Patent Description:
γ-hydroxybutyric acid (GHB) is present in the brain in micromolar concentrations and binds with high affinity to a specific protein (Bay et al. <NUM>), recently identified as CaMK2a, one of the most abundant proteins in the postsynaptic density. CaMK2a is a major regulator of synaptic signaling through its phosphorylation of ion channels and neurotransmitter receptors and is intimately involved in long-term potentiation and synaptic plasticity, and thus higher brain functions such as learning and memory (Hell, <NUM>).

Due to its central role in regulating synaptic function, CaMK2a is involved in most neurodegenerative diseases and a promising drug target, yet unexplored due to the unavailability of small-molecule brain-penetrant ligands with selectivity for the 2a subtype.

CaMK2a is modulated by an inhibitory peptide Tat-CN21, which has shown promise in post-stroke treatment in preclinical studies (Coultrap et al. In addition to potential issues with stability and cell penetrance as well as bioavailability related to general use of peptides, Tat-peptides are further known to cause hypertension. From data obtained by the inventors, GHB and related small-molecule analogues bind directly to CaMK2a, but to a site distinct from CN21.

GHB is highly efficacious in treating cataplexy and excessive daytime sleepiness in relation to narcolepsy and shows better efficacy than the GABAB selective agonist baclofen. Specifically, GHB uniquely works on daytime symptoms (Huang and Guilleminault, <NUM>). Compounds related to GHB may thus have efficacy in narcolepsy through effects on CaMK2a and/or GABAB receptors.

GHB is currently the best treatment for cataplexy.

The inventors have previously published two series of selective ligands with mid to high nanomolar affinity for GHB binding sites i.e. CaMK2a: i) The conformationally restricted.

GHB analogue <NUM>-hydroxycyclopent-<NUM>-enecarboxylic acid (HOCPCA), displaying <NUM> times higher affinity than GHB, and ii) the <NUM>-substituted biaromatic GHB analogues, represented by <NUM>-(<NUM>-benzyloxy)phenyl)-<NUM>-hydroxybutanoic acid (BnOPh-GHB), displaying <NUM> times improved affinity compared to GHB (Bay et al. Overall, the chemical diversity of ligands for the high-affinity GHB binding site is sparse and, in essence, only represented by the core structures of GHB, HOCPCA and (E)-<NUM>-(<NUM>-hydroxy-<NUM>,<NUM>,<NUM>,<NUM>-tetrahydro-<NUM>H-benzo[<NUM>]annulen-<NUM>-ylidene)acetic acid (NCS-<NUM>). GHB, HOCPCA and an analogue of NCS-<NUM> have been shown to work in mouse model of acute stroke. Additional development of novel small-molecule chemical entities is needed to advance and explore CaMK2a as a drug target in brain injuries and central hypersomnias.

<NPL>, discloses the synthesis and SAR studies of <NUM>-aminophthalazinium salts as GABAA receptor antagonists. <NPL>) discloses the synthesis and analgesic activity of imid-azo[<NUM>,<NUM>-b]pyridazine-<NUM>-acetic acid derivatives. <NPL>) discloses a study of the effect of a CAMKII inhibitor GS-<NUM> on diastolic SR Ca leak and CaMKII-dependent pro-arrhythmic activity.

γ-Hydroxybutyric acid (GHB) is a neuromodulator, present in micromolar concentration in the mammalian brain as a metabolite of γ-aminobutyric acid (GABA). GHB is used clinically as a prescribed drug in narcolepsy, and as a recreational drug (e.g. Fantasy). GHB displays low affinity (millimolar) binding to the GABAB receptor and high-affinity (nano- to micromolar) to an abundant highly specific protein (Bay et al. , <NUM>), recently identified to be CaMK2a.

The references to methods of treatment in this description are to be interpreted as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for the treatment of the human (or animal) body by therapy (or for diagnosis).

Until recently now, no small-molecule ligands for selectively targeting CaMK2a have been described. A specific class of GHB-related analogues, herein represented by JON-<NUM> (termed JON-<NUM> analogues) are first-in-class compounds with promise to treat conditions associated with CaMK2a malfunction. In one setting, compounds may be neuroprotective after an acute insult or after lack of oxygen to the brain. In another setting, compounds may improve symptoms associated with sleep disturbances and cataplexies, e.g. in the sleep disorder narcolepsy. Effects may be mediated through effects on CaMK2a autophosphorylation, substrate phosphorylation or kinase activity in general.

As CaMK2a is an intracellular protein, compounds must pass the plasma membrane to act. Being carboxylic acids, GHB ligands are charged at physiological pH. As a consequence, the compounds as such depend on active transport to enter the brain or neurons. At the blood-brain barrier, this is governed by the monocarboxylate transporter <NUM> where HOCPCA and GHB are substrates (Thiesen et al. Neurons express MCT1 and <NUM>, supposedly mediating the uptake here. Another way to promote brain penetration is through the attachment of a chemical pro-moiety so as to promote passive diffusion or target specific transporters involved in brain uptake (e.g. the large amino acid transporter (LAT1)) (Puris et al.

The invention provides compounds having the following general formula I:
<CHM>
wherein X is C, R<NUM> and R<NUM> are different and selected from H, F, I, Cl, -OH, - NH<NUM>, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl wherein butyl, pentyl and hexyl are linear or branched, -O-C<NUM>-C<NUM>-alkyl, -O-benzyl (-O-Bn), wherein C<NUM>-C<NUM>-alkyl is linear or branched and wherein benzyl is unsubstituted or substituted with one or more halogen and/or one or more -C<NUM>-C<NUM>-alkyl or -O-C<NUM>-C<NUM>-alkyl;
or
X is C, R<NUM> and R<NUM> are the same and selected from F, I, Cl, -OH, -NH<NUM>, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl wherein butyl, pentyl and hexyl are linear or branched, -O- C<NUM>-C<NUM>-alkyl, -O-benzyl (-O-Bn), wherein C<NUM>-C<NUM>-alkyl is linear or branched and wherein benzyl is unsubstituted or substituted with one or more halogen and/or one or more C<NUM>-C<NUM>-alkyl or -O-C<NUM>-C<NUM>-alkyl;
and.

In an alternative embodiment, the invention relates to compounds of formula I, wherein:.

The invention also relates to the compounds mentioned herein in any form including geometric isomers, tautomers, enantiomers, racemic mixtures, or deuterated derivatives.

The pharmaceutically acceptable salts include salts of organic or inorganic acids or salts of bases. Specific examples are e.g. salt like hydrobromide, hydrochloride, trifluro-acetate or salts like alkali salts, e.g. sodium or lithium or alkali earth metal salts. C<NUM>-C<NUM>-alkyl is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert. butyl, pentyl, pentyl, hexyl; butyl, pentyl and hexyl may be linear or branched.

Specifically, the present invention provides novel compounds according to formula I, wherein R<NUM> and R<NUM> are different and selected from H, F, I, Cl, -O-C<NUM>-C<NUM>-alkyl and -O-Bn, and R<NUM> and alkyl being as defined above. Specifically, the present invention provides novel compounds according to formula I, wherein R<NUM> and R<NUM> are the same and selected from F, I, Cl, -O-C<NUM>-C<NUM>-alkyl and -O-Bn, and R<NUM> and alkyl being as defined above.

The present invention also provides novel compounds according to formula I, wherein R<NUM> and R<NUM> are different and selected from H, F, I, Cl, -O-C<NUM>-C<NUM>-alkyl and -O-Bn , and R<NUM> is selected from -OH, -O-C<NUM>-C<NUM>-alkyl, -C<NUM>-C<NUM>-alkyl-O-C(=O)-C<NUM>-C<NUM>-alkyl, and -NH-substituted aryl, wherein alkyl and aryl are as defined above. The present invention also provides novel compounds according to formula I, wherein R<NUM> and R<NUM> are the same and selected from F, I, Cl, -O-C<NUM>-C<NUM>-alkyl and -O-Bn , and R<NUM> is selected from -OH, -O-C<NUM>-C<NUM>-alkyl, -C<NUM>-C<NUM>-alkyl-O-C(=O)-C<NUM>-C<NUM>-alkyl, and -NH-substituted aryl, wherein alkyl and aryl are as defined above.

Compounds of the present invention are also compounds according to formula I, wherein:.

and -O-Bn, and R<NUM> is as defined above.

Compounds of particular interest are those wherein X is C.

Within the scope of the present invention are compounds, wherein R<NUM> is selected from - NH<NUM>, -O-C<NUM>-C<NUM>-alkyl, -C<NUM>-C<NUM>-alkyl- O-C(=O)-C<NUM>-C<NUM>-alkyl, -NH-C<NUM>-C<NUM>-alkyl, -NH-aryl, -NH-substituted aryl, wherein -C<NUM>-C<NUM>-alkyl islinear or branched, aryl may be unsubstituted or substituted with one or more amino acid residues to form a compound in the following formula II illustrated with a substituted aryl being represented by phenylalanine, but any other amino acid residue is also within the scope of the present invention; i.e. compounds wherein R<NUM> is not -OH.

In another aspect, the present invention relates to compounds of formula I, where X is C, R<NUM> and R<NUM> are different and is either H or -OH, and R<NUM> is OH. In another aspect, the present invention relates to compounds of formula I, where X is C, R<NUM> and R<NUM> are the same and is -OH, and R<NUM> is OH.

In a further aspect, the invention relates to compounds of formula I, where X is C, R<NUM> and R<NUM> are different and selected from H, -OH, F, I, Cl, NO<NUM> and -O-Bn, and R<NUM> is selected from -O-C<NUM>-C<NUM>-alkyl, -C<NUM>-C<NUM>-alkyl- O-C(=O)-C<NUM>-C<NUM>-alkyl, -NH-C<NUM>-C<NUM>-alkyl. In a further aspect, the invention relates to compounds of formula I, where X is C, R<NUM> and R<NUM> are the same and selected from -OH, F, I, Cl, NO<NUM> and -O-Bn, and R<NUM> is selected from - O-C<NUM>-C<NUM>-alkyl, -C<NUM>-C<NUM>-alkyl- O-C(=O)-C<NUM>-C<NUM>-alkyl, -NH-C<NUM>-C<NUM>-alkyl.

In an even further aspect, the invention relates to compounds of formula I, where:.

Compounds disclaimed from the present invention and known from a previous publication are compounds having formula I, wherein:.

Specific examples of compounds of the invention or for use in accordance with the present invention are given in the examples herein.

The present invention discloses a novel chemical scaffold of formula I (representing inter alia JON-<NUM> (example <NUM>) and the compounds exemplified herein) with affinity for CaMK2a as shown in binding assays to native and recombinant CaMK2a. Furthermore, it is contemplated that the compounds, wherein R<NUM> is -OH or -NH<NUM> are therapeutically active, but there may be compounds that are not capable of entering the brain. For such compounds, the R3 group may be changed to a pro-moiety such as e.g. an ester.

The pro-moiety makes it possible for the compound to enter the brain either passively, or facilitated by a transporter such as LAT1, in which the pro-moiety is cleaved off leaving the therapeutically active compound within the brain. Such prodrugs are also part of the invention and within the scope of formula I and thus part of the present invention as defined in the claims.

As shown in the examples, the present invention provides an ester prodrug (pro-JON-<NUM> (<NUM>); example <NUM>, a prodrug of JON-<NUM>) which after systemic administration displays good plasma stability, freely enters the brain and is cleaved to the parent drug. This is in contrast to JON-<NUM> which is not brain-permeable after intraperitoneal (i. ) injection. JON-<NUM> displays only weak affinity for GABAA receptors, which may be of interest in conditions with an aberrant GABAA receptor activity.

The physico-chemical properties of pro-JON-<NUM>, entrance into the brain, and binding to the target make small molecules of this formula vastly interesting. It is contemplated that the compounds of formula (I) also have acceptable physico-chemical properties. Given that no CaMK2a small-molecule non-peptide selective ligands exist, they may also represent a novel mechanism of action.

The inventors herein demonstrate that JON-<NUM> and novel analogues bind with high affinity to CaMK2a and can be delivered to the brain using the prodrug strategy.

The compounds of formula I may be synthesized in accordance with the following schemes:
<CHM>
<CHM>.

In this specific aspect of the invention, all compounds are contemplated to be useful in the diseases/conditions mentioned in the following; i.e. also the compounds mentioned in the table above. However, some of the compounds wherein R<NUM> is -OH may not reach the brain, but are active if delivered to the brain. If this is the case, the -OH group is changed to one of the other R<NUM> groups mentioned herein so that they contain a pro-moiety that enables transport over the brain barrier and which - after entering the brain - is cleaved off.

One embodiment provides for a compound of general formula I
<CHM>
or a pharmaceutically acceptable salt thereof,.

The compounds of the invention can be used as a medicament for treating diseases. Thus, the invention also relates to compounds of formula I for use as a medicament.

In one embodiment, the compounds of the present invention are contemplated to be useful in the treatment of acute brain injuries. Brain injuries may be caused by an acute primary cerebral or ischemic insult, e.g. traumatic brain injury, stroke, subarachnoid haemorrhage, neonatal hypoxia-ischemia encephalophathy or associated in utero complications, haemodynamic shock with cardiac arrest, and global hypoperfusion during surgery or as a result from heart failure.

In another embodiment, the compounds are contemplated to have beneficial effects in preventing and/or alleviating central hypersomnias and cataplexies. Central hypersomnias include idiopathic hypersomnia, recurrent hypersomnia such as Klein-Levin syndrome and narcolepsy including with cataplexy (narcolepsy type <NUM>; narcolepsy-cataplexy syndrome; NRCLP1; narcolepsy with low hypocretin) and narcolepsy without cataplexy (narcolepsy type <NUM>; narcolepsy with normal hypocretin).

Narcolepsy Type <NUM> and Type <NUM> are sleep disorders characterised by excessive daytime sleepiness and narcolepsy Type <NUM> is further characterised by cataplexy. Cataplexy is characterised by sudden loss of muscle tone. The duration of cataplexy is usually short, ranging from a few seconds to several minutes and recovery is immediate and complete. The loss of muscle tone varies in severity and ranges from a mild sensation of weakness with head drop, facial sagging, jaw drop, slurred speech and buckling of the knees to complete postural collapse, with a fall to the ground. Cataplexy is usually precipitated by emotion that usually has a pleasant or exciting component, such as laughter, elation, pride, anger or surprise.

Besides excessive daytime sleepiness and cataplexy (in narcolepsy type <NUM>), individuals affected by narcolepsy often present symptoms such as sleep fragmentation, abnormal rapid eye movement sleep, nocturnal sleep disruption, paralysis during sleep onset or during awakening, and/or hypnagogic hallucinations. Similar symptoms are shown also by individuals affected by Narcolepsy Due to Medical Condition (NDMC), a group of disorders also known as secondary or symptomatic narcolepsy.

Examples of medical conditions causing narcolepsy symptoms including cataplexy are: tumors, sarcoidosis, arteriovenous malformations affecting the hypothalamus, multiple sclerosis plaques impairing the hypothalamus, paraneoplastic syndrome antt-Ma2 antibodies, Neimann-Pick type C disease or Coffin-Lowry syndrome. Examples of medical conditions commonly causing narcolepsy symptoms without cataplexy are: head trauma, myotonic dystrophy, Prader-Willi syndrome, Parkinson's disease or multisystem atrophy.

Cataplexy is a hallmark of narcolepsy but may also be associated with specific lesions located primarily in the lateral and posterior hypothalamus, as e.g. tumors (astrocytoma, glioblastoma, glioma, craniopharyngioma and subependynoma) and arterio-venous malformations. Conditions in which cataplexy can be seen include ischemic events, multiple sclerosis, head injury, paraneoplastic syndromes, and infections, such as encephalitis. Cataplexy may occur transiently or permanently due to lesions of the hypothalamus that were caused by surgery, especially in difficult tumor resections. In infancy, cataplexy can be seen in association with other neurological syndromes such as Niemann-Pick type C disease.

The term 'acute brain injury' as used herein refers to a primary cerebral or ischemic insult that damages brain tissue in an acute manner, but also initiates cascades of devastating neurotoxic effects. Examples include traumatic brain injury, stroke, subarachnoid haemorrhage, neonatal hypoxia-ischemia encephalopathy or associated in utero complications, haemodynamic shock with cardiac arrest, and global hypoperfusion during surgery or as a result from heart failure.

The term 'autophosphorylation' as used herein refers to the phosphorylation of CaMK2a on residue Thr286.

The term 'CaMK2a' as used herein refers to Calcium/calmodulin-dependent protein kinase type <NUM> alpha.

The term 'cataplexy' is a sudden and transient episode of muscle weakness accompanied by full conscious awareness, typically triggered by emotions such as laughing, crying, or terror.

Disorders of excessive daytime sleepiness related to the central nervous system, i.e., the brain. These disorders share in common the predominant symptom of daytime sleepiness. Various types of central hypersomnias exist, including idiopathic hypersomnia, recurrent hypersomnia such as Klein-Levin syndrome, and narcolepsy.

The term 'JON-<NUM> analogue' as used herein refers to a specific class of GHB-related analogues, typified by <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid.

This refers to the L-type amino acid transporter expressed at the blood-brain-barrier.

The term 'neuroprotective' as used herein refers to the ability of a chemical substance to prevent nerve cell damage such as that induced by acute injury to the brain.

The term 'photothrombotic focal ischemia' as used herein refers to method of introducing a cortical infarction through a photochemical reaction with a light-sensitive dye delivered by i.

A pharmacological inactive substance that is the modified form of a pharmacological active compound to which it is converted e.g. by enzymatic action in the body.

A moiety which is linked to a pharmacological active compound to obtain a prodrug.

The term 'recombinant' as used herein refers to DNA sequences that have been transfected into and expressed as proteins in HEK293T cells.

According to previously published protocols, rat (Sprague-Dawley) cortical synaptic membranes (P2 synaptosomes) were prepared and stored at -<NUM> until the day of assay (Wellendorph et al. Membranes were then washed three times with binding buffer (<NUM> potassium phosphate buffer pH <NUM>). The binding protocol was performed in <NUM>-well microplates. In brief, compounds were incubated with <NUM> [<NUM>H]NCS-<NUM> radioligand and membranes for <NUM> at <NUM>-<NUM>, filtered through GF/C filter plates (PerkinElmer) using a <NUM>-well harvester (Packard), rapidly washed three times with ice-cold binding buffer, and filter plates dried. CPM values were determined using liquid scintillation counting in a TopCount NXT Microplate Scintillation & Luminescence Counter (PerkinElmer). Curves were generated using non-linear regression (GraphPad Prism <NUM>, GraphPad Prism Software, San Diego, CA, USA).

[<NUM>H]HOCPCA binding to recombinant CaMK2a expressed in HEK293T cells HEK293T were cultured using standard conditions, using Dulbecco's modified Eagle Medium with GlutaMax, <NUM>% fetal bovine serum and <NUM>% penicillin-streptomycin, and incubated at <NUM> in a humidified atmosphere of <NUM>% O<NUM> and <NUM>% CO<NUM>. Cells were transfected with cmyc-tagged rat CaMK2a (Origene construct RR201121) using PolyFect (Qiagen, West Sussex, UK) according to the manufacturer's protocol. Whole cell homogenates were prepared <NUM> hr post-transfection by washing the cells with ice-cold 1x PBS and harvesting by scraping. Cells were collected and centrifuged for <NUM> at <NUM> x g. Cell pellets were resuspended in ice-cold 1x PBS and homogenized using <NUM> x <NUM> zirkonium beads in a bullet blender for <NUM> at max speed (NextAdvance, NY, USA). Homogenates were cleared by centrifugation (<NUM>, <NUM>, <NUM> x g). Protein concentration was determined using the Bradford protein assay. <NUM>-<NUM>µg proteins were incubated with <NUM> <NUM>H-HOCPCA (Vogensen et al. , <NUM>) and test compound in <NUM> total volume for <NUM> hr at <NUM>-<NUM>. Nonspecific binding was determined with <NUM>-<NUM> GHB. Proteins were then precipitated by addition of ice-cold acetone (4x of the assay volume), vortexing and incubation at -<NUM> for <NUM> hr. Proteins were filtered rapidly through GF/C unifilters (Whatman) and washed using a <NUM>-well harvester. The dried filters were added scintillation liquid and radioactivity measured on a Tricarb <NUM> Scintillation counter (Packard).

To further confirm direct binding of JON-<NUM> to CaMK2a, SPR was used. SPR measurements were performed at <NUM> using a Pioneer FE instrument from PALL FortéBio. Full-length human recombinant CaMK2a with an N-terminal GST fusion protein (Carna Biosciences, number <NUM>-<NUM>) was immobilized to a biosensor chip by amine coupling to <NUM> response units (RU) using a <NUM> sodium acetate pH <NUM> immobilization buffer. JON-<NUM> was injected in <NUM>-fold serial dilution (ranging from <NUM>-<NUM>) over immobilized CaMK2a using a HBS running buffer pH <NUM> (<NUM> Hepes, <NUM> NaCl, <NUM>% tween <NUM> DTT) supplemented with Ca<NUM>+ (<NUM>). Calmodulin (Sigma) was used as a positive control to evaluate activity of the immobilized protein. Calmodulin was injected in <NUM>-fold serial dilution (ranging from <NUM>-<NUM>) over immobilized CaMK2a. Between calmodulin injections, the biosensor chip surface was regenerated by injections of HBS buffer supplemented with <NUM> EDTA. The data were analyzed using Qdat Data Analysis Tool version <NUM>. <NUM> (PALL FortéBio). The sensorgrams were corrected for buffer bulk effects and unspecific binding of the samples to the chip matrix by blank and reference surface subtraction (flow cell channel activated by injection of EDC/NHS and inactivated by injection of ethanolamine). The equilibrium dissociation constants (KD) were estimated by global non-linear regression analysis and a reversible <NUM>-step interaction reaching steady state at the end of the analyte injection.

Metabolic stability analysis was performed on a Bruker Avance Ultra High Performance Liquid Chromatography (UHPLC) (Bruker Daltonik, Bremen, Germany) equipped with a CTC-PAL-xt autosampler with cooled sample compartments (<NUM>) and a column oven held at <NUM>. The UHPLC eluate was directed to a Bruker Evoq Elite triple quad-ropole MS equipped with a heated electrospray ionization source operated in positive ionization mode. Separation of <NUM>µL sample was obtained using a Phenomenex (Phenomenex, Torrance, CA, USA) Kinetex XB-C<NUM> column (<NUM> × <NUM> i. , <NUM> particle size) equipped with a guard column. The analytes were separated using a solvent system consisting of water/acetonitrile (<NUM>:<NUM>, v/v) (eluent A) and water/acetonitrile (<NUM>:<NUM>, v/v) (eluent B); both acidified with <NUM>% formic acid. The eluent flow rate was maintained at <NUM>/min with the following gradient elution profile: <NUM>: <NUM>% B; <NUM>: <NUM>% B; <NUM>: <NUM>% B, followed by three min equilibration at <NUM>% B. Two ion transitions were monitored for all analytes and internal standard.

The mouse liver microsome assay was performed by transferring <NUM>µL of a <NUM> solution of the test compound (<NUM>% DMSO), dissolved in potassium phosphate monobasic buffer (pH <NUM>) containing <NUM> MgCl<NUM>, to <NUM> wells in a microtiter plate. To each well, <NUM> of a <NUM>/mL mouse liver (Sigma Aldrich) in buffer was added, and the microtiter plate were pre-incubated for <NUM> at <NUM> in a plate shaker at <NUM> rpm. After pre-incubation, <NUM>µL <NUM> NADPH regenerating system, consisting of <NUM> NADP+, <NUM> glucose-<NUM>-phosphate, and <NUM> U/mL glucose-<NUM>-phosphate dehydrogenase dissolved in buffer, was added to start the assay. In triplicates, immediately after adding the NADPH solution, <NUM>µL was transferred to an Eppendorf tube, containing <NUM> ice-cold acetonitrile with <NUM> ng/mL internal standard (<NUM>). Similarly, samples were prepared after <NUM> and <NUM> incubation at <NUM>. The samples were vortexed for <NUM> seconds and centrifuged at <NUM>,<NUM> × g for <NUM> minutes before analyzing the supernatant. The individual analyte responses were normalized to the internal standard. Diazepam was used as positive control.

The mouse live microsome assay was performed by transferring <NUM>µL of a <NUM> solution of the test compound (<NUM>% DMSO), dissolved in PBS buffer (pH <NUM>), to <NUM> wells in a microtiter plate. To each well, <NUM>µL mouse plasma, harvested from in-house mice into K2-EDTA tubes and centrifuged at <NUM>,<NUM> × g, was added. In triplicates, immediately after adding mouse plasma, <NUM>µL was transferred to an Eppendorf tube, containing <NUM>µL ice-cold acetonitrile with <NUM> ng/mL internal standard (<NUM>). Similarly, samples were prepared after <NUM> and <NUM> incubation at <NUM>. The samples were vortexed for <NUM> seconds and centrifuged at <NUM>,<NUM> × g for <NUM> minutes before analyzing the supernatant. The individual analyte responses were normalized to the internal standard. Enalapril was used as positive control.

To evaluate the ability of pro-JON-<NUM> (<NUM>) and JON-<NUM> to penetrate the blood-brain barrier, a brain-to-plasma distribution ratio (B/P ratio) was obtained. All subjects were male C57BL/6JRj mice (<NUM>-<NUM>, Janvier Labs, France) housed in groups of three-four with ad libitum access to food and water. The mice were maintained under diurnal conditions (<NUM>-h light/dark cycle with lights on at <NUM> AM) in a temperature (<NUM>±<NUM>) and humidity-controlled room (<NUM>±<NUM>%). Ethical permission for the procedures used in this in vivo study was granted by the Danish Animal Experiments Inspectorate.

Compounds were dissolved in PEG-<NUM> (Sigma) at <NUM>% of the final required volume. The final volume was then made up by <NUM>% (w/v) hydroxypropyl-beta-cyclodextrin (Sigma Aldrich) in sterile saline. The solution was sonicated for <NUM>. The JON-<NUM> solution was pH-adjusted to <NUM> with <NUM>% <NUM> NaOH.

Both compounds were injected i. at a dose of <NUM>/kg (n=<NUM> for each compound). Two additional mice did not receive any treatment and functioned as 'blank' animals. After <NUM>, the animals were sacrificed by decapitation, trunk blood was collected in EDTA-coated tubes and the brain was gently removed. Blood and tissues were temporarily stored on ice until plasma and brain homogenate preparation. Blood plasma was separated from cells by centrifugation for <NUM> at <NUM>,<NUM> x g at <NUM> and plasma harvested. Brain homogenate was prepared by homogenizing the whole brain with <NUM> volumes (w:v) of ice-cold sterilised water using a bullet blender (NextAdvance). The plasma and brain homogenate were stored on ice.

Before UHPLC-MS analysis, the plasma and brain samples were prepared by mixing <NUM>µL of plasma or brain homogenate with <NUM>µL of blank brain homogenate or blank plasma, respectively. The mixed samples were protein precipitated with <NUM>µL internal standard solution and centrifuged for <NUM> at <NUM>,<NUM> x g at <NUM>-<NUM>. The supernatants were collected and added to HPLC vials for LC-MS analysis, which was performed as described above for metabolic stability analysis.

Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources.

The compounds of the general formula I, wherein R<NUM> is -OH may be prepared as given below from the appropriate substituted pyridazin-<NUM>-amine according to the procedures in Examples <NUM>-<NUM> and Examples <NUM>-<NUM>.

The compounds of the general formula I, wherein R<NUM> is different from -OH may be prepared as given below from the appropriate substituted <NUM>-(imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid according to the procedures in Examples <NUM>-<NUM> and Examples <NUM>-<NUM>.

Step <NUM>: <NUM>-(<NUM>-chlorophenyl)pyridazin-<NUM>-amine (Petrignet et al. <NUM>) (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) were dissolved in EtOH (<NUM>) and heated at <NUM> for <NUM>. Upon cooling to room temperature, the reaction mixture was evaporated in vacuo and the resulting residue was suspended in sat. NaHCO<NUM> (<NUM>) and the mixture was extracted with CH<NUM>Cl<NUM> (<NUM> × <NUM>). The combined organic phases were washed with water (<NUM> × <NUM>), dried over anhydrous Na<NUM>SO<NUM>, filtered, and evaporated in vacuo. Purification by DCVC (Heptane/EtOAc, <NUM>-<NUM>% EtOAc) afforded ethyl <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (1a) (<NUM>, <NUM>%) as white sticky solid. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Ethyl <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) was dissolved in aq. HCl (<NUM>, <NUM>) and heated at <NUM> for <NUM>. Upon cooling to rt. , the reaction mixture was filtered and evaporated in vacuo to afford <NUM>-(<NUM>-(<NUM>-Chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM>%) as light brown solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed a described in example <NUM> (step <NUM>) using <NUM>-(<NUM>-chlorophenyl)pyridazin-<NUM>-amine (Guery et al. <NUM>) (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) in EtOH (<NUM>) Purification by DCVC (Heptane/EtOAc, <NUM>-<NUM>% EtOAc) afforded ethyl <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%), as light brown solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed as describe in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) and aq. HCl (<NUM>, <NUM>) affording <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, quant. ) as brown solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (dt, J = <NUM>, <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed a described in example <NUM> (step <NUM>) using <NUM>-(<NUM>-(<NUM>-benzyloxy)phenyl) pyridazin-<NUM>-amine (Navande et al. <NUM>) (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) in EtOH (<NUM>). Purification by DCVC (Heptane/EtOAc, <NUM>-<NUM>% EtOAc) affording ethyl <NUM>-(<NUM>-(<NUM>-(benzyloxy)phenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) as brown oil. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed as describe in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-(benzyloxy)phenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) in THF (<NUM>), water (<NUM>) and aq. NaOH (<NUM>, <NUM>). Evaporation in vacuo afforded <NUM>-(<NUM>-(<NUM>-benzyloxy)phenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM>%) as brown solid: mp ><NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed a described in example <NUM> (step <NUM>) using <NUM>-(<NUM>-(<NUM>-benzyloxy)phenyl) pyridazin-<NUM>-amine (Iqbal et al. <NUM>) (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) in EtOH (<NUM>). Purification by DCVC (Heptane/EtOAc, <NUM>-<NUM>% EtOAc) afforded ethyl <NUM>-(<NUM>-(<NUM>-benzyloxy)phenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) as light brown solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed as describe in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-(benzyloxy)phenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) in aq. HCl (<NUM>, <NUM>). Upon cooling to rt. , the target compound (<NUM>, <NUM>%) was isolated by filtration as white solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, DMSO-d<NUM>): δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, DMSO-d<NUM>): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed a described in example <NUM> (step <NUM>) using <NUM>-(<NUM>-(<NUM>-methoxy)phenyl) pyridazin-<NUM>-amine (Guery et al. <NUM>) (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) in EtOH (<NUM>) Purification by column chromatography (EtOAC/MeOH <NUM>:<NUM>) afforded ethyl <NUM>-(<NUM>-(<NUM>-methoxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) as sticky light brown solid. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed as describe in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-methoxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) in aq. HCl (<NUM>, <NUM>). Upon cooling to rt. , the reaction mixture was filtered and evaporated in vacuo to afford <NUM>-(<NUM>-(<NUM>-Methoxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM>%) as a brown solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (ddd, J = <NUM>, <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed a described in example <NUM> (step <NUM>) using <NUM>-(<NUM>-(<NUM>-methoxy)phenyl) pyridazin-<NUM>-amine (Petrignet et al. <NUM>) (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) in EtOH (<NUM>). Purification by DCVC (Heptane/EtOAc, <NUM>-<NUM>% EtOAc) afforded ethyl <NUM>-(<NUM>-(<NUM>-methoxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) as sticky off-white sticky solid. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed as describe in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-methoxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) dissolved in aq. HCl (<NUM>, <NUM>). Upon cooling to rt. , the reaction mixture filtered and evaporated in vacuo to afford the target compound (<NUM>, <NUM>%) as white solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: <NUM>-Chloropyridazin-<NUM>-amine (<NUM>, <NUM> mmol), <NUM>,<NUM>-dichlorophenylboronic acid (<NUM>, <NUM> mmol), Pd(PPh<NUM>)<NUM> (<NUM>, <NUM> mmol), and Na<NUM>CO<NUM> (<NUM>, <NUM> mmol) were dissolved in EtOH/water <NUM>:<NUM> (<NUM>). The resulting reaction mixture was flushed with nitrogen for <NUM> minutes and heated at reflux under a nitrogen atmosphere for <NUM> days. The mixture was filtered through a plug celite, diluted with water (<NUM>), and extracted with EtOAc (<NUM> × <NUM>). The combined organic phase were dried over anhydrous Na<NUM>SO<NUM>, filtered, and evaporated in vacuo. Purification by column chromatography (CH<NUM>Cl<NUM>/MeOH <NUM>:<NUM>) afforded <NUM>-(<NUM>,<NUM>-dichlorophenyl)pyridazin-<NUM>-amine (<NUM>, <NUM>%) as white solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, CD<NUM>OD): δ <NUM> (d, J = <NUM>, <NUM>), <NUM>- <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed a described in example <NUM> (step <NUM>) using <NUM>-(<NUM>,<NUM>-dichlorophenyl)pyridazin-<NUM>-amine (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) in EtOH (<NUM>). Purification by DCVC (Heptane/EtOAc, <NUM>-<NUM>% EtOAc) followed by recrystallization from MeCN afforded ethyl <NUM>-(<NUM>-(<NUM>,<NUM>-dichlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) as white solid: mp <NUM>-<NUM>. <NUM>H NMR (<NUM>, DMSO-d<NUM>): δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step <NUM>: Performed as describe in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>,<NUM>-dichlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) in aq. HCl (<NUM>, <NUM>). Upon cooling to rt. , <NUM>-(<NUM>-(<NUM>,<NUM>-dichlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM>%) was isolated by filtration as white solid: mp <NUM>-<NUM>. <NUM>H NMR.

(<NUM>, DMSO-d<NUM>): δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, DMSO-d<NUM>): δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

A mixture of <NUM>-(<NUM>-(<NUM>-Chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM> mmol), K<NUM>CO<NUM> (<NUM>, <NUM> mmol) and KI (<NUM>, <NUM> mmol) in DMF (<NUM>) was stirred at rt. for <NUM> minutes. Chloromethyl acetate (<NUM>, <NUM> mmol) in DMF (<NUM>) was added dropwise, and stirred at <NUM> for <NUM>. Water (<NUM>) was added and the aqueous phase was extracted with EtOAc (<NUM>×<NUM>). The combined organic phasee were dried over anhydrous MgSO<NUM>, filtered, and evaporated in vacuo to dryness. Purification by preparative HPLC (gradient <NUM>-<NUM>% B over <NUM>) afforded the product (<NUM>, <NUM>%) as white solid. <NUM>H NMR (<NUM>, CD<NUM>OD), δ: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD), δ: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

A mixture of <NUM>-(<NUM>-(<NUM>-Chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM> mmol), K<NUM>CO<NUM> (<NUM>, <NUM> mmol) and KI (<NUM>, <NUM> mmol) in DMF (<NUM>) was stirred at room temperature for <NUM> minutes. Chloromethyl pivalate (<NUM>, <NUM> mmol) was added dropwise, and stirred at <NUM> for <NUM>. Water (<NUM>) was added and the aqueous phase was extracted with EtOAc (<NUM>×<NUM>). The combined organic phases were dried over anhydrous MgSO<NUM>, filtered, and evaporated in vacuo to dryness. Purification by column chromatography (EtOAc:Heptane <NUM>:<NUM>) afforded the product (<NUM>, <NUM>%) as off-white solid. <NUM>H NMR (<NUM>, CD<NUM>OD), δ: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD), δ: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

To a solution of <NUM>-(<NUM>-(<NUM>-Chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid (<NUM>, <NUM> mmol, <NUM>) in DMF (<NUM>), tert. butyl <NUM>,<NUM>,<NUM>-trichloroacetimidate (<NUM>, <NUM> mmol) and boron trifluoride diethyl etherate (<NUM>, <NUM> mmol) were added. The mixture was stirred overnight at <NUM>. A solution of saturated NaHCO<NUM> was added and the aqueous phase was extracted with EtOAc. The combined organic phases were dried over anhydrous MgSO<NUM>, filtered, and evaporated in vacuo to dryness. Purification by column chromatography (EtOAc) afforded the product (<NUM>, <NUM>%) as white solid. <NUM>H NMR (<NUM>, CD<NUM>OD), δ: <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, CD<NUM>OD), δ: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

Step1: <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid hydrochloride (<NUM>, <NUM> mmol), N-(<NUM>-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (<NUM>, <NUM> mmol), hydroxybenzotriazole (<NUM>, <NUM> mmol), triethylamine (<NUM>, <NUM> mmol) were dissolved in DMF and stirred for <NUM> minutes under argon. Methyl (S)-<NUM>-(<NUM>-aminophenyl)-<NUM>-((tert. butoxycarbonyl)amino)propanoate <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>) was added, and the mixture was stirred overnight at room temperature. A solution of saturated NH<NUM>Cl was added to the mixture, which then, was washed with EtOAc. The combined organic phase was dried over Na<NUM>SO<NUM>, filtered, and evaporated in vacuo. Purification by column chromatography (EtOAc/MeOH <NUM>:<NUM>) affoprded methyl (S)-<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(<NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetamido)phenyl)propanoate (<NUM>, <NUM>%) as a pink solid. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ: <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>).

Step <NUM>: To a solution of methyl (S)-<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(<NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetamido)phenyl)propanoate (<NUM>, <NUM> mmol) in MeOH (<NUM>) was added with LiOH (<NUM>, <NUM> mmol) in water (<NUM>), and the mixture was stirred overnight at room temperature. The mixture was evaporated to dryness in vacuo, and then, the crude solid was dissolved in DCM (<NUM>) and TFA (<NUM>). It was stirred for <NUM> at room temperature before evaporation. Purification by preparative HPLC (gradient <NUM> - <NUM>% B, eluent A (H<NUM>O/TFA, <NUM>:<NUM>) and eluent B (MeCN/H<NUM>O/TFA, <NUM>:<NUM>:<NUM>) at a flow rate of <NUM> min-<NUM> , over <NUM>) afforded (S)-<NUM>-amino-<NUM>-(<NUM>-(<NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetamido)phenyl)propanoic acid, TFA salt (<NUM>, <NUM>%) as a white solid. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ: <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. HPLC (<NUM>): <NUM>%. UPLC-MS: m/z = <NUM> [M-H]-, <NUM> [M+H]+.

Step1: Performed as described in Example <NUM> (step <NUM>) using <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid hydrochloride (<NUM>, <NUM> mmol), N-(<NUM>-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (<NUM>, <NUM> mmol), hydroxybenzotriazole (<NUM>, <NUM> mmol), triethylamine (<NUM>, <NUM> mmol), methyl (S)-<NUM>-(<NUM>-aminophenyl)-<NUM>-((tert-butoxycarbonyl)amino)propanoate <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>). Purification by column chromatography (EtOAc) afforded methyl (S)-<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(<NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetamido)phenyl)propanoate (<NUM>, <NUM>%) as a pink solid. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ: <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>).

Step <NUM>: Performed as described in Example <NUM> (step <NUM>) using methyl (S)-<NUM>-((tert-butoxycarbonyl)amino)-<NUM>-(<NUM>-(<NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetamido)-phenyl)propanoate (<NUM>, <NUM> mmol), LiOH (<NUM>, <NUM> mmol), MeOH (<NUM>), water (<NUM>), DCM (<NUM>) and TFA (<NUM>). Purification by preparative HPLC (gradient <NUM> - <NUM>% B, eluent A (H<NUM>O/TFA, <NUM>:<NUM>) and eluent B (MeCN/H<NUM>O/TFA, <NUM>:<NUM>:<NUM>) at a flow rate of <NUM> min-<NUM>, over <NUM>) afforded (S)-<NUM>-amino-<NUM>-(<NUM>-(<NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetamido)-phenyl)propanoic acid TFA salt (<NUM>, <NUM>%) as a white solid. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ: <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (tt, J = <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. HPLC (<NUM>): <NUM>%. UPLC-MS: m/z = <NUM> [M-H]-, <NUM> [M+H]+.

JON-<NUM>, related analogues, and pro-JON-<NUM>(<NUM>) were tested in the [<NUM>H]NCS-<NUM> binding assay (Wellendorph et al. A) JON-<NUM> (Ki value of <NUM>) and the analogues <NUM>-<NUM> (respective Kl values of <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) all inhibited binding in a concentration-dependent manner. B) The proform of JON-<NUM>, pro-JON-<NUM> (<NUM>), failed to displace radioligand binding at concentrations up to <NUM> and thus only binds to the binding site after being cleaved into the acid.

Human/rat CaMK2a expressed in HEK cells was assayed in an in-house established [<NUM>H]HOCPCA filtration binding assay performed on whole cell lysates of CaMK2a-transfected HEK293T cells. JON-<NUM> was able to concentration-dependently inhibit radioligand binding (Ki value <NUM>), displaying 14x higher affinity compared to GHB itself (Ki value of <NUM>).

The SPR experiments confirmed an interaction between A) the lower molecular weight compound JON-<NUM> and CaMK2a although at low response level. B) From steady state affinity analysis the KD was estimated to <NUM>. C) As a positive control, calmodulin was similarly injected in a buffer supplemented with Ca<NUM>+ (<NUM>) over immobilized CaMK2a, which revealed a strong complex formation with very slow dissociation rate. The interaction had to be actively broken using a regeneration solution (<NUM> EDTA) so that the signal returned to baseline before new injection of calmodulin. D) From steady state affinity analysis the KD was estimated to <NUM>.

The metabolic stabilities of JON-<NUM> and the prodrugs 1a, <NUM>-<NUM>, <NUM> and <NUM> were determined in vitro in rat plasma and rat liver microsomes. The remaining percentages of the compounds after incubation are summarized. butyl ester (<NUM>) showed high stability in rat liver microsomes and fair stability in rat plasma (t<NUM>/<NUM> ≈ <NUM>), however, prodrugs 1a, <NUM> and <NUM> were highly chemical/metabolic unstable and converted to the desirable parent compound JON-<NUM> instantly. Chemical or enzymatic degradation of JON-<NUM> was not observed during the metabolic stability study, correlating well with the high bioavailability observed in the brain exposure study. The corresponding loss of prodrug compounds and formation of parent drug JON-<NUM> was determined by UHPLC-MS.

Plasma and brain exposure of JON-<NUM> and pro-JON-<NUM> were evaluated in male mice. It was found that JON-<NUM> exhibits seemingly very low brain passage (B/P ratio <NUM> in mice, dose <NUM>/kg i. However, the prodrug, pro-JON-<NUM> enters the brain (B/P ratio <NUM>, dose <NUM>/kg i.

To assess affinity of JON-<NUM> for the GABAB receptor, we employed a GABAB binding assay using rat brain synaptic membranes (Wellendorph et al. A) JON-<NUM> showed no ability to compete with [<NUM>H]GABA at <NUM> (cf. baclofen and GHB). B) JON-<NUM> was further assessed for functional activity using CHO cells stably expressing GABAB(1b,<NUM>) receptors (Rajalu et al. As shown, JON-<NUM> is not a GABAB agonist.

We will assess functional activity of selected analogues in intact CaMK2a-transfected HEK293T cells using the well-described pT286 assay (Kool et al. To this end, HEK293T cells will be transfected with CaMK2a and at <NUM> hrs post-transfection, different concentrations compounds will be bath-applied to the cells alone or together with Ca<NUM>+. After different incubation times, cells will be lysed using 1x RIPA buffer supplemented with phosphatase and protease inhibitors (Phosphatase inhibitor cocktail <NUM> #P0044 (Sigma), Phosphatase inhibitor cocktail <NUM> #P5726 (Sigma) and complete EDTA protease inhibitors (Roche)). Autophosphorylation will be assessed by Western blot analysis, comparing the total level of CaMK2a (quantified using anti-myc-Alexa488, MA1980-A488, ThermoFisher Scientific) to the level of phosphorylated CaMK2a (pThr286: #<NUM>, NewEngland BioLabs; pThr306: #NBP2-<NUM>, Novus Biologicals; goat anti-rabbit HRP: #PI-<NUM> X0126, Vector). Levels of pT286 CaMK2a will be normalized to total CaMK2a to detect changes in autophosphorylation. Cells only treated with vehicle, ionomycin or calcium chelator will be used as controls.

To assess stability of pro-JON-<NUM> and rate of formation of JON-<NUM> following brain delivery of pro-JON-<NUM>, we will determined in vitro stability in mouse brain homogenate following the procedure described for stability test in mouse plasma and liver microsomes. The loss of prodrug compound and formation of parent drug JON-<NUM> will be determined by UHPLC-MS.

To probe the utility of the BBB L-type amino acid transporter <NUM> (LAT1, SLC7A5) for drug delivery, we will synthesize various JON-<NUM> derivatives conjugated with selected amino acid residues. This will be done following the procedure describe in examples <NUM> and <NUM>.

To test selected prodrugs for substrate activity at LAT-<NUM>, we characterised the uptake and efflux of [<NUM>H]leucine in the presence of compounds. For this purpose we used HEK293T cells endogenously expressing a functionally competent LAT-<NUM>, and performed assays according to Chien et al. , <NUM> to measure A) uptake and B) relative exchange efflux rate (L-leucine as control). As shown, JON-<NUM> prodrug <NUM> is a LAT1 inhibitor.

To determine locomotor effects (e.g. sedation or hyperactivity) compounds were assessed after systemic administration to mice. Mice (typically n=<NUM>-<NUM>) were administered selected compounds, e.g. JON-<NUM> and pro-JON-<NUM> and vehicle controls, and placed in transparent cages (L: <NUM> x W: <NUM> x H: <NUM>). Locomotor activity were measured via a camera mounted above the arena. Mice will be recorded for <NUM> and data collected in <NUM>-min intervals. Data will be stored on a computer equipped with Ethovision XT (Noldus). <FIG> shows that JON-<NUM> does not affect locomotor activity.

Using the photothrombotic model for focal ischemia we will test selected compounds, e.g. JON-<NUM> and pro-JON-<NUM> for neuroprotective effects (compared to vehicle and sham animals, all injected i. To this end, a photochemical lesion will be introduced to male C57BL/6J mice (<NUM>-<NUM> weeks) weighing ~ <NUM>-<NUM> as previously described (Clarkson et al. Under anesthesia with isoflurane (<NUM>% to <NUM>% in O<NUM>) mice will be placed in a stereotactic apparatus, the skull exposed through a midline incision, cleared of connective tissue and dried. A cold light source attached to a 40x objective providing a <NUM>-mm diameter illumination will be positioned <NUM> lateral from bregma. Then, <NUM> of Rose Bengal (Sigma-Aldrich, <NUM>/ml in normal saline) is administered i. After <NUM>, the brain is illuminated through the exposed intact skull for <NUM>, while keeping body temperature at <NUM> ± <NUM> degrees using a heating pad (Harvard apparatus, Holliston, MA, USA). <NUM> or <NUM> days later resulting infarct sizes will be measured using histochemistry. Sectioned brains fixed with <NUM>% PFA are cut in µm sections free-floating in anti-freeze media. Sections are mounted, stained for cresyl violet and the infarct volumes determined by measuring every 6th section through the entire infarct as described and reported as infarct volumes (Clarkson et al. Analyses will be performed by an observer blinded to the treatment groups.

Using the DTA mouse model (Tabuchi et al. , <NUM>) we determined changes in sleep-wake EEG/EMG patterns (including cataplexy) at different time points (<NUM> day and <NUM>-<NUM> days) under the influence of JON-<NUM>. After drug cessation day <NUM>, EEG/EMG changes were mapped up to day <NUM>. Under anesthesia with isoflurane (<NUM>% to <NUM>% in O<NUM>) electrodes were placed in the scull and neck muscles of the mice. After <NUM>-<NUM> days recovery the electrodes are connected to a recording system, and EEG/EMG signals are recorded with synchronised video recordings. From the data, sleep/wake parameters and cataplexy episodes are scored and calculated. Analyses were performed by an observer blinded to the treatment groups.

Using the hypocretin knock-out mouse model we will determine changes in sleep-wake EEG/EMG patterns (including cataplexy) at different time points (<NUM> day to <NUM> weeks) under the influence of selected compounds, e.g. JON-<NUM> and pro-JON-<NUM>. After drug cessation, EEG/EMG changes will be further mapped for up to <NUM> weeks. Under anesthesia with isoflurane (<NUM>% to <NUM>% in O<NUM>) electrodes will be placed in the scull and neck muscles of the mice. After <NUM>-<NUM> days recovery the electrodes are connected to a recording system, and EEG/EMG signals are recorded with synchronised video recordings. From the data, sleep/wake parameters and cataplexy episodes are scored and calculated. Analyses will be performed by an observer blinded to the treatment groups.

The sodium salt of <NUM> was prepared by dissolving <NUM> (<NUM>, <NUM> mmol) in ethanol (<NUM>) and NaOH (aq) (<NUM>, <NUM> mmol, <NUM> Tritisol) was added. The solvent was removed in vacuo to give the product (<NUM>) as white solid.

Step1: <NUM>-(<NUM>-nitrophenyl)pyridazine-<NUM>-amine (<NUM>, <NUM> mmol) and ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) were dissolved in <NUM> of ethanol. The reaction mixture was stirred at reflux for <NUM> hours. After evaporating the solvent, aqueous sat NaHCO<NUM> was added. The mixture was extracted with DCM. The combined organic phases were dried over Na<NUM>SO<NUM>, filtered, and evaporated in vacuo. Purification by column chromatography (EtOAc/Heptane <NUM>:<NUM>) furnished ethyl <NUM>-(<NUM>-(<NUM>-nitrophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Step <NUM>: Ethyl <NUM>-(<NUM>-(<NUM>-nitrophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) was dissolved in <NUM> HCl (<NUM>) and stirred overnight at <NUM>. After extraction with ethyl acetate, the aqueous phase was lyophilized to furnish <NUM>-(<NUM>-(<NUM>-nitrophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid, hydrochloride (<NUM>,<NUM>, <NUM> %) as a pale yellow solid: m. p <NUM>-<NUM>. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. HPLC(<NUM>)=<NUM>%.

Step1: Ethyl <NUM>-(<NUM>-(<NUM>-nitrophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) and SnCl<NUM> (<NUM>, <NUM> mmol) were dissolved in <NUM> of methanol and stirred overnight under reflux. After reducing the solvent in vacuo and adding <NUM> of EtOAc, the mixture was alkalized to pH=<NUM> using 5N sodium hydroxide. After filtration through a short pad of celite and evaporation, ethyl <NUM>-(<NUM>-(<NUM>-aminophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) was afforded. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Step2: Performed as described in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-aminophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) and <NUM> HCl (<NUM>). Lyophilization of the aqueous phase furnished <NUM>-(<NUM>-(<NUM>-aminophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid hydrochloride (<NUM>,<NUM>, <NUM> %) as an yellow solid: m. p <NUM>-<NUM>. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM> (d, J = <NUM>), <NUM>, <NUM>. HPLC(<NUM>)=<NUM>%.

Step1: Performed as described in example <NUM>-<NUM> (step <NUM>) using <NUM>-(<NUM>-aminopyridazin-<NUM>-yl)phenol (<NUM>, <NUM> mmol), ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) and ethanol (<NUM>). Purification by column chromatography (EtOAc/Heptane <NUM>:<NUM>) furnished ethyl <NUM>-(<NUM>-(<NUM>-hydroxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Step2: Performed as described in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-hydroxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) and <NUM> HCl (<NUM>). Lyophilization of the aqueous phase furnished <NUM>-(<NUM>-(<NUM>-hydroxyphenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid hydrochloride (<NUM>, <NUM> %) as a white solid: m. p <NUM>-<NUM>. : <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM> (d, J = <NUM>), <NUM> (d, J = <NUM>), <NUM> (d, J = <NUM>), <NUM>, <NUM> (d, J = <NUM>), <NUM> (d, J = <NUM>), <NUM>, <NUM> (d, J = <NUM>), <NUM>, <NUM>, <NUM>. HPLC(<NUM>)=<NUM>%.

Step1: To a thick-wall borosilicate glass vial were added <NUM>-amino-<NUM>-chloropyridazine (<NUM>, <NUM> mmol), K<NUM>CO<NUM> (<NUM>, <NUM> mmol), <NUM>-pyridinylboronic acid (<NUM>, <NUM> mmol), Pd(PPh<NUM>)<NUM> (<NUM>, <NUM>% mmol) in ethanol-water (<NUM>:<NUM>, <NUM>). The mixture was degassed with N<NUM> for <NUM> minutes, which then was irradiated in a microwave reactor at <NUM> for <NUM>. The reaction mixture was concentrated under reduced pressure. Purification by column chromatography (EtOAc/MeOH <NUM>:<NUM>) furnished <NUM>-(pyridin-<NUM>-yl)pyridazin-<NUM>-amine (<NUM>, <NUM>%).

Step2: Performed as described in example <NUM> (step <NUM>) using <NUM>-(pyridin-<NUM>-yl)pyridazin-<NUM>-amine (<NUM>, <NUM> mmol), ethyl <NUM>-chloroacetoacetate (<NUM>µL, <NUM> mmol) and ethanol (<NUM>). Purification by column chromatography (EtOAc/MeOH <NUM>:<NUM>) furnished ethyl <NUM>-(<NUM>-(pyridin-<NUM>-yl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%).

Step3: Performed as described in example <NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(pyridin-<NUM>-yl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) and <NUM> HCl (<NUM>). Lyophilization of the aqueous phase furnished Synthesis of <NUM>-(<NUM>-(pyridin-<NUM>-yl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid hydrochloride (<NUM>, <NUM> %). HPLC(<NUM>)=<NUM>%.

Step1: Performed as described in example <NUM> (step <NUM>) using <NUM>-amino-<NUM>-bromopyridazine (<NUM>, <NUM> mmol), K<NUM>CO<NUM> (<NUM>, <NUM> mmol), <NUM>-chloro-<NUM>-pyridinylboronic acid (<NUM>, <NUM> mmol), Pd(PPh<NUM>)<NUM> (<NUM>, <NUM>% mmol) in ethanol-water (<NUM>:<NUM>, <NUM>) at reflux for overnight. Purification by column chromatography (EtOAc) furnished <NUM>-(<NUM>-chloropyridin-<NUM>-yl)pyridazin-<NUM>-amine (<NUM>, <NUM>%) as a yellow solid. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>).

Performed as described in example <NUM> (step <NUM>) using <NUM>-(<NUM>-chloropyridin-<NUM>-yl)pyridazin-<NUM>-amine (<NUM>, <NUM> mmol), ethyl <NUM>-chloroacetoacetate (<NUM>, <NUM> mmol) and ethanol (<NUM>). Purification by column chromatography (EtOAc/MeOH <NUM>:<NUM>) furnished ethyl <NUM>-(<NUM>-(<NUM>-chloropyridin-<NUM>-yl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Step3: Performed as described in example <NUM>-<NUM> (step <NUM>) using ethyl <NUM>-(<NUM>-(<NUM>-chloropyridin-<NUM>-yl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM> mmol) and <NUM> HCl (<NUM>). Lyophilization of the aqueous phase furnished <NUM>-(<NUM>-(<NUM>-chloropyridin-<NUM>-yl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetic acid hydrochloride (<NUM>, <NUM> %) as an off-white solid. p <NUM>-<NUM>. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM>, <NUM>, <NUM> (d, J = <NUM>), <NUM>, <NUM>, <NUM> (d, J = <NUM>), <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. HPLC(<NUM>)=<NUM>%.

To a solution of <NUM> (JON-<NUM> (Krall et al)) (<NUM>, <NUM> mmol), <NUM>-dimethylaminopyridine (<NUM>, <NUM>% mmol) and <NUM>,<NUM>-dimethylpropan-<NUM>-ol (<NUM>, <NUM> mmol) in DMF (<NUM>) at <NUM> was added N-(<NUM>-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (<NUM>, <NUM> mmol) dissolved in DMF (<NUM>). The mixture was then allowed to room temperature and stirred for overnight. A solution of saturated NH<NUM>Cl was added to the mixture, which then, was extracted with EtOAc. The combined organic phases were washed by brine, dried over Na<NUM>SO<NUM>, filtered, and evaporated in vacuo. Purification by column chromatography (EtOAc/Heptane <NUM>:<NUM>) furnished neopentyl <NUM>-(<NUM>-(<NUM>-chlorophenyl)imidazo[<NUM>,<NUM>-b]pyridazin-<NUM>-yl)acetate (<NUM>, <NUM>%) as a white solid. <NUM>H NMR (<NUM>, Methanol-d<NUM>) δ <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>). <NUM>C NMR (<NUM>, Methanol-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. HPLC (<NUM>): <NUM>%.

Claim 1:
A compound of general formula I:
<CHM>
or a pharmaceutically acceptable salt thereof,
wherein:
X is C, R<NUM> and R<NUM> are different and selected from H, F, I, Cl, -OH, -NH<NUM>, -NO<NUM>, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl wherein butyl, pentyl and hexyl are linear or branched, -O- C<NUM>-C<NUM>-alkyl, -O-benzyl (-O-Bn), wherein C<NUM>-C<NUM>-alkyl is linear or branched and wherein benzyl is unsubstituted or substituted with one or more halogen and/or one or more C<NUM>-C<NUM>-alkyl or -O-C<NUM>-C<NUM>-alkyl;
or
X is C, R<NUM> and R<NUM> are the same and selected from F, I, Cl, -OH, -NH<NUM>, -NO<NUM>, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl wherein butyl, pentyl and hexyl are linear or branched, -O- C<NUM>-C<NUM>-alkyl, -O-benzyl (-O-Bn), wherein C<NUM>-C<NUM>-alkyl is linear or branched and wherein benzyl is unsubstituted or substituted with one or more halogen and/or one or more C<NUM>-C<NUM>-alkyl or -O-C<NUM>-Ce-alkyl;
and
R<NUM> is selected from -OH, -NH<NUM>, -O-C<NUM>-C<NUM>-alkyl, -C<NUM>-C<NUM>-alkyl-O-C(=O)-C<NUM>-C<NUM>-alkyl, -NH-C<NUM>-C<NUM>-alkyl, - O-aryl, O-substituted aryl, -NH-aryl, -NH-substituted aryl, wherein C<NUM>-C<NUM>-alkyl is linear or branched, aryl is unsubstituted or substituted with one or more amino acid residues to form a compound in formula II illustrated with a substituted aryl being represented by phenylalanine:
<CHM>
or a pharmaceutically acceptable salt thereof; provided that the compound is not one of the following, wherein

<TAB>

or hydrochloride or TFA salts of compounds mentioned in the table above.