CN113387947A

CN113387947A - Pyrazolopyridine derivatives that modulate estrogen receptor synthesis activity

Info

Publication number: CN113387947A
Application number: CN202110786668.3A
Authority: CN
Inventors: 马小锋; 刘亚洲; 王阿木; 王飞; 史小可
Original assignee: Chengdu Institute of Biology of CAS
Current assignee: Chengdu Institute of Biology of CAS
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2021-09-14
Anticipated expiration: 2041-07-12
Also published as: CN113387947B

Abstract

The invention relates to the technical field of organic chemistry and pharmaceutical chemistry, in particular to a pyrazolopyridine derivative for regulating the synthetic activity of an estrogen receptor. The specific technical scheme is as follows: substituted 1-aminopyridines are stirred with alpha, beta-unsaturated compounds in the presence of an oxidizing agent under basic conditions to form substituted pyrazolo [1,5-a ] pyridines. The invention adopts a one-step synthesis strategy, takes alpha, beta-unsaturated compounds as raw materials, and efficiently and selectively synthesizes a series of pyrazolo [1,5-a ] pyridine compounds in the presence of organic oxidants.

Description

Pyrazolopyridine derivatives that modulate estrogen receptor synthesis activity

Technical Field

The invention relates to the technical field of organic chemistry and pharmaceutical chemistry, in particular to a pyrazolopyridine derivative for regulating the synthetic activity of an estrogen receptor.

Background

Pyrazole [1,5-a ]]The pyridine compounds are widely present in various clinical drugs, such as compound 1 can be used as adenosine receptor blocker, compound 2 is used as adenosine A1 receptor antagonist, and compound 3 is used as 5-HT₃Receptor blockers, Compound 4 used as dopamine D4 receptor antagonist, Compound 5 used as P₃₈Kinase inhibitor, compound 6, was used as an anti-herpes virus, compound 7, marketed under the trade name ibudilast, is a PDE4 inhibitor, while compound 8, Selpercatinib (chinese name serpatatinib), was the first highly selective RET monokinase inhibitor developed by Loxo Oncology, a firm, marketed at 5 months of 2020, was approved by the FDA, is a RET inhibitor that can inhibit wild-type RET and multiple mutations, and can be used for the treatment of late-RET fusion-positive NSCLC, RET mutant/fusion-positive MTC, etc.

Due to the wide clinical application, the compound can be synthesized with high efficiency and high selectivity under mild conditions, and is always the target of organic chemists and medicinal chemists. Taking Selpercatinib (the Chinese name Selpeptinib) as an example, the parent ring structure of Selpeptinib is pyrazolo [1,5-a ] pyridine-3-carbonitrile (the structural formula is shown in the specification in a bold part). In the synthesis of this compound reported in US2018134702, 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile was used as the main starting material:

6-bromo-4-methoxypyrazole [1,5-a ]]The synthesis of pyridine-3-carbonitrile mainly adopts the following route: firstly, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzene sulfonate is used as a raw material to react with ethyl propiolate under an alkaline condition to formPreparing a mixture of Comp-1 and Comp-2(Comp-1: Comp-2: 4:1), decarboxylating the mixture in 48% HBr, recrystallizing and purifying by column chromatography to obtain Comp-3 and Comp-4, and further separating and purifying by DMF and POCl₃Under the action of (A), synthesizing corresponding aldehyde, reacting the formed aldehyde with hydroxylamine to form oxime, and finally obtaining corresponding 6-bromo-4-methoxy-pyrazole [1,5-a ] through elimination reaction]Pyridine-3-carbonitrile (Comp-7) and 4-bromo-6-methoxypyrazole [1,5-a ]]Pyridine-3-carbonitrile (Comp-8). Although this route allows the target compound to be obtained, the desired route is long (4-step reaction), and the compounds Comp-3 and Comp-4 need to be isolated and not amenable to subsequent synthetic studies. The synthetic route is as follows:

in the subsequent US patent document (US2019106438), researchers made other attempts to synthesize the main intermediate 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile, such as reacting 2-chloroacrylonitrile as a raw material with the corresponding 1-amino-3-bromo-5-methoxypyridine salt, and the compound can be synthesized in one step, but the method is limited by the high cost of the raw material (2-chloroacrylonitrile), the large amount of DBU used, and the low yield of the reaction. The synthetic route is as follows:

chinese patent CN111548349A, 1-amino-3-bromo-5-methoxypyridine hydrogen sulfate as raw material, reacted with ethyl propiolate in 97% yield and 2.4: 1 to give compounds Comp-1 and Comp-2, the subsequent steps being essentially identical to those of US patent US 2018134702. Although this route avoids the use of the more expensive 2,4, 6-trimethylbenzenesulfonyl chloride and BocNHOH to synthesize 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate. However, the synthesis still requires 3 steps to synthesize 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile, and the synthesis of compounds Comp-1 and Comp-2 is not very selective, which ultimately results in a lower overall yield of 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile, as shown in the following synthetic scheme. Therefore, there is a great need for efficient, mild and highly selective methods for the synthesis of such parent ring structures.

Estrogens play an important role in Human Reproduction, bone, blood vessels, central nerves, metabolism, etc. (Brown et al, 33, 2015, semiamines in productive media; Simpson et al, 26, 2005, Endocrine Reviews), which are also associated with the pathophysiology of various diseases, such as breast cancer, ovarian cancer and endometriosis, polycystic ovary syndrome, etc. (Misso et al, 18, 2012, Human Reproduction Update; Patwardhan et al, 115, 2008, British Journal of obsltrics and gynacolology).

In premenopausal women, the major site of estrogen synthesis is the ovary (Stocco, 73, 2008, Steroids), which is formed primarily by the catalysis of androgens in the ovary by aromatase, circulating throughout the body. In postmenopausal women (Patel et al, 102, 2018, Biomedicine & Pharmacotherapy), each tissue site for estrogen synthesis, which is mainly adipose tissue mesenchymal cells, utilizes the synthesis of circulating androgens as estrogens for paracrine and even tissue endocrine factors.

In recent years, the incidence of estrogen disorder-related diseases has increased year by year, and the occurrence and development of the diseases are closely related to estrogen anabolic disorder in vivo. For example, it has been reported that estrogen levels are related to pubertal dysfunctional uterine bleeding, senile vaginitis, etc. and that regulating estrogen levels in vivo can treat these diseases (Lijunqing, et al, 4, 2017, J.J.Utility gynecologic endocrine; Zhang Chihong, et al, 48, 2020, J.Chinese clinicians). Furthermore, elevated estrogen may increase the risk of ovarian cancer in women, while hormone deficiency may lead to osteoporosis by affecting the level of inflammation (Wu et al, 245, 2020, Journal of Endocrinology).

In conclusion, clinical drugs with pyrazolo [1,5-a ] pyridine compounds as the inner core exist widely, but relatively simple and highly selective synthesis methods for the compounds are still unavailable at present. In addition, the change of estrogen level is directly related to the occurrence of various serious diseases, and the development of high-efficiency estrogen regulators can effectively treat the related diseases. Therefore, a simple and highly selective synthesis means is urgently needed to obtain the pyrazolo [1,5-a ] pyridine compound and study the estrogen regulation effect of the compound.

Disclosure of Invention

In response to the deficiencies of the prior art, the present invention provides pyrazolo pyridine derivatives which modulate the synthetic activity of estrogen receptors.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention discloses a compound of formula (I) or a stereoisomer, a tautomer or a salt thereof,

wherein R is¹Is one of hydrogen atom, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclic aryl; the alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl groups are each independently substituted with one or more R^XSubstituted with the substituent(s);

R²is one of hydrogen atom, cyano, nitro, ketone, ester, amide, sulfoxide and sulfone; said ketones, esters, amides, sulfoxides and sulfones being each substituted by one or more R independently of one another^XSubstituted with the substituent(s);

R³is one or more of hydrogen atom, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic aryl, cyano, nitro, hydroxyl, mercapto, amino, substituted amino, ketone, ester, amide, sulfoxide and sulfone; the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic aryl, hydroxy, mercapto, amino, substituted amino, ketone, ester, amide, sulfoxide, and sulfoneEach by one or more independent R^XSubstituted with the substituent(s);

the R is^XIs one of hydrogen atom, halogen, alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxyl and sulfonamide.

Preferably, said R is^XWherein the alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide are substituted with one or more hydrogen atoms, alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide, respectively.

Accordingly, a process for the synthesis of compounds of formula (I) or stereoisomers, tautomers or salts thereof, comprises suspending a substituted 1-aminopyridine in a solvent under basic conditions and stirring with an α, β -unsaturated compound in the presence of an oxidizing agent to form a substituted pyrazolo [1,5-a ] pyridine.

Preferably, the reaction concentration of the solvent is 0.001-100 mol/L, the use amount of the alkali is 0.1-10 equivalents, the use amount of the oxidant is 0.1-10 equivalents, and the reaction temperature is-80-200 ℃.

Preferably, the solvent is one of toluene, 1, 4-dioxane, acetonitrile, methanol, dichloromethane, DMF and THF; the oxidant is TEMPO, nitrogen oxide similar to TEMPO, ammonium oxide salt of the nitrogen oxide, and O₂Air, t-BuOOH, (t-BuO)₂NaOCl, iodobenzene diacetate, iodobenzene bistrifluoroacetate, DDQ, tetrachlorobenzoquinone, MnO₂、SeO₂One of cerium ammonium nitrate, nitric acid, Pd/C and amine nitroxides;

the nitrogen oxides similar to TEMPO are 4-OH-TEMPO, 4-NH₂One of TEMPO, 4-Oxo-TEMPO, ABNO, Keto-CHAMPO and AZADO, wherein the ammonium oxide salt of the nitrogen oxide is [ TEMPO ]][X]、[4-OH-TEMPO][X]、[4-NH₂-TEMPO][X]、[4-Oxo-TEMPO][X]、[ABNO][X]、[Keto-CHAMPO][X]、[AZADO][X])；

[X]Comprises the following steps:

SO₄ ^2-，

PO₃ ^3-，HPO₃ ^2-，

one of (1);

wherein R is⁴Is any one, two, three or four of hydrogen atom, halogen, alkyl, alkoxy, cycloalkyl, cyano, nitro, hydroxyl, sulfydryl, amino, ketone, ester, amide, sulfoxide and sulfone. Wherein the above alkyl, alkoxy, cycloalkyl, hydroxy, mercapto, amino, ketone, ester, amide, sulfoxide, sulfone may be further substituted by one or more groups selected from R^XIs substituted with the substituent(s). R^XAs disclosed above. R⁵Is one of alkyl, (poly) haloalkyl and cycloalkyl, wherein alkyl, (poly) haloalkyl or cycloalkyl may be further substituted with one or more groups selected from R^XIs substituted with the substituent(s). R^XAs disclosed above.

Preferably, the α, β -unsaturated compound includes one of α, β -unsaturated aldehyde, α, β -unsaturated ketone, α, β -unsaturated ester, α, β -unsaturated cyano compound, α, β -unsaturated nitro compound, and α, β -unsaturated amide compound.

Preferably, the α, β -unsaturated aldehyde, β 0, β 1-unsaturated ketone, α, β -unsaturated ester, α, β -unsaturated cyano compound, α, β -unsaturated nitro compound and α, β -unsaturated amide compound are each substituted with one or more independent R^XSubstituted with the substituent(s);

wherein, R is^XIs hydrogen atom, halogen, alkyl, fluoroalkyl, alkenyl, alkynyl, nitroOne of cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy and sulfonamide.

The R is^XWherein the alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide are substituted with one or more hydrogen atoms, alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide, respectively.

Accordingly, a pharmaceutical composition comprising said compound of formula (i) or a stereoisomer, tautomer, or salt thereof, or prodrug molecule thereof, and a pharmaceutically acceptable carrier.

Accordingly, the use of a compound of formula (I) or a stereoisomer, tautomer or salt thereof or prodrug molecule thereof for the manufacture of a medicament for the treatment of a tumor and/or a metabolic disease.

Preferably, the tumor is breast cancer, ovarian cancer; and/or the metabolic diseases are hypoestrogenic dysfunctional uterine bleeding and senile vaginitis, and osteoporosis caused by estrogen deficiency.

The invention has the following beneficial effects:

1. the invention adopts a one-step synthesis strategy, takes alpha, beta-unsaturated compounds as raw materials, efficiently and selectively synthesizes a series of pyrazole [1,5-a ] pyridine compounds in the presence of organic oxidants, and utilizes human ovarian granule-like cell (KGN) cells to evaluate the estrogen biosynthesis activity. The obtained pyrazolo [1,5-a ] pyridine compound shows better activity of stimulating or inhibiting estrogen biosynthesis due to different substituents.

2. The method converts the substituted 1-aminopyridine into the substituted pyrazolo [1,5-a ] pyridine-3-carbonitrile efficiently in one step under mild conditions, thereby greatly reducing the steps (4 steps of reaction) for synthesizing the compounds in the prior patents (such as US2018134702, US2019106438, CN111548349A and the like) and improving the efficiency for synthesizing the compounds. Moreover, the reaction has specific regioselectivity, so that the difficulty of separation and purification is reduced on one hand, and the reaction efficiency is further improved on the other hand. The method can also be well applied to the synthesis of other polysubstituted pyrazolo [1,5-a ] pyridine derivatives, and has the advantages of short reaction steps, mild conditions, high yield, good selectivity, simple operation, high activity and the like in the whole synthesis process.

Drawings

FIG. 1 is a graph showing the cytotoxicity of pyrazolo [1,5-a ] pyridine derivatives on KGN cells;

FIG. 2 is a graph showing the effects of 9 compounds W4, W5, W6, W7, W9, W11, W13, W15 and W19 on estrogen biosynthesis.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art.

1. The invention discloses a compound of formula (I) or a stereoisomer, a tautomer or a salt thereof,

R³is one or more of hydrogen atom, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic aryl, cyano, nitro, hydroxyl, mercapto, amino, substituted amino, ketone, ester, amide, sulfoxide and sulfone; the alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, hydroxy, mercapto, amino, substituted amino, ketone, ester, amide, sulfoxide, and sulfone are each independently substituted with one or more R^XSubstituted with the substituent(s);

2. The invention provides a simple, mild, efficient and high-selectivity method for synthesizing pyrazolopyridine derivatives, which comprises the following specific steps: the substituted 1-aminopyridine is suspended in a solvent with a certain concentration under the alkaline condition and stirred with an alpha, beta-unsaturated compound in the presence of an oxidant to form the substituted pyrazolo [1,5-a ] pyridine compound.

Wherein the reaction concentration of the solvent is 0.001-100 mol/L (based on the amount of the 1-aminopyridine salt), the amount of the base is 0.1-10 equivalents, preferably 2 equivalents, the amount of the oxidant is 0.1-10 equivalents, preferably 1.2 equivalents, and the reaction temperature is-80-200 ℃, preferably 0-100 ℃.

The alkaline condition is organic alkali or inorganic alkali condition, and the organic alkali is an organic alkaline compound, including but not limited to: et (Et)₃N、DBU、DABCO、DIPEA、DMAP、MeONa、t-BuOK、t-BuONa、n-BuLi, LiHMDS, NaHMDS, KHMDS, pyridine, etc., preferably DIPEA, DBU; inorganic bases are inorganic basic compounds including, but not limited to: k₂CO₃、Na₂CO₃、Cs₂CO₃、CaCO₃、KHCO₃、NaHCO₃KOH, NaOH. Preferably Cs₂CO₃. Specifically, the method comprises the following steps: et (Et)₃N (triethylamine), DBU (1, 8-diazabicyclo [5.4.0 ]]Undec-7-ene), DABCO (1, 4-diazabicyclo [2.2.2 ]]Octane), DMAP (4-dimethylaminopyridine), MeONa (sodium methoxide), t-BuOK (potassium t-butoxide), t-BuONa (sodium t-butoxide), n-BuLi (n-butyllithium), LiHMDS (lithium hexamethyldisilazide), NaHMDS (sodium hexamethyldisilazide), KHMDS (potassium hexamethyldisilazide).

② the solvent is an organic solvent, including but not limited to: toluene, 1, 4-dioxane, acetonitrile, methanol, dichloromethane, DMF (N, N-dimethylformamide), THF (tetrahydrofuran), etc., preferably toluene.

③ the oxidizing agent is TEMPO, nitrogen oxides similar to TEMPO (such as 4-OH-TEMPO, 4-NH)₂TEMPO, 4-Oxo-TEMPO, ABNO, Keto-CHAMPO, AZADO), ammonium oxide salts of the above nitroxides (e.g. [ TEMPO ]][X]、[4-OH-TEMPO][X]、[4-NH₂-TEMPO][X]、[4-Oxo-TEMPO][X]、[ABNO][X]、[Keto-CHAMPO][X]、[AZADO][X])、O₂Air, t-BuOOH, (t-BuO)₂NaOCl, iodobenzene diacetate (PIDA), bistrifluoroacetyliodobenzene (PIFA), DDQ, tetrachlorobenzoquinone, MnO₂、SeO₂Cerium ammonium nitrate, nitric acid, Pd/C, amine nitroxide (such as N-Oxide), preferably TEMPO.

Wherein TEMPO (2,2,6, 6-tetramethylpiperidine-1-oxyl), 4-OH-TEMPO (4-hydroxy-2, 2,6, 6-tetramethylpiperidine-1-oxyl) and 4-NH₂TEMPO (4-amino-2, 2,6, 6-tetramethylpiperidin-1-oxyl), 4-Oxo-TEMPO (4-carbonyl-2, 2,6, 6-tetramethylpiperidin-1-oxyl), ABNO (9-azabicyclo [3.3.1 ] s]nonane-N-oxyl radical), Keto-CHAMPO (15-carbonyl-7-azabispiro [5.1.5.3 ]]Hexadecane-7-oxyl radical), N-Oxide (N-methylmorpholine Oxide), AZADO (2-azaadamantane-N-oxyl radical), t-BuOOH (tert-butyl peroxide), (t-BuO)₂(t-butyl peroxy ether), DDQ (2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone).

The ammonium oxide salt of a TEMPO-like nitroxide has the following structural formula:

wherein the content of the first and second substances,

comprises the following steps:

SO₄ ^2-，

PO₃ ^3-，HPO₃ ^2-，

one kind of (1).

Wherein R is⁴Is any one, two, three or four of hydrogen atom, halogen, alkyl, alkoxy, cycloalkyl, cyano, nitro, hydroxyl, sulfydryl, amino, ketone, ester, amide, sulfoxide and sulfone; wherein the alkyl, alkoxy, cycloalkyl, hydroxy, mercapto, amino, ketone, ester, amide, sulfoxide, sulfone may be further substituted with one or more groups selected from R^XIs substituted with the substituent(s). R^XAs disclosed above.

R⁵Is one of alkyl, (poly) haloalkyl and cycloalkyl, wherein alkyl, (poly) haloalkyl (e.g. CF)₃Etc.) or cycloalkyl may be further substituted by one or more groups selected from R^XIs substituted with the substituent(s). R^XAs disclosed above.

Alpha, beta-unsaturated compounds including alpha, beta-unsaturated aldehyde, alpha, beta-unsaturated ketone, alpha, beta-unsaturated aldehydeOne of an ester, an alpha, beta-unsaturated cyano compound, a beta 0, beta 1-unsaturated nitro compound and a beta 2, beta 3-unsaturated amide compound. The beta 4, beta 5-unsaturated aldehyde, beta 6, beta 7-unsaturated ketone, alpha, beta-unsaturated ester, alpha, beta-unsaturated cyano compounds, alpha, beta-unsaturated nitro compounds and alpha, beta-unsaturated amide compounds are respectively substituted by one or more independent R^XSubstituted with the substituent(s);

wherein, R is^XIs one of hydrogen atom, halogen, alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxyl and sulfonamide; the R is^XWherein the alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide are substituted with one or more hydrogen atoms, alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide, respectively.

In the present invention, the general reaction formula of the compound of formula (i) or a stereoisomer, tautomer, or salt thereof is:

wherein R in the reaction formula¹、R²And R³The same as for the compound of formula (I). The substituted 1-aminopyridine is a salt with X, the aminopyridine providing the positive ion moiety and X providing the negative ion moiety.

In the formula (I), the compound is shown in the specification,

comprises the following steps:

S0₄ ^2-，

PO₃ ^3-，HPO₃ ^2-，

one kind of (1).

Wherein R is⁴Is any one, two, three or four of hydrogen atom, halogen, alkyl, alkoxy, cycloalkyl, cyano, nitro, hydroxyl, sulfydryl, amino, ketone, ester, amide, sulfoxide and sulfone; wherein the alkyl, alkoxy, cycloalkyl, hydroxy, mercapto, amino, ketone, ester, amide, sulfoxide, sulfone may be further substituted with one or more groups selected from R^XIs substituted with the substituent(s). R^XAs disclosed above. R⁵Is one of alkyl, (poly) haloalkyl and cycloalkyl, wherein alkyl, (poly) haloalkyl (e.g. CF)₃Etc.) or cycloalkyl may be further substituted by one or more groups selected from R^XIs substituted with the substituent(s). R^XAs disclosed above.

3. The synthetic routes for the compounds of formula (I) in the case of different reaction starting materials are illustrated by the above synthetic methods and reaction formulae.

Firstly, in alkaline solution, substituted 1-aminopyridine compounds are added into organic solvent and react with acrylonitrile at the temperature of-80 ℃ to 200 ℃ in the presence of oxidant to obtain the substituted pyrazolo [1,5-a ] pyridine-3-carbonitrile compounds.

The general reaction formula is as follows:

② in alkaline solution, adding substituted 1-aminopyridine compound into organic solvent, in the presence of oxidant, stirring and reacting together with substituted nitrostyrene compound at-80 deg.C-200 deg.C to obtain substituted pyrazolo [1,5-a ] pyridine-2-aryl or substituted pyrazolo [1,5-a ] pyridine-2-aryl-3-nitro compound.

The general reaction formula is as follows:

thirdly, in alkaline solution, the substituted 1-aminopyridine compound is added into organic solvent, and is stirred and reacted with the substituted alpha, beta-unsaturated ketone compound at the temperature of minus 80 ℃ to 200 ℃ in the presence of oxidant, so as to obtain the substituted pyrazolo [1,5-a ] pyridine compound.

The general reaction formula is as follows:

and fourthly, in alkaline solution, adding the substituted 1-aminopyridine compound into an organic solvent, and stirring and reacting the substituted 1-aminopyridine compound and the substituted alpha, beta-unsaturated ketone compound at the temperature of between 80 ℃ below zero and 200 ℃ in the presence of an oxidant to obtain the substituted pyrazolo [1,5-a ] pyridine compound.

The reaction formula is as follows:

4. to further illustrate the synthesis of pyrazolopyridine derivatives of the present invention, the following materials were selectively synthesized, and the synthesis of each compound was illustrated with reference to specific examples.

Example 14 Synthesis of bromo-7-chloro-6-ethoxypyrazolo [1,5-a ] pyridine-3-carbonitrile (W1)

At room temperature, 1-amino-5-bromo-2-chloro-3-ethoxypyridine 2,4, 6-trimethylbenzenesulfonate (50.0mg,0.11mmol), TEMPO (20.6mg,0.13mmol), acrylonitrile (15.0 μ L,0.22mmol) and toluene (2.0mL) were added to a reaction tube, DIPEA (37.0 μ L,0.22mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 4-bromo-7-chloro-6-ethoxypyrazole [1,5-a ] bipyridine-3-carbonitrile (13.6mg, 61% yield).

The reaction equation is:

and (3) measuring the obtained product on a nuclear magnetic resonance instrument to obtain nuclear magnetic resonance data as follows:

¹H NMR(400MHz,CDCl₃)δ8.32(d,J＝1.2Hz,1H),8.13(s,1H),6.73(d,J＝1.0Hz,1H),4.23(q,J＝7.0Hz,2H),1.58(t,J＝7.0Hz,3H).¹³C NMR(101MHz,CDCl₃)δ149.99,145.28,134.99,122.59,113.52,109.06,108.50,83.04,65.65,14.14.HRMS(ESI+)Calcd.for[C₁₀H₇BrClN₃O]298.94610,Found 299.95285[M+H]⁺.

example Synthesis of 26-bromo-4-ethoxypyrazolo [1,5-a ] pyridine-3-carbonitrile (W2)

At room temperature, 1-amino-3-bromo-5-ethoxypyridine 2,4, 6-trimethylbenzenesulfonate (62.6.2mg,0.15mmol), TEMPO (28.1mg,0.18mmol), acrylonitrile (20.0 μ L,0.20mmol) and toluene (3.0mL) were added to a reaction tube, Diisopropylethylamine (DIPEA) (50.0 μ L,0.30mmol) was further added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 6-bromo-4-ethoxypyrazole [1,5-a ] pyridine-3-carbonitrile (28.0mg, 70% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.31(s,1H),8.13(s,1H),6.73(s,1H),4.23(q,J＝7.0Hz,2H),1.58(t,J＝7.0Hz,3H).¹³C NMR(101MHz,CDCl₃)δ149.99,145.27,134.99,122.59,113.52,109.06,108.50,83.03,65.64,14.13.HRMS(ESI+)Calcd.for[C₁₀H₈BrN₃O]264.98507,Found 265.99177[M+H]⁺.

example Synthesis of 36-bromo-4-benzyloxypyrazolo [1,5-a ] pyridine-3-carbonitrile (W3)

At room temperature, 1-amino-3-bromo-5-benzyloxypyridine 2,4, 6-trimethylbenzenesulfonate (72.0mg,0.15mmol), TEMPO (28.1mg,0.18mmol), acrylonitrile (20.0 μ L,0.30mmol) and toluene (3.0mL) were added to a reaction tube, Diisopropylethylamine (DIPEA) (50.0 μ L,0.30mmol) was further added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 6-bromo-4-benzyloxypyrazole [1,5-a ] pyridine-3-carbonitrile (37.5mg, 76% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.37(s,1H),8.19(s,1H),7.56(d,J＝7.5Hz,2H),7.46(t,J＝7.5Hz,2H),7.38(t,J＝7.2Hz,1H),6.86(s,1H).¹³C NMR(101MHz,CDCl₃)δ149.50,145.40,135.12,134.46,128.87,128.56,126.85,123.05,113.60,109.39,108.88,83.17,71.27.HRMS(ESI+)Calcd.for[C₁₅H₁₀BrN₃O]327.00072,Found 349.99003[M+H]⁺.

example Synthesis of 44-cyanomethylene-pyrazolo [1,5-a ] pyridine-3-carbonitrile (W4)

1-amino-cyanomethylene pyridine 2,4, 6-trimethylbenzenesulfonate (50.0mg,0.15mmol), TEMPO (28.1mg,0.18mmol), acrylonitrile (20.0. mu.L, 0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0. mu.L, 0.30mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 4-cyanomethylene pyrazole [1,5-a ] and pyridine-3-carbonitrile (18.0mg, 65% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.57(d,J＝7.0Hz,1H),8.29(s,1H),7.62(d,J＝7.2Hz,1H),7.09(t,J＝7.1Hz,1H),4.29(s,2H).¹³C NMR(101MHz,CDCl₃)δ146.25,145.99,140.30,130.00,127.36,121.46,118.07,115.38,114.45,20.70.HRMS(ESI+)Calcd.for[C₁₀H₆N₄]182.05925,Found 183.06615[M+H]⁺.

example Synthesis of 54, 7-dibromo-6-methyl-pyrazolo [1,5-a ] pyridine-3-carbonitrile (W5)

1-amino-2, 4-dibromo-3-methylpyridine 2,4, 6-trimethylbenzenesulfonate (70.0mg,0.15mmol), TEMPO (28.1mg,0.18mmol), acrylonitrile (20.0. mu.L, 0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0. mu.L, 0.30mmol) was added at 0 ℃, the reaction mixture was stirred at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 4, 7-dibromo-6-methyl-pyrazolo [1,5-a ] bipyridine-3-carbonitrile (40.8mg, yield 86%).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.32(s,1H),7.52(s,1H),2.51(s,1H).¹³C NMR(101MHz,CDCl₃)δ145.99,140.16,133.77,126.50,120.55,113.38 109.46,86.41,20.71.HRMS(ESI+)Calcd.for[C₉H₅Br₂N₃]312.88502,Found 313.89192[M+H]⁺.

EXAMPLE 6 Synthesis of pyrazolo [1,5-a ] quinoline-3-carbonitrile (W6)

1-amino-quinoline 2,4, 6-trimethylbenzenesulfonate (51.6mg,0.15mmol), TEMPO (28.1mg,0.18mmol), acrylonitrile (20.0. mu.L, 0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0. mu.L, 0.30mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 5:1) to give pyrazolo [1,5-a ] quinoline-3-carbonitrile (32.0mg, 95% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.58(d,J＝8.4Hz,1H),8.26(s,1H),7.85(dd,J＝8.0,1.3Hz,1H),7.77(ddd,J＝8.5,7.2,1.4Hz,1H),7.73(d,J＝9.3Hz,1H),7.62(d,J＝9.3Hz,1H),7.60-7.53(m,1H).¹³C NMR(101MHz,CDCl₃)δ143.89,140.93,134.31,130.84,129.02,128.78,126.33,123.51,116.08,114.26,113.77,85.19.HRMS(ESI+)Calcd.for[C₁₂H₇N₃]193.06400,Found 194.07069[M+H]⁺.

example Synthesis of 74-bromopyrazolo [1,5-a ] pyridine-3-carbonitrile (W7)

At room temperature, 1-amino-3-bromopyridine 2,4, 6-trimethylbenzenesulfonate (55.8mg,0.15mmol), TEMPO (28.1mg,0.18mmol), acrylonitrile (26.0 μ L,0.30mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (50.0 μ L,0.30mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 4-bromopyrazolo [1,5-a ] pyridine-3-carbonitrile (28.6mg, 86% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.55(t,J＝6.6Hz,1H),8.29(s,1H),7.62(d,J＝7.4Hz,1H),6.91(t,J＝7.2Hz,1H).¹³C NMR(101MHz,DMSO-d6)δ147.64,140.12,132.67,130.64,115.90,114.46,110.41,83.32.

EXAMPLE synthesis of 86-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile (W8)

The specific reaction process has the following 4 modes:

1. at room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (40.2mg,0.10mmol), TEMPO (18.8mg,0.12mmol), acrylonitrile (13.0 μ L,0.20mmol) and toluene (2.0mL) were added to a reaction tube, Diisopropylethylamine (DIPEA) (33.0 μ L,0.20mmol) was further added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile (23.4mg, 93% yield).

The reaction equation is:

¹H NMR(400MHz,DMSO-d6)δ8.94(d,J＝1.0Hz,1H),8.59(s,1H),7.24(s,1H),4.03(s,3H).¹³C NMR(101MHz,DMSO-d6)δ149.91,145.92,134.26,123.26,113.91,109.21,109.08,81.20,57.30.

under the same conditions, when DIPEA is used in an amount of 0.2, 0.5, 1.2 and 3.0 equivalents, yields of 32%, 45%, 70% and 93% respectively are obtained.

② when 2.0 equivalents of other base such as Et are used respectively under the same conditions as above₃N、DBU、K₂CO₃、Cs₂CO₃The yields were 25%, 33%, 49%, 90%, 53%, 34% when DIPEA was replaced by MeONa, t-BuOK, respectively.

③ under the same conditions as above, when other solvents such as DMF, THF, CH are used respectively₃CN、CH₂Cl₂When Dioxane was used instead of toluene, the yields were 60%, 40%, 52%, 45%, and 75%, respectively.

Respectively with other oxidizing agents, e.g. O, under the same conditions as above₂When TEMPO is replaced by NaCIO, TBHP (t-BuOOH) and PIDA, the yield is 20%, 32%, 10% and 42% respectively.

Fifthly, under the same conditions, when the amount of TEMPO is 0.2, 0.5, 1.2 and 2.0 equivalents, the yields are respectively 40%, 78%, 93% and 93%.

2. At room temperature, 1-amino-3-bromo-5-methoxypyridine 2, 4-dinitrophenolate (80.0mg,0.20mmol), TEMPO (37.5mg,0.24mmol), acrylonitrile (26.0 μ L,0.40mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (66.0 μ L,0.40mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1 to 5:1) to give 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile (26.7mg, 53% yield).

The reaction equation is:

3. at room temperature, 1-amino-3-bromo-5-methoxypyridine hydrogensulfate (60.0mg,0.20mmol), TEMPO (37.5mg,0.24mmol), acrylonitrile (26.0 μ L,0.40mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (66.0 μ L,0.40mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1 to 5:1) to give 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile (26.2mg, 52% yield).

The reaction equation is:

4. at room temperature, 1-amino-3-bromo-5-methoxypyridine hydrogensulfate (60.0mg,0.20mmol), [ TEMPO ] [ OTf ] (73.3mg,0.24mmol), acrylonitrile (26.0 μ L,0.40mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (66.0 μ L,0.40mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1 to 5:1) to give 6-bromo-4-methoxypyrazole [1,5-a ] pyridine-3-carbonitrile (25.2mg, 50% yield). Note: OTf is shorthand for trifluoromethanesulfonyl.

The reaction equation is:

under the same conditions as above, when [ TEMPO ] [ OTf ] was used in an amount of 0.2 equivalent, the yield was 30%.

EXAMPLE synthesis of 96-bromo-4-methoxy-2- (4-methoxy-phenyl) -pyrazolo [1,5-a ] pyridine (W9)

1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (45.0mg,0.11mmol), TEMPO (20.6mg,0.13mmol), (E) -1-methoxy-4- (2-nitroethylene) benzene (40.0mg,0.22mmol) and toluene (2.0mL) were added to a reaction tube at room temperature, DIPEA (37.0. mu.L, 0.22mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 6-bromo-4-methoxy-3-nitro-2- (4-methoxy-phenyl) -pyrazolo [1,5-a ] bipyridine (22.8mg, yield 59%).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.29(s,1H),7.68(d,J＝8.6Hz,2H),6.99(d,J＝8.6Hz,2H),6.83(s,1H),4.01(s,3H),3.86(s,3H).¹³C NMR(101MHz,CDCl₃)δ160.93,150.19,149.12,130.44,128.65,122.13,121.61,114.04,109.42,108.86,56.73,55.38.HRMS(ESI+)Calcd.For[C₁₅H₁₂BrN₃O₄]377.00112,Found 399.99059[M+Na]⁺.

example 106 Synthesis of bromo-4-methoxy-3-nitro-2- (thiophen-2) -yl-pyrazolo [1,5-a ] pyridine (W10)

At room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), (E) -2- (2-nitroethylene) thiophene (46.5mg,0.30mmol), and toluene (3.0mL) were added to a reaction tube, DIPEA (50.0 μ L,0.30mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 3h, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 20:1) to give 6-bromo-4-methoxy-3-nitro-2- (thiophen-2) -yl-pyrazolo [1,5-a ] bipyridine (35.0mg, 66% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.29(d,J＝1.0Hz,1H),7.89–7.83(m,1H),7.50(dd,J＝5.1,0.8Hz,1H),7.15(dd,J＝5.0,3.9Hz,1H),6.84(s,1H),4.01(s,3H).¹³C NMR(101MHz,CDCl₃)δ150.21,143.52,130.46,130.19,129.03,128.91,127.95,122.02,109.80,109.39,56.79.HRMS(ESI+)Calcd.for[C₁₂H₈BrN₃O₃S]352.94697,Found 375.93631[M+Na]⁺.

example 116 Synthesis of bromo-4-methoxy-2- (4-nitro) -phenyl-pyrazolo [1,5-a ] pyridine (W11)

1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), (E) -1-nitro-4- (2-nitroethylene) benzene (58.2mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0mL,0.30mmol) was added at 0 ℃, the reaction mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 15:1) to give 6-bromo-4-methoxy-2- (4-nitro) -phenyl-pyrazolo [1,5-a ] bipyridine (48.7mg, 83.0% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.32(d,J＝8.7Hz,3H),7.91(d,J＝8.3Hz,2H),6.90(s,1H),4.04(s,3H).¹³C NMR(101MHz,CDCl₃)δ150.60,148.67,135.96,130.25,126.98,124.30,123.81,122.34,110.21,110.14,57.04.HRMS(ESI+)Calcd.for[C₁₄H₉BrN₄O₅]391.97563,Found 392.98268[M+H]⁺.

EXAMPLE 126 Synthesis of bromo-4-methoxy-2- (4-chlorophenyl) -pyrazolo [1,5-a ] pyridine (W12)

1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), (E) -1-chloro-4- (2-nitroethylene) benzene (55.1mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0mL,0.30mmol) was added at 0 ℃, the reaction mixture was stirred at room temperature for 3 hours, the reaction mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 20:1) to give 4, 6-dibromo-2- (4-chlorophenyl) -pyrazolo [1,5-a ] bipyridine (53.1mg, 82% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.30(d,J＝1.1Hz,1H),7.66(d,J＝8.6Hz,1H),7.44(d,J＝8.6Hz,1H),6.86(s,1H),4.01(s,1H).¹³C NMR(101MHz,CDCl₃)δ150.34,148.29,136.18,130.38,128.86,127.88,122.18,109.54,109.36,56.82.HRMS(ESI+)Calcd.for[C₁₄H₉BrClN₃O₃]380.95158,Found 381.95811[M+H]⁺.

EXAMPLE 136 Synthesis of bromo-2- (1H-indol-3-yl) -4-methoxy-pyrazolo [1,5-a ] pyridine (W13)

At room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), (E) -3- (2-nitroethylene) -1H-indole (56.4mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (50.0mL,0.30mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 10:1) to give 6-bromo-2- (1H-indol-3-yl) -4-methoxy-pyrazolo [1,5-a ] bipyridine (37.5mg, 73% yield).

The reaction equation is:

¹H NMR(400MHz,DMSO-d6)δ11.45(s,1H),8.66(s,1H),8.27(d,J＝7.5Hz,1H),8.00(d,J＝2.6Hz,1H),7.45(d,J＝7.6Hz,1H),7.20-7.10(m,1H),7.02(s,1H),6.78(d,J＝0.8Hz,1H),3.99(s,1H).¹³C NMR(101MHz,DMSO-d6)δ150.10,137.04,134.52,125.67,125.40,122.18,121.34,120.22,112.23,108.72,104.77,92.54,56.81.HRMS(ESI+)Calcd.for[C₁₆H₁₂BrN₃O]341.01637,Found 342.02392[M+H]⁺.

EXAMPLE 146 Synthesis of bromo-4-methoxy-2- (1H-pyrrol-2-yl) -pyrazolo [1,5-a ] pyridine (W14)

1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), (E) -2-2- (2-nitroethylene) -1H-pyrrole (41.4mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0mL,0.30mmol) was added at 0 ℃, the reaction was stirred at room temperature for 3H, the reaction was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 10:1) to give 6-bromo-4-methoxy-2- (1H-pyrrol-2-yl) -pyrazolo [1,5-a ] pyridine (30.0mg, 68% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ9.25(s,1H),8.17(t,J＝1.0Hz,1H),6.86(td,J＝2.6,1.4Hz,1H),6.71(d,J＝0.9Hz,1H),6.63(ddd,J＝3.7,2.4,1.4Hz,1H),6.44(d,J＝1.3Hz,1H),6.29(q,J＝2.9Hz,1H),3.95(s,3H).¹³C NMR(101MHz,CDCl₃)δ150.28,146.71,142.9,135.22,125.28,123.34,121.77,118.99,109.76,107.73,105.14,104.20,91.72,56.01.HRMS(ESI+)Calcd.for[C₁₂H₁₀BrN₃O]291.00072,Found 292.00748[M+H]⁺.

example Synthesis of 152-phenyl [ d ] [1,3] dioxol-5-yl-methoxy-pyrazolo [1,5-a ] pyridine (W15)

1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (49.0mg,0.15mmol), TEMPO (28.1mg,0.18mmol), (E) -5- (2-nitroethylene) benzene [ d ] [1,3] dioxole (58.0mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0. mu.L, 0.30mmol) was added at 0 ℃, the reaction mixture was stirred at room temperature for 3h, the reaction mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 20:1) to give 2-phenyl [ d ] [1,3] dioxol-5-yl-methoxy-pyrazolo [1,5-a ] bipyridine (33.6mg, 83.0% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.11(d,J＝6.9Hz,1H),7.46(d,J＝7.2Hz,2H),6.92–6.85(m,1H),6.80(s,1H),6.63(t,J＝7.2Hz,1H),6.36(d,J＝7.6Hz,1H),6.00(s,2H),3.96(s,3H).¹³CNMR(101MHz,CDCl₃)δ152.37,150.71,148.05,147.77,136.58,127.47,121.62,120.25,111.22,108.58,106.92,101.14,99.43,91.79,55.65.HRMS(ESI+)Calcd.for[C₁₅H₁₂N₂O₃]⁺268.08479,Found 269.09156[M+H]⁺.

EXAMPLE 166 Synthesis of bromo-4-methoxy-2-phenylpyrazole [1,5-a ] pyridine-3-carbonitrile (W16)

At room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), cinnamonitrile (38.0 μ L,0.30mmol) and toluene (3.0mL) were added to a reaction tube, Diisopropylethylamine (DIPEA) (33.0 μ L,0.20mmol) was added at 0 ℃, the reaction solution was stirred at 80 ℃ for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 15:1) to give 6-bromo-4-methoxy-2-phenylpyrazole [1,5-a ] pyridine-3-carbonitrile (40.4mg, yield 82.0%).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.34(d,J＝1.2Hz,1H),8.16–8.07(m,2H),7.56-7.46(m,3H),6.76(d,J＝1.2Hz,1H),4.06(s,3H).¹³C NMR(101MHz,CDCl₃)δ150.73,130.29,129.15,127.67,122.77,108.22,56.86.HRMS(ESI+)Calcd.for[C₁₅H₁₀BrN₃O]327.00072,Found328.00718[M+H]⁺.

EXAMPLE 17 Synthesis of methyl 6-bromo-4-methoxy-2-phenylpyrazole [1,5-a ] pyridine-3-carboxylate (W17)

At room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), methyl cinnamate (48.7mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (50.0 μ L,0.30mmol) was added at 0 ℃, the reaction solution was stirred at 80 ℃ for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give methyl 6-bromo-4-methoxy-2-phenylpyrazole [1,5-a ] bipyridine-3-carboxylate (45.5mg, 84% yield).

The reaction equation is:

¹H NMR(600MHz,MeOH-d4)δ8.44(d,J＝1.2Hz,1H),7.67(dd,J＝7.7,1.8Hz,2H),7.43(t,J＝6.2Hz,3H),6.91(d,J＝1.0Hz,1H),3.98(s,3H),3.80(s,3H).¹³C NMR(151MHz,MeOH-d4)δ165.46,152.73,150.69,133.05,131.77,128.66,128.18,128.00,124.75,121.62,110.76,107.64,106.90,103.06,55.85,51.31.HRMS(ESI+)Calcd.for[C₁₆H₁₃BrN₂O₃]360.01096,Found 361.01809[M+H]⁺.

example Synthesis of 186-bromo-4-methoxy-pyrazolo [1,5-a ] pyridin-3-yl-phenyl methanone (W18)

1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), phenylpropenone (39.6mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (37.0. mu.L, 0.22mmol) was added at 0 ℃, the reaction mixture was stirred at 50 ℃ for 3 hours, the reaction mixture was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 15:1) to give 6-bromo-4-methoxy-pyrazolo [1,5-a ] pyridin-3-yl-phenyl ketone (46.6mg, 94% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.43(s,1H),8.17(s,1H),7.88(d,J＝7.5Hz,1H),7.63(t,J＝7.1Hz,1H),7.51(t,J＝7.4Hz,1H),6.76(s,1H),3.82(s,1H).¹³C NMR(101MHz,CDCl₃)δ188.86,151.53,145.18,139.80,132.72,132.49,129.70,128.22,122.69,113.97,108.39,108.11,56.38.HRMS(ESI+)Calcd.for[C₁₅H₁₁BrN₂O₂]330.00039,Found331.00788[M+H]⁺.

example 198 Synthesis of bromo-10-methoxy-3, 4-dihydropyridin [1,2-b ] indazol-1 (2H) -one (W19)

At room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (60.3mg,0.15mmol), TEMPO (28.1mg,0.18mmol), cyclohexenone (28.8mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (50.0 μ L,0.30mmol) was added at 0 ℃, the reaction solution was stirred at 80 ℃ for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 5:1) to give 8-bromo-10-methoxy-3, 4-dihydropyridine [1,2-b ] indazol-1 (2H) -one (33.2mg, 75% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.23(d,J＝1.4Hz,1H),6.77(d,J＝1.4Hz,1H),4.01(s,3H),2.97(t,J＝6.3Hz,2H),2.59(dd,J＝7.1,5.6Hz,2H),2.15(m,2H).¹³C NMR(101MHz,CDCl₃)δ191.64,161.04,151.87,131.98,122.51,110.65,109.52,108.74,56.78,39.85,23.79,23.39.HRMS(ESI+)Calcd.for[C₁₂H₁₁BrN₂O₂]294.00039,Found 295.00730[M+H]⁺.

example Synthesis of 209, 10-dihydroindazol [2,3-b ] quinolin-7 (8H) -one (W20)

1-amino-quinoline 2,4, 6-trimethylbenzenesulfonate (51.6mg,0.15mmol), TEMPO (28.1mg,0.18mmol), cyclohexenone (28.8mg,0.30mmol) and toluene (3.0mL) were added to a reaction tube at room temperature, DIPEA (50.0. mu.L, 0.30mmol) was added at 0 ℃, the reaction solution was stirred at 80 ℃ for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 5:1) to give 9, 10-dihydroindazole [2,3-b ] quinoline-7 (8H) -one (43.6mg, 95% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.57(d,J＝8.5Hz,1H),8.08(d,J＝9.2Hz,1H),7.83(dd,J＝8.0,1.3Hz,1H),7.78–7.70(m,2H),7.57–7.44(m,1H),3.08(t,J＝6.3Hz,2H),2.61(dd,J＝7.1,5.6Hz,2H),2.25(p,J＝6.3Hz,2H).¹³C NMR(101MHz,CDCl₃)δ194.46,159.68,137.43,134.00,130.47,129.36,128.68,125.65,124.07,116.80,116.19,111.18,39.03,24.04,23.60.HRMS(ESI+)Calcd.for[C₁₅H₁₂N₂O₂]236.09496,Found 237.10163[M+H]⁺.

example Synthesis of 214, 6-phenyl-pyrazolo [1,5-a ] pyridine 3-carbonitrile (W21)

Synthesis of Step1.4, 6-dibromopyrazolo [1,5-a ] pyridine-3-carbonitrile

At room temperature, 1-amino-3, 5-dibromopyridine 2,4, 6-trimethylbenzenesulfonate (90.2mg,0.20mmol), TEMPO (37.5mg,0.24mmol), acrylonitrile (26.0 μ L,0.40mmol) and toluene (3.0mL) were added to a reaction tube, DIPEA (66.0 μ L,0.40mmol) was added at 0 ℃, the reaction solution was stirred at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the obtained crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 6:1) to give 4, 6-dibromopyrazole [1,5-a ] bipyridine-3-carbonitrile (52.5mg, yield 87%).

The reaction equation is:

when TEMPO is used in an amount of 0.5 equivalent, 1.2 equivalent and 1.5 equivalent, the yields of the obtained product are 55%, 83% and 95%, respectively.

¹H NMR(400MHz,DMSO-d6)δ9.44(d,J＝1.4Hz,1H),8.76(s,1H),8.21(d,J＝1.4Hz,1H).¹³C NMR(101MHz,CDCl₃)δ147.05,139.40,134.03,129.46,113.20,111.95,108.35.

step2. a reaction tube was charged with 4, 6-dibromopyrazolo [1,5-a ] pyridine-3-carbonitrile (30.0mg,0.10mmol), phenylboronic acid (12.2mg,0.10mmol), potassium carbonate (16.6mg,0.12mmol), tetrakis (triphenylphosphine) palladium (34.7mg,0.03mmol), and ethylene glycol dimethyl ether (DME): water ═ 3:1(4ml) at room temperature, the reaction solution was stirred at 80 ℃ for 12 hours, the reaction solution was concentrated under reduced pressure to give a crude product, and the crude product was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give 4, 6-phenyl-pyrazolo [1,5-a ] pyridine 3-carbonitrile (20.0mg, 67% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.78(s,1H),8.30(s,1H),7.70–7.37(m,11H).¹³C NMR(151MHz,CDCl₃)δ147.20,139.42,135.80,134.90,133.19,129.56,129.42,129.15,128.69,128.34,127.03,125.87,113.92,82.75.HRMS(ESI+)Calcd.for[C₂₀H₁₃N₃]295.11095,Found296.11765[M+H]⁺.

example 22 Synthesis of (E) -1- (6-bromo-4-methoxy-2-phenyl-pyrazolo [1,5-a ] pyrid-in-3-yl) -3-phenylpropenone (W22)

At room temperature, 1-amino-3-bromo-5-methoxypyridine 2,4, 6-trimethylbenzenesulfonate (86.1mg,0.25mmol), TEMPO (39.1mg,0.25mmol), dibenzylideneacetone (23.4mg,0.1mmol) and toluene (5.0mL) were added to a reaction tube, DIPEA (66.0 μ L,0.40mmol) was added at 0 ℃, the reaction solution was stirred at 80 ℃ for 3 hours, the reaction solution was concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (eluent petroleum ether/ethyl acetate ═ 8:1) to give (E) -1- (6-bromo-4-methoxy-2-phenyl-pyrazolo [1,5-a ] pyridopyridin-3-yl) -3-phenylpropenone (45.0mg, 70% yield).

The reaction equation is:

¹H NMR(400MHz,CDCl₃)δ8.39(s,1H),7.76(d,J＝7.2Hz,2H),7.40(dt,J＝21.0,6.4Hz,9H),7.03(t,J＝14.0Hz,1H),6.66(s,1H),3.92(s,3H).HRMS(ESI+)Calcd.for[C₂₃H₁₇BrN₂O₂]432.04734,Found 433.05416[M+H]⁺.

example 23 related content and results of estrogen biosynthesis

Human ovarian granulosa cells (KGN) are obtained from tumor specimens where nuclei were removed from patients who had locally recurred after menopause granulosa cell tumors, and this KGN cell line is very unique and of great importance in research as it maintains most physiological activities, including the expression of functional FSH receptors, and the same pattern of steroidogenesis and Fas-mediated apoptosis observed in normal granulosa cells. The cell also has aromatase activity, and can catalyze androstenedione and testosterone to remove 19-carbon and aromatize A ring to respectively form estrone and estradiol, which are rate-limiting enzymes of estrogen biosynthesis.

Experimental methods

(ii) cell culture

KGN cells were cultured in DMEM/F-12 medium containing 5% fetal bovine serum, 100U/mL penicillin, 0.1mg/mL streptomycin, and the like. Cells were incubated at 37 ℃ with 5% CO₂In an incubator (Tai et al, 35, 2012, Biological)&Pharmaceutical Bulletin)。

② cell proliferation test

KGN cells in logarithmic growth phase were seeded in 96-well plates (5X 10)³One/well), 100. mu.L of complete medium per well. Incubating overnight, replacing with fresh complete culture medium, adding 0.1% DMSO and compounds with different concentrations, setting 3 multiple wells for each group, culturing for 48h, removing old culture medium, adding 100 μ L fresh culture medium containing 10% Cell Counting Kit-8(CCK8) reagent, incubating for 1-4h in Cell incubator, and detecting absorbance at 450nm with microplate reader when color changesPhotometry (Lu et al, 60, 2012, Journal of Agricultural and Food Chemistry).

③ biosynthesis of Estrogen in cells

1. Taking KGN cells in a logarithmic growth phase, inoculating the KGN cells in a 24-well plate, culturing overnight, then replacing the KGN cells with a serum-free culture medium, adding compounds with different concentrations and 1 mu L of DMSO respectively, adding Forskolin (FSK) with a final concentration of 10 mu mol/L and Letrozole (LET) with a final concentration of 10nmol/L as positive controls, arranging 2 multiple wells in each group, after continuously culturing for 24 hours, replacing the culture medium with a new serum-free culture medium, adding testosterone with a final concentration of 10nmol/L as a substrate into each well, continuously culturing for 24 hours, collecting supernatant, and storing at-20 ℃. RIPA lysed cells extracted total protein and concentration detected with BCA kit;

2. the estrogen detection kit is placed at room temperature 30min in advance, and the instrument is preheated. Melting the collected supernatant at room temperature, and centrifuging at 10000rpm for 2 min;

3. respectively taking 30 mu L of supernatant from the standard substance and the experimental sample with the concentration gradient, adding to the bottom of the corresponding test tube, adding 60 mu L of antigen reagent A, uniformly mixing, and performing water bath at 37 ℃ for 15 min; adding 60 μ L of antigen reagent B into each test tube, mixing uniformly, and performing water bath at 37 ℃ for 15 min; adding 30 mu L of the uniformly mixed magnetic beads into each test tube, uniformly mixing by using an oscillator, and carrying out water bath at 37 ℃ for 5 min; placing the test tube rack on a magnetic bead separator, fully contacting the bottom of the test tube with the separator, and standing at room temperature for 2 min; rapidly turning over the magnetic bead separator to remove the upper liquid, then inverting the magnetic bead separator on filter paper, uniformly and vigorously beating the bottom of the magnetic bead separator, and removing the residual liquid; then, a magnetic bead separator is arranged in an upright way, 300 mu L of washing solution is added into each tube, the mixture is uniformly mixed and then stands for 2min, and the step is repeated for 3 times; sequentially placing the test tubes into a semi-automatic chemiluminescence apparatus for detection, and calculating the concentration of E2 according to the obtained luminescence value; the concentration of sample E2 was calibrated using the protein concentration measured above, as the relative E2 content-E2 concentration/protein concentration (schwann et al, 25, 2019, applied and environmental biosciences).

Experimental results

First, 22 compounds were tested for their cytotoxic effect on KGN cells, and as shown in fig. 1, the results showed that 9 compounds W4, W5, W6, W7, W9, W11, W13, W15, and W19 had less than 20% cytotoxic effect on KGN cells at 10 μmol/L. We further examined the effect of these 9 compounds on estrogen biosynthesis. Referring to fig. 2, the results show that W5, W6, W7, W9, W15 and W19 have obvious promotion effect on estrogen biosynthesis, and that W4, W11 and W13 have obvious inhibition effect on the biosynthesis of multiple estrogens. The detailed results are shown in table 1 below.

TABLE 1 influence of pyrazolo [1,5-a ] pyridine derivatives on KGN cell estrogen biosynthesis

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A compound of formula (I) or a stereoisomer, tautomer or salt thereof,

wherein R is¹Is one of hydrogen atom, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclic aryl;

R²is one of hydrogen atom, cyano, nitro, ketone, ester, amide, sulfoxide and sulfone;

R³is hydrogen atom, halogen, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl or arylOne or more of a group, a heterocyclic aryl group, a cyano group, a nitro group, a hydroxyl group, a mercapto group, an amino group, a substituted amino group, a ketone, an ester, an amide, a sulfoxide, and a sulfone.

2. A compound of formula (i) or a stereoisomer, a tautomer, or a salt thereof according to claim 1, wherein: r¹Wherein said alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl are each independently substituted with one or more R^XSubstituted with the substituent(s);

R²wherein said ketone, ester, amide, sulfoxide and sulfone are each independently substituted with one or more R^XSubstituted with the substituent(s);

R³wherein said alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, hydroxy, mercapto, amino, substituted amino, ketone, ester, amide, sulfoxide, and sulfone are each independently substituted with one or more R^XSubstituted with the substituent(s);

3. A compound of formula (i) or a stereoisomer, a tautomer, or a salt thereof according to claim 1, wherein: the R is^XWherein the alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide are substituted with one or more hydrogen atoms, alkyl, fluoroalkyl, alkenyl, alkynyl, nitro, cyano, cycloalkyl, aryl, amino, alkoxy, substituted amino, amido, hydroxy, sulfonamide, respectively.

4. A process for the synthesis of a compound of formula (i) or a stereoisomer, tautomer or salt thereof, characterized in that: substituted 1-aminopyridines are suspended in a solvent under basic conditions and stirred with an alpha, beta-unsaturated compound in the presence of an oxidizing agent to form substituted pyrazolo [1,5-a ] pyridines.

5. A process for the synthesis of a compound of formula (i) or a stereoisomer, a tautomer, or a salt thereof, according to claim 4, wherein: the reaction concentration of the solvent is 0.001-100 mol/L, the dosage of the alkali is 0.1-10 equivalents, the dosage of the oxidant is 0.1-10 equivalents, and the reaction temperature is-80-200 ℃.

6. A process for the synthesis of a compound of formula (i) or a stereoisomer, a tautomer, or a salt thereof, according to claim 4, wherein: the solvent is one of toluene, 1, 4-dioxane, acetonitrile, methanol, dichloromethane, DMF and THF; the oxidant is TEMPO, nitrogen oxide similar to TEMPO, ammonium oxide salt of the nitrogen oxide, and O₂Air, t-BuOOH, (t-BuO)₂NaOCl, iodobenzene diacetate, iodobenzene bistrifluoroacetate, DDQ, tetrachlorobenzoquinone, MnO₂、SeO₂One of cerium ammonium nitrate, nitric acid, Pd/C and amine oxynitride;

wherein the nitrogen oxide similar to TEMPO is 4-OH-TEMPO or 4-NH₂One of TEMPO, 4-Oxo-TEMPO, ABNO, Keto-CHAMPO and AZADO, wherein the ammonium oxide salt of the nitrogen oxide is [ TEMPO ]][X]、[4-OH-TEMPO][X]、[4-NH₂-TEMPO][X]、[4-Oxo-TEMPO][X]、[ABNO][X]、[Keto-CHAMPO][X]、[AZADO][X])；

[X]Comprises the following steps:

one of (1);

wherein R is⁴Is any one, two, three or four of hydrogen atom, halogen, alkyl, alkoxy, cycloalkyl, cyano, nitro, hydroxyl, sulfydryl, amino, ketone, ester, amide, sulfoxide and sulfone; r⁵Is one of alkyl, (poly) haloalkyl and cycloalkyl.

7. A process for the synthesis of a compound of formula (i) or a stereoisomer, a tautomer, or a salt thereof, according to claim 4, wherein: the alpha, beta-unsaturated compound comprises one of alpha, beta-unsaturated aldehyde, alpha, beta-unsaturated ketone, alpha, beta-unsaturated ester, alpha, beta-unsaturated cyano compounds, alpha, beta-unsaturated nitro compounds and alpha, beta-unsaturated amide compounds.

8. A pharmaceutical composition characterized by: a compound of formula (i) as claimed in any one of claims 1 to 4 or a stereoisomer, tautomer or salt thereof, or prodrug molecule thereof, and a pharmaceutically acceptable carrier.

9. Use of a compound of formula (i) or a stereoisomer, tautomer, or salt or prodrug molecule thereof in the manufacture of a medicament for the treatment of a tumour, and/or a metabolic disorder.

10. Use according to claim 9, characterized in that: the tumor is breast cancer and ovarian cancer; and/or the metabolic diseases are hypoestrogenic dysfunctional uterine bleeding and senile vaginitis, and osteoporosis caused by estrogen deficiency.