TrkB positive allosteric regulator

Abstract

The present invention relates to the field of pharmaceutical compositions comprising "LIT-TB" derivatives of formula I. More particularly, the present invention relates to "LIT-TB" derivatives for use in the treatment of neurodegenerative diseases and more particularly for use in the treatment of Huntington's disease. The present invention also relates to said "LIT-TB" derivatives and their preparation.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of pharmaceutical compositions comprising "LIT-TB" derivatives. More particularly, it relates to "LIT-TB" derivatives for use in the treatment of neurodegenerative diseases and more particularly for use in the treatment of Huntington's disease. The present invention also relates to "LIT-TB" derivatives and the preparation thereof.

[0002] In the following description, references between brackets refer to the list of references at the end of the examples. BACKGROUND

[0003] Huntington's disease is an inherited disease that causes nerve cells in the brain to break down (degenerate) over time. Huntington's disease has a wide range of effects on a person's functional abilities and often leads to movement, thinking (cognitive) and psychiatric disorders.

[0004] Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disease characterized by impaired motor control, cognitive dysfunction, behavioral changes, and mood disorders. Progressive neurodegeneration in the striatum and other areas such as the cerebral cortex leads to death of the patient within 10-20 years from the first appearance of symptoms [1].

[0005] Depending on the age of onset, HD can be divided into two forms: the more traditional adult-onset HD and the less common juvenile-onset HD (JHD), also known as the Westphal variant of HD. Patients with adult-onset HD have an average age of onset of symptoms between 30-50 years, while JHD onset occurs before the age of 20. There is some overlap in symptoms between the two forms; however, there is a difference in the pattern of motor dysfunction between adult-onset HD and JHD. Chorea (abnormal, involuntary movements) is usually the first observed symptom in patients with adult-onset HD.

[0006] As the disease worsens, partial or complete loss of muscle movement, known as hypokinesia, becomes more apparent. Conversely, hypokinesia is usually seen in JHD onset, while chorea is less apparent in these patients and can not exist at all in some cases. Seizures are frequently observed in JHD individuals, but not present in adult-onset HD. The severity of symptoms increases over time and the average latency from the time of HD diagnosis to death is 10-20 years for adult-onset HD patients and less than 10 years for JHD patients.

[0007] HD is caused by a genetic defect that results in the expansion of cytosine, adenine, and guanine (CAG) repeat sequences within the huntingtin gene (Htt), leading to the production of mutant huntingtin protein (mHtt). Although the function of wild-type huntingtin protein (Htt) remains to be fully elucidated, it has been demonstrated that mHtt exerts a toxic effect on specific neurons within the brain.

[0008] Htt is ubiquitously expressed in multiple subcellular locations throughout the body. Although the function of Htt remains to be fully determined, studies have shown that it interacts with a range of other proteins involved in several cellular processes, including intracellular signaling, metabolism, and gene transcription. In recent years, there has been increasing evidence that the genetic defect in the huntingtin gene leads to disruption of the normal biological function of Htt and that this can play a role in the pathology of HD in addition to the toxic gain-of-function of mHtt [2-4].

[0009] The huntingtin gene is located on chromosome 4p16.3. The stretch of trinucleotide CAG repeats is at the beginning of this gene in exon 1. Each of these triplet repeats encodes the amino acid glutamine, and thus this CAG triplet repeat encodes a string of glutamines, also known as a polyglutamine tract (Huntington’s Disease Research Collaborative Group, 1993). Normal huntingtin genes have polyglutamine tracts ranging between six and 26 CAG repeats. The number of these CAG repeats is significantly increased in people with HD, and more than 36 repeats is associated with the development of HD [5-6].

[0010] Huntington’s discovery provided a new perspective on the pathogenesis of HD, but the mechanisms leading to selective death and neuronal loss remain unknown.

[0011] While investigations aimed at increasing the understanding of the pathogenesis of HD are being carried out, efforts are also being made to find possible therapies against this devastating disease. In this regard, attention has focused on the use of neurotrophic factors in new therapeutic strategies against human neurodegenerative diseases [7].

[0012] BDNF is a member of the neurotrophin family of growth factors that specifically binds to the TrkB tyrosine kinase receptor, mediating neurotrophic signaling [8-9]. BDNF is the most abundant neurotrophic factor in the adult brain and promotes the survival, growth, and plasticity of various neural cell populations during normal development and after adult brain injury. Given its trophic effects on neurons and its central role in higher cognitive function, BDNF has rapidly become a key factor in the pathophysiology of numerous brain disorders, including neurological disorders, neurodegenerative diseases, and psychiatric disorders.

[0013] The fact that BDNF has pro-survival activity on striatal neurons that die in HD has led to the idea that a reduction in endogenous trophic support may contribute to disease onset and / or progression. This hypothesis has sparked interest in BDNF and / or BDNF mimetics as potential therapeutic agents and has been reinforced by reports of reduced BDNF levels in the cerebral cortex and striatum of humans with HD and in numerous mouse and cell models of the disease [10-12].

[0014] There is a molecular relationship between huntingtin and BDNF, as normal (but not mutant) huntingtin promotes BDNF production and axonal transport.

[0015] Due to reduced BDNF gene transcription .

[0016] Although no underlying molecular mechanism has been proposed to explain the reduced neurotrophic support in other neurological diseases such as Parkinson's disease (PD) or Alzheimer's disease (AD), it is known that the huntingtin mutation in HD reduces the transcriptional activity of the BDNF promoter, thereby reducing transcription of the BDNF gene and reducing protein production in the cerebral cortex.

[0017] This has been confirmed in humans by a study conducted on the cerebral cortex, caudate nucleus, and putamen of HD patients. This study also showed reduced BDNF expression in the caudate and putamen, suggesting that an excess of BDNF may have therapeutic benefits in HD.

[0018] Wild-type huntingtin stimulates BDNF gene transcription by acting at the level of the BDNF promoter II, whereas the presence of the pathological CAG expansion in huntingtin abolishes the ability to maintain BDNF transcription in HD.

[0019] Due to reduced BDNF transport in HD .

[0020] Biochemical studies of mutant huntingtin gene knock-in cells, mice and HD post-mortem tissues suggest that the complex driving BDNF vesicles is altered in HD. Thus, these results can mean that wild-type huntingtin controls the transport of BDNF from the cortex to the striatum and that this transport is affected in HD.

[0021] Many mouse and human studies tend to attribute the deficit of striatal BDNF in HD to a combination of two factors: reduced BDNF production in the cortex and reduced transport of this neurotrophin from the cortex to the striatum. Both processes involving normal huntingtin are simultaneously disrupted in HD.

[0022] Furthermore, it is reported that mutant huntingtin affects TrkB levels in HD by showing reduced TrkB protein content in mutant huntingtin gene knock-in cells and HD mouse models

[13] . A significant reduction of TrkB receptors was also found in the striatum from three HD patients and a reduction of TrkB levels was also detected in cortical samples from four HD subjects. Further investigations are needed to understand the extent and consistency of TrkB downregulation.

[0023] To overcome the problems induced by the BDNF reduction in HD, experiments have been performed on R6 / 1 mice to assess the potential in vivo benefits provided by BDNF

[14] . It was found that BDNF effectively increased the expression of brain-derived neurotrophic factor and the number of striatal cells expressing brain-derived neurotrophic factor, the cells most affected in HD.

[0024] However, despite these promising results, BDNF supplementation also raises many issues: if the amount is too small, it can not be enough to produce the desired effect, if the amount is too large, it can be dangerous. Indeed, uncontrolled BDNF administration can interfere with other mechanisms such as activity-dependent neuronal plasticity and can induce serious side effects such as epileptic activity

[15] .

[0025] Although there are drugs available to help manage the symptoms of Huntington's disease, there is currently a significant unmet need as there is no treatment that can prevent the physical, mental and behavioral decline associated with the condition.

[0026] It is clear that BDNF is one of the key factors missing in HD and that an increase in endogenous BDNF production can produce a therapeutic effect, it is very important to control the central and peripheral concentrations of BDNF. SUMMARY

[0027] The present invention allows a new therapeutic solution based on TrkB-based positive allosteric modulators (PAMs).

[0028] "Positive allosteric modulators" (PAMs), also known as allosteric potentiators or enhancers, mean compounds that induce an amplification of the response effect of the receptor to the primary ligand without directly activating the receptor. Within the present invention, PAMs TrkB activity is related to the effect of BDNF potentiation on the functional activity of the TrkB receptor, measured in vitro or in vivo by specific TrkB receptor phosphorylation assays.

[0029] The compounds and compositions of the present invention have several properties such as effects on neurite outgrowth, BDNF potentiation, BBB (blood brain barrier) penetration, good brain bioavailability, increase in cell survival rate, TrkB selectivity and neuroprotective effects, conferring to this potential PAM an interesting pharmaceutical profile that can address some neurodegenerative diseases such as Huntington's disease, Parkinson's disease and Alzheimer's disease.

[0030] The compounds and compositions of the present invention are able to potentiate TrkB-mediated BDNF functional effects and open a new therapeutic approach to fight HD.

[0031] In a first aspect, the present invention relates to a pharmaceutical composition comprising:

[0032] (a) a LIT-TB compound of formula I:

[0033]

[0034] wherein,

[0035] -R 1 selected from the group comprising H, halogen, C1to C10saturated or unsaturated, substituted or unsubstituted, aliphatic, heteroaliphatic, cyclic, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl, or R 1 is a group of formula la:

[0036]

[0037] wherein,

[0038] R A is a linear C1to C10alkyl chain optionally interrupted by one or more ether or amide functions,

[0039] A 2 is an amide function,

[0040] R B is an optionally branched C1to C6alkyl chain,

[0041] fl is a fluorescent group or a non-fluorescent analogue group thereof,

[0042] -G represents a bond or -G 1 -G2 - a linker, wherein

[0043] • G 1 is a bond or a Ci to C4substituted or unsubstituted alkyl chain optionally comprising a heteroatom such as N or O, and

[0044] • G 2 represents a Ci to Cio saturated or unsaturated, substituted or unsubstituted, aliphatic, heteroaliphatic, cyclic, alicyclic, heteroalicyclic, aryl, heteroaryl, alkylaryl or alkylheteroaryl,

[0045] - X 1 and X 2 are identical or different, independently represent CH or N,

[0046] - X 3 is C or N,

[0047] - X 4 is N or NH,

[0048] - Y represents N or CH,

[0049] - r is an integer from 1 to 3,

[0050] - A is an amide or amine function, preferably A is C(0)NH, NHC(O) or NH,

[0051] - m is equal to 0, 1 or 2,

[0052] - m' is equal to 0, 1 or 2, and m + m' < 3

[0053] - t is an integer from 0 to 5,

[0054] - each R 6 group is identical or different, selected from the group comprising H, fluoride, optionally branched Ci to C6alkyl chain and Ci to C6alkoxy,

[0055] - T 1 and T 2 are identical or different, independently represent CH2, CHR 6 or C=0,

[0056] - Z is selected from the group comprising a bond, H and optionally branched Ci to C3alkyl chain, said optionally branched Ci to C3alkyl chain optionally comprising a heteroatom selected from the group comprising O or N,

[0057] - when Z is H, R 2 is null, or R 2 is selected from the group comprising H and optionally substituted with one or more R 7a group of 5 or 6 membered aromatic or non-aromatic rings or heterocycles, each R 7 groups are the same or different and are selected from the group comprising H, halide, CN, N02, NH2, CONH2, optionally branched C1to C6alkyl chain and optionally branched C1to C6alkoxy, two R 7 groups are optionally covalently bonded to form a ring,

[0058] or a pharmaceutically acceptable salt thereof, and

[0059] (b) a pharmaceutically acceptable excipient or carrier.

[0060] Within the present disclosure, represents a single or double bond, depending on the nature of X 3 and X 4 The adjacent bonds can be single or double bonds, depending on the nature of X

[0061] Within the present disclosure, represents a group and its point of attachment to the parent molecule.

[0062] Pharmaceutically acceptable salts of the compounds of Formula I include acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form nontoxic salts. Examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / diphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate salts, and xinafoate. For a review on suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, Weinheim, Germany, 2002).

[0063] In general, the term "substituted," whether or not it follows the term "optionally," and the substituents contained in the chemical formulae of the present invention, means that the hydrogen radicals in a given structure are replaced with the specified substituent radicals. When more than one position in any given structure can be substituted with more than one substituent selected from a particular group, the substituents may be the same or different at each position. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds.

[0064] As used herein, the term "aliphatic" refers to a non-aromatic group. An aliphatic group can be cyclic. An aliphatic group can be saturated, such as hexane, or unsaturated, such as hexene and hexyne. An open chain group (straight or branched) does not contain any type of ring and is therefore aliphatic. An aliphatic group can be saturated, connected by a single bond (alkanes), or unsaturated, with a double bond (alkenes) or a triple bond (alkynes). A "heteroaliphatic" group is an aliphatic group that carries one or more heteroatoms, the most common of which are oxygen, nitrogen, and sulfur.

[0065] As used herein, the term "alkyl" refers to straight and branched chain alkyl groups. Similar conventions apply to other general terms such as "alkenyl", "alkynyl" etc. In certain embodiments, as used herein, "low carbon number alkyl" is used to represent those alkyl groups (substituted, unsubstituted, branched or unbranched) with about 1-6 carbon atoms. Illustrative alkyl groups include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, part etc., and in addition, they can carry one or more substituents. Alkenyl includes, but is not limited to, for example, vinyl, propenyl, butenyl, 1-methyl-2-butene-1-yl etc. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl etc.

[0066] In general, as used herein, the term "aromatic moiety" or "aryl" refers to a stable, substituted or unsubstituted, unsaturated monocyclic or polycyclic hydrocarbon moiety, preferably having 3 to 14 carbon atoms, containing at least one ring that satisfies the Hackle rule for aromaticity. Examples of aromatic moieties include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, phenanthrenyl, and anthracenyl. "Heteroaryl" is both heterocyclic and aromatic.

[0067] The term "halogen" as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.

[0068] As used herein, the term "independently" refers to the fact that the substituents, atoms or moieties to which these terms refer are independently selected from the list of variables (ie, they may be identical or the same) from one another.

[0069] As the skilled person will appreciate, all numbers, including those expressing quantities of components, properties such as molecular weight, reaction conditions, and so forth, are approximations, and are understood always to be modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by the person utilizing the teachings of the present disclosure. It is further understood that the values inherently contain variability that is caused by for example, standard deviation in the respective testing measurements.

[0070] The skilled person will also readily recognize, where members are grouped together in a common manner, such as in a Markush group, that the present disclosure encompasses not only the entire group listed as a whole, but also each individual member of the group and all possible subgroups of the main group. In addition, the present disclosure encompasses not only the main group, but also the main group without one or more of the group members for all purposes. Thus, the present disclosure contemplates the explicit exclusion of any one or more members of a recited group. Accordingly, provisos can apply to any disclosed class or embodiment, whereby any one or more of the recited elements, species, or embodiments can be excluded from the class or embodiment, for example, as used in an explicit negative limitation.

[0071] Advantageously, the LIT-TB compound can be selected from the group of compounds of formula I, wherein R 1 is selected from the group comprising H, C1 to C10 saturated or unsaturated, substituted or unsubstituted, aliphatic, heteroaliphatic, cyclic, cycloaliphatic, aryl, heteroaryl, alkylaryl or alkylheteroaryl. Preferably, R 1 may be selected from the group comprising H, alkyl (e.g. methyl, ethyl), cycloalkyl (e.g. cyclopropyl, cyclopentyl), aralkyl (e.g. benzyl, phenethyl), heterocyclic aryl (e.g. piperidine) or heteroaryl (e.g. pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, oxazolyl, imidazolyl), R 1 is optionally substituted.

[0072] Advantageously, the LIT-TB compound can be selected from the group of compounds of formula I, wherein R 1 is a fluorescent group fl. The fluorescent group flmay be selected from the group comprising BDP 558 / 568, BDP 581 / 591, BDP 630 / 650, BDP R6G, BDP FL, BDP TMR, BDP TR, coumarin 343, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, cyanine 7.5, DY-647P1, fluorescein, sulfo-cyanine 3, sulfo-cyanine 5, sulfo-cyanine 5.5, sulfo-cyanine 7, sulfo-cyanine 7.5, pyrene, rhodamine X, derivatives thereof or non-fluorescent analogs thereof.

[0073] "Fluorescent group" (or fluorophore) means a group that can re-emit light upon excitation with light. Fluorophores usually contain several aromatic groups in combination, or planar or cyclic molecules with several π bonds.

[0074] As used herein, a "derivative" is a compound or group that is derived from a similar compound by a chemical reaction. For example, a fluorescent group can typically be an NHS ester before attachment. When grafted onto a compound, the fluorescent derivative is the same group, but without the NHS portion.

[0075] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein G represents a bond or -G 1 -G 2 -linker, where G 1 is a bond or a C1 to C4 substituted or unsubstituted alkyl chain optionally containing heteroatoms such as N or O, and G 2 represents a C1 to C10 saturated or unsaturated, substituted or unsubstituted, aliphatic, heteroaliphatic, cyclic, alicyclic aryl, heteroaryl, alkaryl or alkylheteroaryl group. Preferably, G 1 can be a key, and G 2 It may be a saturated or unsaturated, substituted or unsubstituted C2 to C6 aliphatic or heteroaliphatic group or a saturated or unsaturated, substituted or unsubstituted 5-membered, 6-membered or 7-membered ring or heterocyclic ring.

[0076] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein R 1 -G- is linked to the rest of the molecule via a heteroatom, preferably nitrogen.

[0077] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein R 1 -G- is selected from the group comprising radicals of the formula:

[0078]

[0079] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein R 1 -G- is selected from the group comprising radicals of the formula:

[0080]

[0081] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein X 1 and X 2 The same or different ones may independently represent CH or N.

[0082] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein X 3 Can represent C or N.

[0083] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein X 4 Can represent N.

[0084] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein when X 4 When it is N or NH, X 1 、X 2 and X 3 At least one of them is N. In the group of the compound, when X 4 When nitrogen is included, X 1 、X 2 、X 3 May not contain a single carbon atom at the same time.

[0085] Advantageously, X 1 and X 2 It does not mean CH at the same time.

[0086] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein X 4 is N or NH. Preferably, when X 4 When NH, X 3 Preferably, the LIT-TB compound can be selected from the group of compounds represented by formula I, wherein X 3 is N and X 4 is N.

[0087] Advantageously, A may be an amide or amine functional group, preferably A is -C(O)NH-, -NHC(O)- or -NH-. Preferably, A is an amide group.

[0088] Advantageously, m may be equal to 0, 1 or 2, m' may be equal to 0, 1 or 2, and m+m'≤3. Preferably, m=m'=1.

[0089] Advantageously, t may be an integer from 0 to 5. Preferably, t is 0, 1 or 2.

[0090] Advantageously, T 1 and T 2 The same or different, can independently represent CH2, CHR 6 Or C=O.

[0091] Advantageously, the LIT-TB compound may comprise one or more R 6 Group. From R 6 The bond to the center of the ring means that any available position within the ring can carry R6 Groups, including T 1 and T 2 When a carbon atom in the ring carries R 6 When a group is present, it replaces the H bond with the carbon atom. Each R 6 The groups may be identical or different and may be selected from the group consisting of H, fluoride, an optionally branched C1 to C6 alkyl chain and an optionally branched C1 to C6 alkoxy group. Preferably, m=1 and m'=1, t is 0, 1 or 2, and R 6 is F, Cl, Me or OMe, T 1 is CH2 or C=O and T 2 For CH2.

[0092] Advantageously, Z may be selected from the group comprising a bond, H and an optionally branched C1 to C3 alkyl chain, the optionally branched C1 to C3 alkyl chain optionally comprising a heteroatom selected from the group comprising O or N. Preferably, Z is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-, or Z is -(CH2) n -, where n is 1, 2, or 3.

[0093] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein R 2 is selected from the group comprising H, cycloalkyl (e.g. cyclopentyl), aralkyl (e.g. benzyl, phenethyl), heterocyclic aryl (e.g. piperidinyl, piperazinyl) or heteroaryl (e.g. pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, oxazolyl, imidazolyl, furanyl, thienyl, pyrrolyl, thiazolyl, pyrazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl). Optionally, R 2 1, 2 or 3 R 7 Group substitution.

[0094] Advantageously, the LIT-TB compound may be selected from the group of compounds represented by formula I, wherein R 2 is selected from the group consisting of H, cycloalkyl (e.g. cyclopentyl), aralkyl (e.g. benzyl, phenethyl), heteroaryl (e.g. piperidine), or heteroaryl (e.g. pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, oxazolyl, imidazolyl). 2 1, 2 or 3 R 7 Group substitution.

[0095] Advantageously, R 2 Can be selected from the group of formula Ib below:

[0096]

[0097] Among them, each R 7a 、R 7b 、R 7c It can be independently selected from the group consisting of H, F, Cl, Me, OMe, Et, Pr, iPr, Bu, CN, NO2, NH2, CONH2.

[0098] Advantageously, G 1 can be a key, and G 2 Can be -Y 1 (R 4 )-R 3 -Y 2 (R 5 )-, and the LIT-TB compound can be selected from the group of compounds represented by formula II:

[0099]

[0100] in,

[0101] -R 1 、X 1 、X 2 、X 3 、X 4 ,r,A,m,m',t,R 6 、T 1 、T 2 , Z and R 2 As defined above,

[0102] -Y 1 、Y 2 and Y 3 The same or different, independently representing N or CH,

[0103] -R 4 and R 5 are the same or different, independently selected from the group comprising H, optionally branched C1 to C3 alkyl groups, optionally comprising heteroatoms selected from the group comprising O and N, R 4 and R 5 may optionally be covalently bonded together to form a cyclic moiety,

[0104] -R 3 It is a linear or branched C2 to C6 alkyl chain.

[0105] Advantageously, G 1 can be a key, and G 2 Can be -Y 1 (R 4 )-R 3 -Y 2 (R 5 )-, and the LIT-TB compound can be selected from the group of compounds represented by formula IIa:

[0106]

[0107] in,

[0108] -R 1 、X 1 、X 2 、X 3 、X 4 ,r,A,m,m',t,R 6 , Z, R 2 、Y 1 、Y 2 、Y 3 、R 3 、R 4 and R 5 As defined above.

[0109] Advantageously, G 1 can be a key, and G 2 Can be The LIT-TB compound may be selected from the group of compounds represented by formula III:

[0110]

[0111] in,

[0112] -R 1 、X 1 、X 2 、X 3 、X 4 、Y 1 、Y 2 、Y 3 ,r,A,m,m',t,R 6 、T 1 、T 2 , Z and R 2 As defined above.

[0113] Advantageously, G 1 can be a key, and G 2 Can be The LIT-TB compound may be selected from the group of compounds represented by formula IIIa:

[0114]

[0115] in,

[0116] -R 1 、X 1 、X 2 、X 3 、X 4 、Y 1 、Y2 、Y 3 ,r,A,m,m',t,R 6 , Z and R 2 As defined above.

[0117] Advantageously, X 3 and X 4 N, Y 2 NH, G 1 can be a key, and G 2 Can be Y 1 (R 4 )-CH2-CH2-NH, and the LIT-TB compound can be selected from the group of compounds represented by formula IV:

[0118]

[0119] in,

[0120] -R 1 、R 4 、X 1 、X 2 、Y 1 、Y 3 ,r,A,m,m',t,R 6 、T 1 、T 2 , Z and R 2 As defined above.

[0121] Advantageously, X 3 and X 4 N, Y 2 NH, G 1 can be a key, and G 2 Can be Y 1 (R 4 )-R 3 -CH2-CH2-NH-, and the LIT-TB compound can be selected from the group of compounds represented by formula IVa:

[0122]

[0123] in,

[0124] -R 1 、R 4 、X 1 、X 2 、Y 1 、Y 3 ,r,A,m,m',t,R 6 , Z and R 2 As defined above.

[0125] Advantageously, the composition may comprise a pharmaceutically acceptable excipient or carrier.In the context of the present invention, any pharmaceutically acceptable excipient or carrier may be used.

[0126] Advantageously, the composition may be an aqueous composition.

[0127] Advantageously, the pH of the composition may be comprised within the range of 5 to 9.

[0128] Advantageously, the concentration of the LIT-TB compound represented by formula I, II, III or IV in the composition may be comprised within the range of 1 pM to 100 μM.

[0129] In this application, when defining a range, both the lower limit and the upper limit are included.

[0130] Advantageously, the composition of the invention may allow to potentiate the 0.4 nM BDNF response at a 10 nM concentration of the LIT-TB derivative by more than or equal to 10%, preferably more than or equal to 20%, and more preferably more than or equal to 30%.

[0131] Advantageously, the half-maximal effective concentration (EC 50 ) is less than or equal to 10mM.

[0132] Advantageously, the selectivity for positive allosteric modulation of the relevant TrkA and TrkC receptors is greater than or equal to 50.

[0133] In another aspect, the present invention relates to a pharmaceutical composition comprising a LIT-TB compound of formula I, II, IIa, III, IIIa, IV or IVa as defined above for use in a medicine or medicament.

[0134] A third aspect of the present invention is a pharmaceutical composition comprising a LIT-TB compound of formula I, II, IIa, III, IIIa, IV or IVa as defined above for use in treating neurodegenerative diseases, metabolic disorders, mood disorders, spinal cord injury, stroke and ischemia.

[0135] In the context of the present invention, neurodegenerative diseases may be, but are not limited to, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's disease, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, metabolic disorders (which may be, but are not limited to, e.g., obesity, type 2 diabetes), mood disorders (which may be, but are not limited to, e.g., depression, anxiety, schizophrenia, bipolar disorder, autism spectrum disorder).

[0136] The terms "treating / treat / treatment" include (i) preventing the onset of a disease, pathology, or medical condition (e.g., preventing or treating); (ii) inhibiting a disease, pathology, or medical condition or arresting its development; (iii) alleviating a disease, pathology, or medical condition; and / or (iv) alleviating symptoms associated with a disease, pathology, or medical condition. Thus, the terms "treat / treatment / treating" extend to preventing and include preventing / prevention / preventing, reducing, stopping, or reversing the worsening or severity of the condition or symptoms being treated. Thus, the term "treating" includes medical, therapeutic, and / or prophylactic administration, as appropriate.

[0137] "Effective amount" refers to an amount effective to treat a disease, disorder, and / or condition or to produce the recited effect. For example, an effective amount can be an amount effective to reduce the exacerbation or severity of the condition or symptom being treated. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art. The term "effective amount" is intended to include an amount of a compound as described herein, or an amount of a combination of compounds as described herein, e.g., an amount effective to treat or prevent a disease or disorder or to treat a symptom of a disease or disorder in a host. Thus, "effective amount" generally means an amount that provides a desired effect.

[0138] In a fourth aspect, the present invention relates to compounds represented by formula I, II, III or IV as defined above, excluding N-(1-benzyl-4-piperidinyl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide and N-(1-benzyl-4-piperidinyl)-3-[6-(1-piperidinyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide.

[0139] Other advantages may also become apparent to the skilled person when reading the following examples, which are illustrated by the accompanying drawings and are given for illustrative purposes only and are not exhaustive. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] - Figure 1Figure 2 shows the effects of LIT-TB001 on Trk phosphorylation, ERK phosphorylation, and neurite outgrowth in the presence of NGF / TrkA or BDNF / TrkB. Nnr5 PC12-TrkA and nnr5 PC12-TrkB cells are NGF-unresponsive mutant PC12 cells stably transfected with TrkA and TrkB, respectively

[16] . Activation of TrkA and TrkB in nnr5 PC12-TrkB or nnr5 PC12-TrkA cells was assessed by quantifying the amount of phosphorylated Trk at tyrosine 706 (Y706) 15 min after addition of BDNF (1 nM) or NGF (2 nM) in the presence or absence of different concentrations of TB001 (0.1, 10, and 1000 nM), as previously described

[17] . Activation of downstream signaling pathways was assessed by quantifying phosphorylated ERK in the same cells. Neurite outgrowth was determined 48 hours after initial treatment by counting the number of cells bearing neurites with a diameter greater than 2 cells, as described previously

[17] . In all three assays, LIT-TB001 showed high selectivity for TrkB signaling, as demonstrated by increased BDNF-induced, but not NGF-induced, phosphorylated Trk, phosphorylated ERK, and neurite outgrowth.

[0141] - Figure 2 Figure 3. Inhibition of the catalytic activity of 45 kinases (Expression Diversity Kinase Panel, Eurofins Discovery, Product No. P10) by LIT-TB001 at a concentration of 10 μM. For each kinase, the effect of the compound on ATP-induced kinase-mediated substrate phosphorylation was measured using TR-FRET LANCE technology.

[0142] - Figure 3Effect of acute intraperitoneal administration of LIT-TB001 (0, 0.5 and 1.0 mg / kg) on TrkB phosphorylation in the areas of TrkB expression of the mouse brain. Left: Adult C57BL / 6 male mice were intraperitoneally injected with saline (0.9% NaCl) or LIT-TB001 (0.5 or 1 mg / kg). One hour later (unless otherwise stated), mice were decapitated, blood was collected and brains were rapidly removed on ice. Subsequently, the cortex and hippocampus were dissected and tissues were rapidly processed for Western blot analysis using the phospho-Y806-TrkB selective antibody

[17] . Representative Western blots performed in the cortex of mice injected intraperitoneally with saline solution or TB001 (0.5 or 1 mg / kg) 1 hour are shown. Anti-TrkB antibody was used to quantify the total amount of TrkB. Anti-tubulin was used as a loading control. Right: Quantification of phospho-TrkB in the hippocampus and cortex of mice after injection of LIT-TB001 shows a significant TrkB potentiation in vivo compared to saline treatment (*p<0.05, **p<0.01, one-way ANOVA). TrkB phosphorylation levels were calculated as the ratio between phospho-TrkB and total TrkB bands in each area. DETAILED DESCRIPTION

[0143] EXAMPLE

[0144] I. Synthesis methods

[0145] The following synthesis methods and schemes illustrate the general procedures by which the compounds of the application can be prepared. Starting materials have been obtained from commercial sources or prepared by methods well known to those of ordinary skill in the art. For example, the compounds of the application can be prepared according to or analogously to the synthetic routes described in detail in the Examples section. In particular, compounds of general formula (I) and pharmaceutically acceptable salts thereof can be synthesized according to the methods described in the following schemes, wherein X represents a halogen and R represents any group at the corresponding position of general formula (I). While the numbering of the groups R in the following schemes differs from the names of the groups in general formula (I), it is understood that these schemes explain the preparation of compounds of formula (I) and thus these groups R are defined according to the corresponding groups at the same attachment position in general formula (I). Purification of intermediates and final products was performed by normal or reverse phase chromatography using a Dionex UltiMate 300 with the following parameters: flow rate: 0.5 mL / min, column temperature: 30 °C, solvent system: A (MeOH) and B (0.05% TFA in H2O), t = 0 min to 1 min: 50% to 60% B, followed by t = 1 min to t = 10 min: 60% to 100% B, and t = 10 min to t = 15 min: 100% B.

[0146] General procedure A

[0147] Condensation of N-arylalkylpiperidine analogs 1 with cyclic anhydrides 2 affords propionic acid (or homologous) derivatives 4a-c. Following alkaline hydrolysis of the ester group, condensation starting with ethylmalonyl chloride yields 2-[(1-benzylpiperidin-4-yl)carbamoyl]acetic acid 4c. Peptide-type coupling of compound 4 with commercially available 3-chloro-6-hydrazinopyridazine 5 affords hydrazide derivative 6, which is then cyclized to triazolopyridazine 7 under strongly acidic conditions at 135°C. Finally, coupling of 6-chloro-[1,2,4]triazolo[4,3-b]pyridazine derivative 7 with various heterocyclic secondary amines 8 under alkaline conditions affords the final compounds of formulae 9-14 (Scheme 1).

[0148] Scheme 1 (see Formula III)

[0149]

[0150] Conditions: a) succinic anhydride or glutaric anhydride, EtOAc, 25°C, 12h; b) ethylmalonyl chloride, DCM, Et3N, 25°C; c) NaOH / MeOH, followed by 2N HCL→pH 6; d) BOP, NMM, DCM, 12h; e) AcOH, 135°C, 2h; f) 8a-g, Et3N, EtOH, 135°C, 2h or reflux, 12h.

[0151] 4-((1-Benzylpiperidin-4-yl)amino)-4-oxobutanoic acid 4a (m'=1, m=1, n=1, r=1)

[0152] Succinic anhydride 2a (1.5 eq., 394 mg, 3.94 mmol) was dissolved in EtOAc (5 mL). 4-Amino-1-benzylpiperidine 1a (1 eq., 526 mg, 0.566 mL, 2.63 mmol) was added and the reaction mixture was stirred at room temperature overnight (18 h) to give carboxylic acid 4a. The white precipitate was filtered and washed with EtOAc (m = 763 mg, yield = 100%).

[0153] 1 H NMR (400MHz, DMSO-d6) δ7.76(d,J=7.7Hz,1H),7.35-7.22(m,5H),3.51(dtd,J=11.0,7.0,3.9Hz,1H),3.45(s,2H),2.77-2.71(m ,2H),2.42-2.37(m,2H),2.31-2.26(m,2H),2.00(ddd,J=11.8,9.2,2.5Hz,2H),1.68(dd,J=12.9,3.9Hz,2H),1.42-1.31(m,2H). 13CNMR (101 MHz, DMSO-d6) δ 173.8, 170.2, 138.4, 128.8, 128.2, 126.9, 62.1, 51.9, 45.9, 31.5, 30.1, 29.2. N-(1-benzylpiperidin-4-yl)-4-(2-(6-chloropyridazin-3-yl)hydrazine)-4-oxobutanamide (6a) (m'=1, m=1, n=1, r=1)

[0154] [(1-Benzylpiperidin-4-yl)carbamoyl]propanoic acid 4a (1 eq., 285 mg, 0.982 mmol) and BOP (1.2 eq., 520 mg, 1.18 mmol) were suspended in DMF (6.3 mL). NMM (1.5 eq., 148 mg, 0.162 mL, 1.47 mmol) was added and the reaction mixture was stirred at room temperature for 15 min. Subsequently, 3-chloro-6-hydrazinopyridazine 5 (1.2 eq., 170 mg, 1.18 mmol) was added and the reaction was stirred at room temperature overnight (20 h).

[0155] MeOH and silica were added and the crude material was evaporated. The adsorbed compound on silica gel was then purified on silica gel chromatography (eluent MeOH / EtOAc / Et3N; 1 / 9 / 0.3) to afford compound 6a (m=379 mg, yield=93%) as a yellow solid.

[0156] 1 H NMR (500 MHz, methanol-d4) δ 7.47 (d, J = 9.5 Hz, 1H), 7.39-7.31 (m, 5H), 7.13 (d, J = 9.5 Hz, 1H), 3.78-3.70 (m, 3H), 3.03 (d, J = 11.4 Hz, 2H), 2.60-2.40 (m, 6H), 1.94-1.87 (m, 2H), 1.64-1.54 (m, 2H). 13 C NMR (126 MHz, methanol-d4) δ 174.7, 173.8, 161.5, 149.6, 131.28, 131.27, 129.66, 129.65, 129.4, 118.4, 63.2, 52.9, 47.0, 31.5, 31.4, 29.9.

[0157] N-(1-Benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propionamide 7a (m'=1, m=1, n=1, r=1)

[0158] A microwave vial was charged with N-(1-benzylpiperidin-4-yl)-3-[N'-(6-chloropyridazin-3-yl)hydrazinecarbonyl]propanamide 6a (1 eq., 361 mg, 0.866 mmol) and acetic acid (2 mL). The vial was properly capped and the mixing vessel was heated at 135 ° C for 2 h. The mixture was cooled to room temperature and evaporated. The crude material was co-evaporated with cyclohexane and purified by silica gel chromatography (EtOAc / MeOH / Et3N, 9 / 1 / 0.3) to give compound 7a (m=289 mg, yield=84%) as a white solid.

[0159] 1 H NMR (400MHz, methanol-d4) δ8.22(d,J=9.7Hz,1H),7.40(d,J=9.7Hz,1H),7.37-7.27(m,5H),3.73-3.62(m,3H),3.43(t,J=7.4Hz,2H), 2.97(dt,J=12.4,3.9Hz,2H), 2.83(t,J=7.3Hz,2H), 2.36-2.26(m,2H), 1.91-1.83(m,2H), 1.56(dtd,J=13.3,11.2,3.8Hz,2H). 13 CNMR (101 MHz, methanol-d4) δ 173.0, 151.2, 150.9, 144.5, 136.9, 131.0, 129.5, 128.9, 127.2, 124.6, 63.5, 53.0, 47.4, 32.9, 31.7, 21.0.

[0160] LC-MS[M+H] + =399.17

[0161] Example 1 N-(1-Benzylpiperidin-4-yl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 9a (LIT-TB001)

[0162] N-(1-Benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 191 mg, 0.479 mmol) was dissolved in EtOH (2.5 ml). 1-Methylpiperazine 8a (2 eq., 95.9 mg, 0.106 mL, 0.958 mmol) and EtN (2 eq., 96.9 mg, 0.133 mL, 0.958 mmol) were added and the reaction was heated at reflux overnight. The product was evaporated and diluted in MeOH. HCl in EtO (2 M) was added (excess) and the reaction was stirred at room temperature for 1.5 h. The mixture was evaporated and the crude material was purified by silica gel chromatography using a gradient (AcOEt / MeOH / Et3N; 9 / 1 / 0.5 to 5 / 1 / 0.5), salified and lyophilized to afford 9a (LIT-TB001) (m=221.2 mg, yield=86%) as a light yellow solid.

[0163] 1 H NMR (400MHz, methanol-d4) δ7.87(d,J=10.2Hz,1H),7.33-7.23(m,6H),3.66-3.59(m,5H),3.49(s,2H),3.35-3.32(m,2H),2.82(dt,J=12.0,3.6H z,2H),2.75(dd,J=8.0,7.1Hz,2H),2.58(t,J=5.1Hz,4H),2.35(s,3H),2.09(td,J=11.8,2.6Hz,2H),1.82-1.75(m,2H),1.52-1.41(m,2H). 13 C NMR (101 MHz, methanol-d4) δ 173.2, 156.7, 150.0, 143.9, 138.6, 130.7, 129.3, 128.4, 124.7, 116.5, 63.7, 55.4, 53.3, 47.9, 46.4, 46.1, 33.2, 32.3, 21.2.

[0164] LC-MS (ESI) [M+H] + =463.29

[0165] N-(1-Benzylpiperidin-4-yl)-3-[6-(piperidin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 9b (LIT-TB002)

[0166] Following General Procedure A for the synthesis of LIT-TB001 analogs, N-(1-benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 38 mg, 0.0953 mmol), piperidine 8b (2 eq., 16.4 mg, 19 μL, 0.191 mmol) and EtN (2 eq., 19.3 mg, 26.5 μL, 0.191 mmol) in EtOH (0.6 ml) were used. The crude material was evaporated. A (H2O / MeOH; 9 / 1, 1 ml) solution was added to form a solid. The solid was sonicated and triturated in the presence of heptane, then filtered and washed with heptane to give the desired product as a light brown solid. The filtrate was evaporated and purified by reverse phase chromatography (H2O / MeOH) to give another fraction of the product. The two products were combined, salified and lyophilized to give 9b (LIT-TB002) (m = 24.5 mg, yield = 53%) as a light brown solid.

[0167] 1 H NMR (400MHz, methanol-d4) δ7.82(d,J=10.2Hz,1H),7.33-7.23(m,6H),3.66-3.62(m,5H),3.51(s,2H),3.34-3.31(m,2H),2.84(d,J= 11.6Hz, 2H), 2.75 (t, J = 7.7Hz, 2H), 2.11 (t, J = 11.7Hz, 2H), 1.80 (d, J = 13.1Hz, 2H), 1.75-1.67 (m, 6H), 1.47 (q, J = 11.9Hz, 2H). 13 C NMR (101 MHz, methanol-d4) δ 173.3, 156.7, 149.9, 143.8, 138.5, 130.7, 129.3, 128.4, 124.3, 116.9, 64.0, 53.3, 48.0, 47.9, 33.2, 32.3, 26.5, 25.5, 21.2.

[0168] LC-MS (ESI) [M+H] + =448.19

[0169] N-(1-Benzylpiperidin-4-yl)-3-[4-benzylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 9c (LIT-TB005)

[0170] Following General Procedure A for the synthesis of LIT-TB001 analogs, N-(1-benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 100 mg, 0.25 mmol), 1-benzylpiperazine 8c (2 eq., 88.3 mg, 87 μL, 0.5 mmol), and Et3N (2 eq., 50.7 mg, 70 μL, 0.50 mmol) were used in EtOH (1.2 ml). The reaction mixture was heated at 135° C. for 2 h. The crude material was evaporated and purified by flash chromatography on silica gel (EtOAc / MeOH / Et3N: 9 / 1 / 0.5), salified, and lyophilized to afford 9c (LIT-TB005) as a brown solid (m=74 mg, yield=55%).

[0171] 1 H NMR (400MHz, methanol-d4) δ7.76 (d, J = 10.2Hz, 1H), 7.30-7.12 (m, 11H), 3.57-3.51 (m, 4H), 3.49 (s, 2H), 3.45 (s, 2H), 3.22 (t ,J=7.5Hz,2H),2.76(dt,J=12.4Hz,J=2.8Hz,2H),2.64(t,J=7.5Hz,2H),2.55-2.46(m,4H),2.09-2.00(m,2H),1.69(dt J=12.8Hz, J=3.8Hz, 2H), 1.37 (qd, J=11.8Hz, J=2.8Hz, 2H). 13 C NMR (101 MHz, methanol-d4) δ 171.8, 168.7, 164.0, 155.4, 148.6, 145.3, 142.5, 137.1, 137.0, 129.3, 129.2, 128.0, 127.9, 127.1, 127.0, 123.2, 115.2, 62.6, 62.4, 52.1, 51.8, 46.5, 45.2, 31.8, 30.919.7.

[0172] LC-MS(ESI)=538.32[m / z],448.27(-Bn)

[0173] N-(1-Benzylpiperidin-4-yl)-3-[6-(piperidin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 9d (LIT-TB007)

[0174] Following the general procedure A for the synthesis of LIT-TB001 analogues, N-(1- benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 110 mg, 0.276 mmol), piperazine 8d (2 eq., 47.5 mg, 0.552 mmol) and Et3N (2 eq., 55.8 mg, 76.7 μL, 0.552 mmol) in EtOH (2.5 ml). The crude was evaporated and purified by silica gel chromatography (DCM / MeOH / Et3N; 4 / 1 / 0 to 4 / 1 / 0.1) to afford 9d (LIT-TB007) as a light yellow solid (m = 108 mg, yield = 87%).

[0175] 1 H NMR (400 MHz, Chloroform-d) δ 7.78 (d, J = 10.1 Hz, 1H), 7.30 - 7.18 (m, 4H), 6.89 (d, J = 10.1 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 3.78 - 3.70 (m, 1H), 3.52 - 3.48 (m, 4H), 3.44 (s, 2H), 3.33 (t, J = 7.3 Hz, 2H), 2.99 - 2.95 (m, 4H), 2.84 (t, J = 7.2 Hz, 2H), 2.74 (d, J = 11.7 Hz, 2H), 2.05 (t, J = 11.3 Hz, 2H), 1.80 (dd, J = 13.2, 3.8 Hz, 2H), 1.45 (qd, J = 11.2, 3.5 Hz, 2H). 13 CNMR (101 MHz, Chloroform-d) δ 171.1, 155.1, 148.8, 142.7, 138.4, 129.2, 128.3, 127.1, 124.5, 113.6, 63.1, 52.3, 47.1, 46.7, 45.6, 32.7, 32.0, 20.4.

[0176] LC-MS (ES+APCI) [M+H] + = 449.2

[0177] N-(1-benzylpiperidin-4-yl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3- yl]propanamide 9e (LIT-TB030)

[0178] Following the general procedure A for the synthesis of LIT-TB001 analogues, N-(1- benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 38 mg, 0.0953 mmol), 1-phenylpiperazine 8e (2 eq., 31.9 mg, 30 pL, 0.191 mmol) and Et3N (2 eq., 19.3 mg, 26.5 pL, 0.191 mmol) in EtOH (0.6 ml). The crude was evaporated. A solution (H2O / MeOH; 9 / 1, 1 ml) was added to form a solid. The solid was sonicated and triturated in the presence of heptane, then filtered and washed with heptane to give the desired product. The product was salted and lyophilized to give 9e (LIT-TB030) as a light brown solid (m = 25.8 mg, yield = 49%).

[0179] 1 H NMR (400 MHz, Methanol-d4) d 7.89 (d, J = 10.1 Hz, 1H), 7.37 (d, J = 10.2 Hz, 1H), 7.33 - 7.22 (m, 7H), 7.02 (d, J = 8.1 Hz, 2H), 6.87 (t, J = 7.5 Hz, 1H), 3.79 - 3.76 (m, 4H), 3.66 - 3.60 (m, 1H), 3.50 (s, 2H), 3.37 - 3.28 (m, 6H), 2.83 (d, J = 11.8 Hz, 2H), 2.76 (t, J = 7.7 Hz, 2H), 2.10 (t, J = 11.7 Hz, 2H), 1.78 (d, J = 12.8 Hz, 2H), 1.46 (q, J = 11.3, 10.6 Hz, 2H), NH (not visible). 13 C NMR (101 MHz, Methanol-d4) d 173.3, 156.8, 152.6, 150.1, 144.0, 138.2, 130.8, 130.2, 129.3, 128.5, 124.7, 121.5, 117.8, 116.7, 63.9, 53.2, 50.4, 47.9, 46.9, 33.3, 32.2, 21.2.

[0180] LC-MS (ESI) [M+H] + = 525.22

[0181] N-(1-benzylpiperidin-4-yl)-3-(6-(4-(pyrimidin-2-yl)piperazin-1-yl)-[1,2,4]triazolo[4,3- b]pyridazin-3-yl)propanamide 9f (LIT-TB004)

[0182] Following General Procedure A for the synthesis of LIT-TB001 analogs, N-(1-benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 100 mg, 0.25 mmol), 2-(1-piperazinyl)pyrimidine 8f (1 eq., 41.2 mg, 35.5 μL, 0.25 mmol) and Et3N (2 eq., 50.7 mg, 70 μL, 0.50 mmol) were used in EtOH (1.2 ml). The reaction mixture was heated at 135° C. for 2 h. The crude material was evaporated and purified by flash chromatography on silica gel (EtOAc / MeOH / Et3N: 9 / 1 / 0.5), salified, triturated with anhydrous Et2O and lyophilized to afford 9f (LIT-TB004) (m=50 mg, yield=38%) as a brown solid.

[0183] LC-MS[M+H] + =529.2; 551.2 (M+Na)

[0184] 3-(6-([1,4'-bipiperidinyl]-1'-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-N-(1-benzylpiperidin-4-yl)propionamide 9g (LIT-TB003)

[0185] Following General Procedure A for the synthesis of LIT-TB001 analogs, N-(1-benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 100 mg, 0.25 mmol), 4-piperidinylpiperidine 8g (2 eq., 84.4 mg, 0.50 mmol), and Et3N (2 eq., 50.7 mg, 70 μL, 0.50 mmol) were used in EtOH (1.2 ml). The reaction mixture was heated at 135° C. for 2 h. The crude material was evaporated and purified by silica gel flash chromatography (EtOAc / MeOH / Et3N: 9 / 1 / 0.5), triturated with anhydrous Et2O, salted, and lyophilized to afford 9 g (LIT-TB003) as a brown solid (m=100 mg, yield=75%).

[0186] LC-MS[M+H] + =531.4.

[0187] N-(1-Benzylpiperidin-4-yl)-4-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)butanamide 10a (LIT-TB009)

[0188] Following General Procedure A for the synthesis of LIT-TB001 analogs, N-(1-benzylpiperidin-4-yl)-4-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)butanamide 7b (1 eq., 100 mg, 0.24 mmol), 1-methylpiperazine 8a (2 eq., 48.5 mg, 0.48 mmol), and Et3N (2 eq., 49.0 mg, 67 μL, 0.48 mmol) were used in EtOH (1.1 ml). The reaction mixture was heated at 135° C. for 1.5 h. The crude material was evaporated and purified by silica gel flash chromatography (EtOAc / MeOH / Et3N: 9 / 1 / 0.5), triturated with anhydrous Et2O, salted, and lyophilized to afford 10a (LIT-TB009) as a brown solid (m=55 mg, yield=48%).

[0189] 1 H NMR (400 MHz, methanol-d4) δ 7.86 (d, 1H, J = 10.2 Hz), 7.50-7.42 (m, 2H), 7.37-7.30 (m, 3H), 7.29 (d, 1H, J = 10.2 Hz), 3.80-3.60 (m, 5H), 3.40-3.25 (m, 6H), 2.99-3.10 (m, 5H), 2.81 (s, 3H), 2.22 (t, 2H, J = 7.2 Hz), 2.10-1.90 (m, 4H), 1.65-1.80 (m 2H). 13 C NMR (101 MHz, methanol-d4) δ 174.7, 156.2, 150.6, 144.0, 132.4, 131.2, 130.6, 130.4, 125.5116.6, 61.4, 54.0, 52.7, 51.9, 44.7, 44.0, 36.0, 29.6, 24.4, 23.3.

[0190] LC-MS[M+H] + =477.2

[0191] 3-(6-(4-Methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-N-(1-phenylethylpiperidin-4-yl)propanamide 11a (LIT-TB011)

[0192] Following General Procedure A for the synthesis of LIT-TB001 analogs, 3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-N-(1-phenethylpiperidin-4-yl)propanamide 7c (1 eq., 100 mg, 0.24 mmol), 1-methylpiperazine 8a (2 eq., 48.5 mg, 0.48 mmol), and Et3N (2 eq., 49.0 mg, 67 μL, 0.48 mmol) were used in EtOH (1.1 ml). The reaction mixture was heated at 150° C. under microwave irradiation for 1.5 h. The crude material was evaporated and purified by silica gel flash chromatography (EtOAc / MeOH / Et3N: 9 / 1 / 0.5), triturated with anhydrous Et2O, salted, and lyophilized to afford 10a (LIT-TB009) as a light yellow solid (m=70 mg, yield=61%).

[0193] 1 H NMR (400MHz, methanol-d4) δ8.2(d,J=10.2Hz,1H),7.85(d,J=10.2Hz,1H),7.34 -7.07(m,5H),4.58-4.47(m,2H),3.89-3.77(m,1H),3.69-3.57(m,4H),3. 48(t,J=13.3Hz,2H),3.42-3.36(m,2H),3.35-3.22(m,4H),3.06-2.97(m, 4H),2.90(s,3H),2.85-2.81(m,2H),2.10-1.88(m,2H),1.82-1.69(m,2H) 13 C NMR (101 MHz, methanol-d4) 174.6, 156.7, 156.4, 137.5, 137.4, 130.0, 129.8, 128.3, 124.6, 118.5, 59.0, 53.7, 53.0, 45.8, 44.3, 43.6, 32.4, 31.5, 30.2, 20.8.

[0194] LC-MS[M+H] + =477.2

[0195] N-(1-Benzylpiperidin-4-yl)-2-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)acetamide 12a (LIT-TB008) General Procedure A for the synthesis of LIT-TB001 analogs was followed using N-(1-benzylpiperidin-4-yl)-2-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)acetamide 7d (1 eq., 100 mg, 0.26 mmol), 1-methylpiperazine 8a (1.5 eq., 39.0 mg, 0.39 mmol) and Et3N (2 eq., 52.6 mg, 72 μL, 0.52 mmol) in EtOH (0.75 ml). The reaction mixture was heated at 150° C. under microwave irradiation for 1.5 h. The crude material was evaporated and purified by flash chromatography on silica gel (DCM / MeOH / Et3N: 8 / 2 / 0.1), salified and lyophilized to afford 12a (LIT-TB008) (m=70 mg, yield=60%) as a light brown solid.

[0196] 1 H NMR (400MHz, methanol-d4) δ7.96 (d, J = 10.2Hz, 1H), 7.42-7.32 (m, 6H), 4.40-4.29 (m, 2H), 4.20 (s, 2H), 4.05-3.98 (m, 2H), 3.87-3.82 (m, 1H), 3.6 0-3.52(m,2H),3.43(d,J=12.4Hz,2H),3.30-3.25(m,2H),3.01(t,J=12.2Hz,2H),2.86(s,3H),2.12-2.04(m,2H),1.74(q,J=12.2Hz,2H). 13 C NMR (101 MHz, methanol-d4) δ 168.7, 160.1, 156.5, 142.3, 138.6, 132.4, 131.3, 130.4, 125.1117.6, 61.6, 53.7, 52.6, 46.3, 44.3, 43.6, 32.0, 30.0.

[0197] LC-MS[M+H] + =449.2

[0198] Alternatively, compounds 9-14 can be prepared in a three-step sequence as depicted in Scheme 2. Condensation of hydrazinopyridazine 5 with cyclic anhydride 2 in dioxane at 120°C affords triazolopyridazine propionic acid (or homolog) 15 in a single step. Peptide-type coupling of compounds 1 and 15 in the presence of isobutyl chloroformate yields the previously described triazolopyridazinamides 7a-f. Finally, nucleophilic aromatic substitution with piperidine or piperazine derivatives 8a-g, as described in Example 1, affords products of general formula 9-14.

[0199] Scheme 2 (see Formula III)

[0200]

[0201] Conditions: a) succinic anhydride or glutaric anhydride, dioxane, 120°C, 12h; b) isobutyl chloroformate, DIEA, DCM, 25°C, 2h; c) 8a-g, Et3N, EtOH, 150°C, 1h 30.

[0202] Example 2 :N-(1-benzylpiperidin-3-yl)-3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide 13a (m=0, m'=2, n=1, r=1) (LIT-TB055)

[0203] Step 1: 3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanoic acid 15

[0204] Succinic anhydride (1.18 eq., 500 mg, 3.46 mmol) was dissolved in dioxane (5 mL). 3-Chloro-6-hydrazinopyridazine 5 (1.18 eq., 420 mg, 0.566 mL, 4.07 mmol) was added and the reaction mixture was heated for 2 hours to give triazolopyridazinylpropionic acid 15. The white precipitate was filtered and washed with Et2O to give the title compound 15 (m = 437 mg, yield = 56%).

[0205] 1 H NMR (400MHz, DMSO-d6) δ 11.93 (bs, 1H), 8.44 (d, J = 9.6Hz, 1H), 7.49 (d, J = 9.6Hz, 1H), 3.27 (t, J = 7.2Hz, 2H), 2.88 (t, J = 7.2Hz, 2H). 13 C NMR (101MHz, DMSO-d6) δ173.5,149.2,149.0,143.3,127.5,122.9,30.2,19.4.

[0206] Step 2: N-(1-benzylpiperidin-3-yl)-3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide 7e

[0207] 3-(6-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanoic acid 15 (1.0 eq., 119 mg, 0.52 mmol) was suspended in DCM (3 ml), followed by DIEA (2 eq., 129.2 mg, 0.17 ml, 1.05 mmol). Isobutyl chloroformate (1.2 eq., 86.1 mg, 82.2 μL, 0.63 mmol) in DCM (0.5 mL) was then added dropwise to the solution, and the resulting mixture was stirred at room temperature for 30 min. 1-Benzylpiperidin-3-amine (1 eq., 100 mg, 0.52 mmol) was then introduced and stirring was maintained for an additional 2 hours. The volatiles were evaporated, and the crude material was then purified by silica gel column chromatography using DCM / MeOH: 90 / 10 as the eluent to afford the title compound 7e (m = 50 mg, yield = 24%) as a pale yellow solid.

[0208] 1 H NMR (400MHz, methanol-d4) δ8.23(d,J=9.7Hz,1H),7.42(d,J=9.7Hz,1H),7.35-7.30( m,4H),7.29-7.24(m,1H),3.94-3.85(m,1H),3.56(s,2H),3.43(t,J=7.5Hz,2H ),2.84J=7.5Hz,2H),273-2.66(m,1H),2.14(t,J=11.7Hz,1H),2.04-1.94(m,1 H),1.86-1.77(m,1H),1.76-1.68(m,1H),1.66-1.55(m,1H),1.33-1.22(m,1H). 13 C NMR (101 MHz, methanol-d4) δ 129.2, 127.9, 127.0, 125.8, 123.3, 62.6, 57.5, 52.8, 45.9, 31.5, 29.5, 22.9, 19.6.

[0209] Step 3: N-(1-benzylpiperidin-3-yl)-3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide (m=0, m'=2, n=1, r=1)

[0210] The same procedure A for the synthesis of LIT-TB001 analogs described in Example 1 was used, starting with N-(1-benzylpiperidin-3-yl)-3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide 7e (1 eq., 50 mg, 0.12 mmol), 1-methylpiperazine 8a (2 eq., 25.1 mg, 27.8 μL, 0.25 mmol), and Et3N (2 eq., 25.4 mg, 34.8 μL, 0.25 mmol) in EtOH (0.5 ml) at 135° C. for 1.5 h. After salification and lyophilization, the title compound 13a was obtained as a light yellow solid (m=28.6 mg, yield=43%).

[0211] 1 H NMR(400MHz, methanol-d4)δ7.98(d,J=10.2Hz,1H),7.51-7.43(m,5H),7.41(d,J=10 .2Hz,1H),4.16(s,2H),4.06-3.96(m,1H),3.90-3.76(m,4H),3.36(t,J=7.3Hz ,2H),3.29-3.15(m,2H),3.10-3.00(m,4H),2.92-2.67(m,2H),2.81(t,J=12.3 Hz,2H),2.68(s,3H),1.99-1.87(m,2H),1.85-1.74(m,1H),1.59-1.47(m,1H). 13 C NMR (101 MHz, methanol-d4) δ 173.7, 156.5, 150.0, 144.0, 132.0, 130.6, 130.1, 125.1, 116.6, 62.4, 56.15, 54.6, 53.4, 45.9, 45.5, 44.9, 32.8, 29.0, 22.5, 20.9.

[0212] LC-MS[M+H] + =462.28

[0213] Alternatively, compounds 9-14 can also be prepared by reductive amination of N-BOC protected pyridazinotriazoles 17a-f with the aid of sodium cyanoborohydride in the presence of appropriate phenylalkylaldehydes (Scheme 3). Compound 17 is readily obtained from the above carboxylic acid 15 by peptide-type coupling with commercially available N-BOC protected aminopiperidine derivatives (or homologues) 16 using isobutyl chloroformate as an activating agent (Scheme 3).

[0214] Scheme 3 (see Formula III)

[0215]

[0216] Conditions: a) 16, isobutyl chloroformate, DIEA, DCM, 25°C, 2h; b) TFA, DCM, 1h; c) Ph(CH2) n-1 CHO, NaBH3CN, DIEA, EtOH; c) 8a-g, Et3N, EtOH, 135°C, 1h 30.

[0217] Example 3: N-(1-benzylazepan-4-yl)-3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide 14 (m=1, m'=2, n=1, r=1) (LIT-TB056)

[0218] Step 1: tert-Butyl 4-(3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propionylamino)azepane-1-carboxylate 17f (m=1, m'=2, r=1)

[0219] 3-(6-Chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanoic acid 15 (1.0 eq., 116.3 mg, 0.51 mmol) was suspended in DCM (4 ml), followed by DIEA (2 eq., 134.7 mg, 898 μl, 1.04 mmol). Isobutyl chloroformate (1.2 eq., 84.2 mg, 1.20 mL, 0.61 mmol) was dissolved in DCM (0.5 mL) and added dropwise to the previous solution, and the resulting mixture was stirred at room temperature for 30 min. tert-Butyl 4-aminoazepane-1-carboxylate (1 eq., 110 mg, 0.51 mmol) was dissolved in DCM (0.5 mL), added dropwise, and stirring was maintained for an additional 2 hours. The volatiles were evaporated and the crude material was purified by silica gel column chromatography using EtOAc / MeOH: 80 / 20 as eluent to afford the title compound 17 as a light yellow oil (m=129 mg, yield=59%).

[0220] 1H NMR (400 MHz, methanol-d4) δ 8.12 (d, J = 9.7 Hz, 1H), 7.31 (d, J = 9.6 Hz, 1H), 3.69-3.59 (m, 1H), 3.49-3.38 (m, 1H), 3.33 (t, J = 7.5 Hz, 2H), 3.32-3.25 (m, 2H), 3.17-3.06 (m, 1H), 2.71 (2.70) (t, J = 7.5 Hz, 2H), 1.89-1.79 (m, 1H), 1.78-1.67 (m, 2H), 1.69-1.31 (m, 3H), 1.37 (1.36) (s, 9H, cis-trans geometry). 13 C NMR (101 MHz, methanol-d4) δ 172.4, 157.3, 151.2, 150.9, 144.6, 127.3, 124.7, 81.0 (79.9), 51.2 (51.0), 47.4 (46.8), 44.0 (43.6), 35.7 (35.5), 34.2 (33.9), 32.9, 28.7, 25.6 (25.5), 21.0.

[0221] Step 2: N-(1-Benzylazepan-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propionamide 7f (m=1, m'=2, r=1).

[0222] To an ice-cold solution of tert-butyl 4-(3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propionamido)azepane-1-carboxylate 17f (1 eq., 129 mg, 0.30 mmol) in DCM (1.5 mL) was added TFA (0.5 mL) and the resulting mixture was stirred for 2 h. The crude reaction was concentrated in vacuo and TFA was removed azeotropically with heptane. The compound was used in the reductive amination step without further purification. The crude material was dissolved in MeOH (2 mL). Benzaldehyde (2.2 eq., 71.2 mg, 68 μL) was added, followed by NaBHCN (3.6 eq., 69 mg, 1.1 mmol). The resulting mixture was stirred at 25 ° C overnight. The volatiles were evaporated and the crude material was dissolved in EtOAc (25 mL). The organic phase was washed with brine, dried and concentrated in vacuo. The residue was purified by silica gel column chromatography using EtOAc:MeOH (90:10) as eluent to afford 2-(1-benzylpiperidin-4-yl)-4-phenylpyridazin-3(2H)-one (m=92 mg, yield=71%) as a yellow oil.

[0223] 1H NMR (400MHz, methanol-d4) δ8.17(d,J=9.6Hz,1H),7.48-7.39(m,5H),7.35(d,J=9.6Hz,1H),4.21(s,2H),3.93-3.83(m,1H),3 .37(t,J=7.3Hz,2H),3.30-3.08(m,4H),2.77(t,J=7.3Hz,2H),2.07-1.96(m,2H),1.92-1.73(m,3H),1.63-1.50(m,1H). 13 C NMR (101 MHz, methanol-d4) δ 172.9, 151.4, 150.9, 144.7, 132.1, 131.0, 130.4, 127.4, 127.0, 124.8, 62.3, 55.8, 51.5, 50.1, 33.8, 33.0, 30.4, 21.7, 21.0.

[0224] Step 3: N-(1-benzylazepan-4-yl)-3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide 14

[0225] Using the same procedure A described in Example 1 for the synthesis of LIT-TB001 analogs and starting with N-(1-benzylazepan-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7f (1 eq., 92 mg, 0.22 mmol), 1-methylpiperazine 8a (2 eq., 40.2 mg, 44.6 μL, 0.40 mmol) and Et3N (2 eq., 45.2 mg, 62.1 μL, 0.2 mmol) in EtOH (0.5 ml) the title compound 14 was obtained as a light yellow solid (m = 23.5 mg, yield = 11%) after salification and lyophilization.

[0226] 1 H NMR (400MHz, methanol-d4) δ7.79 (d, J=10.2Hz, 1H), 7.36-7.28 (m, 5H), 7.25 (d, J= 10.1Hz,1H),3.89(s,2H),3.87-3.79(m,1H),3.56(t,J=4.5Hz,4H),3.24(t ,J=7.4Hz,2H),2.99-2.75(m,4H),2.66(t,J=7.4Hz,2H),2.51(t,J=5.1Hz, 4H),2.28(s,3H),1.90-1.80(m,2H),1.79-1.59(m,3H),1.54-1.43(m,1H).13 C NMR (101 MHz, methanol-d4) δ 171.6, 155.3, 152.2, 142.6, 129.9, 128.5, 128.4, 123.3, 115.2, 61.4, 54.7, 53.9, 50.3, 48.7, 45.0, 44.6, 32.6, 31.7, 30.7, 21.7, 19.7.

[0227] LC-MS[ESI]:476.30(m / z)

[0228] General Procedure B

[0229] The preparation of compounds of formula 20 carrying various substituted piperidines on the propionamide chain can be carried out using various synthetic routes and conventional methods (Scheme 4). Starting from the readily available 6-chloro-triazolopyridazine N-BOC-protected piperidine 17a, SNAr reaction with 8aj produces the corresponding 6-N-methylpiperazine 18a. Deprotection of the protective BOC group and direct alkylation with an appropriate haloalkyl derivative (Method A, see Example 4) or reductive amination with an appropriate aldehyde (Method B, see Example 5) in the presence of NaBH(OAc)3 provide the examples of the present invention.

[0230] Scheme 4 (see Formula III)

[0231]

[0232] Conditions: a) 8a, Et3N, EtOH, 135°C, 1h 30; b) 4N HCl / dioxane; c) TFA, DCM, 2h; d) RX, K2CO3, DMF, argon, -5°C (30min) → room temperature (overnight); e) RCHO; NaBH(OAc)3, MeOH

[0233] tert-Butyl 4-(3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propionylamino)piperidine-1-carboxylate 17a

[0234] Using the same procedure described for the preparation of 17f and starting with 3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanoic acid 15a (1.0 eq., 200 mg, 0.89 mmol) and 4-amino-1-Bocpiperidine (1.0 eq., 180 mg, 0.89 mmol, CAS number: 87120-72-7), the title compound was obtained as a light brown solid (m=234 mg, yield=64%).

[0235] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=9.7Hz,1H),7.95(d,J=8.0Hz,1H),7.48(d,J=9.7Hz,1H),3.81(d,J=14.3Hz,2H),3.75-3.65(m,1H ), 3.27 (t, J = 7.5Hz, 2H), 2.93-2.75 (m, 2H), 2.68 (t, J = 7.5Hz, 2H), 1.68 (dd, J = 12.9Hz, J = 4.1Hz, 2H), 1.39 (s, 9H), 126-1.14 (m, 2H). 13 C NMR (100MHz, DMSO-d6) δ170.1,154.4,149.4,149.1,143.2,127.4,122.8,79.1,46.1,32.0,31.8,28.5,20.0.

[0236] tert-Butyl 4-(3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propionylamino)piperidine-1-carboxylate 18a

[0237] Following general procedure A for the synthesis of LIT-TB001 analogs, tert-butyl 4-(3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propionamido)piperidine-1-carboxylate 17a (1 eq., 50 mg, 0.12 mmol), 1-methylpiperazine 8a (2 eq., 16.4 mg, 19 μL, 0.191 mmol), and EtN (2 eq., 24.75 mg, 34 μL, 0.24 mmol) were used in EtOH (0.8 ml). The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH) to give the title compound (m=45 mg, yield=78%) as a white solid.

[0238] 1 H NMR (400MHz, methanol-d4) δ7.90(d,J=10.2Hz,1H),7.36(d,J=10.2Hz,1H),3.98(d, J=13.7Hz,2H),3.81(tt,J=10.8,4.1Hz,1H),3.68(t,J=5.2Hz,4H),3.36(dd,J =7.9,7.2Hz,2H),2.96-2.85(m,2H),2.78(t,J=7.5Hz,2H),2.62(t,J=5.1Hz,4 H), 2.38 (s, 3H), 1.80 (dd, J = 13.1, 3.8Hz, 2H), 1.46 (s, 9H), 1.37-1.25 (m, 2H).13 C NMR (101 MHz, methanol-d4) δ 173.2, 156.8, 156.4, 150.0, 144.0, 124.7, 116.6, 81.1, 55.4, 47.9, 46.46, 46.44, 46.1, 33.2, 32.6, 28.7, 21.1.

[0239] 3-(6-(4-Methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-N-(piperidin-4-yl)propionamide 19 (LIT-TB021)

[0240] Tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (67 mg, 0.14 mmol) was dissolved in DCM (0.7 mL). 4N HCl in dioxane (10 eq., 1.42 mmol, 0.35 ml) was added, and the reaction mixture was stirred at room temperature for 30 min. The precipitate was collected, washed three times with anhydrous EtO, and dried (m = 27 mg, yield = 43%).

[0241] 1 H NMR (400MHz, methanol-d4) δ7.89(d,J=10.2Hz,1H),7.34(d,J=10.2Hz,1H),3.79-3.68( m,1H),3.66(t,J=5.1Hz,4H),3.34(t,J=7.6Hz,2H),3.03(dt,J=12.7Hz,J=4.1Hz, 2H),2.76(t,J=7.6Hz,2H),2.65(td,J=12.2Hz,J=2.8Hz,2H),2.60(t,J=5.1Hz,4 H), 2.36 (s, 3H), 1.81 (dd, J = 12.9Hz, J = 3.8Hz, 2H), 1.37 (qd, J = 12.0Hz, J = 6.0Hz). 13 CNMR (101MHz, methanol-d4)δ173.3,156.9,150.2,144.1,124.9,116.8.55.5,48.0,46.6,46.3,45.8,33.3,33.1,21.3.

[0242] LC-MS[M+H] + =372.24

[0243] Example 4N-{1-[(4-methoxyphenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20a (LIT-TB017)

[0244] Tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (17.2 mg, 0.0364 mmol) was dissolved in DCM (0.3 mL). TFA (10 eq., 41.5 mg, 27 μL, 0.364 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The crude material was evaporated and then co-evaporated twice with DCM / heptane. After drying, the crude material was dissolved in anhydrous DMF under argon. K2CO3 (5 eq., 25.2 mg, 0.182 mmol) was added and the reaction mixture was stirred at -5 °C for 30 min. 1-(Bromomethyl)-4-methoxybenzene (1 eq., 7.32 mg, 5.25 μL, 0.0364 mmol) was added, and the mixture was stirred at -5 ° C for 0.5 h, then stirred at room temperature overnight. Water (a few drops) was added, and the crude material was directly purified by reverse phase chromatography (HO / MeOH). The product was evaporated and diluted in MeOH. EtO (2M) containing HCl (excess) was added, and the reactants were stirred at room temperature for 1.5 h. The mixture was evaporated, diluted in water and lyophilized. The title compound 20a (m=6.9 mg, yield=30%) was obtained as a light yellow solid.

[0245] 1 H NMR (400 MHz, methanol-d4) δ 7.89 (d, J = 10.2 Hz, 1H), 7.35 (d, J = 10.2 Hz, 1H), 7.24 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 8.1 Hz, 2H), 3.79 (s, 3H), 3.68-3.60 (m, 5H), 3.52 (s, 2H), 3.36-3.3 1(m,2H),2.88(d,J=11.6Hz,2H),2.76(t,J=7.7Hz,2H),2.61-2.58(m,4H),2.37(s,3H),2.18(t,J=11.6Hz,2H),1.81(d,J=13.0Hz,2H),1.48(q,J=12.0Hz,2H),NH(invisible). 13C NMR (126 MHz, methanol-d4) δ 173.3, 160.8, 156.8, 150.1, 143.9, 132.2, 129.5, 124.7, 116.6, 114.8, 63.1, 55.7, 55.4, 53.0, 47.7, 46.4, 46.1, 33.2, 32.0, 21.2.

[0246] LC-MS (ESI) [M+H] + =493.20

[0247] N-{1-[(3-chlorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20b (LIT-TB018)

[0248] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 14.9 mg, 0.0315 mmol), 3-chlorobenzyl bromide (1.1 eq., 7.35 mg, 4.69 μL, 0.0347 mmol), and KCO (5 eq., 21.8 mg, 0.158 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20b as a light yellow solid (m = 9.4 mg, yield = 52%).

[0249] 1 H NMR (400Mhz, methanol-d4) δ7.89 (d, J = 10.1Hz, 1H), 7.39-7.22 (m, 5H), 3.67-3.50 (m, 5H), 3.50 (s, 2H), 3.33 (t, J = 11.0Hz, 2H), 2.82 (d, J = 11. 8Hz, 2H), 2.76 (t, J = 7.6Hz, 2H), 2.61-2.58 (m, 4H), 2.36 (s, 3H), 2.12 (t, J = 11.8Hz, 2H), 1.80 (d, J = 12.9Hz, 2H), 1.47 (q, J = 11.9Hz, 2H). 13 C NMR (126 MHz, methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 141.3, 135.3, 130.8, 130.4, 128.9, 128.5, 124.7, 116.6, 63.2, 55.4, 53.3, 47.9, 46.4, 46.1, 33.2, 32.4, 21.2.

[0250] LC-MS (ESI) [M+H] + =497.17

[0251] N-{1-[(2-chlorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20c (LIT-TB019)

[0252] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 14.8 mg, 0.0313 mmol), 2-chlorobenzyl bromide (1.1 eq., 7.08 mg, 4.47 μL, 0.0344 mmol), and KCO (5 eq., 21.6 mg, 0.157 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20c as a light yellow solid (m = 11.2 mg, yield = 63%).

[0253] 1 H NMR (400MHz, methanol-d4) δ7.88(d,J=10.0Hz,1H),7.47(d,J=6.9Hz,1H),7.42(d,J=6.8Hz,1H),7.38-7.27(m,3H),3.83(s,2H),3.72-3.69(m,5H) ,3.35-3.31(m,2H),3.02(d,J=11.8Hz,2H),2.80-2.69(m,6H),2.49-2 .43(m,2H),2.45(s,3H),1.86(d,J=12.9Hz,2H),1.57(q,J=11.5Hz,2H. 13 C NMR (101 MHz, methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 132.4, 130.5, 129.7, 127.9, 124.7, 116.5, 60.1, 55.4, 53.4, 47.9, 46.4, 46.1, 33.2, 32.5, 21.2.

[0254] LC-MS (ESI) [M+H] + =497.16

[0255] N-{1-[(4-Fluorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20d (LIT-TB020)

[0256] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 16.3 mg, 0.0345 mmol), 4-fluorobenzyl chloride (1.1 eq., 5.49 mg, 4.52 μL, 0.0379 mmol), and KCO (5 eq., 23.8 mg, 0.172 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20d as a light yellow solid (m = 5.1 mg, yield = 27%).

[0257] 1 H NMR (400MHz, methanol-d4) δ7.88(d,J=10.2Hz,1H),7.35-7.31(m,3H),7.04(t,J=8.6Hz,2H),3.67-3.62(m,5H),3.50(s,2H),3.35-3.30(m,2H),2.83( d,J=11.5Hz,2H),2.75(t,J=7.6Hz,2H),2.60-2.58(m,4H),2.35(s,3H) ,2.12(t,J=11.8Hz,2H), 1.79(d,J=12.9Hz,2H), 1.46(q,J=12.0Hz,2H). 13 C NMR (126MHz, Methanol-d4) δ 173.2, 163.6 (d, J = 244.1Hz), 156.8, 150.1, 143.9, 134.6 (d, J = 3.2Hz), 132. 5(d,J=8.0Hz),124.7,116.6,115.9(d,J=21.5Hz),63.0,55.4,53.2,47.9,46.4,46.1,33.2,32.4,21.2. 19 F NMR (376 MHz, methanol-d4) δ -117.5.

[0258] LC-MS (ESI) [M+H] + =481.18

[0259] N-{1-[(2-fluorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20e (LIT-TB022)

[0260] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 16.3 mg, 0.0345 mmol), 2-fluorobenzyl bromide (1.1 eq., 7.17 mg, 4.58 μL, 0.0379 mmol), and KCO (5 eq., 23.8 mg, 0.172 mmol) were used in DMF (0.3 ml). The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20e (10.9 mg, 57% yield) as a pale yellow solid.

[0261] 1 H NMR (400Mhz, methanol-d4) δ7.88 (d, J = 10.1Hz, 1H), 7.42-7.27 (m, 3H), 7.15 (t, J = 7. 6Hz,1H),7.08(t,J=9.4Hz,1H),3.67-3.65(m,5H),3.59(s,2H),3.35-3.31(m ,2H),2.86(d,J=11.8Hz,2H),2.75(t,J=7.7Hz,2H),2.60-2.58(m,4H),2.36( s, 3H), 2.17 (t, J = 11.7Hz, 2H), 1.79 (d, J = 12.9Hz, 2H), 1.47 (q, J = 12.0Hz, 2H). 13 C NMR (126MHz, methanol-d4) δ 173.2, 162.9 (d, J = 245.2Hz), 156.8, 150.1, 143.9, 133.3 (d, J = 4.2Hz), 130.6 (d, J = 8.3Hz), 125.1 (d ,J=3.6Hz),125.0,124.7,116.6,116.2(d,J=22.6Hz),56.0(d,J=1.9Hz),55.4,53.1,47.8,46.4,46.1,33.2,32.3,21.2. 19 F NMR (376 MHz, methanol-d4) δ -119.35.

[0262] LC-MS (ESI) [M+H] + =481.19

[0263] 3-[6-(4-Methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N-[1-(1-phenylethyl)piperidin-4-yl]propanamide 20f (LIT-TB023)

[0264] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 19.4 mg, 0.0411 mmol), (1-bromoethyl)benzene (1.1 eq., 8.36 mg, 6.19 μL, 0.0452 mmol), and KCO (5 eq., 28.4 mg, 0.205 mmol) were used in DMF (0.3 ml). The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20f (m=14.4 mg, yield=64%) as a light yellow solid. 1 H NMR (400 MHz, methanol-d4) δ 7.88 (d, J = 10.0 Hz, 1H), 7.36-7.22 (m, 6H), 3.66-3.64 (m, 4H), 3.57 (t, J = 11.4 Hz, 1H), 3.48 (q, J = 6.7 Hz, 1H), 3.35-3.31 (m, 2H), 3.07 (d, J = 11. 6Hz,1H),2.80-2.72(m,3H),2.60-2.57(m,4H),2.35(s,3H),2.08(dt,J=34.5,11. 9Hz, 2H), 1.78 (dd, J = 34.3, 13.0Hz, 2H), 1.56-1.38 (m, 2H), 1.41 (d, J = 6.7Hz, 3H). 13 C NMR (126 MHz, methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 143.4, 129.4, 129.0, 128.5, 124.7, 116.6, 66.4, 55.4, 51.0, 50.4, 47.9, 46.4, 46.1, 33.2, 32.4, 21.2, 19.7.

[0265] LC-MS (ESI) [M+H] + =477.21

[0266] N-{1-[(2-methylphenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20g (LIT-TB024)

[0267] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 17.7 mg, 0.0375 mmol), 2-methylbenzyl bromide (1.1 eq., 7.62 mg, 5.52 μL, 0.0412 mmol), and KCO (5 eq., 25.9 mg, 0.187 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20g (m=13.3 mg, yield=65%) as a light yellow solid.

[0268] 1 H NMR (400Mhz, methanol-d4) δ7.88 (d, J = 10.2Hz, 1H), 7.33 (d, J = 10.1Hz, 1H), 7.23-7.21 (m,1H),7.14-7.10(m,3H),3.67-3.64(m,5H),3.49(s,2H),3.35-3.31(m,2H),2. 85(d,J=11.5Hz,2H),2.75(t,J=7.6Hz,2H),2.61-2.58(m,4H),2.36(s,3H),2.3 5(s,3H),2.15(t,J=11.8Hz,2H),1.78(d,J=12.8Hz,2H),1.45(q,J=11.9Hz,2H). 13 C NMR (126 MHz, methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 138.7, 137.0, 131.4, 131.2, 128.4, 126.6, 124.7, 116.6, 61.4, 55.4, 53.5, 48.0, 46.4, 46.1, 33.2, 32.5, 21.2, 19.5.

[0269] LC-MS (ESI) [M+H] + =477.24

[0270] 3-[6-(4-Methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N-[1-(pyridin-4-ylmethyl)piperidin-4-yl]propanamide 20h (LIT-TB025)

[0271] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 20.5 mg, 0.0434 mmol), 4-(chloromethyl)pyridine hydrochloride (1.1 eq., 7.83 mg, 0.0477 mmol), and KCO (5 eq., 30 mg, 0.217 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20h as a light yellow solid (m = 13.9 mg, yield = 56%).

[0272] 1 H NMR (500MHz, methanol-d4) δ8.49-8.43(m,2H),7.88(d,J=10.2Hz,1H),7.43-7.40( m,2H),7.33(d,J=10.2Hz,1H),3.68-3.60(m,5H),3.56(s,2H),3.35-3.31(m ,2H),2.80(d,J=11.9Hz,2H),2.75(t,J=7.6Hz,2H),2.61-3.58(m,4H),2.36 (s,3H),2.14(td,J=11.8,2.5Hz,2H),1.83-1.76(m,2H),1.53-1.44(m,2H). 13 C NMR (126 MHz, methanol-d4) δ 173.2, 156.8, 150.3, 150.1, 150.0, 144.0, 125.8, 124.7, 116.6, 62.5, 55.4, 53.5, 47.9, 46.4, 46.1, 33.2, 32.5, 21.2.

[0273] LC-MS (ESI) [M+H] + =464.18

[0274] N-{1-[(3,4-dichlorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20i (LIT-TB026)

[0275] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 19.2 mg, 0.0406 mmol), 3,4-dichlorobenzyl chloride (1.1 eq., 8.74 mg, 6.2 μL, 0.0447 mmol), and KCO (5 eq., 28.1 mg, 0.203 mmol) were used in DMF (0.3 ml). The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20i as a light yellow solid (m = 15.6 mg, yield = 64%).

[0276] 1 H NMR (500 MHz, methanol-d4) δ 7.88 (d, J = 10.1 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 10.2 Hz, 1H), 7.24 (dd, J = 8.2, 2.0 Hz, 1H), 3.69-3.58 (m, 5H), 3.47 (s, 2H ),3.35-3.31(m,2H),2.83-2.77(m,2H),2.75(t,J=7.6Hz,2H),2.60-2.57(m,4H),2.35 (s,3H),2.11(td,J=11.8,2.6Hz,2H),1.79(dd,J=12.9,3.9Hz,2H),1.53-1.41(m,2H). 13 C NMR (126 MHz, methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 140.1, 133.2, 132.3, 132.0, 131.4, 130.2, 124.7, 116.6, 62.5, 55.4, 53.3, 47.9, 46.4, 46.1, 33.2, 32.4, 21.2.

[0277] LC-MS (ESI) [M+H] + =531.11

[0278] N-(1-Benzoylpiperidin-4-yl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide hydrochloride 20j (LIT-TB027)

[0279] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 20.2 mg, 0.0427 mmol), 3-benzoyl chloride (1.1 eq., 6.61 mg, 5.46 μL, 0.047 mmol), and KCO (5 eq., 29.5 mg, 0.214 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20j as a light yellow solid (m = 8.2 mg, yield = 32%).

[0280] 1 H NMR (400 MHz, methanol-d4) δ 7.91 (d, J = 10.2 Hz, 1H), 7.48-7.44 (m, 3H), 7.41-7.35 (m, 3H), 4.47 (d, J = 13.4 Hz 1H), 3.93 (tt, J = 10.5, 4.2 Hz, 1H), 3.77-3.63 (d, J = 5.3 Hz, 5H), 3.36 (t, J = 7.4 Hz, 2H), 3.23-3.02 (m, 2H), 2.83-2.76 (m, 6H), 2.50 (s, 3H), 2.00-1.73 (m, 2H), 1.51-1.30 (m, 2H). 13 C NMR (126 MHz, methanol-d4) δ 173.3, 172.5, 156.8, 150.0, 144.0, 137.0, 131.1, 129.8, 127.8, 124.7, 116.6, 55.4, 47.8, 46.4, 46.1, 42.1, 33.2, 32.2, 21.1.

[0281] LC-MS (ESI) [M+H] + =477.17

[0282] N-{1-[(4-chlorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20k (LIT-TB028)

[0283] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 19.8 mg, 0.0419 mmol), 4-chlorobenzyl chloride (1.1 eq., 9.47 mg, 0.0461 mmol) (1.1 eq., 6.61 mg, 5.46 μL, 0.047 mmol), and KCO (5 eq., 29 mg, 0.209 mmol) in DMF (0.3 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20k as a light yellow solid (m = 10.8 mg, yield = 45%).

[0284] 1 H NMR (500Mhz, methanol-d4) δ7.88 (d, J=10.1Hz, 1H), 7.33 (d, J=10.2Hz, 1H), 7.31 -7.29(m,4H),3.69-3.58(m,5H),3.48(s,2H),3.35-3.31(m,2H),2.81(d, J=11.8Hz,2H),2.74(t,J=7.5Hz,2H),2.60-2.57(m,4H),2.35(s,3H),2.1 0(td,J=11.8,2.5Hz,2H),1.78(dd,J=13.5,3.7Hz,2H),1.54-1.41(m,2H). 13 C NMR (126 MHz, methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 137.5, 134.2, 132.2, 129.4, 124.7, 116.6, 63.0, 55.4, 53.2, 47.9, 46.4, 46.1, 33.2, 32.4, 21.2.

[0285] LC-MS (ESI) [M+H] + =497.16

[0286] N-[1-(Cyclohexylmethyl)piperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 201 (LIT-TB031)

[0287] Following the general procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4- methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoylamino}piperidine-1- carboxylate 18a (1 eq., 19.8 mg, 0.0419 mmol), KI (1 eq., 7.73 mg, 0.0466 mmol) and 4-methylbenzene-1-sulfonic acid cyclohexylmethyl ester (1.1 eq., 13.7 mg, 0.0512 mmol) and K2CO3 (5 eq., 32.2 mg, 0.233 mmol) in DMF (0.3 ml) at 85 °C overnight. The crude was evaporated and purified by reverse phase chromatography (H2O / MeOH), salted and lyophilized to give 20l as a light yellow solid (m = 4.1 mg, yield = 16%).

[0288] 1 H NMR (500 Mhz, Methanol-d4) δ 7.92 (d, J = 10.2 Hz, 1H), 7.38 (d, J = 10.2 Hz, 1H), 3.74 - 3.61 (m, 5H), 3.38 (t, J = 7.6 Hz, 2H), 2.87 (d, J = 11.8 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 2.64 - 2.62 (m, 4H), 2.40 (s, 3H), 2.18 (d, J = 6.8 Hz, 2H), 2.06 (t, J = 11.6 Hz, 2H), 1.84 - 1.70 (m, 7H), 1.35 - 1.21 (m, 3H), 1.37 - 1.17 (m, 3H), 0.98 - 0.90 (m, 2H). 13 C NMR (126 MHz, Methanol-d4) δ 173.2, 156.8, 150.1, 144.0, 124.7, 116.6, 66.9, 55.4, 54.0, 48.1, 46.4, 46.1, 36.4, 33.2, 33.2, 32.3, 27.7, 27.2, 21.2.

[0289] LC-MS (ESI) [M+H] + = 469.24

[0290] N-{1-[(5-Methyl-1H-imidazol-4-yl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)- [1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20m (LIT-TB032)

[0291] Following the general procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4- methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamido}piperidine-1- carboxylate 18a (1 eq., 22 mg, 0.0466 mmol), KI (1 eq., 7.73 mg, 0.0466 mmol) and 4-(chloromethyl)-5-methyl-1H-imidazole (1.1 eq., 6.69 mg, 0.0512 mmol) and K2CO3 (5 eq., 32.2 mg, 0.233 mmol) in DMF (0.5 ml) at 85 °C for 5 h. The crude was evaporated and purified by reverse phase chromatography (H2O / MeOH), salted and lyophilized to give 20m as a light yellow solid (m = 7.8 mg, yield = 30%).

[0292] 1 H NMR (500 Mhz, Methanol-d4) δ 7.95 (d, J = 10.2 Hz, 1H), 7.57 (s, 1H), 7.40 (d, J = 10.2 Hz, 1H), 3.74-3.72 (m, 4H), 3.70-3.63 (m, 1H), 3.55 (s, 2H), 3.42-3.39 (m, 2H), 2.93 (d, J = 11.6 Hz, 2H), 2.82 (t, J = 7.6 Hz, 2H), 2.67-2.65 (m, 4H), 2.43 (s, 3H), 2.27 (s, 3H), 2.26-2.21 (m, 2H), 1.87 (dd, J = 13.2, 3.8 Hz, 2H), 1.57-1.50 (m, 2H). 13 C NMR (126 MHz, Methanol-d4) δ 173.2, 156.8, 150.1, 143.9, 134.8, 124.7, 116.6, 55.4, 52.9, 47.8, 46.4, 46.1, 33.2, 32.3, 21.2.

[0293] LC-MS (ESI) [M+H] + = 467.21

[0294] N-{1-[(2,4-Difluorophenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)- [1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20n (LIT-TB040)

[0295] Following the general procedure B for the synthesis of 20a, using tert-butyl 4-{3-[6-4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamido}piperidine-1 - carboxylate 18a (1 eq., 26 mg, 0.055 mmol), 2,4-difluorobenzyl bromide (1.1 eq., 12.5 mg, 7.78 μί, 0.0605 mmol) and K2CO3(5 eq., 38 mg, 0.275 mmol) in DMF (0.3 ml). The crude was evaporated and purified by reverse phase chromatography (H2O / MeOH), salted and lyophilized to give 20n as a light yellow solid (m = 25.6 mg, yield = 81 %).

[0296] 1 H NMR (500 Mhz, Methanol-d4) δ 7.93 (d, J = 10.2 Hz, 1 H), 7.49 - 7.44 (m, 1 H), 7.38 (d, J = 10.2 Hz, 1 H), 7.01 - 6.95 (m, 2 H), 3.72 - 3.70 (m, 4 H), 3.70 - 3.63 (m, 1 H), 3.60 (s, 2 H), 3.40 - 3.37 (m, 2 H), 2.88 (d, J = 11.9 Hz, 2 H), 2.80 (t, J = 7.6 Hz, 2 H), 2.65 - 2.63 (m, 4 H), 2.41 (s, 3 H), 2.23 - 2.18 (m, 2 H), 1.88 - 1.80 (m, 2 H), 1.56 - 1.48 (m, 2 H). 13 C NMR (126 MHz, Methanol-d4) δ 173.2, 163.9 (dd, J = 247.0, 12.0 Hz), 162.9 (dd, J = 248.0, 12.5 Hz), 156.8, 150.1, 143.9, 134.3 (dd, J = 9.6, 5.9 Hz), 124.7, 121.5 (dd, J = 14.7, 3.7 Hz), 116.5, 112.1 (dd, J = 21.6, 3.8 Hz), 104.4 (dd, J = 26.8, 25.7 Hz), 55.5, 55.4, 53.0, 47.8, 46.5, 46.1, 33.2, 32.4, 21.2. 19 F NMR (376 MHz, Methanol-d4) δ -113.2, -114.8.

[0297] LC-MS (ESI) [M+H] + = 499.21

[0298] N-{1-[(4-fluoro-2-methylphenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 20o (LIT-TB044)

[0299] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 24.7 mg, 0.0523 mmol), 1-(bromomethyl)-4-fluoro-2-methylbenzene (1.1 eq., 11.7 mg, 8.02 μL, 0.0575 mmol), and KCO (5 eq., 36.1 mg, 0.261 mmol) were used in DMF (0.4 ml). The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20o as a light yellow solid (m = 14.8 mg, yield = 50%).

[0300] 1 H NMR (500Mhz, methanol-d4) δ7.87(d,J=10.2Hz,1H),7.33(d,J=10.2Hz,1H),7.21(dd,J=8.4,6.0Hz,1H), 6.88(dd,J=9.9,2.7Hz,1H),6.83(td,J=8.5,2.8Hz,1H),3.67-3.60(m,5H),3.42(s,2H),3.35-3. 31(m,2H),2.80(d,J=11.6Hz,2H),2.75(t,J=7.6Hz,2H),2.58(t,J=5.1Hz,4H),2.35(s,3H),2.35 (s, 3H), 2.09 (td, J = 11.7, 2.5Hz, 2H), 1.76 (dd, J = 13.5, 4.0Hz, 2H), 1.42 (qd, J = 11.6, 3.8Hz, 2H). 13 C NMR (126MHz, methanol-d4) δ 173.2, 163.3 (d, J = 243.4Hz), 156.8, 150.1, 143.9, 141.4 (d, J = 7.7Hz), 133.4 (d, J = 2.9Hz), 132.8 (d, J = 8.3Hz), 124.7, 117.7 (d, J = 21.0Hz), 116.6, 112.9 (d, J = 20.9Hz), 60.8, 55.4, 53.4, 48.1, 46.4, 46.1, 33.2, 32.6, 21.2, 19.5. 19F NMR (471 MHz, methanol-d4) δ -118.5.

[0301] LC-MS (ESI) [M+H] + =495.28

[0302] N-{1-[(4-methoxy-2-methylphenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide dihydrochloride 20p (LIT-TB045)

[0303] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 25 mg, 0.0529 mmol), 1-(bromomethyl)-4-methoxy-2-methylbenzene (1.2 eq., 13.7 mg, 0.0635 mmol), and KCO (5 eq., 36.6 mg, 0.265 mmol) in DMF (0.4 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20p as a light yellow solid (m = 11.5 mg, yield = 38%).

[0304] 1 H NMR (500Mhz, methanol-d4) δ7.97(d,J=10.1Hz,1H),7.42(d,J=10.2Hz,1H),7.21(d,J=8.4Hz,1H), 6.82(d,J=2.6Hz,1H),6.78(dd,J=8.3,2.7Hz,1H),3.86(s,3H),3.78-3.70(m,5H),3.50(s,2 H),3.46-3.41(m,2H),2.92(d,J=11.8Hz,2H),2.85(t,J=7.6Hz,2H),2.69(t,J=5.1Hz,4H),2 .46(s,3H),2.43(s,3H),2.22-2.16(m,2H),1.90-1.84(m,2H),1.53(qd,J=11.5,3.7Hz,2H). 13C NMR (126 MHz, methanol-d4) δ 173.2, 160.3, 156.8, 150.1, 143.9, 140.1, 132.5, 129.4, 124.7, 116.9, 116.6, 111.6, 60.9, 55.6, 55.4, 53.4, 48.2, 46.4, 46.1, 33.3, 32.6, 21.2, 19.7.

[0305] LC-MS (ESI) [M+H] + =507.31

[0306] N-{1-[(2-fluoro-4-methoxyphenyl)methyl]piperidin-4-yl}-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide dihydrochloride 20q (LIT-TB046)

[0307] Following General Procedure B for the synthesis of 20a, tert-butyl 4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 18a (1 eq., 26 mg, 0.055 mmol), 1-(bromomethyl)-2-fluoro-4-methoxybenzene (1.4 eq., 16.9 mg, 0.077 mmol), and KCO (5 eq., 38 mg, 0.275 mmol) in DMF (0.4 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 20q as a light yellow solid (m = 16.5 mg, yield = 51%).

[0308] 1 H NMR (400Mhz, methanol-d4) δ7.89 (dd, J=10.3, 2.9Hz, 1H), 7.34 (dd, J=10.5, 2.8Hz, 1H), 7.27 (dd, J= 10.0,7.5Hz,1H),6.73(d,J=8.6Hz,1H),6.68(d,J=12.1Hz,1H),3.79(s,3H),3.69-3.57(m,5 H),3.52(s,2H),3.36-3.33(m,2H),2.85(d,J=11.6Hz,2H),2.75(t,J=7.8Hz,2H),2.62-2.58 (m, 4H), 2.36 (s, 3H), 2.14 (t, J = 11.8Hz, 2H), 1.79 (d, J = 12.8Hz, 2H), 1.46 (q, J = 12.1Hz, 2H). 13C NMR (101MHz, methanol-d4) δ 173.2, 163.5 (d, J = 244.9Hz), 162.2 (d, J = 11.2Hz), 156.8, 150.1, 143.9, 133.9 (d, J = 6.2Hz), 124.7, 116. 56,116.55(d,J=15.6Hz),110.9(d,J=2.9Hz),102.2(d,J=26.6Hz),56.1,55.6,55.4,52.8,47.8,46.4,46.1,33.2,32.3,21.2. 19 FNMR (376 MHz, methanol-d4) δ-116.9.

[0309] LC-MS (ESI) [M+H] + =511.26

[0310] Example 5: 3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N-[1-(1,3-oxazol-4-ylmethyl)piperidin-4-yl]propanamide 20r (LIT-TB050)

[0311] 4-{3-[6-(4-methylpiperazine-1-yl)-[1,2,4]triazolo[4,3-b]pyridazine-3-yl]propionamido}piperidine-1-carboxylic acid tert-butyl ester 18a (18.6 mg, 0.039 mmol) was dissolved in DCM (0.3 mL). TFA (10 eq., 41.5 mg, 27 μL, 0.364 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The crude material was evaporated and then co-evaporated twice with DCM / heptane. After drying, the crude material was dissolved in saturated KCO solution and extracted twice with DCM. The organic phase was dried over NaSO, filtered and evaporated. The crude material (13 mg, 0.035 mmol) was used in the next step without further purification.

[0312] Dissolve 3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N- (piperidin-4-yl)propanamide 19 (1 eq., 13 mg, 0.0349 mmol) in anhydrous MeOH (0.5 ml) under argon. Add 1,3-oxazole-4-carboxaldehyde (2 eq., 6.78 mg, 0.0698 mmol) and stir the reaction mixture at room temperature for 10 min. Dissolve NaBH(OAc)3 (2 eq., 15.6 mg, 0.0698 mmol) in anhydrous MeOH (0.5 ml) and add to the reaction mixture. Stir the reaction at room temperature for 40 h. Add water and purify the crude material directly by reverse phase chromatography (H20 / MeOH), salt with aqueous HC1 (2M) and lyophilize to give 20r as a white solid (m = 3.7 mg, yield = 20%).

[0313] 1 H NMR (500 MHz, Methanol-d4) δ 8.16 (d, J = 0.9 Hz, 1H), 7.90 (d, J = 10.2 Hz, 1H), 7.86 (d, J = 0.9 Hz, 1H), 7.36 (d, J = 10.2 Hz, 1H), 3.69 - 3.65 (m, 4H), 3.65 - 3.58 (m, 1H), 3.52 (s, 2H), 3.37 - 3.33 (m, 2H), 2.90 (d, J = 11.8 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 5.1 Hz, 4H), 2.37 (s, 3H), 2.22 - 2.12 (m, 2H), 1.81 (dd, J = 13.4, 3.8 Hz, 2H), 1.52 - 1.44 (m, 2H). 13 CNMR (126 MHz, Methanol-d4) δ 173.2, 156.8, 153.4, 150.1, 144.0, 139.0, 137.2, 124.7, 116.6, 55.4, 53.8, 53.1, 47.8, 46.4, 46.1, 33.2, 32.3, 21.2.

[0314] LC-MS (ESI) [M+H]+= 454.24

[0315] General procedure C

[0316] In general procedure C, the hydrazinyl pyridazine 5 was diacylated followed by cyclization under acidic conditions 22 to give the ethyl propionate triazolopyridazine 23 (Scheme 5). Reaction with secondary amines produced triazolopyridazines 24 with various amine substitutions in position 6. Hydrolysis of the carboxylate ester and coupling with primary amines 25 produced the final analogs 26 with another point of diversity on the six-membered aliphatic ring (Scheme 5).

[0317] Scheme 5 (see formula II and III)

[0318]

[0319] Conditions: a) 21, Na2S04, DIEA, DMF, 48 h, rt; b) AcOH, 135 °C, overnight; c) NR1R2, Et3N, EtOH, reflux overnight; d) LiOH, THF / H20, 1 h, rt; e) HATU, NEt3, DMF, overnight.

[0320] 4-[2-(6-chloropyridazin-3-yl)-2-(4-ethoxy-4-oxo-butyryl)hydrazino]-4-oxo- butyric acid ethyl ester 22

[0321] 3-chloro-6-hydrazinopyridazine 5 (1 eq., 600 mg, 4.15 mmol) was dissolved in dry DMF (10 ml). Na2S04(50 mg) and DIEA (2.2 eq., 1180 mg, 1.51 mL, 9.13 mmol) were added and the reaction mixture was cooled to 0 °C and stirred for 15 min. Then, ethyl succinyl chloride 21 (1.2 eq., 819 mg, 0.708 mL, 4.98 mmol) was added dropwise and the reaction mixture was stirred at room temperature over the weekend. DMF was evaporated and the crude material was purified by silica gel chromatography (EtOAc / heptane, 1 / 1, 5 / 1 to 1 / 0) to give 22 as a white solid (m = 1 g, yield = 61 %).

[0322] 1 H NMR (400 Mhz, Methanol-d4) δ 7.51 (d, J = 9.4 Hz, 1H), 7.15 (d, J = 9.4 Hz, 1H), 4.15 (qd, J = 7.1, 6.0 Hz, 4H), 2.72 - 2.54 (m, 8H), 1.26 (td, J = 7.1, 1.9 Hz, 6H). 13 C NMR (101 MHz, Methanol-d4) δ 174.4, 174.3, 174.1, 173.3, 161.6, 149.6, 131.3, 118.2, 61.8, 61.7, 30.0, 29.9, 29.32, 29.28, 14.48, 14.46.

[0323] 3-{6-chloro-[l,2,4]triazolo[4,3-b]pyridazin-3-yl}propionic acid ethyl ester 23

[0324] By 4- [2- (6- chloropyridazine -3- bases) -2- (4- ethoxy -4- oxo - butyryl) hydrazine] -4- oxo - butyric acid ethyl ester 22 (1 eq., 960 mg, 3.52 mmol) was dissolved in acetic acid (38.6 eq., 8157 mg, 7.78 mL, 135 mmol) and the reaction was heated at 135 ° C overnight. The crude material was cooled to room temperature and evaporated. The crude material was purified by silica gel chromatography (heptane / EtOAc, 1 / 1, 1 / 5 to 0 / 1) to obtain compound 23 (m = 586 mg, yield = 96%) as a white solid.

[0325] 1 H NMR (400 MHz, methanol-d4) δ 8.26 (d, J = 9.7 Hz, 1H), 7.45 (d, J = 9.7 Hz, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.47 (t, J = 7.3 Hz, 2H), 3.04 (t, J = 7.3 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13 CNMR (101 MHz, methanol-d4) δ 173.5, 151.2, 150.6, 144.6, 127.3, 124.6, 61.9, 31.2, 20.4, 14.4.

[0326] Ethyl 3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoate 24a

[0327] 3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazine-3-yl}ethyl propionate 23 (1 eq., 586 mg, 2.3 mmol) was dissolved in EtOH (2.5 ml). 1-Methylpiperazine (2 eq., 460 mg, 0.51 mL, 4.6 mmol) and Et3N (2 eq., 465 mg, 0.64 mL, 4.6 mmol) were added and the reactants were heated under reflux overnight. The crude material was cooled to room temperature and evaporated. The crude material was purified by silica gel chromatography (EtOAc / MeOH / Et3N; 9 / 1 / 0.5 to 7 / 1 / 0.5) to give 24a (m=728 mg, yield=99%) as a light yellow solid.

[0328] 1H NMR (400 MHz, methanol-d4) δ 7.97 (d, J = 10.2 Hz, 1H), 7.39 (d, J = 10.2 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.90-3.85 (m, 4H), 3.36 (t, J = 7.4 Hz, 2H), 3.24-3.19 (m, 4H), 2.97 (t, J = 7.4 Hz, 2H), 2.80 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, methanol-d4) δ 173.7, 156.4, 149.9, 144.0, 125.3, 116.5, 61.9, 54.3, 31.3, 20.5, 14.4.

[0329] Example 6: 3-[6-(4-Methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N-(1-methyl-piperidin-4-yl)propionamide 26a (LIT-TB016)

[0330] Ethyl 3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoate 24a (1 eq., 30 mg, 0.0942 mmol) was diluted in a THF / H2O mixture (1 / 1; 6 ml). LiOH (5 eq., 19.8 mg, 0.471 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The crude material was acidified with HCl (2 M), evaporated and diluted in anhydrous DMF (0.5 ml). HATU (2.5 eq., 89.6 mg, 0.236 mmol) and Et3N (2.5 eq., 23.8 mg, 32.7 μL, 0.236 mmol) were added and the reaction mixture was stirred at room temperature for 15 min. Then, 1-methylpiperidin-4-amine 25a (1.2eq., 13.3mg, 14.6 μ L, 0.113mmol) is added, and the reaction mixture is stirred at room temperature overnight. The crude material is directly purified by reverse phase chromatography (MeOH / H o) to produce a viscous oil. Purification is performed for the second time to produce the desired compound. The product is evaporated and diluted in MeOH. Et o (excessive) containing 2M-HCl is added, and the reactant is stirred at room temperature for 1.5h. The mixture is evaporated, diluted in water and lyophilized to obtain 26a (m=2.9mg, yield=7%) as a white solid.

[0331] 1H NMR (500 MHz, methanol-d4) δ 7.88 (d, J = 10.2 Hz, 1H), 7.34 (d, J = 10.2 Hz, 1H), 3.68-3.63 (m, 5H), 3.33 (t, J = 7.5 Hz, 2H), 2.93-2.85 (m, 2H), 2.76 (t, J = 7.5, 2H), 2.61-2.57 (m, 4H), 2.36 (s, 3H), 2.34 (s, 3H), 2.25 (t, J = 11.8 Hz, 2H), 1.88-1.83 (m, 2H), 1.54-1.47 (m, 2H). 13 C NMR (126 MHz, methanol-d4) δ 173.4, 156.8, 150.1, 144.0, 124.7, 116.6, 68.9, 55.4, 46.4, 46.1, 45.8, 33.1, 31.9, 26.5, 21.1.

[0332] LC-MS (ESI) [M+H] + =387.17

[0333] N-(1-Benzyl-4-piperidinyl)-3-[6-[2-(dimethylamino)ethylamino]-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 26b (LIT-TB051)

[0334] Ethyl 3-[6-[2-(dimethylamino)ethylamino]-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoate 24b (1 eq., 18 mg, 0.0588 mmol) was diluted in a THF / H2O mixture (1 / 1; 6 ml). LiOH (5 eq., 12.3 mg, 8.62 μL, 0.294 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The crude material was acidified with HCl (2 M), evaporated, and diluted in anhydrous DMF (0.5 ml). The sulfate salt was added to the mixture and stirred for 5 min. HATU (1.2 eq., 26.8 mg, 0.0705 mmol) and Et3N (2.5 eq., 14.9 mg, 20.4 μL, 0.147 mmol) were added and the reaction mixture was stirred at room temperature for 15 min. Then, 4-amino-1-benzylpiperidine 25b (1.5 eq., 16.8 mg, 18 μ, 0.0881 mmol) was added and the reaction mixture was stirred at 60 ° C for 3 h. The crude material was filtered through a pad of celite and washed with MeOH. The filtrate was evaporated and purified by reverse phase chromatography (MeOH / HO), salted with aqueous HCl (2M) and lyophilized to give 26b (m = 14.3 mg, yield = 46%) as a white solid.

[0335] 1 H NMR (400 MHz, Methanol-d4) δ 7.74 (d, J = 9.9 Hz, 1H), 7.34 - 7.24 (m, 5H), 6.81 (d, J = 9.9 Hz, 1H), 3.68 - 3.60 (m, 1H), 3.58 - 3.53 (m, 4H), 3.35 - 3.29 (m, 2H), 2.87 (d, J = 11.7 Hz, 2H), 2.78 - 2.73 (m, 4H), 2.41 (s, 6H), 2.18 - 2.12 (m, 2H), 1.81 (dd, J = 13.4, 3.9 Hz, 2H), 1.55 - 1.42 (m, 2H). 13 C NMR (101 MHz, Methanol-d4) δ 173.2, 155.8, 149.9, 144.3, 138.2, 130.8, 129.4, 128.6, 124.0, 119.4, 63.9, 58.2, 53.2, 47.8, 45.4, 39.7, 33.2, 32.2, 21.1.

[0336] LC-MS (ESI) [M+H] = 451.26 +

[0337] N-(l-benzyl-2-oxopiperidin-4-yl)-3-[6-(4-methylpiperazin-l-yl)- [l,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 26c (LIT-TB033)

[0338] Following the general procedure C for the synthesis of 26a, using 3-[6-(4- methylpiperazin- 1 -yl)- [ 1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoic acid ethyl ester 11a (1.5 eq., 54.9 mg, 0.173 mmol) and LiOH (5 eq., 24.1 mg, 0.575 mmol) in THF / H2O (1 / 1 ; 6 ml). The crude was treated with HATU (1.2 eq., 52.5 mg, 0.138 mmol), Et3N (5 eq., 58.2 mg, 80 μL, 0.575 mmol) and 4-amino-l-benzylpiperidin-2-one 25c (1 eq., 23.5 mg, 0.115 mmol) in dry DMF (1 ml).

[0339] The crude was directly purified by reverse phase chromatography (MeOH / H2O). Semi-preparative chromatography (MeOH / H2O + 0.05% HC1) was performed to isolate the product. The compound was salted and lyophilized to give 26c as a light yellow solid (m = 8.5 mg, yield = 14%).

[0340] 1 ​H NMR (500 Mhz, Methanol-d4) δ 7.78 (d, J = 10.2 Hz, 1H), 7.26 - 7.20 (m, 3H), 7.17 - 7.14 (m, 3H), 4.56 - 4.41 (m, 2H), 4.00 (tdd, J = 9.1, 5.7, 3.3 Hz, 1H), 3.57 - 3.55 (m, 4H), 3.27 - 3.16 (m, 4H), 2.68 (t, J = 7.5 Hz, 2H), 2.62 (ddd, J = 17.4, 5.7, 1.6 Hz, 1H), 2.51 - 2.49 (m, 4H), 2.26 (s, 3H), 2.23 (dd, J = 17.9, 9.2 Hz, 1H), 1.89 (ddt, J = 13.0, 4.8, 3.1 Hz, 1H), 1.68 - 1.59 (m, 1H). 13 C NMR (126 MHz, Methanol-d4) δ 173.6, 170.5, 156.8, 150.0, 144.0, 138.1, 129.7, 129.0, 128.6, 124.7, 116.6, 55.4, 50.9, 46.4, 46.1, 45.5, 45.1, 38.5, 33.0, 29.2, 21.0.

[0341] LC-MS (ESI) [M+H] + = 477.19

[0342] N-(4-benzylcyclohexyl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3- b]pyridazin-3-yl]propanamide 26d (LIT-TB034)

[0343] Following the general procedure C for the synthesis of 26a, 3-[6-(4- methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoic acid ethyl ester 24a (1.5 eq., 50.2 mg, 0.158 mmol) and LiOH (5 eq., 22.1 mg, 0.526 mmol) in THF / H2O (1 / 1; 6 ml). The crude was treated with HATU (1.2 eq., 48 mg, 0.126 mmol), Et3N (5 eq., 53.2 mg, 73 μL, 0.526 mmol) and 4-benzylcyclohex-1-amine 25d (1 eq., 19.9 mg, 0.105 mmol) in dry DMF (1 ml). The crude was directly purified by reverse phase chromatography (MeOH / H2O). A semi-preparative chromatography (MeOH / H2O + 0.05% HC1) was performed to isolate the product. The compound was salted and lyophilized to give 26d as a light yellowish solid (m = 11.3 mg, yield = 22%).

[0344] 1 H NMR (500 MHz, methanol-d4) δ 8.26 (d, J = 9.7 Hz, 1H), 7.93 (d, J = 9.8 Hz, 1H), 7.22-7.19 (m, 2H), 7.14-7.07 (m, 3H), 4.59 (d, J = 14.2 Hz, 2H), 3.66 (d, J = 11.5 Hz, 2H), 3.60-3.48 (m, 3H), 3.43 (t, J = 6.5 Hz, 2H), 3.35-3.28(m,2H),2.96(s,3H),2.85-2.82(m,2H),2.46(d,J=7.0Hz,2H),1.81(d,J=9.3Hz,2H), 1.70(d,J=11.0Hz,2H), 1.47(ddt,J=11.3,7.7,3.8Hz,1H), 1.19-1.11(m,2H), 1.07-0.96(m,2H). 13 C NMR (126 MHz, methanol-d4) δ 172.1, 157.5, 150.5, 142.1, 141.0, 130.1, 129.2, 126.8, 122.6, 122.6, 53.8, 50.2, 44.4, 44.1, 43.7, 40.3, 33.5, 32.7, 31.9, 20.7.

[0345] LC-MS (ESI) [M+H] + =462.20

[0346] 3-[6-(4-Methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N-(1-phenylpiperidin-4-yl)propanamide 26e (LIT-TB035)

[0347] Following General Procedure C for the synthesis of 26a, ethyl 3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoate 24a (1.5 eq., 60 mg, 0.188 mmol) and LiOH (1.5 eq., 60 mg, 0.188 mmol) in THF / H2O (1 / 1; 6 ml) were used. The crude material was treated with HATU (1.2 eq., 57.3 mg, 0.151 mmol), Et3N (5 eq., 63.6 mg, 87.3 μL, 0.628 mmol) and 1-phenylpiperidin-4-amine 25e (1 eq., 22.1 mg, 0.126 mmol; CAS 63921-23-3) in anhydrous DMF (1 ml). The crude material was directly purified by reverse phase chromatography (MeOH / H2O).The compound was salified and lyophilized to afford 26e (m=20.9 mg, yield=34%) as a light yellow solid.

[0348] 1 H NMR (500 MHz, methanol-d4) δ 7.88 (d, J = 10.1 Hz, 1H), 7.34 (d, J = 10.2 Hz, 1H), 7.23-7.17 (m, 2H), 6.99-6.94 (m, 2H), 6.81 (tt, J = 7.3, 1.1 Hz, 1H), 3.77 (tt, J = 10.8, 4.2 Hz, 1H),3.69-3.63(m,4H),3.61-3.56(m,2H),3.35(t,J=7.6Hz,2H),2.82-2.74(m,4 H), 2.59 (t, J = 5.1Hz, 4H), 2.35 (s, 3H), 1.92-1.88 (m, 2H), 1.61-1.53 ​​(m, 2H), NH. 13 C NMR (126 MHz, methanol-d4) δ 173.3, 156.8, 152.8, 150.1, 144.0, 130.0, 124.7, 121.1, 118.2, 116.6, 55.4, 50.2, 48.0, 46.4, 46.1, 33.2, 32.5, 21.2.

[0349] LC-MS (ESI) [M+H] + =449.17

[0350] General Procedure D for the Preparation of 3-Fluoro-4-aminopiperidine Analogs of LIT-TB001

[0351] Scheme 6 (see Formula I)

[0352]

[0353] Conditions: a) TFA, DCM, 2 h, RT; b) RX, K2CO3, DMF, Ar, -5°C (30 min) → RT (overnight). β-Fluoropiperidine analogs 27a-d were obtained by peptide-type coupling of enantiopure 4-amino-3-fluoropiperidine with ethyl 3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanoate 24a according to General Procedure C (Compound 23 → Compound 26).

[0354] tert-Butyl (3S,4R)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 27a

[0355] 1 H NMR (500MHz, methanol-d4) δ7.90(d,J=10.2Hz,1H),7.36(d,J=10.2Hz,1H),4.64(d,J=48.9Hz,1H),4.3 5(s,1H),4.14(d,J=12.6Hz,1H),4.00(dddd,J=30.8,12.3,4.9,2.2Hz,1H),3.67(dd,J=6.2,4.1 Hz, 4H), 3.39-3.34 (m, 2H), 2.83 (t, J = 7.6 Hz, 2H), 2.61 (t, J = 5.1 Hz, 4H), 2.38 (s, 3H), 1.74 (qd, J = 12.7, 4.5 Hz, 1H), 1.62 (ddd, J = 10.1, 5.2, 2.6 Hz, 1H), 1.46 (s, 9H), 1.35-1.29 (m, 2H), NH (invisible). 13 C NMR (126 MHz, methanol-d4) δ 173.5, 156.9, 156.8, 150.0, 144.0, 124.7, 116.6, 88.5 (d, J = 177.3 Hz), 81.4, 55.4, 50.1 (d, J = 18.9 Hz), 46.4, 46.1, 33.0, 32.9, 28.6, 23.7, 21.1, 14.4.

[0356] 19 F NMR (471 MHz, methanol-d4) δ -205.7.

[0357] Example 7 N-[(3S,4R)-1-Benzyl-3-fluoropiperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 28a (LIT-TB047)

[0358] Tert-butyl (3S,4R)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 27a (1 eq., 30.4 mg, 0.062 mmol) was dissolved in DCM (0.7 mL). TFA (10 eq., 70.7 mg, 46 μL, 0.62 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The crude material was evaporated and then co-evaporated with DCM / heptane (3 times). After drying, the crude material was dissolved in anhydrous DMF under argon. K2CO3 (5 eq., 42.8 mg, 0.31 mmol) was added and the reaction mixture was stirred at -5 °C for 30 min. Benzyl bromide (1.1 eq., 11.7 mg, 8.15 μL, 0.0682 mmol) was added, and the mixture was stirred at -5°C for 0.5 h, then at room temperature overnight. Water (a few drops) was added, and the crude material was directly purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to give the title compound 28a (m = 18.8 mg, yield = 55%) as a light yellow solid.

[0359] 1 H NMR (400 MHz, methanol-d4) δ 7.88 (d, J = 10.2 Hz, 1H), 7.37-7.22 (m, 6H), 4.61 (d, J = 49.3 Hz, 1H), 3.84 (dd, J = 30.4, 12.2 Hz, 1H), 3.65 (t, J = 4.8 Hz, 4H), 3.63-3.48 (m, 2H), 3.37-3.31 (m, 2H ),3.11(t,J=11.8Hz,1H),2.90(d,J=11.7Hz,1H),2.85-2.77(m,2H),2.59(t,J=4.8Hz,4 H), 2.36 (s, 3H), 2.29-2.15 (m, 2H), 1.89 (q, J = 13.0, 12.5Hz, 1H), 1.63 (d, J = 13.0Hz, 1H). 13 C NMR (101 MHz, methanol-d4) δ 173.5, 156.8, 150.0, 144.0, 138.3, 130.5, 129.3, 128.4, 124.7, 116.6, 89.0 (d, J = 177.1 Hz), 63.3, 56.3 (d, J = 18.9 Hz), 55.4, 52.7, 50.0 (d, J = 18.5 Hz), 46.4, 46.1, 32.9, 27.0, 21.1. 19 F NMR (376 MHz, methanol-d4) δ -201.6.

[0360] LC-MS (ESI) [M+H] + =481.25

[0361] N-[(3S,4S)-1-Benzyl-3-fluoropiperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 28b (LIT-TB048)

[0362] Following General Procedure D for the synthesis of 28a, tert-butyl (3S,4S)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 27b (1 eq., 26 mg, 0.053 mmol), benzyl bromide (1.1 eq., 9.97 mg, 6.97 μL, 0.0583 mmol), and KCO (5 eq., 36.6 mg, 0.265 mmol) in DMF (0.5 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 28b as a light yellow solid (m=13.0 mg, yield=44%).

[0363] 1 H NMR (400Mhz, methanol-d4) δ7.88 (d, J = 10.3Hz, 1H), 7.35-7.25 (m, 6H), 4.46-4.21 (m, 1H),3.88-3.75(m,1H),3.65(t,J=4.9Hz,4H),3.61-3.53(m,2H),3.37-3.33(m ,2H),3.10(dd,J=11.0,5.7Hz,1H),2.82-2.76(m,3H),2.59(t,J=4.9Hz,4H),2 .36(s,3H),2.16-2.06(m,2H),1.89(d,J=12.5Hz,1H),1.46(q,J=11.7Hz,1H). 13 C NMR (101 MHz, methanol-d4) δ 173.9, 156.8, 150.0, 144.0, 138.7, 130.4, 129.4, 128.5, 124.7, 116.6, 90.6 (d, J = 177.8 Hz), 63.2, 57.1 (d, J = 25.0 Hz), 55.4, 52.6 (d, J = 18.4 Hz), 52.4, 46.4, 46.1, 33.3, 30.4 (d, J = 6.9 Hz), 21.1. 19 F NMR (376 MHz, methanol-d4) δ -189.7.

[0364] LC-MS (ESI) [M+H] + =481.25

[0365] N-[(3R,4R)-1-Benzyl-3-fluoropiperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 28c (LIT-TB049)

[0366] Following General Procedure D for the synthesis of 28a, tert-butyl (3R,4R)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 17c (1 eq., 22.4 mg, 0.0457 mmol), benzyl bromide (1.1 eq., 8.59 mg, 6.01 μL, 0.0502 mmol), and KCO (5 eq., 31.6 mg, 0.228 mmol) were used in DMF (0.5 ml). The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 28c as a light yellow solid (m = 13.4 mg, yield = 54%).

[0367] 1 H NMR (500 MHz, methanol-d4) δ 7.88 (d, J = 10.1 Hz, 1H), 7.36-7.25 (m, 6H), 4.34 (dtd, J = 49.7, 9.4, 4.7 Hz, 1H), 3.80 (tdd, J = 11.2, 9.2, 5.0 Hz, 1H), 3.68-3.64 (m, 4H), 3.61-3.53 (m, 2H), 3.38-3. 34(m,2H),3.13-3.06(m,1H),2.82-2.75(m,3H),2.59(t,J=5.1Hz,4H),2.36(s,3H),2.16- 2.08(m,2H),1.89(dtt,J=13.6,5.8,3.0Hz,1H),1.46(dtdd,J=12.9,11.7,4.2,1.0Hz,1H). 13 C NMR (126MHz, methanol-d4) δ173.9,156.8,150.0,144.0,138.6,130.4,129.4,128.5,124.7,116.6,90.6(d,J=177 .9Hz), 63.2, 57.1 (d, J = 25.0Hz), 55.4, 52.6 (d, J = 18.5Hz), 52.4, 46.4, 46.1, 33.3, 30.4 (d, J = 6.8Hz), 21.1. 19F NMR (471 MHz, methanol-d4) δ -189.7.

[0368] LC-MS (ESI) [M+H] + =481.26

[0369] N-[(3R,4S)-1-Benzyl-3-fluoropiperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 18d (LIT-TB054)

[0370] Following General Procedure D for the synthesis of 28a, tert-butyl (3R,4S)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamido}piperidine-1-carboxylate 17d (1 eq., 24 mg, 0.0489 mmol) (1.1 eq., 9.2 mg, 6.44 μL, 0.0538 mmol) and KCO (5 eq., 33.8 mg, 0.245 mmol) in DMF (0.5 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 28d as a light yellow solid (m = 13.4 mg, yield = 49%).

[0371] 1 H NMR (400 MHz, methanol-d4) δ 7.88 (d, J = 10.2 Hz, 1H), 7.36-7.24 (m, 6H), 4.61 (ddd, J = 49.3, 3.8, 2.1 Hz, 1H), 3.84 (dddd, J = 30.2, 12.3, 5.0, 2.5 Hz, 1H), 3.66 (t, J = 5.1 Hz, 4H), 3.55 (dd, J = 42.3, 13.0 Hz, 1H). z,2H),3.37-3.33(m,2H),3.15-3.08(m,1H),2.93-2.88(m,1H),2.82(t,J=7.6Hz,2H),2.59(t, J=5.1Hz,4H),2.36(s,3H),2.31-2.14(m,2H),1.94-1.84(m,1H),1.63(dd,J=13.0,3.9Hz,1H). 13C NMR (101 MHz, methanol-d4) δ 173.54, 156.78, 150.02, 143.95, 138.28, 130.55, 129.31, 128.43, 124.71, 116.57, 89.02 (d, J = 177.1 Hz), 63.26, 56.29 (d, J = 19.0 Hz), 55.39, 52.73, 50.02 (d, J = 18.5 Hz), 46.43, 46.11, 32.94, 27.04 (d, J = 1.7 Hz), 21.11. 19 F NMR (376 MHz, methanol-d4) δ -201.62.

[0372] LC-MS (ESI) [M+H] + =481.23

[0373] N-[(3S,4S)-3-Fluoro-1-[(4-methoxyphenyl)methyl]piperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 29b (LIT-TB052)

[0374] Following General Procedure D for the synthesis of 28a, tert-butyl (3S,4S)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 27b (1 eq., 34 mg, 0.0693 mmol), 4-methoxybenzyl chloride (1.1 eq., 12.2 mg, 10.5 μL, 0.0762 mmol), and KCO (5 eq., 47.9 mg, 0.347 mmol) in DMF (0.7 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 29b (m=15.2 mg, yield=58%) as a white solid.

[0375] 1H NMR (400 MHz, methanol-d4) δ 7.88 (d, J = 10.2 Hz, 1H), 7.34 (d, J = 10.2 Hz, 1H), 7.24-7.19 (m, 2H), 6.90-6.85 (m, 2H), 4.33 (dtd, J = 49.7, 9.4, 4.7 Hz, 1H), 3.84-3.73 (m, 1H), 3.78 (s, 3H), 3.66 (t, J = 5.1 Hz, 4H), 3.55-3.47 (m,2H),3.37-3.33(m,2H),3.11-3.06(m,1H),2.80(t,J=7.7Hz,2H),2.80-2.74(m,1H),2.59(t,J=5.1H z,4H),2.36(s,3H),2.12-2.05(m,2H),1.89(dtd,J=10.7,5.4,2.8Hz,1H),1.45(qd,J=12.0,3.9Hz,1H). 13 C NMR (101 MHz, methanol-d4) δ 173.89, 160.6, 156.8, 150.0, 144.0, 131.6, 130.4, 124.7, 116.6, 114.7, 90.7 (d, J = 177.8 Hz), 62.6, 57.0 (d, J = 24.9 Hz), 55.7, 55.4, 52.6 (d, J = 18.4 Hz), 52.3, 46.4, 46.1, 33.3, 30.4 (d, J = 7.0 Hz), 21.1. 19 F NMR (376 MHz, methanol-d4) δ -189.7.

[0376] LC-MS (ESI) [M+H] + =511.27

[0377] N-[(3R,4R)-3-Fluoro-1-[(4-methoxyphenyl)methyl]piperidin-4-yl]-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 29c (LIT-TB053)

[0378] Following General Procedure D for the synthesis of 28a, tert-butyl (3R,4R)-3-fluoro-4-{3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionylamino}piperidine-1-carboxylate 27c (1 eq., 48.3 mg, 0.0985 mmol), 4-methoxybenzyl chloride (1.1 eq., 17.3 mg, 15 μL, 0.108 mmol), and KCO (5 eq., 68 mg, 0.492 mmol) in DMF (0.7 ml) were used. The crude material was evaporated and purified by reverse phase chromatography (HO / MeOH), salified, and lyophilized to afford 29c as a white solid (m = 17.3 mg, yield = 66%).

[0379] 1 H NMR (400 MHz, methanol-d4) δ 7.88 (d, J = 10.2 Hz, 1H), 7.34 (d, J = 10.2 Hz, 1H), 7.24-7.19 (m, 2H), 6.90-6.85 (m, 2H), 4.33 (dtd, J = 49.7, 9.4, 4.7 Hz, 1H), 3.84-3.74 (m, 1H), 3.79 (s, 3H), 3.66 (t, J = 5.1 Hz, 4H), 3.55-3.47 (m,2H),3.37-3.33(m,2H),3.12-3.06(m,1H),2.80(t,J=7.7Hz,2H),2.80-2.74(m,1H),2.59(t,J=5.1H z,4H),2.36(s,3H),2.12-2.05(m,2H),1.89(dtd,J=10.7,5.4,2.8Hz,1H),1.46(qd,J=12.0,3.9Hz,1H). 13 C NMR (101 MHz, methanol-d4) δ 173.9, 160.6, 156.8, 150.0, 144.0, 131.6, 130.4, 124.7, 116.6, 114.7, 90.7 (d, J = 177.8 Hz), 62.6, 57.0 (d, J = 25.0 Hz), 55.7, 55.4, 52.6 (d, J = 18.4 Hz), 52.3, 46.4, 46.1, 33.3, 30.4 (d, J = 6.9 Hz), 21.1. 19 F NMR (376 MHz, methanol-d4) δ -189.7.

[0380] LC-MS (ESI) [M+H] + =511.25

[0381] Preparation of triazolopyridine

[0382] Alternatively, the carbaisostere of compound 9a (LIT-TB001) has been prepared as reported in Scheme 7. Starting from the known hydrazine-bromopyridine derivative 35, reaction with propionic acid 4a in the presence of isobutyl chloroformate afforded hydrazide 36, which was subsequently cyclized to triazolopyridine 37 under Mitsunobu conditions in the presence of TMSN3. Final compound 38 was obtained under Buchwald cross coupling reaction conditions.

[0383] Option 7

[0384]

[0385] Conditions: NH2-NH2, 100°C, see Synthesis, 47(20), 3169-3178; 2015; b) 4a, isobutyl chloroformate, DIEA, THF, 25°C, 12h; c) DIAD, PPh3, TMSN3, THF, 12h; d) Pd(OAc)2, Binap, Cs2CO3, dioxane, 105°C, 12h.

[0386] Example 8 :N-(1-benzylpiperidin-4-yl)-3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)propanamide 38 (LIT-TB006)

[0387] Step 1: N-(1-benzylpiperidin-4-yl)-4-(2-(5-bromopyridin-2-yl)hydrazinyl)-4-oxobutanamide 36

[0388] 4-((1-benzylpiperidin-4-yl)amino)-4-oxobutanoic acid 4a (1.0 eq., 300 mg, 1.56 mmol) was suspended in THF (6 ml), followed by NMM (1.2 eq., 193.7 mg, 0.21 ml). Isobutyl chloroformate (0.5 g, 0.49 mL) was then added dropwise to the solution, and the resulting mixture was stirred at room temperature for 30 min. 5-Bromo-2-hydrazinopyridine (1 eq., 300 mg, 1.59 mmol) was then added and stirring was maintained for an additional hour. The volatiles were evaporated and the crude material was dissolved in EtOAc (30 mL). The organic phase was washed once with 1N NaCO (15 mL), water (15 mL), and brine (20 mL), and dried over NaSO, filtered, and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography using a gradient of 0% to 3% NEt3 / EtOAc:MeOH 9:1 to give the title compound (212 mg, 29%) as a white solid.

[0389] 1 H NMR (400MHz, CDCl3) δ5.62 (s, 1H), 8.11 (s, 1H), 7.50 (d, 1H, J = 8.0Hz), 7.29-7 .20(m,5H);6.96(s,1H),6.54(d,1H,J=8.0Hz),5.93(d,1H,J=4.0Hz),3.73-3. 65(m,1H),3.45(s,2H),2.76(d,2H,J=4.0Hz),2.49(dd,2H,J=8.0Hz,J=4.0Hz) ,2.04(t,2H,J=12.0Hz), 1.79(d,2H,J=12Hz), 1.40(dq,2H,J=12Hz,J=4.0Hz). 13 C NMR (101MHz, CDCl3) δ172.5,171.3,158.1,148.7,140.5,129.3,128.4,127.3,110.9,108.3,63.1,52.3,46.9,32.1,31.4,29.7.

[0390] Step 2: N-(1-Benzylpiperidin-4-yl)-3-(6-bromo-[1,2,4]triazolo[4,3-a]pyridin-3-yl)propanamide 37

[0391] A solution of DIAD (109, 8 g, 107.7 μL, 2.5 equiv) and TMS-N3 (62.56 mg, 0.54 mmol, 72.08 μl) in THF (0.4 mL) was slowly added to a solution of triphenylphosphine (142.4, 0.53 mmol, 2.5 equiv), N-(1-benzylpiperidin-4-yl)-4-(2-(5-bromopyridin-2-yl)hydrazinyl)-4-oxobutanamide (100 mg, 0.21 mmol) in THF (1.2 mL), and the resulting turbid mixture was stirred at room temperature overnight. Silica gel was added to the mixture and the volatiles were evaporated. The crude product was flash chromatographed using a gradient of 0% to 3% Et3N / EtOAc-MeOH 9:1 to give the title compound (m = 53.2 mg, yield = 55%) as a light yellow solid.

[0392] 1 H NMR (400MHz, methanol-d4) δ8.68(s,1H),7.62(d,1H,J=8.0Hz),7.48(d,1H,J=8.0Hz),7.33-7.25(m,5H),3.67-3.61(m,1H),3.65( s, 2H), 2.90 (d, 2H, J = 12.0Hz), 2.79 (t, 2H, J = 8.0Hz), 2.24 (t, 1H, J = 12.0Hz), 1.80 (m, 2H), 2.26 (dq, 2H, J = 12.0Hz, J = 4.0Hz). 13 C NMR (101 MHz, methanol-d4) δ 173.1, 149.6, 148.3, 137.2, 133.1, 130.9, 129.4, 128.8, 125.3, 116.9, 109.8, 63.6, 53.0, 47.5, 33.6, 31.8, 21.1.

[0393] Step 3: N-(1-Benzylpiperidin-4-yl)-3-(6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)propanamide 38 (LIT-TB006)

[0394] A microwave vial (oven dried and under argon) was charged with N-(1-benzylpiperidin-4-yl)-3-(6-bromo-[1,2,4]triazolo[4,3-a]pyridin-3-yl)propanamide 37 (100 mg, 0.23 mmol, 1 eq), 1-methylpiperazine (22.64 mg, 25 μL, 0.23 mmol), CsCO (147.3 mg, 0.45 mmol, 2 eq) and Pd(OAc) (1.02 mg, 2 mol%) and Binap (8.45 mg, 6 mol%) followed by dioxane (1.05 mL). The vial was capped appropriately and the mixing vessel was evacuated and backfilled with argon (the process was repeated 3 times) and heated at 105° C. overnight. After cooling to room temperature, silica gel was added and the resulting mixture was evaporated to dryness. The crude material was flash chromatographed using EtOAc / MeOH / Et3N 8:2:0.3 as eluent to afford the title compound (m=40 mg, yield=38%).

[0395] LC-MS (ESI) [M+H] + =462,2979

[0396] Preparation of imidazopyridine

[0397] The present invention also provides a method for preparing imidazopyridine derivatives of general formula 44. An illustrative general synthetic method is given in Scheme 8. A three component Michael-type (3CC) reaction involving bromo-imidazopyridine, Meldrum acid, and formaldehyde produces the corresponding 3-imidazo[1,2-a]pyridin-3-ylpropionic acid using known procedures

[18] . The reaction is carried out in the presence of a catalytic amount of L-proline to give the corresponding "Michael-type" Yonemitsu adduct 41, which is first converted to a stable ester 42 by ethanolysis and concomitant copper-catalyzed decarboxylation, and then converted to the corresponding amide 43 after sequential base hydrolysis and classical peptide coupling reactions. Finally, a Buchwald-type cross-coupling reaction is performed to produce the target compound 44 (LIT-TB013).

[0398] Scheme 8 (see Formula III)

[0399]

[0400] Conditions: a) 5 mol% L-proline, MeCN, 50°C, 10 h; b) Cu, pyridine-EtOH 10:1, reflux 3 h; c) KOH, EtOH-H2O, 50°C, 10 h, 1N HCl (pH = 6); d) 1, BOP, NMM, DCM, 12 h; e) Pd(OAc)2, Binap, Cs2CO3, dioxane, 105°C, 12 h.

[0401] Example 9 N-(1-Benzylpiperidin-4-yl)-3-(6-(4-methylpiperazin-1-yl)imidazo[1,2-a]pyridin-3-yl)propanamide 44 (LIT-TB013)

[0402] Step 1: Ethyl 3-(6-bromoimidazo[1,2-a]pyridin-3-yl)propanoate 42

[0403] 6-Bromoimidazo[1,2-a]pyridine (1.50 g, 7.61 mmol, 1 equivalent), Michaelis' acid (1 eq., 1.10 g, 7.61 mmol), paraformaldehyde (1 eq., 228.6 mg, 7.61 mmol), and L-proline (43.8 mg, 5 mol%) were suspended in acetonitrile (29.23 mL), and the reaction mixture was stirred overnight at 50°C under a nitrogen atmosphere. The precipitated product was collected by filtration and thoroughly washed with diethyl ether. The solid was dried (m = 1.83 g, 5.18 mmol, yield = 68%). The resulting compound 41 (1 eq., 1.50 g, 4.25 mmol) was dissolved in pyridine / EtOH (10:1 v / v, 5.5 mL), copper powder (12.75 mg, 0.20 mmol) was added, and the mixture was refluxed for 3 h. The solvent was removed under reduced pressure. The crude material was flash chromatographed using EtOAc as eluent to afford the title compound 42 (m=500 mg, yield=40%).

[0404] 1 H NMR (400MHz, CDCl3) δ8.04 (d, 1H, J = 1.2Hz), 7.43 (d, 1H, J = 9.2Hz), 7.36 (s, 1H), 7.15 (dd, 1H, J = 9.2Hz, J=1.2Hz), 4.09(q,2H,J=7.2Hz), 3.10(t,2H,J=15.2Hz), 2.72(t,2H,J=14.8Hz), 1.19(t,2H,J=7.2Hz). 13 C NMR (101MHz, CDCl3) δ172.5,151.6,131.8,126.9,123.2,123.1118.7,112.6,107.1,60.9,32.0,19.4,14.2.

[0405] Step 2: N-(1-benzylpiperidin-4-yl)-3-(6-bromoimidazo[1,2-a]pyridin-3-yl)propanamide 43

[0406] Ethyl 3-(6-bromoimidazo[1,2-a]pyridin-3-yl)propanoate 42 (1 eq., 500 mg, 1.68 mmol) was dissolved in EtOH (10 mL) at 0°C and then treated with potassium hydroxide (2 eq., 189 mg, 3.36 mmol in 1 mL of H2O). The resulting mixture was stirred at ambient temperature for 1 hour. The volatiles were evaporated, and the crude material was dissolved in H2O (20 mL) and extracted with EtOAc (15 mL). The organic solvent was removed, and the remaining aqueous solution was acidified with 1N HCl until the pH reached approximately 4. The resulting solid was filtered and dried under reduced pressure to give 3-(6-bromoimidazo[1,2-a]pyridin-3-yl)propanoic acid (m = 340 mg, yield = 75%).

[0407] The obtained acid (200 mg, 0.74 mmol, 1 eq) and BOP (349.5 mg, 0.74 mmol) were suspended in DCM (5.0 mL). NMM (112.8 mL, 122 μL, 1.11 mmol, 1.5 eq) was added and the reaction mixture was stirred at room temperature for 15 min. Subsequently, 1-benzylpiperidin-4-amine (141.5 mg, 0.74 mmol, 1 eq) was added and the reaction was stirred at room temperature overnight (20 h). MeOH and silica were added and the crude material was evaporated. The adsorbed compound on silica gel was then purified on silica gel chromatography (eluent MeOH / AcOEt8 / 2) to give the yellow title compound 43 (m=379 mg, yield=93%).

[0408] Step 3: N-(1-benzylpiperidin-4-yl)-3-(6-(4-methylpiperazin-1-yl)imidazo[1,2-a]49-pyridazin-3-yl)propanamide 44 (LIT-TB013)

[0409] A microwave vial (oven dried and under argon) was charged with N-(1-benzylpiperidin-4-yl)-3-(6-bromoimidazo[1,2-a]49-pyridazin-3-yl)propanamide 43 (1 eq., 50 mg, 0.11 mmol), methylpiperazine (12.5 mg, 13.8 μL, 0.12 mmol), Cs2CO3 (2 eq., 73.8 mg, 0.23 mmol), Pd(OAc)2 (0.8 mg, 3 mol%) and Binap (4.2 mg, 6 mol%), followed by dioxane (1.0 mL). The vial was capped appropriately, and the mixing vessel was evacuated and backfilled with argon (the process was repeated 3 times) and heated at 105°C overnight. After cooling to room temperature, silica gel was added and the resulting mixture was evaporated to dryness. The crude material was subjected to first flash chromatography using EtOAc / MeOH / Et3N 8:2:0.3 followed by reverse phase C18 flash chromatography (10% to 100% MeOH / H2O + 0.05% HCl) to afford the title compound 44 (m = 7 mg, yield = 13%).

[0410] LC-MS[M+H] + =461.2

[0411] Preparation of imidazopyridazine

[0412] The previous Michael-type (3CC) reaction using Michaelis acid and formaldehyde can be extended to imidazopyridazine derivatives (Scheme 9). The reaction can form the corresponding propionic acid 47 in the presence of an electron-donating group (OMe) at position 6 of the imidazopyridine moiety (cpd 46). Demethylation in the presence of LiCl and p-toluenesulfonic acid generates 6-chloroimidazole-pyridazinamide 49 after chlorination using POCl3 followed by peptide coupling with 1. Finally, as previously described, the final compound of formula 50 is obtained by coupling 49 with various heterocyclic secondary amines 8 under basic conditions.

[0413] Scheme 9 (see Formula III)

[0414]

[0415] Conditions: a) MeONa, MeOH, 18h; b) 5 mol% L-proline, MeCN, 50℃, 36h; b) LiCl, pTsOH hydrate, DMF, 150℃, 16h; c) POCl3, cat DMF, 150℃, 16h; d) 1, BOP, NMM, DCM, 12h; e) EtOH, microwave, 150℃, 2h.

[0416] Example 10Preparation of N-(1-benzylpiperidin-4-yl)-3-(6-(4-methylpiperazin-1-yl)imidazo[1,2-b]pyridazin-3-yl)propanamide 50 (LIT-TB014)

[0417] Step 1: 6-Methoxyimidazo[1,2-b]pyridazine 46

[0418] Sodium methoxide (7.35 eq., 7.76 g, 143.6 mmol) was added to a solution of 6-chloroimidazo[1,2-bb]pyridazine (3.0 g, 19.54 mmol) in anhydrous methanol (8 ml) at ambient temperature, and the reaction mixture was stirred for 18 hours. The volatiles were removed by evaporation, and the yellow oily residue was dissolved in dichloromethane (100 ml). The solution was washed with water (5 × 100 ml) until the aqueous washings became neutral. The organic solution was dried (MgSO 4 ) and the solvent was removed. The title compound (m=8.87 g, yield=91%) was obtained as a white solid.

[0419] 1 H NMR(400MHz,DMSO-d6)δ7.36(d,J=9.3Hz,1H),6.85(d,J=9.3Hz,1H),6.61(s,1H) 13 C NMR (101MHz, CDCl3) δ160.2,137.3,132.4,127.3,116.8,112.1,54.4.

[0420] Step 2: 3-(6-methoxyimidazo[1,2-b]pyridazin-3-yl)propanoic acid 47

[0421] 6-Methoxyimidazo[1,2-b]pyridazine (1 eq., 1.0 g, 6.7 mmol), Michaelis' acid (1 eq., 0.97 g, 6.70 mmol), paraformaldehyde (1 eq., 201.3 mg, 6.70 mmol) and L-proline (38.6 mg, 5 mol%) were suspended in acetonitrile (30 mL), and the reaction mixture was stirred at 50° C. under a nitrogen atmosphere for 36 h. The precipitated product was collected by filtration, washed thoroughly with diethyl ether, and dried to give the title compound as a white solid (m=1.0 g, yield=67%).

[0422] 1 H NMR (400MHz, DMSO-d6) δ 12.71-12.01 (bs, 1H), 7.96 (d, J = 9.6Hz, 1H), 7.43 (s, 1H, J = 9.6Hz), 6.81 (d, J = 9.6Hz, 1H), 3.97 (s, 3H). 13C NMR (101 MHz, CDC13) δ 173.5, 159.5, 136.5, 129.9, 127.6, 127.5, 110.3, 54.3, 31.1, 18.8.

[0423] Step 3: 3-(6-hydroxyimidazo[l,2-b]pyridazin-3-yl)propanoic acid 48

[0424] The obtained acid (1 eq., 920 mg, 4.16 mmol) was suspended in DMF (11.5 mL). LiCl (5 eq., 881.6 mg, 20.8 mmol) was added, followed by pTsOH hydrate (5 eq., 3.95 g, 20.79 mmol) and the resulting mixture was heated at 150 °C under nitrogen atmosphere overnight. DMF was evaporated and the crude material was suspended in water. The precipitated product was collected by filtration and thoroughly washed with diethyl ether and dried, yielding the title compound 48 (m = 600 mg, yield = 70%).

[0425] 1 H NMR (400 MHz, DMSO-d6) δ 12.71 - 11.68 (bs, 1H), 7.96 (d, J = 9.6 Hz, 1H), 7.43 (s, 1H, J = 9.6 Hz), 6.83 (d, J = 9.6 Hz, 1H), 3.10 (t, J = 7.1 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H).

[0426] LC-MS [M+H] + = 208.0

[0427] Step 4: N-(l-benzylpiperidin-4-yl)-3-(6-(4-methylpiperazin-l-yl)imidazo[l,2- b]pyridazin-3-yl)propanamide 50 (LIT TB014)

[0428] 3-(6-hydroxyimidazo[l,2-b]pyridazin-3-yl)propanoic acid (1 eq., 200 mg, 0.96 mmol) and N(Me)4Cl (1 eq. 105.8 mg, 0.96 mmol) were suspended in POCl3(1.1 mL) and the resulting mixture was heated under nitrogen atmosphere overnight. After cooling at room temperature, DMF was evaporated and the crude material was purified by flash chromatography using EtOAc / MeOH / AcOH (8:2:0.5) as eluent, yielding 3-{6-chloroimidazo[l,2-b]pyridazin-3-yl}propanoic acid (100 mg, 46%). LC-MS (ES+APCI): 282.2 [M+Na + ], 208.0 [M+H] +

[0429] The above product (1 eq., 50 mg, 0.22 mmol), BOP (1.2 eq., 117.6 mg, 0.22 mmol) and NMM (1.5 eq., 33.6 mg, 0.33 mmol) were suspended in DCM (1.5 mL), and the reaction mixture was stirred at room temperature for 15 min. Then, 4-amino-1-benzylpiperidine (42.17 mg, 45.3 μL, 0.22 mmol) was added, and the reaction was stirred at room temperature overnight (20 h). Water (15 mL) was then added to the resulting mixture, and the aqueous solution was extracted twice with DCM (3 × 8 mL). The organic phases were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting oil was purified by flash chromatography on silica gel using EtOAc / MeOH 8 / 2 as eluent to yield N-(1-benzylpiperidin-4-yl)-3-{6-chloroimidazo[1,2-b]pyridin-3-yl}propanamide 49 (65 mg, 74%). LC-MS [M+H] + =398.2

[0430] Using the same procedure A as described for 9a (LIT-TB001) and starting from the above product 49 (1 eq. 40 mg, 0.10 mmol) and 1-methylpiperazine (20.14 mg, 22.3 μL, 0.20 mmol, 2 equiv), the title compound was obtained in 65% yield.

[0431] LC-MS (ES+APCI): 484.2[M+Na + ],462.2[M+H + ].

[0432] Preparation of triazolopyridazines

[0433] The present invention also provides a method for preparing an appropriate N-substituted triazolo[4,3-b]pyridazin-3-yl)propylpiperidin-4-amine of formula 56 (Scheme 10). Starting from N-benzylpiperidin-4-one 51, an amination reaction with methyl 4-aminobutyrate in the presence of NaBH3CN affords N-benzylpiperidin-4-amino-ethylbutyrate 52. To avoid intramolecular cyclization, 53 is first N-Boc protected (cpd 53) and then, after saponification, proceeds to a peptide-type coupling reaction with hydrazine pyridazine 5 under conditions well known in the art. Cyclization under strongly acidic conditions (135°C) followed by a SNAr-type amination reaction in the presence of 8a-g yields the desired product 56.

[0434] Scheme 10 (see Formula III)

[0435]

[0436] Conditions: a) H2N-(CH2)3CO2Et, AcOH, NaBH(AcO)3, DCM, 25 °C, 12 h; b) BOC2O, DCM, Et3N, 24 h; c) NaOH, MeOH, followed by 1 N HC1 (pH = 6); d) BOP, NMM, DCM, 12 h; e) AcOH, 150 °C, 2 h; f) EtOH, 150 °C, microwave, 1 h.

[0437] Example 11 1 -Benzyl- N-(3-(6-(4-methylpiperazin- 1 -yl)- [ 1,2,4] triazolo [4,3 -b] pyridazin-3- yl)propyl)piperidin-4-amine 56a (LIT-TB015) preparation

[0438] Step 1 : 4-((l-Benzylpiperidin-4-yl)amino)butanoic acid ester 52

[0439] To an ice-cold solution of l-benzylpiperidin-4-one 51 (1 eq., 1.00 g, 5.28 mmol) in CH2CI2(35 ml) was added methyl 4-aminobutanoate hydrochloride (1 eq., 0.88 g, 5.28 mmol), acetic acid (3.5 eq., 1.1 ml, 18.49 mmol), Et3N (1.5 eq., 802 mg, 1.1 mL, 3 mmol) and sodium triacetoxyborohydride (3 eq., 3.5 g, 3 mmol). The mixture was allowed to reach room temperature and stirred for 16 h. At that time the solution was washed with saturated potassium carbonate solution, dried (Na2S04), filtered and concentrated. The crude material was purified by flash chromatography using EtOAc-MeOH (8:2) to yield ethyl 4-((l-benzylpiperidin-4-yl)amino)butanoate 52 (m = 1.15 g, yield = 71 %). 4) and concentrated. The crude material was purified by flash chromatography using EtOAc-MeOH (8:2) to yield ethyl 4-((l-benzylpiperidin-4-yl)amino)butanoate 52 (m = 1.15 g, yield = 71 %).

[0440] 1 H NMR (400 MHz, CDC13) δ 7.25-7.21 (m, 4H), 7.20-7.14 (m, 1H), 4.05 (q, 2H, J = 7.0 Hz). 343 (s, 2H), 2.82-2.75 (m, 2H), 2.62-2.61 (bs, 1H), 2.59 (t, 2H, J = 7.2 Hz), 2.43-2.36 (m, 1H), 2.28 (t, 2H, J = 7.2 Hz), 1.93-1.63 (m, 4H), 1.33 (dq, 2H, J = 11.8 Hz, J = 3.6 Hz). 13C NMR (101MHz, CDCl3) δ174.6,138.3,129.2,128.3,127.0,62.9,52.7,48.7,42.7,31.4,29.0,18.1.

[0441] Step 2: Ethyl 4-((1-benzylpiperidin-4-yl)(tert-butoxycarbonyl)amino)butanoate 53

[0442] To a stirred solution of ethyl 4-((1-benzylpiperidin-4-yl)amino)butanoate (1 eq., 1.2 g, 3.94 mmol) in DCM (15 mL) was added EtN (2 eq., 797.7 mg, 7.88 mmol) followed by BocO (1.5 eq., 1.29 g, 1.26 mmol) and the resulting mixture was stirred overnight. After that time, the solution was washed with water, dried (NaSO) and concentrated. The crude material was purified by flash chromatography to yield the title compound 53 (m = 1.35 g, yield = 85%).

[0443] 1 H NMR (400MHz, CDCl3) δ7.28-7.11(m,5H),4.06(q,2H,J=7.2Hz),3.96-3.79(m,1H),3.41(s,2H),3.11-2.99(m,2H),2.98(d,2H, J=12.0Hz),2.20(t,2H,J=7.7Hz),2.02-1.91(m,2H),1.79-1.70(m,2H),1.68-1.63(m,4H),1.39(s,8H),1.19(t,3H,J=7.2Hz) 13 CNMR(101MHz, CDCl3)δ173.2,155.6,129.1,128.2,127.0,79.5,63.0,60.3,53.3,42.2,31.9,30.1,25.8,14.3.

[0444] Step 3: tert-Butyl (1-benzylpiperidin-4-yl)(4-(2-(6-chloropyridazin-3-yl)hydrazinyl)-4-oxobutyl)carbamate 54

[0445] Ethyl 4-((1 -benzylpiperidin-4-yl)(tert-butoxycarbonyl)amino)butanoate 53 (1 eq. 1.3 g, 3.21 mmol) was diluted in MeOH (5 mL). 1 N NaOH (15 mL) was added and the reaction mixture was stirred at room temperature overnight. The crude was acidified with 2 N HC1 to pH = 6 and evaporated. The crude product (1 eq., 1.0 g, 2.66 mmol), BOP (1.2 eq. 1.4 g, 2.66 mmol) and NMM (2.5 eq. 0.67 g, 730 μΐ, 6.64 mmol) were suspended in DCM (1.5 mL) and the reaction mixture was stirred at room temperature for 15 min. Then, 3-chloro-6-hydrazinylpyridazine 5 (1 eq. 384 mg, 2.66 mmol) was added and the reaction was stirred at room temperature overnight (20 h). After evaporation of the volatiles, the crude was directly purified by flash chromatography on silica gel using EtOAc / MeOH / Et3N 8 / 2 / 0.3 as eluent to yield the title compound (m = 1.0 g, yield = 75%).

[0446] 1 H NMR (400 MHz, CDC13) δ 8.50 (bs, 1H), 7.52 (bs, 1H), 7.42-729 (m, 5H), 7.27 (d, 1H, J = 9.5 Hz), 7.04 (d, 1H, J = 9.9 Hz), 4.30-4.13 (m, 2H), 4.04-3.89 (m, 1H), 3.7 (t, 2H, J = 4.9 Hz), 3.45 (bs, 2H), 3.15-3.04 (m, 2H), 2.83 (t, 2H, J = 12.1 Hz), 2.27 (t, 2H, J = 7.2 Hz), 1.85-1.77 (m, 4H), 1.36 (s, 9H).

[0447] LC-MS (ES+APCI): 501 (M-H + ), 401 (-Boc)

[0448] Step 4: 1 -Benzyl-N-(3-(6-(4-methylpiperazin-1 -yl)-[1,2,4]triazolo[4,3- b]pyridazin-3-yl)propyl)piperidin-4-amine 56a (LIT-TB015)

[0449] A microwave vial was charged with ethyl 4-((1-benzylpiperidin-4-yl)(tert-butoxycarbonyl)amino)butanoate 54 (1 eq., 400 mg, 0.82 mmol) and acetic acid (1.87 mL). The vial was properly capped and the mixing vessel was heated at 110 °C for 2 h. The mixture was cooled to room temperature and evaporated. The crude material was co-evaporated with cyclohexane and triturated with cold ether. A white solid (210 mg, LC / MS 385.2 [M+H]) was collected by filtration to produce compound 55, which was used in the next step without further purification.

[0450] Using the same procedure A described for 9a (LIT-TB001) and starting with compound 55 (1 eq., 100 mg, 0.25 mmol) and 1-methylpiperazine 8a (2 eq., 100.1 mg, 57.6 μl), the title compound 56a (m = 40 mg, yield = 34%) was obtained under microwave irradiation.

[0451] LC-MS[M+H] + =449.2; 471.2 (M+Na)

[0452] Preparation of 57(LIT-TB-058)

[0453] The present invention also provides a method for the reductive dehalogenation of 6-chlorotriazolopyridazine derivatives. In particular, 7a-f are used as substrates in the presence of Pd(PPh3)4 and HCOOH as reducing agents in a halogen / metal exchange (see Scheme 11).

[0454] Scheme 11 (see Formula I)

[0455]

[0456] Conditions: a) Pd(PPh3)4 (4 mol%), HCOOH (1 eq.), TEA (12 eq.), DMF, 100°C, 45 min, microwave.

[0457] Example 12 :3-([1,2,4]triazolo[4,3-b]pyridazin-3-yl)-N-(1-benzylpiperidin-4-yl)propionamide 57a (LIT-TB058) preparation

[0458] To a solution of N-(1-benzylpiperidin-4-yl)-3-{6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl}propanamide 7a (1 eq., 100 mg, 0.25 mmol) in anhydrous DMF (2 mL) was added TEA (12 eq., 314.6 mg, 0.43 mL, 3.1 mmol) and Pd(PPh3)4 (4 mol%, 11.6 mg). The vial was properly capped and degassed, and the contents were stirred at room temperature for 10 min. A solution of formic acid (1 eq., 11.54 mg, 9.5 μl, 1 mmol) in anhydrous DMF (0.4 mL) was then added, and the reaction mixture was heated at 100°C for 45 min via microwave irradiation. After it was cooled, the reaction mixture was concentrated and purified by flash chromatography on silica gel using DCM / MeOH, 90 / 10+2% NH 3 to give, after salification, the title compound as a yellow solid (m=26 mg, yield=26%).

[0459] 1 H NMR (400Mhz, methanol-d4) δ8.58 (dd, J=4.2Hz, J=1.6Hz, 1H), 8.2 (dd, J=9.5Hz, J=1.6Hz, 1H), 7 .36(dd,J=9.5Hz,J=4.3Hz),7.35-7.31(m,4H),7.31-7.25(m,1H),3.71-3.60(m,1H),3. 52(s,2H),3.49(t,J=7.5Hz,2H),2.92-2.80(m,2H),2.84(t,J=7.5Hz,2H),2.13(dt,J=1 1.6Hz, J=2.0Hz, 2H), 1.82 (dd, J=13.1Hz, J=3.5Hz), 1.5 (dq, J=11.9Hz, J=3.5Hz, 2H).). 13 C NMR (101 MHz, methanol-d4) δ 171.7, 149.5, 146.0, 144.4, 137.1, 129.4, 127.9, 127.0, 123.9, 121.1, 62.6, 51.9, 46.5, 31.6, 30.9, 19.7.

[0460] LC-MS[M+H] + =365.20

[0461] Preparation of analogs 60a-f

[0462] The present invention also provides a method for the direct introduction of the 4-methyltetrahydropyridine moiety at position 6 via N-methyl-piperidin-3-en-4-yl boronate 58 under Suzuki-Miyaura conditions, followed by hydrogenation over Pd / C (Scheme 12).

[0463] Scheme 12 (see Formula I)

[0464]

[0465] Conditions: a) PdCl2dppf.CH2Cl2, K2CO3, DMF / H2O; b) H2, Pd / C, MeOH

[0466] Example 13 :Preparation of N-(1-benzylpiperidin-4-yl)-3-(6-(1-methylpiperidin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propanamide 60a (LIT-TB059)

[0467] N-(1-benzyl-4-piperidinyl)-3-(6-chloro-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)propionamide 7a (200 mg, 0.50 mmol, 1.0 eq.) was dissolved in dimethylformamide (10 mL). After adding pinacol borate 58 (110 mg, 0.50 mmol, 1.0 eq.), potassium carbonate (210 mg, 1.50 mmol, 3.0 eq.) and 2 drops of water, the reaction mixture was degassed by bubbling argon for 20 minutes. Palladium complex PdCl2dppf.CH2Cl2 (41 mg, 0.05 mmol, 0.1 eq.) was added in portions, and the reaction vessel was sealed and heated at 80°C for 18 h. After cooling, the solvent was removed in vacuo, and the product was purified by flash chromatography [ column The residue was purified by HPLC [24 g; eluent: EtOAc / MeOH; gradient: 100 / 0→100 / 0 (2CV), 100 / 0→70 / 30 (12CV), then 70 / 30→70 / 30 (3CV)] to give compound 59 (120 mg, 52% yield) as a dark red powder. LCMS: m / z=460.2 (M+H) confirmed.

[0468] N-(1-benzyl-4-piperidinyl)-3-[6-(1-methyl-3,6-dihydro-2H-pyridin-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]acrylamide 59 (120 mg, 0.26 mmol, 1.0 eq.) was dissolved in methanol (30 mL). After adding 10% palladium on activated carbon (145 mg, 0.14 mmol, 0.5 eq.), the reaction mixture was hydrogenated under hydrogen pressure (4 bar) at 20 ° C for 6 h. The reaction mixture was filtered through a pad of Celite® and the solvent was evaporated under vacuum. column 4 g; eluent: DCM / MeOH; gradient: 90 / 100→80 / 20 (10CV)], the residue was purified to give compound 8 (53 mg, 44% yield) as a light brown powder, which was further lyophilized to remove traces of solvent.

[0469] 1 H NMR (300MHz, CDCl3) δ7.98(d,J=9.6Hz,1H),7.33-7.22(m,5H),7.02(d,J=9.6Hz,1H),6.08(d,J=7.7Hz,1H),3.82-3.72(m,1H),3.49-3.43(m,4H),3 .04-2.99(m,2H),2.89(t,J=7.1Hz,2H),2.79-2.74(m,3H),2.35(s,3H),2 .17-2.06(m,4H),1.98-1.93(m,4H),1.93-1.83(m,2H),1.53-1.39(m,2H).

[0470] 13 C NMR (75MHz, CDCl3) δ170.7,160.2,149.3,143.8,138.3,129.1(2C),128.2(2C),127.0,12 4.7,119.9,63.0,55.3(2C),52.2(2C),46.6,46.3,41.7,32.5,32.0(2C),30.8(2C),20.3.

[0471] LCMS: m / z=462.2 (M+H).

[0472] Preparation of pyrazolopyridine

[0473] Alternatively, in a 4-step sequence, the triazolopyridazine ring can be replaced by the pyrazolopyridine ring of general structure 66 as depicted in Scheme 13 below.

[0474] Scheme 13 (see Formula I)

[0475]

[0476] Conditions: a) 3,4-dihydro-2H-pyran, pTsOH, THF; b) PdCl2dppf.CH2Cl2, K2CO3, toluene / EtOH; c) NMe-piperazine, MeCN, 160°C, 4H microwave; d) Pd / C (10%), H2, EtOH; e) HCl 6N, MeCN; f) EDCI, HOBT, H2O, Et3N, DCM.

[0477] Example 14 :N-(1-benzylpiperidin-4-yl)-3-(5-(4-methylpiperazin-1-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl)propanamide 66a (LIT-TB060) preparation

[0478] Step 1: 5-Chloro-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[4,3-b]pyridine 62

[0479] 5-Chloro-3-iodo-1H-pyrazolo[4,3-b]pyridine 1 (1.0 g, 3.60 mmol, 1.0 eq.), 3,4-dihydro-2H-pyran (650 mg, 7.70 mmol, 0.7 mL, 2.1 eq.) and p-toluenesulfonic acid (150 mg, 0.80 mmol, 0.2 eq.) were dissolved in THF (10 mL) and stirred at 60 ° C for 18 h. After cooling to room temperature, a saturated solution of NaHCO 3 (50 mL) was added and the mixture was extracted with ethyl acetate (3×75 mL). The organic layer was dried over magnesium sulfate and evaporated in vacuo. The product was purified by flash chromatography [ column 80 g; eluent: cyclohexane / DCM; gradient: 100 / 0→100 / 0 (3CV), 100 / 0→0 / 100 (20CV)], and the residue was purified to give compound 3 (1.30 g, 99% yield) as a colorless gum.

[0480] Step 2: (E)-ethyl 3-(5-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl)acrylate 64

[0481] 5-Chloro-3-iodo-1-tetrahydropyran-2-yl-pyrazolo[4,3-b]pyridine 62 (1.0 g, 2.75 mmol, 1.0 eq.) was dissolved in a mixture of toluene (10 mL) and ethanol (5 mL). After adding pinacol borate 63 (810 mg, 3.58 mmol, 1.3 eq.) and potassium carbonate (2M) aqueous solution (5.60 mmol, 2.8 mL, 2.0 eq.), the reaction mixture was degassed by bubbling argon for 20 minutes. Palladium complex (115 mg, 0.14 mmol, 0.05 eq.) was added in batches, and the reaction vessel was sealed and heated at 110 ° C for 18 h. After cooling to room temperature, water (20 mL) was added and the mixture was extracted with ethyl acetate (3×50 mL). The organic layer was dried over magnesium sulfate and evaporated in vacuo. The product was purified by flash chromatography [ column 80 g; eluent: cyclohexane / EtOAc; gradient: 90 / 10→60 / 40 (20 cv)], the residue was purified to give compound 64 (m=475 mg, yield=51%) as a white solid. Confirmed by LCMS: m / z=336.3 (M+H).

[0482] Step 3: 3-(5-(4-methylpiperazin-1-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl)propanoic acid 65

[0483] (E)-3-(5-chloro-1-tetrahydropyran-2-yl-pyrazolo[4,3-b]pyridin-3-yl)prop-2-enoic acid ethyl ester 64 (470 mg, 1.40 mmol, 1.0 eq.) was dissolved in a mixture of N-methylpiperazine 6 (5 mL) and MeCN (5 mL). The reaction mixture was heated at 160 ° C for 4 h under microwave irradiation. The solvent was evaporated in vacuo and the product was purified by flash chromatography [ column The residue was purified by HPLC-MS / MS [compound 7] (24 g; eluent: DCM / MeOH; gradient: 90 / 10→80 / 20 (20 CV) to give compound 7 (340 mg, yield 60%) as a brown oil. LCMS: m / z=400.50 (M+H) confirmed the reaction.

[0484] (E)-3-[5-(4-methylpiperazin-1-yl)-1-tetrahydropyran-2-yl-pyrazolo[4,3-b]pyridin-3-yl]prop-2-enoic acid ethyl ester (330 mg, 0.83 mmol, 1.0 eq.) was dissolved in ethanol (30 mL). After adding 10% palladium on activated carbon (100 mg, 0.09 mmol, 0.1 eq.), the reaction mixture was hydrogenated under hydrogen pressure (4 bar) at 50 ° C for 24 h. The reaction mixture was filtered through a pad of Celite® and the solvent was evaporated in vacuo to give 3-[5-(4-methylpiperazin-1-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl]propanoic acid (m=335 mg, yield=99%) as a brown oil. Confirmed by LCMS: m / z=402.1 (M+H).

[0485] 3-[5-(4-methylpiperazine-1-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl]propionic acid (330 mg, 0.83 mmol, 1.0 eq.) was dissolved in acetonitrile (5 mL). After adding HCl (6N) aqueous solution (5.0 mL), the reaction mixture was heated at 100 ° C for 30 minutes under microwave irradiation. The solvent was evaporated in vacuo, and the aqueous residue was washed with dichloromethane (3×20 mL). The aqueous layer was evaporated and dried in vacuo to obtain compound 65 mixed with a salt complex. The residue was used in the next step without any further purification. Confirmed by MS: m / z=290.25 (M+H).

[0486] Step 4: N-(1-Benzylpiperidin-4-yl)-3-(5-(4-methylpiperazin-1-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl)propanamide 66a

[0487] Crude 3-[5-(4-methylpiperazin-1-yl)-1H-pyrazolo[4,3-b]pyridin-3-yl]propanoic acid 65 (crude material, 0.83 mmol theoretical, 1.0 eq.) and 1-benzylpiperidin-4-amine 10 (280 mg, 1.47 mmol, 0.30 mL, 1.8 eq.) were dissolved in dimethylformamide (10 mL). EDCI-HCl (315 mg, 1.66 mmol, 2.0 eq.), HOBt (225 mg, 1.66 mmol, 2.0 eq.), and Et3N (725 mg, 7.17 mmol, 1.0 mL, 8.6 eq.) were added to the reaction mixture, which was stirred at room temperature for 24 h. The reaction mixture was filtered and the filtrate was evaporated to dryness under high vacuum. Water (10 mL) was added to the residue. The residual aqueous solution was washed with ethyl acetate (3 x 20 mL) and then with dichloromethane (3 x 20 mL). The aqueous layer was evaporated and dried in vacuo. The residue was dissolved in isopropanol and precipitated by diisopropyl ether. After trituration and filtration, the filtrate was evaporated in vacuo. Trituration in dichloromethane followed by filtration resulted in the detection of the target compound 11 in the filtrate. The product was purified by flash chromatography [ column 24 g; eluent: EtOAc / MeOH; gradient: 100 / 0→100 / 0 (3CV), 100 / 0→70 / 30 (15CV), then 70 / 30→70 / 30 (15CV), then DCM / NH3(7N) in MeOH; gradient: 100 / 0→100 / 0 (3CV), 100 / 070 / 30 (15CV), then 70 / 30→70 / 30 (5CV)] The residue containing 11 was purified to give compound 11 mixed with an EDCI derivative. A second purification was performed by semi-preparative HPLC (Gilson PLC 2020, column C8 Princeton SPHER. 60-10 μm, gradient: water / acetonitrile (0.1% HCOOH) 95 / 5→95 / 5, 10 min and 95 / 5→0 / 100, 25 min) followed by direct lyophilization to afford pure compound 66 (22 mg, 7% yield) (0.3 eq. formate salt) as a light brown powder. The hydrochloride salt of 66 was prepared by dissolving in dioxane (5.0 mL) and adding a solution of HCl (4 N) in dioxane (5.0 mL). After stirring at room temperature for 1 h, the solvent was evaporated and the residue was lyophilized to afford 66a (m=22 mg, yield=5%) as a light brown powder as a hydrochloride salt.

[0488] 1 H NMR (300MHz, DMSO-d6): δ7.78(d,J=7.6Hz,1H),7.71(d,J=9.2Hz,1H),7.33-7.22(m,5H),7.02(d,J=9.2Hz,1H),3.65-3.30(m,7H ),3.05-2.98(m,2H),2.80-2.72(m,2H),2.65-2.50(m,5H),2.31(s,3H),2.11-2.25(m,2H),1.70-1.65(m,2H),1.43-1.35(m,2H). 13 C NMR (75MHz, DMSO-d6): δ170.8,163.3,155.4,137.5,136.4,129.3,129.0,128 .2,127.1,120.6,109.4,61.7,54.0,51.7,45.5,45.3,45.0,34.1,31.1,21.7.

[0489] LCMS: m / z=462.2 (M+H).

[0490] Synthesis of fluorescent analogue (LIT-TB043)

[0491] As indicated in Scheme 14, fluorescent analogs of compound 9a (LIT-TB001) can be prepared by coupling a fluorescent probe (eg, DY-647P1-NHS-ester) to an appropriately substituted primary amine.

[0492] Scheme 14 (see Formula Ia)

[0493]

[0494] Conditions: a) 67, K2CO3, DMF, 80°C, 16h; b) PPh3, MeOH / H2O, room temperature overnight; c) DY-647P1-NHS-ester, DIEA, DMSO, room temperature, overnight.

[0495] (2E)-1-[6-[2-[2-[2-[4-[3-[3-[(1-benzyl-4-piperidinyl)amino]-3-oxo-propyl]-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]piperazin-1-yl]ethoxy]ethoxy]ethylamino]-6-oxo-hexyl]-2-[(2E,4E)-5-[1-(2-methoxyethyl)-3,3-dimethyl-5-sulfonato-indol-1-ium-2-yl]penta-2,4-dienylidene]-3,3-dimethyl-indoline-5-sulfonate; dihydrochloride (LIT-TB043)

[0496] Step 1: 3-(6-(4-(2-(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)-N-(1-benzylpiperidin-4-yl)propanamide hydrochloride 68

[0497] N-(1-Benzylpiperidin-4-yl)-3-[6-(piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide 9d (1 eq., 10.4 mg, 0.0232 mmol), 2-[2-(2-azidoethoxy)ethoxy]ethyl methanesulfonate 67 (1.5 eq., 8.81 mg, 0.0348 mmol) and KCO (2 eq., 6.41 mg, 0.0464 mmol) were dissolved in anhydrous DMF (0.2 ml). The reaction was flushed with argon three times, and the mixture was stirred at 80° C. for 16 h. The crude material was filtered through a pad of celite and washed with MeOH. The filtrate was evaporated to give a light yellow solid (compound 69), which was dissolved in a mixture of MeOH / H O (3 / 1, 1 ml). To the mixture was added PPh (2.5 eq., 15.2 mg, 0.058 mmol) and stirred overnight at room temperature. DMSO was added to the crude material and the mixture was evaporated. The remaining DMSO phase was purified by reverse phase chromatography (H O + 0.05% HCl / MeOH) to give the compound as a white solid (m = 7.0 mg, yield = 44%).

[0498] Step 2: (2E)-1-[6-[2-[2-[2-[4-[3-[3-[(1-benzyl-4-piperidinyl)amino]-3-oxo-propyl]-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]piperazin-1-yl]ethoxy]ethoxy]ethylamino]-6-oxo-hexyl]-2-[(2E,4E)-5-[1-(2-methoxyethyl)-3,3-dimethyl-5-sulfonato-indol-1-ium-2-yl]penta-2,4-dienylidene]-3,3-dimethyl-indoline-5-sulfonate; dihydrochloride 69 (LIT-TB043)

[0499] 3-[6-(4-{2-[2-(2-aminoethoxy)ethoxy]ethyl}piperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]-N-(1-benzylpiperidin-4-yl)propanamide hydrochloride 68 (1 eq., 0.855 mg, 0.00124 mmol) and DY-647P1-NHS-ester (1 eq., 1 mg, 0.00124 mmol) were dissolved in anhydrous DMSO (0.3 ml). DIEA (5 eq., 0.802 mg, 1.03 μL, 0.0062 mmol) was added and the reaction was flushed with Ar three times. The reaction was stirred at room temperature overnight. The crude material was directly purified by reverse phase chromatography (H2O + 0.05% HCl / MeOH) to afford LIT-TB043 (m = 1.58 mg, yield = 98%) as a blue solid.

[0500] LC-MS [2Na(m / 2)] = 646

[0501] II. Results

[0502] Material

[0503] Recombinant human BDNF and NGF were obtained from Peprotech. Recombinant human TrkB ECD -Fc was obtained from R&D Systems, and BDNF-biotin was purchased from Alomone Labs. AAV-GCAMP6F virus was produced at the U Penn vector core. Phosphatase inhibitor cocktail 2 was purchased from Roche, and protease inhibitor Complete ultra cocktail was purchased from Sigma. Antibodies were obtained from different sources as follows: polyclonal anti-TrkB, anti-phosphotyrosine (4G10), and anti-pY816-TrkB were from Millipore; monoclonal anti-TrkB was from BD Biosciences, anti-phospho-S473Akt, anti-AKT, anti-phospho-ERK1 / 2, anti-ERK1 / 2, anti-pY516-TrkB, and anti-pY706 / 707-TrkB were from Cell Signaling, HRP-conjugated streptavidin was from Amersham Biosciences, and anti-βIII-tubulin was from Millipore.

[0504] Intraperitoneal administration to mice

[0505] Adult C57BL / 6 male mice were intraperitoneally injected with saline (0.9% NaCl) or LIT-TB001 (dissolved in saline solution) at doses ranging from 0.1 to 5.0 mg / kg. A volume of 10 μl / g body weight was injected. One hour later (unless otherwise stated), the mice were decapitated, blood was collected, and the brains were quickly removed on ice. Subsequently, the cortex and hippocampus were dissected, and the tissues were quickly washed in ice-cold PBS and transferred to ice-cold solubilization buffer at 4°C, followed by homogenization. The samples were centrifuged at 10,000 × g for 10 min at 4°C. Protein concentration was determined, equal amounts of protein were loaded, and Western blotting was performed as described above.

[0506] TrkB selectivity

[0507] The development of Trk canonical (allosteric) agonists is limited by a lack of selectivity for the receptors because there are three most common and similar types of Trk receptors: TrkA, TrkB, and TrkC. Each of these receptors has a different binding affinity for certain types of neurotrophins. The differences in signaling initiated by these different types of receptors are crucial for generating different biological responses.

[0508] TrkB PAMs may have some advantages in terms of selectivity. Therefore, the selectivity of LIT-TB001 as a potentiating TrkBPAM against TrkB has been evaluated in vitro ( Figure 1 ).

[0509] The selectivity of LIT-TB001 for signaling activation and biological function was tested in PC12-TrkB or PC12-TrkA cells in the presence of BDNF (TrkB) or NGF (TrkA). Key experiments were recapitulated in cells expressing TrkA or TrkB to test TB selectivity: Trk phosphorylation, ERK phosphorylation, and neurite outgrowth ( Figure 1 ).

[0510] In PC12-TrkA cells, LIT-TB001 did not induce ERK or TrkA phosphorylation in the presence or absence of NGF. In PC12-TrkB cells, ERK and TrkB phosphorylation was induced only in the presence of BDNF. Similar observations were made at the functional level regarding neurite outgrowth.

[0511] In summary, LIT-TB001 potentiated BDNF- but not NGF-dependent signaling pathways (pERK and pTrkB) and biological functions (neurite outgrowth). These results demonstrate the selectivity of TB compounds for the Trk family.

[0512] Next, kinase profiles were performed to test the selectivity of LIT-TB001 against other kinases. A kinase profile with 45 kinases showed good selectivity for TrKB, as LIT-TB001 did not activate or block the catalytic activity of the tested kinases at a concentration of 10 μM (among which TrkA is the most similar to TrkB, confirming our previous results) ( Figure 2 ).

[0513] In vitro activity of LIT-TB derivatives in TrkB phosphorylation assays

[0514] The in vitro activities of LIT-TB derivatives in the TrkB phosphorylation assay are listed in Table 1 below:

[0515]

[0516]

[0517] a In vitro potentiation of TrkB phosphorylation induced by BDNF at 10 nM or 0.4 nM PAM concentrations in cortical neurons (+: <20%, ++: 20-35%, +++: >35%). For comparison, a 10-fold increase in BDNF concentration (0.4 to 4 nM) resulted in a 55% potentiation in the assay.

[0518] In vivo target engagement

[0519] Following peripheral injection, we evaluated TrkB engagement in the mouse brain by LIT-TB001 in vivo. C57B16 male mice received intraperitoneal injections of 0.5 and 1 mg / kg 1 hour after which their brains were carefully removed and their cortex and hippocampus sub-dissected. BDNF and TrkB are known to play key roles in these two regions. TrkB phosphorylation levels at tyrosine 816 ( Figure 3 These results clearly demonstrate that low doses (0.5 and 1 mg / kg, intraperitoneal) of LIT-TB001 effectively increase TrkB activation in the brain 1 h after systemic administration in mice.

[0520] References

[0521] [1] Vonsattel JP, DiFiglia M., Journal of Neuropathology and Experimental Neurology, 57, 369-384, 1998.

[0522] [2]Li SH, Li XJ. Multiple pathways contribute to the pathogenesis of Huntington disease. Molecular Neurodegeneration, 1, 19, 2006.

[0523] [3] Harjes P., Wanker EE, The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem. Sci, 28, 425-433, 2003.

[0524] [4] Schulte J., Littleton JT. The biological function of the Huntingtin protein and its relevance to Huntington's disease pathology. Current Trends in Neurology, 5, 65-78, 2011.

[0525] [5] The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell, 72, 971-983, 1993.

[0526] [6] Roos RAC, Huntington's disease: a clinical review; Orphanet Journal of Rare Diseases, 5, 40, 2010.

[0527] [7] Koliatsos VE, Mocchetti I., Cell Death and Diseases of the Nervous System, Humana Press, Totowa, NJ, 545–591, 1999.

[0528] [8] Barbacid M., Structural and functional properties of the Trk family of neurotrophin receptors, Ann. NY Acad. Sci., 766, 442-458, 1995.

[0529] [9] Leibrock J., Lottspeich F., Hohn A., Hofer H., Hengerer B., Masiakowski P., Thoenen H., Barde YA. Molecular cloning and expression of brain-derived neurotrophic factor, Nature, 341, 149-152, 1989.

[0530]

[10] Ferrer I., Goutan E., Marin C., Rey MJ, Ribalta T., Brain-derived neurotrophic factor in Huntington disease, Brain Research, 866, 257-261, 2000.

[0531]

[11] Zuccato C., Ciammola A., Rigamonti D., Leavitt BR, Goffredo D., Conti L., MacDonald ME, Friedlander RM, Silani V., Hayden MR, Timmusk T., Sipione S., Cattaneo E., Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease. Science, 293, 493-498, 2001.

[0532]

[12] Gauthier LR, Charrin BC, Borrell-Pagès M., Dompierre JP, Rangone H., Cordelières FP, De Mey J., MacDonald ME, Lessmann V., Humbert S., Saudou F. Huntingtin controls neutrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell, 1, 118, 127-138, 2004.

[0533]

[13] Ginés S., Bosch M., Marco S., Gavaldà N., Díaz-Hernández M, Lucas J.J., Canals JM, Alberch J., Reduced expression of the TrKB receptor in Huntington's disease mouse models and in human brain. Eur. J. Neurosci., 23, 649-658, 2006.

[0534]

[14] Canals JM, Pineda JR, Torres-Peraza JF, Bosch M., R., MT, Mengod G., Ernfors P., Alberch J., Brain-derived neurotrophic factor regulates the onset and severity of motor dysfunction associated with enkephalinergic neuronal degeneration in Huntington's disease. J. Neurosci, 24, 7727-7739, 2004.

[0535]

[15] Binder DK, Croll SD, Gall CM, Scharfman HE, BDNF and epilepsy: too much of a good thing? Trends Neurosci, 24, 47–53, 2001.

[0536]

[16] Green, SH, Rydel, RE, Connolly, JL, Greene, LA. PC12 cell mutants that possess low-but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. J. Cell. Biol. 102, 830-843, 1986.

[0537]

[17] Cazorla, M., Premont, J., Mann, A., Girard, N., Kellendonk, C., Rognan, D., Identification of a low-molecular weight TrkB antagonist with anxiolytic and antidepressant activity in mice. J. Clin. Invest., 121, 1846-1857, 2011.

[0538]

[18] Gerencsér, J., Panka, G., Nagy, T., Egyed, O., Dorman, G., Urge, L. and Darvas, F., Procedure for the parallel preparation of 3-Imidazo[1,2-a]pyridin-3-yl-propionic acid derivatives involving Meldrum's acid. J. Comb. Chem., 7, 530–538, 2005.

Claims

1. A pharmaceutical composition comprising (a) LIT-TB compound represented by formula I: in, -R 1 -G- is selected from the group consisting of groups of the formula: -X 1 and X 2 The same or different, independently represent CH or N, -X 3 For N, -X 4 For N, -Y represents N or CH, -r is an integer from 1 to 3, -A is C(O)NH or NH, -m is equal to 0, 1 or 2, -m' is equal to 0, 1, or 2, and m+m'≤3 -t is an integer from 0 to 5, -Each R 6 The groups are identical or different and are selected from the group consisting of H, fluoride, an optionally branched C1 to C6 alkyl chain and a C1 to C6 alkoxy group, -T 1 and T 2 The same or different, independently represent CH2, CHR 6 or C=O, - Z is selected from the group consisting of a bond, H and an optionally branched C1 to C3 alkyl chain, optionally comprising a heteroatom selected from the group consisting of O or N, -When Z is H, R 2 Empty, or R 2 Selected from H and optionally one or more R 7 A 5- or 6-membered aromatic ring or heterocyclic ring optionally substituted by one or more R 7 A group consisting of a 5- or 6-membered non-aromatic ring or heterocyclic ring substituted by a group, each R 7 The groups are identical or different and are selected from the group consisting of H, halide, CN, NO2, NH2, CONH2, optionally branched C1 to C6 alkyl chains and optionally branched C1 to C6 alkoxy groups, or a pharmaceutically acceptable salt thereof, Excludes: -N-(1-benzyl-4-piperidinyl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide and -N-(1-benzyl-4-piperidinyl)-3-[6-(1-piperidinyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide; and (b) a pharmaceutically acceptable excipient.

2. The composition according to claim 1, wherein The LIT-TB compound is selected from the group of compounds represented by formula I, wherein X 1 and X 2 At least one of them is N.

3. The composition according to claim 1 or 2, wherein The LIT-TB compound is selected from the group of compounds represented by formula I, wherein R 2 Selected from H and optionally 1, 2 or 3 R 7 A 5- or 6-membered aromatic ring or heterocyclic ring optionally substituted by 1, 2 or 3 R 7 A group consisting of a 5- or 6-membered non-aromatic ring or heterocyclic ring substituted by a group.

4. Use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament: in, -R 1 -G- is selected from the group consisting of groups of the formula: -X 1 and X 2 The same or different, independently represent CH or N, -X 3 For N, -X 4 For N, -Y represents N or CH, -r is an integer from 1 to 3, -A is C(O)NH or NH, -m is equal to 0, 1 or 2, -m' is equal to 0, 1, or 2, and m+m'≤3 -t is an integer from 0 to 5, -Each R 6 The groups are identical or different and are selected from the group consisting of H, fluoride, an optionally branched C1 to C6 alkyl chain and a C1 to C6 alkoxy group, -T 1 and T 2 The same or different, independently represent CH2, CHR 6 or C=O, - Z is selected from the group consisting of a bond, H and an optionally branched C1 to C3 alkyl chain, optionally comprising a heteroatom selected from the group consisting of O or N, -When Z is H, R 2 Empty, or R 2 Selected from H and optionally one or more R 7 A 5- or 6-membered aromatic ring or heterocyclic ring optionally substituted by one or more R 7 A group consisting of a 5- or 6-membered non-aromatic ring or heterocyclic ring substituted by a group, each R 7 The groups are identical or different and are selected from the group consisting of H, halide, CN, NO2, NH2, CONH2, optionally branched C1 to C6 alkyl chains and optionally branched C1 to C6 alkoxy groups, Excludes: -N-(1-benzyl-4-piperidinyl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide and -N-(1-benzyl-4-piperidinyl)-3-[6-(1-piperidinyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide.

5. Use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a TrkB positive allosteric modulator: in, -R 1 -G- is selected from the group consisting of groups of the formula: -X 1 and X 2 The same or different, independently represent CH or N, -X 3 For N, -X 4 For N, -Y represents N or CH, -r is an integer from 1 to 3, -A is C(O)NH or NH, -m is equal to 0, 1 or 2, -m' is equal to 0, 1, or 2, and m+m'≤3 -t is an integer from 0 to 5, -Each R 6 The groups are identical or different and are selected from the group consisting of H, fluoride, an optionally branched C1 to C6 alkyl chain and a C1 to C6 alkoxy group, -T 1 and T 2 The same or different, independently represent CH2, CHR 6 or C=O, - Z is selected from the group consisting of a bond, H and an optionally branched C1 to C3 alkyl chain, optionally comprising a heteroatom selected from the group consisting of O or N, -When Z is H, R 2 Empty, or R 2 Selected from H and optionally one or more R 7 A 5- or 6-membered aromatic ring or heterocyclic ring optionally substituted by one or more R 7 A group consisting of a 5- or 6-membered non-aromatic ring or heterocyclic ring substituted by a group, each R 7 The groups are identical or different and are selected from the group consisting of H, halide, CN, NO2, NH2, CONH2, optionally branched C1 to C6 alkyl chains and optionally branched C1 to C6 alkoxy groups, Excludes: -N-(1-benzyl-4-piperidinyl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide and -N-(1-benzyl-4-piperidinyl)-3-[6-(1-piperidinyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide.

6. A compound represented by formula I: in, -R 1 -G- is selected from the group consisting of groups of the formula: -X 1 and X 2 The same or different, independently represent CH or N, -X 3 For N, -X 4 For N, -Y represents N or CH, -r is an integer from 1 to 3, -A is C(O)NH or NH, -m is equal to 0, 1 or 2, -m' is equal to 0, 1, or 2, and m+m'≤3 -t is an integer from 0 to 5, -Each R 6 The groups are identical or different and are selected from the group consisting of H, fluoride, an optionally branched C1 to C6 alkyl chain and a C1 to C6 alkoxy group, -T 1 and T 2 The same or different, independently represent CH2, CHR 6 or C=O, - Z is selected from the group consisting of a bond, H and an optionally branched C1 to C3 alkyl chain, optionally comprising a heteroatom selected from the group consisting of O or N, -When Z is H, R 2 Empty, or R 2 Selected from H and optionally one or more R 7 A 5- or 6-membered aromatic ring or heterocyclic ring optionally substituted by one or more R 7 A group consisting of a 5- or 6-membered non-aromatic ring or heterocyclic ring substituted by a group, each R 7 The groups are identical or different and are selected from the group consisting of H, halide, CN, NO2, NH2, CONH2, optionally branched C1 to C6 alkyl chains and optionally branched C1 to C6 alkoxy groups, or a pharmaceutically acceptable salt thereof, Excludes: -N-(1-benzyl-4-piperidinyl)-3-[6-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propionamide and -N-(1-benzyl-4-piperidinyl)-3-[6-(1-piperidinyl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl]propanamide.

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