CN113712964A - Application of 9, 10-dihydrophenanthrene compound in preparation of coronavirus 3CL protease inhibitor - Google Patents

Abstract

The invention provides an application of a 9, 10-dihydrophenanthrene compound in preparation of a coronavirus 3CL protease inhibitor. The 9, 10-dihydrophenanthrene compound has good inhibition effect on coronavirus 3CL protease, wherein the 9, 10-dihydrophenanthrene compound has the structure shown in formula I, and the structure is IC₅₀The value is in the range of 1-70. mu.M; in particular, IC of 9,10 dihydrophenanthrenes having the structure shown in formula II-C₅₀The value can reach 1.5-6 mu M range.

Description

Application of 9, 10-dihydrophenanthrene compound in preparation of coronavirus 3CL protease inhibitor

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an application of a 9, 10-dihydrophenanthrene compound in preparation of a coronavirus 3CL protease inhibitor.

Background

Coronaviruses (Coronaviruses, CoVs) belong to the order Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), a class of enveloped positive-strand RNA (+ ssRNA) viruses that are widely found in nature. The virus particles are spherical or elliptical and have polymorphism and diameters of about 60-220 nm. The family of coronaviridae is divided into four genera of alpha, beta, gamma and delta, wherein beta-CoVs are the most serious in harm to human beings, can cause lower respiratory tract diseases such as pneumonia and the like in different degrees, and can also affect gastrointestinal tract systems, central nervous systems, hearts, kidneys, livers and the like to cause multi-organ failure. SARS-CoV-2 is a novel type of beta-CoV, and this virus has spread globally. 3CL proteases, also known as 3CL^proOr M^proIs a cysteine protease that is capable of processing the polyprotein precursors pp1a and pp1ab at not less than 11 conserved positions. It is therefore responsible for the release of most non-structural functional proteins (NSPS), involved in the replicative transcription process of the virus. 3CL^proThe key role in the viral life cycle and the absence of homologous proteins in human cells allows 3CL^proBecomes a very attractive target for resisting SARS-CoV-2. At present, 3CL is used for a plurality of people^proInhibitors that are targeted are reported.

CN112574104A discloses a target M for SARS-CoV-2^proAcetamide compounds of inhibitors, but IC of said compounds₅₀Values generally between 15-100. mu.M, did not appear to be M for the novel coronavirus^proParticularly excellent inhibitory action.

CN111773240A discloses the application of natural sulfated polysaccharide from marine organisms as an anti-coronavirus and disease-causing drug. The marine organism-derived natural sulfated polysaccharide provided by the invention can inhibit the combination of Spike protein (Spike) on the surface of coronavirus and host cells, and can inhibit the activities of main protease (Mpro) and papain-like (PLpro), thereby effectively blocking coronavirus from infecting host cells; however, the natural sulfated polysaccharide provided by the invention has a complex structure, separation and extraction preparation process.

In summary, SARS-CoV-23CL is now disclosed^proThe inhibitor has the problems of complex preparation process and low activity. Therefore, there is a need to develop a safe, effective coronavirus 3CL with simple preparation process^proAnd (3) an inhibitor.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the application of a 9, 10-dihydrophenanthrene compound in preparing a coronavirus 3CL protease inhibitor, and provides a new strategy for preparing a medicament for treating diseases caused by novel coronaviruses and/or novel coronavirus mutant strains. The 9, 10-dihydrophenanthrene compound has good inhibition effect on coronavirus 3CL protease, wherein the 9, 10-dihydrophenanthrene compound has the structure shown in formula I, and the structure is IC₅₀The value is in the range of 1-70. mu.M; in particular, IC of 9,10 dihydrophenanthrenes having the structure shown in formula II-C₅₀The value can reach 1.5-6 mu M range. In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a use of a 9,10 dihydrophenanthrene compound in the preparation of a coronavirus 3CL protease inhibitor.

In the application of the invention, the 9, 10-dihydrophenanthrene compound has a structure shown as the following formula I:

in the formula I, R is¹-R⁷Each is independently selected from any one of hydrogen, cyano, nitro, hydroxyl, aldehyde group, carboxyl, sulfonic group, ester group, acyl, sulfone group, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amino, substituted or unsubstituted heterocyclic group and substituted or unsubstituted aryl; or, R¹-R⁷Each independently forms a saturated or unsaturated carbocyclic or heterocyclic ring with an adjacent benzene ring through a covalent bond;

in the formula I, R is⁸Selected from substituted or unsubstituted nitrogen-containing heterocyclic groups;

in the formula I, R is⁹And is selected from any one of hydroxyl substituted alkyl, ester substituted alkyl or heteroatom substituted alkyl.

Wherein R is¹-R⁷Each independently forms a saturated or unsaturated carbocyclic or heterocyclic ring with the adjacent benzene ring by a covalent bond, and is referred to as R¹-R⁷Any two adjacent substitution positions form a ring-merging structure through covalent bonds; the saturated or unsaturated carbocyclic ring may be an alkyl ring, alkenyl ring, alkynyl ring, phenyl ring, or the like; the heterocyclic ring may be a pyridine ring, an imidazole ring, or the like.

Specifically, the formation of a saturated or unsaturated carbocyclic or heterocyclic ring by covalent bond includes, but is not limited to, the structures shown below:

in the present invention, said R¹-R⁷Each independently selected from any one of substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy, substituted amino, substituted heterocyclic radical or substituted aryl, and the substituents of the alkyl, alkenyl, alkynyl, alkoxy, amino, heterocyclic radical and aryl are each independently selected fromIs selected from any one of cyano, nitryl, amino, hydroxyl, aldehyde group, carboxyl, sulfonic group, ester group, acyl, sulfone group or halogen.

Preferably, said R is¹Any one selected from hydrogen, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, preferably unsubstituted C1-C6 alkyl (for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl may be mentioned), halogen-substituted C1-C6 alkyl (for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl may be mentioned), cyano-substituted C1-C6 alkyl (for example, C1 alkyl, C2 alkyl or C2 alkyl may be mentioned), unsubstituted C2-C2 aryl (for example, C2 aryl or C2 aryl may be mentioned), halogen-substituted C2 aryl (for example, C2 aryl or C2 aryl may be mentioned), and C2-C2 aryl (for example, C2 aryl may be mentioned, C10 aryl group, C12 aryl group, etc.), and more preferably, it is any of unsubstituted C1 to C3 alkanyl group (for example, it may be C1 alkanyl group, C2 alkanyl group, or C3 alkanyl group), unsubstituted C5 to C8 cycloalkyl group (for example, it may be C5 cycloalkyl group, C6 cycloalkyl group, C7 cycloalkyl group, or C8 cycloalkyl group), unsubstituted phenyl group, halogen-substituted phenyl group, or cyano-substituted phenyl group.

Preferably, said R is²、R³Or R⁵-R⁷Each independently selected from hydrogen, halogen, substituted or unsubstituted alkyl or substituted or unsubstituted alkoxy, preferably hydrogen, halogen, unsubstituted C1-C6 alkyl (which may be, for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl), or unsubstituted C1-C6 alkoxy (which may be, for example, C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, or C6 alkoxy).

Preferably, said R is²Or R³Selected from hydrogen.

Preferably, said R is⁵Selected from hydrogen, halogen, unsubstituted C1-C6 alkyl (such as C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl) or unsubstituted C1-C6 alkoxy (such as C1 alkoxy, C5 alkyl or C6 alkyl),C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy or C6 alkoxy).

Preferably, said R is⁶Selected from hydrogen or unsubstituted C1-C6 alkyl (which may be, for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl).

Preferably, said R is⁷Selected from hydrogen or unsubstituted C1-C6 alkyl (which may be, for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl).

Preferably, said R is⁴Selected from hydroxy or unsubstituted C1-C6 alkoxy (which may be, for example, C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy or C6 alkoxy).

Preferably, said R is⁸And a substituent selected from any one of substituted or unsubstituted pyridyl, substituted or unsubstituted quinolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted pyrrolopyrimidyl, substituted or unsubstituted pyrimidyl, or substituted or unsubstituted benzimidazolyl.

Preferably, said R is⁸Selected from nitrogen-containing heterocycles as shown in the following structure, wherein,

represents a substituent attachment position:

wherein R is^aSelected from hydrogen, C1-C6 alkyl (which may be, for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl), C2-C6 alkenyl (which may be, for example, C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, or C6 alkenyl), cyano, halogen, C1-C6 alkoxy (which may be, for example, C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, or C6 alkoxy), C1-C6 acyl (which may be, for example, C1 acyl, C2 acyl, C3 acyl, C4 acyl, C5 acyl, or C6 acyl), C1-C6 sulfonyl (which may be, for example, C1 sulfonyl, C2 sulfonyl, C3 sulfonyl, C4 sulfonyl, C5 sulfonylOr a C6 sulfonyl group), a C6-C12 aryl group (for example, a C6 aryl group, a C8 aryl group, a C10 aryl group, a C12 aryl group, or the like), an alkyl-substituted amino group, an aryl-substituted amino group, or a C3-C6 nitrogen-containing heterocyclic group (for example, a C3 nitrogen-containing heterocyclic group, a C4 nitrogen-containing heterocyclic group, a C5 nitrogen-containing heterocyclic group, or a C6 nitrogen-containing heterocyclic group).

Preferably, said R is⁹Selected from ester group substituted C1-C6 alkyl (for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl) or hydroxyl substituted C1-C6 alkyl (for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl), preferably the structure shown below,

represents a substituent attachment position:

in the invention, the 9, 10-dihydrophenanthrene compound is selected from any one of the following A1-A28:

in the present invention, the 9,10 dihydrophenanthrene compound has a structure represented by the following formula II:

wherein R is¹⁰Is halogen, R¹¹Is hydrogen or unsubstituted C1-C6 alkyl (for example, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl), R¹²Is hydrogen or unsubstituted C1-C6 acyl (e.g. maySo as to be any one of C1 acyl group, C2 acyl group, C3 acyl group, C4 acyl group, C5 acyl group or C6 acyl group), R¹³An unsubstituted C1-C6 alkyl group (which may be, for example, C1 alkyl group, C2 alkyl group, C3 alkyl group, C4 alkyl group, C5 alkyl group, or C6 alkyl group), an unsubstituted C1-C6 acyl group (which may be, for example, C1 acyl group, C2 acyl group, C3 acyl group, C4 acyl group, C5 acyl group, or C6 acyl group), an unsubstituted C1-C6 sulfonyl group (which may be, for example, C1 sulfonyl group, C2 sulfonyl group, C3 sulfonyl group, C4 sulfonyl group, C5 sulfonyl group, or C6 sulfonyl group), an unsubstituted C6-C12 aryl group (which may be, for example, a C6 aryl group, C8 aryl group, or the like), or a C8-C8 nitrogen-C8 heterocyclic group (which may be, for example, a C8 nitrogen-containing heterocyclic group).

Preferably, the 9,10 dihydrophenanthrene compound has a structure shown as II-B or II-C:

preferably, the II-B is selected from any one of the following B1-B20:

preferably, the II-C is selected from any one of the following C1-C7:

in a second aspect, the present invention provides the use of a 9,10 dihydrophenanthrene compound in the manufacture of a medicament for the treatment of diseases caused by a novel coronavirus and/or a novel mutant coronavirus.

In the present invention, the 3CL protease inhibitor further comprises a pharmaceutically acceptable adjuvant.

In the invention, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of vitamin C, sorbitol, mannitol, xylitol, fructose, amino acid, meglumine, dextrin, magnesium stearate or sucrose.

In the invention, the dosage form of the 3CL protease inhibitor comprises any one of an injection preparation, an oral preparation or a freeze-dried powder injection.

In the invention, the dosage form of the 3CL protease inhibitor is an oral preparation, and the oral preparation comprises any one of tablets, powder, capsules or granules.

In the present invention, the 3CL protease inhibitor further comprises a pharmaceutically acceptable excipient.

Preferably, the pharmaceutically acceptable excipient comprises any one of a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer or a colorant, or a combination of at least two thereof.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "substituted or unsubstituted" means that a hydrogen in the structure is substituted with the substituent or that the hydrogen is unsubstituted. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, or more than one (to the substitutable position on the substituted structure) position in the structure may be substituted.

In the present invention, "C1-C6", "C6-C12" and the like before the specific group indicate the number of carbon atoms contained in the group, for example, "C1-C6" indicates a group whose number of carbon atoms may be 1, 2, 3, 4, 5 or 6, "C6-C12" indicates a group whose number of carbon atoms may be 6, 7, 8, 9,10, 11 or 12, and the like.

Radical definition

The term "alkyl" denotes a saturated alkanyl or cycloalkyl group.

The term "alkanyl" denotes a saturated straight or branched alkyl group, which may be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

The term "cycloalkyl" denotes a saturated monocyclic hydrocarbon ring group containing more than 3 carbon atoms, which may be, for example

(cyclopropyl) to give,

(cyclobutyl) or

(cyclopentyl), and the like, wherein,

represents a substituent attachment position.

The term "alkoxy" means an alkyl group attached to the rest of the molecule through an oxygen atom and may be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy and the like.

The term "halogen" denotes F, Cl, Br or I.

The term "aryl" denotes a carbocyclic ring system having aromatic character, which refers to monocyclic, bicyclic and tricyclic carbocyclic ring systems containing aromatic character, and may be, for example, phenyl, naphthyl, anthracenyl and the like.

The term "alkenyl" denotes an unsaturated hydrocarbon group containing a carbon-carbon double bond, and may be, for example, vinyl, propenyl, allyl, isopropenyl, n-butenyl, isobutenyl, n-pentenyl, isopentenyl, n-hexenyl, isohexenyl, and the like.

The term "heterocyclyl" denotes a heteroatom containing heterocycloalkyl or heterocycloaryl which may be azacycloalkyl, oxacycloalkyl or thiacycloalkyl, and may be, for example, tetrahydrofuranyl, hexahydropyridanyl, dioxanyl, dithianyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, or the like; the heterocyclic aryl group may be azaheterocyclic aryl, oxaheterocyclic aryl or thiaheterocyclic aryl, and may be, for example, furyl, thienyl, pyrrolyl, thiazolyl, imidazolyl, pyrazolyl, pyranyl, pyridyl, pyrimidinyl, quinolyl, purinyl, isoquinolyl, benzimidazolyl, pyrrolopyrimidyl, thienyl, thiazolyl, oxazolyl, pyridazinyl, phenylpropfuranyl, benzothiazolyl, or the like.

The term "acyl" denotes a compound having

A group of structures; wherein the content of the first and second substances,

represents a substituent attachment position, and unless otherwise limited, in the present invention, the R' means an alkyl group or an aryl group having 1 to 20 carbon atoms.

The term "sulfonyl" denotes a compound having

A group of structures; wherein the content of the first and second substances,

represents a substituent attachment position, and unless otherwise limited, in the present invention, the R "means an alkyl group or an aryl group having 1 to 20 carbon atoms.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention applies the 9, 10-dihydrophenanthrene compound to the preparation of the coronavirus 3CL protease inhibitor for the first time, and provides a new strategy for preparing the medicine for treating diseases caused by the novel coronavirus and/or the novel coronavirus mutant strain.

(2) The 9, 10-dihydrophenanthrene compound has good inhibition effect on coronavirus 3CL protease, wherein the 9, 10-dihydrophenanthrene compound has the structure shown in formula I, and the structure is IC₅₀The value is in the range of 1-70. mu.M; in particularIC of 9,10 dihydrophenanthrene compounds having the structure shown in formula II-C₅₀The value can reach 1.5-6 mu M range.

Drawings

FIG. 1 shows that B1-B20 is directed to SARS-CoV-23CL^proSchematic representation of the inhibition of (1).

FIG. 2 shows that C1-C7 is directed to SARS-CoV-23CL^proSchematic representation of the inhibition of (1).

FIG. 3 shows that C1 is directed to SARS-CoV-23CL^proDose inhibition profile of (a).

FIG. 4 shows the comparison of C1 for SARS-CoV-23CL^proLineweaver-Burk plot of inhibition.

FIG. 5 shows the comparison of C2 for SARS-CoV-23CL^proDose inhibition profile of (a).

FIG. 6 shows that C2 is against SARS-CoV-23CL^proLineweaver-Burk plot of inhibition.

FIG. 7 is a graph of the metabolic stability test of C1 and C2 in the human liver microsome stage I metabolic system.

FIG. 8 is a graph of the metabolic stability tests of C1 and C2 in the human liver microsomal phase II metabolic system.

FIG. 9 shows the combination of compound C1 and SARS-CoV-23CL^proDimeric interface molecular docking map of proteins.

FIG. 10 shows the combination of compound C1 and SARS-CoV-23CL^proMolecular docking of the substrate binding domain of the protein.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The sources of instrumentation and reagents in the following examples are as follows:

the preparation methods of the compound A1-A4, A6-A28 and the compound B1 are disclosed in CN111303017A and are not described in detail.

Preparation example 1

Preparation of methyl (8-cyclohexyl-5-hydroxy-1- (4-phenylpyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (A5)

First step Synthesis of 2, 4-Diphenylpyridine (2a) Phenylboronic acid (2a) (146mg,1.2mmol), 2-bromo-4-phenylpyridine (3a) (234mg,1.0mmol), Pd (PPh) under Nitrogen protection₃)₂Cl₂(3.5mg,0.5mol％)、K₂CO₃(387mg,2.8mmol) and DME/H₂O(6mL,V_DME:V_H2O3:1) mixing; the mixture was stirred at 80 ℃ for about 14 hours and the completion of the reaction was monitored by TLC. After cooling to room temperature, the mixture was extracted with ethyl acetate, the organic phase was washed with brine, then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography to give a colorless oily liquid with a yield of 57.0%.¹H NMR(600MHz,CDCl₃)δ8.71(d,J＝5.1Hz,1H),8.04(d,J＝7.5Hz,2H),7.90(s,1H),7.65(d,J＝7.5Hz,2H),7.49–7.45(m,4H),7.43(d,J＝7.4Hz,1H),7.40(d,J＝6.8Hz,2H).ESI-HRMS[M+H]⁺calcd for C₁₇H₁₄N:232.1121,found:232.1131.

Second step synthesis of compound a 5: under the protection of nitrogen, compound 3a (0.4mmol), compound 4a (60mg,0.2mmol) and [ Cp + RhCl ] are added₂]₂(8mg,5mol％)、NaBAr^F4(55mg,24 mol%) in CH₃COOH (1.0mL) were mixed, the mixture was stirred at 100 ℃ for 8 hours, and the reaction was monitored by TLC for completion. After cooling to room temperature, NaHCO was added₃The pH of the aqueous solution is adjusted to be alkalescent. The mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The white solid is obtained by column chromatography separation, and the yield is 45.0%.¹H NMR(600MHz,CDCl₃)δ8.79(d,J＝5.2Hz,1H),8.16(d,J＝7.7Hz,1H),7.71(d,J＝7.2Hz,2H),7.67(d,J＝1.0Hz,1H),7.56(dd,J＝5.2,1.8Hz,1H),7.56(dd,J＝5.2,1.8Hz,1H),7.51(t,J＝7.4Hz,2H),7.47(d,J＝7.2Hz,1H),7.32(dd,J＝7.5,0.8Hz,1H),7.24(d,J＝7.7Hz,1H),7.00(d,J＝8.6Hz,1H),6.74(d,J＝8.5Hz,1H),3.87(dd,J＝10.8,5.7Hz,1H),3.48(t,J＝10.5Hz,1H),3.43–3.37(m,1H),2.92(dd,J＝15.5,2.0Hz,1H),2.78–2.69(m,2H),1.86–1.67(m,8H),1.57–1.51(m,1H),1.44–1.33(m,2H),1.29–1.22(m,2H).¹³C NMR(150MHz,CDCl₃)δ170.78,160.42,151.69,149.05,148.99,139.79,137.99,137.05,135.64,133.24,132.79,129.31,129.23,128.08,128.01,127.10,126.07,125.92,122.61,121.71,119.87,116.46,63.65,38.97,35.90,33.62,33.08,28.15,27.10,26.25,20.51.ESI-HRMS[M+H]⁺ calcd for C₃₄H₃₄NO₃:504.2533,found:504.2540.

Preparation example 2

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (4-phenylpyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B2)

See preparation example 1 for reaction conditions and workup.

Compound B2 was a white solid in 68.5% yield.¹H NMR(600MHz,CDCl₃)δ8.78(d,J＝5.3Hz,1H),8.26(d,J＝7.7Hz,1H),7.70(d,J＝7.2Hz,2H),7.63(s,1H),7.58(dd,J＝5.3,1.6Hz,1H),7.52–7.45(m,6H),7.24(d,J＝7.7Hz,1H),7.11(d,J＝8.3Hz,2H),6.86(d,J＝8.3Hz,1H),6.71(d,J＝8.3Hz,1H),3.65(dd,J＝10.8,5.6Hz,1H),3.49(t,J＝10.5Hz,1H),3.13–3.09(m,1H),2.88(dd,J＝15.6,4.5Hz,1H),2.77(dd,J＝15.6,2.2Hz,1H),1.50(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.42,153.43,148.86,140.48,137.81,136.27,133.13,132.78,132.44,131.23,131.19,129.71,129.42,129.26,128.48,128.09,127.07,125.91,122.56,121.89,120.97,120.01,118.12,116.25,63.46,34.14,28.30,20.42.ESI-HRMS[M+H]⁺calcd for C₃₄H₂₇BrNO₃:576.1169,found:576.1186.

Preparation example 3

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (4-methylpyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B3)

See preparation example 1 for reaction conditions and workup.

Intermediate 4-methyl-2-phenylpyridine (2b) was a white solid in 42.0% yield.¹H NMR(600MHz,CDCl₃)δ8.55(d,J＝5.0Hz,1H),7.97(d,J＝7.4Hz,2H),7.55(s,1H),7.47(t,J＝7.6Hz,2H),7.40(t,J＝7.3Hz,1H),7.06(d,J＝4.9Hz,1H),2.42(s,3H).ESI-HRMS[M+H]⁺ calcd for C₁₂H₁₂N:170.0964,found:170.0965.

Compound B3 was a white solid with a yield of 40.9%.¹H NMR(600MHz,DMSO-d₆)δ10.12(s,1H),8.56(d,J＝5.0Hz,1H),8.49(d,J＝7.8Hz,1H),7.64(d,J＝8.4Hz,2H),7.42(t,J＝7.7Hz,1H),7.37(dd,J＝7.6,1.2Hz,1H),7.35(s,1H),7.33(d,J＝8.4Hz,2H),7.26(d,J＝5.0Hz,1H),7.04(q,J＝8.4Hz,2H),3.54(dd,J＝10.8,6.2Hz,1H),3.44(t,J＝10.2Hz,1H),3.24–3.19(m,1H),2.92-2.84(m,2H),2.43(s,3H),1.64(s,3H).¹³C NMR(150MHz,DMSO-d₆)δ170.00,159.24,155.01,149.13,147.43,140.93,140.65,136.57,133.26,132.51,132.13,131.62,131.49,130.28,128.80,128.60,125.96,125.36,123.26,121.12,120.61,115.99,63.25,33.82,28.25,21.00,20.60.ESI-HRMS[M+H]⁺ calcd for C₂₉H₂₅BrNO₃:514.1012,found:514.1020.

Preparation example 4

Preparation of methyl (8- (4-bromophenyl) -1- (4-chloropyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B4)

See preparation example 1 for reaction conditions and workup.

Intermediate 4-chloro-2-phenylpyridine (2c) was a colorless oily liquid in 54.0% yield.¹H NMR(600MHz,CDCl₃)δ8.57(d,J＝5.1Hz,1H),7.96(d,J＝7.3Hz,2H),7.72(s,1H),7.49–7.42(m,3H),7.23(d,J＝3.6Hz,1H).ESI-HRMS[M+H]⁺ calcd for C₁₁H₉ClN:190.0418,found:190.0421.

Compound B4 was a white solid in 27.3% yield.¹H NMR(600MHz,Acetone-d₆)δ9.03(s,1H),8.66(d,J＝5.4Hz,1H),8.59(dd,J＝5.4,3.8Hz,1H),7.62(d,J＝8.3Hz,2H),7.60(d,J＝1.8Hz,1H),7.48(dd,J＝5.3,1.9Hz,1H),7.42(d,J＝1.8Hz,1H),7.41(s,1H),7.33(d,J＝8.3Hz,2H),7.06(q,J＝8.3Hz,2H),3.65(dd,J＝10.9,5.8Hz,1H),3.52(t,J＝10.5Hz,1H),3.31–3.26(m,1H),2.98(dd,J＝15.7,2.3Hz,1H),2.91–2.88(m,1H),1.66(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.42,153.43,140.48,137.81,137.73,136.27,136.23,133.13,132.78,132.44,132.33,131.19,129.71,129.26,128.48,127.07,125.91,122.56,121.89,120.97,120.01,116.25,63.46,34.14,28.30,20.42.ESI-HRMS[M+H]⁺ calcd for C₂₈H₂₂BrClNO₃:534.0466,found:534.0475.

Preparation example 5

Preparation of methyl (8- (4-bromophenyl) -1- (4-cyanopyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B5)

See preparation example 1 for reaction conditions and workup.

Intermediate 2-phenylisonicotinic acid nitrile (2d) was a white solid in 77.8% yield.¹H NMR(600MHz,CDCl₃)δ8.78(d,J＝4.3Hz,1H),7.92(dd,J＝8.1,1.4Hz,2H),7.86(s,1H),7.45–7.39(m,3H),7.37(dd,J＝4.9,1.3Hz,1H).ESI-HRMS[M+H]⁺ calcd for C₁₂H₉N₂:181.0760,found:181.0772.

Compound B5 was a white solid in 69.9% yield.¹H NMR(600MHz,Acetone-d₆)δ9.05(d,J＝16.6Hz,1H),8.61(dd,J＝7.4,1.4Hz,1H),8.33(dd,J＝8.2,2.1Hz,1H),7.78(d,J＝8.2Hz,1H),7.61(d,J＝8.3Hz,2H),7.46–7.42(m,2H),7.32(d,J＝8.3Hz,2H),7.08–7.02(m,2H),3.64(dd,J＝10.9,5.9Hz,1H),3.49(t,J＝10.4Hz,1H),3.30–3.25(m,1H),3.00(dd,J＝15.7,2.2Hz,1H),2.94(dd,J＝13.5,4.5Hz,1H),1.62(s,3H).¹³C NMR(150MHz,Acetone-d₆)δ171.11,164.81,156.25,153.61,142.61,141.50,140.93,138.50,135.36,134.69,134.00,133.43,132.98,131.92,131.61,130.30,127.59,126.25,122.92,122.32,118.60,117.44,109.53,64.54,35.90,29.80,21.33.ESI-HRMS[M+Na]⁺ calcd for C₂₉H₂₁BrN₂O₃Na:547.0628,found:547.0635.

Preparation example 6

Preparation of methyl (8- (4-bromophenyl) -1- (4-formylpyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B6)

See preparation example 1 for reaction conditions and workup.

Intermediate 2-phenylisonicotinal (2e) was a colorless oily liquid with a yield of 95.0%.¹H NMR(600MHz,CDCl₃)δ10.02(s,1H),8.83(d,J＝4.8Hz,1H),8.02(s,1H),7.96(d,J＝7.3Hz,2H),7.52(dd,J＝4.8,1.1Hz,1H),7.41(t,J＝7.4Hz,2H),7.37(d,J＝7.2Hz,1H).ESI-HRMS[M+H]⁺ calcd for C₁₂H₁₀NO:184.0757,found:184.0758.

Compound B6 was a yellow solid in 35.8% yield.¹H NMR(600MHz,Acetone-d₆)δ10.23(s,1H),9.03(s,1H),8.97(d,J＝4.9Hz,1H),8.60(d,J＝7.7Hz,1H),7.96(s,1H),7.80(d,J＝4.8Hz,1H),7.60(d,J＝8.1Hz,2H),7.48–7.40(m,2H),7.31(d,J＝8.1Hz,2H),7.04(q,J＝8.3Hz,2H),3.64(dd,J＝10.8,5.9Hz,1H),3.51(t,J＝10.4Hz,1H),3.31–3.23(m,1H),3.00(d,J＝15.6Hz,1H),2.94–2.90(m,1H),1.57(s,3H).¹³C NMR(150MHz,Acetone-d₆)δ192.34,169.32,161.24,154.49,150.52,142.34,140.88,139.77,136.73,133.43,132.75,132.16,131.65,131.17,130.01,129.30,128.48,125.69,122.84,121.30,120.48,119.83,115.63,62.80,48.89,34.15,19.49.ESI-HRMS[M+Na]⁺ calcd for C₂₉H₂₂BrNO₄Na:550.0624,found:550.0631.

Preparation example 7

Preparation of methyl (8- (4-bromophenyl) -1- (4- (dimethylamino) pyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B7)

First step synthesis of N, N-dimethyl-2-phenylpyridin-4-amine (2 f): phenylboronic acid (146mg,1.2mmol), 2-bromo-N, N-dimethylpyridin-4-amine (1f) (234mg,1.0mmol), Pd (PPh) under nitrogen₃)₄(1.0mol％)、K₂CO₃(2.0mmol) of C₂H₅And (4) mixing the OH solution. The mixture was stirred at 75 ℃ for about 15 hours and the reaction was monitored by TLC for completion. After cooling to room temperature, the mixture was extracted with ethyl acetate. The organic layer was washed with brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo, and chromatographed on silica gel column to give a white solid in 83.0% yield.¹H NMR(600MHz,CDCl₃)δ8.25(d,J＝6.0Hz,1H),7.89(d,J＝8.1Hz,2H),7.40(t,J＝7.5Hz,2H),7.35(d,J＝7.2Hz,1H),6.81(s,1H),6.38(d,J＝5.9Hz,1H),2.93(s,6H).ESI-HRMS[M+H]⁺ calcd for C₁₃H₁₅N₂:199.1230,found:199.1231.

Synthesis of second step B7, reaction conditions and workup refer to the second step of preparation example 1. White solid, yield 36.0.¹H NMR(600MHz,CDCl₃)δ8.30(s,1H),8.26(d,J＝5.8Hz,1H),7.46(d,J＝7.7Hz,2H),7.09(d,J＝7.7Hz,2H),7.00(d,J＝3.7Hz,2H),6.77(s,2H),6.60(d,J＝4.4Hz,,1H),6.54(s,1H),3.60(dd,J＝10.6,5.7Hz,1H),3.46(t,J＝9.8Hz,1H),3.10(s,7H),2.88(d,J＝14.9Hz,1H),2.56(d,J＝15.1Hz,1H),1.63(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.37,155.76,155.00,141.05,135.58,133.32,132.70,131.39,131.23,131.04,129.46,127.48,125.32,121.54,120.62,116.97,107.22,105.24,64.07,39.55,33.98,29.71,28.44,20.64.ESI-HRMS[M+H]⁺ calcd for C₃₀H₂₈BrN₂O₃:543.1278,found:543.1288.

Preparation example 8

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (4- (pyrrolidin-1-yl) pyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B8)

See preparation example 1 for reaction conditions and workup.

Intermediate 2-phenyl-4- (pyrrolidin-1-yl) pyridine (2g) was a white solid in 75.0% yield.¹H NMR(600MHz,CDCl₃)δ8.31(d,J＝5.9Hz,1H),7.93(d,J＝7.6Hz,2H),7.45(t,J＝7.5Hz,2H),7.40(t,J＝7.3Hz,1H),6.76(d,J＝2.0Hz,1H),6.38(dd,J＝5.9,2.1Hz,1H),3.39(t,J＝6.5Hz,4H),2.06(t,J＝6.6Hz,4H).ESI-HRMS[M+H]⁺ calcd for C₁₅H₁₈N₂:225.1386,found:225.1388.

Compound B8 was a white solid in 68.5% yield.¹H NMR(600MHz,CDCl₃)δ8.41–8.36(m,1H),8.11(d,J＝6.0Hz,1H),7.46(d,J＝8.3Hz,2H),7.09(d,J＝8.3Hz,2H),7.07(d,J＝4.1Hz,2H),7.03(d,J＝8.0Hz,1H),6.82(d,J＝8.3Hz,1H),6.55(dd,J＝6.8,2.2Hz,1H),6.43(d,J＝2.2Hz,1H),3.60(dd,J＝10.9,5.9Hz,1H),3.46(d,J＝10.5Hz,5H),3.12–3.16(m,1H),3.02(d,J＝13.6Hz,1H),2.55(d,J＝14.3Hz,1H),2.09(s,4H),1.62(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.36,154.85,153.92,140.77,135.56,133.76,132.74,131.47,131.38,131.12,130.62,130.04,127.64,126.05,120.96,120.74,116.92,107.77,105.99,64.05,48.14,33.98,28.62,25.24,20.68.ESI-HRMS[M+Na]⁺ calcd for C₃₂H₂₉BrN₂O₃Na:591.1254,found:591.1259.

Preparation example 9

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (4-morpholinopyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B9)

See preparation example 1 for reaction conditions and workup.

Intermediate 4- (2-phenylpyridin-4-yl) morpholine (2h) was a colorless oily liquid, yield 61.0%.¹H NMR(600MHz,CDCl₃)δ8.40(d,J＝5.9Hz,1H),7.91(d,J＝7.6Hz,2H),7.45(t,J＝7.5Hz,2H),7.39(t,J＝7.3Hz,1H),7.08(d,J＝2.4Hz,1H),6.65(dd,J＝5.9,2.5Hz,1H),3.90–3.85(m,4H),3.39–3.33(m,4H).ESI-HRMS[M+H]⁺ calcd for C₁₅H₁₇N₂O:241.1335,found:241.1338.

Compound B9 was a white solid in 23.0% yield.¹H NMR(600MHz,CDCl₃)δ8.41(d,J＝6.0Hz,1H),8.26(d,J＝7.8Hz,1H),7.47(d,J＝8.3Hz,2H),7.09(d,J＝8.3Hz,2H),7.05–6.98(m,2H),6.76–6.71(m,3H),6.63(d,J＝8.2Hz,1H),3.87–3.82(m,4H),3.56(dd,J＝10.8,6.0Hz,1H),3.49(t,J＝10.1Hz,1H),3.41–3.35(m,4H),3.06(s,1H),2.86(dd,J＝15.5,4.0Hz,1H),2.59(d,J＝14.1Hz,1H),1.65(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.35,156.05,154.52,141.03,135.76,132.89,131.52,131.35,131.06,129.25,127.62,125.09,121.87,120.67,116.75,108.66,106.49,66.33,64.09,46.10,34.01,28.39,20.71..ESI-HRMS[M+H]⁺ calcd for C₃₂H₃₀BrN₂O₄:585.1383,found:585.1396.

Preparation example 10

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (5-methylpyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B10)

See preparation example 1 for reaction conditions and workup.

Compound B10 was a white solid in 44.6% yield.¹H NMR(600MHz,CDCl₃)δ8.57(s,1H),8.20(t,J＝4.6Hz,1H),7.62(d,J＝7.8Hz,1H),7.48(d,J＝8.3Hz,2H),7.31(d,J＝7.9Hz,1H),7.16(d,J＝4.4Hz,2H),7.09(d,J＝8.3Hz,2H),6.82(d,J＝8.3Hz,1H),6.67(d,J＝8.3Hz,1H),3.64(dd,J＝10.9,5.9Hz,1H),3.44(t,J＝10.4Hz,1H),3.12–3.07(m,1H),2.82(dd,J＝15.5,4.5Hz,1H),2.70(dd,J＝15.5,2.0Hz,1H),2.44(s,3H),1.66(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.39,153.44,148.78,140.54,137.28,136.31,133.06,132.69,132.33,131.66,131.26,131.17,129.63,128.51,127.78,125.80,123.98,121.91,120.92,116.18,63.66,34.08,28.35,20.63,18.25.ESI-HRMS[M+H]⁺ calcd for C₂₉H₂₅BrNO₃:514.1012,found:514.1020.

Preparation example 11

Preparation of methyl (8- (4-bromophenyl) -1- (5-fluoropyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B11)

See preparation example 1 for reaction conditions and workup.

Intermediate 4-fluoro-2-phenylpyridine (2j) was a white solid in 82.9% yield.¹H NMR(600MHz,CDCl₃)δ8.46(d,J＝2.9Hz,1H),7.85(d,J＝7.4Hz,2H),7.63(dd,J＝8.7,4.3Hz,1H),7.42–7.36(m,3H),7.33(t,J＝7.3Hz,1H).ESI-HRMS[M+H]⁺ calcd for C₁₁H₉FN:174.0714,found:174.0716.

Compound B11 was a white solid in 36.0% yield.¹H NMR(600MHz,CDCl₃)δ8.57(d,J＝2.6Hz,1H),8.21(dd,J＝6.8,2.0Hz,1H),7.50(d,J＝8.1Hz,3H),7.42(dd,J＝8.6,4.4Hz,1H),7.33–7.29(m,2H),7.13(d,J＝8.2Hz,2H),6.94(d,J＝8.3Hz,1H),6.77(d,J＝8.3Hz,1H),3.69(dd,J＝10.9,5.8Hz,1H),3.48(t,J＝10.4Hz,1H),3.17(s,1H),2.88–2.78(m,2H),1.68(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.47,155.90,153.06,140.27,139.16,137.11,136.96,136.42,133.05,132.81,131.28,131.22,129.96,128.71,128.02,126.19,125.32,123.39,123.26,121.69,121.11,115.99,63.50,34.13,28.34,20.64.ESI-HRMS[M+Na]⁺calcd for C₂₈H₂₁BrFNO₃Na:540.0581,found:540.0590.

Preparation example 12

Preparation of methyl (8- (4-bromophenyl) -1- (5-chloropyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B12)

See preparation example 1 for reaction conditions and workup.

Intermediate 2k was a colorless oily liquid, yield 82.0%.¹H NMR(600MHz,CDCl₃)δ8.56(d,J＝2.4Hz,1H),7.89–7.86(m,2H),7.64(dd,J＝8.5,2.5Hz,1H),7.59(d,J＝8.5Hz,1H),7.40(t,J＝7.5Hz,2H),7.36–7.33(m,1H).ESI-HRMS[M+H]⁺ calcd for C₁₁H₉NCl:190.0418,found:190.0420.

Compound B12 was a white solid with a yield of 25.5%.¹H NMR(600MHz,CDCl₃)δ8.67(d,J＝2.1Hz,1H),8.22(dd,J＝6.9,1.7Hz,1H),7.76(dd,J＝8.3,2.4Hz,1H),7.51(d,J＝8.2Hz,2H),7.38(d,J＝8.3Hz,1H),7.34–7.29(m,2H),7.13(d,J＝8.2Hz,2H),6.94(d,J＝8.3Hz,1H),6.77(d,J＝8.3Hz,1H),6.71(s,1H),3.69(dd,J＝11.0,5.8Hz,1H),3.47(t,J＝10.4Hz,1H),3.17(s,1H),2.88–2.80(m,2H),1.68(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.44,157.72,152.96,147.78,140.21,139.07,136.45,136.17,133.04,132.84,132.79,131.27,131.19,130.64,130.01,128.67,128.11,126.28,125.12,121.61,121.11,115.96,63.47,34.11,28.36,20.64.ESI-HRMS[M+H]⁺ calcd for C₂₈H₂₂BrClNO₃:534.0466,found:534.0475.

Preparation example 13

Preparation of methyl (8- (4-bromophenyl) -1- (5-bromopyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B13)

See preparation example 1 for reaction conditions and workup.

Compound B13 was a yellow solid in 20.0% yield.¹H NMR(600MHz,CDCl₃)δ8.77(d,J＝2.0Hz,1H),8.22(dd,J＝7.2,1.6Hz,1H),7.90(d,J＝8.3Hz,1H),7.51(d,J＝8.3Hz,2H),7.33(t,J＝8.7Hz,3H),7.14(d,J＝8.2Hz,2H),6.96(dd,J＝8.3,1.8Hz,1H),6.80(dd,J＝8.3,2.5Hz,1H),6.40(s,1H),3.69(dd,J＝10.9,5.7Hz,1H),3.48(t,J＝10.4Hz,1H),3.19(s,1H),2.86(d,J＝3.3Hz,2H),1.69(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.41,157.98,152.86,150.05,140.16,138.96,136.53,133.04,132.90,132.84,131.29,131.19,130.09,128.69,128.00,127.13,126.40,125.59,121.59,121.14,119.26,115.93,63.44,34.13,28.38,20.65.ESI-HRMS[M+Na]⁺ calcd for C₂₈H₂₁Br₂NO₃Na:599.9780,found:599.9788.

Preparation example 14

Preparation of methyl (8- (4-bromophenyl) -1- (5-cyanopyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B14)

See preparation example 1 for reaction conditions and workup.

Intermediate 5-phenyl nicotinonitrile (2m) was a white solid in 93.4% yield.¹H NMR(600MHz,CDCl₃)δ8.94(dd,J＝2.1,0.7Hz,1H),8.04(dd,J＝7.9,1.7Hz,2H),8.01(dd,J＝8.3,2.2Hz,1H),7.85(dd,J＝8.3,0.8Hz,1H),7.53–7.50(m,3H).ESI-HRMS[M+H]⁺ calcd for C₁₂H₉N₂:181.0760,found:181.0763.

Compound B14 was a white solid in 26.9% yield.¹H NMR(600MHz,Acetone-d₆)δ9.05(s,1H),8.94(d,J＝5.0Hz,1H),8.61(d,J＝7.6Hz,1H),7.92(s,1H),7.75(dd,J＝5.0,1.3Hz,1H),7.61(d,J＝8.3Hz,2H),7.46–7.41(m,2H),7.32(d,J＝8.3Hz,2H),7.07–7.03(m,2H),3.66(dd,J＝10.9,5.8Hz,1H),3.50(t,J＝10.4Hz,1H),3.30–3.25(m,1H),2.99–2.90(m,2H),1.63(s,3H).¹³C NMR(150MHz,Acetone-d₆)δ169.34,160.87,154.52,150.32,140.84,138.93,136.66,133.50,132.80,132.15,131.65,131.18,130.09,129.62,128.50,125.88,125.76,123.30,121.16,120.50,120.35,116.61,115.65,62.78,34.13,27.89,19.55.ESI-HRMS[M+H]⁺ calcd for C₂₉H₂₂BrN₂O₃:525.0808,found:525.0822.

Preparation example 15

Preparation of methyl (8- (4-bromophenyl) -1- (5-formylpyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B15)

See preparation example 1 for reaction conditions and workup.

Intermediate 5-phenyl nicotinaldehyde (2n) was a white solid, 79.1% payee.¹H NMR(600MHz,CDCl₃)δ10.16(s,1H),9.15(d,J＝1.9Hz,1H),8.26(dd,J＝8.2,2.2Hz,1H),8.11(dd,J＝8.0,1.5Hz,2H),7.93(d,J＝8.2Hz,1H),7.56–7.51(m,3H).ESI-HRMS[M+H]⁺ calcd for C₁₂H₁₀NO:184.0757,found:184.0765.

Compound B15 was a white solid in 28.3% yield.¹H NMR(600MHz,CDCl₃)δ10.18(s,1H),9.17(s,1H),8.29–8.26(m,2H),7.62(d,J＝8.0Hz,1H),7.51(d,J＝8.0Hz,2H),7.43–7.36(m,2H),7.14(d,J＝7.9Hz,2H),6.97(d,J＝8.2Hz,1H),6.81(d,J＝8.2Hz,1H),6.40(s,1H),3.71(dd,J＝10.8,5.7Hz,1H),3.49(t,J＝10.4Hz,1H),3.19(s,1H),2.94–2.86(m,2H),1.66(s,3H).¹³C NMR(150MHz,CDCl₃)δ190.36,170.36,164.75,152.84,151.49,140.07,139.26,136.54,136.09,133.25,133.04,132.98,131.32,131.17,130.20,129.67,128.87,128.75,126.51,124.86,121.46,121.20,115.96,63.40,34.11,28.51,20.62.ESI-HRMS[M+Na]⁺ calcd for C₂₉H₂₂BrNO₄Na:550.0624,found:500.0632.

Preparation example 16

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (5- (methylsulfonyl) pyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B16)

See preparation example 1 for reaction conditions and workup.

Intermediate 4-, (Methylsulfonyl) -2-phenylpyridine (2o) was a white solid in 99.0% yield.¹H NMR(600MHz,CDCl₃)δ9.20(d,J＝2.3Hz,1H),8.27(dd,J＝8.4,2.4Hz,1H),8.07(dd,J＝7.7,1.9Hz,2H),7.92(d,J＝8.4Hz,1H),7.55–7.50(m,3H),3.15(s,3H).ESI-HRMS[M+H]⁺ calcd for C₁₂H₁₂NO₂S:234.0583,found:234.0591.

Compound B16 was a white solid, 39.0% payee.¹H NMR(600MHz,Acetone-d₆)δ9.17(s,1H),9.04(s,1H),8.62(d,J＝7.6Hz,1H),8.40(dd,J＝8.2,2.2Hz,1H),7.81(d,J＝8.2Hz,1H),7.60(d,J＝8.2Hz,2H),7.47–7.42(m,2H),7.31(d,J＝8.3Hz,2H),7.07–7.03(m,2H),3.64(dd,J＝10.9,5.9Hz,1H),3.50(t,J＝10.4Hz,1H),3.29(s,4H),3.00(d,J＝15.7Hz,1H),2.93(dd,J＝15.7,4.5Hz,1H),1.61(s,3H).¹³C NMR(150MHz,,Acetone-d₆)δ170.29,165.03,155.44,148.65,141.80,140.11,137.69,136.53,136.50,134.52,133.92,133.17,132.61,132.15,131.08,130.72,129.51,126.75,125.50,122.11,121.48,116.63,63.79,44.73,35.06,29.01,20.54.ESI-HRMS[M+H]⁺ calcd for C₂₉H₂₅BrNO₅S:578.0631,found:578.0636.

Preparation example 17

See preparation example 1 for reaction conditions and workup.

Preparation of methyl (1- (5-acetylpyridin-2-yl) -8- (4-bromophenyl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B17)

Intermediate 1- (6-methylpyridin-3-yl) -ethanone (2p) was a white solid in 75.0% yield.¹H NMR(600MHz,CDCl₃)δ9.26(d,J＝1.9Hz,1H),8.32(dd,J＝8.3,2.3Hz,1H),8.09(d,J＝7.0Hz,2H),7.87(d,J＝8.3Hz,1H),7.55–7.49(m,3H),2.69(s,3H).ESI-HRMS[M+H]⁺ calcd for C₁₃H₁₂NO:198.0913,found:198.0915.

Compound B17 was a yellow solid, 33.8% collected.¹H NMR(600MHz,CDCl₃)δ9.27(s,1H),8.33(d,J＝5.9Hz,1H),8.28(d,J＝6.5Hz,1H),7.56(d,J＝8.1Hz,1H),7.50(d,J＝8.3Hz,2H),7.31(q,J＝4.7Hz,2H),7.11(d,J＝8.3Hz,2H),6.91(d,J＝8.3Hz,1H),6.73(d,J＝8.3Hz,1H),3.68(dd,J＝11.0,5.8Hz,1H),3.47(t,J＝10.4Hz,1H),3.19–3.14(m,1H),2.91(dd,J＝15.6,4.5Hz,1H),2.81(d,J＝15.6Hz,1H),2.70(s,3H),1.66(s,3H).¹³C NMR(150MHz,CDCl₃)δ196.40,170.45,163.70,153.22,149.11,140.25,138.99,136.36,136.08,133.06,133.04,132.62,131.27,131.19,130.55,129.95,128.88,128.57,126.18,124.46,121.53,121.09,116.04,63.53,34.05,28.46,26.82,20.65.ESI-HRMS[M+H]⁺ calcd for C₃₀H₂₅BrNO₄:542.0961,found:542.0979.

Preparation example 18

Preparation of methyl (8- (4-bromophenyl) -1- (5- (dimethylamino) pyridin-2-yl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B18)

See preparation example 1 for reaction conditions and workup.

Intermediate N, N-dimethyl-6-phenylpyridin-3-amine (2q) was a white solid in 83.0% yield.¹H NMR(600MHz,Acetone-d₆)δ8.08(s,1H),7.91–7.84(m,2H),7.60(d,J＝8.8Hz,1H),7.28–7.25(m,2H),7.15(t,J＝6.6Hz,1H),7.04(dd,J＝9.6,2.3Hz,1H),2.90(s,6H).ESI-HRMS[M+H]⁺calcd for C₁₃H₁₅N₂:199.1230,found:199.1231.

Compound B18 was a white solid in 48.1% yield.¹H NMR(600MHz,CDCl₃)δ8.24(s,1H),8.18(d,J＝6.5Hz,1H),7.46(d,J＝6.5Hz,2H),7.24(s,1H),7.14–7.04(m,5H),6.75(d,J＝8.2Hz,1H),6.59(d,J＝8.2Hz,1H),3.61–3.59(m,1H),3.44(t,J＝10.2Hz,1H),3.06(s,7H),2.85(d,J＝13.0Hz,1H),2.70(d,J＝15.5Hz,1H),1.66(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.48,154.12,147.61,145.06,140.97,138.80,136.05,133.11,132.78,132.51,131.70,131.34,131.06,129.20,128.22,127.88,125.21,124.50,122.18,120.69,119.67,116.45,64.00,40.19,34.10,28.38,20.69.ESI-HRMS[M+H]⁺ calcd for C₃₀H₂₈BrN₂O₃:543.1278,found:543.1290.

Preparation example 19

Preparation of methyl (8- (4-bromophenyl) -5-hydroxy-1- (5-phenylpyridin-2-yl) -9, 10-dihydrophenanthren-9-yl) acetate (B19)

See preparation example 1 for reaction conditions and workup.

Intermediate 2, 5-diphenylpyridine (2r) was a colorless oily liquid, yield 44.2%.¹H NMR(600MHz,CDCl₃)δ8.86(d,J＝2.3Hz,1H),7.97(d,J＝7.1Hz,2H),7.88(dd,J＝8.2,2.4Hz,1H),7.73(dd,J＝8.2,0.6Hz,1H),7.56(d,J＝7.1Hz,2H),7.42(t,J＝7.6Hz,4H),7.37–7.32(m,2H).ESI-HRMS[M+H]⁺ calcd for C₁₇H₁₄N:232.1121,found:232.1172.

Compound B19 was a white solid in 43.8% yield.¹H NMR(600MHz,CDCl₃)δ8.99(s,1H),8.25(d,J＝7.7Hz,1H),8.02(d,J＝7.6Hz,1H),7.79(s,1H),7.67(d,J＝7.3Hz,2H),7.56–7.44(m,6H),7.34–7.27(m,2H),7.12(d,J＝7.5Hz,2H),6.89(d,J＝8.2Hz,1H),6.75(d,J＝8.2Hz,1H),3.71–3.65(m,1H),3.49(t,J＝10.3Hz,1H),3.16(s,1H),2.88(dd,J＝44.9,15.4Hz,2H),1.67(s,3H).¹³C NMR(150MHz,CDCl₃)δ170.42,153.45,140.45,136.31,135.19,133.10,132.90,132.42,131.25,131.20,129.77,129.28,128.56,128.45,128.34,127.10,126.02,124.62,121.78,120.97,116.22,63.63,34.10,28.45,20.66.ESI-HRMS[M+H]⁺calcd for C₃₄H₂₇BrNO₃:576.1169,found:576.1186.

Preparation example 20

Synthesis of methyl (1- (5- (benzylamino) pyridin-2-yl) -8- (4-bromophenyl) -5-hydroxy-9, 10-dihydrophenanthren-9-yl) acetate (B20)

See preparation example 1 for reaction conditions and workup.

Intermediate N-benzyl-6-phenylpyridin-3-amine (2s) was a brown oily liquid in 48.0% yield.¹H NMR(600MHz,CDCl₃)δ8.16(d,J＝2.8Hz,1H),7.89(d,J＝7.2Hz,2H),7.54(d,J＝8.6Hz,1H),7.42(t,J＝7.7Hz,2H),7.40–7.35(m,4H),7.33–7.29(m,2H),6.95(dd,J＝8.6,2.9Hz,1H),4.39(s,2H).ESI-HRMS[M+Na]⁺ calcd for C₁₈H₁₆N₂Na:283.1206,found:283.1209.

Compound B20 was a yellow solid in 87.0% yield.¹H NMR(600MHz,MeOD)δ8.45(d,J＝7.9Hz,1H),7.96(d,J＝2.7Hz,1H),7.53(d,J＝8.2Hz,2H),7.38(d,J＝7.5Hz,2H),7.31(t,J＝7.7Hz,3H),7.25–7.21(m,2H),7.20–7.16(m,3H),7.07(dd,J＝8.5,2.8Hz,1H),6.96(d,J＝8.3Hz,1H),6.90(d,J＝8.3Hz,1H),4.40(d,J＝2.0Hz,2H),3.63(dd,J＝10.8,5.4Hz,1H),3.40(t,J＝10.6Hz,1H),3.17–3.12(m,1H),2.79(s,2H),1.56(s,3H).¹³C NMR(150MHz,MeOD)δ170.80,154.53,146.72,143.93,140.78,139.63,139.11,136.15,133.22,132.28,131.86,131.19,130.91,129.54,128.31,128.23,127.87,126.87,126.73,125.28,124.88,121.46,120.50,119.49,115.06,62.88,46.55,34.31,27.66,19.16.ESI-HRMS[M+H]⁺calcd for C₃₅H₃₀BrN₂O₃:605.1434,found:605.1443.

Preparation example 21

Preparation of 1- (4-bromophenyl) -10- (hydroxymethyl) -8- (4-phenylpyridin-2-yl) -9, 10-dihydrophenanthren-4-ol (C1)

The synthesis steps are as follows: aqueous NaOH (1M) (3mL,3.00mmol), CH₃OH (3mL) and 0.2mmol of compound B2 were mixed and stirred for 2 hours. The methanol was then removed in vacuo and the residue was acidified with HCl (1M) to a pH of 2 or less. It is then extracted with ethyl acetate, and the combined organic solvents are dried over sodium sulfate and concentrated in vacuo. Separating with silica gel column chromatography to obtainC1 white solid, yield 78.8%.¹H NMR(600MHz,MeOD)δ8.59(d,J＝5.3Hz,1H),8.56(d,J＝7.8Hz,1H),7.89(s,1H),7.80(d,J＝7.3Hz,2H),7.68(dd,J＝5.3,1.6Hz,1H),7.54(d,J＝8.3Hz,2H),7.51(t,J＝7.4Hz,2H),7.46(d,J＝7.1Hz,1H),7.38(t,J＝7.7Hz,1H),7.35(d,J＝7.5Hz,1H),7.21(d,J＝8.3Hz,2H),6.95(d,J＝8.3Hz,1H),6.88(d,J＝8.3Hz,1H),4.60(s,1H),3.29–3.27(m,1H),3.21(d,J＝14.3Hz,1H),3.11–3.03(m,2H),2.72(dd,J＝15.1,3.4Hz,1H).¹³C NMR(150MHz,MeOD)δ160.19,154.61,149.82,148.39,141.03,139.66,137.59,137.47,133.96,132.52,131.79,131.25,130.88,129.60,129.11,128.91,128.01,126.85,125.37,122.50,121.16,120.44,119.88,114.69,60.29,37.69,26.77.ESI-HRMS[M+H]⁺ calcd for C₃₂H₂₅BrNO₂,534.1063,found,534.1078.

Preparation example 22

Preparation of 2- (8- (4-bromophenyl) -5-hydroxy-9- (hydroxymethyl) -9, 10-dihydrophenanthren-1-yl) isonicotinal (C2)

See preparation 21 for reaction conditions and workup. Compound C2 was a colorless oily liquid, yield 37.0%.¹H NMR(600MHz,CDCl₃)δ10.16(s,1H),8.82(d,J＝5.0Hz,1H),8.38(d,J＝7.9Hz,1H),7.96(s,1H),7.72(d,J＝5.0Hz,1H),7.51(d,J＝8.2Hz,2H),7.44(t,J＝7.8Hz,1H),7.32(d,J＝7.6Hz,1H),7.17(d,J＝8.2Hz,2H),6.99(d,J＝8.2Hz,1H),6.83(d,J＝8.2Hz,1H),6.17(s,1H),3.47–3.40(m,2H),3.27(t,J＝11.4Hz,1H),3.13–3.11(m,1H),2.55(dd,J＝14.7,3.4Hz,1H).¹³C NMR(150MHz,CDCl₃)δ191.09,161.55,152.96,149.62,143.08,140.11,138.63,134.09,133.22,133.07,131.33,131.12,130.25,129.15,128.31,126.69,123.09,121.55,121.30,120.78,115.30,60.91,37.91,26.88.ESI-HRMS[M+Na]⁺ calcd for C₂₇H₂₀BrNO₃Na:508.0519,found:508.0534.

Preparation example 23

Preparation of 1- (4-bromophenyl) -10- (hydroxymethyl) -8- (5-methylpyridin-2-yl) phenanthren-4-ol (C3)

See preparation 21 for reaction conditions and workup. Compound C3 was a white solid in 99.0% yield.¹H NMR(600MHz,CDCl₃)δ8.37(s,1H),8.28(d,J＝7.9Hz,1H),7.66(dd,J＝7.9,1.5Hz,1H),7.50(d,J＝8.2Hz,2H),7.44(d,J＝7.9Hz,1H),7.39(t,J＝7.8Hz,1H),7.25(s,1H),7.17(d,J＝8.2Hz,2H),6.97(d,J＝8.2Hz,1H),6.82(d,J＝8.2Hz,1H),6.26(s,1H),3.52(dd,J＝14.6,2.6Hz,1H),3.41(dd,J＝11.6,5.1Hz,1H),3.27(t,J＝11.4Hz,1H),3.12-3.07(m,1H),2.50(dd,J＝14.6,3.4Hz,1H),2.37(s,3H).¹³C NMR(150MHz,CDCl₃)δ156.68,152.91,148.35,140.22,139.70,138.89,138.34,133.82,133.18,133.15,131.82,131.26,131.14,130.06,129.11,127.19,126.43,123.76,121.78,121.20,115.17,60.75,38.11,26.96,18.19.ESI-HRMS[M+H]⁺ calcd for C₂₇H₂₃BrNO₂ 472.0907,Found 472.0914.

Preparation example 24

Preparation of 1- (4-bromophenyl) -8- (5-fluoropyridin-2-yl) -10- (hydroxymethyl) -9, 10-dihydrophenanthren-4-ol (C4)

See preparation 21 for reaction conditions and workup. C4 was a white solid in 83.0% yield.¹H NMR(600MHz,CDCl₃)δ8.42(d,J＝2.3Hz,1H),8.30(d,J＝7.9Hz,1H),7.60–7.53(m,2H),7.52(d,J＝8.2Hz,2H),7.41(t,J＝7.8Hz,1H),7.24(s,1H),7.16(d,J＝8.2Hz,2H),7.00(d,J＝8.2Hz,1H),6.84(d,J＝8.2Hz,1H),5.73(s,1H),3.46–3.37(m,2H),3.23(t,J＝11.4Hz,1H),3.10(dd,J＝7.0,3.8Hz,1H),2.52(dd,J＝14.7,3.4Hz,1H).¹³C NMR(150MHz,CDCl₃)δ152.65,140.09,138.81,136.62,136.46,133.83,133.39,133.23,131.33,131.11,130.20,129.14,127.53,126.55,125.38,124.67,121.65,121.31,115.26,60.86,37.99,26.80.ESI-HRMS[M+H]⁺ calcd for C₂₆H₂₀BrFNO₂:476.0656,found:476.0667.

Preparation example 25

Preparation of 1- (4-bromophenyl) -8- (5-chloropyridin-2-yl) -10- (hydroxymethyl) -9, 10-dihydrophenanthren-4-ol (C5)

See preparation 21 for reaction conditions and workup. C5 was a white solid in 84.4% yield.¹H NMR(600MHz,CDCl₃)δ8.52(d,J＝2.3Hz,1H),8.32(d,J＝7.9Hz,1H),7.83(dd,J＝8.3,2.5Hz,1H),7.50(t,J＝8.2Hz,3H),7.41(t,J＝7.8Hz,1H),7.25(d,J＝8.1Hz,1H),7.16(d,J＝8.3Hz,2H),6.99(d,J＝8.2Hz,1H),6.82(d,J＝8.3Hz,1H),6.02(s,1H),5.27(s,1H),3.45(dd,J＝14.8,2.7Hz,1H),3.39(s,1H),3.24(t,J＝11.4Hz,1H),3.13–3.09(m,1H),2.53(dd,J＝14.8,2.5Hz,1H).¹³C NMR(150MHz,CDCl₃)δ157.82,152.83,147.15,140.10,138.71,138.58,137.54,133.93,133.27,133.11,131.32,131.11,130.81,130.23,129.03,127.89,126.59,125.20,121.59,121.29,115.26,60.88,37.93,26.84.ESI-HRMS[M+H]⁺ calcd for C₂₆H₂₀BrClNO₂:492.0360,found:492.0373.

Preparation example 26

Preparation of 6- (8- (4-bromophenyl) -5-hydroxy-9- (hydroxymethyl) -9, 10-dihydrophenanthren-1-yl) nicotinonitrile (C6)

See preparation 21 for reaction conditions and workup. C6 was a white solid in 80.0% yield.¹H NMR(600MHz,MeOD)δ8.93(s,1H),8.58(d,J＝7.7Hz,1H),8.23(d,J＝8.1Hz,1H),7.79(d,J＝8.2Hz,1H),7.54(d,J＝8.0Hz,2H),7.42–7.34(m,2H),7.21(d,J＝8.0Hz,2H),6.96(d,J＝8.3Hz,1H),6.88(d,J＝8.3Hz,1H),3.25(dd,J＝10.3,4.1Hz,1H),3.09(d,J＝15.3Hz,1H),3.02(s,1H),2.97(t,J＝10.6Hz,1H),2.76(dd,J＝15.3,3.7Hz,1H).¹³C NMR(150MHz,MeOD)δ163.20,154.58,151.25,140.95,140.17,138.47,137.36,134.16,132.81,131.76,131.23,130.90,129.75,127.97,125.48,124.84,120.84,120.47,116.28,114.71,107.97,60.28,37.61,26.88.ESI-HRMS[M+H]⁺calcd for C₂₇H₂₀BrN₂O₂:483.0703,found:483.0724.

Preparation example 27

Preparation of 1- (4-bromophenyl) -8- (5- (dimethylamino) pyridin-2-yl) -10- (hydroxymethyl) -9, 10-dihydrophenanthren-4-ol (C7)

See preparation 21 for reaction conditions and workup. Compound C7 was a white solid in 81.3% yield.¹H NMR(600MHz,CDCl₃)δ8.21(d,J＝7.8Hz,1H),8.04(d,J＝2.9Hz,1H),7.50(d,J＝8.2Hz,2H),7.37(dd,J＝18.0,8.3Hz,2H),7.25(d,J＝7.7Hz,1H),7.17(d,J＝8.2Hz,2H),7.13(dd,J＝8.7,2.9Hz,1H),6.97(d,J＝8.2Hz,1H),6.84(d,J＝8.2Hz,1H),3.57(d,J＝14.2Hz,1H),3.40(dd,J＝11.6,5.1Hz,1H),3.26(t,J＝11.4Hz,1H),3.13–3.07(m,1H),2.99(s,6H),2.49(dd,J＝14.5,3.3Hz,1H).¹³C NMR(150MHz,CDCl₃)δ152.85,144.97,140.30,139.02,133.71,133.30,133.19,131.24,131.16,129.94,129.09,126.37,124.07,121.95,121.17,115.18,60.73,40.06,38.23,27.07ESI-HRMS[M+H]⁺ calcd for C₂₈H₂₆BrN₂O₂:501.1172,found:501.1180.

Example 1

For SARS-CoV-23CL^pro(coronavirus 3CL protease) inhibition test

Testing a sample: compound A1-A28, compound B1-B20 and compound C1-C7

The test method comprises the following steps:

preparing a pre-incubation solution: mu.L of compound solutions of different concentrations, 10. mu.L of SARS-CoV-23CL^proAnd 78 μ L of phosphate buffer (pH 7.4, containing 1mM ethylenediaminetetraacetic acid) to obtain the pre-incubation solution.

The testing steps are as follows: adding 90 mu L of pre-incubation liquid into a black 96-well plate, pre-incubating for 30min at 37 ℃, adding Dabcyl-KNSTLQSGLRKE-Edans (fluorescent substrate) to initiate reaction, continuously detecting for 20min (excitation wavelength is 340nm and emission wavelength is 490nm) in a multifunctional microplate reader, collecting the fluorescence value of hydrolysate, taking disulfiram as a positive drug, and setting 3 times of repeated tests for each compound. And (3) drawing an inhibition curve by taking the concentration of the compound to be detected as a horizontal coordinate and the residual enzyme activity as a vertical coordinate, and carrying out data processing by Graph Pad Prism 7.0 software. Wherein the calculation formula of the residual enzyme activity is as follows:

the residual enzyme activity (F0-F1)/F0X 100%

F0 is the fluorescence intensity value measured when the compound to be detected is not added in the pre-incubation liquid, and F1 is the fluorescence intensity value measured when each compound to be detected is added in the pre-incubation liquid; computing IC using software₅₀The value is obtained.

The test results of the A series compounds (A1-A28) are shown in Table 1; the test results of the B series compounds (B1-B20) are shown in Table 2; the test results of the C series compounds (C1-C7) are shown in Table 3.

TABLE 1

Compound (I)	IC50±SD(μM)	Compound (I)	IC50±SD(μM)
				A1	61.15±3.60	A15	58.91±4.58
A2	33.06±8.58	A16	68.16±2.64
				A3	29.46±1.64	A17	5.65±1.40
A4	9.06±0.34	A18	66.80±2.74
				A5	6.44±0.64	A19	35.77±2.96
A6	19.32±1.92	A20	18.50±0.76
				A7	11.39±0.64	A21	14.17±1.30
A8	>100	A22	11.97±0.68
				A9	>100	A23	13.82±0.90
A10	>100	A24	>100
				A11	85.58±2.36	A25	>100
A12	>100	A26	>100
				A13	57.67±2.78	A27	>100
A14	53.81±8.49	A28	>100

TABLE 2

Compound (I)	IC50±SD(μM)	Compound (I)	IC50±SD(μM)
				B1	8.54±0.94	B11	4.29±0.62
B2	2.46±0.14	B12	2.72±0.23
				B3	9.82±1.16	B13	6.73±1.29
B4	4.79±0.22	B14	2.78±0.18
				B5	10.26±0.66	B15	8.89±3.34
B6	3.32±0.25	B16	6.65±0.26
				B7	10.00±0.47	B17	5.30±0.34
B8	11.23±0.70	B18	3.66±0.17
				B9	7.39±0.11	B19	8.28±1.88
B10	5.25±0.24	B20	3.24±0.43

TABLE 3

As is clear from the data in tables 1, 2 and 3, SARS-CoV-23CL is treated^proIC of A series compound, B series compound and C series compound₅₀The value is basically between 1 and 70 mu M; IC of the A series of compounds₅₀The value is basically 10-100 mu M; IC of B series compound₅₀The value is basically between 2.7 and 12 mu M; IC of C series compound₅₀The value is substantially 1.5-6. mu.M.

As can be seen from Table A, among the A series of compounds, A17 is against SARS-CoV-23CL^proHas the strongest inhibiting effect, IC₅₀The value was 5.65. mu.M, indicating R⁸Is phenyl substituted pyridine, and is useful for improving SARS-CoV-23CL of compound^proInhibition of (d); as can be seen from Table B, among the B series of compounds, B2, B12 and B14 were directed against SARS-CoV-23CL^proHas the strongest inhibiting effect, IC₅₀The value was around 2. mu.M, indicating that R¹³The phenyl substituted pyridine, the chlorine substituted pyridine or the cyano substituted pyridine can help to improve the SARS-CoV-23CL of the compound^proInhibition of (d); as can be seen from Table C, in the C series of compounds, compounds C1 and C2 were directed against SARS-CoV-23CL^proHas the strongest inhibiting effect, IC₅₀The value was about 1.5. mu.M, indicating that R¹³Is phenyl substituted pyridine or aldehyde substituted pyridine, and is helpful for improving SARS-CoV-23CL of compound^proThe inhibitory action of (1).

As shown in FIG. 1, B1-B20 is directed to SARS-CoV-23CL^proThe inhibition effect of B1-B20 on SARS-CoV-23CL is shown in the figure, compared with that of the blank control^proHas obvious inhibiting effect, and the residual enzyme activity is below 40 percent for most compounds at the concentration of 10 mu M. As shown in FIG. 2, C1-C7 is shown for SARS-CoV-23CL^proThe inhibition effect of (A) is shown in the figure, and the residual enzyme activity is about 30% for C1 and C2 and about SARS-CoV-23CL at the concentration of 10 mu M^proShows obvious inhibiting effect.

Example 2

Determination of the inhibition constant Ki

Testing a sample: compound C1 and compound C2

The test method comprises the following steps: adding 90 mu L of pre-incubation liquid into a black 96-well plate, pre-incubating the pre-incubation liquid at 37 ℃ for 30min, respectively adding a series of Dabcyl-KNSTLQSGLRKE-Edans (fluorescent substrates) with different concentrations to initiate reaction, continuously detecting for 20min (excitation wavelength is 340nm and emission wavelength is 490nm) by a multifunctional microplate reader, and collecting the fluorescence value of the hydrolysate. The reciprocal of the substrate concentration is used as the abscissa and the reciprocal of the fluorescence value of the hydrolysate is used as the ordinate, the data are processed by GraphPad Prism 7.0 software, the inhibition pattern of the measured object is determined by Lineweaver-Burk mapping, and the corresponding K is calculated by Lineweaver-Burk quadratic mapping_iThe value is obtained.

Fitting data using competitive inhibition equation (1), non-competitive inhibition equation (2), and mixed inhibition equation (3):

V＝(V_maxS)/[K_m(+I/K_i)+S] (1)

V＝(V_maxS)/[(K_m+S)(1+I/K_i)] (2)

V＝(V_maxS)/((K_m+S)(1+I/αK_i)) (3)

v is the speed of the reaction; k_iIs an inhibition constant describing the affinity of the enzyme inhibitor; s and I are respectively substrate concentration and inhibitor concentration; v_maxIs the maximum speed; k_mIs the mikeley constant (substrate concentration of 0.5V)_max). The goodness of fit parameter is used to determine the most appropriate type of inhibition kinetics.

C1 vs SARS-CoV-23CL as shown in FIG. 3^proThe dose inhibition curve of (A) shows that when the concentration of C1 is 0-20. mu.M, the concentration of the compound is opposite to that of SARS-CoV-23CL^proThe influence of the inhibition effect is great, and the residual enzyme activity is sharply reduced along with the increase of the concentration; when the concentration of C1 exceeds 40 mu M, the residual enzyme activity is basically unchanged; c1 vs SARS-CoV-23CL as shown in FIG. 4^proLineweaver-Burk plot of inhibition, where the abscissa "fluorogenic substrate" is Dabcyl-KNSTLQSGLRKE-Edans; c1 dose-dependently inhibits SARS-CoV-23CL by means of mixed inhibition^pro，K_iThe value was 6.09. mu.M.

C2 vs SARS-CoV-23CL as shown in FIG. 5^proThe dose inhibition graph can be seen from the figure that when the concentration of C2 is 0-10. mu.M, the concentration of the compound is opposite to that of SARS-CoV-23CL^proThe influence of the inhibition effect is great, and the residual enzyme activity is sharply reduced along with the increase of the concentration; when the concentration of C2 exceeds 20 mu M, the residual enzyme activity is basically unchanged; FIG. 6 shows that C2 is against SARS-CoV-23CL^proLineweaver-Burk plot of inhibition, in which the abscissa "fluorogenic substrate" is Dabcyl-KNSTLQSGLRKE-Edans, C2 dose-dependently inhibits SARS-CoV-23CL by means of mixed inhibition^pro,K_iThe value was 7.64. mu.M.

Example 3

Phase I metabolism (hcPPs) stability assay

Testing a sample: compound C1 and compound C2

The test method comprises the following steps: add 4. mu.M test compound (C1 and C2) to 1.5mL EP tubes, respectively; the buffer and various cofactors were added to the EP tube as in Table 4, mixed by gentle shaking, and then pre-incubated for 3 minutes at 37 ℃ in a constant temperature mixer. Reaction cofactors were added at 30s intervals to initiate the reaction (NADP +). After the reaction was carried out for various times, 100. mu.L of the sample was taken from the incubation EP tube and placed in a solution containing 100. mu.L of glacial acetonitrile, and the reaction was terminated by vigorous shaking. The stop solution was centrifuged at 20000g (14300r/min) at 4 ℃ for 20 minutes and the supernatant was aspirated for liquid phase analysis.

TABLE 4

Wherein, NADP⁺Is nicotinamide adenine dinucleotide phosphate, G-6-P is glucose-6-phosphate, G-6-PDH is glucose-6-phosphate dehydrogenase, and HLM is human liver microsome.

The results are shown in figure 7, where C1 is hardly metabolized by hCYPs, whereas C2 is easily metabolized by hCYPs, with an in vitro half-life of 32.09min, compared to testosterone, which is a positive drug.

Example 4

Phase II metabolism (hcPPs) stability assay

The test method comprises the following steps: HLM and Brij 58 (punching agent) are mixed in an EP tube according to the volume ratio of 1:1, and placed in ice for punching for 20 min. The compound to be tested, the buffer solution and various cofactors were added to 1.5mL of EP tubes at the concentrations and volumes shown in Table 5, mixed by gentle shaking, and pre-incubated for 3 minutes at 37 ℃ in a constant temperature mixer. The reaction was initiated by adding the reaction cofactors (UDPGA) at 30s intervals. After the reaction was carried out for various times, 100. mu.L of the sample was taken from the incubation EP tube and placed in a solution containing 100. mu.L of acetonitrile, and the reaction was terminated by vigorous shaking. The stop solution was centrifuged at 20000g (14300r/min) at 4 ℃ for 20 minutes and the supernatant was aspirated for liquid phase analysis.

TABLE 5

Wherein UDPGA uridine diphosphate glucuronate and Tris-HCl are Tris hydrochloride.

The experimental result is shown in fig. 8, compared with the positive drug 7-hydroxycoumarin, C1 is hardly metabolized by hUGTs, while C2 is easily metabolized by hUGTs, and the in vitro half-life is 81.88 min. The results of combining example 3 and example 4 show that C1 has outstanding liver metabolic stability and is a novel oral SARS-CoV-2-3CL^proGreat potential for inhibitor development.

Example 5

C1 and SARS-CoV-23CL^proProtein molecule docking

The butt joint method comprises the following steps: molecular docking software is adopted to carry out the alignment of C1 and SARS-CoV-23CL^proMolecular docking simulation of the substrate binding region and dimer interface of the protein, SARS-CoV-23CL^proThe PDB of the protein is PBE 7.

As shown in FIG. 9, in the dimer interface region, Compound C1 was bound to SARS-CoV-23CL^proGlu290 of the protein can form 1 hydrogen bond interaction with the distance of

Can form a cation-pi action with Lys 5; furthermore, the compounds can form a hydrophobic interaction with Phe3, Tyr126 in this region.

As shown in FIG. 10, in the substrate binding region, the phenolic hydroxyl group of compound C1 acts as a hydrogen bond acceptor to form two hydrogen bonds with Cys145 and Gly143 at respective distances of

The alcoholic hydroxyl group is used as a hydrogen bond donor to form a hydrogen bond with Gln189 at a distance of

In addition, the compounds formed hydrophobic interactions with Leu167, Pro168 in this region.

The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims (10)

1. An application of 9, 10-dihydrophenanthrene compound in preparing coronavirus 3CL protease inhibitor is disclosed.

2. The use according to claim 1, wherein the 9,10 dihydrophenanthrene compound has the structure shown in formula I below:

in the formula I, R is¹-R⁷Each is independently selected from any one of hydrogen, cyano, nitro, hydroxyl, aldehyde group, carboxyl, sulfonic group, ester group, acyl, sulfone group, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amino, substituted or unsubstituted heterocyclic group and substituted or unsubstituted aryl; or, R¹-R⁷Each independently forms a saturated or unsaturated carbocyclic or heterocyclic ring with an adjacent benzene ring through a covalent bond;

in the formula I, R is⁸Selected from substituted or unsubstituted nitrogen-containing heterocyclic groups;

in the formula I, R is⁹And is selected from any one of hydroxyl substituted alkyl, ester substituted alkyl or heteroatom substituted alkyl.

3. Use according to claim 2, wherein R is¹-R⁷Each independently selected from substituted alkyl groups,Any one of substituted alkenyl, substituted alkynyl, substituted alkoxy, substituted amino, substituted heterocyclic radical or substituted aryl, wherein the substituents of the alkyl, alkenyl, alkynyl, alkoxy, amino, heterocyclic radical and aryl are respectively and independently selected from any one of cyano, nitro, amino, hydroxyl, aldehyde group, carboxyl, sulfonic group, ester group, acyl, sulfone group or halogen;

preferably, said R is¹Any one selected from hydrogen, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, preferably any one selected from unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, cyano substituted C1-C6 alkyl, unsubstituted C6-C12 aryl, halogen substituted C6-C12 aryl or cyano substituted C6-C12 aryl, more preferably any one selected from unsubstituted C1-C3 alkyl, unsubstituted C5-C8 cycloalkyl, unsubstituted phenyl, halogen substituted phenyl or cyano substituted phenyl;

preferably, said R is²、R³Or R⁵-R⁷Each independently selected from any one of hydrogen, halogen, substituted or unsubstituted alkyl and substituted or unsubstituted alkoxy, preferably any one of hydrogen, halogen, unsubstituted C1-C6 alkyl or unsubstituted C1-C6 alkoxy;

preferably, said R is²Or R³Selected from hydrogen;

preferably, said R is⁵Any one selected from hydrogen, halogen, unsubstituted C1-C6 alkyl or unsubstituted C1-C6 alkoxy;

preferably, said R is⁶Selected from hydrogen or unsubstituted C1-C6 alkyl;

preferably, said R is⁷Selected from hydrogen or unsubstituted C1-C6 alkyl;

preferably, said R is⁴Selected from hydroxy or unsubstituted C1-C6 alkoxy;

preferably, said R is⁸Selected from substituted or unsubstituted pyridyl, substituted or unsubstituted quinolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted pyrrolopyrimidyl, substituted or unsubstituted pyrimidyl orAny one of substituted or unsubstituted benzimidazolyl;

preferably, said R is⁸Selected from nitrogen-containing heterocycles as shown in the following structure, wherein,

represents a substituent attachment position:

wherein R is^aAny one selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, cyano, halogen, C1-C6 alkoxy, C1-C6 acyl, C1-C6 sulfonyl, C6-C12 aryl, alkyl-substituted amino, aryl-substituted amino or C3-C6 nitrogen-containing heterocyclic group;

preferably, said R is⁹Selected from ester group substituted C1-C6 alkyl or hydroxyl substituted C1-C6 alkyl, preferably the structure shown in the specification, wherein,

represents a substituent attachment position:

4. the use according to any one of claims 1 to 3, wherein the 9,10 dihydrophenanthrene compound is selected from any one of the following A1-A28:

5. the use according to any one of claims 1 to 4, wherein the 9,10 dihydrophenanthrene compound has the structure shown in formula II below:

wherein R is¹⁰Is halogen, R¹¹Is any one of hydrogen or unsubstituted C1-C6 alkyl, R¹²Is any one of hydrogen or unsubstituted C1-C6 acyl, R¹³Is any one of cyano, halogen, alkyl substituted amino, aryl substituted amino, unsubstituted C1-C6 alkyl, unsubstituted C1-C6 acyl, unsubstituted C1-C6 sulfonyl, unsubstituted C6-C12 aryl or C3-C6 nitrogen-containing heterocyclic group;

preferably, the 9,10 dihydrophenanthrene compound has a structure shown as II-B or II-C:

preferably, the II-B is selected from any one of the following B1-B20:

preferably, the II-C is selected from any one of the following C1-C7:

6. use according to any one of claims 1 to 5, wherein the 9,10 dihydrophenanthrene compound is used for the preparation of a medicament for the treatment of diseases caused by novel coronaviruses and/or novel mutant strains of coronaviruses.

7. The use of any one of claims 1-6, wherein the 3CL protease inhibitor further comprises a pharmaceutically acceptable adjuvant;

preferably, the pharmaceutically acceptable auxiliary materials comprise any one or a combination of at least two of vitamin C, sorbitol, mannitol, xylitol, fructose, amino acid, meglumine, dextrin, magnesium stearate or sucrose.

8. The use of any one of claims 1-7, wherein the 3CL protease inhibitor is in a dosage form comprising any one of an injectable formulation, an oral formulation, or a lyophilized powder for injection.

9. The use according to any one of claims 1 to 8, wherein the 3CL protease inhibitor is in the form of an oral formulation comprising any one of a tablet, powder, capsule or granule.

10. The use of any one of claims 1-9, wherein the 3CL protease inhibitor further comprises a pharmaceutically acceptable excipient;

preferably, the pharmaceutically acceptable excipient comprises any one of a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer or a colorant, or a combination of at least two thereof.

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