阅读说明：本技术 杂环化合物及其制备方法和应用 (Heterocyclic compounds, their preparation and use ) 是由 彭晓梅 刘健平 于 2020-04-20 设计创作，主要内容包括：本发明涉及医药化学领域,公开了杂环化合物及其制备方法和应用,本发明的杂环化合物为结构如下式I所示的化合物、或其药学上可接受的盐、药学上可接受的以氨基酸为载体形成的类似氨基酸化合物、异构体、溶剂化物、同位素变体、水合物、多晶型物或前药,其中,T为取代或未取代的五元杂环、取代或未取代的六元杂环、或取代或未取代的苯环；M-(1)和M-(2)各自独立地选自C或N；R-(1)、R-(2)和R-(3)各自独立地选自C-(1)-C-(4)烷基、羟烷基、氨基、连接有保护基的氨基、羧基或卤素。本发明的杂环化合物具有很好的体内镇痛和止痒效果。(The invention relates to the field of medicinal chemistry, and discloses a heterocyclic compound and a preparation method and application thereof, wherein the heterocyclic compound is a compound with a structure shown as a formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable similar amino acid compound formed by taking amino acid as a carrier, isomer, solvate, isotopic variant, hydrate, polymorph or prodrug thereof, wherein T is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring or a substituted or unsubstituted benzene ring; m 1 And M 2 Each is independently selected from C or N; r 1 、R 2 And R 3 Each independently selected from C 1 ‑C 4 Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen. The heterocyclic compound has good in-vivo analgesic and antipruritic effects.)

1. A heterocyclic compound is characterized in that the heterocyclic compound is a compound with the structure as shown in the formula I, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable similar amino acid compound formed by taking an amino acid as a carrier, an isomer, a solvate, an isotopic variant, a hydrate, a polymorphic substance or a prodrug,

wherein T is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring, or a substituted or unsubstituted benzene ring; m₁And M₂Each is independently selected from C or N; r₁、R₂And R₃Each independently selected from C₁-C₄Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen.

2. The heterocyclic compound according to claim 1, wherein T is a five-membered cycloalkane or a six-membered cycloalkane containing O and/or N, or T is a halogen-substituted benzene ring;

and/or, R₁、R₂And R₃At least one of (A) is amino, C₁-C₄Hydroxyalkyl or halogen.

3. The heterocyclic compound according to claim 1, wherein T is M₁And M₂Are all N; r₁And R₂Are each methyl, hydroxymethyl, chlorine or fluorine, R₃Is amino;

preferably, T isM₁And M₂Are all N; r₁And R₂Are each methyl, R₃Is an amino group.

4. A method of preparing a heterocyclic compound, the method comprising:

(a) carrying out a first contact reaction on a compound shown as a formula a and a compound shown as a formula b to obtain a compound shown as a formula c,

(b) carrying out a second contact reaction on the compound shown in the formula c and ammonium acetate to obtain a compound shown in a formula I;

wherein T' is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring, or a substituted or unsubstituted benzene ring; m₁' and M₂' each is independently selected from C or N; r₁’、R₂' and R₃' each is independently selected from C₁-C₄Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen; x is halogen.

5. The method according to claim 4, wherein T 'is a five-or six-membered cycloalkane containing O and/or N, or T' is a halogen-substituted benzene ring;

and/or, R₁’、R₂' and R₃At least one of' is amino, C₁-C₄Hydroxyalkyl or halogen;

preferably:

t' is M₁' and M₂' are both N; r₁' and R₂' are each methyl, hydroxymethyl, chloro or fluoro, R₃' is amino;

more preferably:

t' isM₁' and M₂' are both N; r₁' and R₂' are both methyl, R₃' is amino.

6. A pharmaceutical composition comprising the heterocyclic compound of any one of claims 1-3 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable carrier is selected from at least one of starch, microcrystalline cellulose, lactose, sucrose, mannitol, inorganic salts, hydroxypropyl cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, sodium alginate, agar, hydroxypropyl methylcellulose, hydroxyethylcellulose, carbopol, polyvinyl alcohol, acrylic resin, chitosan, beeswax, and stearic acid.

8. Use of a heterocyclic compound according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 6 or 7 for the preparation of a medicament for the treatment of a disorder caused by an abnormality of a voltage-gated sodium ion channel.

9. The use of claim 8, wherein the voltage-gated sodium ion channel is Na_v1.7 and/or Na_v1.8。

10. Use according to claim 8 or 9, wherein the condition is selected from pain and/or itching.

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a heterocyclic compound and a preparation method and application thereof.

Background

Voltage-gated sodium ion channel (Na)_v)Na_V1.7 and Na_V1.8 plays an important role in the transmission of itch and pain, is widely present on the cell membranes of excitable cells such as neurons, skeletal muscle cells, and is a type of transmembrane glycoprotein complex composed of an alpha subunit and several beta subunits. For example, code for Na_V1.7 gain-of-function mutations in SCN9A of the alpha subunit cause hereditary erythema myalgia and idiopathic small fiber neuropathic pain (SFN), whereas the Na deficiency_V1.7 causes congenital insensitivity to pain in humans and Na knock-out_V1.7 mice did not have any pain (including inflammatory pain and neuropathic pain). Code for Na_VGain-of-function mutations of SCN10A of the alpha subunit of 1.8 are associated with SFN, neuropathic pain and diabetic peripheral neuropathic pain, whereas loss-of-function mutations of SCN10A may reduce the sensitivity of humans to mechanical pain, and SCN10A knockout mice have a higher threshold to noxious mechanical and thermal stimuli (a. kanelopoulos et al, Voltage-gated sodium channels and pain-related disorders. clin sci (lond). Gene knockout experiments also confirmed Na_V1.7 and Na_V1.8 are also essential for itch conduction, indicating that they are targets for itch treatment (H.K uhn et al, comparative roles of murine NaV1.7, NaV1.8 and NaV1.9 in acute matter signaling Sci Rep.2020Feb 11; 10(1): 2326.). Recently reported Na_V1.7 and Na_V1.8 are co-expressed in sensory neurons in a portion of the dorsal root ganglia and require simultaneous inhibition of Na_V1.7 and Na_V1.8 (D.Jurcakova et al, Voltage-Gated Sodium Channels Regulating Action Potential Generation in Itch-, Nociceptive-, and Low-Threshold transduction C-fibers, mol Pharmacol 94, 1047-. It may therefore be desirable to suppress N simultaneouslya_V1.7 and Na_V1.8 can achieve a sufficiently good analgesic effect.

In addition, it was previously thought that Na_V1.7 and Na_V1.8 are expressed only in neurons of the peripheral nervous system of the pain conductance pathway, whereas the first generation of sodium channel inhibitor drugs bring about side effects due to lack of selection specificity, so the guiding principles followed in recent development of the second generation of sodium channel inhibitor drugs are: 1) screening for single ion channel specific small molecule inhibitors, 2) designed to be impermeable to the blood brain barrier (a. kanelopoulos et al, Voltage-gated sodium channels and pain-related disorders. clin sci (lond). 130(24):2257-2265). At present, the reported ability to target Na_V1.7 small molecule compounds include: GNE-0439 and PF-05089771; can target Na_V1.8 small molecule compounds include: a-803467, the concrete structure is as follows:

GNE-0439：

PF-05089771：

A-803467：

the prior small molecule compounds mostly specifically inhibit Na_V1.7 or Na_V1.8, Na cannot be inhibited simultaneously_V1.7 and Na_V1.8; but also completely impermeable to the blood-brain barrier (such as PF-05089771) or poorly soluble (such as a-803467). However, researchers believe that Na is present_V1.7 and Na_V1.8 Small molecule inhibitors require blood brain barrier permeability to achieve optimal analgesic effect, since Na_V1.7 and Na_V1.8 is expressed in the spinal cord and is regulated by the miR-96 family. Moreover, PF-05089771 failed in clinical studies for the treatment of diabetic neuropathic pain (A.McDonnell., Efficacy of the Nav1.7 packer PF-050897)71in a random, placebo-controlled, double-blind, clinical study in subjects with a pa in clinical diagnostic neurological therapy. pain.2018Aug; 159(8) 1465-1476.) may include the property of being impermeable to the blood-brain barrier and inhibiting Na only singly_V1.7. Whereas a-803467 was not pushed into clinical studies by developers due to poor water solubility. In summary, the second generation of recently developed inhibitory Na_V1.7 or Na_V1.8 has not been developed as a drug for treating pain due to the above-mentioned disadvantages.

In conclusion, the miR-96 knockout mouse is a good analgesic drug screening model, and the ideal targeting Na_V1.7 and Na_V1.8 Small molecule Compounds for the treatment of pain and itch need to have a certain ability to penetrate the blood brain Barrier, good solubility and at the same time inhibit Na_V1.7 and Na_V1.8, the existing small molecule compounds can not meet the requirement.

Disclosure of Invention

The invention aims to solve the problem that only single targeting can be realized in the prior art, and discovers a compound with good pain relieving and itching relieving effects by utilizing the latest conceptual design and the optimal pain animal model screening, and provides a preparation method thereof.

In order to obtain a small molecular compound which has good effects of relieving pain and itching and inhibits a sodium ion channel, the inventor designs Na which has good water solubility and potential permeability to a blood brain barrier and inhibits the Na_V1.7 and Na_V1.8, screening a mouse model and a rat model which are sensitive to pain and can represent miR-96 knockout of a wide range of pain types, and verifying the analgesic effect of the small molecular compound on chronic pain by using rodent models such as neuropathic pain, inflammatory pain and the like to finally obtain the effect of simultaneously inhibiting Na_V1.7 and Na_V1.8, good water solubility and better analgesic effect than PF-05089771. A tricyclic compound (shown as an exemplary structural formula) containing a nitrogen heterocyclic ring and two aromatic rings has good analgesic and antipruritic effects. Accordingly, in order to achieve the above object, the present invention provides, in a first aspect, a heterocyclic compound which is a compound having a structure represented by the following formula I, or a drug thereofPharmaceutically acceptable salt, pharmaceutically acceptable similar amino acid compound formed by taking amino acid as a carrier, isomer, solvate, isotopic variant, hydrate, polymorph or prodrug,

wherein T is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring, or a substituted or unsubstituted benzene ring; m₁And M₂Each is independently selected from C or N; r₁、R₂And R₃Each independently selected from C₁-C₄Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen.

In a second aspect, the present invention provides a process for preparing a heterocyclic compound, which comprises:

(a) carrying out a first contact reaction on a compound shown as a formula a and a compound shown as a formula b to obtain a compound shown as a formula c,

(b) carrying out a second contact reaction on the compound shown in the formula c and ammonium acetate to obtain a compound shown in a formula I;

wherein T' is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring, or a substituted or unsubstituted benzene ring; m₁' and M₂' each is independently selected from C or N; r₁’、R₂' and R₃' each is independently selected from C₁-C₄Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen; x is halogen.

In a third aspect, the present invention provides a pharmaceutical composition comprising a heterocyclic compound as described above and a pharmaceutically acceptable carrier.

In a fourth aspect, the invention provides the use of a heterocyclic compound or pharmaceutical composition as described above for the preparation of a medicament for the treatment of a condition caused by an abnormality of a voltage-gated sodium ion channel.

Through the technical scheme, the heterocyclic compound has good in-vivo analgesic effect, and the reasons include but are not limited to 1) the heterocyclic compound is obtained by testing and screening a mouse model containing central nociception, and 2) the heterocyclic compound can simultaneously inhibit Na_V1.7 and Na_V1.8, 3) good solubility, and 4) low effective dose. In addition, due to Na_V1.7 and Na_V1.8 are involved in itch transmission, so the heterocyclic compound of the present invention also has an antipruritic effect.

Drawings

FIG. 1: HPLC detection scheme after purification of compound PJ 103;

FIG. 2: MS detection map after purification of compound PJ 103;

FIG. 3: h-NMR check of the purified compound PJ 103;

FIG. 4: MiR-96 remission by compound PJ103^+/-Effect of hypersensitivity to mechanical stimulation in mice, wherein data are presented as mean ± SEM, n is 5-12 · P<0.01,***P<0.001，T-test；

FIG. 5: compound PJ103 at miR-96^+/-The dose-response profile of analgesia in mice;

FIG. 6: different compounds are in miR-96^-/+Difference plot of analgesic effect on mice;

FIG. 7: an analgesic aging curve of compound PJ103 on miR-96KO SD rats;

FIG. 8: analgesic aging profile of compound PJ103 on SNI rats;

FIG. 9: compound PJ103 significantly reduced the characterization of inflammatory pain, where data are presented as mean ± SEM, n-5, × P <0.05, T-test.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a heterocyclic compound, which is a compound with a structure shown in the formula I, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable similar amino acid compound formed by taking amino acid as a carrier, an isomer, a solvate, an isotopic variant, a hydrate, a polymorph or a prodrug,

wherein T is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring, or a substituted or unsubstituted benzene ring; m₁And M₂Each is independently selected from C or N; r₁、R₂And R₃Each independently selected from C₁-C₄Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen.

In the present invention, the substituted or unsubstituted five-membered heterocyclic ring, substituted or unsubstituted six-membered heterocyclic ring, or substituted or unsubstituted benzene ring may be halogen or C₁-C₄The heteroatom in the alkyl group, the five-membered heterocycle or the six-membered heterocycle may be O, N, S or the like.

In the present invention, the term "C" is used₁-C₄The alkyl group "may be linear or branched. Said C is₁-C₄Examples of the alkyl group may include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.

In the context of the present invention, reference to "hydroxyalkyl" generally refers to a group of the formula OH-R-, wherein R may be C₁-C₄An alkylene group.

In the present invention, the protecting group attached to the amino group may be various common protecting groups for amino groups, such as t-butyloxycarbonyl group.

In the present invention, the "halogen" may be F, Cl, Br or I, preferably Cl.

In a preferred embodiment of the present invention, T is a five-membered cycloalkane or a six-membered cycloalkane containing O and/or N, or T is a halogen-substituted benzene ring.

In a preferred embodiment of the invention, R₁、R₂And R₃At least one of (A) is amino, C₁-C₄Hydroxyalkyl or halogen.

In a more preferred embodiment of the invention, T is

In a more preferred embodiment of the invention, M₁And M₂Are all N.

In a more preferred embodiment of the invention, R₁And R₂Are each methyl, hydroxymethyl, chlorine or fluorine, R₃Is an amino group.

According to a particular embodiment of the invention, the heterocyclic compound is one of the compounds having the structure shown below:

PJ103：

PJ124：

PJ301：

PJ302：

"pharmaceutically acceptable salts" may be prepared from inorganic or organic acids. Salts derived from inorganic acids include hydrochloric, sulfuric, hydrobromic, nitric, phosphoric, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malonic acid, citric acid, maleic acid, tartaric acid, pamoate salts, and the like.

The term "pharmaceutically acceptable amino acid-like compound formed by using amino acid as carrier" refers to a compound in which the amine group on the compound and the carboxyl group on the amino acid form an amino acid-like compound, such as a compound which forms an amino acid-like compound with valine, threonine, phenylalanine, etc.

"isomers" include, but are not limited to: stereoisomers, enantiomers and diastereomers.

"solvate" refers to a complex formed by combining a compound represented by formula I or a derivative thereof and a solvent.

"isotopic variants" include, but are not limited to, deuterated variants.

"hydrate" refers to a compound containing water, which may be coordinately bound to a moiety of formula I or covalently bound thereto.

"polymorphs" refer to the different crystal structures of a crystalline compound. Different polymorphic compounds may be due to differences in the stacking of the crystalline forms (stacking polymorphisms) or differences in the stacking between different conformers of the same molecule (conformational polymorphisms).

The term "prodrug" is also referred to as prodrug, etc., and refers to a compound obtained by modifying a chemical structure of a drug, which is inactive or less active in vitro and releases the active drug by enzymatic or nonenzymatic conversion in vivo to exert its pharmacological effect. The amino group in the formula I is structurally modified to form compounds such as amides, amino esters, amidines and the like.

According to the structural formula of the heterocyclic compound of the present invention, the heterocyclic compound can be obtained synthetically by those skilled in the art, and therefore, the present invention does not limit the preparation method of the heterocyclic compound, but in one embodiment, the present invention also provides a method for preparing the heterocyclic compound, characterized in that the method comprises:

(a) carrying out a first contact reaction on a compound shown as a formula a and a compound shown as a formula b to obtain a compound shown as a formula c,

(b) carrying out a second contact reaction on the compound shown in the formula c and ammonium acetate to obtain a compound shown in a formula I;

wherein T' is a substituted or unsubstituted five-membered heterocyclic ring, a substituted or unsubstituted six-membered heterocyclic ring, or a substituted or unsubstituted benzene ring; m₁' and M₂' each is independently selected from C or N; r₁’、R₂' and R₃' each is independently selected from C₁-C₄Alkyl, hydroxyalkyl, amino with attached protecting groups, carboxyl or halogen; x is halogen.

Preferably, T 'is a five-or six-membered cycloalkane containing O and/or N, or T' is a halogen-substituted benzene ring.

Preferably, R₁’、R₂' and R₃At least one of' is amino, C₁-C₄Hydroxyalkyl or halogen.

More preferably, T' is

More preferably, M₁' and M₂' are both N.

More preferably, R₁' and R₂' are each methyl, hydroxymethyl, chloro or fluoro, R₃' is amino.

According to the invention, in step (a), the first contact reaction is carried out on Cs₂CO₃The temperature of the first contact reaction can be room temperature (20-30 ℃), and the time of the first contact reaction can be 6-12 h. The conditions of the first contact reaction further comprise: an inert atmosphere. The first contact reaction is carried out in the presence of a third solvent, which is a solvent for every mg of the compound represented by the formula aThe amount of (B) can be 0.01-0.05 ml. The third solvent may be Dimethylformamide (DMF), and the compound represented by formula a is contacted with the compound represented by formula b at 0-5 ℃.

According to the present invention, in the step (b), the temperature of the second contact reaction may be room temperature (20-30 ℃), and the time of the second contact reaction may be 6-12 h. The conditions of the first contact reaction may be the same as or different from the conditions of the second contact reaction. The second contact reaction is carried out in the presence of a fourth solvent, which may be used in an amount of 0.01 to 0.05ml per mg of the compound represented by formula c. The solvent used for the second contact reaction may be toluene.

According to a particular embodiment of the invention, the heterocyclic compound (PJ103) is prepared by a process comprising:

(1) contacting 5-chloro-1, 3, 4-thiadiazole-2-carboxylic acid ethyl ester with morpholine to carry out a first reaction to obtain a compound shown as a formula (1);

(2) contacting the compound shown in the formula (1) with chloroiodomethane to carry out a second reaction to obtain a compound shown in a formula (2);

(3) contacting the compound shown in the formula (2) with 2- (tert-butyloxycarbonylamino) -2-methylpropanoic acid to carry out a third reaction to obtain a compound shown in a formula (3);

(4) contacting the compound shown in the formula (3) with ammonium acetate to carry out a fourth reaction to obtain a compound shown in a formula (4);

(5) removing the protecting group on the compound shown in the formula (4).

The temperature of the first reaction in the step (1) may be 120-. The first reaction is carried out in a first solvent, which may be used in an amount of 0.02 to 0.05ml per mg of ethyl 5-chloro-1, 3, 4-thiadiazole-2-carboxylate. The first solvent can be ethanol, the product of the first reaction is washed and then subjected to the next reaction, and the solution used for washing is saturated sodium bicarbonate solution.

The temperature of the second reaction in the step (2) can be-90 ℃ to-70 ℃, and the time of the second reaction can be 1-5 h. The second reaction is carried out in a second solvent, which may be used in an amount of 0.01 to 0.05ml per mg of the compound represented by formula (1). The solvent used for the second reaction may be DMF. The second reaction is preferably carried out in the presence of Lithium Diisopropylamide (LDA).

In step (3), the third reaction is preferably carried out in Cs₂CO₃The temperature of the third reaction may be room temperature (20-30 ℃), and the time of the third reaction may be 6-12 h. The conditions of the third reaction further comprise: an inert atmosphere. The third reaction is carried out in the presence of a third solvent, which may be used in an amount of 0.01 to 0.05ml per mg of the compound represented by formula (3). The third solvent may be Dimethylformamide (DMF), and the compound represented by formula (3) is contacted with 2- (tert-butoxycarbonylamino) -2-methylpropanoic acid at 0-5 ℃.

The temperature of the fourth reaction in the step (4) may be room temperature (20-30 ℃), and the time of the fourth reaction may be 6-12 h. The fourth reaction is carried out in the presence of a fourth solvent, which may be used in an amount of 0.01 to 0.05ml per mg of the compound represented by formula (3). The solvent used in the fourth reaction may be toluene.

The method for deprotecting the group in step (5) is not particularly limited, and can be carried out according to a conventional method, and will not be described herein again.

The invention also provides a pharmaceutical composition, which is characterized by containing the heterocyclic compound and a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutically acceptable carrier includes any solvent, excipient, dispersion medium, coating, isotonic agent and/or absorption delaying agent, and the like. For example, it may be selected from at least one of starch, microcrystalline cellulose, lactose, sucrose, mannitol, inorganic salts (e.g., calcium sulfate, calcium hydrogen phosphate, calcium carbonate, calcium sulfate dihydrate, etc.), hydroxypropyl cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, sodium alginate, agar, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, carbopol, polyvinyl alcohol, acrylic resin, chitosan, beeswax, and stearic acid.

In the present invention, the weight ratio of the heterocyclic compound to the pharmaceutically acceptable carrier may be 1 to 100: 100.

in addition, the invention also provides the heterocyclic compound or the pharmaceutical composition as described above for preparing the medicine for treating the voltage-gated sodium ion channel (especially Na)_v1.7 and/or Na_v1.8) disorders caused by abnormalities.

In addition, the invention relates to the treatment of voltage-gated sodium ion channels (in particular Na)_v1.7 and/or Na_v1.8) a method of treating a disorder caused by an abnormality, the method comprising: administering to the subject a heterocyclic compound as described above. Where administration may be by conventional means, routes of administration may include, but are not limited to: oral, buccal, injection, respiratory, skin, eye, nasal mucosa, rectum, vagina, ear, dialysis, etc. The subject may be an animal, including a mammal (particularly a primate, such as a human) or a rodent (such as a mouse). The medical staff can select the administration dosage according to the physical condition of the subject, and the administration dosage can be 0.01-20mg/kg in adult.

According to a preferred embodiment of the invention, the condition is selected from pain and/or itching.

Pain can be classified into inflammatory pain (inflammatory pain) and neuropathic pain (neuropathic pain) according to the pathophysiological characteristics of pain production. Inflammatory pain refers to pain caused by various mediators produced by tissue injury, such as prostaglandin, adenosine and the like, stimulating sensory nerve endings, and neuropathic pain generally has no tissue injury and is often caused by injury of the central nervous system or peripheral nervous system or functional disorder after injury. The pain referred to herein includes inflammatory pain and/or neuropathic pain, and may be selected from at least one of migraine, dental pain, trigeminal neuralgia, cancer-related pain, post-operative and traumatic pain, musculoskeletal pain (e.g., arthralgia), dysmenorrhea, visceral pain, and pain caused by diabetic peripheral neuropathy.

The pruritus refers to the pruritus caused by the skin itch and comprises the pruritus caused by the skin itch and the pruritus caused by only skin itch without primary skin damage, and the pruritus mentioned in the invention can comprise at least one of histamine-dependent chronic pruritus and histamine-independent chronic pruritus, pruritus after fungal infection and pruritus caused by eczema.

According to the invention, the term "treatment" means: therapeutic as well as prophylactic or inhibitory measures for a disease or condition include those that cause any clinically desirable or beneficial effect, including but not limited to alleviation or alleviation of one or more symptoms, regression, slowing or cessation of disease or condition progression.

The present invention will be described in detail below by way of examples. All of the following animal experiments were in compliance with the guidelines of the international society for pain research and were reviewed and approved by the institutional animal ethics committee of the Suzhou biomedical engineering and technology research institute of Chinese academy of sciences.

In each step, the "yield" means: the weight of the product obtained is a percentage of the total weight of the reaction substrate used.

Preparation example 1

Designing, synthesizing and purifying compounds

The heterocyclic compound PJ103 is synthesized according to the following synthetic route:

the method comprises the following specific steps:

first step of

Taking a compound 1: ethyl 5-chloro-1, 3, 4-thiadiazole-2-carboxylate (900mg,4.672mmol), compound 2: morpholine (1.3g, 14.952mmol), dissolved in EtOH (22ml), was refluxed at 120 ℃ for 1.5h and evaporated to dryness. The residue was dissolved in EtOAc (40mL) and washed with saturated NaHCO₃(20 ml. times.3) and 0.9% (w/v) NaCl solution (20 ml. times.3), filtered, and the filtrate was washed with Na₂S₂O₄Evaporating and drying to obtain a yellow solid as a target product (a compound 3): 5-morpholine-1, 3, 4-thiadiazole-2-carboxylic acid ethyl ester, yield 1.073g, 94.5%.¹H NMR(400MHz,CDCl₃)δ4.45(q,J＝7.1Hz,2H),3.89-3.79(m,4H),3.67-3.58(m,4H),1.42(t,J＝7.1Hz,3H).

Second step of

Taking a compound 3: 5-morpholine-1, 3, 4-thiadiazole-2-carboxylic acid ethyl ester (850mg, 3.494 mmol) compound 4: chlorodomethyl (1.848g, 10.482m mol), 2ml of DMF in which LDA (lithium diisopropylamide, 10.482m mol) was dissolved were added at-78 ℃ and the resulting mixture was stirred at the same temperature for 2h, NH was added₄The reaction was stopped with Cl solution (5ml) and extracted with DCM (20 ml. times.3). The organic layer was washed with brine, Na₂S₂O₄Drying, filtering, concentrating under reduced pressure to obtain a crude product, and performing flash chromatography purification by using 100-200-mesh silica gel (PLC) as a filler (ethyl acetate/petroleum ether (PE/EA) ═ 2:1 as an eluent) to obtain a target compound 5: 2-chloro-1- (5-morpholine-1, 3, 4-thiadiazol-2-yl) ethan-1-one, yield 220mg, 25.5%. 1H NMR (400MHz, CDCl)₃)δ4.91(s,2H),3.89-3.82(m,4H),3.73-3.65(m,4H).

The third step

At 0 ℃, to a mixture containing compound 6: 2- (tert-Butoxycarbonyl) -2-methylpropionic acid (197mg, 0.969 mmol)) in a reaction flask filled with nitrogen was added 5: 2-chloro-1- (5-morpholine-1, 3, 4-thiadiazol-2-yl) ethan-1-one (200mg, 0.807mmol) with compound 7: cs₂CO₃(212mg,0.807mmol)), the reaction mixture was stirred at room temperature overnight, and the organic layer was washed with Na₂SO₄Drying, vacuum filtration and evaporation, and purifying the organic layer with PLC as the filler (PE/EA ═ 5: 1 as the eluent) to obtain the target product 8: 2- (5-morpholine-1, 3, 4-thiadiazole-2-oxoethyl 2- (tert-butoxycarbonyl) amino) -2-methylpropionate in a yield of 0.25g and 42.5%. LC-MS found M/z 322.11,366.10,422.23[ M + H ]]⁺.¹H NMR(400MHz,CDCl₃)δ5.50(s,2H),5.05(s,1H),3.90-3.79(m,4H),3.73-3.63(m,4H),1.60(d,J＝14.0Hz,6H),1.45(d,J＝7.1Hz,9H).

The fourth step

Compound 8: 2- (5-Morpholine-1, 3, 4-thiadiazole-2-oxoethyl) -2- (tert-butoxycarbonyl) amino-2-methylpropionate (200mg, 0.482m mol), Compound 9: NH₄OAc (557.93mg, 7.237m mol), toluene (3ml) were added to the reaction flask in succession, the reaction mixture was refluxed overnight, the reaction mixture was evaporated under reduced pressure, the reaction mixture was diluted with water (20ml), extracted with DCM (3X 20ml), the extracted organic combination was washed with water (3X 20ml), Na₂SO₄Drying, filtration under reduced pressure and evaporation gave crude compound 10, which was purified by PLC (PE: EA ═ 2:1 as eluent) to afford target product 10: tert-butyl (2- (5-morpholine-1, 3, 4-thiadiazol-2-yl) -1H-imidazol-2-yl) alanine, yield 150mg, 78.9%.

The fifth step

Mixing compound 10: tert-butyl (2- (5-morpholine-1, 3, 4-thiadiazol-2-yl) -1H-imidazol-2-yl) alanine (120mg, 0.304m mol) was dissolved in DCM (1m l), TFA (0.25ml) was added and the reaction mixture stirred at room temperature for 1H and evaporated under reduced pressure with 1,2 dichloroethane to give the title compound 11: 2- [ [4- (5-morpholine) -1,3, 4-thiadiazol-2-yl group]-1H-imidazol-2-yl]Propan-2-amine. Yield: 50mg (55.8%) LC-MS found M/z 295.01, [ M + H]⁺.¹H NMR(400MHz,DMSO-d₆)δ7.53(s,1H),3.80-3.66(m,4H),3.49-3.39(m,4H),1.38(s,6H).

The purity of PJ103 was greater than 98% by HPLC (FIG. 1), the molecular weight of the compound was determined by LC-MS (FIG. 2), and the structural formula of the synthesized compound was 2- [ [4- (5-morpholine) 1,3, 4-thiadiazol-2-yl ] -1H-imidazol-2-yl ] propan-2-amine (FIG. 3) determined by HNMR.

Example 1

Compound pair miR-96^+/-Determination of analgesic Effect in mice

Chronic pain is a complex disease in which multiple genes are involved, and up to several hundred genes have been found to be associated with chronic pain, while the pain genes involved in different types of chronic pain are different. This may be one of the major reasons why drug development at a single target has not been successful. Prior studies found that the miR-183-96-182 family systemically regulates neuropathic pain by inhibiting 80% of genes in the neuropathic pain gene regulatory network including 7 transcription factors at the target and core of the clinical drug gabapentin in dorsal-heel neurons (C.Peng et al, miR-183 scales mechanical pain sensitivity by regulating basic and neuropathic pain genes, science 356, 1168-. Recently, the prediction of miR-96, miR-183 and miR-182 through biological software shows that the Na can be regulated and controlled by the miR-96, miR-183 and miR-182_V1.7 and Na_V1.8, and single cell RNA sequencing found that a small amount of Na was expressed by a portion of neurons in the dorsal horn of the spinal cord involved in itch and pain transmission_V1.7 and Na_V1.8 (M.Haring et al, neural atlas of the nasal horn definitions and links sensory input to transcriptional cell type. Nat Neurosci 21, 869-. Therefore, the following is by miR-96^+/-The analgesic effect of the compounds was determined in a mouse model.

Adult male miR-96^+/-Mice (manufactured by shanghai square model biotechnology, ltd.) were transferred from SPF animal rooms to behavioral testing rooms and were adapted to be reared for one week. Mice were acclimated for half an hour in a clear plexiglass chamber on a wire mesh, and then miR-96 was measured with von Frey fibers (von Frey hairs; Danmic Global, USA)^+/-Basal mechanical pain sensitivity threshold of mice (the minimum pressure value that causes the mice to contract the paw is the machinePain sensitivity threshold). The test compound is dissolved in DMSO to prepare a high-concentration stock solution, and then the compound is diluted to working solutions with different concentrations by using DMSO/physiological saline solution with the lowest concentration capable of completely dissolving the compound. Injecting PJ103 with low dose (0.15mg/kg), medium dose (0.3mg/kg) and high dose (0.6mg/kg) into abdominal cavity respectively, and measuring miR-96 after one hour^+/-Threshold for mechanical pain sensitivity after administration to mice. Statistical results show that the miR-96 can be remarkably improved by injecting PJ103 with the dose of 0.15mg/kg^+/-Mice were threshold for mechanical pain sensitivity, whereas high doses of PJ103(0.6mg/kg) had an analgesic effect that was comparable to 2mg/kg Na_V1.7 the inhibitor PF-05089771 (positive control) was found to work well (FIG. 4). The analgesic effect of PJ103 was further tested at 1.2mg/kg and 2.4mg/kg doses, n is 12, and the PJ103 was calculated and plotted at miR-96^+/-The analgesic effect profile in mice (fig. 5) shows that PJ103 has a very good analgesic effect over a wide dose range. The dose-response curve chart is used for calculating the corresponding dose of the mouse 1/2Emax to be about 0.3mg/kg according to the dose-response relation of PJ103 analgesia, and other compounds are tested by the same method, and the results are shown in Table 1. The analgesic effect of the compounds by different group substitutions was different (see fig. 6), the maximum analgesic effect values of PJ301 and PJ302 were 0.35g and 0.46g, respectively, and the maximum analgesic effect value (0.56g) was high without PJ 103. The difference between PJ103, PJ301 and PJ302 is the number of carbon atoms substituted by nitrogen atoms in the formula, indicating that substitution of carbon atoms by nitrogen atoms in the structure of PJ302 enhances its analgesic effect. This result also suggests that further increasing the number of N atoms on PJ103 may enhance its analgesic effect.

TABLE 1

Compound numbering	Emin	Emax	1/2Emax	Test concentration Range
					PJ103	0.15mg/kg	0.6mg/kg	0.3mg/kg	0.15-2.4mg/kg
PJ301	0.2mg/kg	1.8mg/kg	0.9mg/kg	0.15-2.4mg/kg
					PJ302	0.3mg/kg	1.6mg/kg	0.8mg/kg	0.15-2.4mg/kg
PJ124	0.1mg/kg	0.6mg/kg	0.3mg/kg	0.15-2.4mg/kg

Example 2

PJ103 pair miR-96^+/-Determination of analgesic Effect in rats

In the same manner as above, the PJ103 was detected in the miR-96 of the adult male sensitive to mechanical pain^+/-Rat (manufactured by trusted society of Biotechnology, Inc.)To) analgesic effect. n is 12. After intraperitoneal injection of 0.3mg/kg PJ103, miR-96 is detected every half hour after injection^+/-Mechanical pain threshold in rats, up to 2.5 hours. The difference in elevated mechanociceptive Threshold for the PJ103 compound (Δ PTW, Paw withawal Threshold) was then obtained by subtracting the basal mechanociceptive Threshold from the post-dose mechanociceptive Threshold, and then plotting the PJ103 at miR-96^+/-Time profile of analgesia in rats (figure 7).

As can be seen from FIG. 7, the heterocyclic compound of the present invention has very good analgesic effect for rats sensitive to mechanical pain in half an hour, reaches a peak in one hour, and has significant effect up to 2.5 hours,

example 3

Determination of analgesic Effect of PJ103 on SNI rats

Adult-200 g SD rats (Shanghai Stick laboratory animals Co., Ltd.) were anesthetized with isoflurane, and a neuropathic pain model of peripheral Nerve Injury (round Nerve input, SNI) was prepared using the method reported in the references "C.Peng et al, miR-183cluster scales mechanical pain sensitivity by regulating basal and neuropathic pain genes, science 356, 1168-. Rats were tested for mechanical pain threshold on day 14 post-surgery, followed by intraperitoneal injection of PJ103 at a dose of 0.3mg/kg, with mechanical pain threshold being tested every half hour up to 2.5 hours after injection. The effect of PJ103 compounds on the inhibition of mechanical pain (Δ PTW, Paw withawal Threshold) was calculated and then a graph of the time course of pain relief of PJ103 in SNI SD rats was plotted (fig. 8).

As can be seen from FIG. 8, the analgesic effect of the heterocyclic compound of the present invention on the neuropathic pain model rats appeared after half an hour, peaked at 1.5 hours, and had a certain effect at 2.5 hours.

Example 4

Detection of effect of PJ103 on inhibition of inflammatory pain

Adult (9 week old) C57/BJ6 mice (SHOWA-DERIVED NEW MEDICINE SCHOOL, Inc.) were anesthetized with chloral hydrate and then injected plantar with 5% Freund's complete adjuvant (Beyotime Biotechnology), and all mice developed inflammation-induced hypersensitivity mechanociceptive pain after 24 hours. Intraperitoneal injection of PJ103 at a dose of 0.6mg/kg significantly reduced inflammatory pain (fig. 9).

As can be seen in fig. 9, a 0.6mg/kg dose of PJ103 significantly reduced CFA-induced chronic inflammatory pain.

Example 5

Detection of inhibition rates of Nav1.7 and Nav1.8 activity by PJ103

HEK293 cells stably expressing hNav1.7 were cultured in 90% DMEM, 10% FBS,100U/mL Penicilin-Streptomyces, 20mM HEPES and 400. mu.g/mL of G418. Cells were tested at 5X 10⁵The density of individual cells/6 cm cell culture dish was plated on a glass cover. The PJ103 pairs hNa were detected at 5 concentrations (from 0.3. mu.M to 30. mu.M) using manual patch clamp (HEKA EPC 10) technique_V1.7, followed by detection of 300nM Na_V1.7 inhibitors PF-05089771 vs hNa_V1.7 inhibitory Effect of Activity. Calculate PJ103 pairs hNa_V1.7 (see table 2), the inhibition was calculated as (1- (compound peak current-positive control peak current)/(blank peak current-positive control peak current)) × 100%.

Similarly, hNa was stably expressed using manual patch clamp (HEKA EPC 10) technology_V1.8 detection of 5 concentrations (from 0.3. mu.M to 30. mu.M) of PJ103 vs hNa on CHO-K1 cells_V1.8 inhibition of channel activity. The PJ103 pair hNa is obtained by calculation_V1.8 inhibition efficiency (see table 2). Although PJ103 was on Na at a concentration of 30. mu.M_V1.7 and Na_VThe inhibition rate of 1.8 is only 11% and 34%, respectively, but the optimum dose for analgesia in mice and rats is only 0.6mg/kg and 0.3mg/kg, respectively. These results indicate that PJ103 weakly inhibits Na_V1.7 and Na_V1.8, the activity of the composition produces remarkable synergistic analgesic and antipruritic effects. And PJ103 on Na at optimal analgesic dose_V1.7 and Na_V1.8 inhibition of activity by less than 50%, indicating that it is on expression of Na alone_V1.7 or Na_V1.8 cell and tissue effects are minimal, i.e., potentially Na_V1.7 and Na_V1.8 associated side effects are less likely to occur.

TABLE 2

Compound numbering	Concentration (μ M)	Inhibition ratio (%) of hNaV1.7	Inhibition efficiency (%) for hNaV1.8
				PJ103	30.00	10.99±8.15	33.5±5.8

Example 6

Detection of hERG Activity inhibition Rate by PJ103

The inhibitory effect of PJ103 on hERG channel activity was examined at 5 concentrations (from 0.3 μ M to 30 μ M) on HEK293 cells stably expressing hERG using manual patch clamp (HEKA EPC 10) technique (n 2) as in example 5. Using the following equation: the inhibition efficiency of hERG by PJ103 was calculated by (%) inhibition ratio (control peak current-compound peak current)/control peak current. times.100% (see Table 3)

TABLE 3

As can be seen from table 3, compound PJ103 inhibited the hERG potassium channel at high IC50 doses, indicating that PJ103 blocked hERG with little potential to cause cardiotoxicity.

Example 7

5 adult male miR-96^+/-Mice were placed in a 60 cm long by 40 cm wide paper box, their activity was recorded by taking a video of 5 minutes, and then the effect of intraperitoneal injections of 0.6mg/kg, 1.2mg/kg, or 2.4mg/kg of PJ103 on mouse movement and behavior was tested from low to high doses. Half and one hour after injection of each dose of PJ103, 5 mice were placed in a paper box and their activity was recorded by taking a 5 minute video. By comparison, the locomotion and social behavior of mice after injection of 2.4mg/kg (four-fold optimal dose) of PJ103 was not different from the locomotion and behavior of mice before injection of PJ 103. These results indicate that PJ103 has no effect on mouse movement and social behavior, and that PJ103 is safe.

Example 8

The results of the assay to test the solubility of PJ103 in water and DMSO show that the solubility of PJ103 in DMSO is greater than 340mM and the solubility in water is about 0.05 mM. The optimal analgesic dose of PJ103 in mice was 0.6mg/kg, so that only a very low DMSO content (2.5%) was required to formulate the optimal analgesic dose of PJ103 solution (0.61 mM). The solubility and effective dose of PJ103 were much better than those of A-803467, and the comparative data are shown in Table 4.

TABLE 4

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.