CN113563341B - Substituted pyrazolo [1,5-a ] pyrimidine compounds as Trk inhibitors - Google Patents

Abstract

The invention relates to a compound with a general formula (I) or pharmaceutically acceptable salt thereof, a pharmaceutical composition and application. The pyrazolo [1, 5-a) with the structure of the general formula (I)]The pyrimidine derivative is more convenient to synthesize and has good Trk family protein tyrosine kinase inhibitory activity.

Description

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

Trk (tropiomycin-related kinase) is a high affinity receptor tyrosine kinase activated by a group of soluble growth factors called Neurotrophins (NTs). The Trk receptor family has 3 members: TrkA, TrkB and TrkC. Among the neurotrophins are (i) Nerve Growth Factor (NGF) which activates TrkA; (ii) brain Derived Neurotrophic Factor (BDNF) and NT-4/5 that activate TrkB; and (iii) NT3 activating TrkC. Trk is widely expressed in neuronal tissue and is involved in neuronal cell retention, signaling and survival (Current Opinion in Neurobiology,2001,11: 272-.

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-. In addition, recent literature has shown that BDNF content and TrkB signaling increase in dorsal root ganglia following inflammation (Brain Research,1997,749:358), and several studies have shown that antibodies that reduce signaling via the BDNF/TrkB pathway can inhibit neuronal hypersensitivity and related 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-, Cancer (Clin. Cancer Res.1998, 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 Larotretinoib (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, and after four months, the lung tumor mass basically disappears and no obvious adverse reaction is found during the clinical treatment (Cancer Discov,2015,5: 1049-. LOXO-101 has significant therapeutic effects on 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 carcinoma, cholangiocarcinoma, kidney cancer, salivary gland cancer, appendiceal cancer, pancreatic cancer, peripheral nerve sheath tumor, spindle cell tumor, etc., and the drug has few side effects (New England Journal of Medicine,2018,378(8):731 and 739.). 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 rearrangement, and the results showed that 72% of patients responded to Entrectinib (J Target Ther 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 is 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 binding 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 (Expert opin. 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. However, the general structure of the compound contains a nitrogenous five-membered ring, so that the synthesis is relatively complex, the reaction conditions are harsh, and the cost is high. In particular, the preparation of compounds of this general structural formula generally requires the initial synthesis of the key intermediate (R) -2- (2, 5-difluorophenyl) pyrrolidine. However, the following synthetic routes are mainly reported for the synthesis of (R) -2- (2, 5-difluorophenyl) pyrrolidine:

1)

this process requires too low a temperature (-78 deg.C) and also requires sec-butyllithium, which is too air sensitive, and expensive palladium acetate and sparteine base.

2)

The route is constructed by 5 steps to form an R-configuration pyrrole ring. In the preparation process, expensive Grignard reagent magnesium isopropyl chloride needs to be used, dangerous lithium triethylborohydride needs to be used, and low-temperature reaction at-78 ℃ needs to be carried out.

3)

This route uses air sensitive n-butyllithium ethyl and low temperature conditions of-78 ℃ and in addition the overall yield is relatively low.

Therefore, it is a current problem to provide a novel substituted pyrazolo [1,5-a ] pyrimidine compound as Trk kinase inhibitor, which is relatively convenient to synthesize, low in cost, and suitable for industrialization, and has good pharmaceutical properties and metabolic stability.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a compound with a general formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition and application.

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

wherein:

R¹selected from H or (1-3C) alkyl;

R²selected from NR^aR^b(1-4C) alkyl or (1-4C) hydroxyalkyl;

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

R^bselected from H, (1-4C) alkyl, (1-4C) hydroxyalkyl, NHMe or N (Me)₂Substituted (1-4C) alkyl, hetero Ar or phenyl, wherein the phenyl is optionally independently selected from hydroxy, halo, CF₃、CHF₂、CH₂F. CN is substituted by one or more substituents;

or NR^aR^bForming a 4-to 6-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;

heteroar is 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;

R³is (i) phenyl optionally independently selected from halo, (1-4C) alkoxy, CF₃、CHF₂、CHF₂Substituted with one or more substituents of (a); or (ii) a 5 to 6 membered heteroaryl ring having a heteroatom selected from N and S, wherein the heteroaryl ring is optionally substituted with one or more halogen atoms;

R⁴selected from hydrogen, (1-3C) alkyl, deuterated (1-3C) alkyl;

x is selected from

R⁵Selected from OH, CN, (CH)₂)_nOH, wherein n independently represents 1,2 or 3;

y is selected from NH.

Preferably, R¹Selected from H, CH₃、CD₃. Preferably, R²Is selected from NR^aR^bAnd (1-4C) alkyl. Further preferably, R²Selected from NR^aR^bWherein said NR is^aR^bIn R^aSelected from H or (1-6C) alkyl, R^bSelected from (1-4C) alkyl, NHMe or N (Me)₂Substituted (1-4C) alkyl or phenyl, wherein said phenyl is optionally independently selected from hydroxy, halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a 4-to 6-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. Even more preferably, the NR is^aR^bIn R^aSelected from H, methyl, ethyl, n-propyl or isopropyl, R^bSelected from methyl, ethyl, n-propyl, isopropyl or phenyl, wherein the phenyl group is optionally substituted by hydroxy, halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a 4 to 6 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.

Preferably, R³Selected from phenyl, optionally independently selected from halogen, CF₃、CHF₂Is substituted with one or more substituents of (a). Even more preferably, R³Selected from phenyl substituted by one or two fluorine or chlorine atoms or by one fluorine atom and CF₃A substituted phenyl group.

Preferably, R⁴Selected from hydrogen, CH₃Or CD₃。

In some embodiments of the invention, the compounds of the invention are compounds having the general formula (I) described above, or a pharmaceutically acceptable salt thereof, wherein,

R¹is selected from H or CH₃；

R²Selected from NR^aR^b(ii) a Wherein R is^aSelected from H, methyl, ethyl, n-propyl or isopropyl; r is^bSelected from methyl, ethyl, n-propyl, isopropyl or phenyl, said phenyl being optionally independently selected from hydroxy, halogen, CF₃、CHF₂、CH₂One or two substituents of F; or NR^aR^bForming a five-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, preferably NR^aR^bForming a five-membered heterocyclic ring having a ring nitrogen atom, the heterocyclic ring being substituted with one hydroxyl group;

R³is phenyl, optionally independently selected from halogen, CF₃、CHF₂、CH₂One or two substituents of F, further preferably R³Is phenyl substituted by two halogen atoms;

R⁴is selected from H or CH₃；

X is selected from

Preferably X is selected from

R⁵Selected from OH and CN;

y is selected from NH.

Further, in some preferred embodiments, the compounds of the present invention are compounds having the general formula (I) above or a pharmaceutically acceptable salt thereof, wherein R is¹Is selected from H or CH₃；R²Selected from pyrrolidinyl optionally substituted with hydroxy, preferably substituted with one hydroxy;

R³is phenyl optionally substituted by two halogen atoms; r is⁴Is selected from H or CH₃；

X is selected from

R⁵Is selected fromOH and CN; y is selected from NH.

Further, the pharmaceutically acceptable salt is an inorganic salt or an 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 present invention provides a compound of the structure:

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 invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof for 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 myeloleukemia, 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 a simpler structure and a more convenient synthesis method under the condition of ensuring good Trk inhibitory activity and metabolic stability. The reaction process of the invention avoids the low temperature of minus 78 ℃, avoids the use of reagents which are sensitive to air and expensive, has low synthesis cost, has better overall yield than the construction of commercial products such as (R) -2- (2, 5-difluorophenyl) pyrrolidine in the larotretinib, and is more suitable for industrialization.

Detailed Description

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

In the present invention, "(1-3C) alkyl" means a saturated branched or branched monovalent hydrocarbon group of 1 to 3 carbon atoms, respectively. Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl. (1-6C) alkyl can be defined by analogy, specific examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, and the like.

In the present invention, "deuterated (1-3C) alkyl" refers to a saturated branched or branched monovalent hydrocarbon group of 1 to 3 carbon atoms having hydrogen atoms partially or fully substituted with deuterium.

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, "alkoxy" refers to an alkyl group attached to the rest of the compound through an oxygen atom. 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.

In the present invention, "halogen" means fluorine, chlorine, bromine and iodine.

In the present invention "heterocycle" means an aliphatic saturated or partially saturated ring having no aromatic character, at least one ring member of which is a heteroatom selected from N, O and S.

By "pharmaceutically acceptable salts" in the context of the present invention is meant those salts which retain the biological effectiveness and properties of the parent compound. The term "salt" refers to any salt of a compound according to the invention prepared from an inorganic or organic acid or base and an internally formed salt. Typically, such salts have a physiologically acceptable anion or cation.

In the present invention "aromatic ring" refers to a moiety wherein the constituent carbon atoms form an unsaturated ring system, all atoms in the ring system being sp2 hybridized, the total number of pi-electrons being equal to 4n +2, where n is an integer. In the present invention, "heteroaromatic ring" or "heteroar" refers to an aromatic ring as defined above wherein 1 to 4 carbon atoms are independently substituted with a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur. Non-limiting examples of heteroaryl ring groups are, for example, pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl and the like.

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

"optional" or "optionally" in the present context means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F) Polysubstituted (e.g. CHFCH)₂F、CH₂CHF₂Etc.) or completely substituted (CF₂CF₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern that is sterically impossible and/or cannot be synthesized.

Reference throughout this specification to "an embodiment" or "in another embodiment" or "in certain embodiments" or "in portions of this application" means that a particular reference element, structure or feature described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated meaning, i.e. the word "comprising" and "comprises", in an open-ended, inclusive sense, i.e. to mean "including but not limited to".

It should be understood that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

< 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 pharmaceutically acceptable salt thereof, wherein the structural formula of the compound is shown as a general formula (I):

wherein:

R¹selected from H or (1-3C) alkyl;

R²is selected from NR^aR^b(1-4C) alkyl or (1-4C) hydroxyalkyl;

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

R^bis H, (1-4C) alkyl, (1-4C) hydroxyalkyl, NHMe or N (Me)₂Substituted (1-4C) alkyl, hetero Ar or phenyl, wherein the phenyl is optionally independently selected from hydroxy, halo, CF₃、CHF₂、CH₂F. CN is substituted by one or more substituents;

or NR^aR^bForming a 4-to 6-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;

heteroar is 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;

R³is (i) phenyl optionally independently selected from halogen, (1-4C) alkoxy, CF₃、CH₂F、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;

R⁴selected from hydrogen, (1-3C) alkyl or deuterated (1-3C) alkyl;

x is selected from

R⁵Selected from OH, CN, (CH)₂)_nOH, wherein n independently represents 1,2 or 3;

y is selected from NH.

In certain embodiments of the invention, R¹Selected from H or (1-3C) alkyl. Further, R¹Selected from H, methyl or ethyl. Specific examples, R¹Is H or methyl, and the inventors have found that such groups are relatively more favorable for the formation of their active conformation due to the small steric hindrance.

Preferably, R²Selected from NR^aR^bAnd (1-4C) alkyl. In certain embodiments of the invention, R²Selected from NR^aR^bFurther said NR^aR^bIn R^aSelected from H or (1-6C) alkyl, R^bSelected from (1-4C) alkyl, NHMe or N (Me)₂Substituted (1-4C) alkyl or phenyl, wherein said phenyl is optionally independently selected from hydroxy, halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a 4-to 6-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. Even more preferably, the NR is^aR^bIn R^aSelected from H, methyl, ethyl, n-propyl or isopropyl, R^bSelected from methyl, ethyl, n-propyl, isopropyl or phenyl, wherein phenyl is optionally substituted by hydroxy, halogen, CF₃Is substituted with one or more substituents of (a), or the NR is^aR^bForming a five-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, and in certain embodiments, the heterocycle is optionally substituted with hydroxy.

Further preferably, R²Selected from NR^aR^b，NR^aR^bIncluding pyrrolidinyl optionally substituted with hydroxy, preferably substituted with one hydroxy.

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

in some preferred embodiments of the invention, the compound is selected from NR^aR^bThe group represented refers to the following structure:

in certain embodiments of the invention, R³Is (i) phenyl, optionally independently selected from halogen, CF₃、CHF₂Is substituted with one or more substituents of (a). Further, the air conditioner is provided with a fan,R³selected from phenyl substituted by one or two fluorine atoms or by one fluorine atom and CF₃A substituted phenyl group. Specific example, R³Is that

In certain embodiments of the invention, R⁴Selected from hydrogen, (1-3C) alkyl, deuterated (1-3C) alkyl, and further, R⁴Selected from hydrogen, methyl or deuterated methyl, further, R⁴Selected from hydrogen or methyl.

In certain embodiments of the invention, X is

Wherein R is⁵Selected from OH or CN.

In certain embodiments of the invention, Y is selected from NH to better enhance the metabolic stability and activity of the compound.

In some preferred embodiments of the invention, the compounds of the invention are compounds having the general formula (I) above or a pharmaceutically acceptable salt thereof, wherein R¹Is selected from H or CH₃；R²Selected from pyrrolidinyl optionally substituted with hydroxy, preferably substituted with one hydroxy; or R²Selected from dialkylamino groups, the dialkyl groups are each (1-4C) alkyl groups; r³Is phenyl optionally substituted by two halogen atoms; r⁴Is selected from H or CH₃(ii) a X is selected from

R⁵Selected from OH and CN; y is selected from NH.

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

the compound provided by the invention has a simpler skeleton structure, part of compounds have no chiral center, the synthesis convenience is obviously improved relatively, and the synthesis cost is relatively lower.

In order to obtain better Trk inhibitory activity, in certain embodiments of the present 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 the group consisting of acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, sulfonate, benzenesulfonate, salicylate. In certain embodiments of the invention, pharmaceutically acceptable salts described herein include hydrochloride and sulfate salts.

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:

preparation of intermediate (formula (II)):

in some embodiments of the invention, the intermediates include the following three: compound (a), compound (B) and compound (C), each of which is prepared by the following scheme:

production scheme of compound (a) (reaction formula 1):

reaction scheme 1

As shown in a reaction formula 1,2, 5-difluorobenzaldehyde and methylamine are subjected to reduction ammoniation to obtain an intermediate 1; nucleophilic substitution is carried out on the intermediate 1 and 5-chloropyrazolo [1,5-a ] pyrimidine at high temperature to obtain an intermediate 2; nitrifying the intermediate 2 with nitric acid to obtain an intermediate 3; and reducing the intermediate 3 with zinc powder to obtain the compound (A).

Production scheme of compound (B) (reaction formula 2):

reaction formula 2

As shown in a reaction formula 2, dehydrating raw materials of 2, 5-difluoroacetophenone and R- (+) tert-butyl sulfenamide to obtain an intermediate 4; reducing the intermediate 4 by sodium borohydride at low temperature (-50 ℃) to obtain an intermediate 5; hydrolyzing the intermediate 5 under an acidic condition to obtain an intermediate 6; nucleophilic substitution of the intermediate 6 and 5-chloropyrazolo [1,5-a ] pyrimidine at high temperature to obtain an intermediate 7; nitrifying the intermediate 7 with nitric acid to obtain an intermediate 8; and reducing the intermediate 8 with zinc powder to obtain a compound (B).

Production scheme of compound (C) (reaction formula 3):

reaction formula 3

As shown in a reaction formula 3, the intermediate 6 and formaldehyde are reduced and aminated to obtain an intermediate 9; nucleophilic substitution of the intermediate 9 and 5-chloropyrazolo [1,5-a ] pyrimidine occurs at high temperature to obtain an intermediate 10; nitrifying the intermediate 10 with nitric acid to obtain an intermediate 11; and reducing the intermediate 11 with zinc powder to obtain a compound (C).

The preparation process of the intermediate does not need too low temperature (-78 ℃) and avoids using air sensitive and expensive reagents, and compared with n-butyllithium, sec-butyllithium or Grignard reagents, the synthesis conditions of the invention are relatively safer. For the compound A, the synthesis convenience is greatly improved compared with the prior art because the compound A has no chiral center. For the compound B and the compound C, certain optimization is realized in the aspects of synthesis cost and synthesis reaction conditions.

Preparation of the compound:

for compounds in which X is

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

wherein R is⁶Independently selected from halogen, hydroxyl, 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

Of formula (I) with HNR^aR^bIn the presence of a coupling reagent; or

(a) For R in the formula²Is NR^aR^bAnd R is^aA compound of formula (I) which is H, reacting a corresponding compound of formula (II) with a compound having the formula O ═ C ═ NR^bReacting the compound (1); or

(b) For R in the formula²Is NR^aR^bAnd NR^aR^bA compound of formula (I) which forms a 4 to 6 membered heterocyclic ring having a ring nitrogen atom is reacted with a corresponding compound of formula (II) in the presence of a tertiary amine base in the presence of a coupling reagent with a substituted 4 to 6 membered heterocyclic ring of a ring nitrogen atom.

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

For compounds in which X is

Of formula (I) with HNR^aR^bWith amino-substituted R after formation of urea or thiourea⁵Reaction to obtain X of

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.

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), triethylamine 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, alginates, gelatin, polyvinyl pyrrolidone, sucrose, gum arabic, and the like; (3) humectants, such as glycerin 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, etc.; (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 a composition 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 commonly 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 compound of the present invention or a pharmaceutically acceptable salt 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 a compound of a general formula (I) or pharmaceutically acceptable salts 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 formula (I) or a 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 compounds of the present invention or pharmaceutically acceptable salts thereof may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents in combination with other anti-tumor 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 DNA chemical structures, 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 paclitaxel, 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 which act 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. If not otherwise stated, the reagents were purchased from commercial suppliers and used without further purification. The structure of the final product, intermediates and starting materials is confirmed by standard analytical methods, such as spectroscopic characterisation, MS, NMR. Abbreviations used are those conventional in the art.

Intermediate A

N⁵- (2, 5-difluorobenzyl) -N⁵-methylpyrazolo [1,5-a]Process for preparing pyrimidine-3, 5-diamine (A)

Step a: preparation of 1- (2, 5-difluorobenzyl) -N-methylmethanamine

Methylamine hydrochloride (632mg,9.1mmol), K₂CO₃(646mg,4.69mmol) was suspended in 20mL of methanol, stirred at room temperature for 30 minutes, 2, 5-difluorobenzaldehyde (1g,7.0mmol) was added dropwise, the reaction was stirred overnight, then the reaction was cooled to 0 deg.C and NaBH was added in portions₄(453mg, 12mmol), after the addition was completed, stirring for 1 hour, moving to room temperature, stirring for reaction for 2 hours, monitoring by TLC that the reaction of the raw materials was completed, suction filtration, washing of the filtrate with water, drying of the organic phase over anhydrous sodium sulfate, and concentration to obtain a yellow oily liquid (860mg, yield 77%). MS (ESI) M/z 158.1(M +1)⁺。

Step b: preparation of N- (2, 5-difluorobenzyl) -N-methylpyrazolo [1,5-a ] pyrimidin-5-amine

1- (2, 5-difluorobenzyl) -N-methylaminoamine (816mg,5.2mmol,1eq), 5-chloropyrazole [1,5-a ]]Pyrimidine (874mg,5.7mmol,1.1eq) is suspended in 0.8mL n-butanol, 1.7mL DIPEA is added, the tube is sealed, the reaction is stirred at 160 ℃ for 4 hours, TLC monitors that the raw material reaction is finished, 5mL water is added into the reaction system, ethyl acetate is extracted, organic phase anhydrous sodium sulfate is dried, brown black oily liquid is obtained by concentration, and white powdery solid (722mg, 51%) is obtained by column chromatography purification. MS (ESI) M/z 275.1(M +1)⁺。

Step c: preparation of N- ((2, 5-difluorobenzyl) -N-methyl-3-nitropyrazolo [1,5-a ] pyrimidin-5-amine

Reacting N- (2, 5-difluorobenzyl) -N-methylpyrazole [1,5-a ]]Pyrimidin-5-amine (712mg,2.6mmol), dissolved in 7.1mL TFA, 0.71mL nitric acid, stirred at room temperature for 20 minutes, TLC monitored the completion of the reaction of the starting material, added dropwise to ice water to precipitate a white solid, filtered with suction, and dried to obtain an off-white solid (590mg, 71% yield). Step d: n is a radical of⁵- (2, 5-difluorobenzyl) -N⁵-methylpyrazolo [1,5-a ]]Preparation of pyrimidine-3, 5-diamine (A)

Reacting N- ((2, 5-difluorobenzyl) -N-methyl-3-nitropyrazole [1,5-a ]]Dissolving pyrimidine-5-amine (200mg,0.62mmol) in a mixed solvent of 2mL methanol and 2mL dichloromethane, adding zinc powder (394mg,6mmol), adding saturated ammonium chloride solution 2mL while stirring, stirring vigorously at room temperature for 3 hours, monitoring by TLC that the reaction of raw materials is finished, adding 5mL dichloromethane, filtering, washing filtrate with water, drying organic phase anhydrous sodium sulfate, and concentrating to obtain a red solid N⁵- (2, 5-difluorobenzyl) -N⁵-methylpyrazolo [1,5-a]Pyrimidine-3, 5-diamine (150mg, yield 82%). MS (ESI) M/z 290.1(M +1)⁺。

Intermediate B

(R)-N⁵- (1- (2, 5-difluorophenyl) ethyl) pyrazolo [1,5-a]Process for preparing pyrimidine-3, 5-diamine (B)

Step e: preparation of (R, E) -N- (1- (2, 5-difluorophenyl) ethylene) -2-methylpropyl-2-sulfinamide

2, 5-difluoroacetophenone (2.93g,18.77mmol,1eq), R- (+) tert-butylsulfinamide (2.71g,22.5mmol, 1.2eq), Ti (OEt)₄(5.14g,22.52mmol,1.2eq) was dissolved in 30mL of tetrahydrofuran, heated under reflux for 5 hours, monitored by TLC for completion of the reaction, 30mL of saturated ammonium chloride solution was added, 30mL of ethyl acetate was stirred for 30 minutes, extracted with ethyl acetate after suction filtration, washed with saturated brineWashing, drying over anhydrous sodium sulfate and concentrating to obtain yellow oily liquid (4.7g, 96% yield). MS (ESI) M/z 260.1(M +1)⁺。

Step f: preparation of (R) -N- ((R) -1- (2, 5-difluorophenyl) ethyl) -2-methylpropyl-2-sulfinamide

(R, E) -N- (1- (2, 5-difluorophenyl) ethylene) -2-methylpropyl-2-sulfinamide (4.73g,18.12mmol,1eq), 30mL of tetrahydrofuran and 0.6mL of water were added to a reaction flask, the temperature was reduced to-50 deg.C, and NaBH was added portionwise₄(617mg, 16.31mmol,0.9eq), stirring for 30 min after addition, moving to room temperature and stirring for 3 h, TLC monitoring the completion of the reaction of the raw materials, adding 20mL of ice water for quenching, extracting 3 times with 20mL of ethyl acetate, drying with anhydrous sodium sulfate and concentrating to obtain yellow oily liquid (4.0g, yield 84%). MS (ESI) M/z 262.1(M +1)⁺。

Step g: preparation of (R) -1- (2, 5-difluorophenyl) ethylamine

(R) -N- ((R) -1- (2, 5-difluorophenyl) ethyl) -2-methylpropyl-2-sulfinamide (4.0g,15.3mmol, 1eq), 150mL of a 4mol/L dioxane solution of hydrogen chloride was stirred overnight, TLC was used to monitor the completion of the reaction, 1, 4-dioxane was removed by rotary evaporation, 50mL of water was added, impurities were removed by extraction with ethyl acetate, the aqueous layer was made alkaline with a 2mol/L NaOH solution, extracted 3 times with 30mL of ethyl acetate, and dried over anhydrous sodium sulfate and concentrated to give a colorless transparent liquid (1.9g, yield 79%). MS (ESI) M/z 158.1(M +1)⁺。

Step h: preparation of (R) -N- (1- (2, 5-difluorophenyl) ethyl) pyrazolo [1,5-a ] pyrimidin-5-amine

The compound (R) -N- (1- (2, 5-difluorophenyl) ethyl) pyrazolo [1, 5-a) is prepared as a yellow oily liquid by the method in the step b]Pyrimidin-5-amine (301 mg). MS (ESI) M/z 275.1(M +1)⁺。

Step i: preparation of (R) -N- (1- (2, 5-difluorophenyl) ethyl) 3-nitropyrazolo [1,5-a ] pyrimidin-5-amine

Preparation of (R) -N- (1- (2, 5-difluorophenyl) ethyl) 3-nitropyrazolo [1,5-a ] pyrimidin-5-amine (177mg) as a pale yellow solid was prepared according to the procedure in step c.

Step j: (R) -N⁵- (1- (2, 5-phenyl) ethyl) pyrazolo [1,5-a]Preparation of pyrimidine-3, 5-diamine (B)

Preparing light red solid (R) -N according to the method of the step d⁵- (1- (2, 5-phenyl) ethyl) pyrazolo [1,5-a]Pyrimidine-3, 5-diamine (B) (163 mg). MS (ESI) M/z 290.1(M +1)⁺。

Intermediate C

(R)-N⁵- (1- (2, 5-difluorophenyl) ethyl) -N⁵-methylpyrazolo [1,5-a]Process for preparing pyrimidine-3, 5-diamine (C)

Step k: preparation of (R) -1- (2, 5-difluorophenyl) -N-methylethylamine

(R) -1- (2, 5-difluorophenyl) ethylamine (500mg,3.18mmol,1eq) was dissolved in 10mL of methanol, formaldehyde (251mg,3.18mmol,1eq, 38% w/w) was added dropwise, stirring was carried out at room temperature for 20 minutes, and NaBH was added in portions₄(482mg,12.74mmol, 4eq), 20 min, TLC to monitor completion, quenching with 5mL water, extraction with 10mL ethyl acetate 3 times, washing with saturated brine, drying over anhydrous sodium sulfate and concentration to give a yellow oily liquid (475mg, 87% yield). MS (ESI) M/z 172.1(M +1)⁺。

Step l: (R) -N- (1- (2, 5-difluorophenyl) ethyl) -N-methylpyrazolo [1,5-a]Preparation of pyrimidin-5-amine the compound (R) -N- (1- (2, 5-difluorophenyl) ethyl) pyrazolo [1, 5-a) was prepared as a yellow oily liquid according to the procedure in step b]Pyrimidin-5-amine (155 mg). MS (ESI) M/z 289.1(M +1)⁺。

Step m: preparation of (R) -N- (1-2, 5-difluorophenyl) ethyl) -N-methyl-3-nitropyrazolo [1,5-a ] pyrimidin-5-amine

Prepared according to the procedure of step c to give (R) -N- (1-2, 5-difluorophenyl) ethyl) -N-methyl-3-nitropyrazolo [1,5-a ] pyrimidin-5-amine (136mg) as a pale yellow solid.

Step n: (R) -N⁵- (1- (2, 5-difluorophenyl) ethyl) -N⁵-methylpyrazoleAnd [1,5-a ]]Preparation of pyrimidine-3, 5-diamine (C)

Preparing light red solid (R) -N according to the method of the step d⁵- (1- (2, 5-difluorophenyl) ethyl) -N⁵-methylpyrazolo [1,5-a]Pyrimidine-3, 5-diamine (113 mg). MS (ESI) M/z 304.1(M +1)⁺。

Example 1

(S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione

Structural formula (xvi):

dissolving the intermediate A (60mg,0.21mmol,1eq), (S) -3-ethoxy-4- (3-hydroxypyrrolidin-1-yl) but-3-ene-1, 2-dione (purchased from Yancheng Zhengchi Biotechnology Co., Ltd.) (53mg,0.25mmol,1.2eq) in 5mL of ethanol, refluxing at 80 ℃ for 12 hours, cooling to room temperature, concentrating the reaction solution to obtain a solid, and purifying the crude product by column chromatography to obtain a tan solid (S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] (a)]Pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione (30mg, yield 32%).¹H-NMR(400MHz,DMSO-d₆)δ：9.07(s,1H),8.64 (d,J＝8.0Hz,1H),7.87(s,1H),7.30–7.00(m,3H),6.66(d,J＝8.0Hz,1H),5.06(s,1H), 4.85(s,2H),4.26(s,1H),4.02-3.33(m,4H),3.17(s,3H),2.04–1.73(m,2H).MS(ESI)m/z 455.2[M+1]⁺。

Example 1A

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

Structural formula (xvi):

to a solution of (S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione (0.46g, 1.0mmol, 1eq) in methanol (15mL) was added sulfuric acid (5mL, 1mmol,1eq) in methanol at room temperature. The resulting solution was stirred for 2 hours and then concentrated to give (S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione sulfate as a yellow solid (0.41g, yield 72%).

Example 1B

(S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-en-1, 2-dione hydrochloride

Structural formula (xvi):

to a solution of (S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione (0.46g, 1.0mmol, 1eq) in methanol (15mL) at room temperature was added a solution of hydrogen chloride in methanol (5mL, 1 mmol). The resulting solution was stirred for 1 hour and then concentrated to give (S) -3- ((5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) amino) -4- (3-hydroxypyrrolidin-1-yl) cyclobut-3-ene-1, 2-dione hydrochloride as a yellow solid (0.42g, yield 86%).

Examples 2-3 (see table 1) were synthesized following a procedure similar to the basic procedure of example 1 to give the desired product.

TABLE 1 structural and Mass Spectrometry data for examples 2-3

Example 4

(S) -N- (5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide structural formula:

dissolving intermediate A (64mg,0.22mmol,1eq) in 2mL dichloromethane, slowly adding N, N' -Carbonyldiimidazole (CDI) (71mg,0.44mmol,2eq) at room temperature, stirring at room temperature for 0.5 h, (S) -3-hydroxypyrrolidine hydrochloride (56mg,0.44mmol,2eq), triethylamine (56mg,0.55mmol,2.5eq) are suspended in 1mL dichloromethane, and added dropwise to the reaction solution, stirring at room temperature for 1 h, monitoring by TLC, quenching with 2mL water, extracting with DCM/MeOH (v/v: 10/1), drying the organic phase with anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain (S) -N- (5- ((2, 5-difluorophenyl) (methyl) amino) pyrazolo [1,5-a ] as a red solid]Pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide (25mg, yield 28%).¹H-NMR(400MHz,CDCl₃)δ：8.03(s,2H),7.36(s,2H),7.09(s,2H),5.35(t,J＝6.0Hz,2H), 4.61(s,1H),3.90(q,J＝8.0Hz,2H),3.82–3.69(m,4H),3.62(d,J＝12Hz,2H),2.01(d,J＝ 6.4Hz,2H).MS(ESI)m/z 403.2[M+1]⁺。

Example 4A

(S) -N- (5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide sulfate

Structural formula (la):

example 4A was synthesized as example 4 following a procedure similar to the basic procedure of example 1A.

Example 4B

(S) -N- (5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide hydrochloride

Structural formula (xvi):

example 4B was synthesized as example 4 following a procedure similar to the basic procedure for example 1B.

Example 5

(S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide

Structural formula (I)

Intermediate B (40mg,0.14mmol,1eq) was dissolved in 1.5mL of dichloromethane, n, N' -Carbonyldiimidazole (CDI) (45mg,0.28mmol,2eq) was added portionwise, stirred for 2 hours, and (3) monitoring by TLC, dropwise adding (S) -3-hydroxypyrrolidine hydrochloride (34.2mg,0.28mmol,2eq) and a dichloromethane (1mL) solution of triethylamine (35mg,0.35mmol,2.5eq) into the system, stirring at room temperature for reacting for 30 minutes, monitoring by TLC that the raw materials react completely, adding 5mL of ice water for quenching, extracting by 5mL of dichloromethane for 3 times, drying and concentrating by anhydrous sodium sulfate to obtain a yellow oily liquid, and purifying by column chromatography to obtain a light yellow solid (S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) amino) pyrazolo [1, 5-a).]Pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide (20mg, yield 36%).¹H-NMR(400MHz,CDCl₃)δ：8.25(s,1H),8.10(d,J＝7.5Hz, 1H),7.11–6.96(m,2H),6.93–6.86(m,1H),6.14(d,J＝7.6Hz,1H),5.95(d,J＝7.5Hz, 1H),5.27(d,J＝14.6Hz,2H),4.58(s,1H),3.71–3.51(m,4H),2.29–1.94(m,2H),1.57(d, J＝6.6Hz,3H).MS(ESI)m/z 403.2[M+1]⁺。

Example 5A

(S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide sulfate

Structural formula (I)

Example 5A was synthesized as example 5 following a procedure similar to the basic procedure of example 1A.

Example 5B

(S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide hydrochloride

Structural formula (I)

Example 5B was synthesized as example 5 following a procedure similar to the basic procedure for example 1B.

Example 6

(S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide

Structural formula (I)

Intermediate C (50mg,0.16mmol,1eq) was dissolved in 2mL of dichloromethane, N' -Carbonyldiimidazole (CDI) (53mg,0.33mmol,2eq) was added in portions, stirred at room temperature for 2 hours, monitored by TLC, then (S) -3-hydroxypyrrolidine hydrochloride (59.3mg,0.48mmol,3eq) and a dichloromethane (0.5mL) solution of triethylamine (41mg,0.41mmol,2.5eq) are added, stirring is carried out for 30 minutes at room temperature, TLC is used for monitoring the completion of the raw material reaction, 5mL ice water is added for quenching, 5mL dichloromethane is used for extraction for 3 times, anhydrous sodium sulfate is dried and concentrated to obtain 40mg of yellow oily liquid, and column chromatography purification is carried out to obtain the reddish-brown bubble solid (S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) methyl) amino) pyrazolo [1, 5-a).]Pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide (20mg, yield 29%).¹H-NMR(400MHz,CDCl₃)δ：8.22(d,J＝3.4Hz,1H), 8.18(dd,J＝7.9,1.4Hz,1H),7.03–6.85(m,3H),6.24(d,J＝7.9Hz,1H),5.96(d,J＝7.4 Hz,1H),4.51(s,1H),3.65–3.54(m,5H),2.96(d,J＝4.6Hz,3H),1.81(s,2H),1.56(dd,J＝ 7.0,3.9Hz,3H).MS(ESI)m/z 417.2[M+1]⁺。

Example 6A

(S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide sulfate

Structural formula (I)

Example 6A was synthesized as example 6 following a procedure similar to the basic procedure of example 1A.

Example 6B

(S) -N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -3-hydroxypyrrolidine-1-carboxamide hydrochloride

Structural formula (I)

Example 6B was synthesized as example 6 following a procedure similar to the basic procedure for example 1B.

Examples 7-17 (see table 2) were synthesized in a procedure similar to the basic procedure of example 4, using the corresponding intermediates or starting materials, to give the desired products.

Table 2 structural and mass spectral data for examples 7-17

Intermediate D

Preparation method of (R, E) -methyl N' - (5- ((1- (2, 5-difluorophenyl) ethyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidine-3-yl) -N-isocyano isothiourea (D)

Step o: preparation of (R, E) -methyl N' - (5- ((1- (2, 5-difluorophenyl) ethyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-3-yl) -N-isocyanoisothiourea (D)

Intermediate C (100mg,0.30mmol,1eq) was dissolved in 1mL of DMF, and a solution of cyanoiminodithiocarbonate (46.4mg,0.32mmol,1eq) in 1mL of DMF was added dropwise, followed by addition of cesium carbonate (155mg,0.448mmol,1.5eq) and reaction at 100 ℃ overnight. TLC monitoring indicated the reaction was complete, 10mL water was added, extracted 3 times with 10mL ethyl acetate, washed 3 times with saturated brine, dried and concentrated to give the crude product, which was purified on a column to give a yellow solid (27mg, 21% yield). MS (ESI) M/z 402.1[ M +1 ]]⁺。

Intermediate E, F

According to the preparation method of the step o, the following components are prepared:

(R, E) -methyl N' - (5- ((1- (2, 5-difluorophenyl) ethyl) amino) pyrazolo [1,5-a]Pyrimidin-3-yl) -N-isocyanoisothourea (E)31mg, MS (ESI) M/z 388.1[ M +1 ]]⁺。

(E) -methyl N' - (5- ((2, 5-difluorobenzyl) (methyl) amino) pyrazolo [1,5-a]Pyrimidin-3-yl) -N-isocyanoisothiourea (F)53mg, MS (ESI) M/z 388.1[ M + 1%]⁺。

Example 18

(S, E) -N' -cyano-N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-2-yl) -3-hydroxypyrrolidine-1-carboxamidine

Structural formula (I)

Intermediate D (28mg,0.07mmol,1eq) was dissolved in 1mL ethanol and (S) -3-hydroxypyrrole hydrochloride (41mg,0.35mmol,5eq), pyridine (20mg,0.35mmol,5eq), protected with nitrogen, was added and refluxed at 80 ℃ overnight. TLC monitoring of the completion of the reaction of the starting materials, decreaseThe ethanol was evaporated under pressure, extracted 3 times with 5mL of water and 5mL of dichloromethane, dried, concentrated, and purified by column to give a yellow solid (12mg, yield 41%).¹H-NMR(400MHz,CDCl₃)δ:8.21(d,J＝8.0Hz, 1H),8.05(s,1H),7.03-6.91(m,3H),6.31(d,J＝8.0Hz,1H),6.03(s,1H),4.46(s,1H), 3.73-3.50(m,4H),3.11-2.48(m,4H),2.22-1.90(m,5H).MS(ESI)m/z 441.2[M+1]⁺。

Examples 19-20 (see table 3) were synthesized using intermediates E or F, respectively, in a procedure similar to the basic procedure of example 18, to afford the desired products.

Table 3 structural and mass spectral data for examples 19-20

Example 21

(S, E) -N' -hydroxy-N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) (methyl) amino) pyrazolo [1,5-a ] pyrimidin-2-yl) -3-hydroxypyrrolidine-1-carboxamidine

Structural formula (I)

Intermediate D (176mg,0.44mmol,1eq) was dissolved in 2mL ethanol, hydroxylamine hydrochloride (152mg,2.2mmol, 5eq) and pyridine (172mg,2.2mmol,5eq) were added and refluxed at 80 ℃ overnight. TLC (thin layer chromatography) monitoring the completion of the raw material reaction, evaporating ethanol to dryness, adding 5mL of water, extracting with 5mL of ethyl acetate for 3 times, drying, concentrating to obtain a dark brown oily liquid, and purifying with a column to obtain a brown yellow solid (S, E) -N' -hydroxy-N- (5- (((R) -1- (2, 5-difluorophenyl) ethyl) (methyl) amino) pyrazolo [1,5-a ] as a raw material]Pyrimidin-2-yl) -3-hydroxypyrrolidine-1-carboxamidine (80mg, 42% yield).¹H NMR(400MHz,CDCl₃)δ:8.22(d,J＝8.0Hz, 1H),8.06(s,1H),7.03-6.90(m,3H),6.32(d,J＝8.0Hz,1H),6.04(s,1H),4.46(s,1H), 3.74-3.49(m,4H),3.12-2.48(m,4H),2.23-1.90(m,5H).MS(ESI)m/z 432.2[M+1]⁺。

Examples 22-23 (see table 4) were synthesized using intermediates E or F, respectively, in a procedure analogous to the basic procedure of example 21, to give the desired product.

TABLE 4 structural and Mass Spectrometry data for examples 22-23

Example 24

TrkA, TrkB and TrkC ELISA assay

TrkA, TrkB, and TrkC kinase activity was evaluated using an enzyme-linked immunosorbent assay (ELISA) in the presence of the inhibitor. Taking the TrkA kinase activity assay as an example: 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 was composed of 15mM MOPS pH7.5, 0.005% (v/v) Triton X-100 and 5mM MgCl₂And (4) forming. 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. TrkB, TrkC kinase activity was determined using a procedure similar to the basic procedure for TrkA kinase activity assay.

In the above activity assay, the compounds of the invention have an average IC of less than 1000nM₅₀. Some compounds have an average IC of less than 100nM₅₀The compound of the invention has simpler structure than that of the commercially available compound Larotrectinib (LOXO-101), and the average IC of the compounds of the invention can reach or be lower than that of the commercially available compound Larotrectinib (LOXO-101)₅₀. Table 5 provides specific ICs tested in this assay for compounds of the invention and the commercially available compound Larotrectinib (LOXO-101)₅₀The value is obtained.

TABLE 5 specific IC's of the Compounds of the invention and of the commercially available Compounds₅₀Value of

Example 25

The stability of the compounds was evaluated using human liver microsomes.

Measurement System: the metabolic stability of the compound of the present invention was tested using 1mM NADPH for liver microparticles mixed in 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 in 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 sampled at 0,2, 4, 8, 16, 24, 36, 48 hours, positive control (5 μ M testosterone) was incubated in parallel and sampled 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 satisfied an acceptable internal standard.

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 6 for gradient program.

TABLE 6 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 4,5, 6, 9, 10 showed a metabolic half-life of more than 30 hours, and examples 7, 11, 12, 13, 14, 15, 16, 17 showed a metabolic half-life between 24-30 hours, which was higher than Larotrectinib (LOXO-101)23 hours. 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.

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

Claims (5)

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

2. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an inorganic salt selected from the group consisting of a hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, 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.

3. A pharmaceutical composition comprising a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

4. Use of a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer, pain, inflammation, neurodegenerative disease, trypanosoma cruzi infection, or osteolytic disease in a mammal.

5. The use of claim 4, wherein the cancer is selected from the group consisting of 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 selected from the group consisting of cancer, surgery, bone fracture, bone pain due to tumor metastasis, visceral pain, inflammatory pain, migraine, chronic low back pain, bladder pain syndrome, and neuropathic pain; wherein said inflammation is selected from the group consisting of asthma, interstitial cystitis, ulcerative colitis, Crohn's disease, atopic dermatitis, eczema, and psoriasis.