CN112661775A

CN112661775A - Substituted macrocyclic quinoxaline compound, pharmaceutical composition and application thereof

Info

Publication number: CN112661775A
Application number: CN202110127555.2A
Authority: CN
Inventors: 王义汉; 赵九洋
Original assignee: Shenzhen Targetrx Inc
Current assignee: Shenzhen Targetrx Inc
Priority date: 2016-05-16
Filing date: 2017-05-11
Publication date: 2021-04-16
Also published as: WO2017198102A1; CN108368130B; CN108368130A

Abstract

The invention provides a substituted macrocyclic quinoxaline compound, a pharmaceutical composition and an application thereof, wherein the macrocyclic quinoxaline compound is a compound shown as a formula (I), or a polymorphism, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvent compound thereof. The compound can be used as a hepatitis C virus inhibitor, has better hepatitis C virus protein NS3A inhibition activity, better pharmacodynamics/pharmacokinetics performance, good applicability and high safety, can be used for preparing a medicament for treating hepatitis C virus infection, and has good market development prospect.

Description

Substituted macrocyclic quinoxaline compound, pharmaceutical composition and application thereof

The application is a divisional application of an invention patent application with the application date of 2017, 05 and 11, the application number of 201780004350.7 and the invention name of 'a substituted macrocyclic quinoxaline compound and a pharmaceutical composition and application thereof'.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a hepatitis C virus inhibitor, a pharmaceutical composition and application thereof.

Background

HCV (Hepatitis C Virus) is an RNA Virus belonging to the genus Hepatitis C Virus (Hepacivirus genus) in the family Flaviviridae (Flaviviridae family). The encapsulated HCV virions contain a positive strand RNA genome that encodes all known virus-specific proteins in a single uninterrupted open reading frame. The open reading frame comprises approximately 9500 nucleotides and encodes a single large polyprotein of about 3000 amino acids. The polyprotein includes the core protein, the envelope proteins E1 and E2, the membrane bound protein P7, and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS 5B.

Sofosbuvir is the first good medicine in the world at present and can completely cure hepatitis C in a short period. It directly reaches the focus by oral administration, has simple method and little side effect, and is deeply touted by patients. Sofosbuvir is produced by Gilidide, USA, is marketed in 2013, and can effectively treat type-1, type-2, type-3 and type-4 hepatitis C through clinical tests, including clinical tests on liver transplantation, liver cancer and HCV/HIV-1 combined infection. The breakthrough brings good news to hepatitis C patients all over the world.

HCV infection is associated with progressive liver disease symptoms, including cirrhosis and hepatocellular carcinoma. Grazoprevir is an NS3 inhibitor developed by mazzuon. In 2016, month 1, the united states Food and Drug Administration (FDA) approved the drug elbasvir/gradoprevir (zepatier) combination of the mersandeast (Merck & Co) for the treatment of chronic Hepatitis C (HCV) gene type 1 and type 4 infections. The combination does not require the concomitant use of interferon, and avoids all serious adverse events of interferon therapy.

Therefore, there is still a need in the art to develop compounds having inhibitory activity or better pharmacodynamic properties against the hepatitis c virus protein NS 3.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a hepatitis c virus inhibitor, a pharmaceutical composition and use thereof, which has better hepatitis c virus protein NS5A inhibitory activity and/or better pharmacodynamic/pharmacokinetic properties.

In contrast, the technical scheme adopted by the invention is as follows:

a hepatitis C virus inhibitor is a macrocyclic quinoxaline compound shown as a formula (I), or a polymorphism, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvent compound thereof,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹、R³⁰、R³¹、R³²、R³³、R³³、R³⁴、R³⁵、R³⁶、R³⁷、R³⁸、R³⁹、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴、R⁴⁵、R⁴⁶、R⁴⁷Each independently is hydrogen, deuterium, halogen or trifluoromethyl;

with the proviso that R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹、R³⁰、R³¹、R³²、R³³、R³³、R³⁴、R³⁵、R³⁶、R³⁷、R³⁸、R³⁹、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴、R⁴⁵、R⁴⁶And R⁴⁷At least one of which is deuterated or deuterium.

By adopting the technical scheme, the shape and the volume of deuterium in a drug molecule are basically the same as those of hydrogen, and if the hydrogen in the drug molecule is selectively replaced by deuterium, the original biological activity and selectivity of the deuterium-substituted drug can be generally kept. Meanwhile, the inventor proves that the combination of carbon and deuterium bonds is more stable than the combination of carbon and hydrogen bonds, and the absorption, distribution, metabolism, excretion and other properties of some medicines can be directly influenced, so that the curative effect, safety and tolerance of the medicines are improved.

Preferably, the deuterium isotope content of deuterium at the deuterated position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹、R³⁰、R³¹、R³²、R³³、R³³、R³⁴、R³⁵、R³⁶、R³⁷、R³⁸、R³⁹、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴、R⁴⁵、R⁴⁶And R⁴⁷The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

Preferably, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹、R³⁰、R³¹、R³²、R³³、R³³、R³⁴、R³⁵、R³⁶、R³⁷、R³⁸、R³⁹、R⁴⁰、R⁴¹、R⁴²、R⁴³、R⁴⁴、R⁴⁵、R⁴⁶And R⁴⁷Preferably, at least one of R comprises deuterium, more preferably two of R comprises deuterium, more preferably three of R comprises deuterium, more preferably four of R comprises deuterium, more preferably five of R comprises deuterium, more preferably six of R comprises deuterium, more preferably seven of R comprises deuterium, more preferably eight of R comprises deuterium, more preferably nine of R comprises deuterium, more preferably ten of R comprises deuterium, more preferably eleven of R comprises deuterium, more preferably twelve of R comprises deuterium, more preferably thirteen of R comprises deuterium, more preferably fourteen of R comprises deuterium, more preferably fifteen of R comprises deuterium, more preferably sixteen of R comprises deuterium, more preferably seventeen of R comprises deuterium, more preferably eighteen of R comprises deuterium, more preferably nineteen of R comprises deuterium, more preferably twenty of R comprises deuteriumPreferably twenty one R, more preferably twenty two R, more preferably twenty three R, more preferably twenty four R, more preferably twenty five R, more preferably twenty six R, more preferably twenty seven R, more preferably twenty eight R, more preferably twenty nine R, more preferably thirty five R, more preferably thirty one R, more preferably thirty two R, more preferably thirty three R, more preferably thirty four R, more preferably thirty five R, more preferably thirty six R, more preferably thirty seven R, more preferably thirty eight R, more preferably thirty nine R, more preferably forty four R, more preferably forty five R, more preferably forty one forty R, more preferably forty two R, more preferably forty three R, more preferably forty-four R, more preferably forty-five R, more preferably forty-six R, more preferably forty-seven R.

As a further improvement of the invention, R¹、R²And R³Each independently is deuterium or hydrogen.

In another preferred embodiment, R¹、R²、R³Is deuterium.

As a further improvement of the invention, R⁴、R⁵And R⁶Each independently is deuterium or hydrogen.

In another preferred embodiment, R⁴、R⁵、R⁶Is deuterium.

As a further improvement of the invention, R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹And R²⁰Each independently is deuterium or hydrogen.

In another preferred embodiment, R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰Is deuterium.

As an originalIn a further development of the invention, R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹And R³⁰Each independently is deuterium or hydrogen.

In another preferred embodiment, R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹、R³⁰Is deuterium.

As a further improvement of the invention, R³¹、R³²、R³³、R³³、R³⁴、R³⁵And R³⁶Each independently is deuterium or hydrogen.

In another preferred embodiment, R³¹、R³²、R³³、R³³、R³⁴、R³⁵、R³⁶Is deuterium.

As a further improvement of the invention, R³⁷、R³⁸And R³⁹Each independently is deuterium or hydrogen.

In another preferred embodiment, R³⁷、R³⁸、R³⁹Is deuterium.

As a further improvement of the invention, R⁴⁰、R⁴¹And R⁴²Each independently is deuterium or hydrogen.

In another preferred embodiment, R⁴⁰、R⁴¹、R⁴²Is deuterium.

As a further improvement of the invention, R⁴³、R⁴⁴、R⁴⁵、R⁴⁶And R⁴⁷Each independently is deuterium or hydrogen.

In another preferred embodiment, R⁴³、R⁴⁴、R⁴⁵、R⁴⁶、R⁴⁷Is deuterium.

As a further improvement of the present invention, the compound is selected from the following compounds or pharmaceutically acceptable salts thereof:

in another preferred embodiment, the compound does not include non-deuterated compounds.

The invention also discloses a pharmaceutical composition which contains a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor, or a polymorphism, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof.

As a further improvement of the present invention, it also comprises other active compounds including, but not limited to, other HCV antivirals, anti-infectives, immunomodulators, antibiotics or vaccine combinations.

As a further improvement of the invention, the immunomodulator is an interferon drug compound.

As a further improvement of the invention, the quinoxaline macrocyclic compounds of the invention may be used in combination therapy involving one or more additional therapeutic agents. Additional therapeutic agents include those that also target HCV, target different pathogenic agents, or enhance the immune system. Agents that enhance the immune system include those that generally enhance immune system function and those that generate specific immune responses against HCV. Additional therapeutic agents targeting HCV include agents that target NS3 and agents that target other HCV activities, such as NS5A and NS5B, and agents that target host cell activities involved in HCV replication.

Other examples of therapeutic agents that may be present in the combination include ribavirin, levovirin, viramidine, thymosin alpha-1, interferon-beta, interferon-alpha, pegylated interferon-alpha (PEG interferon-alpha), a combination of interferon-alpha and ribavirin, a combination of PEG interferon-alpha and ribavirin, a combination of interferon-alpha and levovirin, and a combination of PEG interferon-alpha and levovirin. Interferon- α includes recombinant interferon- α 2a (e.g., Roferon interferon available from Hoffmann-LaRoche, Nutley, NJ), pegylated interferon- α 2a (pegasys), interferon- α 2b (e.g., Intron-a interferon available from Schering corp., Kenilworth, NJ), pegylated interferon- α 2b (pegintron), recombinant consensus interferon (e.g., interferon alphacon-1), and purified interferon- α products. The individual components of the combination may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.

For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with the antiviral agent amantadine (1-aminoadamantane). For a complete description of this agent, see J.Kirschbaum,12anal. profiles Drug Subs.1-36 (1983).

For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with the antiviral agent polymerase inhibitor R7128 (Roche).

For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with an HCV NS5B polymerase inhibitor. Such HCV NS5B polymerase inhibitors that may be used as combination therapies include, but are not limited to, international patent application publications WO 02/057287, WO 02/057425, WO 03/068244, WO 2004/000858, WO 04/003138, and WO 2004/007512; U.S. patent No.6,777,392 and U.S. patent application publication US2004/0067901 (the contents of each of which are incorporated herein by reference in their entirety). Other such HCV polymerase inhibitors include, but are not limited to, valopicitabine (NM-283; Idenix) and 2 '-F-2' - β -methylcytidine (see also WO 2005/003147).

As a further improvement of the invention, the pharmaceutically acceptable carrier comprises at least one of a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersing agent, a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent, or an emulsifier.

As a further improvement of the present invention, the pharmaceutical composition is a tablet, pill, capsule, powder, granule, paste, emulsion, suspension, solution, suppository, injection, inhalant, gel, microsphere or aerosol.

Typical routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: mixing a pharmaceutically acceptable carrier and the hepatitis C virus inhibitor or the crystal form, the pharmaceutically acceptable salt, the hydrate or the solvate thereof to form the pharmaceutical composition.

The invention also discloses an application of the hepatitis C virus inhibitor, which is characterized in that: the application of the compound in preparing the medicine for treating hepatitis C virus infection.

NS3 inhibitors may also be useful in the preparation and implementation of screening assays for antiviral compounds. For example, such compounds can be used to isolate enzyme mutants, which are excellent screening tools for more potent antiviral compounds. In addition, these compounds can be used to establish or determine binding sites for other antiviral agents to HCV protease, for example, by competitive inhibition.

The compounds of the present invention, optionally in salt form, may be administered by contacting the active agent with the site of action of a drug for the purpose of inhibiting the HCV NS3 protease and treating HCV infection and/or reducing the likelihood or severity of symptoms of HCV infection. They may be administered as separate therapeutic agents or combinations of therapeutic agents by conventional means which may be used in conjunction with a drug. They may be administered alone, but will generally be administered with a pharmaceutical carrier selected in accordance with the chosen route of administration and standard pharmaceutical practice.

The HCV includes its various genotypes and various gene subtypes, e.g. 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6 a.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Herein, "halogen" means F, Cl, Br, and I, unless otherwise specified. More preferably, the halogen atom is selected from F, Cl and Br.

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The invention also includes isotopically-labeled compounds (also referred to as "isotopic variations"), equivalent to those disclosed herein for the original compound. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of formula (I) of the present invention, or polymorphs, pharmaceutically acceptable salts, prodrugs, stereoisomers, isotopic variations, hydrates or solvates thereof, containing the aforementioned isotopes or other isotopic atoms are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. In addition, heavier isotopes such as deuterium, i.e.²H, due to its good metabolic stability, may be advantageous in certain therapies, such as increased half-life in vivo or reduced dose, and therefore, may be preferred in certain circumstances. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "polymorphs" refers to the different arrangements of chemical drug molecules, typically expressed as the presence of the drug material in a solid state. One drug can exist in a plurality of crystal form substances, and different crystal forms of the same drug can be dissolved and absorbed in vivo differently, so that the dissolution and release of the preparation can be influenced.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

The term "prodrug" refers to a compound that is converted in vivo by hydrolysis, for example in the blood, to its active form with a medicinal effect. A prodrug is any covalently bonded carrier that releases a compound of the invention in vivo when such prodrug is administered to a patient. Prodrugs are typically prepared by modifying functional groups such that the prodrug is cleaved in vivo to yield the parent compound. Prodrugs include, for example, compounds of the present invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, cleaves to form a hydroxy, amino, or sulfhydryl group. Thus, representative examples of prodrugs include, but are not limited to, covalent derivatives of the compounds of the present invention with acetic acid, formic acid, or benzoic acid through a hydroxy, amino, or mercapto functional group therein. In addition, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, and the like may be used. The ester itself may be active and/or may hydrolyze under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable esters include those which readily break down in the human body to release the parent acid or salt thereof.

The compounds of the invention may include one or more asymmetric centers, and thus may exist in a variety of "stereoisomeric" forms, e.g., enantiomeric and/or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from mixtures by methods known to those skilled in the art, including: chiral High Pressure Liquid Chromatography (HPLC) and the formation and crystallization of chiral salts; alternatively, preferred isomers may be prepared by asymmetric synthesis.

Compared with the prior art, the invention has the beneficial effects that: first, the compounds of the present invention have excellent inhibitory activity against the hepatitis c virus protein NS 3. Second, the metabolism of compounds in organisms is altered by deuteration, which results in compounds with better pharmacokinetic parameters. In this case, the dosage can be varied and a long acting formulation formed, improving the applicability. Thirdly, deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body is improved, and the medicine curative effect is improved. Fourthly, the replacement of hydrogen atoms in the compound by deuterium can inhibit certain metabolites and improve the safety of the compound.

Detailed Description

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 100 ℃, preferably from 0 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

Example 1a substituted macrocyclic quinoxaline compound G-1 is prepared having the formula:

the synthesis was carried out using the following route:

step 1. Synthesis of diethyl [2- (dimethylamino) -2-oxoethyl ] phosphate (Compound 3).

Compound 2(10.5g, 63.65mmol) was added to a three-necked flask under nitrogen and heated to 110 ℃. After dropwise addition of Compound 1(10g, 82.65mmol), the reaction mixture was reacted at 110 ℃ for 3 to 4 hours, concentrated at 65 ℃ to give a crude product, which was purified by column chromatography to give 7.7g of Compound 3, yield 54.5%, LC-MS (APCI) M/z 224(M +1)⁺，¹H NMR(300MHz,CDCl₃)δ4.22-4.08(m,4H),3.11(s,3H),3.08(d,J＝2.4Hz,2H),2.99-2.91(m,3H),1.32(t,J＝7.1Hz,6H)。

Step 2.7-chloro-2-hydroxy-1-heptene (Compound 6) synthesis.

10mL of dimethyltetrahydrofuran was added under nitrogen, magnesium (2g, 82.29mmol) was added, 90mg of iodine was added, and Compound 4(10g, 70.0) was slowly added dropwise4mmol), reacting at 70 ℃ for 3-4 hours after completion of dropwise addition, cooling to room temperature for standby, adding compound 3(5.26g, 56.82mmol), CuI (530mg, 2.78mmol) and 28mL of dimethyltetrahydrofuran into a three-necked bottle under the protection of nitrogen, cooling to-60 ℃, slowly dropwise adding the above format reagent, controlling the reaction system not to exceed-50 ℃, stirring and reacting for 1 hour after completion of dropwise addition, detecting by TLC that the raw materials are completely reacted, adding an ammonium chloride aqueous solution to quench the reaction, stirring for 30 minutes after raising to room temperature, extracting for three times by using tert-butyl methyl ether, combining organic phases, washing by using saturated salt solution, drying by using sodium sulfate, concentrating to obtain 9g of anhydrous compound 6, wherein the yield is 95.14%, and the anhydrous compound is directly used for the next step.¹H NMR(300MHz,CDCl₃)δ5.81(m,J＝16.9,10.2,6.7Hz,1H),5.09-4.91(m,2H),3.80(t,J＝7.4Hz,1H),3.69-3.61(m,1H),3.49(m,J＝11.1,7.2Hz,1H),2.16(t,J＝5.2Hz,1H),2.09(t,J＝6.6Hz,2H),1.58-1.49(m,3H)。

Step 3 Synthesis of (1R,2R) -2-pent-4-enyl-N, N-dimethylcyclopropylcarboxamide (Compound 7).

Adding compound 6(3.579g, 24.182mmol) and compound 3(5.39g, 24.18mmol) into a three-reaction-port bottle under the protection of nitrogen, adding 70mL of dimethyl tetrahydrofuran, cooling to-35 ℃, slowly adding n-butyl lithium (29mL, 72.546mmol) dropwise, keeping the temperature of a reaction system not to exceed-20 ℃, dropwise adding for about 1 hour, naturally heating to room temperature after reacting for 20 minutes, reacting for 1 hour after heating to 72 ℃, changing the reaction liquid into light yellow, detecting that the raw materials are completely reacted by TLC, cooling to room temperature, dropwise adding 10% sodium chloride solution to quench the reaction, extracting with ethyl acetate for three times, combining organic phases, drying with anhydrous sodium sulfate, concentrating, purifying by column chromatography to obtain 1.83g of compound 8, wherein the yield is 41.9%,¹H NMR(300MHz,CDCl₃)δ5.90-5.70(m,1H),5.05-4.84(m,2H),3.14(d,J＝11.1Hz,3H),2.99-2.91(m,3H),2.14-2.02(m,2H),1.60-1.42(m,3H),1.41-1.25(m,3H),1.15(m,J＝6.4,4.5Hz,1H),0.95-0.85(m,1H),0.60(m,J＝5.2,3.7Hz,1H)。

step 4 Synthesis of (1R,2R) -2-pent-4-enyl cyclopropylmethyl ketone (Compound 8).

Methyl magnesium chloride (16.9mL, 50.94mmol) is added into a three-mouth reaction bottle under the protection of nitrogen and heated to 60 ℃, dimethyl tetrahydrofuran solution (4.16g, 25.47mmol, 20mL) of the compound 7 is slowly dripped at the temperature, the mixture reacts for 2 hours at 60 ℃ after the dripping is finished, TLC detects that the raw material is completely reacted, the mixture is quenched by ammonium chloride aqueous solution, extracted by n-hexane for three times, organic phases are combined, washed by 1M hydrochloric acid, washed by saturated sodium chloride aqueous solution, dried by anhydrous sodium sulfate and concentrated to obtain 4.37g of the compound 8, and the yield is 100%. Directly used for the next reaction.

Step 5 Synthesis of (1R,2R) -2- (4, 5-dibromopentyl) cyclopropylmethyl ketone (Compound 9).

Adding compound 8(1.394g, 9.96mmol) into a reaction bottle under the protection of nitrogen, adding 12mL of dichloromethane for dissolution, cooling to-45 to-50 ℃, dropwise adding bromine (3.2g, 19.92mmol) twice at the temperature, reacting for 40 minutes after completion of dropwise adding, detecting by TLC that the raw material is completely reacted, adding N, N-diisopropylethylamine (DIPEA, 321mg, 2.49mmol), quenching with sodium thiosulfate, extracting with N-hexane three times, combining organic phases, washing with a saturated sodium chloride aqueous solution, drying with anhydrous sodium sulfate, and purifying by column chromatography to obtain 1.01g of compound 9 with a yield of 38.4%, LC-MS (APCI): M/z 313(M +1)⁺，¹H NMR(400MHz,CDCl₃)δ4.22-4.10(m,1H),3.91-3.79(m,1H),3.67-3.58(m,1H),2.25(d,J＝2.0Hz,3H),2.20-2.12(m,1H),1.82(m,J＝6.1,5.1,3.0Hz,1H),1.77-1.67(m,2H),1.53(m,J＝18.6,12.2,6.4Hz,1H),1.27(m,J＝8.5,4.2Hz,1H),0.83-0.73(m,1H)。

Step 6 Synthesis of (1R,2R) -2- (4, 5-dibromopentyl) cyclopropylacetate (Compound 10).

Adding compound 9(963mg, 3.086mmol) into a reaction bottle, adding 10mL of ethyl acetate for dissolving, cooling to 0 ℃, adding urea hydrogen peroxide complex (UHP, 1.16g, 12.346mmol) for three times, dropwise adding trifluoroacetic anhydride (TFAA, 2.673g, 12.73mmol) to control the temperature to be 0-3 ℃, dropwise adding for 1 hour approximately, clarifying the reaction solution, heating and refluxing for 16 hours, detecting the reaction completion by TLC, cooling to room temperature, adjusting the pH to 7-9 with 20% sodium bicarbonate, extracting with ethyl acetate for three times, combining organic phases, washing with sodium thiosulfate solution, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, concentrating, and carrying out column chromatography to obtain 419mg of compound 10, wherein the yield is 41.4％。¹H NMR(300MHz,CDCl₃)δ4.17(m,J＝13.3,9.4,3.9Hz,1H),3.94-3.75(m,2H),3.71-3.50(m,1H),2.24-2.08(m,1H),2.03(s,2H),1.92-1.64(m,2H),1.59-1.47(m,1H),1.45-1.15(m,2H),1.03(m,J＝16.5,6.9,2.6Hz,1H),0.91-0.79(m,1H),0.66-0.47(m,1H)。

Step 7 Synthesis of (1R,2R) -2-pent-4-ynyl cyclopropanol (Compound 11).

Adding diallyl phthalate (DAP, 18.11mL) into a three-necked flask under the protection of nitrogen, cooling to 0 ℃, slowly dropwise adding n-butyl lithium (20.4mL, 52mmol, 2.5M) into the reaction flask, controlling the temperature to be lower than 5 ℃, stirring at the temperature for 30 minutes after the dropwise adding is finished, dissolving compound 10(2.84g, 8.66mmol) into 5mL THF, slowly dropwise adding into the reaction flask, controlling the temperature to be 0-2 ℃, generating yellow precipitates during the dropwise adding, stirring at 0 ℃ for 30 minutes after the dropwise adding is finished, detecting that raw materials completely react by TLC, adding an ammonium chloride aqueous solution, quenching, adding methyl tert-butyl ether, extracting for three times, adding 1M NaOH into an aqueous phase to be alkaline, extracting for two times by using the methyl tert-butyl ether, combining organic phases, drying by using anhydrous sodium sulfate, concentrating at the temperature of lower than 30 ℃, and obtaining 730mg of compound 11 with the yield of 67.5%.¹H NMR(300MHz,CDCl₃)δ4.24-4.08(m,1H),3.86(m,J＝10.2,4.3Hz,1H),3.62(t,J＝10.1Hz,1H),2.24(d,J＝1.3Hz,3H),2.20-2.08(m,1H),1.78(m,J＝21.9,18.2,9.2,6.2Hz,3H),1.53(m,J＝13.2,6.3Hz,1H),1.45-1.34(m,3H),1.27(m,J＝7.9,3.5Hz,2H),0.77(m,J＝7.5,4.9,2.8Hz,1H)。

Step 8. Synthesis of Compound 13.

Compound 11(630mg, 5.08mmol) was added to the reaction flask, DIPEA (2.35g, 18.28mmol) was added, N' N-carbonyldiimidazole (CDI, 864mg, 5.334mmol) was added in two portions, the reaction was carried out at room temperature for one hour, Compound 12(800mg, 6.1mmol) was added, the temperature was raised to 90 ℃ for 4 hours, TLC was used to detect completion of the reaction of the starting material, 20mL of water was added to the reaction solution, and the pH was adjusted to 1.5-2.0 with 1M hydrochloric acid. Extraction with methyl tert-butyl ether (MTBE) was carried out three times and then concentration gave 1.56g, compound 12 in 100% yield, which was used directly in the next reaction.

Step 9.2, synthesis of 3-dihydroxy-7-methoxyquinoxaline (compound 15).

Adding compound 14(1g, 7.24mmol), oxalyl chloride (1.2g, 10.136mmol) under nitrogen, adding 10mL of 3N hydrochloric acid, reacting at 90 deg.C for 7 hr, cooling to room temperature, standing at 0 deg.C for 5 hr to precipitate black solid, filtering, washing the filter cake with water, washing with small amount of methanol, vacuum drying below 50 deg.C for 18 hr to obtain 1.08g, compound 15, yield 77.6%, LC-MS (APCI): M/z 193(M +1)⁺，¹H NMR(300MHz,DMSO)δ11.80(d,J＝13.7Hz,2H),7.03(d,J＝8.6Hz,1H),6.68(m,J＝4.6,2.6Hz,2H),3.70(s,3H)。

Step 10.2, Synthesis of 3-dichloro-7-methoxyquinoxaline (Compound 16).

To a reaction flask under nitrogen atmosphere was added compound 15(5.2g, 27.06mmol) and POCl was added₃(8mL), heated to 98 ℃ for 20 hours, cooled to 80 ℃ and then added with acetonitrile 25mL, cooled to 10-15 ℃ and then added with ice water to the reaction flask, the reaction system is lower than 25 ℃, blue solid is separated out, filtered, the filter cake is washed with water, washed with 5% sodium bicarbonate to pH 8-9, and dried under vacuum at 50 ℃ for 24 hours to obtain 4.65g of compound 16, the yield is 74.5%, LC-MS (APCI): M/z 230(M +1)⁺。

Step 11. Synthesis of Compound 19.

Glycine methyl ester hydrochloride (7.142g, 0.057mmol), anhydrous sodium sulfate (4.57g, 0.032mmol), triethylamine (6g, 0.059mmol) and methyl tert-butyl ether (100 mL) were added to the reaction flask and reacted at room temperature for 18 hours, TLC detected complete reaction of starting materials, filtered and concentrated to give intermediate 18.

Adding lithium tert-butoxide (10.07g,0.125mmol) into a reaction flask, adding 100mL of anhydrous toluene, adding 50mL of a toluene solution of compound 18 and 1, 4-dibromo-2-butene (8.57g, 0.04mmol) dropwise into an ice-water bath, reacting at room temperature for 18 hours after the dropwise addition is completed, detecting that the raw materials completely react by TLC, adding 20mL of water, adding 4N hydrochloric acid to adjust the pH to 1-2, extracting with water for 3 times, combining aqueous phases, extracting with methyl tert-butyl ether for three times, adjusting the pH of the aqueous phase with 1N sodium hydroxide to 11-12, extracting with ethyl acetate for 3 times, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, and concentrating to obtain 1.54g of compound 19. The yield is 19.25%

Step 12. Synthesis of Compound 20.

Adding compound 19(2g, 12.89mmol) into a reaction bottle, adding 10mL of dichloromethane for dissolving, adding triethylamine (5g, 48mmol), adding Boc anhydride (4.3g, 19.33mmol), reacting at room temperature for 3 hours, detecting by TLC that the raw material is completely reacted, concentrating the reaction solution, and performing column chromatography to obtain 2.2g of compound 20 with the yield of 64.7%

Step 13. Synthesis of Compound 21.

To a reaction flask was added compound 20(2.2g, 9.12mmol) and tetrahydrofuran: dissolving in methanol (1: 140 mL), adding lithium hydroxide (873g, 36.47mmol), heating to 40 deg.C, reacting for 18 h, detecting by TLC that the raw material is completely reacted, concentrating the reaction solution, adding 10mL of water into the reaction flask, extracting with ethyl acetate 4 times, combining the organic phases, drying with anhydrous sodium sulfate, concentrating to obtain 0.968g of compound 21, the yield is 46.7%

Step 14. Synthesis of Compound 22.

Adding compound 21(678mg, 2.986mmol) into a reaction bottle, dissolving with tetrahydrofuran 12mL, adding CDI (628mg, 3.88mmol), heating under reflux for 1 hr, cooling to room temperature, dissolving cyclopropanesulfonamide (469mg, 3.88mmol) with tetrahydrofuran 2mL, adding into the reaction bottle, adding 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU, 658mg, 4.329mmol), reacting at room temperature for 18 hr, detecting by TLC that the raw material has reacted completely, adding 1M hydrochloric acid to adjust pH to 1-2, extracting with ethyl acetate for 3 times, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, and performing column chromatography to obtain compound 22(495 mg) with yield of 49.6%

Step 15. Synthesis of Compound 23.

Compound 22(495mg, 1.39mmol) was added to the reaction flask, and 10mL of ethyl acetate was added to dissolve the compound, 5mL of methanol (4M) hydrochloride solution was added to react at 40 ℃ for 4 hours, TLC detection showed that the starting material was completely reacted, and the reaction solution was concentrated to give 400mg of compound 23, which was used directly in the next reaction.

Step 16. Synthesis of Compound 25.

Under nitrogen protection, compound 16(2g, 8.73mmol), compound 24(2.3 mmol) were added to the flask6g, 9.6mmol), adding 10mL of Dimethylacetamide (DMAC), adding DBU (2g, 13.09mmol), heating to 50 ℃ for reaction for 20-30 hours, cooling to room temperature, adding 20mL of water and 30mL of MTBE, filtering, removing black insoluble substances, extracting with MTBE three times, combining organic phases, drying with anhydrous sodium sulfate, concentrating, purifying by column chromatography to obtain 1.2g of compound 25, recovering the raw materials with a yield of 31.4%,¹H NMR(300MHz,CDCl₃)δ7.81(d,J＝9.1Hz,1H),7.23(m,J＝9.1,2.6Hz,1H),7.16(m,J＝7.7,2.5Hz,1H),5.73(d,J＝20.8Hz,1H),4.57(m,J＝22.7,7.8Hz,1H),4.02-3.89(m,5H),3.78(d,J＝9.0Hz,4H),2.68(d,J＝5.6Hz,1H),2.51-2.35(m,1H),1.46(d,J＝8.6Hz,10H)。

step 17. Synthesis of Compound 26.

To reaction flask 1 was added compound 25(1.866g, 4.27mmol) dissolved by adding 10mL of cyclopentyl methyl ether, and compound 13(1.44g, 5.124mmol) dissolved by 10mL of cyclopentyl methyl ether was added to the reaction flask, and the mixture was stirred under nitrogen for 30 minutes. Palladium acetate (32mg, 0.145mmol), P (t-Bu) was added to reaction flask 2₃BF₄(74mg, 0.256mmol), potassium carbonate (1.47g, 1.067mmol), acetonitrile 10mL, nitrogen gas introduction and stirring for 15 minutes, charging it into a reaction flask 1, nitrogen gas introduction and stirring for 15 minutes, heating to 85 ℃ and reacting for 3 hours, TLC detecting the completion of the reaction of the raw materials, adding 30mL of water, adjusting pH to 1-2 with 2M phosphoric acid, extracting with ethyl acetate for 3 times, combining the organic phases, drying with anhydrous sodium sulfate, concentrating, column chromatography to obtain 1.06g of compound 26 with a yield of 36.5%. LC-MS (APCI) M/z 683(M +1)⁺。¹H NMR(300MHz,CDCl₃)δ7.89-7.78(m,1H),7.19(d,J＝9.1Hz,1H),7.11(d,J＝7.4Hz,1H),5.68(s,1H),5.31(d,J＝7.5Hz,1H),4.49(t,J＝7.5Hz,1H),4.02-3.89(m,4H),3.79(s,6H),2.60(d,J＝6.4Hz,3H),2.45(m,J＝12.9,6.7Hz,1H),1.89-1.71(m,2H),1.44(d,J＝5.8Hz,11H),1.26(s,1H),1.01(s,11H),0.89(m,J＝10.6,5.9Hz,3H),0.56(d,J＝6.3Hz,1H)。

Step 18. Synthesis of Compound 27.

Adding compound 26(445mg, 0.651mmol) into a reaction flask, adding 5mL of deuterated methanol to dissolve, adding 80mg of 10% palladium-carbon, introducing deuterium, reacting at 40 ℃ for 18 hours, detecting by LCMS that the raw materials are completely reacted, cooling to room temperature, adding kieselguhr, filtering, washing a filter cake with methanol, and concentrating to obtain 380mg of compound 27 with the yield of 84.4%

Step 19. Synthesis of Compound 28.

Adding compound 27(380mg, 0.55mmol) into a reaction bottle, adding 3mL acetonitrile to dissolve, adding trifluoroacetic acid (156mg, 1.375mmol), heating to 40 ℃, reacting for 18 hours, detecting the complete reaction of raw materials by TLC, concentrating to remove acetonitrile and excessive trifluoroacetic acid, adjusting pH to 8-9 with DIPEA, adding acetonitrile 3mL, adding HATU (282mg, 0.74mmol), reacting for 8 hours at room temperature, detecting the complete reaction of raw materials by TLC, concentrating the reaction solution, purifying by column chromatography to obtain 211mg of compound 28, the yield is 66.9%

Step 20. Synthesis of Compound 29.

To a reaction flask was added compound 28(211mg, 0.368mmol) 5mL, tetrahydrofuran, lithium hydroxide (73mg, 3.68mmol) dissolved in 2mL of water and added to the reaction, which was heated to 40 ℃ for 1 hour, TLC detected that the reaction of the starting material was complete, extracted 3 times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give 114mg of compound 29 with a yield of 45.6%.

Step 21. Synthesis of Compound G-1.

Compound 29(114mg, 0.205mmol), compound 23(61mg, 0.266mmol) were added to the reaction flask at room temperature and dissolved in 5mL of acetonitrile. Pyridine (229mg, 0.87mmol) is added, the reaction is carried out for 15 minutes at room temperature, EDCI (62.4mg, 0.328mmol) is added, the reaction is carried out for 1.5 hours at room temperature, the reaction solution becomes clear, 2mL of 2N hydrochloric acid solution is added, the reaction is stirred for 20 minutes, TLC is used for detecting that the raw materials are completely reacted, the reaction solution is concentrated, 31mg of compound G-1 is prepared by TLC,¹H NMR(400MHz,CDCl₃)δ7.81(d,J＝9.1Hz,1H),7.18(m,J＝9.0,2.8Hz,1H),7.12(d,J＝2.7Hz,1H),7.04(s,1H),5.96(s,1H),5.81(s,1H),5.67(s,1H),5.33(m,J＝13.5,9.1Hz,1H),5.12(s,1H),4.51(d,J＝11.3Hz,1H),4.45(d,J＝9.8Hz,1H),4.37(s,1H),4.08(d,J＝6.7Hz,1H),3.92(d,J＝5.3Hz,2H),3.80-3.73(m,1H),2.93-2.84(m,1H),2.93-2.83(m,1H),2.74(m,J＝13.3,9.1Hz,1H),2.57(m,J＝13.8,7.3Hz,1H),2.41(s,1H),2.22-2.15(m,1H),1.83(s,1H),1.53(m,J＝36.0,16.6Hz,3H),1.28(s,3H),1.06(s,9H),1.00-0.88(m,2H),0.67(d,J＝6.8Hz,1H),0.46(q,J＝6.3Hz,1H)。

example 2a substituted macrocyclic quinoxaline compound G-2 is prepared having the formula:

the synthesis adopts the following route:

step 1.2, 3-dichloro-7-hydroxyquinoxaline (compound 30).

Adding 40mL of toluene into a reaction bottle at 0 ℃, adding aluminum trichloride (1.96g, 14.67mmol), adding compound 16(1.4g, 6.11mmol), heating to 80 ℃ for reaction for 5 hours, detecting that the raw materials completely react by TLC, cooling to room temperature, separating out solids, adding 40mL of water into the reaction solution, adding 50mL of ethyl acetate, heating until the solids are dissolved, extracting with ethyl acetate for 4 times, combining organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain 1.3g of compound 30 with the yield of 100%.

Step 2.2 Synthesis of 2, 3-dichloro-7-d 3-methoxyquinoxaline (Compound 31).

Adding compound 30(1g, 4.6mmol) into a reaction bottle, adding potassium carbonate (1.6g, 11.6mmol), adding 30mL of DMF to dissolve, adding deuterated iodomethane (1.65g, 11.6mmol), heating to 80 ℃, reacting for 3 hours, detecting that the raw materials are completely reacted by TLC, cooling to room temperature, adding 50mL of water into the reaction solution, extracting for 3 times by ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain 781mg of compound 31 with the yield of 72.3%.

Step 3. Synthesis of Compound 32.

Adding compound 31(780mg, 3.36mmol), compound 24(906mg, 3.69mmol), DMAC30 mL, DBU (664g, 4.368mmol), heating to 50 deg.C for 18 hr, detecting by TLC that the raw materials are completely reacted, cooling to room temperature, adding 40mL water, extracting with ethyl acetateCollecting 3 times, mixing organic phases, drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain 645mg of compound 32 with a yield of 45.3%, MS (APCI): M/z 441(M +1)⁺。

Step 4. Synthesis of Compound 33.

To reaction flask 1 was added compound 32(463mg, 1.06mmol), dissolved in 1mL of cyclopentyl methyl ether, and compound 13(357mg, 1.27mmol) dissolved in 1.5mL of cyclopentyl methyl ether and added to the reaction flask, which was stirred under nitrogen for 30 minutes. To reaction flask 2 was added palladium acetate (8mg, 0.036mmol), P (t-Bu)₃BF₄(18mg, 0.064mmol), potassium carbonate (365g, 2.65mmol), acetonitrile 2mL, nitrogen gas for 15 minutes, added to the reaction flask 1, nitrogen gas for 15 minutes, heated to 85 ℃ for 2.5 hours, TLC detecting the completion of the reaction of the raw materials, adjusting pH to 1-2 with 2M phosphoric acid, extracting 3 times with ethyl acetate, combining the organic phases, drying with anhydrous sodium sulfate, concentrating, column chromatography to obtain 370mg of compound 33 with a yield of 55.4%. LC-MS (APCI): M/z ═ 686(M +1)⁺。

Step 5. Synthesis of Compound 34.

Adding compound 33(0.185mg, 0.219mmol) into a reaction flask, adding 2mL of methanol to dissolve the compound, adding 5mL of isopropanol to dissolve the compound, adding 20mg of 10% palladium carbon, introducing hydrogen to react at 40 ℃ for 18 hours, detecting by LC-MS that the raw material reaction is complete, cooling to room temperature, adding kieselguhr to filter, washing a filter cake with methanol, and concentrating to obtain 191mg of compound 34 with the yield of 95.5% LC-MS (APCI): M/z ═ 690(M +1)⁺。

Step 6. Synthesis of Compound 35.

Adding compound 34(191mg, 0.29mmol) into a reaction flask, adding 3mL of dichloromethane to dissolve, adding 2mL of trifluoroacetic acid, reacting at room temperature for 18 hours, detecting the completion of the reaction by TLC, concentrating to remove dichloromethane and excess trifluoroacetic acid, adjusting pH to 8-9 with DIPEA, adding 3mL of acetonitrile, adding HATU (148.9mg, 0.39mmol), reacting at room temperature for 3 hours, detecting the completion of the reaction by TLC, concentrating the reaction solution, and purifying by column chromatography to obtain 54mg of compound 35 with a yield of 32.9%, LC-MS (APCI): M/z 572(M +1)⁺。

Step 7. Synthesis of Compound 36.

Compound 35(54mg, 0.094mmol) was added to the reaction flask, tetrahydrofuran 4mL was added, lithium hydroxide (22mg, 0.944mmol) was dissolved in 2mL water and added to the reaction, the temperature was raised to 40 ℃ for reaction for 1 hour, TLC detected that the raw material was completely reacted, extracted 3 times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give 74mg of compound 36, which was used in the next step without further purification. LC-MS (APCI) M/z 556(M +1)^-。

And 8, synthesizing a compound G-2.

Compound 36(74mg, 0.135mmol), compound 23(40.6mg, 0.177mmol) were added to the reaction flask at room temperature and dissolved in 4mL of acetonitrile. Pyridine (152mg, 1.902mmol) was added, the reaction was carried out at room temperature for 15 minutes, EDCI (41mg, 0.217mmol) was added, the reaction was carried out at room temperature for 1.5 hours, the reaction solution became clear, 2mL of 2N hydrochloric acid solution was added, the reaction was stirred for 20 minutes, the reaction of the starting materials was completed by TLC, the reaction solution was concentrated, and 27mg of Compound G-2, LC-MS (APCI): M/z 770(M +1) was prepared by TLC⁺，¹H NMR(500MHz,CDCl₃)δ7.82(d,J＝9.0Hz,1H),7.19(m,J＝9.0,2.8Hz,1H),7.12(d,J＝2.7Hz,1H),6.98(s,1H),5.98(s,1H),5.85-5.76(m,1H),5.62(d,J＝7.9Hz,1H),5.41-5.32(m,1H),5.14(m,J＝23.0,9.9Hz,1H),4.53(d,J＝11.4Hz,1H),4.46(d,J＝9.9Hz,1H),4.35(m,J＝25.6,12.8Hz,1H),4.07(m,J＝11.6,5.8Hz,1H),3.83-3.71(m,1H),2.91-2.84(m,2H),2.62-2.53(m,2H),2.46-2.36(m,1H),2.19(m,J＝16.1,7.6Hz,2H),2.06-1.96(m,1H),1.84(m,J＝8.3,5.3Hz,1H),1.76-1.65(m,3H),1.57-1.42(m,3H),1.36-1.30(m,4H),1.08(s,9H),0.95(m,J＝10.1,8.4,3.0Hz,3H),0.69(s,1H),0.49-0.41(m,1H)。

Example 3a substituted macrocyclic quinoxaline compound G-3 is prepared having the formula:

the synthesis adopts the following route:

step 1. Synthesis of Compound 37.

Adding compound 33(0.185mg, 0.219mmol) into a reaction flask, adding 5mL of deuterated methanol for dissolving, adding 20mg of 10% palladium carbon, introducing deuterium gas, reacting at 40 ℃ for 18 hours, detecting by LC-MS that the raw materials are completely reacted, cooling to room temperature, adding kieselguhr for filtering, washing a filter cake with methanol, and concentrating to obtain 211mg of compound 37 with the yield of 100%. LC-MS (APCI) M/z 692(M +1)⁺。

Step 2. Synthesis of Compound 38.

Adding the compound 37(211mg, 0.306mmol) into a reaction bottle, adding 3mL of dichloromethane for dissolving, adding 2mL of trifluoroacetic acid, reacting at room temperature for 18 hours, detecting the raw material reaction completion by TLC, concentrating to remove dichloromethane and excess trifluoroacetic acid, adjusting pH to 8-9 by DIPEA, adding acetonitrile 3mL, adding HATU (157mg, 0.414mmol), reacting at room temperature for 3 hours, detecting the raw material reaction completion by TLC, concentrating the reaction solution, and purifying by column chromatography to obtain 63mg of the compound 38 with the yield of 36%.

Step 3. Synthesis of Compound 39.

To a reaction flask was added compound 38(63mg, 0.109mmol) 4mL tetrahydrofuran, lithium hydroxide (26mg, 1.09mmol) was dissolved in 2mL water and added to the reaction, which was warmed to 40 ℃ for 1 hour, TLC detected the completion of the reaction of the starting material, extracted 3 times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give 76mg compound 39, which was used in the next step without further purification.

And 4, synthesizing a compound G-3.

To a reaction flask, compound 39(74mg, 0.135mmol), compound 23(40.6mg, 0.177mmol) were added at room temperature and dissolved in 4mL of acetonitrile. Pyridine (152mg, 1.902mmol) is added, the reaction is carried out for 15 minutes at room temperature, EDCI (41mg, 0.22mmol) is added, the reaction is carried out for 1.5 hours at room temperature, the reaction solution becomes clear, 2mL of 2N hydrochloric acid solution is added, the reaction is stirred for 20 minutes, TLC detection is carried out to detect that the raw material completely reacts, the reaction solution is concentrated, and 28mg of compound G-3 is prepared by TLC,¹H NMR(500MHz,CDCl₃)δ7.82(d,J＝9.0Hz,1H),7.19(dd,J＝9.0,2.8Hz,1H),7.12(d,J＝2.7Hz,1H),6.98(s,1H),5.98(s,1H),5.84-5.74(m,1H),5.61(d,J＝9.9Hz,1H),5.38–5.32(m,1H),5.16(d,J＝11.3Hz,1H),4.53(d,J＝11.4Hz,1H),4.46(d,J＝9.9Hz,1H),4.36(m,J＝10.6,6.6Hz,1H),4.06(m,J＝11.7,4.1Hz,1H),3.80-3.73(m,1H),2.89(m,J＝12.9,8.2,4.9Hz,2H),2.70(d,J＝19.5Hz,1H),2.62-2.53(m,1H),2.44(m,J＝10.6,3.8Hz,1H),2.19(m,J＝16.1,7.6Hz,2H),1.84(m,J＝8.3,5.3Hz,1H),1.78-1.65(m,4H),1.52-1.42(m,3H),1.38-1.31(m,3H),1.08(s,8H),0.94(m,J＝13.6,12.7,7.8Hz,4H),0.69(s,1H),0.48-0.42(m,1H)。

example 4a substituted macrocyclic quinoxaline compound G-4 is prepared having the formula:

the synthesis adopts the following route:

step 1. Synthesis of Compound 41.

Adding 3mL of ethylene glycol dimethyl ether into a reaction bottle, adding cyclopropanesulfonyl chloride (1.5g, 1.07mmol), cooling by using an ice water bath, diluting sodium deuteroxide (40%, 3mL, 0.03mol) with 3mL of heavy water, then dropwise adding into the reaction bottle, reacting at room temperature for 18 hours after dropwise adding, cooling, adjusting the pH to acidity by using deuterated hydrochloric acid, concentrating to remove the solvent, and obtaining 3.19g of a crude product of the compound 41 which is directly used in the next step without further purification.

Step 2. Synthesis of Compound 42.

To a reaction flask was added compound 41(1.6g, 11.2mmol) and 10mL of thionyl chloride, DMF 5 drops was added, the reaction was refluxed at 60 ℃ for 4 hours, cooled to room temperature, and concentrated to remove excess thionyl chloride, to give 2.1g of compound 42 as a crude product, which was used in the next step without further purification.

Step 3. Synthesis of Compound 43.

Adding 2.1g of the crude product of the compound 42 into a reaction bottle, adding 100mL of anhydrous tetrahydrofuran, cooling to-5 ℃, introducing ammonia gas for 20 minutes while stirring, reacting at room temperature for 18 hours, concentrating to remove tetrahydrofuran, adding 20mL of water, extracting for 4 times by using ethyl acetate, combining organic phases, washing by using saturated sodium chloride, drying by using anhydrous sodium sulfate, and concentrating to obtain 525mg of a compound 43, wherein the yield of the three steps is 98%.

Step 4. Synthesis of Compound 44.

Compound 21(430mg, 1.894mmol) was added to a reaction flask, dissolved in 8mL of tetrahydrofuran, CDI (398mg, 2.46mmol) was added, the reaction was refluxed for 1 hour at elevated temperature, cooled to room temperature, compound 43(300mg, 2.46mmol) was dissolved in 2mL of tetrahydrofuran and added to the reaction flask, DBU (417mg, 2.48mmol) was added, the reaction was carried out at room temperature for 17 hours, TLC starting material was completely reacted, 1M hydrochloric acid was added to adjust pH to 1-2, extraction was carried out 3 times with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and column chromatography gave 326mg of compound 44 with a yield of 40.9%.

And 5, synthesizing a compound 45.

To a reaction flask, compound 44(710mg, 2.14mmol) was added and dissolved in 10mL of dichloromethane, 5mL of dioxane (4M) hydrochloride solution was added, the reaction was carried out at 40 ℃ for 4 hours, the TLC detection showed that the starting material had reacted completely, and the reaction solution was concentrated to give 338mg of compound 45, which was used directly in the next reaction.

Step 6. Synthesis of Compound 46.

Adding compound 26(109mg, 0.159mmol) into a reaction flask, adding 1mL of methanol to dissolve the compound, adding 2mL of isopropanol to dissolve the compound, adding 7mg of palladium carbon, introducing hydrogen to react at 40 ℃ for 18 hours, detecting the completion of the raw material reaction by LCMS, cooling to room temperature, adding kieselguhr to filter, washing a filter cake with methanol, and concentrating to obtain 191mg of compound 46 with the yield of 63.5 percent (LC-MS (APCI): M/z-687 (M +1)⁺。

Step 7. Synthesis of Compound 47.

To a reaction flask was added compound 46(1g, 1.45mmol), dissolved in 10mL of acetonitrile, and added trifluoroacetic acid (573 mg)3.6mmol), heating to 40 ℃, reacting for 18 hours, detecting by TLC that the raw materials completely react, concentrating to remove acetonitrile and excess trifluoroacetic acid, adjusting the pH to 8-9 with DIPEA, adding 3mL acetonitrile, adding HATU (751mg, 1.97mmol) and reacting for 4 hours at room temperature, detecting by TLC that the raw materials completely react, concentrating the reaction solution, and purifying by column chromatography to obtain 300mg of compound 47 with a two-step yield of 36.2%, LC-ms (apci): M/z ═ 569(M +1)⁺。

Step 8. Synthesis of Compound 48.

Adding 1mL of compound 47(100mg, 0.152mmol) into a reaction flask, adding 1mL of tetrahydrofuran, dissolving lithium hydroxide (76mg, 1.5mmol) with 2mL of water, adding into the reaction, heating to 40 ℃ for reaction for 1 hour, detecting by TLC that the raw material is completely reacted, cooling to room temperature, adjusting the pH to acidity with 1N hydrochloric acid, extracting with ethyl acetate for 3 times, drying with anhydrous sodium sulfate, and concentrating to obtain 90mg of compound 48 with the yield of 92.7%.

And 9, synthesizing a compound G-4.

Compound 48(100mg, 0.18mmol), compound 45(53.8mg, 0.23mmol) were added to the reaction flask at room temperature and dissolved in 5mL of acetonitrile. Pyridine (200mg, 2.25mmol) is added, the reaction is carried out for 15 minutes at room temperature, EDCI (55mg, 0.288mmol) is added, the reaction is carried out for 1.5 hours at room temperature, the reaction solution becomes clear, 2mL of 2N hydrochloric acid solution is added, the reaction is stirred for 20 minutes, TLC detection is carried out to ensure that the raw materials are completely reacted, the reaction solution is concentrated, and 10mg of compound G-4 is prepared by TLC,¹H NMR(500MHz,CDCl₃)δ7.83(d,J＝9.0Hz,1H),7.20(m,J＝9.0,2.7Hz,1H),7.14(d,J＝2.7Hz,1H),7.08(s,1H),5.99(s,1H),5.87-5.79(m,1H),5.38(s,1H),5.21(d,J＝17.2Hz,1H),5.09(d,J＝10.1Hz,1H),4.51(d,J＝11.0Hz,1H),4.42(d,J＝9.9Hz,1H),4.37(s,1H),4.05(d,J＝7.4Hz,1H),3.94(d,J＝8.4Hz,3H),3.79(d,J＝6.7Hz,1H),2.98(d,J＝6.6Hz,2H),2.88(s,1H),2.57(s,1H),2.47(s,1H),2.07(d,J＝8.5Hz,1H),1.76(d,J＝14.1Hz,2H),1.66-1.61(m,2H),1.57-1.48(m,3H),1.36-1.31(m,3H),1.09(d,J＝5.4Hz,9H),0.89(m,J＝13.3,6.6Hz,4H),0.68(s,1H),0.48(d,J＝6.5Hz,1H)。

example 5a substituted macrocyclic quinoxaline compound G-5 is prepared having the formula:

the synthesis adopts the following route:

to a reaction flask was added compound 36(77mg, 0.138mmol), compound 45(41.4mg, 0.179mmol) at room temperature, and dissolved with 5mL of acetonitrile. Pyridine (153mg, 0.192mmol) is added, the reaction is carried out for 15 minutes at room temperature, EDCI (43mg, 0.219mmol) is added, the reaction is carried out for 1.5 hours at room temperature, the reaction solution becomes clear, 2mL of 2N hydrochloric acid solution is added, the reaction is stirred for 20 minutes, TLC detection is carried out to detect that the raw materials are completely reacted, the reaction solution is concentrated, and TLC is used for preparing 11mg of compound G-5,¹H NMR(500MHz,CDCl₃)δ7.83(d,J＝9.1Hz,1H),7.20(m,J＝9.1,2.8Hz,1H),7.13(d,J＝2.7Hz,1H),6.85(s,1H),6.00(s,1H),5.79(m,J＝17.4,8.8Hz,1H),5.43(d,J＝9.7Hz,1H),5.22(d,J＝17.3Hz,1H),5.13(d,J＝10.0Hz,1H),4.65(s,1H),4.53(d,J＝11.3Hz,1H),4.34-4.29(m,1H),4.07-4.02(m,1H),3.77(d,J＝6.9Hz,1H),2.87(dd,J＝18.5,11.2Hz,2H),2.79(d,J＝4.5Hz,1H),2.58(m,J＝12.8,7.9Hz,1H),2.46(d,J＝10.4Hz,1H),2.08(d,J＝8.5Hz,1H),1.75(d,J＝15.1Hz,4H),1.53-1.47(m,3H),1.33(d,J＝7.0Hz,3H),1.09(s,9H),0.89(dd,J＝13.4,6.6Hz,4H),0.70(d,J＝18.8Hz,1H),0.48(m,J＝12.6,6.1Hz,1H)。

and (4) evaluating the biological activity.

To verify the effect of the compounds described herein on HCV, the inventors used the HCV Replicon System (HCV replication System) as an evaluation model. Since the first report in Science1999, the HCV replicon system has become one of the most important tools for studying HCV RNA replication, pathogenicity, and virus persistence, for example, the minimal 5' -NCR region necessary for HCV RNA replication has been successfully demonstrated using the replicon, and the HCV replicon system has been successfully used as an evaluation model for antiviral drugs. The inventors of the present invention performed the verification according to the methods described in Science, 1999, 285(5424), 110-3, and j.virol, 2003, 77(5), 3007-19.

(1) Detection of Compound Activity against HCV 1a and 1b genotype replicons

HCV-1a and HCV-1b stably transfected replicon cells were used to detect the inhibitory activity of the compounds of hepatitis C virus genotype 1a and 1b replicons. This experiment will use the NS3 inhibitor MK-5172 as a positive control compound.

The method comprises the following steps: compounds were serially diluted 1:3 into 8 concentration spots, double-plated and added to 96-well plates. DMSO was set as no compound added control. The final concentration of DMSO in the cell culture broth was 0.5%.

Step two: HCV-1a and 1b cells were suspended in culture medium containing 10% FBS, respectively, and seeded into compound-containing 96-well plates at a density of 8,000 cells per well. Cells were in 5% CO₂And cultured at 37 ℃ for 3 days.

Step three: cytotoxicity of compounds against GT1b replicon was determined using CellTiter-Fluor (Promega).

Step four: luciferase assay Compounds were assayed for anti-hepatitis C virus activity using Bright-glo (Promega).

Step five: data were analyzed using GraphPad Prism software, curves were fitted and EC calculated₅₀Value sum CC₅₀The value is obtained.

TABLE 1 comparison of anti-HCV genotype replicon activity of examples 1-4 and control MK-5172

Numbering	HCV GT1a EC₅₀(nM)	HCV GT1b EC₅₀(nM)	HCV CC₅₀(nM)
				MK-5172	0.967	0.98	>1000
G-1	2.188	1.863	>1000
				G-2	1.172	0.963	>1000
G-3	1.404	0.884	>1000
				G-4	3.370	2.613	>1000

As shown in table 1, the compounds of the present invention inhibit multiple genotypes of HCV, which in turn can be used for the inhibition of hepatitis c virus.

(2) Metabolic stability evaluation

Microsome experiment: human liver microsomes: 0.5mg/mL, Xenotech; rat liver microsomes: 0.5mg/mL, Xenotech; coenzyme (NADPH/NADH): 1mM, Sigma Life Science; magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

Preparing a stock solution: an amount of the compound of example 1-4 was weighed out precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L of pre-cooled stop solution was added to each well of a 96-well deep-well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30 min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

The compounds of the invention and compounds without deuteration were tested simultaneously and compared to evaluate their metabolic stability in human and rat liver microsomes. The half-life and intrinsic hepatic clearance as indicators of metabolic stability are shown in table 2. The non-deuterated compound MK-5172 was used as a control sample in Table 2. As shown in Table 2, the compounds of the present invention, particularly G-4, can significantly improve metabolic stability by comparison with the non-deuterated compound MK-5172, and thus are more suitable as hepatitis C virus inhibitors.

TABLE 2 comparison of metabolic stability of examples 1-4 with MK-5172 controls

Claims

1. A substituted macrocyclic quinoxaline compound, characterized by: a compound shown as a formula (I), or a polymorphism, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variation, a hydrate or a solvate thereof,

2. The macrocyclic quinoxaline compound according to claim 1, wherein: r¹、R²And R³Each independently is deuterium or hydrogen.

3. The macrocyclic quinoxaline compound according to claim 1, wherein: r⁴、R⁵And R⁶Each is independentAnd is independently deuterium or hydrogen.

4. The macrocyclic quinoxaline compound according to claim 1, wherein: r⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹And R²⁰Each independently is deuterium or hydrogen.

5. The macrocyclic quinoxaline compound according to claim 1, wherein: r²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷、R²⁸、R²⁹And R³⁰Each independently is deuterium or hydrogen.

6. The macrocyclic quinoxaline compound according to claim 1, wherein: r³¹、R³²、R³³、R³³、R³⁴、R³⁵And R³⁶Each independently is deuterium or hydrogen.

7. The macrocyclic quinoxaline compound according to claim 1, wherein: r³⁷、R³⁸And R³⁹Each independently is deuterium or hydrogen.

8. The macrocyclic quinoxaline compound according to claim 1, wherein: r⁴⁰、R⁴¹And R⁴²Each independently is deuterium or hydrogen.

9. The macrocyclic quinoxaline compound according to claim 1, wherein: r⁴³、R⁴⁴、R⁴⁵、R⁴⁶And R⁴⁷Each independently is deuterium or hydrogen.

10. The macrocyclic quinoxaline compound according to claim 1, wherein: the compound is selected from the following compounds or pharmaceutically acceptable salts thereof:

11. a pharmaceutical composition characterized by: comprising a pharmaceutically acceptable carrier and a macrocyclic quinoxaline compound according to any one of claims 1 to 10, or a polymorph, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotopic variant, a hydrate or a solvate thereof.

12. The pharmaceutical composition of claim 11, wherein: it further comprises an additional active compound which can be selected from HCV protease inhibitors, HCV NS5A inhibitors, and HCV NS5B polymerase inhibitors.

13. Use of a macrocyclic quinoxaline compound according to any one of claims 1 to 10, wherein: the application of the compound in preparing the medicine for treating hepatitis C virus infection.

14. Use of a pharmaceutical composition according to any one of claims 11 and 12, characterized in that: for the manufacture of a medicament for inhibiting HCV NS3 protease activity in a subject in need thereof.