forms of SMA often die from respiratory diseases due to weakness of the muscles that support breathing. Children with milder forms of SMA survive longer, but they may require extensive medical support, especially those at the more severe end of the spectrum. The clinical spectrum of SMA symptoms has been divided into the following five groups.

(a) Type 0 SMA (in utero SMA) is the most severe form of the disease and begins before birth. Typically, the first symptom of type 0 SMA is decreased fetal movement, which can be first observed between 30 and 36 weeks of gestation. After birth, these newborns have little movement and difficulty swallowing and breathing.

(b) Type 1 SMA (infantile SMA or Werdnig-Hoffmann disease) presents between 0 and 6 months of age and is also very severe. Patients are never able to sit up, and death usually occurs within the first 2 years due to the absence of respiratory support.

(c) Type 2 SMA (transitional SMA) presents between 7 and 18 months of age. Patients are able to sit up without support but are unable to stand or walk independently. The prognosis for this group is largely dependent on the degree of respiratory distress.

(d) Type 3 SMA (juvenile SMA or Kugelberg-Welander disease) is usually diagnosed after 18 months of age. Individuals with Type 3 SMA are able to walk independently at some point during the course of the disease but are typically confined to a wheelchair during adolescence or adulthood.

(e) Type 4 SMA (adult-onset SMA). Weakness usually begins in the tongue, hands, or feet during late adolescence and then spreads to other areas of the body. Adult-onset SMA progresses more slowly and has little or no effect on life expectancy.

The SMN gene map has been derived through linkage analysis of a complex region on chromosome 5q. In humans, this region contains an approximately 500,000 base pair (kb) inverted duplication, resulting in two nearly identical copies of the SMN gene. SMN1 is caused by inactivating mutations or deletions in the telomeric copies of the gene (SMN1) on both chromosomes, leading to loss of SMN1 gene function. However, all patients retain the centromeric copy of the gene (SMN2), and the number of SMN2 copies in SMA patients generally correlates negatively with disease severity; that is, patients with less severe SMA have more SMN2 copies. Despite this, SMN2 cannot fully compensate for the loss of SMN1 function due to alternative splicing of exon 7 caused by a translationally silent C-to-T mutation in exon 7. Consequently, the majority of transcripts produced by SMN2 lack exon 7 (Δ7 SMN2) and encode a truncated SMN protein with impaired function and rapid degradation.

The SMN protein is thought to play a role in RNA processing and metabolism, with a well-characterized function in mediating the assembly of a specific class of RNA-protein complexes known as snRNPs. In motor neurons, SMN may have other functions; however, its role in preventing selective motor neuron degeneration is less well established.

In most cases, SMA is diagnosed based on clinical symptoms and the presence of at least one SMN1 gene copy. However, in approximately 5% of cases, SMA is caused by mutations in genes other than SMN1 inactivation, some of which are known and others are yet to be identified. In some cases, when SMN1 gene testing is not feasible or does not show any abnormalities, other tests such as electromyography (EMG) or muscle biopsy may be indicated.

Currently, medical care for SMA patients is limited to supportive therapies, including respiratory, nutritional, and rehabilitation care; there are no known medications that address the underlying cause of the disease. Current treatment for SMA consists of preventing and managing the secondary effects of chronic motor unit loss. The primary management issue in type 1 SMA is the prevention and early treatment of lung problems, which are the cause of death in the majority of cases. Although some infants with SMA survive into adulthood, the life expectancy of patients with type 1 SMA is less than two years.

Several SMA mouse models have been developed. Specifically, the SMN Δ exon 7 (Δ7 SMN) model (Le et al., Hum. Mol. Genet., 2005, 14:845) carries several copies of the SMN2 gene and Δ7 SMN2 cDNA and recapitulates many phenotypic features of type 1 SMA. The Δ7 SMN model can be used for SMN2 expression studies and assessment of motor function and survival. The C/C-allele mouse model (Jackson Laboratory strain #008714, The Jackson Laboratory, Bar Harbor, ME) provides a less severe SMA disease model in which mice have low levels of full-length SMN2 (FL SMN2) mRNA and SMN protein. The C/C-allele mouse phenotype has both the SMN2 gene and an alternatively spliced hybrid mSMN1-SMN2 gene, but does not have overt muscle weakness. The C/C-allele mouse model is used for SMN2 expression studies.

As understanding of the genetic basis and pathophysiology of SMA has increased, several therapeutic strategies have been developed, but none have yet been shown to be successful in the clinic.

Gene replacement of SMN1 using viral delivery vectors and cell replacement using differentiated SMN1 ^+/+ stem cells have shown efficacy in animal models of SMA. Before these approaches can be applied to humans, more studies are needed to determine safety and immune responses and to address the requirement to start treatment in the neonatal stage.

Correction of alternative splicing of SMN2 in cultured cells has also been achieved using synthetic nucleic acids as therapeutic agents: (i) antisense oligonucleotides that target sequence elements in SMN2 pre-mRNA and divert the outcome of the splicing reaction to produce full-length SMN2 mRNA (Passini et al., Sci. Transl. Med., 2011, 3:72ra18; and Hua et al., Nature, 2011, 478:123); and (ii) trans-splicing RNA molecules that provide a fully functional RNA sequence that replaces the mutant fragment during splicing and produces full-length SMN1 mRNA (Coady and Lorson, J Neurosci., 2010, 30:126).

Other approaches under investigation include finding drugs that increase SMN levels, enhance residual SMN function, or compensate for its loss. Aminoglycosides have been shown to enhance the expression of stable SMN protein produced from Δ7 SMN2 mRNA by promoting translational readthrough of abnormal stop codons, but they have poor central nervous system penetration and are toxic after repeated doses. Chemotherapeutic agents such as aclarubicin have been shown to increase SMN protein in cell culture; however, the toxicity profile of these drugs prohibits long-term use in SMA patients. Some drugs under clinical investigation for the treatment of SMA include transcriptional activators such as histone deacetylase ("HDAC") inhibitors (e.g., butyrate, valproic acid, and hydroxyurea) and mRNA stabilizers (e.g., the mRNA decapping inhibitor RG3039 from Repligen), which aim to increase the amount of total RNA transcribed from the SMN2 gene. However, the use of HDAC inhibitors or mRNA stabilizers does not address the underlying cause of SMA and may lead to global increases in transcription and gene expression in humans, which has potential safety concerns.

In an alternative approach, neuroprotective agents such as olesoxime have been selected for investigation. These strategies are not aimed at using SMN for the treatment of SMA, but rather are being developed to protect SMN-deficient motor neurons from neurodegeneration.

International Patent Application WO 2009/151546 A1 describes a system designed to identify compounds that increase the inclusion of SMN exon 7 in RNA transcribed from the SMN2 gene, and certain benzoxazole and benzisoxazole compounds identified thereby. International Patent Applications WO 2010/019236 A1 and WO 2013/119916 A2 describe a system designed to identify compounds that cause ribosomal frameshifting to produce stabilized SMN protein from Δ7 SMN2 mRNA, and certain isoindolinone compounds identified thereby.

Despite advances in understanding the genetic basis and pathophysiology of SMA, there remains a need to identify compounds that modify the course of spinal muscular atrophy, one of the most devastating childhood neurological diseases.

Detailed Description of the Invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

All publications, patent applications, patents, and references mentioned herein are incorporated by reference in their entirety.

Unless otherwise indicated, the nomenclature used in this application is based on the IUPAC systematic nomenclature.

Unless otherwise indicated, any open valence appearing on a carbon, oxygen, sulfur, or nitrogen atom in structures herein represents the presence of a hydrogen.

Regardless of whether the terms in question appear alone or in combination, the definitions described herein apply. It is contemplated that the definitions described herein can be appended to form chemically related combinations, such as, for example, "heterocycloalkylaryl,""haloalkylheteroaryl,""arylalkylheterocycloalkyl," or "alkoxyalkyl." The last member of the combination is the group to which the remainder of the molecule is attached. The other members of the combination are attached to the attached group in reverse order of the letter, for example, the combination amino- _{Ci_7} -alkyl refers to a _{Ci_7} -alkyl substituted by an amino group, or the combination arylalkylheterocycloalkyl refers to a heterocycloalkyl substituted by an alkyl substituted by an aryl group.

The term "moiety" refers to an atom or a chemically bonded group of atoms that is attached to another atom or molecule by one or more chemical bonds, thereby forming a part of a molecule. For example, the variables A, R ¹ , R ² and R ³ of formula (I) refer to moieties that are attached to the core structure of formula (I) by covalent bonds.

When indicating the number of substituents, the term "one or more" refers to a range from one substituent to the highest possible number of substitution, ie, replacement of one hydrogen by a substituent to replacement of all hydrogens by substituents.

The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "substituent" refers to an atom or group of atoms that replaces a hydrogen atom on the parent molecule.

The term "substituted" means that the specified group carries one or more substituents. Where any group can carry multiple substituents and a variety of possible substituents are provided, the substituents can be independently selected and need not be the same. The term "unsubstituted" means that the specified group does not carry a substituent. The term "optionally substituted" means that the specified group is unsubstituted or substituted with one or more substituents independently selected from the group of possible substituents. When indicating the number of substituents, the term "one or more" means from one substituent to the maximum possible number of substitutions, i.e., replacement of one hydrogen by a substituent to replacement of all hydrogens by a substituent.

The terms "compounds of this invention" and "compounds of the present invention" refer to compounds as disclosed herein, as well as stereoisomers, tautomers, solvates, and salts (eg, pharmaceutically acceptable salts) thereof.

When the compounds of the invention are solids, those skilled in the art understand that these compounds and their solvates and salts may exist in different solid forms (particularly different crystalline forms), all of which are contemplated within the scope of the invention and the specified molecular formula.

The term "pharmaceutically acceptable salts" refers to salts that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include acid and base addition salts.

The term "pharmaceutically acceptable acid addition salt" refers to pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from the group consisting of aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic acid types, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.

The term "pharmaceutically acceptable base addition salt" refers to those pharmaceutically acceptable salts formed with organic or inorganic bases. Examples of useful inorganic bases include sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include 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, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and salts of polyamine resins.

Stereochemical definitions and conventions used herein generally follow those of S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center. The substituents attached to the chiral center under consideration are ordered according to the Cahn, Ingold, and Prelog order rules. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511). The prefixes D and L or (+) and (-) are used to indicate the sign of rotation of plane-polarized light of a compound, where (-) or L indicates that the compound is levorotatory. Compounds prefixed with (+) or D are dextrorotatory.

The term "chiral center" refers to a carbon atom bound to four non-identical substituents. The term "chiral" refers to an embodiment that is non-superimposable on its mirror image, while the term "achiral" refers to an embodiment that is superimposable on its mirror image. Chiral molecules are optically active, i.e., they are able to rotate the plane of plane-polarized light.

The compounds of the present invention may have one or more chiral centers and may exist as optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates, or mixtures of diastereomeric racemates. When a chiral center is present in a chemical structure, it is intended that the present invention includes all stereoisomers associated with the chiral center.

The terms "halo," "halogen," and "halide" are used interchangeably herein and refer to fluorine, chlorine, bromine, or iodine. A specific example of a halogen is fluorine.

The term "alkyl" refers to a monovalent straight or branched chain saturated hydrocarbon radical of 1 to 12 carbon atoms. In particular embodiments, the alkyl radical has 1 to 7 carbon atoms, and in more particular embodiments, has 1 to 4 carbon atoms. Examples of alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Particular examples of alkyl radicals are methyl and ethyl.

The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom of the alkyl group has been replaced by the same or different halogen atoms, in particular fluorine atoms. Examples of haloalkyl groups include monofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, such as 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl or trifluoromethyl, etc. The term "perhaloalkyl" refers to an alkyl group in which all hydrogen atoms of the alkyl group have been replaced by the same or different halogen atoms.

The term "bicyclic ring system" refers to two rings fused to each other via a common single or double bond (annelated bicyclic ring system), via a sequence of three or more common atoms (bridged bicyclic ring system) or via a single common atom (spirobicyclic ring system). Bicyclic ring systems may be saturated, partially unsaturated, unsaturated or aromatic. Bicyclic ring systems may contain heteroatoms selected from N, O and S.

The term "cycloalkyl" refers to a saturated monocyclic or bicyclic hydrocarbon radical having 3 to 10 ring carbon atoms. In particular embodiments, a cycloalkyl radical refers to a monovalent saturated monocyclic hydrocarbon radical having 3 to 8 ring carbon atoms. Bicyclic refers to a radical consisting of two saturated carbocyclic rings that share one or more carbon atoms. Particular cycloalkyl radicals are monocyclic. Examples of monocyclic cycloalkyl radicals are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. Examples of bicyclic cycloalkyl radicals are bicyclo[2.2.1]heptyl or bicyclo[2.2.2]octyl. A particular example of a cycloalkyl radical is cyclopropyl.

The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic, bicyclic or tricyclic ring system of 3 to 9 ring atoms, which contains 1, 2 or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon. In a particular embodiment, heterocycloalkyl is a monovalent saturated monocyclic ring system of 4 to 7 ring atoms, which contains 1, 2 or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon. Examples of monocyclic saturated heterocycloalkyl groups are aziridine, oxirane, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl or oxazepanyl. Examples of bicyclic saturated heterocycloalkyl groups are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, or 3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocycloalkyl groups are dihydrofuranyl, imidazolinyl, dihydro-oxazolyl, tetrahydro-pyridinyl, or dihydropyranyl. Specific examples of heterocycloalkyl groups are 1,4-diazacycloheptyl, hexahydropyrrolo[1,2-a]pyrazinyl, piperidinyl, piperazinyl, and pyrrolidinyl. More specific examples of heterocycloalkyl groups are hexahydropyrrolo[1,2-a]pyrazinyl and piperazinyl.

The term "N-heterocycloalkyl" refers to a heterocycloalkyl group containing at least one nitrogen ring atom, and wherein the point of attachment of the heterocycloalkyl group to the rest of the molecule is through the nitrogen ring atom. Specific examples of N-heterocycloalkyl groups are 1,4-diazepanyl, hexahydropyrrolo[1,2-a]pyrazinyl, piperidinyl, piperazinyl, and pyrrolidinyl. More specific examples of N-heterocycloalkyl groups are hexahydropyrrolo[1,2-a]pyrazinyl and piperazinyl.

The term "basicity" as used herein in connection with a compound is expressed as the decimal logarithm of the negative conjugate acid's acidity constant (pKa = -log Ka). The greater the pKa of the conjugate acid, the stronger the base (pKa + pKb = 14). In this application, an atom or functional group is designated as "basic" if it is suitable for accepting a proton and if the calculated pKa of its conjugate acid is at least 7, more particularly if the calculated pKa of its conjugate acid is at least 7.8, and most particularly if the calculated pKa of its conjugate acid is at least 8. pKa values are calculated in silico as described in F. Milletti et al., J. Chem. Inf. Model (2007) 47: 2172-2181.

The term "alkylene" refers to a straight-chain saturated divalent hydrocarbon group of 1 to 7 carbon atoms or a divalent branched saturated hydrocarbon group of 3 to 7 carbon atoms. Examples of alkylene groups include methylene, ethylene, propylene, 2-methylpropylene, butylene, 2-ethylbutylene, pentylene, and hexylene. Specific examples of alkylene groups are ethylene, propylene, and butylene.

The term "amino" refers to a group of formula -NR'R" where R' and R" are independently hydrogen, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or as described herein. Alternatively, R' and R" together with the nitrogen to which they are attached can form a heterocycloalkyl. The term "primary amino" refers to a group where R' and R" are both hydrogen. The term "secondary amino" refers to a group where R' is hydrogen and R" is a group other than hydrogen. The term "tertiary amino" refers to a group where neither R' nor R" is hydrogen. Particular secondary and tertiary amines are methylamine, ethylamine, propylamine, isopropylamine, aniline, benzylamine, dimethylamine, diethylamine, dipropylamine, and diisopropylamine.

The term "active pharmaceutical ingredient" (or "API") refers to the compound or molecule in a pharmaceutical composition that possesses a particular biological activity.

The terms "pharmaceutical composition" and "pharmaceutical formulation" (or "formulation") are used interchangeably and refer to a mixture or solution containing a therapeutically effective amount of an active pharmaceutical ingredient and a pharmaceutically acceptable excipient that is administered to a mammal (e.g., a human) in need thereof.

The term "pharmaceutically acceptable" refers to the property of materials useful in preparing pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and that are acceptable for veterinary as well as human pharmaceutical uses.

The terms "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," and "therapeutically inert excipient" are used interchangeably and refer to any pharmaceutical ingredient in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, or lubricant used to formulate a drug product.

The term "subject" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

The term "therapeutically effective amount" refers to an amount of a compound or molecule of the invention which, when administered to a subject, (i) treats or prevents a particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. The therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

The term "treating" or "treating" a disease state includes inhibiting the disease state, ie, arresting the development of the disease state or its clinical symptoms, or relieving the disease state, ie, causing temporary or permanent regression of the disease state or its clinical symptoms.

The term "spinal muscular atrophy" (or SMA) refers to a disease caused by an inactivating mutation or deletion of the SMN1 gene on both chromosomes, resulting in loss of SMN1 gene function.

Symptoms of SMA include muscle weakness, low muscle tone, weak cry, weak cough, limp or tendency to fall, difficulty sucking or swallowing, difficulty breathing, accumulation of secretions in the lungs or throat, clenched fists and sweaty hands, twitching/vibrating tongue, head that often tilts to one side (even when lying down), legs that tend to be weaker than the arms, legs that often assume a "frog-leg" position, difficulty feeding, increased susceptibility to respiratory infections, bowel/bladder weakness, lower than normal weight, inability to sit unsupported, inability to walk, inability to crawl, and hypotonia, areflexia, and multiple congenital contractures (joint contractures) associated with loss of anterior hom cells.

The term "treating spinal muscular atrophy (SMA)" or "treatment of spinal muscular atrophy (SMA)" includes one or more of the following effects: (i) reducing or ameliorating the severity of SMA; (ii) delaying the onset of SMA; (iii) inhibiting the progression of SMA; (iv) reducing hospitalizations of subjects; (v) reducing the length of hospitalizations of subjects; (vi) increasing survival of subjects; (vii) improving the quality of life of subjects; (viii) reducing the number of SMA-associated symptoms; (ix) reducing or ameliorating the severity of one or more symptoms associated with SMA; (x) shortening the duration of symptoms associated with SMA; (xi) preventing the recurrence of symptoms associated with SMA; (xii) inhibiting the development or onset of SMA symptoms; and/or (xiii) inhibiting the progression of symptoms associated with SMA.

More specifically, the term "treating SMA" refers to one or more of the following beneficial effects: (i) reducing muscle strength impairment; (ii) increasing muscle strength; (iii) reducing muscle atrophy; (iv) reducing motor function loss; (v) increasing motor neurons; (vii) reducing motor neuron loss; (viii) protecting SMN-deficient motor neurons from degeneration; (ix) improving motor function; (x) improving lung function; and/or (xi) reducing lung function loss.

In more detail, the term "treating SMA" refers to the functional ability of a human infant or human toddler to sit unassisted or a human infant, human toddler, human child or human adult to stand unassisted, walk unassisted, run unassisted, breathe unassisted, turn over unassisted during sleep or swallow unassisted, or the maintenance of such functional ability.

The term "EC _1.5x concentration that produces full-length SMN2 minigene mRNA" (or "EC _1.5x minigene") is defined as the concentration of test compound that effectively increases the amount of full-length SMN2 minigene mRNA to 1.5-fold compared to the level in vehicle-treated cells.

The term "EC _1.5x concentration for SMN protein expression" (or "EC _1.5x SMN protein") is defined as the concentration of test compound effective to produce 1.5-fold the amount of SMN protein in SMA patient fibroblasts compared to the amount produced by vehicle control.

In detail, the present invention relates to compounds of formula (I)

in

R ¹ is hydrogen or C _1-7 -alkyl;

R ² is hydrogen, cyano, C _1-7 -alkyl, C _1-7 -haloalkyl or C _3-8 -cycloalkyl;

R ³ is hydrogen, C _1-7 -alkyl or C _3-8 -cycloalkyl;

A is N-heterocycloalkyl or NR ¹² R ¹³ , wherein N-heterocycloalkyl contains 1 or 2 nitrogen ring atoms and is optionally substituted with 1, 2, 3 or 4 substituents selected from R ¹⁴ ;

R ¹² is heterocycloalkyl containing 1 nitrogen ring atom, wherein the heterocycloalkyl is optionally substituted with 1, 2, 3 or 4 substituents selected from R ¹⁴ ;

R ¹³ is hydrogen, C _1-7 -alkyl or C _3-8 -cycloalkyl;

R ¹⁴ is independently selected from hydrogen, C _1-7 -alkyl, amino, amino-C _1-7 -alkyl, C _3-8 -cycloalkyl and heterocycloalkyl or two R ¹⁴ together form C _1-7 -alkylene;

Provided that, if A is an N-heterocycloalkyl group containing only 1 nitrogen ring atom, at least one R ¹⁴ substituent is amino or amino-C _1-7 -alkyl;

and pharmaceutically acceptable salts thereof.

Particular embodiments of the present invention are compounds of formula (I) and pharmaceutically acceptable salts thereof.

Furthermore, it is to be understood that each embodiment as disclosed herein relating to a specific A, R ¹ , R ² or R ³ may be combined with any other embodiment as disclosed herein relating to an additional A, R ¹ , R ² or R ³ .

A particular embodiment of the invention relates to compounds of formula (I), wherein

R ¹ is hydrogen or C _1-7 -alkyl;

R ² is hydrogen, cyano, C _1-7 -alkyl, C _1-7 -haloalkyl or C _3-8 -cycloalkyl;

R ³ is hydrogen, C _1-7 -alkyl or C _3-8 -cycloalkyl;

A is an N-heterocycloalkyl group containing 1 or 2 nitrogen ring atoms, wherein the N-heterocycloalkyl group is optionally substituted with 1, 2, 3 or 4 substituents selected from R ¹⁴ ;

R ¹⁴ is independently selected from hydrogen, C _1-7 -alkyl, amino, amino-C _1-7 -alkyl, C _3-8 -cycloalkyl and heterocycloalkyl or two R ¹⁴ together form C _1-7 -alkylene;

Provided that, if A is an N-heterocycloalkyl group containing only 1 nitrogen ring atom, at least one R ¹⁴ substituent is amino or amino-C _1-7 -alkyl;

and pharmaceutically acceptable salts thereof.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ¹ is C _1-7 -alkyl, in particular methyl.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ² is hydrogen or C _1-7 -alkyl, in particular hydrogen or methyl.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ³ is hydrogen or C _1-7 -alkyl, in particular hydrogen or methyl.

A particular embodiment of the present invention relates to compounds of formula (I), wherein R ¹² is piperidinyl optionally substituted with 1, 2, 3 or 4 substituents selected from R ¹⁴ .

A particular embodiment of the invention relates to compounds of formula (I), wherein R ¹³ is hydrogen or C _1-7 -alkyl, in particular hydrogen or methyl.

A particular embodiment of the present invention relates to compounds of formula (I), wherein R ¹⁴ is independently selected from C _1-7 -alkyl and heterocycloalkyl or two R ¹⁴ together form C _1-7 -alkylene.

A particular embodiment of the present invention relates to compounds of formula (I), wherein R ¹⁴ is independently selected from methyl, ethyl and pyrrolidinyl or two R ¹⁴ together form an ethylene group.

A particular embodiment of the invention relates to compounds of formula (I), wherein A is a saturated monocyclic or bicyclic N-heterocycloalkyl group containing 1 or 2 nitrogen atoms and optionally substituted by 1, 2, 3 or 4 substituents selected from R ¹⁴ .

A particular embodiment of the present invention relates to compounds of formula (I), wherein N-heterocycloalkyl in A or heterocycloalkyl in R ¹² as defined herein is substituted by 1 or 2 substituents selected from R ¹⁴ .

A particular embodiment of the invention relates to compounds of formula (I), wherein the N-heterocycloalkyl group in A as defined herein is further characterized in that one of the ring nitrogen atoms is basic.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is

X is N or CH;

R ⁴ is hydrogen, C _1-7 -alkyl or -(CH ₂ ) _m -NR ⁹ R ¹⁰ ;

R ⁵ is hydrogen or C _1-7 -alkyl;

R ⁶ is hydrogen or C _1-7 -alkyl;

R ⁷ is hydrogen or C _1-7 -alkyl;

R ⁸ is hydrogen or C _1-7 -alkyl;

R ⁹ and R ¹⁰ are independently selected from hydrogen, C _1-7 -alkyl and C _3-8 -cycloalkyl;

R ¹³ is hydrogen, C _1-7 -alkyl or C _3-8 -cycloalkyl;

n is 0, 1, or 2;

m is 0, 1, 2, or 3;

or R ⁴ and R ⁵ together form a C _1-7 -alkylene group;

or R ⁴ and R ⁷ together form a C _1-7 -alkylene group;

or R ⁵ and R ⁶ together form a C _2-7 -alkylene group;

or R ⁵ and R ⁷ together form a C _1-7 -alkylene group;

or R ⁵ and R ⁹ together form a C _1-7 -alkylene group;

or R ⁷ and R ⁸ together form a C _2-7 -alkylene group;

or R ⁷ and R ⁹ together form a C _1-7 -alkylene group;

or R ⁹ and R ¹⁰ together form a C _2-7 -alkylene group;

Provided that, if X is CH, then R ⁴ is -(CH ₂ ) _m -NR ⁹ R ¹⁰ ; and

Provided that, if X is N and R ⁴ is -(CH ₂ ) _m -NR ⁹ R ¹⁰ , then m is 2 or 3.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is

X is N or CH;

R ⁴ is hydrogen, C _1-7 -alkyl or -(CH ₂ ) _m -NR ⁹ R ¹⁰ ;

R ⁵ is hydrogen or C _1-7 -alkyl;

R ⁶ is hydrogen or C _1-7 -alkyl;

R ⁷ is hydrogen or C _1-7 -alkyl;

R ⁸ is hydrogen or C _1-7 -alkyl;

R ⁹ and R ¹⁰ are independently selected from hydrogen, C _1-7 -alkyl and C _3-8 -cycloalkyl;

n is 0, 1, or 2;

m is 0, 1, 2, or 3;

or R ⁴ and R ⁵ together form a C _1-7 -alkylene group;

or R ⁴ and R ⁷ together form a C _1-7 -alkylene group;

or R ⁵ and R ⁶ together form a C _2-7 -alkylene group;

or R ⁵ and R ⁷ together form a C _1-7 -alkylene group;

or R ⁵ and R ⁹ together form a C _1-7 -alkylene group;

or R ⁷ and R ⁸ together form a C _2-7 -alkylene group;

or R ⁷ and R ⁹ together form a C _1-7 -alkylene group;

or R ⁹ and R ¹⁰ together form a C _2-7 -alkylene group;

Provided that, if X is CH, then R ⁴ is -(CH ₂ ) _m -NR ⁹ R ¹⁰ ; and

Provided that, if X is N and R ⁴ is -(CH ₂ ) _m -NR ⁹ R ¹⁰ , then m is 2 or 3.

It has been found that brain penetration is improved when at least one of ^R4 , ^R5 , ^R6 , ^R7 and ^R8 is other than hydrogen.

In a particular embodiment of the present invention, at least one of ^R4 , ^R5 , ^R6 , ^R7 and ^R8 is not hydrogen.

A particular embodiment of the invention relates to compounds of formula (I), wherein X is N.

A particular embodiment of the invention relates to compounds of formula (I), wherein n is 1.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ⁴ is hydrogen, methyl or —(CH ₂ ) _m —NR ⁹ R ¹⁰ , more particularly hydrogen.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ⁵ is hydrogen, methyl or ethyl, more particularly methyl.

A particular embodiment of the invention relates to compounds of formula (I), wherein ^R6 is hydrogen or methyl, more particularly hydrogen.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ⁷ is hydrogen or methyl.

A particular embodiment of the invention relates to compounds of formula (I), wherein R ⁸ is hydrogen.

A particular embodiment of the invention relates to compounds of formula (I), wherein m is 0.

A particular embodiment of the present invention relates to compounds of formula (I), wherein R ⁴ and R ⁵ together form a propylene group.

A particular embodiment of the present invention relates to compounds of formula (I), wherein R ⁵ and R ⁶ together form an ethylene group;

A particular embodiment of the invention relates to compounds of formula (I), wherein R ⁹ and R ¹⁰ together form butylene.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of:

wherein R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ¹³ are as defined herein and wherein R ¹¹ is hydrogen or C _1-7 -alkyl.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of:

wherein R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are as defined herein and wherein R ¹¹ is hydrogen or C _1-7 -alkyl.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of piperazinyl, diazepanyl, pyrrolidinyl and hexahydropyrrolo[1,2-a]pyrazinyl, each of which is optionally substituted with 1, 2, 3 or 4 substituents selected from R ¹⁴ as defined herein.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of piperazin-1-yl, 1,4-diazepan-1-yl, pyrrolidin-1-yl and hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, each of which is optionally substituted by 1 or 2 substituents selected from R ¹⁴ as defined herein.

A particular embodiment of the invention relates to compounds of formula (I), wherein A is NR ¹² R ¹³ , wherein R ¹² and R ¹³ are as defined herein.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is wherein R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ¹³ are as defined herein.

A particular embodiment of the invention relates to compounds of formula (I), wherein A is wherein R ¹³ is hydrogen or methyl.

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of:

A particular embodiment of the present invention relates to compounds of formula (I), wherein A is selected from the group consisting of:

A particular embodiment of the present invention relates to compounds of formula (I), wherein R ¹ is methyl, R ² is hydrogen or methyl, R ³ is hydrogen, and A is

Particular compounds of formula (I) of the present invention are those selected from the group consisting of:

2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aR)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aS)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aR)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-(1,4-diazepan-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-(1,4-diazepan-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aS)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

and pharmaceutically acceptable salts thereof.

Particular compounds of formula (I) of the present invention are those selected from the group consisting of:

7-[(8aR)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aS)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aR)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-[(8aS)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one;

7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

and pharmaceutically acceptable salts thereof.

The compounds of formula (VI) are suitable as intermediates for the preparation of compounds of formula (I).

Another embodiment of the present invention relates to compounds of formula (VI)

wherein R ¹ , R ² and R ³ are as defined herein;

Y is halogen or triflate;

and its salts.

A particular embodiment of the invention relates to compounds of formula (VI), wherein Y is fluorine, chlorine, bromine, iodine or triflate, in particular fluorine.

Particular compounds of formula (VI) of the present invention are those selected from the group consisting of:

7-Fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one;

7-Fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one;

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one;

and its salts.

Preparation method

The compounds of formula (I) and their pharmaceutically acceptable salts as defined above can be prepared following standard methods known in the art.

As shown in Scheme 1, commercially available amino-pyridines of formula (II) can be reacted with malonates to provide intermediates of formula (III), wherein Y and R ³ are as defined herein and R is C _1-2 -alkyl, particularly methyl. The compound of formula (III) is then treated with a chlorinating agent (such as POCl ₃ and the like) to provide compounds of formula (IV). The compound of formula (IV) is then reacted with a compound of formula (V) (wherein R 1 and R 2 are as defined herein and Z is B(OH) 2 or C _1-7 -alkyl borate such as 4,4,5,5-tetramethyl- _1,3,2 -dioxaborolan- ₂ -yl) in a Suzuki cross-coupling reaction in the presence of a catalyst (such as (1,1′-bis(diphenylphosphino)ferrocene)palladium(II) dichloride (Pd(dppf)Cl ² ) and a base (such as K ₂ CO 3 and the _like ⁾ in a suitable solvent (such as DMF and the like) to provide a compound of formula (VI). Finally, the compound of formula (VI) is reacted with compound MA in the presence of:

a) in an aromatic nucleophilic substitution reaction (particularly if Y is fluorine), by heating at a temperature of 80°C to 200°C; or

b) Buchwald-Hartwig amination in the presence of a palladium catalyst (e.g., tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ ) or bis(dibenzylideneacetone)palladium (Pd(dba) ₂ )) by heating at a temperature of 20° C. to 100° C.;

Reaction in a solvent such as dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) or dimethylformamide (DMF) provides a compound of formula (I), wherein A is as defined herein, M is hydrogen, sodium or potassium, in particular hydrogen, and wherein M is attached to A via its nitrogen atom.

Solution 1.

In one embodiment, the present invention relates to a process for preparing a compound of formula (I) as defined above and a pharmaceutically acceptable salt thereof, said process comprising reacting a compound of formula (VI) with a compound M-A in the presence of:

a) in an aromatic nucleophilic substitution reaction (particularly if Y is fluorine), by heating at a temperature of 80°C to 200°C; or

b) Buchwald-Hartwig amination in the presence of a palladium catalyst (e.g., tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ ) or bis(dibenzylideneacetone)palladium (Pd(dba) ₂ )) by heating at a temperature of 20° C. to 100° C.;

The reaction in a solvent such as dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) or dimethylformamide (DMF), wherein A, Y, R ¹ , R ² and R ³ are as defined herein, M is hydrogen, sodium or potassium, in particular hydrogen, and wherein M is attached to A via its nitrogen atom.

A particular embodiment of the present invention relates to a process for preparing compounds of formula (I) as defined above and pharmaceutically acceptable salts thereof, said process comprising an aromatic nucleophilic substitution reaction between a compound of formula (VI) as described above and a compound of formula MA by heating in a solvent, wherein A, R ¹ , R ² , R ³ and Y are as defined above, M is hydrogen, sodium or potassium, and wherein M is attached to A via the nitrogen atom of A.

A particular embodiment of the present invention relates to a process for the preparation of compounds of formula (I) and pharmaceutically acceptable salts thereof as defined above, wherein the aromatic nucleophilic substitution reaction is carried out at a temperature of 80°C to 200°C.

A particular embodiment of the present invention relates to a process for preparing compounds of formula (I) as defined above and pharmaceutically acceptable salts thereof, wherein the solvent for the aromatic nucleophilic substitution reaction is selected from dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) and dimethylformamide (DMF).

A particular embodiment of the present invention relates to a process for the preparation of compounds of formula (I) as defined above, and pharmaceutically acceptable salts thereof, wherein M is hydrogen.

In particular, the compounds of formula (I) and pharmaceutically acceptable salts thereof can be prepared according to the methods described in the Examples herein.

Pharmaceutical composition

Another embodiment provides pharmaceutical compositions or medicaments comprising a compound of the invention and a therapeutically inert carrier, diluent, or pharmaceutically acceptable excipient, and methods of using the compounds of the invention to prepare such compositions and medicaments.

The compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of drug delivery, the method of administration, the timing of administration, and other factors known to practitioners.

The compounds of the present invention can be administered by any suitable means, including oral administration, topical administration (including buccal and sublingual administration), rectal administration, vaginal administration, transdermal administration, parenteral administration, subcutaneous administration, intraperitoneal administration, intrapulmonary administration, intradermal administration, intrathecal administration and epidural administration and intranasal administration and (if necessary for local treatment) intralesional administration. Parenteral injection includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration or subcutaneous administration.

The compounds of the present invention can be administered in any convenient form of administration, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, and the like. Such compositions may contain conventional components of pharmaceutical preparations, such as diluents, carriers, pH regulators, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts for varying osmotic pressure, buffers, masking agents, antioxidants, and other active agents. They may also contain other therapeutically valuable substances.

Typical formulations are prepared by mixing the compound of the invention with a carrier or excipient. Suitable carriers and excipients are known to those skilled in the art and are described in detail in, for example, Ansel H.C. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams & Wilkins, Philadelphia; Gennaro A.R. et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia; and Rowe R.C., Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavorings, diluents and other known additives to provide an elegant appearance of the drug (i.e., the compound of the present invention or its pharmaceutical composition) or to facilitate the production of the drug product (i.e., pharmaceutical product).

The applicable dosage of compound of the present invention can change in a wide range and certainly will be suitable for the individual needs in each specific case.Usually, in the case of oral administration, the daily dosage of the compound of general formula (I) of about 0.01 to 1000mg/ people should be suitable, but also can exceed the above upper limit when needed.

An example of a suitable oral dosage form is a tablet containing about 100 mg to 500 mg of a compound of the invention compounded with about 30 to 90 mg of anhydrous lactose, about 5 to 40 mg of croscarmellose sodium, about 5 to 30 mg of polyvinylpyrrolidone (PVP) K30, and about 1 to 10 mg of magnesium stearate. The powder ingredients are first mixed together and then mixed with a solution of PVP. The resulting composition can be dried, granulated, mixed with magnesium stearate, and compressed into tablet form using conventional equipment.

An example of an aerosol formulation can be prepared by dissolving, for example, 10 to 100 mg of a compound of the invention in a suitable buffer solution (e.g., phosphate buffer) and, if necessary, adding a tonicity adjuster, e.g., a salt such as sodium chloride. The solution can be filtered, for example, using a 0.2 μm filter to remove impurities and contaminants.

use

As described above, the compounds of formula (I) and pharmaceutically acceptable salts thereof have valuable pharmacological properties and have been found to enhance the inclusion of exon 7 of SMN1 and/or SMN2 into the mRNA transcribed from the SMN1 and/or SMN2 genes, thereby increasing the expression of SMN protein in human subjects in need thereof.

The compounds of the present invention can be used alone or in combination with other drugs to treat or prevent diseases caused by inactivating mutations or deletions of the SMN1 gene and/or associated with loss of SMN1 gene function or defects. These diseases include but are not limited to spinal muscular atrophy (SMA).

A particular embodiment of the invention relates to a pharmaceutical composition comprising a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof as defined above and one or more pharmaceutically acceptable excipients.

A particular embodiment of the present invention relates to a pharmaceutical composition comprising a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients, for treating or preventing diseases caused by inactivating mutations or deletions of the SMN1 gene and/or associated with loss of SMN1 gene function or defects, in particular for treating or preventing SMA.

A particular embodiment of the present invention relates to a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for use as a therapeutically active substance, in particular for use as a therapeutically active substance for the treatment or prevention of diseases caused by inactivating mutations or deletions of the SMN1 gene and/or associated with loss of function or deficiency of the SMN1 gene, in particular for the treatment or prevention of spinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for use in treating or preventing diseases caused by inactivating mutations or deletions of the SMN1 gene and/or associated with loss of SMN1 gene function or deficiency, in particular for treating or preventing spinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to a method for treating or preventing diseases caused by inactivating mutations or deletions of the SMN1 gene and/or associated with loss of SMN1 gene function or deficiency, in particular for treating or preventing spinal muscular atrophy (SMA), the method comprising administering to a subject a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof.

A particular embodiment of the present invention relates to the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof for treating or preventing diseases caused by an inactivating mutation or deletion of the SMN1 gene and/or associated with a loss of SMN1 gene function or defect, in particular for treating or preventing spinal muscular atrophy (SMA).

A particular embodiment of the present invention relates to the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating or preventing diseases caused by an inactivating mutation or deletion of the SMN1 gene and/or associated with a loss of SMN1 gene function or defect, in particular for treating or preventing spinal muscular atrophy (SMA). Such a medicament comprises a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

Example

The present invention will be more fully understood by reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention.

Abbreviations used

ACN: acetonitrile; CH ₂ Cl ₂ : dichloromethane (DCM); DIPEA: diisopropylethylamine; DMA: dimethylacetamide; TEA: triethylamine; RT: room temperature; B ₂ (pin) ₂ : bis(pinacol)diboron; Pd(dppf)Cl ₂ : (1,1′-bis(diphenylphosphino)ferrocene)palladium(II) dichloride; PPTS: pyridinium p-toluenesulfonate

Intermediate 1

7-Fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

a) 2-Chloro-7-fluoro-pyrido[1,2-a]pyrimidin-4-one

A mixture of 2-amino-5-fluoropyridine (11.20 g, 0.10 mol) and dimethyl malonate (57.0 mL, 0.50 mol) was heated at 230° C. for 1.5 h. After cooling to room temperature, the precipitate was filtered and washed with ACN (3×) to provide 7-fluoro-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one as a dark solid (14 g), which was used directly in the next step. MS m/z 181.3 [M+H] ⁺ .

A dark mixture of crude 7-fluoro-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one ( ₁₄ g, 77 mmol) in POCl (50 mL) and DIPEA (13.3 mL, 77 mmol) was heated at 110° C. for 15 hours. The solvent was removed and the dark residue was treated with ice water, washed with water (3×) and dried to provide a brown solid. The crude brown solid was chromatographed (5% MeOH in CH ₂ Cl ₂ ) to provide 2-chloro-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one as a yellow solid (9.84 g, 50% over 2 steps), MS m/z 199.2 [M+H] ⁺ .

b) 2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b] Pyridazine

A mixture of 6-chloro-2-methylimidazo[1,2-b]pyridazine (900 mg, 5.37 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (1.36 g, 5.37 mmol, 1.0 equiv), KOAc (1.05 g, 10.7 mmol) and Pd(dppf) _Cl₂ · _{CH₂Cl₂ (} 393 mg, 0.54 mmol) in dioxane (50 mL) was degassed and heated at 95° C. under _N₂ . After 15 h, the mixture was diluted with EtOAc, filtered through celite and concentrated in vacuo to provide 2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine, which was used directly in the next step.

c) 7-Fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

To a solution of 2-chloro-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one (750 mg, 3.78 mmol) in ACN (36 mL) was added 2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine (1.17 g, 4.53 mmol, eq: 1.2), Pd(Ph ₃ P) ₄ (218 mg, 0.189 mmol, 0.05 eq) and K ₂ CO ₃ in water (3.78 mL, 7.55 mmol, 2.0 eq). The mixture was degassed and heated at 105° C. under argon overnight. The reaction was cooled to RT and filtered. The precipitate was washed with _Et2O and then water, dried in vacuo to provide 250 mg (22%) of 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one as a light brown solid. MS m/z 296.1 [M+H] ⁺ .

Intermediate 2

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one

a) 2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1, 2-b]pyridazine

In a sealed flask, 3,6-dichloro-4-methylpyridazine (27 g, 161 mmol) was suspended in aqueous ammonia (25%, 300 mL). The reaction mixture was heated at 110°C for 48 hours (became a solution after 1 hour ₎ . After cooling to room temperature, the reaction was poured into _CH2Cl2 , and the organic phase was separated, dried _{over Na2SO4} , and concentrated under vacuum to provide 22.4 g of 6-chloro-4-methyl-pyridazin-3-amine and 6-chloro-5-methyl-pyridazin-3-amine as a mixture of regioisomers, which was used directly in the next step.

A mixture of regioisomers, 6-chloro-4-methyl-pyridazin-3-amine and 6-chloro-5-methyl-pyridazin-3-amine (22.4 g), was suspended in 2-propanol (300 mL). 1-Bromo-2,2-dimethoxypropane (36.0 g, 26.6 mL, 193 mmol, 1.2 eq) and PPTS (2.96 g, 11.6 mmol, 0.0725 eq) were added, and the resulting solution was heated at 105° C. overnight. The solvent was removed in vacuo, and the residue was taken up in CH ₂ Cl ₂ and washed with NaHCO ₃ . The organic _phase was dried over _Na2SO4 , concentrated in vacuo and the crude light brown solid was chromatographed (EtOAc/heptane 1/2 to 1/1) to provide 6.1 g of 6-chloro-2,8-dimethyl-imidazo[1,2-b]pyridazine as a white solid MS m/z 182.1 [M+H] ⁺ (21%) and 5.9 g of 6-chloro-2,7-dimethyl-imidazo[1,2-b]pyridazine as a white solid MS m/z 182.1 [M+H] ⁺ (20%), respectively.

A mixture of 6-chloro-2,8-dimethylimidazo[1,2-b]pyridazine (0.9 g, 4.96 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (1.26 g, 4.96 mmol, 1.0 equiv), KOAc (0.97 g, 9.91 mmol) and Pd(dppf) _Cl₂ · _{CH₂Cl₂ (} 363 mg, 0.49 mmol) in dioxane (50 mL) was degassed and heated at 110° C. under _N₂ . After 15 h, the mixture was diluted with EtOAc, filtered through celite and concentrated in vacuo to provide 2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine, which was used directly in the next step.

b) 2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one

To a solution of 2-chloro-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one (750 mg, 3.78 mmol, described above) in ACN (36 mL) was added 2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine (1.24 g, 4.53 mmol, 1.2 eq), Pd(Ph ₃ P) ₄ (218 mg, 0.189 mmol, 0.05 eq) and K ₂ CO ₃ in water (3.78 mL, 7.55 mmol, 2.0 eq). The mixture was degassed and heated under argon at 100° C. for 6 h. The reaction was cooled to RT and filtered. The precipitate was washed with _Et2O and then water, dried in vacuo to provide 700 mg (60%) of 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one as a light brown solid. MS m/z 310.1 [M+H] ⁺ .

Intermediate 3

7-Fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

a) 2-Chloro-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one

A mixture of 5-fluoro-3-methylpyridin-2-amine (3.3 g, 26.2 mmol) and dimethyl malonate (15.0 mL, 0.13 mol, 5.0 equiv) was heated at 210° C. for 1.5 hours. After cooling to room temperature, the precipitate was filtered and washed with ACN (3×) to provide 7-fluoro-2-hydroxy-9-methyl-pyrido[1,2-a]pyrimidin-4-one as a dark solid (2.3 g), which was used directly in the next step. MS m/z 195.1 [M+H] ⁺ .

A mixture of crude 7-fluoro-2-hydroxy-9-methyl-pyrido[1,2-a]pyrimidin-4-one (2.3 g, 11.8 mmol) in POCl ₃ (7.7 mL, 82.9 mmol) and DIEA (2.07 mL, 11.8 mmol) was heated at 110° C. for 15 hours. The solvent was removed and the residue was treated with ice water, washed with water (3×) and dried to provide a brown solid. The crude brown solid was chromatographed (5% MeOH in CH ₂ Cl ₂ ) to provide 2-chloro-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one as a yellow solid (1.77 g, 70% over 2 steps), MS m/z 213.1 [M+H] ⁺ .

b) 7-Fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

To a solution of 2-chloro-7-fluoro-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (2.2 g, 10.3 mmol) in ACN (80 mL) was added 2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine (3.22 g, 12.4 mmol, 1.2 eq, described above), Pd(Ph ₃ P) ₄ (1.20 g, 1.03 mmol, 0.1 eq) and an aqueous solution of K ₂ CO ₃ (10.3 mL, 20.7 mmol, 2.0 eq). The mixture was degassed and heated under argon at 100° C. for 6 h. The reaction was cooled to RT and filtered. The precipitate was washed with _Et2O and then water, dried in vacuo to provide 1.80 g (56%) of 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one as a light brown solid. MS m/z 310.1 [M+H] ⁺ .

Intermediate 4

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one

To a solution of 2-chloro-7-fluoro-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (0.98 g, 4.61 mmol, described above) in ACN (50 mL) was added 2,8-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-b]pyridazine (1.51 g, 5.53 mmol, 1.2 equiv, described above), Pd(Ph ₃ P) ₄ (0.32 g, 0.277 mmol, 0.06 equiv) and K ₂ CO ₃ in water (4.61 mL, 9.22 mmol, 2.0 equiv). The mixture was degassed and heated under argon at 100° C. for 6 hours. The reaction was cooled to RT and filtered. The precipitate was washed with _Et2O and water, then dried in vacuo to provide 0.89 g (60%) of 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one as a light brown solid. MS m/z 324.4 [M+H] ⁺ .

Example 1

2-(2-Methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 35 mg, 0.119 mmol) and 1-methylpiperazine (47.5 mg, 0.474 mmol, 4 equiv) were stirred in DMSO (1 mL) at 120° C. overnight. LC-MS showed complete conversion. The solvent was removed under high vacuum. The crude product was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 9/1) to provide the title product (25 mg, 56%) as a light yellow solid. MS m/z 376.3 [M+H ⁺ ].

Example 2

7-[(8aR)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 125 mg, 0.426 mmol) and (R)-octahydropyrrolo-[1,2-a]pyrazine (160 mg, 1.27 mmol, 3 equiv) were stirred in DMSO (5 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=98/2 to 95/5) to provide the title product (65 mg, 38%) as a light yellow solid. MS m/z 402.5 [M+H ⁺ ].

Example 3

7-[(8aS)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 200 mg, 0.647 mmol) and (S)-octahydropyrrolo-[1,2-a]pyrazine (286 mg, 2.26 mmol, 3.5 equiv) were stirred in DMSO (5 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=98/2 to 95/5) to provide the title product (115 mg, 43%) as a light yellow solid. MS m/z 416.3 [M+H ⁺ ].

Example 4

7-[(8aR)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 200 mg, 0.647 mmol), DIPEA (0.113 mL, 0.67 mmol, 1 eq) and (R)-octahydropyrrolo[1,2-a]pyrazine (245 mg, 1.95 mmol, 3.0 eq) were stirred in DMSO (2.5 mL) at 125°C overnight. The solvent was removed under high vacuum. The residue was taken _up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} , and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=98/2 to 95/5) to afford the title product (132 mg, 49%) as a light yellow solid. MS m/z 416.3 [M+H ⁺ ].

Example 5

7-[(8aS)-8a-Methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 90 mg, 0.291 mmol), DIPEA (0.05 mL, 0.29 mmol, 1 eq) and (S)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (81 mg, 0.58 mmol, 2.0 eq) were stirred in DMSO (2.5 mL) at 125°C overnight. The solvent was removed under high vacuum. The residue was taken _up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} , and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to afford the title product (55 mg, 44%) as a light yellow solid. MS m/z 430.3 [M+H ⁺ ].

Example 6

7-[(8aR)-8a-Methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 90 mg, 0.291 mmol), DIPEA (0.05 mL, 0.29 mmol, 1 eq) and (R)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (81 mg, 0.58 mmol, 2.0 eq) were stirred in DMSO (2.5 mL) at 125°C overnight. The solvent was removed under high vacuum. The residue was taken _up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} , and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to afford the title product (50 mg, 40%) as a light yellow solid. MS m/z 430.4 [M+H ⁺ ].

Example 7

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 50 mg, 0.162 mmol) and cis-2,6-dimethylpiperazine (74 mg, 0.647 mmol, 4.0 equiv) were stirred in DMSO (1.5 mL) at 110°C overnight. The solvent was removed under high vacuum. The residue was taken _up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated and dried _{over Na2SO4} and concentrated in vacuo. The crude material was purified by column chromatography ( _SiO2 , _CH2Cl2 /MeOH = _95/5 to 90/10) to provide the title product (32 mg, 49%) as a light yellow solid. MS m/z 404.4 [M+H ⁺ ].

Example 8

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 33 mg, 0.107 mmol) and (S)-2-methylpiperazine (43 mg, 0.427 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 120° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (18 mg, 43%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 9

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 85 mg, 0.275 mmol) and (R)-2-methylpiperazine (110 mg, 1.10 mmol, 4.0 equiv) were stirred in DMSO (5 mL) at 120° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (35 mg, 33%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 10

7-(1,4-diazepan-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 33 mg, 0.107 mmol) and 1,4-diazepane (32 mg, 0.320 mmol, 3.0 equiv) were stirred in DMSO (2 mL) at 120 ° C overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (20 mg, 48%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 11

2-(2-Methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol) and (S)-2-methylpiperazine (68 mg, 0.677 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 110° _C overnight. The solvent was removed under high vacuum. The residue was taken up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} , and concentrated in vacuo. The crude material was purified by column chromatography ( _SiO2 , _CH2Cl2 /MeOH = _95/5 to 90/10) to provide the title product (40 mg, 63%) as a light yellow solid. MS m/z 376.2 [M+H ⁺ ].

Example 12

2-(2-Methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol) and (R)-2-methylpiperazine (68 mg, 0.677 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 110° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated, dried over Na ₂ SO ₄ , and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (48 mg, 75%) as a light yellow solid. MS m/z 376.3 [M+H ⁺ ].

Example 13

7-(1,4-diazepan-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol) and 1,4-diazepane (68 mg, 0.677 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 110°C overnight. The solvent was removed under high vacuum. The residue was taken _up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated and dried _{over Na2SO4} and concentrated in vacuo. The crude material was purified by column chromatography ( _SiO2 , _CH2Cl2 /MeOH = _95/5 to 90/10) to provide the title product (41 mg, 65%) as a light yellow solid. MSm/z 376.2 [M+H ⁺ ].

Example 14

7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol) and cis-2,6-dimethylpiperazine (77 mg, 0.677 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 110° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (41 mg, 62%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 15

7-[(8aS)-3,4,6,7,8,8a-Hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol) and (S)-octahydropyrrolo[1,2-a]pyrazine (85 mg, 0.677 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (36 mg, 53%) as a light yellow solid. MS m/z 402.3 [M+H ⁺ ].

Example 16

7-[(8aS)-8a-Methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol) and (S)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (95 mg, 0.677 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (45 mg, 64%) as a light yellow solid. MS m/z 416.3 [M+H ⁺ ].

Example 17

7-[(8aR)-8a-Methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 100 mg, 0.339 mmol) and (R)-8a-methyloctahydropyrrolo[1,2-a]pyrazine (190 mg, 1.35 mmol, 4.0 equiv) were stirred in DMSO (4 mL) at 125 ° C overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (45 mg, 64%) as a light yellow solid. MS m/z 416.3 [M+H ⁺ ].

Example 18

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a microwave reactor, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 45 mg, 0.145 mmol), (R)-1,3′-dipyrrolidine dihydrochloride (62 mg, 0.291 mmol, 2.0 equiv) and DIPEA (0.20 mL, 1.16 mmol, 8 equiv) were stirred in NMP (3 mL) at 220° C. for 1 hour. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=98/2 to 90/10) to provide the title product (25 mg, 40%) as a light yellow solid. MS m/z 430.3 [M+H ⁺ ].

Example 19

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 50 mg, 0.169 mmol), DIPEA (0.24 mL, 1.35 mmol, 8 equiv) and 4,7-diazaspiro[2.5]octane dihydrochloride (62.7 mg, 0.339 mmol, 2.0 equiv) were stirred in DMSO (2 mL) at 125 ° C for 2 days. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (22 mg, 33%) as a light yellow solid. MS m/z 388.3 [M+H ⁺ ].

Example 20

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 50 mg, 0.162 mmol), DIPEA (0.22 mL, 1.29 mmol, 4 equiv) and 4,7-diazaspiro[2.5]octane dihydrochloride (32 mg, 0.320 mmol, 3.0 equiv) were stirred in DMSO (2 mL) at 130 ° C for 48 hours. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=98/2 to 95/5) to provide the title product (12 mg, 18%) as a light yellow solid. MS m/z 402.3 [M+H ⁺ ].

Example 21

2-(2-Methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 40 mg, 0.135 mmol), DIPEA (0.19 mL, 1.08 mmol, 8 equiv) and (R)-1,3′-bipyrrolidine dihydrochloride (58 mg, 0.271 mmol, 2.0 equiv) were stirred in DMSO (4 mL) and heated in a microwave at 220° C. for 40 minutes. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated, dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=98/2 to 90/10) to afford the title product (30 mg, 53%) as a light yellow solid. MS m/z 416.3 [M+H ⁺ ].

Example 22

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 40 mg, 0.129 mmol) and 2,2-dimethylpiperazine (59 mg, 0.517 mmol, 4.0 equiv) were stirred in DMSO (1.6 mL) at 130 ° C overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 9/1) to provide the title product (29 mg, 55%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 23

7-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 40 mg, 0.135 mmol) and 2,2-dimethylpiperazine (62 mg, 0.542 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 130° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated, dried over Na ₂ SO ₄ , and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (26 mg, 49%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 24

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one (Intermediate 4; 50 mg, 0.155 mmol) and (S)-2-methylpiperazine (62 mg, 0.619 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (45 mg, 72%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 25

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one (Intermediate 4; 50 mg, 0.155 mmol) and (R)-2-methylpiperazine (62 mg, 0.619 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (40 mg, 70%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 26

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one (Intermediate 4; 50 mg, 0.155 mmol) and cis-2,6-dimethylpiperazine (70 mg, 0.619 mmol, 4.0 equiv) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (26 mg, 40%) as a light yellow solid. MS m/z 418.3 [M+H ⁺ ].

Example 27

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one (Intermediate 4; 50 mg, 0.155 mmol) and 2,2-dimethylpiperazine (35 mg, 0.309 mmol, 2.0 equiv) were stirred in DMSO (2 mL) at 125° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (36 mg, 56%) as a light yellow solid. MS m/z 418.3 [M+H ⁺ ].

Example 28

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one (Intermediate 4; 50 mg, 0.155 mmol), DIPEA (0.21 mL, 1.24 mmol, 8 equiv) and 4,7-diazaspiro[2.5]octane dihydrochloride (57 mg, 0.309 mmol, 2.0 equiv) were stirred in DMSO ₍ 2 mL) at 125°C for 2 days. The solvent was removed under high vacuum. The residue was taken up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to afford the title product (17 mg, 26%) as a light yellow solid. MS m/z 416.3 [M+H ⁺ ].

Example 29

2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 50 mg, 0.162 mmol), TEA (0.18 mL, 1.29 mmol, 8 equiv) and (2S,6S)-2,6-dimethylpiperazine dihydrochloride (90 mg, 0.485 mmol, 3.0 equiv) were stirred in DMSO (2 mL) at 140 ° C overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 9/1) to provide the title product (20 mg, 30%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 30

2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one (Intermediate 2; 50 mg, 0.162 mmol), DIPEA (0.22 mL, 1.29 mmol, 8 equiv) and (S)-1,3′-bipyrrolidine dihydrochloride (103 mg, 0.485 mmol, 3.0 equiv) were stirred in NMP (2 mL) at 140° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 9/1) to provide the title product (22 mg, 32%) as a light yellow solid. MS m/z 430.3 [M+H ⁺ ].

Example 31

2-(2-Methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidin-1-ylpyrrolidin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 75 mg, 0.254 mmol), TEA (0.28 mL, 2.03 mmol, 8 equiv) and (S)-1,3′-bipyrrolidine dihydrochloride (162 mg, 0.762 mmol, 3.0 equiv) were stirred in NMP (4 mL) and heated in a microwave at 220° C. for 1 hour. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (12 mg, 11%) as a light yellow solid. MS m/z 416.2 [M+H ⁺ ].

Example 32

7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 75 mg, 0.254 mmol), TEA (0.28 mL, 2.03 mmol, 8 equiv) and (2S,6S)-2,6-dimethylpiperazine dihydrochloride (143 mg, 0.762 mmol, 3.0 equiv) were stirred in DMSO (3 mL) and heated at 140° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to provide the title product (10 mg, 10%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 33

9-Methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (Intermediate 3; 250 mg, 0.808 mmol) and (S)-2-methylpiperazine (405 mg, 4.04 mmol, 5.0 equiv) were stirred in DMSO (6 mL) and heated at 130° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 85/15) to provide the title product (135 mg, 43%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 34

9-Methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (Intermediate 3; 250 mg, 0.808 mmol) and (R)-2-methylpiperazine (405 mg, 4.04 mmol, 5.0 equiv) were stirred in DMSO (6 mL) and heated at 130° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 85/15) to provide the title product (100 mg, 32%) as a light yellow solid. MS m/z 390.3 [M+H ⁺ ].

Example 35

7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (Intermediate 3; 250 mg, 0.808 mmol) and (2S,6R)-2,6-dimethylpiperazine (461 mg, 4.04 mmol, 5.0 equiv) were stirred in DMSO (6 mL) and heated at 130° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated and dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 85/15) to provide the title product (101 mg, 31%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 36

7-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (Intermediate 3; 250 mg, 0.808 mmol) and 2,2-dimethylpiperazine (461 mg, 4.04 mmol, 5.0 equiv) were stirred in DMSO (6 mL) and heated at 130° C. overnight. The solvent was removed under high vacuum. The residue was taken up in CH ₂ Cl ₂ and washed with a saturated aqueous solution of NaHCO _3. The organic layer was separated, dried over Na ₂ SO ₄ and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 85/15) to provide the title product (120 mg, 36%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 37

7-(4,7-diazaspiro[2.5]octan-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (Intermediate 3; 125 mg, 0.404 mmol), _K2CO3 (223 _mg , 1.62 mmol, 4 eq) and 4,7-diazaspiro[2.5]octane dihydrochloride (112 mg, 0.606 mmol, 1.5 eq) were stirred in DMA ₍ 2 mL) and heated at 130°C overnight. The solvent was removed under high vacuum. The residue was taken up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to afford the title product (75 mg, 46%) as a light yellow solid. MS m/z 402.2 [M+H ⁺ ].

Example 38

7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one (Intermediate 3; 125 mg, 0.404 mmol), _K2CO3 (223 _mg , 1.62 mmol, 4 eq) and (2S,6S)-2,6-dimethylpiperazine dihydrochloride (113 mg, 0.606 mmol, 1.5 eq) were stirred in DMA ₍ 2 mL) and heated at 130°C overnight. The solvent was removed under high vacuum. The residue was taken up in _CH2Cl2 and washed with a saturated aqueous solution of _NaHCO3 . The organic layer was separated, dried _{over Na2SO4} and concentrated in vacuo. The crude material was purified by column chromatography (SiO ₂ , CH ₂ Cl ₂ /MeOH=95/5 to 90/10) to afford the title product (50 mg, 31%) as a light yellow solid. MS m/z 404.3 [M+H ⁺ ].

Example 39

7-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

In a sealed tube, 7-fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (Intermediate 1; 200 mg, 0.677 mmol) _{, K2CO3} (374 mg, 2.71 mmol, 4 eq) and (R)-2-ethylpiperazine dihydrochloride (238 mg, 0.606 mmol, 1.5 eq) were stirred in DMA (3 mL) at 100°C for 4 days. The solvent was removed under high vacuum. The crude material was purified by column chromatography ( _SiO2 , _CH2Cl2 / _MeOH = 95/5 to 8/2) to provide the title product (168 mg, 64%) as a light yellow solid. MS m/z 390.2 [M+H ⁺ ].

Biological assays

To describe in more detail and aid in understanding the present specification, the following non-limiting biological examples are provided to more fully illustrate the scope of the specification and are not to be considered as specifically limiting its scope. Such variations of the present specification that are now known or that may be developed later (which will be within the scope of those skilled in the art) are considered to fall within the scope of the present specification and as claimed below. These examples illustrate in vitro and/or in vivo testing of certain compounds described herein and demonstrate the usefulness of the compounds for treating SMA by enhancing the inclusion of exon 7 of SMN2 in the mRNA transcribed from the SMN2 gene. The compounds of formula (I) enhance the inclusion of exon 7 of SMN2 in the mRNA transcribed from the SMN2 gene and increase the level of SMN protein produced by the SMN2 gene, and therefore can be used to treat SMA in human subjects in need thereof. These examples also illustrate in vitro and/or in vivo testing of certain compounds described herein and demonstrate the usefulness of the compounds for enhancing the inclusion of exon 7 of SMN1 in the mRNA transcribed from the SMN1 gene. Thus, the compounds of formula (I) also enhance the inclusion of exon 7 of SMN1 in the mRNA transcribed from the SMN1 gene and increase the level of SMN protein produced by the SMN1 gene.

Determination 1

RT-qPCR Assay of SMN2 Minigene mRNA Splicing in Cultured Cells

A reverse transcription-quantitative PCR (RT-qPCR)-based assay was used to quantify the levels of full-length SMN2 minigene (herein referred to as "FL SMN2mini") mRNA containing SMN2 exon 7 in HEK293H cell lines stably transfected with the minigene and treated with test compounds. The materials used and their respective sources are listed in Table 1 below.

Table 1. Materials used and their sources in the RT-qPCR assay for SMN2 minigene mRNA splicing in cultured cells.

The SMN2-A minigene construct was prepared as described in International Patent Application WO 2009/151546 A1 , page 145, paragraph [00400] to page 147, paragraph [00412] (including Figures 1 and 3 therein).

HEK293H cells stably transfected with the SMN2-A minigene construct (10,000 cells/well) were seeded in 200 μL of cell culture medium (DMEM plus 10% FBS and 200 μg/mL hygromycin) in 96-well flat-bottom plates and immediately vortexed to ensure proper cell dispersion and formation of a uniform cell monolayer. Cells were allowed to attach for 6 hours. Test compounds were serially diluted 3.16-fold in 100% DMSO to generate a 7-point concentration curve. A solution of the test compound (1 μL, 200x in DMSO) was added to each well containing cells and the plates were incubated in a cell culture incubator (37°C, 5% _CO2 , 100% relative humidity) for 24 hours. Two replicates were prepared for each test compound concentration. Cells were then lysed in Cells-To-Ct lysis buffer and the lysates were stored at -80°C.

The full-length SMN2-A minigene and GAPDH mRNA were quantified using the primers and probes indicated in Table 2. Primer SMN forward A (SEQ ID NO. 1) hybridizes to a nucleotide sequence in exon 7 (nucleotides 22 to 40), primer SMN reverse A (SEQ ID NO. 2) hybridizes to a nucleotide sequence in the coding sequence of firefly luciferase, and SMN probe A (SEQ ID NO. 3) hybridizes to nucleotide sequences in exon 7 (nucleotides 50 to 54) and exon 8 (nucleotides 1 to 21). This combination of three oligonucleotides detects only the SMN1 or SMN2 minigene (RT-qPCR) and does not detect the endogenous SMN1 or SMN2 gene.

Table 2. ¹ Primers and probes designed by PTC Therapeutics, Inc.; ² Commercially available from Life Technologies, Inc. (formerly Invitrogen).

SMN forward and reverse primers were used at a final concentration of 0.4 μM. SMN probe was used at a final concentration of 0.15 μM. GAPDH primers were used at a final concentration of 0.2 μM and the probe was used at a final concentration of 0.15 μM.

The SMN2-minigene GAPDH mix (15 μL total volume) was prepared by combining 7.5 μL of 2x RT-PCR buffer, 0.4 μL of 25x RT-PCR enzyme mix, 0.75 μL of 20x GAPDH primer-probe mix, 4.0075 μL of water, 2 μL of 10-fold diluted cell lysate, 0.06 μL of 100 μM SMN forward primer, 0.06 μL of 100 μM SMN reverse primer, and 0.225 μL of 100 μM SMN probe.

PCR was performed at the following temperatures for the indicated times: step 1: 48°C (15 min); step 2: 95°C (10 min); step 3: 95°C (15 sec); step 4: 60°C (1 min); and then steps 3 and 4 were repeated for a total of 40 cycles.

Each reaction mixture contained both the SMN2-A minigene and the GAPDH primer/probe set (multiplex design), allowing for simultaneous measurement of the levels of both transcripts.

Using a modified ΔΔCt method (as described in Livak and Schmittgen, Methods, 2001, 25:402-8), real-time PCR data was used to determine the increase in FL SMN2 mini mRNA abundance relative to cells treated with a vehicle control. The amplification efficiency, E, was calculated from the slopes of the amplification curves for FL SMN2 mini and GAPDH alone. The abundance of FL SMN2 mini and GAPDH mRNA was then calculated as (1 + E) ^{- Ct} , where Ct is the threshold for each amplicon. The abundance of FL SMN2 mini mRNA was normalized to that of GAPDH mRNA. The normalized FL SMN2 mini mRNA abundance from samples treated with the test compound was then divided by the normalized FL SMN2 mini mRNA abundance from cells treated with the vehicle to determine the level of FL SMN2 mini mRNA relative to the vehicle control.

Table 3 provides the EC _1.5x concentrations for full-length SMN2 minigene mRNA production obtained from 7-point concentration data generated according to the above procedure for particular compounds of the invention.

Particular compounds of the invention exhibited an EC _1.5x concentration of ≤ 1 μM for full-length SMN2 minigene mRNA production.

More particularly, compounds of the present invention exhibit an EC _1.5x concentration of ≤ 0.1 μM for full-length SMN2 minigene mRNA production.

Most particular compounds of the invention exhibited EC 1.5x concentrations of ≤ 0.02 μM for full-length SMN2 minigene mRNA production.

Table 3. EC _1.5x concentrations for full-length SMN2 minigene mRNA production.

Determination 2

SMN protein assay in cultured cells

SMN HTRF (homogeneous time-resolved fluorescence) assay was used to quantify the levels of SMN protein in SMA patient fibroblasts treated with test compounds. The materials used and their respective sources are listed in Table 4 below.

Table 4. Materials used in the SMN protein assay in cultured cells and their sources.

The cells were thawed and cultured in DMEM-10% FBS for 72 hours. The cells were trypsinized, counted and resuspended in DMEM-10% FBS to a concentration of 25,000 cells/mL. The cell suspension was plated in a 96-well microtiter plate at 5,000 cells/well and incubated for 3 to 5 hours. The test compound was serially diluted 3.16 times in 100% DMSO to generate a 7-point concentration curve. 1 μL of the test compound solution was transferred to the well containing the cells and the cells were incubated in a cell culture incubator (37°C, 5% _CO2 , 100% relative humidity) for 48 hours. Three replicates were set for each test compound concentration. After 48 hours, the supernatant was removed from the well and 25 μL of RIPA lysis buffer containing protease inhibitors was added to the well and incubated at room temperature for 1 hour with shaking. 25 μL of diluent was added, and then 35 μL of the resulting lysate was transferred to a 384-well plate, where each well contained 5 μL of the antibody solution (1:100 dilution of anti-SMN d2 and anti-SMN kryptate in SMN reconstitution buffer). The plate was centrifuged for 1 minute to allow the solution to reach the bottom of the wells and then incubated overnight at room temperature. The fluorescence of each well of the plate was measured at 665 nm and 620 nm on an EnVision multilabel plate reader (Perkin-Elmer).

The normalized fluorescence signal for each sample, blank, and vehicle control well was calculated by dividing the signal at 665 nm by the signal at 620 nm. The normalized signal accounts for possible fluorescence quenching due to matrix effects of the lysate. The ΔF value for each sample well (a measure of SMN protein abundance as a percentage value) was calculated by subtracting the normalized mean fluorescence of the blank control wells from the normalized fluorescence of each sample well, then dividing this difference by the normalized mean fluorescence of the blank control wells, and multiplying the resulting value by 100. The ΔF value for each sample well represents the SMN protein abundance from the sample treated with the test compound. The ΔF value for each sample well was divided by the ΔF value for the vehicle control well to calculate the fold increase in SMN protein abundance relative to the vehicle control. Table 5 provides the EC _1.5x concentration for SMN protein expression, which was obtained from 7-point concentration data generated according to the above procedure for a particular compound of the present invention.

Particular compounds of the present invention showed an EC _1.5x concentration of ≤ 1 μM against SMN protein expression.

More particularly the compounds of the present invention showed an EC _1.5x concentration of ≤ 100 nM against SMN protein expression.

Most particular compounds of the present invention showed an EC _1.5x concentration of ≤ 30 nM against SMN protein expression.

Table 6 provides the maximum fold increase in SMN protein obtained from 7-point concentration data generated according to the above procedure for particular compounds of the invention.

Particular compounds of the invention showed a maximum fold increase of >1.5.

More particularly compounds of the invention showed a maximum fold increase of >1.7.

Most particular compounds of the invention showed a maximum fold increase of >1.8.

Table 5. EC _1.5x concentrations for SMN protein expression.

Table 6. Maximum fold increase in SMN protein.

Claims (47)

1. Compounds of formula (I) in ^R1 is hydrogen or _C1-7 -alkyl; ^R2 is hydrogen, cyano, _C1-7 -alkyl, _C1-7 -haloalkyl, or _C3-8 -cycloalkyl; ^R3 is hydrogen, _C1-7 -alkyl, or _C3-8 -cycloalkyl; A is one of them. X is N or CH; ^R4 is hydrogen, _C1-7 -alkyl, or -( _CH2 ) _m - ^{NR9R10 ; R5} is hydrogen or _C1-7 -alkyl; ^R6 is hydrogen or _C1-7 -alkyl; R ⁷ is hydrogen or _C1-7 -alkyl; ^R8 is hydrogen or _C1-7 -alkyl; ^R9 and ^R10 are independently selected from hydrogen, _C1-7 -alkyl, and _C3-8 -cycloalkyl; n is 0, 1, or 2; m is 0, 1, 2, or 3; Or ^R4 and ^R5 together form a _C1-7 -alkylene group; Or ^R4 and ^R7 together form a _C1-7 -alkylene group; Or ^R5 and ^R6 together form a _C2-7 -alkylene group; Or ^R5 and ^R7 together form a _C1-7 -alkylene group; Or ^R5 and ^R9 together form a _C1-7 -alkylene group; Or ^R7 and ^R8 together form a _C2-7 -alkylene group; Or ^R7 and ^R9 together form a _C1-7 -alkylene group; Or ^R9 and ^R10 together form a _C2-7 -alkylene group; The premise is that if X is CH, then ^R4 is -( _CH2 ) _m - ^NR9R10 ; ^and The premise is that if X is N and ^R4 is -( _CH2 ) _m ^{- NR9R10} , then m is 2 or 3; And its medicinal salts. 2. The compound according to claim 1, wherein ^R1 is a _C1-7 -alkyl group. 3. The compound according to any one of claims 1 to 2, wherein R ¹ is a methyl group. 4. The compound according to any one of claims 1 to 2, wherein ^R2 is hydrogen or _C1-7 -alkyl. 5. The compound according to any one of claims 1 to 2, wherein ^R2 is hydrogen or methyl. 6. The compound according to any one of claims 1 to 2, wherein ^R3 is hydrogen or _C1-7 -alkyl. 7. The compound according to any one of claims 1 to 2, wherein ^R3 is hydrogen or methyl. 8. The compound according to any one of claims 1 to 2, wherein X is N. 9. The compound according to any one of claims 1 to 2, wherein n is 1. 10. The compound according to any one of claims 1 to 2, wherein ^R4 is hydrogen, methyl or -( _CH2 ) _m ^{- NR9R10} . 11. The compound according to any one of claims 1 to 2, wherein R ⁴ is hydrogen. 12. The compound according to any one of claims 1 to 2, wherein R ⁵ is hydrogen, methyl or ethyl. 13. The compound according to any one of claims 1 to 2, wherein ^R5 is methyl. 14. The compound according to any one of claims 1 to 2, wherein R ⁶ is hydrogen or methyl. 15. The compound according to any one of claims 1 to 2, wherein R ⁶ is hydrogen. 16. The compound according to any one of claims 1 to 2, wherein R ⁷ is hydrogen or methyl. 17. The compound according to any one of claims 1 to 2, wherein R ⁸ is hydrogen. 18. The compound according to any one of claims 1 to 2, wherein m is 0. 19. The compound according to any one of claims 1 to 2, wherein ^R4 and ^R5 together form a propylidene group. 20. The compound according to any one of claims 1 to 2, wherein ^R5 and ^R6 together form an ethylene. 21. The compound according to any one of claims 1 to 2, wherein ^R9 and ^R10 together form a butylene group. 22. The compound according to any one of claims 1 to 2, wherein A is selected from the group consisting of: ^R4 , ^R5 , ^R6 , ^R7 and ^R8 are defined as in any one of claims 1 to 2. 23. The compound according to any one of claims 1 to 2, wherein A is selected from the group consisting of: ^R4 and ^R6 are defined as in any one of claims 1 to 2. 24. The compound according to any one of claims 1 to 2, wherein A is selected from the group consisting of: ^R4 , ^R6 and ^R7 are defined as in any one of claims 1 to 2. 25. The compound according to any one of claims 1 to 2, wherein A is selected from the group consisting of: 26. The compound according to any one of claims 1 to 2, wherein A is selected from the group consisting of: 27. The compound according to any one of claims 1 to 2, wherein the compound is selected from the group consisting of: 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-(4-methylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-(1,4-diazacycloheptane-1-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-(1,4-diazacycloheptane-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aS)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aR)-8a-methyl-1,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidine-1-ylpyrrolidine-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-pyrrolidine-1-ylpyrrolidine-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(3,3-dimethylpiperazin-1-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-(3,3-dimethylpiperazin-1-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidine-1-ylpyrrolidine-1-yl]pyrido[1,2-a]pyrimidin-4-one; 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-pyrrolidine-1-ylpyrrolidine-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 9-Methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 9-Methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)-7-[(3R)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(3,3-dimethylpiperazin-1-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(3S,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(3R)-3-ethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; And its medicinal salts. 28. The compound according to any one of claims 1 to 2, wherein the compound is selected from the group consisting of: 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-[(3S,5R)-3,5-dimethylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-[(8aS)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-7-[(3S)-3-methylpiperazin-1-yl]pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one; 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 7-(4,7-diazaspiro[2.5]oct-7-yl)-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; And its medicinal salts. 29. The compound according to any one of claims 1 to 2, wherein the compound is 7-[(8aR)-3,4,6,7,8,8a-hexahydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof. 30. The compound according to any one of claims 1 to 2, wherein the compound is 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, or a pharmaceutical salt thereof. 31. The compound according to any one of claims 1 to 2, wherein the compound is 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, or a pharmaceutical salt thereof. 32. The compound according to any one of claims 1 to 2, wherein the compound is 7-(4,7-diazaspiro[2.5]oct-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-9-methyl-pyrido[1,2-a]pyrimidin-4-one, or a pharmaceutical salt thereof. 33. The compound according to any one of claims 1 to 2, wherein the compound is 7-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one, or a pharmaceutically acceptable salt thereof. 34. The compound according to any one of claims 1 to 2, wherein the compound is 35. Compounds of formula (VI) Wherein ^R1 , ^R2 and ^R3 are as described in any one of claims 1 to 6; Y is a halogen or trifluoromethanesulfonate; And its salt. 36. The compound of formula (VI) according to claim 35, wherein Y is fluorine, chlorine, bromine, iodine or trifluoromethanesulfonate. 37. The compound of formula (VI) according to claim 35, wherein Y is fluorine. 38. The compound of formula (VI) according to any one of claims 35 to 37, wherein the compound is selected from the group consisting of: 7-Fluoro-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-Dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-pyrido[1,2-a]pyrimidin-4-one; 7-Fluoro-9-methyl-2-(2-methylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one; 2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)-7-fluoro-9-methyl-pyrido[1,2-a]pyrimidin-4-one; And its salt. 39. A method for preparing a compound of formula (I) according to any one of claims 1 to 28, the method comprising an aromatic nucleophilic substitution reaction between a compound of formula (VI) according to any one of claims 35 to 38 and a compound of formula MA by heating in a solvent, wherein A is defined as any one of claims 1 to 28, ^R1 , ^R2 and ^R3 are defined as any one of claims 1 to 6, Y is defined as any one of claims 35 to 38, M is hydrogen, sodium or potassium, and wherein M is attached to A via a nitrogen atom of A. 40. The method according to claim 39, wherein the aromatic nucleophilic substitution reaction is carried out at a temperature of 80°C to 200°C. 41. The method according to any one of claims 39 or 40, wherein the solvent for the aromatic nucleophilic substitution reaction is selected from dimethyl sulfoxide, N-methylpyrrolidone, and dimethylformamide. 42. The method according to any one of claims 39 or 40, wherein M is hydrogen. 43. A compound of formula (I) according to any one of claims 1-28 that can be obtained by the method according to any one of claims 39 to 42. 44. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1-34, or a pharmaceutical salt thereof, and one or more pharmaceutical excipients. 45. A compound of formula (I) according to any one of claims 1-34, or a pharmaceutical salt thereof, used as a therapeutic active substance. 46. A compound of formula (I) according to any one of claims 1-34 or a pharmaceutical salt thereof for the treatment or prevention of spinal muscular atrophy (SMA). 47. Use of a compound of formula (I) according to any one of claims 1-34 or a pharmaceutical salt thereof for the preparation of a medicament for the treatment or prevention of spinal muscular atrophy (SMA).