CN110294761B

CN110294761B - Substituted pyrazolo [1,5-a ] pyrimidine compounds as Trk kinase inhibitors

Info

Publication number: CN110294761B
Application number: CN201810586447.XA
Authority: CN
Inventors: 范晶晶; 唐春雷; 范为正
Original assignee: Nanjing Leizheng Pharmaceutical Technology Co ltd
Current assignee: Nanjing Leizheng Pharmaceutical Technology Co ltd
Priority date: 2018-06-08
Filing date: 2018-06-08
Publication date: 2020-09-08
Anticipated expiration: 2038-06-08
Also published as: CN110294761A

Abstract

The present invention relates to a substituted pyrazolo [1,5-a ] as Trk kinase inhibitors]Pyrimidine compounds, or pharmaceutically acceptable salts, pharmaceutical compositions and uses thereof. The pyrazolo [1, 5-a) with the structure of the general formula (I)]The pyrimidine derivative has good Trk family protein tyrosine kinase inhibition activity and metabolic stability.

Description

Substituted pyrazolo [1,5-a ] pyrimidine compounds as Trk kinase inhibitors

Technical Field

The invention belongs to the field of chemical medicine, and relates to a series of substituted pyrazolo [1,5-a ] pyrimidine compounds, a pharmaceutical composition containing the compounds and application of the compounds.

Background

Tropomyosin-related kinase (Trk) is a class of nerve growth factor receptors, and the family thereof consists of highly homologous tropomyosin-related kinase a (TrkA), tropomyosin-related kinase b (trkb), tropomyosin-related kinase c (trkc), encoded by NTRK1, NTRK2, and NTRK3, respectively. Among the neurotrophic factors are (i) Nerve Growth Factor (NGF) which activates TrkA, (ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activate TrkB, and (iii) NT3 which activates TrkC. Trk is widely expressed in neuronal tissue and is involved in the maintenance, signaling and survival of neuronal cells (Current Opinion in neurobiology,2001,11: 272-280).

Trk kinases were originally thought to be involved in neuronal cell growth, differentiation, apoptosis, etc., and inhibitors of the Trk/neurotrophin pathway were effective in a variety of preclinical animal models of pain. For example, antagonistic TrkA/NGF pathway antibodies (e.g., RN-624) have been shown to be effective in inflammatory and neuropathic pain animal models and in human clinical trials (Neuroscience,1994,62: 327-331; J.pain,2004,5: 157-163; Nat.Med.1995,1: 774-780; Neuroreport,1997,8: 807-810). In addition, recent literature shows that BDNF content and TrkB signaling increase in dorsal root ganglia following inflammation (Brain Research,1997,749:358), and several studies show that antibodies that reduce signaling via the BDNF/TrkB pathway can inhibit neuronal hypersensitivity and associated Pain (Molecular Pain,2008,4: 27). Recent literature also shows that overexpression, activation, amplification and/or mutation of Trk are associated with a number of cancers, including melanoma (Journal of Investigative surgery, 2008,128(8):2031-, 4(8) 1887-. In preclinical models of Cancer, non-selective small molecule inhibitors of Trk A, B and C and Trk/Fc chimeras are effective in inhibiting tumor growth and preventing tumor metastasis (Cancer Letters,2001,169: 107-114; Cancer Letters,2006,232: 90-98; Cancer Res,2008,68 (2) 346-351). In a cancer clinical test, a LMNA-NTRK1 gene fusion type soft tissue sarcoma metastasis patient is treated by a small molecular Trk kinase inhibitor Larotretinib (LOXO-101), the lung sarcoma volume is obviously reduced within four weeks, the metastasis and diffusion of cancer cells are slowed down, the dyspnea of the patient is relieved, the lung tumor mass basically disappears after four months, and no obvious adverse reaction is found during the clinical treatment (cancer D dicov, 2015,5: 1049-. LOXO-101 has significant efficacy in adult/pediatric cancer patients who carry NTRK1/2/3 fusion positive, such as soft tissue malignancies, thyroid cancer, salivary gland cancer, gastrointestinal stromal tumors, childhood fibrosarcoma, non-small cell carcinomas, cholangiocarcinoma, renal cancer, salivary gland cancer, appendiceal cancer, pancreatic cancer, peripheral nerve sheath tumors, spindle cell tumors, etc., and the drug has low side effects (New England Journal of Medicine,2018,378(8): 731-. The Trk kinase inhibitor Entrectinib (NMS-P626) was studied in clinical trials in 18 patients with solid tumors with NTRK1/2/3, ROS1 or ALK gene rearrangements, and the results showed that 72% of patients responded to Entrectinib (J targetTher Cancer,2015,12: 34-37).

In addition, inhibition of the neurotrophin/Trk pathway has been shown to be effective in treating inflammatory diseases in a preclinical mode. For example, inhibition of the neurotrophin/Trk pathway is associated with preclinical patterns of the following diseases: inflammatory lung diseases including asthma (Pharmacology & Therapeutics,2008,117(1):52-76), interstitial cystitis (the Journal of Urology,2005,173(3): 1016-.

The neurotrophin/Trk pathway, and particularly the BDNF/TrkB pathway, is also implicated in the etiology of neurodegenerative diseases, including multiple sclerosis, Parkinson's disease, and Alzheimer's disease (Frontiers in Neuroendocrinology,2006,27(4): 404-414). Modulation of the neurotrophin/Trk pathway may be useful in the treatment of these and related diseases.

Studies have shown that the TrkA receptor is crucial for the disease process in the infection of Trypanosoma cruzi (Typanosoma cruzi) (Chagasdisease) by parasitic infections in human hosts (Cell Host & Microbe,2007,1(4): 251-261). Thus, inhibition of TrkA may be useful in the treatment of chagas disease and related protozoal infections.

Also, Trk inhibitors may be useful in the treatment of diseases associated with imbalance in regulation of bone remodeling, such as osteoporosis, rheumatoid arthritis, and bone metastases. Bone metastases are a frequent complication of cancer which can occur in up to 70% of patients with advanced breast or prostate cancer and in about 15 to 30% of patients with lung, colon, stomach, bladder, uterus, rectum, thyroid or kidney cancer. Osteolytic metastases can cause severe pain, pathological fractures, life-threatening hypercalcemia, spinal cord compression, and other nerve compression syndromes. Therefore, drugs that induce apoptosis of proliferative osteoblasts would be extremely advantageous. Expression of the TrkA receptor and TrkC receptor has been observed in the osteogenic region in a fractured mouse model (Bone,2000,26(6): 625-633). In addition, NGF distribution was observed in almost all osteoblasts. The pan-Trk inhibitor can inhibit tyrosine signaling activated by neurotrophins bound to all 3 Trk receptors in human hFOB osteoblasts. These data support the theory of using Trk inhibitors for the treatment of bone remodeling diseases, such as bone metastasis in cancer patients.

Several small molecule inhibitors of Trk kinases are known to be useful in cancer or pain (expetpopin. ther. patents,2009,19(3): 305-319).

International patent applications WO2006115452 and WO2006087538 describe several classes of small molecules, known as inhibitors or Trk kinases, which can be used to treat pain or cancer.

International patent application WO2007025540 discloses certain substituted imidazo [1,2-b ] pyridazines, which have a secondary amino group or a BOC-protected piperazinyl group in the 6-position. These compounds are disclosed as inhibitors of Protein Kinase C (PKC).

International patent application WO2008052734 discloses (R) -4- (6- (2- (3-fluorophenyl) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazin-3-yl) benzonitrile, i.e. imidazo [1,2b ] pyridazine compounds having an aryl-substituted heterocyclic group at the 6-position and a benzonitrile group at the 3-position. The compounds are said to be suitable for the treatment of diseases mediated by the PI3K receptor, the JAK-2 receptor and the Trk receptor.

International patent application WO2007013673 discloses 1-phenyl-3- (6- (1-phenylethylamino) imidazo [1,2-b ] pyridazin-3-yl) urea and N- (6- (4-hydroxycyclohexylamino) imidazo [1,2-b ] pyridazin-3-yl) benzamide, i.e. imidazo [1,2b ] pyridazine compounds, which have an amino group in the 6-position and an amide or urea moiety in the 3-position. These compounds are known as Trk inhibitors. There is a continuing need for new and more effective treatments for alleviating pain, particularly chronic pain. Inhibitors of TrkA and other Trk kinases may provide an effective treatment for chronic pain states, as TrkA and other Trk kinases may act as mediators of NGF-driven biological responses.

U.S. patent publication No. US20110195948 describes substituted pyrazolo [1,5-a ] pyrimidine compounds as Trk inhibitors.

In recent years, there is an urgent clinical need for Trk inhibitors with better therapeutic efficacy, fewer side effects, high metabolic stability and good bioavailability. The invention provides a novel substituted pyrazolo [1,5-a ] pyrimidine compound serving as a Trk kinase inhibitor, which aims to overcome the problems of insufficient stability, activity and the like in the prior art and meet the increasing clinical requirements.

Disclosure of Invention

Problems to be solved by the invention

The present invention provides a novel substituted pyrazolo [1,5-a ] pyrimidine compound as a Trk kinase inhibitor. The structural compound has better pharmacodynamic property and higher metabolic stability.

Further, the invention provides a compound with a general formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition and application.

Means for solving the problems

The invention firstly provides a compound with a general formula (I) or a pharmaceutically acceptable salt thereof,

wherein:

R¹is (i) phenyl optionally independently selected from halogen, (1-4C) alkoxy, CF₃、CHF₂Substituted with one or more substituents of (a); or (ii) a 5 to 6 membered aromatic heterocycle having a heteroatom selected from N and S, wherein the aromatic heterocycle is optionally substituted with one or more halogen atoms;

R²selected from NR^aR^bAlkyl (1-4C), (1-4C) fluoroalkyl, (1-4C) hydroxyalkyl, - (1-4C alkyl) hetero Ar¹Hetero Ar²Hetero Cyc¹Hetero Cyc²Or optionally F, CF₃、OH、NH₂OMe, OEt, NHMe substituted (3-6C) cycloalkyl;

R^ais H or (1-6C) alkyl;

R^bis H, (1-4C) alkyl, (1-4C) hydroxyalkyl, hetero Ar³Or phenyl, wherein said phenyl is optionally independently selected from halogen, CF₃CN is substituted by one or more substituents;

or NR^aR^bForming a 4-membered heterocyclic ring having a ring nitrogen atom, wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from: halogen, OH, (1-4C) alkyl, (1-4C) alkoxy;

or NR^aR^bForming a 5 to 6 membered heterocyclic ring with the ring nitrogen atom and optionally with a ring nitrogen atom selected from N, O and SO₂Wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: OH, halogen, CF₃Alkyl (1-4C), NH₂、COOH；

Hetero Ar¹Is a 5-membered heteroaromatic ring having 1 to 3 ring nitrogen atoms;

hetero Ar²Is a 5 to 6 membered heteroaromatic ring having at least one nitrogen ring atom therein and optionally having a second ring heteroatom independently selected from N and S, wherein the heteroaromatic ring is optionally substituted with one or more substituents independently selected from (1-4C) alkyl, halogen;

miscellaneous Cyc¹Is a carbon-linked 4-to 6-membered nitrogen heterocycle, optionally independently selected from (1-4C) alkyl, CO₂(1-4C alkyl) substituted with one or more substituents;

miscellaneous Cyc²Is a pyridone or pyridazine ring optionally substituted by a substituent selected from (1-4C) alkyl;

hetero Ar³Is a 5 to 6 membered heteroaromatic ring having 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from (1-4C) alkyl;

x is selected from

R³Selected from OH, CN, (CH)₂)_nOH、(CH₂)_nNH₂Wherein n independently represents 1,2 or 3;

y is selected from O, NH or NR⁴；

R⁴Selected from (1-6C) alkyl.

Preferably, R²Selected from NR^aR^bAlkyl (1-4C), fluoroalkyl (1-4C), hetero Ar²Hetero Cyc¹Hetero Cyc². Even more preferably, R²Selected from NR^aR^bHetero Ar²Wherein said NR is^aR^bIn R^aSelected from H, R^bSelected from phenyl optionally substituted by halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a 4-membered heterocyclic ring having a ring nitrogen atom, wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: halogen, OH, (1-4C) alkyl, (1-4C) alkoxy, or NR^aR^bForming a 5-to 6-membered heterocyclic ring, forming a heterocyclic ring having a ring nitrogen atom, the heterocyclic ring optionally substituted with one or more substituents independently selected from: OH, halogen, CF₃(1-4C) alkyl, wherein said hetero Ar²Is a 6 membered heteroaromatic ring having at least one ring nitrogen atom therein and optionally having a second ring heteroatom independently selected from N and S.

Preferably, R¹Selected from phenyl, optionally independently selected from halogen, CF₃、CHF₂Is substituted with one or more substituents of (a). Even more preferably, R¹Selected from phenyl substituted by one or two fluorine or chlorine atoms or by one fluorine and CF₃A substituted phenyl group.

Preferably, the compound is selected from:

preferably, the pharmaceutically acceptable salt of the invention is an inorganic salt or an organic salt, and the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, benzenesulfonate, salicylate.

The invention also provides a pharmaceutical composition, which comprises the compound or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or diluent.

Furthermore, the present invention provides the use of a compound of the present invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer, pain, inflammation, neurodegenerative disease, trypanosoma cruzi infection, or osteolytic disease in a mammal. Preferably, wherein said cancer comprises melanoma, non-small cell lung cancer, thyroid cancer, acute myeloid leukemia, glioblastoma, astrocytoma and medulloblastoma, colon cancer, neuroblastoma, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, multiple myeloma, and large cell neuroendocrine tumors; wherein said pain is associated with cancer, surgery, bone fractures, bone pain due to tumor metastasis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, interstitial cystitis, chronic pancreatitis, visceral pain, inflammatory pain, migraine, chronic low back pain, bladder pain syndrome, and neuropathic pain; wherein said inflammation comprises asthma, interstitial cystitis, ulcerative colitis, Crohn's disease, atopic dermatitis, eczema, and psoriasis.

ADVANTAGEOUS EFFECTS OF INVENTION

The substituted pyrazolo [1,5-a ] pyrimidine compound with the structure shown in the general formula (I) has good Trk inhibitory activity and excellent pharmacodynamic performance, and is higher in metabolic stability and relatively low in production cost.

Detailed Description

The technical solution of the present invention will be described in detail with reference to the following examples.

In the present invention, "(1-4C) alkyl" means a saturated straight-chain or branched monovalent hydrocarbon group of 1 to 4 carbon atoms, respectively. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, and 2-methyl-2-propyl.

In the present invention "(1-4C) alkoxy" means a saturated, straight or branched monovalent radical of 1 to 4 carbon atoms each, wherein the radical is on an oxygen atom.

In the present invention "(1-4C) hydroxyalkyl" means a saturated, straight-chain or branched, monovalent alkyl group of 1 to 4 carbon atoms, respectively, in which 1 hydrogen atom is substituted by an OH group.

The term "fluoroalkyl" as used in the present invention refers to an alkyl group in which one or more hydrogen atoms are replaced with a fluorine atom. Examples of fluoroalkyl groups include-CHF₂、–CH₂CF₃Or perfluoroalkyl groups such as-CF₃or-CF₂CF₃。

In the present invention "administering" or "administering" an individual compound means providing a compound of the invention to an individual in need of treatment.

< Compound or pharmaceutically acceptable salt thereof >

The invention provides a novel substituted pyrazolo [1,5-a ] pyrimidine compound serving as a Trk kinase inhibitor or a pharmaceutically acceptable salt thereof, wherein the structural formula of the compound is shown as a general formula (I):

wherein:

R^ais H or (1-6C) alkyl, examples of (1-6C) alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl;

R^bis H, (1-4C) alkyl, (1-4C) hydroxyalkyl, hetero Ar³Or phenyl; wherein (1-4C) alkyl is exemplified by methyl; examples of (1-4C) hydroxyalkyl include CH₂CH₂OH or CH₂CH₂CH₂OH; hetero Ar³Is a 5 to 6 membered heteroaromatic ring having 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from (1-4C) alkyl, specific examples include isoxazole ring, dimethyl isoxazolyl, and the like; wherein said phenyl is optionally independently selected from halogen, CF₃CN is substituted by one or more substituents;

or NR^aR^bForming a 4-membered heterocyclic ring having a ring nitrogen atom, wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: halogen, OH, (1-4C) alkyl, (1-4C) alkoxy;

hetero Ar²Is a 5 to 6 membered heteroaromatic ring having at least one ring nitrogen atom therein and optionally having a second ring heteroatom independently selected from N and S, wherein the heteroaromatic ring is optionally substituted with one or more substituents independently selected from (1-4C) alkyl, halogen;

x is selected from

y is selected from O, NH or NR⁴；

R⁴Selected from (1-6C) alkyl.

In certain embodiments of the invention, R¹Selected from phenyl, optionally independently selected from halogen, CF₃、CHF₂Is substituted with one or more substituents of (a). Further, R¹Selected from phenyl substituted by one or two fluorine atoms or by one fluorine atom and CF₃A substituted phenyl group. Specific example, R¹Is p-difluorophenyl.

In certain embodiments of the invention, R²Selected from NR^aR^bPreferably, said NR^aR^bIn R^aSelected from H, R^bSelected from phenyl optionally substituted by halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a 4-membered heterocyclic ring having a ring nitrogen atom, wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: halogen, OH, (1-4C) alkyl, (1-4C) alkoxy, or NR^aR^bForming a 5-to 6-membered heterocyclic ring, forming a heterocyclic ring having a ring nitrogen atom, the heterocyclic ring optionally substituted with one or more substituents independently selected from: OH, halogen, CF₃And (1-4C) alkyl.

In a particular embodiment, from NR^aR^bThe groups represented include the following structures:

in view of further improving the inhibitory activity or drug-forming property, NR^aR^bPreferably selected from

In certain embodiments of the invention, R²Is (1-4C) hydroxyAn alkyl group. Specific examples include C (CH)₃)₂OH and C (CH)₃)₂CH₂OH。

In certain embodiments of the invention, R²Is- (1-4C alkyl) Ar¹. Wherein Ar is hetero¹Is a 5-membered heteroaromatic ring having 1 to 3 ring nitrogen atoms. Hetero Ar¹Examples of (a) are triazole rings, such as 1,2, 4-triazole rings. Examples of (1-4C) alkyl moieties include methylene, ethylene, dimethylmethylene, and the like.

In certain embodiments of the invention, R²Is hetero Ar². The hetero Ar²Is a 6 membered heteroaromatic ring having at least one ring nitrogen atom therein and optionally having a second ring heteroatom independently selected from N and S. Hetero Ar²Examples of (b) include pyridyl, pyrimidyl, pyrazinyl, pyrazolyl, imidazolyl and thiazolyl rings. From hetero Ar²R of²The specific structure of (a) includes the following structures:

preferably, from hetero Ar²R of²Is composed of

In certain embodiments of the invention, R²Is miscellaneous Cyc¹. Miscellaneous Cyc¹Examples of (a) include carbon-linked azetidinyl, pyrrolidinyl and piperidinyl rings, optionally independently selected from (1-4C alkyl), CO₂(1-4C alkyl) is substituted with one or more substituents. Examples of substituents include methyl, ethyl, propyl, CO₂Me、CO₂Et and CO₂C(CH₃)₃. In one embodiment, the heterocyclic Cyc¹Optionally substituted with one or two of the substituents.

In certain embodiments of the invention, R²Is miscellaneous Cyc². Miscellaneous Cyc²Examples of (B) include optionally substituted alkyl (e.g. methyl or ethyl) groups selected from (1-4C)Group) or a pyridazinone ring.

In certain embodiments of the invention, Y is selected from O, NH or NCH₃、NCH₂CH₃. To improve the metabolic stability and activity of the compound, preferably Y is NH.

In certain embodiments of the invention, X is selected from

Preferably, for further enhancing pharmacodynamic properties, X is selected from

In contrast, when X is selected from

When the compound is used, the IC50 value is large.

In certain embodiments of the invention, the compounds have the following structure and spatial configuration:

in order to achieve higher pharmacodynamic and metabolic stability properties, in certain embodiments of the invention, it is preferred that the compounds have the following structure and spatial configuration:

the compounds of formula (I) include pharmaceutically acceptable salts thereof. The pharmaceutically acceptable salt is inorganic salt or organic salt, and the inorganic salt comprises hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate and acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, benzenesulfonate, salicylate.

Further, the compounds of formula I or their salts may be isolated in the form of solvates, and thus any such solvate is within the scope of the present invention.

The compounds of formula (I) also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds of the present invention include compounds in which one or more hydrogen atoms are replaced by deuterium or tritium or one or more carbon atoms are replaced by 13C-or 14C-enriched carbon, which are within the scope of the present invention.

< preparation method of Compound >

The present invention also provides a process for the preparation of a compound of formula (I) comprising:

for compounds in which X is

Reacting the corresponding compound of formula II with a compound having formula III in the presence of a coupling agent and/or a base to give a compound of formula IVa;

wherein R is⁵Independently halogen, hydroxy, methoxy, ethoxy, propoxy, OTs, OMs, and the like.

Formula IVa and formula HNR^aR^bWherein X is

A compound of the general formula (I).

If desired, any protecting groups are added or removed, and if desired, salts or solvent compounds are formed.

For compounds in which X is

The compound of formula (I) of (I), reacting the corresponding compound of formula II to form an isothiocyanate with a compound having the formula HNR^aR^bOr with HNR after the compound of formula II has reacted with thiophosgene^aR^bBy reaction of compounds of (a) or under the action of other condensing agents to give X

A compound of the general formula (I).

For compounds in which X is

Of formula (I) reacting a corresponding compound of formula (II) with HNR^aR^bWith amino-substituted R after formation of urea or thiourea³Reaction to obtain X of

A compound of the general formula (I).

For compounds in which X is

Of formula (I) with a corresponding compound of formula (II) and a compound of formula (HNR)^aR^bUnder the action of sulfinyl chloride or sulfonyl chloride, X is

A compound of the general formula (I).

For compounds in which X is

Of formula I, of formula IIThe corresponding compound and the compound with the formula VI react under the action of a coupling agent and/or a base to obtain X

A compound of the general formula (I).

R⁶Independently halogen, hydroxyl, OTs, OMs, and the like.

Coupling agents mentioned in the present invention include 2- (7-benzotriazol-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), benzotriazol-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU), O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU), N, N-Dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (DIEC) and any other amide coupling agent known to the person skilled in the art. Suitable bases include tertiary amine bases such as Diisopropylethylamine (DIEA) and triethylamine, inorganic bases, and the like.

< pharmaceutical composition >

The invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined above, and a pharmaceutically acceptable carrier, excipient or diluent.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated as solid formulations for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like. In these solid dosage forms, the compounds of general formula (I) according to the invention as active ingredient are mixed with at least one customary inert excipient (or carrier), for example with sodium citrate or dicalcium phosphate. Or mixing with the following components: (1) fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, silicic acid, and the like; (2) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic and the like; (3) humectants, such as glycerol and the like; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and the like; (5) a slow solvent such as paraffin and the like; (6) absorption accelerators such as quaternary ammonium compounds and the like; (7) wetting agents such as cetyl alcohol and glyceryl monostearate and the like; (8) adsorbents, for example, kaolin, and the like; (9) lubricants, for example, talc, calcium stearate, solid polyethylene glycols, sodium lauryl sulfate, and the like, or mixtures thereof. Capsules, tablets, pills, etc. may also contain buffering agents.

The solid dosage forms, e.g., tablets, dragees, capsules, pills, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other crystalline forms of materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active ingredient may also be in microencapsulated form with one or more of the above excipients.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated in liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, and the like. In addition to the compounds of formula (I) or pharmaceutically acceptable salts thereof as active ingredients, the liquid dosage forms may contain inert diluents conventionally employed in the art, such as water and other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil and the like or mixtures of such materials and the like. In addition to these inert diluents, the liquid dosage forms of the present invention may also include conventional adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, perfuming agents and the like.

Such suspending agents include, for example, ethoxylated stearyl alcohol, polyoxyethylene sorbitol, and sorbitan, microcrystalline cellulose, agar, and the like, or mixtures of these materials.

The compounds of the present invention and pharmaceutically acceptable salts thereof may be formulated for parenteral injection in dosage forms including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated into dosage forms for topical administration, including, for example, ointments, powders, suppositories, drops, sprays, inhalants and the like. The compounds of the general formula (I) according to the invention or their pharmaceutically acceptable salts as active ingredients are mixed under sterile conditions with physiologically acceptable carriers and optionally preservatives, buffers and, if desired, propellants.

The pharmaceutical composition comprises the compound of the general formula (I) or pharmaceutically acceptable salt thereof as an active ingredient, and pharmaceutically acceptable carriers, excipients and diluents. In preparing the pharmaceutical compositions, the compounds of formula (I) or pharmaceutically acceptable salts thereof of the present invention are typically mixed with a pharmaceutically acceptable carrier, excipient or diluent. Wherein the content of the compound of the general formula (I) or the pharmaceutically acceptable salt thereof may be 0.01-1000mg, for example, 0.05-800mg, 0.1-500mg, 0.01-300mg, 0.01-200mg, 0.05-150mg, 0.05-50mg, etc.

< use >

The invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer, pain, inflammation, neurodegenerative disorders, trypanosoma cruzi infection or osteolytic disorders in a mammal.

A compound of general formula (I), or a pharmaceutically acceptable salt thereof, for use in a method of treatment of cancer, pain, inflammation, neurodegenerative disorders, trypanosoma cruzi infection or osteolytic disorders in a mammal, including a human, by inhibition of tropomyosin kinase (Trk) activity, which method comprises administering to said mammal, including a human, a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, as defined above.

A "therapeutically effective amount" is an amount of a compound of the invention effective to produce a biological or medical response (e.g., decrease or inhibit enzymatic protein activity, or ameliorate symptoms, alleviate a condition, slow or delay disease progression, or prevent disease) in an individual.

Cancers of the present invention include melanoma, non-small cell lung cancer, thyroid cancer, acute myeloid leukemia, glioblastoma, astrocytoma and medulloblastoma, colon cancer, neuroblastoma, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, multiple myeloma, and large cell neuroendocrine tumors.

Pain referred to herein relates to cancer, surgery, bone fractures, bone pain caused by tumor metastasis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, interstitial cystitis, chronic pancreatitis, visceral pain, inflammatory pain, migraine, chronic low back pain, bladder pain syndrome, and neuropathic pain.

The inflammation mentioned in the invention includes asthma, interstitial cystitis, ulcerative colitis, Crohn's disease, atopic dermatitis, eczema and psoriasis.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be administered to mammals including humans, either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments, drops) or intratumorally.

The compound or the pharmaceutically acceptable salt thereof can be independently administered or combined with other pharmaceutically acceptable therapeutic agents to be combined with other antitumor drugs. Such combination therapy may be achieved by the simultaneous, sequential or separate use of the individual components of the therapy. Such therapeutic agents include, but are not limited to: antineoplastic drugs acting on the chemical structure of DNA, such as cisplatin, antineoplastic drugs affecting nucleotide synthesis, such as methotrexate, 5-fluorouracil and the like, antineoplastic drugs affecting nucleic acid transcription, such as doxorubicin, epirubicin, aclacinomycin and the like, antineoplastic drugs affecting tubulin synthesis, such as taxol, vinorelbine and the like, aromatase inhibitors, such as aminoglutethimide, letrozole, rennin and the like, cell signaling pathway inhibitors, such as epidermal growth factor receptor inhibitor Imatinib (Imatinib), Gefitinib (Gefitinib), Erlotinib and the like. Anti-tumor monoclonal antibodies, immunosuppressants PD-1, PD-L1 and the like, and the components to be combined can be administered simultaneously or sequentially, in a single preparation form or in different preparation forms. Such combinations include not only combinations of one or other active agents of the compounds of the present invention, but also combinations of two or more other active agents of the compounds of the present invention.

In combination with one or more other drugs acting by the same or different mechanisms of action. Such combination therapy may be achieved by the simultaneous, sequential or separate use of the individual components of the therapy. Such therapeutic agents include, but are not limited to: dexamethasone, cortisone, and fluticasone as steroids; analgesics, aspirin, ibuprofen, indomethacin and opioids.

Examples

The following examples illustrate but do not limit the synthesis of the compounds of formula (I). The temperatures are given in degrees Celsius. All evaporation was performed under reduced pressure if not otherwise stated. If not otherwise stated, the reagents were purchased from commercial suppliers and used without further purification. The structure of the final products, intermediates and starting materials is confirmed by standard analytical methods, such as elemental analysis, spectroscopic characterization, e.g., MS, NMR. Abbreviations used are those conventional in the art. Intermediate A

Preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4-methoxycyclobut-3-ene-1, 2-dione

Methanol (50mL) was added to a 100mL flask, followed by addition of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-amine (6.4g, 20.5mmol) and 3, 4-dihydroxy-3-cyclobutene-1, 2-dione (2.32g, 16.3mmol) and heating under reflux for 6-8h to gradually precipitate a solid, which was cooled and filtered to obtain 2.5g (yield 28.6%) of a yellow solid. The structural formula is as follows:

intermediate B

Preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4-ethoxycyclobut-3-ene-1, 2-dione

Ethanol (50mL) was added to a 100mL flask, followed by addition of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-amine (6.4g, 20.5mmol) and 3, 4-dihydroxy-3-cyclobutene-1, 2-dione (2.32g, 13.6mmol) and heating under reflux for 6-8h to gradually precipitate a solid, which was cooled and filtered to obtain 2.7g (yield 30.9%) of a yellow solid. The structural formula is as follows:

intermediate C

Preparation of (R) -1- ((5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) carbamoyl) cyclopropylcarboxylic acid

1, 1-Cyclobutyldicarboxylic acid (10.0g, 69.4mmol) was added to anhydrous THF (100 mL). Triethylamine (7.02g, 69.4mmol, 1.0eq) was added dropwise under nitrogen and stirred at 0 ℃ for 30 minutes, followed by the addition of thionyl chloride (8.25g, 69.4mmol, 1.0eq) and stirring at 0 ℃ for 30 minutes. To the reaction was added dropwise a further (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-amine (21.7g, 76.3mmol) in anhydrous THF (50mL) under nitrogen and stirred at 10-15 ℃ for 10 h. The reaction was diluted with ethyl acetate and extracted with 2N NaOH (pH > 10). The aqueous phase was adjusted to pH 2-3 by dropwise addition of 2N HCl and then extracted with ethyl acetate. The organic phase was dried and suspended to give 23.0g of an off-white solid (yield 71%). The structural formula is as follows:

intermediate D

Preparation of (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-isothiocyanatopyrazolo [1,5-a ] pyrimidine

Carbon dichlorosulfide (0.76g, 10mmol) was added to a solution of dichloromethane (8mL) in a three-necked flask, and (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-amine (2.83g, 9mmol) in dichloromethane (10mL) was added thereto with cooling to 0 ℃. Sodium hydroxide (1.2g, 30mmol) was added to the reaction solution. The reaction mixture was stirred at room temperature for 3 hours, washed with water, dried, and the solvent was evaporated under reduced pressure to give 2.1g of a product (yield 63%). The structural formula is as follows:

example 1

3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione

The intermediate A is (R) -5- (2- (2, 5-difluorophenyl) pyrrolidine-1-yl) pyrazolo [1,5-a]Dissolving pyrimidine-3-yl) amino) -4-methoxycyclobut-3-ene-1, 2-dione (0.5g, 1.18mmol) or an equivalent amount of intermediate B in methanol (10mL), adding (S) -3-hydroxypyrrolidine hydrochloride (0.2g, 2.36mmol), reacting in a closed container under microwave conditions at 80 ℃ for 1.5h, cooling to room temperature to generate a solid, filtering, suspending the filtrate, adding a small amount of dichloromethane, standing to precipitate a solid, and filtering to obtain 0.12g of a product (yield 21%).¹H-NMR(400MHz，CDCl₃)：6.31(s,1H),8.17(d,2H),7.0(m,1H),6.91(m,1H),6.76(m,1H),6.22(s,1H),5.97(s，1H),5.30(s,1H),4.56(d,1H),3.91-3.53(m,5H),2.5(m,1H),2.13-2.08(m,6H),MS m/z 481.2(M+1)⁺. The structural formula is as follows:

example 1A

3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione sulfate

To a solution of 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione (0.48g, 1.0mmol) in methanol (20mL) was added sulfuric acid in methanol (5mL, 1mmol) at room temperature. The resulting solution was stirred for 1 hour, then concentrated to give 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione sulfate as a yellow solid 0.42g (yield 72%). The structural formula is as follows:

example 1B

3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-en-1, 2-dione hydrochloride

To a solution of 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione (0.48g, 1.0mmol) in methanol (20mL) was added a solution in hydrogen chloride in methanol (5mL, 1mmol) at room temperature. The resulting solution was stirred for 1 hour, then concentrated to give 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-en-1, 2-dione hydrochloride 0.44g as a yellow solid (yield 86%).

Example 2

Reacting (R) -5- (2- (5-fluoro-2-trifluorophenyl) pyrrolidine-1-yl) pyrazolo [1,5-a]Pyrimidin-3-yl) amino) -4-ethoxycyclobut-3-ene-1, 2-dione (commercially available, 0.36g, 1.0mmol) was dissolved in methanol (10mL), and (S) -3-hydroxypyrrolidine hydrochloride (0.17g, 2.0mmol) was added, and the mixture was reacted in a closed vessel under microwave conditions at 80 ℃ for 1.5 hours, cooled to room temperature, to produce a solid, filtered, and after the filtrate was suspended to dryness, a small amount of dichloromethane was added, and after standing, a solid precipitated, and filtered to obtain 0.10g of a product (yield 22%). MS M/z 531.2(M +1)⁺. The structural formula is as follows:

examples 3-9 (see table 1) were synthesized in a similar operation to the basic operation of example 1 to give the desired product.

TABLE 1 structural and Mass Spectrometry data for examples 3-8

Example 9

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1- ((S) -3-hydroxypyrrolidine-1-carboxy) cyclopropylcarboxamide

The intermediate C, namely (R) -1- ((5- (2- (2, 5-difluorophenyl) pyrrolidine-1-yl) pyrrolo [1, 5-a)]Pyrimidin-3-yl) amino) carbamoyl) cyclopropylcarboxylic acid (21.4g, 50mmol) was dissolved in 150mL of THF, 0.2mL of DMF was added dropwise, and (COCl) was added dropwise to the solution in an ice-water bath₂(6.3g, 50mmol), the temperature does not exceed 15 ℃ in the dropping process, after the dropping is finished, the mixture is stirred for 4 hours at 15 ℃, and then the mixture is transferred into a dropping funnel for standby. (S) -3-hydroxypyrrolidine hydrochloride (4.9g, 40mmol) and potassium carbonate (27.6g, 200mmol) are sequentially added into a mixed solution of 150mL of water and 300mL of THF, the acyl chloride is dropwise added, the temperature in the process is not more than 25 ℃, after the dropwise addition is finished, the mixture is stirred at room temperature for 2h, water (500mL) is added, the mixture is stirred at room temperature for 8h, solid is separated out, suction filtration is carried out to obtain off-white solid, the off-white solid is directly added into a mixed solution of 500mL of THF and 500mL of water, the reflux is carried out for 1h, the cooling is carried out to the room temperature, the solid is separated out, the suction filtration is carried out.

¹H-NMR(400MHz，CDCl₃)：6.33(s,1H),8.17(d,2H),7.16(m,1H),6.92(m,1H),6.70(m,1H),6.23(s,1H),5.97(s，1H),5.28(s,1H),4.57(d,1H),3.91-3.53(m,5H),2.5(m,1H),2.12-2.08(m,6H),1.49-1.47(m,4H).MS m/z 497.2(M+1)⁺. The structural formula is as follows:

example 9A

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1- ((S) -3-hydroxypyrrolidine-1-carboxy) cyclopropylcarboxamide sulfate

To a solution of 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione (0.50g, 1.0mmol) in methanol (20mL) was added sulfuric acid in methanol (5mL, 1mmol) at room temperature. The resulting solution was stirred for 1 hour, then concentrated to give 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione sulfate as a yellow solid 0.42g (yield 72%). The structural formula is as follows:

example 9B

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1- ((S) -3-hydroxypyrrolidine-1-carboxy) cyclopropylcarboxamide hydrochloride

To a solution of N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolo [1,5-a ] pyrimidin-3-yl) -1- ((S) -3-hydroxypyrrolidin-1-carboxy) cyclopropylcarboxamide (0.50g, 1.0mmol) in methanol (20mL) at room temperature was added a solution of hydrogen chloride in methanol (5mL, 1mmol) and the resulting solution was stirred for 1 hour and then concentrated to give 3- ((5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrrolo [1,5-a ] pyrimidin-3-yl) amino) -4- ((S) -3-hydroxypyrrolidin-1-yl) cyclobut-3-en-1 as a yellow solid, 2-diketone hydrochloride 0.45g (yield 83%). The structural formula is as follows:

example 9C

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -1- ((S) -3-hydroxypyrrolidine-1-carboxy) cyclopropylcarboxamide L-malate salt

N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrolo [1,5-a ] pyrimidin-3-yl) -1- ((S) -3-hydroxypyrrolidine-1-carboxy) cyclopropylcarboxamide (0.50g, 1.0mmol) was dissolved in 10mL of butanone, L-malic acid (0.16g,1.2mmol) was dissolved in 2mL of water, the aqueous solution of malic acid was slowly added to the butanone solution, stirred at an internal temperature of 50-55 ℃ for 2 hours, refluxed with stirring for 4-5 hours, cooled to room temperature, stirred for 24 hours, filtered, the filter cake was washed with 2mL of water, 2mL of butanone, and vacuum dried to give 0.5g (yield 80%) of a white solid having the following structural formula:

example 10

(S) -N- (5- ((R) -2- (2, 5-difluorophenyl) pyrrol-1-yl) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrole-1-thiourea

Intermediate D, i.e. (R) -5- (2- (2, 5-difluorophenyl) pyrrolidin-1-yl) -3-isothiocyanatopyrazolo [1, 5-a) -was added to sodium hydride (60% oil dispersion, 240mg, 6.0mmol) previously washed with n-hexane in DMF (10mL) at 0 deg.C]Pyrimidine (3mmol) in DMF (5 mL). After stirring at 0 ℃ under argon for 15min, a DMF solution of (S) -3-hydroxypyrrolidine hydrochloride (5mL, 3mmol) was added dropwise at 0 ℃ and stirred at room temperature for 1 h. The reaction was stopped by adding 0.5M sodium dihydrogen phosphate. The mixture was extracted with ethyl acetate, washed with water and dried over sodium sulfate, and the crude product was recrystallized from methanol to give the objective compound 0.99g (yield 75%), MS M/z 445.2(M +1)⁺. The structural formula is as follows:

examples 11-14 (see Table 2) were synthesized in a procedure similar to the basic procedure of example 9 to give the desired product.

TABLE 2 structural and Mass Spectrometry data for examples 11-14

Intermediate E

At 0 deg.C, argon gas and under stirringExample 13(1.0g, 2.3mmol) and triethylamine (1mL, 6.9mmol) in dichloromethane was added methanesulfonyl chloride (4.6mmol) dropwise. Stirring at 0 deg.C for 15 min. And (5) detecting the reaction is finished. The mixture was purified by column chromatography (eluting with dichloromethane) to give the objective compound 0.7g (yield 73%), MS M/z 417.2(M +1)⁺. The structural formula is as follows:

example 15

To a mixture of cyanamide (560mg, 13.33mmol) and Huinig base (2.6mL) in acetonitrile was added dropwise, with stirring, a solution of intermediate E (0.7g, 1.69mmol) in acetonitrile (30 mL). The reaction was stirred at room temperature for 15min, then the solvent was removed under reduced pressure and the residue was hydrolyzed with 0.5M sodium dihydrogen phosphate. The ethyl acetate extract of the aqueous mixture was washed with 0.5M sodium dihydrogen phosphate and brine, dried, filtered and distilled to give a crude product, and column chromatography was performed to give the desired compound 0.46g (yield 61%), MS M/z 445.2(M +1) +. The structural formula is as follows:

examples 16-18 (see Table 3) were synthesized in a procedure similar to the basic procedure of example 15 to give the desired products.

TABLE 3 structural and Mass Spectrometry data for examples 16-18

Example 19

TrkA ELISA assay

TrkA kinase activity was assessed using an enzyme-linked immunosorbent assay (ELISA) in the presence of the inhibitor. Immulon4HBX 384-well microtiter plates (Thermo) were coated with 0.025mg/mL poly (Glu, Ala, Tyr; 6:3: 1; Sigma P3899) solution. Each concentration of test compound, 2.5nM TrkA (Invitrogen, histidine-tagged recombinant human Trk, cytoplasmic domain) and 500 μmol ATP were incubated in coated plates at ambient temperature for 25 min while shaking. The assay buffer consisted of 15mM MOPS pH7.5, 0.005% (v/v) Triton X-100 and 5mM MgCl 2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween (Tween) 20. Phosphorylation reaction products were detected using a 0.2. mu.g/mL phosphotyrosine-specific monoclonal antibody conjugated to horseradish peroxidase (clone PY20) in combination with the TMB peroxidase material system (KPL). After addition of 1M phosphoric acid, the chromogenic substance color intensity was quantified via absorbance at 450 nm. IC50 values were calculated using a 4 or 5-parameter logarithmic (logistic) curve fit.

In this assay, the compounds of the invention have an average IC of less than 1000nM₅₀. Some compounds have an average IC of less than 100nM₅₀. Table 4 provides the specific IC of the compounds of the invention tested in this assay₅₀The value is obtained.

TABLE 4 specific IC's of the compounds of the invention₅₀Value of

Example numbering	TrkA ELISA enzyme IC₅₀(nM)
		1	3.7
2	6.5
		3	10.3
4	87.5
		5	3.7
6	2.0
		7	28.4
8	47.1
		9	97.8
10	117.6
		11	239.4
12	317.6
		13	129.5
14	137.2
		15	23.1
16	36.8
		17	181.5
18	233.1

Example 20

Evaluation of compound stability was performed using human liver microsomes.

The liver microsomal enzyme stability of the example compounds was compared to Larotrectinib (LOXO-101).

Measurement System: the metabolic stability of the compounds of the present invention was tested using 1mM NADPH for liver microparticles mixed by men and women. The samples were analyzed using a mass spectrometer. HRMS was used to determine peak area response ratios (peak area corresponding to test compound or control divided by peak area of the analytical internal standard) without running a standard curve. In order to detect all possible metabolites, HRMS scans were performed at the appropriate m/z range.

The measurement conditions were as follows: the assay was performed with one incubation (N ═ 1). Test compounds were incubated at 37 ℃ in buffer containing 0.5 mg/ml liver microsomal protein. Reactions were initiated by addition of cofactors and samples taken at 0,2, 4, 8, 16, 24, 36, 48 hours, positive controls (5 μ M testosterone) were incubated in parallel and samples taken at 0,2, 4, 8, 16, 24, 36, 48 hours.

And (3) measuring quality control: the control compound testosterone was performed in parallel to confirm the enzymatic activity of the (liver) microsomes. After the final time point, the addition of NADPH to the reaction mixture was confirmed using fluorimetry. The T1/2 of the control met acceptable internal standards.

The analysis method comprises the following steps:

liquid chromatography column: thermo BDS Hypersil C1830X2.0 mM, 3 μm, with guard column M.P., buffer 25mM formic acid buffer, pH 3.5;

aqueous phase (a): 90% water, 10% buffer;

organic phase (B): 90% acetonitrile, 10% buffer;

flow rate: 300 microliter/min

Automatic sample injector: injection volume 10 microliter

See table 5 for gradient program.

TABLE 5 gradient program

Time (minutes)	％A	％B
			0.0	100	0
1.5	0	100
			2.0	0	100
2.1	100	0
			3.5	100	0

By using human liver microsomes, examples 1,2, 4,5, 6, 7,8, 16 showed a metabolic half-life of more than 48 hours, examples 3,9, 17, 18 showed a metabolic half-life between 36-48 hours, significantly more than larotryptinib (LOXO-101)23 hours, example 15 showed a metabolic half-life between 24-36 hours, slightly more than larotryptinib (LOXO-101) metabolic half-life, as described in the present invention. The results show that the relatively long metabolic half-life makes them potentially useful for lowering therapeutic doses and extending the time interval between administrations.

Specific IC from the Compounds of examples 1-18₅₀Values and metabolic half-lives data show that for compounds of the general formula (I), linking groups and substituents such as X groups, R²Selection of radicals and R¹The substituent and the position thereof in (A) have important influence on the pharmacodynamic performance and the metabolic stability of the compound.

While the invention has been illustrated by the foregoing specific embodiments, it is not to be construed as being limited thereby; but that the present invention encompass the generic aspects previously disclosed. Various modifications and embodiments can be made without departing from the spirit and scope of the invention.

Claims

1. A compound having the general formula (I) or a pharmaceutically acceptable salt thereof,

wherein:

R^ais H or (1-6C) alkyl;

or NR^aR^bForm with a ring nitrogen atomA 4-membered heterocyclic ring of a subgroup, wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: halogen, OH, (1-4C) alkyl, (1-4C) alkoxy;

or NR^aR^bForming a 5 to 6 membered heterocyclic ring having a ring nitrogen atom and optionally having a ring nitrogen atom selected from N, O and SO₂Wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: OH, halogen, CF₃Alkyl (1-4C), NH₂、COOH；

x is selected from

y is selected from NH or NR⁴；

R⁴Selected from (1-6C) alkyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is as defined in claim 1In that R²Selected from NR^aR^bAlkyl (1-4C), fluoroalkyl (1-4C), hetero Ar²Hetero Cyc¹Hetero Cyc²。

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is²Selected from NR^aR^bHetero Ar²Wherein said NR is^aR^bIn R^aSelected from H, R^bSelected from phenyl optionally substituted by halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a 4-membered heterocyclic ring having a ring nitrogen atom, wherein the heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of: halogen, OH, (1-4C) alkyl, (1-4C) alkoxy, or NR^aR^bForming a 5-to 6-membered heterocyclic ring, forming a heterocyclic ring having a ring nitrogen atom, the heterocyclic ring optionally substituted with one or more substituents independently selected from: OH, halogen, CF₃(1-4C) alkyl, wherein said hetero Ar²Is a 6 membered heteroaromatic ring having at least one ring nitrogen atom therein and optionally having a second ring heteroatom independently selected from N and S.

4. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R is¹Selected from phenyl, optionally independently selected from halogen, CF₃、CHF₂Is substituted with one or more substituents of (a).

5. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R is¹Selected from phenyl substituted by one or two fluorine or chlorine atoms or by one fluorine and CF₃A substituted phenyl group.

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof,

the compound is selected from:

7. the compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, and the inorganic salt comprises a hydrochloride, a hydrobromide, a hydroiodide, a sulfate, a bisulfate, a nitrate, a phosphate, an acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate.

8. A pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

9. Use of a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating cancer, pain, inflammation, neurodegenerative disease, trypanosoma cruzi infection, or osteolytic disease in a mammal.

10. The use of claim 9, wherein the cancer comprises melanoma, non-small cell lung cancer, thyroid cancer, acute myeloid leukemia, glioblastoma, astrocytoma and medulloblastoma, colon cancer, neuroblastoma, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, multiple myeloma, and large cell neuroendocrine tumors; wherein said pain is associated with cancer, surgery, bone fractures, bone pain due to tumor metastasis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, interstitial cystitis, chronic pancreatitis, visceral pain, inflammatory pain, migraine, chronic low back pain, bladder pain syndrome, and neuropathic pain; wherein said inflammation comprises asthma, interstitial cystitis, ulcerative colitis, Crohn's disease, atopic dermatitis, eczema, and psoriasis.